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Geographic and host-induced variations of Haematoloechus buttensis and a re-evaluation of representatives… Kennedy, Murray James 1978

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G E O G R A P H I C AND HOST—INDUCED V A R I A T I O N S OF _ M _ _ _ _ _ _ C H 2 S B U T T E N S I S AND A R E - E V A L U A T I O N OF R E P R E S E N T A T I V E S OF T H E GENUS I N CANADA AND THE U N I T E D S T A T E S MURRAY J A M E S KENNEDY B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 7 1 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1974 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF THE REQUI R E M E N T S FOR T H E DEGREE OF DOCTOR OF P H I L O S O P H Y i n T H E DEPARTMENT OF ZOOLOGY l e a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H C O L U M B I A M a r c h , 1 9 7 8 ( cP) M u r r a y James Kennedy, 1978 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f - " g o The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 D a t e - ^ W / Z*0,/??? ABSTRACT Of t i e f i f t e e n s p e c i e s of f l u k e s r e f e r r e d t o t h e genus Haeffiatoloechus Looss. 1899, i n Canada and the U n i t e d S t a t e s , nine are c o n s i d e r e d t o be synonyms of one or another o f the s i x s p e c i e s c o n s i d e r e d to be v a l i d . S p e c i e s i d e n t i f i c a t i o n i s p r i m a r i l y based on the r a t i o of the t r a n s v e r s e diameter of the o r a l sucker t o the acetabulum (0/A), the a n t e r i o r extent of the e x t r a c a e c a l loops along the ovary or t e s t e s , and o r i e n t a t i o n of the /testes.. ; The s p e c i e s c o n s i d e r e d v a l i d are: H. l o n g i p l e x u s S t a f f o r d . 1902: H. b r e v i p l e x u s S t a f f o r d , 1902; H. v a r i o p l e x u s S t a f f o r d , 1902 <= H. s i m i l i p i e x u s . = H. p a r v i p l e x u s . = •H.,v|rf;b-ufetea-si-s,,- =- H. f l o e d a e , = • fl. u n i p l e x u s ); H. medjoplexus S t a f f o r d . 1902: B. complexus f S e e l y . 1906) f= H. co l o r a dens i s . = cpnfusus.- • ~ •>• ii. / o x y o r c h i s - - ); >"H.-^k'ey-tteas'is - I n g l e s , 19 32 (= H. tumidus ) . Flukes are d i s c u s s e d u s i n g the name p r e v i o u s l y c o n s i d e r e d v a l i d . r H « v b u t t e n i S l s . e x p e r i m e n t a l l y reared i n the l a b o r a t o r y , was used t o study m o r p h o l o g i c a l v a r i a t i o n s r e s u l t i n g from changes i n t e c h n i c a l procedures and environment. Technigues used t o prepare study specimens of f l u k e s a f f e c t e d some taxonomic c h a r a c t e r s p r e v i o u s l y used to s e p a r a t e s p e c i e s . The presence o f s p i n e s , p o s i t i o n o f o r a l sucker, and s i z e of f l u k e s were a f f e c t e d by temperature of f i x a t i v e , and the use of d i s t i l l e d water. Pre s s u r e added to the c c v e r s l i p a f f e c t e d l e n g t h and width of ovary, t e s t e s , sucker, and body., The s i z e o f f l u k e s , e x p e r i m e n t a l l y r e a r e d i n the f r o g , R. p r e t j o s a , was a f f e c t e d by temperature at which the host was maintained, the numbers of metacercariae f e d t o f r o g s , and the age of the worm. Host s i z e and sex had no apparent i n f l u e n c e on f l u k e morphology. S i x t y - d a y - o l d f l u k e s were ex p e r i m e n t a l l y r e a r e d i n e i t h e r R. p r e t i o s a . B , • o r e a s , R, c l a m i t a n s . or R. a u r o r a , The e f f e c t s of d eveloping i n d i f f e r e n t f r o g hosts were to a l t e r the a n t e r i o r extent of the e x t r a c a e c a l u t e r i n e l o o p s r e l a t i v e to the p o s t e r i o r t e s t i s , the d i s t r i b u t i o n of. •the v i t e l l a r i a , and the s i z e and shape of ovary, t e s t e s , and s i z e of the body and suckers. The 0/& r a t i o remained r e l a t i v e l y uniform i n f l u k e s from a l l hosts. Flukes recovered from f r o g s fed i n f e c t e d d r a g o n f l i e s had a l a r g e r body s i z e (length and width), l a r g e r t e s t e s and o v a r i e s , and had e x t r a c a e c a l l o o p s r e a c h i n g f a r t h e r along the p o s t e r i o r t e s t i s than d i d f l u k e s recovered from f r o g s fed i n f e c t e d d a m s e l f l i e s . The 0/a r a t i o s f o r •• H. long i p l e x us. H.. fare v i plexus, H.= .com plexus, H, b u t t e n s i s , H. c o l o r a d e n s i s , and H. medioplexus did not vary among specimens c o l l e c t e d from d i f f e r e n t l o c a l i t i e s . No s i g n i f i c a n t d i f f e r e n c e i n t h i s r a t i o occurred between f l u k e s t h a t had i n h a b i t e d more than one host. The 0/a r a t i o o f H. p a r v i p l e x u s was s i g n i f i c a n t l y g r e a t e r when R. s y l v a t i c a was the d e f i n i t i v e host than when R. c a t e s b e i a n a was the host. T h i s r a t i o a l s o d i f f e r e d s i g n i f i c a n t l y between specimens c o l l e c t e d from two l o c a l i t i e s i n Nebraska, even though the host i n both l o c a l i t i e s was R. c a t e s b e i a n a . Pooled data f o r H. v a r i o p l e x u s and H. s i m i l i p l e x u s i n d i c a t e d t h a t f l u k e s from R. c a t e s b e i a n a . R. c l a m i t a n s . i v R. p i p i e j j s , and B. woodhousei d i f f e r e d i n t h e i r 0/A r a t i o s and egg l e n g t h s and widths. Ovary and t e s t e s may be lobed or unlobed. U t e r i n e loops extend from the p o s t e r i o r p o r t i o n of the worm t o near the a n t e r i o r border of the a n t e r i o r t e s t i s i n H. v a r i o p l e x u s , H. b u t t e n s i s . and H. par v i p l e x u s . T h e ext r a caeca1 l o o p s reach t o the p o s t e r i o r border o f the ovary i n H. b r e v i p l e x u s and beyond the a n t e r i o r border o f t h e ovary • i n •fiy^loii^jpi^gaca-s.::..-In f l u k e s c o n t a i n i n g e x t r a c a e c a l u t e r i n e loops, the l e f t loop vas absent i n 4.2% of the H. p a r v i p l e x u s and 8.3% of H. v a r i o p l e x u s examined, the r i g h t l oop was absent i n 9.051 of •: H. b u t t e n s i s examined.- ,• ,. Egg s i z e ( l e n g t h and width) d i d not vary g e o g r a p h i c a l l y i n H. l c n g i p l e x u s , H. complexus. H. b r e v i p l e x u s , H. buttensis„ H. c o l o r a d e n s i s . o r fl. medioplexus. Egg l e n g t h s o f H. p a r v i p l e x u s d i f f e r e d i n f l u k e s from R. s y l v a t i c a and R. c a t e s b e i a n a . TABLE OF CONTENTS Abs t r a c t •• *'.y»:» »y'.«-. • y y»• • »•«• • • • • • •» v> • • •« *:»•.• .•  » .. »-.•».,:•ii Table of Contents .....................«...•••......* v L i s t of Tables .".y. • vy vv v v> «' v« v«' «• v «- « i »:»-v «• • L i s t of F i g u r e s and Baps , * . . . . . . . . . . . . . . . . . . . . y y . . i x L i s t of Appendices ..... ».y . . . y •*.,» , » x i v Acknowledgements .... ...... ............ •-• ..•....,.«,. x v i I n t r o d u c t i o n . • '.-.•-.yyy v.yy''.*y:y.':v.''. *••.••. •,'*•'..•» v . 1 L i f e C y c l e of Hae matolpec bu s b u t t e n s i s ... .......... 7 I n t r o d u c t ion';''vy>->rvy»v .y/vyyy.'vv^ «.*»..y«'«« .yv»». .• 7 M a t e r i a l s and Methods .. .. ,..*............... , 7 Re s u l t s and D i s c u s s i p n 9 Part I. M o r p h o l o g i c a l v a r i a t i o n . . . . y ^ » . . , , . . , . . . . . , . 13 1. E f f e c t s o f F i x a t i o n and F l a t t e n i n g on A d u l t Morphology • •••>^y;'»>:yyy;i.:y.y>v.;yyy:yyf.yy.:;.>;.iyyy.;-v» ••./» .... »••• *:••» . . 13 In t r o d u c t i o n ' ...".y . v.'V.y'Vyy:'.yyyyv.y"../. , 13 M a t e r i a l s and Methods• v . y y y y y y y y y M : . v . ' , v . , , . - , , . . ; . , f ' 14 P r e p a r a t i o n Method I .y i y y . . . .. ...... ^ ....... ..... 15' Re s u l t s of Method I «»*« • 1 7 P r e p a r a t i o n Method I I 17 R e s u l t s of Method I I 18 P r e p a r a t i o n Method I I I .'.>'V.^.:.^-.V'.'.;r.i.y.y.y»:;*.•,; 23 R e s u l t s of ! Method I I I . . . . . . . . . . . . i .yy.... . . y y * v. 23 P r e p a r a t i P n Methpd IV 26 Re s u l t s pf Method IV i . . . .. . 26 • 2. V a r i a t i o n i n f l u k e s developed i n the n a t u r a l d e f i n i t i v e host, B. p r e t i o s a ........................ 28 i ,Introduction , v - . : » . • ^ • l y * : * ; ^ ^ 28 M a t e r i a l s and Methods 29 Re s u l t s 31 3. V a r i a t i o n i n f l u k e s developed i n R . p r e t i p s a maintained a t d i f f e r e n t temperatures ......,..,,».... 43 I n t r o d u c t i o n .............,..-,....... v........... 43 M a t e r i a l s and Methods 43 .Results-' yyy.;:V.vy;y. . .yy.yv .TWv.*:v.vyv.v. v . . " . . > *. 4.3 4. E f f e c t s of host s i z e on development of H. b u t t e n s i s . .... ...... ..... .y v. . . ... ,-. 49 I n t r o d u c t i o n 49 M a t e r i a l s and Methods 49 Resul t s 49 5. E f f e c t s of host sex on development of H. b u t t e n s i s 51 . I n t r o d u c t i o n ,r ry>yy^yVvvvw^ 51 M a t e r i a l s and Methods 51 R e s u l t s ................ 51 6. E f f e c t s of worm burder on development of H. b u t t e n s i s vy.-vyyyyv-^ , 53 I n t r o d u c t i o n 53 M a t e r i a l s and Methods ........................v.. 53 . Results ................. *. . . .............., . • 53 7.,., V a r i a t i o n i n worms developed i n d i f f e r e n t d e f i n i t i v e hosts ...................... .... .......... 57 Introduction-..••yyy^.....v. .• v»>-.. .y. • 57' M a t e r i a l s and Methods 57 Res u l t s • 60 v i i 8. V a r i a t i o n due to development i n d i f f e r e n t i n s e c t i n t e r m e d i a t e h o s t s ...,.,....,.......... ...... 72 I n t r o d u c t i o n ... 72 M a t e r i a l s and Methods 72 Re s u l t s 73 Morphological v a r i a t i o n i n H . b u t t e n s i s w he n c e r c a r i a e are developed i n v a r i o u s s n a i l hosts ...... 76 : I n t r o d u c t i o n .... ": 76 M a t e r i a l s and Methods , 76 Result s . ..... 77 10. D i s c u s s i o n and C o n c l u s i o n s frem Experimental R e s u l t s :. . y . y y v.y V* vs.-*"* . - . . . . . . . . ..7.. v-. vv. ... ... .... ....... .. ,y 78 • Part I I . taxonomy and M o r p h o l o g i c a l V a r i a t i o n ....... 88 , i n t r o d u c t i o n - . 88 M a t e r i a l s and Methods . 88 Re s u l t s » .... .... . ' 89 Generic Diagnosis 93 V a l i d S p e c i e s Group I 95 V a l i d Species Group I I . y.ivv.•'.•.•••.»•.''",:V.vv,;.131 D i s c u s s i o n and Co n c l u s i o n s 1 4 4 A. D i f f e r e n t i a t i o n o f s p e c i e s .. 141 B. , Examining Type Sp ec linens .,, .y . .... ........ ..... . 153 Part I I I , Taxonomic D i s c u s s i o n 156 A Key t o the Haematoloecbus sp. i n Canada and the United -States'^.v.vv..yy.v<f^ . Summary-:.;.-y.:yy.,>:.y,.y?.:.;,-;.^  ./.v.^VvY,i.V:.-v.,;. *.....:.• 171 L i t e r a t u r e C i t e d .................................... 177 Appendix- ••.^.:V'.:r*--"^ 185 LIST OP TABLES v i i i Table 1 E f f e c t of p r e — f i x a t i v e s o l u t i o n on H. b u t t e n s i s .. 19 2 E f f e c t o f temperature on l e n g t h and width o f H. b__.tensis 21 3 E f f e c t of temperature and f i x a t i v e on morphology ;• of H. b u t t ^ n s i s •-. ,-..vvv .'.yy-.'.yyvvywr ... .24 4 E f f e c t s of crowding on H., but t e n s i s .................... 55 5 C o l l e c t i n g s i t e s f o r u n i n f e c t e d d e f i n i t i v e hosts 59 6 Age at ma t u r i t y o f H. b a t t e n s i s developed i n d i f f e r e n t d e f i n i t i v e hosts 61 ,7 Some morphological v a r i a t i o n i n H. but ten s i s reared i n d i f f e r e n t i n s e c t hosts 74 8 Summary of amphibian hosts examined f o r n a t u r a l i n f e c t i o n s o f Haematoloechus spp. . . v . i . . . .y« .91 LIST OF FIGURES AND MAPS Fi g u r e 1 L i f e c y c l e o f Haematoloechus b u t t e n s i s . . . . y . . . . . . » . . . . . It 2 Dlgraffloatic f i g u r e o f fl a e ma to1oechus sp. ..»...,,...,... 16 3 Percent i n c r e a s e i n body s i z e o f f l u k e s maintained i n d i s t i l l e d water 20 4 p e r c e n t i n c r e a s e i n body s i z e of f l u k e s maintained a t d i f f e r e n t temperatures 22 5; E f f e c t o f f i x a t i o n and temperature on body s i z e , of---_..^butten.sis--w 25 6 E f f e c t o f f l a t t e n i n g on Jf. b u t t e n s i s . 27 7 Change i n body p r o p o r t i o n o f H. b u t t e n s i s developed i n R. p r e t i o s a . .... ..«...,«. . 34 8 M e t a c e r c a r i a from i s c h n u r a perparva .................... 37 9 F i v e - d a y — o l d f l u k e developed i n R. p r e t i o s a ............ 37 10 Fourteen-day o l d f l u k e developed i n R. p r e t i o s a ........ 37 11 Twenty—one day o l d f l u k e developed i n R. p r e t j o s a ...... 37 12 Twenty—eight day o l d f l u k e developed i n R. ; p r e t i o s a ...« 37 13 s i x t y - d a y o l d f l u k e developed i n R. p r e t i o s a ;.«:^v.v;4;.>.'.,37 14—18 v a r i a t i o n i n s i z e , shape and p o s i t i o n o f ovary and t e s t e s of 60-day o l d worms developed i n B. p r e t i o s a 38 19 Change i n O/A r a t i o with age of H. b u t t e n s i s ........... 41 20, R e l a t i o n s h i p between worm age and sucker diameter ,? ^ of: H. b u t t e n s i s . . s . v v v > v , ^ . ; •>,-••;• *V,/.wy.y.;^ 21 I n f l u e n c e o f temperature and age of worm on H. b u 11 en s i s d eveioped i n R. fifjgt i o s a .. ............ y ^6 X 22 E f f e c t of temperature on gonads of H. b u t t e n s i s developed i n R. p r e t i o s a .. . 47 23j R e l a t i v e growth o f the hind body of H. b u t t e n s i s ....... 48 24 Change i n l e n g t h and width o f H. b u t t e n s i s developed . i n d i f f e r e n t • s i z e s o f R. p r e t i o s a •;. .«/,:V,.4yY ,^'Y;yi'yy>:y>y>i,-. . 50 25 I n f l u e n c e of host sex on H. b u t t e n s i s .................. 52 26 E f f e c t s of crowding on H. b u t t e n s i s .................... 56 27 Body s i z e o f fl. b u t t e n s i s developed i n d i f f e r e n t f r o g h o s t s •'. -> Y*"*M':*:^»-:** •., y • »;vi-.! ./>;>•......... 62 28 E f f e c t on sucker s i z e o f J . b t i i i e n g l s developed i n d i f f e r e n t f r o g h o s t s 64 29 Fluke developed i n R. p r e t j p s a . 66 30 Fluke, developed i n R.•• aucora ..vi.y/»y;;.,..yv*-. .- ...yy.yyy .y> v.v 66 31 Fluke developed i n R. c l a m i t a n s . ........ .y .... 66 32 , Fluke developed i n j}. boreas .............. ........ ..... 66 33 Fluke developed i n R. c l a m i t a n s •.yr,:>w.>.--.-i..-.v..W.y'.v.y;. . 6 6 34 ,,: Fluke developed i n 1 1 . aurora ••'.w«?y»**y;V:V^ 35 The e f f e c t o f d i f f e r e n t amphibian hosts on gonads 67 36 D i s t r i b u t i o n o f v i t e l l a r i a i n worms developed ._ i n R. p r e t i o s a . . v.:.'.v.. Y . . . .Y:.iV:-.y..y>y;*..-. y . y y . .. . 69 37 D i s t r i b u t i o n of v i t e l l a r i a i n worms developed I. i n R. aurora ,.:':.vv../.!...vv. 69 38 D i s t r i b u t i o n of v i t e l l a r i a i n worms developed i n •R-. :!-j;jclamitans . 6 9 39 V a r i a t i o n s i n H. b u t t e n s i s r e a r e d i n d i f f e r e n t i n s e c t i n t e r m e d i a t e hosts - v . . . . . 75 40 ; H. paryipj.exus redrawn from type specimen .............. 97 41 J . b r e v i p l e x u s redrawn from S t a f f o r d (1902) 97 x i 42 ^.--H.^lpnqiplexus redrawn from S t a f f o r d (1 902) 97 43 H.: y l p ^ g i p l e x a s showing v a r i a t i o n i n e x t r a c a e c a l 44 H. l o n g i p l e x n s showing v a r i a t i o n i n e x t r a c a e c a l 45 Abnormal ext e n t o f e x t r a c a e c a l u t e r i n e loops 46 . Abnormal extent o f e x t r a c a e c a l u t e r i n e l o o p s 47 Abnormal t e s t e s p o s i t i o n i n H. l o n q j p l e x u s 101 48 v a r i a t i o n i n egg measurements of• • H.rjlonqlplex :us•• from d i f f e r e n t ; l o c a l i t i e s 1 ................................... 102 49 C/A r a t i o of - fl. p ar v ji p i e x us from c o l l e c t i o n s made i n B r i t i s h Columbia and Nebraska ......111 50-52 Some v a r i a t i o n i n shape of o v a r i e s and t e s t e s and i n e x t e n t o f e x t r a c a e c a l u t e r i n e l o o p s i n H. p a r v i p l e x u s 53 Lenqth and width o f eggs of H. p a r v i p l e x u s from 54 0/A r a t i o of _{. y a r i o p l e x u s and J . s i m i l i p l e x u s from ,£5;. ^ l e n g t h ; and width o f eggs of H. v a r i o p l e x u s and H. s i m l l i p l e x u s from d i f f e r e n t l o c a l i t i e s ......119 56-59 V a r i a t i o n i n exte n t o f e x t r a c a e c a l u t e r i n e loops 60 Lcbed o v a r i e s and ov e r l a p of t e s t e s i n H. b u t t e n s i s .... 122 61 Lobed o v a r i e s and o v e r l a p o f t e s t e s i n fl.vbutt^nsis ..v.122 ^2 Extent of e x t r a c a e c a l u t e r i n e loops i n H.•%,-b,Ut-tenisis--..y;. .122 x i i 63 fl. b u t t e n s i s redrawn from type m a t e r i a l 125 64 H. s i r o i l i p l e x u s redrawn from S t a f f o r d {1902) . 1 2 5 65 H. v a r i p p l e x u s redrawn from S t a f f o r d (1 902) ........... . 125 66 fl. f l o g d a g redrawn from the type specimen .............,125 67 .:> H.' u n i p l e x u s redrawn from the type specimen 125 68 ,H. complexus redrawn from S e e l y (1906) ..,..,....,».... .128 69 fl. ke r h e n s j s redrawn from the type specimen ..,. ,...... .128 70 H. tumidus redrawn from the type specimen »v.:.yyyv•>•• 128 71. O/fi r a t i o of fl. medioplexus from d i f f e r e n t l o c a l i t i e s .,134 72. H. o x y o r c h i s redrawn from the type specimen .136 73 fl. confusus redrawn from the type specimen ...v.y.......136 74 H. c o l o r a d e n s i s redrawn from Cort (1915a) ..............136 75. >-,fl..:-/medioplex-us redrawn from S t a f f o r d {1902)^--yV>y,:>;:#:,.^y*..136 76 Length and width of eggs of H. c g i e l e x u s from • d i f f e r e n t l o c a l i t i e s - - . : y v v > y.-V« > Y > / . Y y ^ ^ 140 77 The shape of p o s t e r i o r u t e r i n e c o i l s o f H. c o l o r a d e n s i s when the f l u k e i s r e c o v e r e d from B. p i g l e n s ............ 141 78 The shape of p o s t e r i o r u t e r i n e c o i l s of a . c o l o r a d e n s i s , : when the f l u k e i s recovered from- R. b l a i r i 141 79-80 Some v a r i a t i o n s i n u t e r i n e l o o p s of---•p./..colora'densis~::.141 81 A proposed d e r i v a t i o n of the body types which c o n s t i t u t e , fl. sowglgxjs {=H. miS&SMs* f . ^ JL* a&as, 82—83 D e l i n e a t i o n of some worm measurements . y-wyy.y.y>.Y:ICY4;vWl88 84 V a r i a t i o n i n s i z e and shape of eggs taken from d i f f e r e n t p a r t s o f the uterus of H, b u t t e n s i s .,195 x i i i Map 1 Variation in 0/A r a t i o between populations of H. ioriqiplexus . ..... -.. v.'.v..v. v . v.;. v.;.. 100 Map 2 Areas providing samples of H. '.:>breylp'lex:u:s-v.. v . . .v4v.<106 Map 3 The d i s t r i b u t i o n of the Rana pipiens Complex (After Pace, 1958} and t h e i r lung flukes i n North America: vvvyVV -y^vvV^ Map 4 The d i s t r i b u t i o n of R. catesbeiana and R. clamitans (After conant, 1958) and t h e i r lung flukes in' -North' America1" .>/«?y\,;^  x i v LIST OF APPENDICES 1 Summary of measurements used .......185 2 Recipes f o r f i x a t i v e s and s o l u t i o n s 189 3 Flow sheet f o r p r e p a r i n g study s p e c i m e n s ...vvvyv.vv. ;vy.y1,90 .ft,/-.Effect •of - f l a t t e n i n g on' H. b u t t e n s i s .... ...w .......... .191 5 Average measurements of H. b u t t e n s i s developed i n ft. p r e t i o s a ........ 192 6 .Heasurements and s t a n d a r d i z e d measurement system .......193 7 Average measurements of H. b u t t e n s i s maintained a t 12°C 212 8 Average measurements of H. 'buttensis maintained a t 20°C 213 9 Average measurements of H. b u t t e n s i s maintained at 27°C 211 ,10 JJ.,,j^ i n B. p r e t i o s a (30-35 mm) 215 11 B. b u t t e n s i s developed i n B. p r e t i o s a (45-50 mm) .......216 12 H. b u t t e n s i s developed i n R. p r e t i o s a (55-60 mm) .217 13 H. b u t t e n s i s developed i n R . . p r e t i o s a (65-70 mm) .......218 14 H. b u t t e n s i s developed i n female R. p r e t i o s a .......... .219 15 H. b u t t e n s i s developed i n male R. p r e t i o s a 220 16 E f f e c t of crowding on H. b u t t e n s i s . 221 17 Average measurements of H. b u t t e n s i s developed i n R. p r e t i o s a . . v y . y".yy y .••i'-:*:y>.';. . i y v ^ v y y vvVy 222 18 Average measurements of H. b u t t e n s i s developed i n B. aurora 223 19 Average measurements of H. b u t t e n s i s developed i n , R . c l ami tans . .'y-.>.y'.?y.^  ..-.224 20 Average measurements of fl. b u t t e n s i s developed i n • _. borease ....................v.-*. v.". yyvyvyy.. . .. ..... . 225 X V 21 a v e r a g e measure-sent o f H. b u t t e n s i s o f v a r y i n g a g e s d e v e l o p e d i n R. p r e t i o s a when P.; n u t t g i l l i was t h e s n a i l i n t e r m e d i a t e h o s t . . . v . . , , ; . - . • • . . . . . . . . . . i/i . . . . . . . . . . . . , . 22 6 22 Summary o f some m e a s u r e m e n t s g i v e n i n t y p e d e s c r i p t i o n s 227 23 Known h o s t s o f a d u l t H a e m a t o l o e c h u s s p p . i n C a n a d a and . t h e . U n i t e d S t a t e s ' ^^y^-vVv^ i ?^ . ; .^ '^ .231 x v i ACKNOWLEDGEMENTS I would l i k e to thank my s u p e r v i s o r . Dr. J.B. Adams, f o r h i s f i n a n c i a l and moral support, and f o r h i s great p a t i e n c e . I would a l s o l i k e t o thank D r s . J . D . Lynch, B.B. Babero, G. J . Bowers, M. L. Eberhard, B, Campbell as w e l l as Mr. D a n i e l Brooks and Mr. Mike Baker f o r sending me mounted or preserved specimens. : The f o l l o w i n g people c o n t r i b u t e d l i v e specimens: Drs. J.B. Adams, B.J.G. L e s t e r and h i s wi f e , Judy, Dr. Hi / Ching, Ms. L. K i l l i c k , Ms. M. Westoli, Mr. and Mrs. 1. field, Mr. and Mrs. Bonnyman, Mr. And Mrs. Barsaloux, Hr. B. R u s s e l l , and Mr., D. Edamura. I thank them a l l . S p e c i a l thanks go to Dr. B. L i c h t e n f e l s a t the U.S. N a t i o n a l Museum who provided study space and permiss i o n t o examine type specimens, and t o Dr. M.H. P r i t c h a r d f o r l e n d i n g me numerous s l i d e s of lung f l u k e s from the Manter c o l l e c t i o n . Mrs. H.Fi€. / Smith, N a t i o n a l Museums, Ottawa, checked' the i d e n t i f i c a t i o n s of the s n a i l s , Mr. Bob Cannings, P r o v i n c i a l Museum, V i c t o r i a , i d e n t i f i e d the d a m s e l f l y and d r a g o n f l y naiads, and Ms. D. Hards made c r o s s - s e c t i o n s of some of the i n t e r m e d i a t e : h o s t s . I would a l s o l i k e t o thank the f o l l o w i n g people f o r c r i t i c a l r e a d i n g of the manuscript: Drs. J.R. Adams, D. C h i t t y , H. Ching, H. Kasinsky, J.D. S c P h a i l , C O . Person, G.G.E. Scudder, and J . Mary T a y l o r . 1 INTBOO0CTZO8 Morphology i s the p r i n c i p a l standard used by p a r a s i t o l o g i c a l taxonomists i n naming, i d e n t i f y i n g and c l a s s i f y i n g most groups of p a r a s i t i c organisms (Crites,1962}. P h y s i o l o g i c a l changes d e v e l o p i n g i n an organism as a r e s u l t of environmental v a r i a t i o n may p a r a l l e l morphological changes, although the l a t t e r may or may not be obvious (Chandler, 1923). As pointed out by Haley (1962), t h e r e has long been a need f o r s t u d i e s to determine the i n f l u e n c e of d i f f e r e n t host environments on p a r a s i t e s t r u c t u r e , p h y s i o l o g y and behaviour. I n t r a s p e c i f i c v a r i a t i o n may r e s u l t from the i n f l u e n c e of v a r y i n g environments encountered by a p a r a s i t e among i n d i v i d u a l s of a s i n g l e host s p e c i e s and, t o a g r e a t e r degree, from t h a t o f the v a r i o u s environments encountered by a p a r a s i t e i n hosts of d i f f e r e n t s p e c i e s . I n t r a s p e c i f i c v a r i a t i o n among p a r a s i t i c animals occurs f r e q u e n t l y and, a c c o r d i n g t o Wharton (1957) , i s s i m i l a r to t h a t known to e x i s t among f r e e - l i v i n g animals. Wharton a l s o noted t h a t d i f f e r e n c e s r e s u l t i n g from i n t r a s p e c i f i c v a r i a b i l i t y present taxoncmic problems. According to Stunkard (1957), among hermaphroditic s e l f - f e r t i l i z i n g organisms such as helminths, the " g e n e t i c " s p e c i e s i s v a l u e l e s s , and c l a s s i f i c a t i o n of these organisms must depend on the use of other c r i t e r i a . "The concept of s p e c i e s " , he s t a t e s , "must be based on a c o r r e l a t i o n o f l a r v a l s t r u c t u r e , l i f e h i s t o r y , p h y s i o l o g y , host r e l a t i o n s h i p s and m o r p h o l o g i c a l comparisons of adults.'* However, he s t a t e s t h a t "morphology i s the main f a c t o r d e f i n i n g s p e c i e s of 2 p a r a s i t e s . " Wright (I960) disagreed with. Stunkard i n t h a t he c o n s i d e r e d acceptance o f the " g e n e t i c " s p e c i e s to be a more c o n s t r u c t i v e approach toward trematode taxonomy. Mayr (1969) d e f i n e d a s p e c i e s as: "groups o f i n t e r b r e e d i n g n a t u r a l p o p u l a t i o n s t h a t are r e p r o d u c t i v e l y i s o l a t e d from other such groups." Cross- and s e l f - i n s e m i n a t i o n have been demonstrated f o r some s p e c i e s o f trematodes ( N o l l e n , 1968; Moseley and N o l l e n ^ 1973). Reproductive i s o l a t i o n i n c oncurrent i n f e c t i o n s has a l s o been demonstrated ( N o l l e n , Pyne, Hoseley and Bunker, 1975). T h e r e f o r e , the b i o l o g i c a l s p e c i e s concept c o u l d be a p p l i e d t o trematode taxonomy; but, the sheer volume of experimental study r e q u i r e d f o r i t s adequate a p p l i c a t i o n makes i t d i f f i c u l t t o apply .•to more than a few thoroughly s t u d i e d organisms. The use of morphology i n d e f i n i n g s p e c i e s o f helminth p a r a s i t e s w i l l remain f o r q u i t e some time. The genus Haematqloechus comprises a group o f widely d i s t r i b u t e d p l a q i o r c h i o i d f l u k e s , p a r a s i t i c i n the lunqs of Amphibia. So l e s s than 56 s p e c i e s of trematodes have been r e f e r r e d to t h i s qenus world-wide and, at one time or another, have been a l l o c a t e d t o as many as f i v e qenera. Odeninq (1958) assigned a l l s p e c i e s to the qenus Haematoloechus and r e c o q n i z e d t h r e e major qroups: 1) Old World; 2) A u s t r a l i a ; and 3) America, the l a t t e r d i v i d e d i n t o three subgroups: North, South, and C e n t r a l America, He l a t e r (Odening, 1960b) employed f o u r genera f o r the same assembly of s p e c i e s . The v a l i d i t y of s e v e r a l s p e c i e s i n the North American subgroup has been questioned by C o r t (1915a) , Banter (1938) , 3 and Harwood (193-2). These authors i n d i c a t e d the d e s i r a b i l i t y of e s t a b l i s h i n g e x p e r i m e n t a l i n f e c t i o n s i n the l a b o r a t o r y t o a s c e r t a i n s p e c i e s v a l i d i t y . The need f o r such s t u d i e s on i n t r a s p e c i f i c v a r i a t i o n s was a l s o emphasized by Haley (1562). S t u d i e s o f t h i s type have been undertaken f o r s e v e r a l genera e.g. T e l o r c h i s by B a t e r t o r (1965); ProsthogOnimus by Boddeke (196 0 a, b) and others . The u n c e r t a i n taxonomy of s p e c i e s of Haematoloechus seems to have a r i s e n from primary r e l i a n c e on morp h o l o g i c a l f e a t u r e s of s e x u a l l y mature worms f o r which the degree o f v a r i a t i o n was unknown; many s p e c i e s o f t h i s genus are known from o n l y one type specimen. These taxonomic c h a r a c t e r s , d e f i n e d by Mayr (1969: 121) as "any a t t r i b u t e o f a member o f a taxon by which i t d i f f e r s or may d i f f e r from a member o f a d i f f e r e n t taxon." are s u b j e c t t o v a r i e d i n t e r p r e t a t i o n s and t e c h n i c a l m a n i p u l a t i o n s . ; The complex l i f e h i s t o r i e s o f d i g e n e t i c trematodes i n c l u d e , i n a d d i t i o n t o t h e s e x u a l l y mature a d u l t i n the d e f i n i t i v e host, s e v e r a l l a r v a l stages whose morphology and developmental aspects provide a d d i t i o n a l taxonomic i n f o r m a t i o n . The development of "haematoloechid" taxonomy i s hindered by the f a c t t h a t l i f e h i s t o r i e s have been determined f o r only ten s p e c i e s . K r u l l (1930, 1931) d e s c r i b e d the l i f e h i s t o r i e s of H. medioplexus S t a f f o r d , 1902 and H. p a r v i p l e x u s (Irwin, 1929). In 1932 K r u l l r e p o r t e d on p a r t of the l i f e c y c l e of H. IgigiEjexus S t a f f o r d , 1902, and, i n 1933 and 1934 on a l l stages of H. complexus (Seelv, 1906). The l i f e h i s t o r i e s -of .- H. o x y o r c h i s I n g l e s , 1932, H.::breviplexus S t a f f o r d ^ 1902 and H. complexus were d e s c r i b e d by I n g l e s (1933), S c h e l l (1965), and Kronen (1975) r e s p e c t i v e l y . 4 The l i f e h i s t o r y of H. s i m i l i s (LOoss, 1899) was d e s c r i b e d by Grabda-Kazubska (1960), G i n e t s i n k a y a and D o b r o v o l s k i j (1968), and Smirnova and Ibrasheva (1967). D o l l f u s , Doby and Laurent (1960) i n v e s t i g a t e d the l i f e c y c l e of H. bombynae (Zeder, 1800). The l i f e h i s t o r y o f fl. asjger Looss, 1899 was d e s c r i b e d by D o b r o v o l s k i j (1965), and by G i n e t s i n k a y a and D o b r o v o l s k i j (1968). Many of t h e above l i f e c y c l e s are i n c o m p l e t e l y known, and three are European. Specimens of Haematoloechus sp. c o l l e c t e d by the author i n the; s p r i n g and summer of 1974 i n B r i t i s h Columbia i n d i c a t e d a great d e a l o f mo r p h o l o g i c a l v a r i a t i o n i n c h a r a c t e r s used t o separate s p e c i e s i n t h i s genus. My attempts to i d e n t i f y the worms' l e d to the c o n c l u s i o n t h a t e i t h e r as many as s i x s p e c i e s or as few as th r e e s p e c i e s occurred here. The purpose of t h i s t h e s i s i s t o examine some of the v a r i a t i o n s i n morphology of the f i f t e e n s p e c i e s o f Haematoloechus d e s c r i b e d from Canada and the Onited S t a t e s and to use these data t o assess the v a l i d i t y o f the d e s c r i b e d s p e c i e s . T h i s w i l l be done i n two p a r t s . Part I : Experimental Laboratory experiments i n v e s t i g a t e the morphological v a r i a t i o n which occur i n t h i s genus by using H. b u t t e n s i s I n g l e s , 1936 as a model. Rana p r e t i p s a B a i r d and G i r a r d , 1853 the usual d e f i n i t i v e host of fl. b u t t e n s i s i n B r i t i s h Columbia, i s used t o determine the e f f e c t s on important taxonomic c h a r a c t e r s of ho s t ambient temperature, s i z e , sex and number of worms present. V a r i a t i o n i n 5 f l u k e s , developed i n d i f f e r e n t d e f i n i t i v e h o s t s , i s a l s o examined. As w e l l , v a r i a t i o n s i n f l u k e s are examined when d i f f e r e n t i n s e c t second i n t e r m e d i a t e hosts or molluscan f i r s t i n t e r m e d i a t e h o s t s are used. Aspects of p r e p a r i n g specimens f o r examination are i n v e s t i g a t e d to e s t a b l i s h t h e i r e f f e c t s on a d u l t morphology. Part I I : A. Geographical V a r i a t i o n : Specimens of some s p e c i e s c o l l e c t e d from v a r i o u s l o c a l i t i e s i n Canada and the United S t a t e s are compared and analyzed f o r morphological v a r i a t i o n . Data from t h i s p a r t o f the t h e s i s have been d e r i v e d by examining specimens from new c o l l e c t i o n s made by the author or co o p e r a t i n g c o l l e a g u e s . These data are supplemented by examining specimens borrowed from museums and other u n i v e r s i t i e s . Obvious e r r o r s i n the i d e n t i f i c a t i o n o f borrowed specimens have been c o r r e c t e d . B. Examination o f type specimens: A re—examination o f 9 out of the 15 type specimens a v a i l a b l e has been done to determine the accuracy o d e s c r i p t i o n s of the type m a t e r i a l . A l l s p e c i e s are i n i t i a l l y c o n s i d e r e d v a l i d and are d i s c u s s e d under t h e i r a p p r o p r i a t e names. A t e n t a t i v e taxonomic r e v i s i o n i s made at the end o f the t h e s i s . The r e s u l t s from the above i n v e s t i g a t i o n s should provide a p r a c t i c a l c l a s s i f i c a t i o n of the s p e c i e s from the United S t a t e s and Canada. I t should a l s o c o n t r i b u t e i n f o r m a t i o n on f a c t o r s important i n the d e l i n e a t i o n of s p e c i e s w i t h i n other genera o f 6 trematodes, as w e l l as w i t h i n the genus gafe'-m'atg;le^ eG.hqs. 7 LIFE CYCLE OF H AE MA'TOLOECH 0 S ---BilTT EM SIS I n t r o d u c t i o n : Haematoloechus b u t t e n s i s I n g l e s , 1936 vas recovered from Rana p r e t i o s a c o l l e c t e d from a l a k e 6 miles e a s t o f Greenwood, B.C., d u r i n g the summer o f 1975. Frogs c o n t a i n e d from one t o ten lung f l u k e s each. Examination o f damselfly and d r a g o n f l y naiads from the same l a k e , r e v e a l e d t h a t only the d a m s e l f l y naiad (Ischnura perparva S e l y s , 1876) vas i n f e c t e d with a metacercaria s i m i l a r t o t h a t of other Haematoloechus spp. Naiads of I. c e r y u l a S e l y s , 1876 and the d r a g o n f l y , AeseJiiaa. palmata Bagen. 1856 i n the same l a k e , were not i n f e c t e d . Physa n u t t a l l i . Lea, 1864 c o l l e c t e d from the same l o c a t i o n , c o n t a i n e d c e r c a r i a e , i n s p o r o c y s t s , which f i t the g e n e r a l d e s c r i p t i o n of c e r c a r i a e p r e v i o u s l y d e s c r i b e d f o r other f r o g lung f l u k e s . St a g n i c o l a elode s (Say, 1821) and Helisoma t r i v o l v i s (Say. 1816) were recovered from the l a k e , but d i d not c o n t a i n any trematode i n f e c t i o n s . These o b s e r v a t i o n s suggested that H. b u t t e n s i s had as i n t e r m e d i a t e hosts the s n a i l P. nutt a l i i and the naiad I. perparva i n B.C. Since the l i f e h i s t o r y o f H. butfre~sis has not been r e p o r t e d , I had to determine the l i f e - c y c l e b e f o r e I c o u l d do o t h e r experimental work on host—induced v a r i a b i l i t y . The r e s u l t s are presented here i n b r i e f . A d e t a i l e d account w i l l be p u b l i s h e d separately.,, M a t e r i a l s and Methods: Sources of Experimental Animals Resources were not a v a i l a b l e f o r e s t a b l i s h i n g and breeding 8 l a b o r a t o r y - r e a r e d s t o c k s o f a l l the hosts needed f o r the experimental work. However, p r e c a u t i o n s were taken to s e l e c t s t o c k s of hosts which were f r e e o f p a r a s i t e s . T h i s was done by examining p o t e n t i a l hosts from many l o c a l i t i e s i n B r i t i s h Columbia. , L o c a l i t i e s found t o c o n t a i n u n i n f e c t e d s t o c k s o f p o t e n t i a l h o s t s were sampled on at l e a s t two et h e r subseguent o c c a s i o n s . The c l o s e s t l o c a l i t i e s t o Vancouver which c o n t a i n e d u n i n f e c t e d hosts were s e l e c t e d as sources f o r experimental animals. P a r t o f the sample was rechecked every time a new c o l l e c t i o n was made. The l o c a l i t i e s and numbers of hosts examined are given below. Physa n u t t a l l i was c o l l e c t e d from a stream 15 miles south of P e n t i c t o n . One-hundred s n a i l s were s e l e c t e d at random and examined f o r trematode i n f e c t i o n s . No i n f e c t i o n was found. Naiads of Ischnura perparva were c o l l e c t e d from a stream 15 miles e a s t of Lumby. F i f t y naiads were examined f o r trematode i n f e c t i o n s . A l l were n e g a t i v e f o r trematode i n f e c t i o n s of any kind. !» firetiosa was c o l l e c t e d from a stream 15 miles east o f Lumby, B. C. F i f t e e n f r o g s were examined f o r lung f l u k e i n f e c t i o n s . A l l were negative. Frogs were kept an a d d i t i o n a l two weeks t o allow any f l u k e s t h a t might have been a c g o i r e d n a t u r a l l y t o mature enough t o be separated from experimental i n f e c t i o n s . Damselfly and d r a g o n f l y l a r v a e were i d e n t i f i e d by Robert Cannings, P r o v i n c i a l Museum, V i c t o r i a , S n a i l s were I d e n t i f i e d by Mrs. M.F.I. Smith, N a t i o n a l Museum of Canada, Ottawa. 9 Methods f o r I n f e c t i n g Hosts F i f t y P. n u t t a l l i were i n f e c t e d by a l l o w i n g them t o i n g e s t eggs of H. b u t t e n s i s from Manning Park f o r one hour, a f t e r which s n a i l s were washed to removed excess eggs and placed i n f i n g e r bowls c o n t a i n i n g d e c h l o r i n a t e d water. Two s n a i l s were d i s s e c t e d one hour a f t e r f e e d i n g on eggs. Two more s n a i l s were examined every hour f o r the f i r s t f i v e hours, every t h r e e hours f o r the next 18 hours, then every f i v e days u n t i l c e r c a r i a e emerged. S n a i l s were maintained i n a g u a r i a on a d i e t of t o i l e d l e t t u c e . Ten c e r c a r i a e , shed from e x p e r i m e n t a l l y i n f e c t e d Phys a n u t t a l l i , were p l a c e d i n each o f 50 p e t r i d i s h e s , each c o n t a i n i n g one I. perparva naiad. Two n a i a d s were examined a f t e r f i v e hours, and an a d d i t i o n a l two every day f o r 10 days. Ten f r o g s were each fed ten metacercariae, which had been d i s s e c t e d from e x p e r i m e n t a l l y i n f e c t e d naiads. Two f r o g s were necr o p s i e d at 5, 7, 21, and 30 days, a l l f r o g s were maintained at 20°C on a d i e t of earthworms. R e s u l t s and D i s c u s s i o n ; F i r s t Intermediate Host The eggs of H. b u t t e n s i s were 0. 024-0.02 9 (0.026) mm l o n g by 0.012-0.017 (0.015) wide, o p e r c u l a t e , l i g h t brown, and contained m i r a c i d i a when l a i d . G r a v i d worms d e p o s i t embryonated eggs i n the lung c a v i t y of Rana p r e t i o s a . Eggs are c a r r i e d from the lungs up the b r o n c h i o l e s and through the g l o t t i s by c i l i a r y a c t i o n of the lung and b r o n c h i o l e c e l l s ( K r u l l , 1931). Eggs are swallowed and were found i n faeces upon p a s s i n g through the d i g e s t i v e t r a c t . 10 They s i n k t o the bottom of the pond, but do not hatch u n t i l they are swallowed by the s n a i l P^jrsa^nuttalli----•(•Fig. 1). E g g s h e l l s and some m i r a c i d i a a r e passed i n the faec e s . , M i r a c i d i a i n P. n u t t a l l i migrate through the i n t e s t i n a l w a l l . Mother s p o r o c y s t s develop i n the i n t e s t i n a l w a l l o f the s n a i l , o r may be found f r e e i n the haemocoel. C o r t e t a l . (1954) noted t h a t the development of mother s p o r o c y s t s i n t h i s way t y p i c a l l y occurs i n members of the s u p e r f a m i l y P l a g i o r c h i o i d e a . Mother s p o r o c y s t s have been observed i n only three other s p e c i e s o f Haem a t o l o e c h u s : s c h e l l (1965) r e p o r t e d a mother s p o r o c y s t belonging t o H. b r e v i p l e x u s i n the i n t e s t i n a l w a l l of the s n a i l Gyraulus s i m i l a r i s (Baker, 1919): fiankin (1939) demonstrated t h e e x i s t e n c e of a mother s p o r o c y s t stage i n H. merchant! ; and i n H. •uc.plpradensiSy where Dronen (1975) demonstrated the development of a mother s p o r o c y s t on the i n t e s t i n a l s u r f a c e i n the s n a i l Physa v i r g a t a . Daughter s p o r o c y s t s developed i n the s n a i l ' s h e p a t i c gland and produced c e r c a r i a e i n approximately 30 days. Op to 250 c e r c a r i a e were shed per n i g h t by a s i n g l e h e a v i l y i n f e c t e d s n a i l . C e r c a r i a e swam a c t i v e l y and su r v i v e d up t o 36 hours at 20 °C. Fiqure 1. L i f e c y c l e o f Haeaatoloec%us _ u t t e n s i s . 11A c xlphidlocercarla 12 Second Intermediate Host Under experimental c o n d i t i o n s c e r c a r i a e i n f e c t e d the l a r v a l s tages o f d a m s e l f l i e s by p e n e t r a t i n g the abdominal w a l l and passing through the the haemocoel, where they d i d not encyst. M e t a c e r c a r i a e were a l s o recovered from the head r e g i o n , but were not seen to p e n e t r a t e the c u t i c l e of t h i s area. Metacercariae never encysted on the r e c t a l g i l l s of l a r v a e . My f i e l d c o l l e c t i o n s of p o t e n t i a l i n t e r m e d i a t e hosts suggested t h a t the damselfly Ischnura perparva i s the usual i n s e c t host i n B r i t i s h Columbia. Adult d a m s e l f l i e s may harbor up to 73 metacer c a r i a e , though t h i s i s r a r e . My f i e l d o b s e r v a t i o n s i n d i c a t e t h a t two to t h r e e metacercariae per i n f e c t e d d a m s e l f l y i s more common. I n f e c t i o n o f D e f i n i t i v e Host Frogs were e x p e r i m e n t a l l y i n f e c t e d through e a t i n g i n f e c t e d n a i a d s , but they can a l s o become i n f e c t e d by e a t i n g i n f e c t e d a d u l t d a m s e l f l i e s or d r a g o n f l i e s ( K r u l l , 1931) . The author has o f t e n seen t e n e r a l s , r e s t i n g on stems of v e g e t a t i o n , captured and eaten by f r o g s . Experimental o b s e r v a t i o n s demonstrated t h a t , upon being r e l e a s e d from the damselfly, the young f l u k e s migrate forward through the oesophagus, pass through the g l o t t i s and enter the lun g s by way of the b r o n c h i . Flukes matured i n about 14 t o 21 days. Lung f l u k e s may remain i n f r o g s f o r up t o 15 months, a f t e r which they are l o s t and new ones take t h e i r p l a c e ( K r u l l , 1930, 1931). I have found up t o 74 H. b u t t e n s i s i n a s i n g l e lung of R. p r e t i o s a . 13 PART -I. MORPHOLOGICAL VARIATION 1.. EFFECTS OF FIXATION AND FLATTENING ON ASOIT MORPHOLOGY I n t r o d u c t i o n Measurements of p a r a s i t e s have long been c o n s i d e r e d of s i g n i f i c a n c e i n the c h a r a c t e r i z a t i o n of new s p e c i e s (Ulmer, 1950). References t o measurements i n the l i t e r a t u r e o f t e n omit p r e c i s e i n f o r m a t i o n about the technigues employed. F l a t t e n i n g trematodes p r i o r t o measurement, or f i x i n g worms i n s o l u t i o n s of d i f f e r e n t temperatures, may have a profound e f f e c t on t h e i r morphology. These changes i n morphology may r e s u l t i n new s p e c i e s being d e s c r i b e d by d i f f e r e n t authors using d i f f e r e n t technigues. I t i s t h e r e f o r e important t o examine the e f f e c t s of v a r i o u s p r e p a r a t o r y technigues on f l u k e morphology. Se v e r a l authors who d e s c r i b e d new s p e c i e s o f Haeroatoloechus, d i d not r e c o r d t h e i r p r e f i x a t i v e and f i x a t i v e technigues (Seely, 1906; Irwi n , 1929; Harwood, 1932; I n g l e s , 1932, 1936). C o r t (1915a) u s u a l l y t r a n s f e r r e d f l u k e s i n t o d i s t i l l e d water be f o r e k i l l i n g them. T h i s was followed by k i l l i n g the worm i n e i t h e r c o l d or i n hot f l u i d . H.W.Manter (communication to G.J.Spencer, 1934) a l s o used d i s t i l l e d water, worms were then k i l l e d by p l a c i n g them on a s l i d e , adding a drop of k i l l i n g s o l u t i o n (A.F.A.) t o the worm, a p p l y i n g a c o v e r s l i p and e x e r t i n g pressure on the c o v e r g l a s s t o f l a t t e n the specimen. K r u l l (1931: 222) placed f l u k e s i n c o l d water t o r e l a x them and t o get them to shed eggs b e f o r e k i l l i n g them. T h i s r e g u i r e d from ten to s i x t y minutes. He s t a t e d t h a t " T h i s treatment causes no s e r i o u s changes i n s t r u c t u r e , except t h a t p a r t s of the 14 c u t i c l e slough o f f . ? Worms were then plunged i n t o hot 70S a l c o h o l . S t a f f o r d {1902: 597) s t a t e s o n l y t h a t "worms are k i l l e d i n a mixture of g l a c i a l a c e t i c a c i d and alcohol.'? C h a r a c t e r s c o n s i d e r e d by p r e v i o u s authors to be important i n d e l i n e a t i n g s p e c i e s i n t h i s group are : the presence and extent o f e x t r a c a e c a l l o n g i t u d i n a l f o l d s of the u t e r u s , egg s i z e , degree of l o b a t i o n o f t e s t i s and ovary, general shape and p o s i t i o n of the t e s t e s , degree o f s p i n a t i o n o f the tegument, and the r a t i o between the t r a n s v e r s e diameters of the o r a l sucker and the acetabulum ( F i g . 2 ) . A d e s c r i p t i o n o f these and other measurements i s given i n Appendix 1. M a t e r i a l s and Methods To determine the e f f e c t s of f i x a t i o n and f l a t t e n i n g on the a d u l t morphology of JH. b u t t e n s i s I fed metacercariae, developed i n naiads of the da m s e l f l y i s c h n u r a Perparva, to each of ten male, a d u l t Rana p r e t i o s a of approximately the same l e n g t h {55-59 mm snout u r o s t y l e l e n g t h ) . Twenty-one days l a t e r , 157 g r a v i d a d u l t s were recovered from the lungs of these e x p e r i m e n t a l l y i n f e c t e d d e f i n i t i v e hosts. One—hundred t h i r t y — f i v e of these worms were used t o determine the e f f e c t s v a r i o u s p r e p a r a t o r y methods have on some morphological c h a r a c t e r s . A l l specimens were measured and drawn to s c a l e . A l l drawings were made with t h e a i d o f a Bausch and Lomb microprorjector. Measurements are i n m i l l i m e t e r s u n l e s s otherwise s t a t e d . 15 P r e p a r a t i o n Method I T h i s method i n v e s t i g a t e s morphological changes i n f l u k e s kept i n one of two commonly used p r e — f i x a t i v e s o l u t i o n s : d i s t i l l e d water and amphibian Ringer*s S o l u t i o n (Appendix 2 ) . Three worms were p l a c e d i n each o f e i g h t 90 mm round pyrex p e t r i d i s h e s . Four o f the d i s h e s contained 25 ml each of d i s t i l l e d water and the remaining f o u r d i s h e s c o n t a i n e d 25 ml each of Ringer's s o l u t i o n (Appendix 2). A l l of the d i s h e s were kept at 6°c. A sample of three worms from a d i s h of each s o l u t i o n was taken at 3, 5, 7 and 10 minutes. These samples were f i x e d i n hot 70% e t h a n o l , s t a i n e d by the method given i n Appendix 3 and mounted u n f l a t t e n e d on a s l i d e u s ing a number 1 cover g l a s s (18 mm s g ) . The percent i n c r e a s e i n a measurement i s c a l c u l a t e d by s u b t r a c t i n g the measurement i n Frog S i n g e r ' s s o l u t i o n (FS) from the corresponding measurement i n d i s t i l l e d water (DB), d i v i d i n g the d i f f e r e n c e by FR, then m u l t i p l y i n g the guotient by 100 31. F i g u r e 2. Diagrammatic f i g u r e o f a d u l t Haematoloechus showing p r i n c i p a l p a r t s of anatomy. 16A Oral Sucker Pharynx Intestinal Caecum Vitellaria Anterior Test i s Posterior Testis Uterus Extracaecal Uterine 17 R e s u l t s of Method I Twenty—one-day o l d worms f i x e d and s t a i n e d under s i m i l a r c o n d i t i o n s , but d i f f e r i n g o n l y i n r e s p e c t to treatment i n d i f f e r e n t p r e - f i x a t i v e s o l u t i o n s , showed c o n s i d e r a b l e v a r i a t i o n i n body l e n g t h and width and the presence of s p i n e s on the tegument. Body l e n g t h and width o f a d u l t s t r e a t e d i n d i s t i l l e d water are g r e a t e r than those of f l u k e s placed i n Ringer's s o l u t i o n by as much as QOSIand 100% r e s p e c t i v e l y . Frog R i n g e r ' s s o l u t i o n d i d not a f f e c t s p i n a t i o n , but e u t i c u l a r s p i n e s of worms maintained i n d i s t i l l e d water were l o s t (Table 1). D i f f e r e n c e s i n measurements o f f l u k e s maintained i n d i s t i l l e d water compared with those of f l u k e s i n Ringer's s o l u t i o n i n c r e a s e d with time ( F i g . 3). The p r o g r e s s i v e d i f f e r e n c e i n l e n g t h and width i s a r e s u l t of an i n c r e a s e i n these dimensions f o r f l u k e s maintained i n d i s t i l l e d water. No s i g n i f i c a n t i n c r e a s e i n s i z e occurred i n f l u k e s kept i n f r o g Ringer's s o l u t i o n f o r 3, 5, 7 o r 10 minutes. P r e p a r a t i o n Method I I , 1 t e s t e d the e f f e c t of the temperature of d i s t i l l e d water and of Ringer's s o l u t i o n on the morphology of H. b u t t e n s i s by p l a c i n g three worms i n each o f 24 p e t r i d i s h e s ( d e s c r i b e d i n method I ) . Groups of four d i s h e s were kept at e i t h e r 6<>C, 20<>C or 40°C. One d i s h from each group was sampled at 3, 5, 7 and 10 minutes. P o s t — f i x a t i v e handling was as f o r Method I . 18 R e s u l t s of method I I Body; l e n g t h and width d i d not d i f f e r i n f l u k e s maintained i n Frog S i n g e r ' s s o l u t i o n at d i f f e r e n t temperatures ( f a b l e 2 ) . Wo s i g n i f i c a n t i n c r e a s e i n length or width occurred when f l u k e s from d i s t i l l e d water but at d i f f e r e n t temperatures were compared at c orresponding times. Flukes maintained i n d i s t i l l e d water a t 6 ° C or 2 0 ° C l o s e t h e i r s p i n e s a f t e r three minutes. Spines were l o s t p r i o r to t h r e e minutes i n d i s t i l l e d water kept at 40 ° C . The body l e n g t h and width o f worms i n d i s t i l l e d water i n c r e a s e d with time. The percent i n c r e a s e i n length or width o f f l u k e s maintained i n d i s t i l l e d water, compared with t h a t of worms sampled at a cor r e s p o n d i n g time and temperature, but from Frog Ringer's s o l u t i o n , i n c r e a s e d with time ( F i g . 4 ) . .Worms, kept i n Ringer's s o l u t i o n , and sampled a t 3 , 5, 7 and 10 minutes, had s p i n e s over t h e i r e n t i r e tegument. Table 1. Effect of pre-fixative solution on some characters of 21-day-old H. buttensis. F r o g R inger's T i m e (min. ) Character Spines Body Length Body Width Dis t i l l e d water T i m e (min. ) Character Spines Body Length Body Width present 3.14(2.78-3.29) 0. 66(0.60-0.81) present 3. 33(2.91-3. 50) 0. 68(0.57-0.75) present 3. 28(2. 81-3.44) 0.63(0.61-0.71) absent present 2.95(2.73-3. 30) 0.68(0.64-0.77) absent 3.41(3.17-3. 56) 3. 56(3.40-3. 89) 0. 71(0. 63-0.80) 0. 89(0.76-0.98) 10 present 3. 26(2.90-3.50) 0.65(0.53-0.70) 10 absent 4.57(4.30-4.81) 1. 35(1.12 -1.44) A l l solutions were used at 6 C. 2 0 F i g . 3 Percent i n c r e a s e i n body length and width of f l u k e s maintained i n d i s t i l l e d water over those maintained i n Frog Ringer's s o l u t i o n . The percent i n c r e a s e i s c a l c u l a t e d by s u b t r a c t i n g the measurement i n Frog Ringer's s o l u t i o n (FR) from the corresponding measurement i n d i s t i l l e d water (DW) , d i v i d i n g the d i f f e r e n c e by FR, then m u l t i p l y i n g t h e - q u o t i e n t by 1 0 0 . %1= (DH - FR) /FR x 1 0 0 Time (min.) Table 2. The effect of temperature on morphology of 21-day old H. buttensis reared ln R. pretiosa maintained at 6 °C, 20 °C or 40 °C. Frog Singer's Time (mln.) 3 5 7 10 Character Spines 6 present present present present 20 present present • present present 40 present present present present Body Length 6 3.47(3-26-3 .67) 3.61(3-40-3.93) 3.39(3.17 -3 .66) 20 3.52(3.33-3-71) 3.57(3-33-3.77) 3.35(3.18-3.58) 3.46(3-21-3.72) 40 ' 3.26(3.19-3.51) 3.49(3.37-3-71) 3.55(3-25-3.79) 3.57(3-17-3.70) Body Width 6 0.72(0.71-0.65) 0.69(0.60-0.73) 0.76(0.64-0.83) 0.66(0.59-0.75) 20 0.75(0.69-0.87) 0.65(0.55-0.77) 0.67(0.61-0.76) 0.70(0.59-0.73) 40 0.77(0.65-0.80) 0.73(0.61-0.81) 0.73(0.63-0.79) 0.71(0.65-0.88) D i s t i l l e d Water Spines 6 20 present present absent absent absent absent absent absent 40 absent absent absent absent Body Length 6 3.69(3.41-3.92) 3.88(3.59-3-99) 4 .11(3-76-4 .23) 4.6K4.24-4 .7D 20 3.81(3.61-4.12) 4.00(3.72-4.18) 4.22 (3-96-4 .38) 4.73(4.66-5.03) 40 3.77(3.52-4.18) 3.98(3-67-4.11) 4.33(4.01-4.45) 4.67(4.39-4.84) Body Width 6 0 . 7 7 ( 0 . 6 8 - 0 . 8 9 ) 0.81(0.70-0.86) 1.01(0.89-1.11) 1.27(1.15-1.35) 20 0 . 8 1 ( 0 . 7 3 - 0 . 8 8 ) 0.79(0.67-0.86) 1.13(1.05-1.22) 1 . 3 K 1 . 1 9 - 1 . 3 9 ) 40 0 . 8 3 ( 0 . 7 5 - 0 . 8 8 ) 0.80(0.71-0.94) 1.07(0:99-1.14) 1 . 3 5 ( 1.28 - 1 . 4 7 ) 22 F i g . 4 Percent i n c r e a s e i n body l e n g t h and width of f l u k e s maintained at d i f f e r e n t temperatures i n d i s t i l l e d water over those maintained at a cor r e s p o n d i n g temperature but i n Frog Ringer's s o l u t i o n . The percent i n c r e a s e i s c a l c u l a t e d by s u b t r a c t i n g t h e measurement i n Frog Ringer's s o l u t i o n {FH) from the corresponding measurement i n d i s t i l l e d water (DH) , d i v i d i n g the d i f f e r e n c e by FR, then m u l t i p l y i n g the q u o t i e n t by 1 0 0 . . % 1= (DH - FR) / FR X 100 22A 23 P r e p a r a t i o n Method I I I The e f f e c t s o f d i f f e r e n t f i x a t i v e s used at 6°C or 70°C on f l u k e morhphology were i n v e s t i g a t e d . Flukes were washed f o r one minute i n Ringer's s o l u t i o n , and three worms were placed i n each of t e n p e t r i d i s h e s . The 10 di s h e s were sep a r a t e d i n t o two egual groups. Each group c o n t a i n e d a d i s h with one of the f o l l o w i n g f i v e f i x a t i v e s o l u t i o n s : AvF.lu (Appendix 2) 70S; e t h a n o l Schaudinn's f i x a t i v e (Appendix 2) Bouin's f i x a t i v e (Appendix 2) 105£ f o r m a l i n F i x a t i v e s i n the f i r s t and second groups were used at 6°C and 70°G, r e s p e c t i v e l y . The p o s t - f i x a t i o n process was the same i n a l l cases (Appendix 3 ) . Re s u l t s of Method I I I Measurements of worms f i x e d i n d i f f e r e n t s o l u t i o n s , but a t the same temperature, d i d not d i f f e r s i g n i f i c a n t l y . However, worms from hot (70°C) f i x a t i v e s were longer and narrower than worms f i x e d i n the same s o l u t i o n at 6°c (Fig.5 and Table 3 ) . Twenty percent (3/15) of the worms f i x e d a t 6°C had s u b t e r m i n a l suckers. The O/A and O/P r a t i o s , l o b i h g of t e s t e s and ovary, and extent of e x t r a c a e c a l l o o p s were not a f f e c t e d by f i x a t i v e s o l u t i o n or temperature. 3 Table 3. Effec t of temperature and fixative on length and width of H. buttensi Fixative Solution 10% F o r m a l i n ' A. F. A. 70% Ethanol Schaudin's Bouin 1 Length Cold 3. 02(2. 80- 3.14) 3.16(3.00- 3.25) 3. 32(3. 23-•3.47) 2. 84(2.76- 3. 05) 3. 23(3.11- 3.41) Hot 4. 57(4. 21-4. 62) 5.04(4. 85- 5.17) 4. 83(4. 67-4.92) 4. 70(4. 55- 4. 83) 4. 74(4. 65--5.02) Width Cold 0. 63(0. 59- 0. 68) 0.65(0. 62- 0. 70) 0.61(0.57- 0. 63) 0. 66(0. 62- 0. 73) 0. 59(0. 56-- 0 .68) Hot 0. 41(0. 37- 0.45) 0. 39(0. 32- 0.41 ) 0.45(0.40- 0. 52) 0. 42(0. 33- 0. 45) 0. 44(0. 40-•0. 51 ) ro 25 F i g . 5 The e f f e c t s of f i x a t i v e and temperature on body length and width of 21-day—old H. b u t t e n s i s r e a r e d i n B. p r e t i o s a . 25A 6°C 7 0 ° C 10&FORMALIN 6°C 70°C A F A 6°C 70°C 70&ETHANOL 6°C 7 0 ° C SCHAUDINN'S 70 ° c BOUINS Fixative 26 P r e p a r a t i o n Method IV Th i s method i n v e s t i g a t e d the e f f e c t s on f l u k e morphology o f weight added t o the c o v e r s l i p w h i l e d r y i n g s l i d e s . Nine worms were prepared f o r mounting on s l i d e s i n the manner d e s c r i b e d i n Appendix 3. Each of the nine worms was mounted i n permount on a separate s l i d e . The s l i d e s were d i v i d e d i n t o three e g u a l groups. A 5g weight was p l a c e d on t h e c o v e r s l i p o f each s l i d e i n group 1, a 10g weight on those of group 2 and no weight i n group 3. S l i d e s were d r i e d f o r f i v e days and measurements were taken on 15 c h a r a c t e r s {Appendix 4). R e s u l t s of Method IV The s i z e o f a c h a r a c t e r i n c r e a s e d with i n c r e a s e d weight on the c o v e r s l i p d u r i n g d r y i n g ( F i g . 6 ) . B o d y l e n g t h ranged from a mean of 3.05 i n u n f l a t t e n e d worms t o 5.43 I n worms f l a t t e n e d with 10g. Body widths v a r i e d from 0.63 to 1.51, a n t e r i o r t e s t i s l e n g t h ranged from 0.6 3 t o 0.83. Measurements f o r other c h a r a c t e r s are giv e n i n Appendix 4. The s i z e r a t i o of the o r a l sucker to acetabulum diameters d i d not change markedly with i n c r e a s e d weight d u r i n g d r y i n g . T h i s r a t i o v a r i e d between 2.5 and 2.8 . 27 F i g . 6 The e f f e c t of f l a t t e n i n g on some c h a r a c t e r s of 21—day-old H. b u t t e n s i s . 27A Charac ter 28 2, VARIATION IN FLUKES DEVELOPED IN THE NATURAL DEFINITIVE HOST RANA PRETIOSA. I n t r o d u c t i o n More i n f o r m a t i o n i s needed about host—dependent v a r i a t i o n i n p a r a s i t i c helminths i n order b e t t e r to a s s e s s the s t a b i l i t y of taxonoraic c h a r a c t e r s used i n i d e n t i f i c a t i o n (Haley, 1962). The i n f l u e n c e s of d i f f e r e n t h o s t s , as w e l l as of i n d i v i d u a l s of a s i n g l e host s p e c i e s , on v a r i a t i o n s i n p a r a s i t e s t r u c t u r e has r e c e i v e d i n c r e a s e d a t t e n t i o n over the past f i f t e e n y ears. S c h i l l e r (1959), Haley (1962) , and o t h e r s , have p o i n t e d out t h a t t h i s v a r i a t i o n has caused c o n s i d e r a b l e c o n f u s i o n i n c l a s s i f y i n g helminth p a r a s i t e s . Despite the c a u t i o n i n g o f the above authors, many taxonomists continue to d e s c r i b e new s p e c i e s from a s m a l l number of specimens, A s m a l l sample s i z e could r e s u l t i n a narrow range of v a r i a t i o n i n any p a r t i c u l a r c h a r a c t e r . Subsequent samples from the same or other host s p e c i e s c o u l d r e s u l t i n specimens with v a r i a t i o n s i n c h a r a c t e r s d i f f e r e n t from those of the f i r s t sample. T h i s would be most pronounced i f t h e host i n some way i n f l u e n c e d p a r a s i t e development.. A c c o r d i n g l y , watertor (1967) noted t h a t the developmental r a t e s o f e x p e r i m e n t a l l y reared Te l g r c f a i s bonnerensis Waitz, 1960 d i f f e r e d markedly when r e a r e d in d i f f e r e n t amphibian or r e p t i l i a n hosts. Blankespoor (1974) experimenting with P l a g i o r c h i s n o b l e i Park, 1936, Boddeke (1960a) with P r ost h og d n i mus p va t u s . and o t h e r s , have noted s i m i l a r host-dependent v a r i a t i o n s i n development. F a c t o r s such as host age, number of worms present, and 29 temperatures may a l s o a l t e r f l u k e morphology (Haley, 1962). The purpose of t h i s s e c t i o n o f the t h e s i s was t o i n v e s t i g a t e m o r p h o l o g i c a l v a r i a t i o n i n H. b u t t e n s i s developed i n i t s normal host R. p r e t i o s a . T h i s study should provide i n f o r m a t i o n on the c h a r a c t e r s most s u i t a b l e f o r use i n s e p a r a t i n g s p e c i e s w i t h i n the genus. As w e l l , i t should p r o v i d e guide l i n e s f o r s t u d y i n g v a r i a t i o n i n H. b u t t e n s i s developed i n d i f f e r e n t host s p e c i e s . M a t e r i a l s and Methods Source of Experimental Animals Large numbers of u n i n f e c t e d or " c l e a n " f r o g s , s n a i l s and naiads of Odonata were needed f o r t h i s and other experiments. I t was c o n s i d e r e d i m p r a c t i c a l t o t r y t o r e a r these animals i n the l a b o r a t o r y and so n a t u r a l sources o f u n i n f e c t e d animals were sought. U n i n f e c t e d sna11s. Phvsa n u t t a l l i . were c o l l e c t e d on four separate o c c a s i o n s , over a two-year p e r i o d , from a pond f i f t e e n m i les south of P e n t i c t o n / B.C. F i f t y s n a i l s from each sample were examined f o r n a t u r a l i n f e c t i o n s with trematodes, but none was found. Some o f the s n a i l s remaining from the f i r s t two c o l l e c t i o n s were used as a source of " c l e a n " hosts i n subseguent experiments. The l a s t two c o l l e c t i o n s were examined as an a d d i t i o n a l assurance that s n a i l s from the c o l l e c t i n g s i t e were not i n f e c t e d . A source of u n i n f e c t e d d a m s e l f l y n a i a d s . Ischnura perparva* was d i s c o v e r e d i n a stream f i f t e e n m iles e a s t of Lumby, B. C. 30 F i f t y naiads were c o l l e c t e d and examined f o r n a t u r a l trematode i n f e c t i o n s . M e t a c e r c a r i a e are v i s i b l e when l i v e n aiads are i l l u m i n a t e d from beneath and observed under a s t e r e o s c o p i c microscope, n e c r o p s i e s were performed on the f i f t y naiads to assure no metacercariae were present. No metacercariae were found. In a d d i t i o n , each naiad was m i c r o s c o p i c a l l y examined p r i o r t o use i n a l l experiments. T h i s assured that no n a t u r a l i n f e c t i o n s occurred i n n a i a d s used f o r experiments. A stream l o c a t e d f i f t e e n m i l e s e a s t of Lumby, B. C. served as a source o f u n i n f e c t e d Rana p r e t i o s a . F i f t e e n f r o g s , frcm a l a r g e r sample, were examined f o r helminth i n f e c t i o n s of the lungs, but none was found. Subseguent examination o f R. p r e t i o s a from the same l o c a l i t y i n 1975 and 1976 demonstrated t h a t lung f l u k e s were not present i n f r o g s i n t h a t area. Frogs to be used i n experiments were kept an a d d i t i o n a l two weeks at 20°C. F a e c a l smears were taken d u r i n g the two-week p e r i o d and examined f o r f l u k e eggs. No eggs were pres e n t . The two—week qu a r a n t i n e would have allowed any f l u k e s t h a t were a l r e a d y i n the lungs to mature enough t o be separated from t h o s e from experimental i n f e c t i o n s . I n f e c t e d R a na t apretiosa were c o l l e c t e d from a beaver pond one mile e a s t o f Manning Park Lodge i n Manning Park. Frogs were sampled from t h i s pond on f i v e d i f f e r e n t o c c a s i o n s d u r i n g the s p r i n g and summer of 1974. Examination of these samples r e v e a l e d t h a t only one s p e c i e s o f lung f l u k e was p r e s e n t . The morphology o f t h i s f l u k e c o i n c i d e d with the d e s c r i p t i o n of H. b u t t e n s i s p u b l i s h e d by I n g l e s i n 1936. Eggs from these worms served as the source f o r experimental i n f e c t i o n s . 31 Experimental I n f e c t i o n s Adult H. b u t t e n s i s , o b tained from the lungs of 8. p r e t i o s a . were placed i n a d i s h of pond water f o r r e l e a s e of eggs. Groups of 25 s n a i l s were i n f e c t e d by a l l o w i n g them t o i n g e s t eggs f o r one hour, a f t e r which the s n a i l s were removed and placed i n aqu a r i a c o n t a i n i n g pond water, where they were l e f t f o r approximately 30 days f o r the c e r c a r i a e t o develop and emerge. I n f e c t e d s n a i l s were maintained at 20°C. Emerging c e r c a r i a e were p i p e t t e d from f i s h tanks, and 200 c e r c a r i a e were p l a c e d i n each of 10 f i n g e r bowls f i l l e d with pond water. Ten naiads were p l a c e d i n each bowl and l e f t f o r 24 hours, a f t e r which they were t r a n s f e r r e d to p l a s t i c c o n t a i n e r s and maintained at 20°C.,After f i v e days naiads were d i s s e c t e d and metacercariae removed. Frogs were i n f e c t e d by wrapping ten metacercariae i n moistened bread, s t r i n g i n g a t h r e a d through the bread and lowering the wad i n t o a tank c o n t a i n i n g one f r o g . Frogs u s u a l l y a te the bread w i t h i n t h r e e minutes. F i f t e e n f r o g s were i n f e c t e d i n t h i s way. Samples of three f r o g s were examined and lung f l u k e s c o l l e c t e d a t 5, 14, 21, 28 and 60 days. Specimens were prepared u s i n g the s t a n d a r d i z e d procedure g i v e n i n Appendix 3. Drawings of s e l e c t e d f l u k e s were made with the a i d of a Bausch and Lomb microprorjector and camera l u c i d a . A l l measurements were made as d e s c r i b e d i n Appendix 1 and are i n m i l l i m e t e r s u n l e s s otherwise s t a t e d . R e s u l t s The experiment y i e l d e d /f0l0 specimens of H. b u t t e n s i s o f known ages. Comparative measurements are presented i n 32 Appendix 5. Comparisons of the morphological f e a t u r e s used by I n g l e s (1936) i n h i s d e s c r i p t i o n of H. b u t t e n s i s are presented with my exp e r i m e n t a l r e s u l t s . V a r i a t i o n s with Age, Growth and M a t u r i t y : 1. M a t u r i t y : A l l worms were mature by 14 days a f t e r i n f e c t i n g f r o g s . M a t u r i t y i s here d e f i n e d as being reached when worms c o n t a i n eggs i n t h e i r uterus. My experiments i n d i c a t e t h a t worms may become mature when l e s s than 2.0 mm long (Appendix 5). I n g l e s (193.6) d i d not mention the s i z e a t which H. b u t t e n s i s becomes mature. His type d e s c r i p t i o n i s based on ten worms t h a t v a r i e d i n l e n g t h from 3.2 mm t o 10 mm. T h e r e f o r e , they were probably a l l a d u l t s . 2. Body S i z e : Type D e s c r i p t i o n : "Length averages 7.4 mm and v a r i e s from 3.2 mm t o 10 ram. l i d t h averages 1.3 mm and v a r i e s between 0.7 mm and 2.2 mm" ( I n g l e s , 1936, pp. 78-80) . Experimental R e s u l t s : Flukes recovered from R . p r e t i o s a 60 days a f t e r i n i t i a l exposure were on average 3.4 times l o n g e r (6.01) than fourteen-^day—old mature worms (1.75). Average body width i n c r e a s e d from 0.51 i n fo u r t e e n - d a y - o l d worms t c 1.26 i n 60-day-old worms. The f a s t e s t r e l a t i v e growth occurred between the me t a c e r c a r i a l stage and f i v e - d a y — o l d worms, and between 14 and 21 days ( F i g . 7 ) . Adu l t f l u k e s thus ranged from 1.61 to 6.67 mm l o n g . 33 3. V i t e l l a r i a : Type D e s c r i p t i o n : " V i t e l l a r i a l a t e r a l and d o r s a l to caeca; extend a c r o s s d o r s a l r e g i o n a n t e r i o r t o ovary; seventeen groups were counted i n one young f l u k e . " {Ingles, 1936, p.80). Experimental R e s u l t s ; V i t e l l a r i a are not e v i d e n t u n t i l 14 days post i n f e c t i o n . They extend a c r o s s the d o r s a l r e g i o n a n t e r i o r to the ovary and down the l a t e r a l margins of the worms. The r i g h t v i t e l l a r i a l e n g t h i s c o n s i s t e n t l y g r e a t e r than the l e f t . However, the percent d i f f e r e n c e between the two changes with age, which i n d i c a t e s a d i f f e r e n c e i n growth r a t e between the l e f t and r i g h t v i t e l l a r i a . At 14, 21, 28 and 60 days the percent d i f f e r e n c e i s 21*4, 7.0y 34.0 and 39.6, r e s p e c t i v e l y . T h e r e f o r e , the l e f t v i t e l l a r i a grew much f a s t e r r e l a t i v e t o the r i g h t s i d e between 14 and 21 days, which corresponds t o the f a s t e s t growth period o f the f l u k e . The r i g h t s i d e develops f a s t e r than the l e f t s i d e a f t e r 21 days. 4. Ovary and T e s t e s : Type D e s c r i p t i o n : "Ovary kidney—shapad, never l o b e d , averages 0.44 i n l e n g t h by 0.32 i n width; i t s range v a r i e s from 0.26 to 0.55 and from 0.20 t o 0.37 r e s p e c t i v e l y ; u s u a l l y on r i g h t s i d e . , . . . , , . , Testes n e a r l y same s i z e and shape; l e n g t h averages 0.82; width averages 0.64; range from 0.45 to 1.03 and 0.48 t o 0.87 r e s p e c t i v e l y . " ( I n g l e s , 19 36, p. 80) . 34 P i g . 7 R e l a t i o n s h i p between body l e n g t h , body width, and r e l a t i v e growth, and age o f worms developed i n Sana p r e t i o s a . The mean and range i s given f o r the l e n g t h and width of worms of d i f f e r e n t age groups. Size (mm) Relative Growth YK 35 Experimental R e s u l t s : the r a t i o of the l e n g t h of the t e s t e s (sum of both t e s t e s l e n g t h s ) t o that of the ovary remained f a i r l y c onstant f o r 14, 21 and 28—day—old f l u k e s , but i n c r e a s e d f o r 60-day-old worms, the r a t i o s being 2.7:1, 2.6:1, 2.5:1 and 3.3:1, r e s p e c t i v e l y . T h i s suggests an i n c r e a s e i n exjg p r o d u c t i o n at t h i s time. The ovary changed from a smooth round organ a t f o u r t e e n days to an e l l i p t i c a l , h i g h l y lobed form by 60 days ( F i g s . 8 t o 13). The ovary i n c r e a s e d i n l e n g t h from 0.08 to 0.71 and i n width from 0.08 to 0.36 d u r i n g the study p e r i o d . Testes changed from e l l i p t i c a l , smooth organs to e l o n g a t e forms which e i t h e r remained smooth or developed some degree of l o b i n g ( F i g s . 14 to 18). The a n t e r i o r t e s t i s i n c r e a s e d i n l e n g t h from an average of 0.34 i n 14— day—old worms to 1.12 i n 60-day-old worms, and 0.22-0.48 i n width. The p o s t e r i o r t e s t i s i n c r e a s e d frem 0.31-1.22 and 0.22-0.52 f o r l e n g t h and width r e s p e c t i v e l y . 5. L o n g i t u d i n a l F o l d s of the Uterus; Type D e s c r i p t i o n : "Uterus runs p o s t e r i o r l y between t e s t e s ; e x t r a c a e c a l l y the f o l d s may extend to a n t e r i o r l e v e l of a n t e r i o r t e s t i s on the same s i d e as the ovary; on the o p p o s i t e s i d e they extend only h a l f as f a r ; i t r e t u r n s t o a n t e r i o r p a r t of the body between the t e s t e s and has many f o l d s a n t e r i o r to the acetabulum." ( I n g l e s , 1936, p.80). Experimental R e s u l t s : the r i g h t e x t r a c a e c a l u t e r i n e loop extends forward a d i s t a n c e o f 1/4 t o 3/4 the l e n g t h along the p o s t e r i o r t e s t i s . The l e f t l o o p extends forward 1/2 the d i s t a n c e 3 6 to the p o s t e r i o r t e s t i s , from the end of the worm., to 3/4 the d i s t a n c e along the p o s t e r i o r t e s t i s . The r e l a t i v e l e ngths of both u t e r i n e f o l d s (body l e n g t h / u t e r i n e l o o p length) decreased with age. The r i g h t f o l d decreased i n r e l a t i v e l e n g t h from 16.1:1 a t 14 days t o 4.0:1 at 60 days. The l e f t f o l d decreased i n r e l a t i v e l e n g t h from 19.6:1 at 14 days t o 5.6:1 a t 60 days. 37 F i g . 8 . M e t a c e r c a r i a from Ischnura perparva. F i g . 9 F i v e - d a y - o l d f l u k e developed i n Rana p r e t i o s a . F i g . 10 Fourteen—day—old f l u k e developed i n Rana p r e t i o s a . F i g . 11 Twenty-one—day-old worm developed i n Rana p r e t i o s a . F i g . 12 Twenty-eight—day-old worm developed i n 'Rana' p r e t i o s a . F i g . 13 S i x t y — d a y - o l d f l u k e developed i n Rana p r e t i o s a . > 38 F i g u r e s 14 t o 18. V a r i a t i o n i n s i z e , shape and p o s i t i o n of ovary and t e s t e s o f 60—day-old worms developed i n Rana p r e t i o s a . 38A 39 6. S p i n a t i o n : Type D e s c r i p t i o n : " C u t i c l e armed wi t h s p i n e s p o s t e r i o r l y to the acetabulum.: ( I n g l e s , 1936, p. 78) . Experimental R e s u l t s : Flukes of a l l ages had spi n e s . .Spines were u n i f o r m l y d i s t r i b u t e d over the e n t i r e body s u r f a c e . Spines appeared t o be l e s s numerous and were s m a l l e r on the p o s t e r i o r p o r t i o n o f the body. 7. Sucker r a t i o : Type D e s c r i p t i o n : " O r a l sucker s u b t e r m i n a l averages 0.33 mm i n l e n g t h by 0.46 mm i n width; i t ranges from 0.18 to 0.14 and from 0.29 t o 0.47 r e s p e c t i v e l y f s i c 1. Acetabulum l o c a t e d i n f i r s t h a l f of body and s m a l l e r than o r a l sucker; i t averages 0.26 i n l e n g t h by 0.31 i n width; i t ranges from 0.15 to 0.36 i n l e n g t h and 0.24 t o 0.37 i n width. R a t i o of l e n g t h o f o r a l sucker to acetabulum i s 1:0.7, and range o f v a r i a t i o n of t h i s r a t i o i s 1:0. 8 t o 1:0.6 " ( I n g l e s , 1936, pp.78^80) . Experimental R e s u l t s : The o r a l sucker i n 60-day-old wcrms was t e r m i n a l and averaged 0.25 long by 0.29 wide. The acetabulum was median and always c l o s e r t o the ovary than to the a n t e r i o r end. : Acetabulum diameter i n c r e a s e d from an average of 0.07 i n four t e e n - d a y ^ o l d f l u k e s t o 0.11 i n 60-day—old worms. O r a l sucker diameter i n c r e a s e d from 0*18 to 0.29 durin g the same time. The sucker r a t i o f o r post metacercarialworms remained constant f o r a l l age groups. F i v e — d a y — o l d worms had only a s l i g h t l y s m a l l e r r a t i o (2.35:1) than mature worms (2.5:1 t o 2.6: 1) ( F i g . 19). Met a c e r c a r i a e have an o/A r a t i o s i g n i f i c a n t l y s m a l l e r than t h a t i n worms recovered from f r o g s ( F i g . 19). The sm a l l e r 0/A r a t i o 40 f o r m etacercariae i s a r e f l e c t i o n of the r e l a t i v e l y l a r g e r acetabulum. The diameter of the acetabulum decreases f o r f i v e — d a y - o l d worms, then begins t o i n c r e a s e i n s i z e again ( F i g . 20). 8. Eggs: Type D e s c r i p t i o n : "Eggs o p e r c u l a t e , brown with completely formed m i r a c i d i a when l a i d ; average 0.027 by 0.014; range 0.025 to 0.030 and 0.011 to 0.017 f o r len g t h and width r e s p e c t i v e l y " ( I n g l e s , 1936, p. 80). Experimental R e s u l t s : Eggs a r e present f i r s t i n area "C" o f the uterus (Appendix 6, F i g . 8T:) 14 days a f t e r i n i t i a l exposure and average 0.017 by 0.015. Eggs i n c r e a s e i n s i z e as they pass along the uterus u n t i l , at 21 days, they average 0.024 by 0.016 and are found i n area "A" (Appendix 6, F i g . 8#) . T h e r e a f t e r egg s i z e remains constant r e g a r d l e s s of f l u k e age. 41 Fig.19 Change i n O/fl r a t i o with age of worm. The Bean and range i s given f o r each age group. 41A Worm Age (Days) 42 Fig.20 R e l a t i o n s h i p between age of worms developed i n Rana p r e t i o s a and diameter of o r a l sucker and acetabulum. The p o s i t i o n of the measurements f o r metacercariae has been staggerd f o r c l a r i t y . The mean and range i s g i v e n f o r each age group. Age of Worm (Days) 43 3.,; VARIATION IN FLUKES DEVELOPED IN SANA PfiETIOSA HAINTAINED AT DIFFERENT TEMPERATURES INTRODUCTION The temperature range at which l a r v a l and a d u l t helminths l i v e and develop v a r i e s c o n s i d e r a b l y from s p e c i e s t o s p e c i e s . A d i r e c t r e l a t i o n s h i p e x i s t s between temperature and r a t e of growth and development t o a c e r t a i n p o i n t , a f t e r which v a r i o u s unfavourable e f f e c t s may occur (Watertor, 1965). T h i s study examines the e f f e c t of temperature on the morphology o f H. butt ens i s when r e a r e d i n R. p r e t i o s a . kept at 120c, 20°C or 27oc. M a t e r i a l s and Methods Three groups of 15 f r o g s each were f e d 10 metacercariae each. Each group was maintained i n aguari a (50 cm by 25 cm by 30 cm) at e i t h e r 12°C, 20»Cor 27<>C. Set paper towels were kept on the bottom of the tanks to provide moisture f o r the f r o g s . The tops o f ' t h e tanks were covered with g l a s s sheets t o prevent e x c e s s i v e e v a p o r a t i o n . Samples of three f r o g s were examined from each group a t 5, 14, 21, 28 and 60 days p o s t - i n f e c t i o n . Physa n u t t a l l i and Ischnura Perparva served as the i n t e r m e d i a t e hosts. R e s u l t s Growth and development o f 204 e x p e r i m e n t a l l y - r e a r e d worms r e s u l t i n g from the f e e d i n g experiments with a d u l t R. p r e t i o s a were s t u d i e d and measured. Measurements of tax o n o m i c a l l y s i g n i f i c a n t c h a r a c t e r s are g i v e n i n Appendices 7 t o 9 i n c l u s i v e . Worms maintained a t a higher temperature were longer and wider than worms o f a comparable age but developed a t a lower temperature. Worms rea r e d a t 27°C a t t a i n e d the l a r g e s t s i z e s (length and width) o f a l l age groups s t u d i e d (Pig. 21). Worm le n g t h and width i n c r e a s e d with age o f worms.maintained a t 20°€ and 279C. Worms at 12°C d i d not show t h i s a g e - r e l a t e d i n c r e a s e ( F i g . 21) Flukes r e a r e d at 27°c had the l a r g e s t {length and width) o r a l suckers f o r each age group s t u d i e d ( F i g . 21). The onl y e x c e p t i o n was i n 14-day-old worms. Worms i n t h i s age group, developed a t 2G°C and 27°C, had s i m i l a r o r a l sucker widths (Appendix 8 and 9). The l e n g t h and width of the o r a l sucker i n worms r e a r e d a t 20<»C and 27«c i n c r e a s e d with age. Worms a t 12°C d i d not show an a g e - r e l a t e d i n c r e a s e i n these measurements ( F i g . 21) . The diameter of the acetabulum d i d not i n c r e a s e i n worms developed a t 12®C. However, t h i s measurement showed an i n c r e a s e with age i n f l u k e s r e a r e d a t 20°C and 27»C ( F i g . 21). F i v e - and 14-day-old worms, r e a r e d at 12°C had wider a c e t a b u l a than d i d s i m i l a r l y aged worms developed at 20°C or 27«C. By 21 days, the acetabula of a l l f l u k e s were the same s i z e , but by 28 and 60 days worms developed at 20°C and 27°C had wider acetabula than d i d worms developed a t the lowest temperature. Older worms had l a r g e r t e s t e s at a l l temperatures s t u d i e d . There was a marked i n c r e a s e i n l e n g t h o f t e s t e s as w e l l as r a t e of growth i n worms .^^SS^p-v at 2 0°C compared with those a t 45 12°C. Worms at 27°C had skoYt«.r t e s t e s than those at 20°C ( F i g . 22) . The ovary d i d not develop i n worms maintained at 12°C. The ovary l e n g t h i n f l u k e s developed a t 2 0°C was g r e a t e r than t h a t develope 27«C ( P i g . 22) . a l l f l u k e s r e a r e d at 20<>C and 27°C were g r a v i d by 14 days, norms maintained at 12°C d i d not mature. The i n c r e a s e i n l e n g t h of the p o s t e r i o r p o r t i o n o f the worm was g r e a t e s t i n worms developed at higher temperatures. T h i s i n c r e a s e i s expressed by comparing the d i s t a n c e of the p o s t e r i o r margin o f the a n t e r i o r as w e l l as the p o s t e r i o r t e s t i s f r o a the p o s t e r i o r end of the worm ( F i g . 23). The g r e a t e s t r a t e o f i n c r e a s e o c c u r r e d at 27 °C between 14 and 21-day-old worms. T h i s corresponds to the h i g h e s t r a t e of i n c r e a s e i n s i z e of 'the ovary and t e s t e s , and the i n c r e a s e i n egg production i n worms at t h i s time The O/fi r a t i o o f f l u k e s d i d not d i f f e r with age of worm or when the host was maintained at 20°C or 27°C. Worms of a l l ages r e a r e d a t 12°C had a s i g n i f i c a n t l y lower o/ft r a t i o , e s s e n t i a l l y t h a t of the meta c e r c a r i a e . 46 F i g . 21 The i n f l u e n c e o f t e m p e r a t u r e and age o f worm on some c h a r a c t e r s o f H. b u t t e n s i s d e v e l o p e d i n R. p r e t i o s a . 8.0-7.0-6.0-5.0-4 .0 -3.0 2.0-L 1.0-T 11 5 14 21 28 60 BODY LENGTH Temperature 5 14 21 28 60 BODY WIDTH 5 14 21 28 60 5 14 21 28 60 ORAL SUCKER LENGTH ORAL SUCKER WIDTH 5 14 21 28 60 ACETABULUM WIDTH Age of Worm (Days)and Character Measured ON > The e f f e c t o f temperature on the le n g t h of the ovary, a n t e r i o r t e s t i s , and p o s t e r i o r t e s t i s of worms reared i n R. p r e t i o s a . 47A Age (Days) 4 8 F i g . 23 R e l a t i v e growth of the hind body. Distance between the p o s t e r i o r margin of the a n t e r i o r and p o s t e r i o r t e s t i s from the end o f the worm. 48A Age (Days) 49 4. EFFECT OF HOST SIZE ON DEVELOPMENT OF H. BUTTENSIS I n t r o d u c t i o n R e l a t i o n s h i p s between s i z e and form of p a r a s i t e s and s i z e (age) o f d e f i n i t i v e hosts have been observed by many p a r a s i t o l o g i s t s , but few have examined these r e l a t i o n s h i p s i n d e t a i l . D o g i e l (1966) and ot h e r s have summarized the l i t e r a t u r e on t h i s r e l a t i o n s h i p . , M a t e r i a l s and Methods The experiments u t i l i z e d 15 specimens of B. p r e t i o s a i n each of 4 s i z e groups: 30-35 mm; 45-50 mm; 55-60 mm; and €5-70 mm. Ten metacercariae from I . perparva were f e d to each f r o g i n a l l g r o u p s . Frogs were maintained at 20°C f o r 60 days. Samples of t h r e e f r o g s were taken from each group a t 5, 14, 21, 28 and 60 days p o s t - i n f e c t i o n and examined f o r lung f l u k e s . R e s u l t s Measurements o f a l l specimens r e c o v e r e d from these experiments i n d i c a t e that l e n g t h (age) of R. p r e t i o s a does not a f f e c t the s i z e or shape of ad u l t H. b u t t e n s i s (Appendices 10 to 13). worm l e n g t h or width d i d not d i f f e r i n samples from f r o g s of d i f f e r e n t s i z e groups ( F i g . 24). The i n c r e a s e i n s i z e of a l l c h a r a c t e r s measured d i d not d i f f e r i n f l u k e s developed i n f r o g s of d i f f e r e n t s i z e s . 50 F i g . 24 The r e l a t i o n s h i p between the l e n g t h {&) and width <B) of H. b u t t e n s i s developed i n d i f f e r e n t s i z e groups of R. p r e t i o s a . S-7 i s the snout-vent l e n g t h of the f r o g . Millimeters 51 5. EFFECT OF HOST SEX OS MORPHOLOGY 0F jg. SJOTTENSIS I n t r o d u c t i o n another area o f i n t e r e s t t o p a r a s i t o l o g i s t s i s the i n f l u e n c e o f the sex o f the host on trematodes. H o l l i s (1972) found a s i g n i f i c a n t d i f f e r e n c e i n seasonal pj^'^vji^ . o f Haematploechus medioplexus between male and female Rana p r e t j o s a . He concluded t h a t female gonadotropins were i n v o l v e d i n the seasonal prevalence. However, no mention was made o f d i f f e r e n c e s i n morphology of f l u k e s recovered from male or female f r o g s . Such d i f f e r e n c e s as do e x i s t between male and female f r o g s may a l t e r the morphology of developing f l u k e s . The o b j e c t i v e o f t h i s study was to i n v e s t i g a t e the e f f e c t s of host sex on the morphology of H. b u t t e n s i s . M a t e r i a l s and Methods F i f t e e n male and f i f t e e n female £. p r e t i o s a were each i n f e c t e d with 10 metacercariae which had developed i n I . p e r p a r v a . Frogs were maintained at 20°C, and samples of three f r o g s were taken from each group at i n t e r v a l s of 5, 14, 21, 28 and 60 days., R e s u l t s a d u l t H. b u t t e n s i s developed i n e i t h e r male or female f r o g s show no s i g n i f i c a n t d i f f e r e n c e s i n morphology., F i g u r e 25 i l l u s t r a t e s t h i s f o r body l e n g t h , body width, and O/A r a t i o . Values f o r other c h a r a c t e r s measured can be found i n appendices 14 and 15. 52 F i g . 25 The i n f l u e n c e of host sex on body l e n g t h , body width, and 0/& r a t i o o f f l u k e s developed i n e i t h e r male or i n f e o a l e Rana p r e t i o s a . < 52A Age (Days) 53 6. E F F E C T OF ROHM BUBDEH* ON DEVELOPMENT OF H. B U T T E N S I S I n t r o d u c t i o n Worm b u r d e n r e s u l t i n g i n c r o w d i n g c a n be a n i m p o r t a n t f a c t o r i n r e g u l a t i n g t h e s i z e o f f l u k e s ( B l a n k e s p o o r , 1 9 7 0 ) . T h i s e x p e r i m e n t w a s c o n d u c t e d t o d e t e r m i n e t h e e f f e c t o f v a r y i n g n u m b e r s o f worms o n s i z e o f a d u l t H. b u t t e n s i s . M a t e r i a l s a n d M e t h o d s F i v e g r o u p s o f t h r e e m a l e R. p r e t i o s a , o f a p p r o x i m a t e l y t h e same s i z e ( 4 5 - 5 0 ) , w e r e f e d e i t h e r 1 0 , 2 0 , 4 0 , 80 o r 160 m e t a c e r c a r i a e . I n f e c t e d f r o g s w e r e m a i n t a i n e d f o r 6 0 d a y s a t 20°C c n a d i e t o f b e e f h e a r t a n d e a r t h w o r m s . R e s u l t s T h e e f f e c t o f d e g r e e o f " c r o w d i n g " o n 9 3 0 e x p e r i m e n t a l w o r m s r e a r e d i n R. p r e t i o s a i s s u m m a r i z e d i n T a b l e 4 f r o m d a t a i n A p p e n d i x 1 6 . T h e n u m b e r o f w o r m s r e c o v e r e d f r o m f r o g s i n c r e a s e d w i t h i n c r e a s e d n u m b e r o f m e t a c e r c a r i a e f e d . H o w e v e r , t h e p e r c e n t a g e o f g r a v i d w o r m s d e c r e a s e d s i g n i f i c a n t l y . An a n a l y s i s o f t h e d a t a , u s i n g l i n e a r r e g r e s s i o n , g a v e a c o e f f i c i e n t , b = - 0 . 1 9 w h i c h h a d a s i g n i f i c a n c e o f 0.05 when a t — t e s t w a s u s e d . T h e p e r c e n t s u r v i v a l o f m e t a c e r c a r i a e d e c r e a s e d i n i t i a l l y a n d t h e n i n c r e a s e d s l i g h t l y ( T a b l e 4 ) . T h e n u m b e r s o f worms r e c o v e r e d f r o m t h e l e f t l u n g w e r e t h e s a m e a s t h a t f o r t h e r i g h t l u n g . T h e p e r c e n t g r a v i d f l u k e s f o r l e f t a n d r i g h t l u n g lWorm burden i s the number o f worms recovered from the host 54 d i d not d i f f e r s i g n i f i c a n t l y (X 2, P>0.05%). The average body s i z e of f l u k e s decreased with i n c r e a s e d numbers of metacercariae fed ( F i g . 26). Worms re c o v e r e d from f r o g s fed 160 metacercariae were on an average 1/3 the s i z e as those recovered from f r o g s f e d only 10 metacercariae., Table 4 . Effects of crowding on 60-day-old H. buttensis in Rana pretiosa metace rcariae # worms # worms % recovered survival Lung Left Right # gravid worms % gravid worms Lung Left Right Lung Left Right 'average body size (LxW)/2 30 23 76. 7 14 9 14 9 100 100 3.77(2. 38-5. 29) 60 46 76. 7 25 21 21 21 84. 0 100 3.36(2. 13-4. 73) 120 84 70.0 41 43 28 27 68. 3 63. 0 2.91(1. 63-3. 75) 240 122 50. 8 62 60 32 34 51. 6 56. 7 1.95(1. 31-2. 63) 480 306 63. 8 148 158 59 52 39.9 32.9 1.26(0. 89-1.80) Total 290 291 154 143 measurements in square millimeters 56 Fig.26 Decrease i n body s i z e o f H. b u t t e n s i s a f t e r 60 days o f development i n R. p r e t i o s a which were f e d e i t h e r 30, 60, 120, 240 or 480 metacercariae. The mean and ranges are graphed. 56A 57 7. V A R I A T I O N I N WORMS DE V E L O P E D I N D I F F E R E N T D E F I N I T I V E HOSTS I n t r o d u c t i o n I n c r e a s i n g a t t e n t i o n h a s b e e n f o c u s s e d i n r e c e n t y e a r s o n t h e v a r i a t i o n s i n a p a r a s i t e ' s s t r u c t u r e , p h y s i o l o g y , a n d b e h a v i o u r r e s u l t i n g f r o m i t s d e v e l o p m e n t i n d i f f e r e n t h o s t s p e c i e s { K i n s e l l a , 1971). T h e f o l l o w i n g e x p e r i m e n t s w e r e c o n d u c t e d t o d e t e r m i n e i f m o r p h o l o g i c a l c h a n g e s i n a d u l t S« b u t t e n s i s o c c u r when t h e y a r e r e a r e d i n d i f f e r e n t a m p h i b i a n h o s t s . N i n e t y a m p h i b i a n s r e p r e s e n t i n g s i x s p e c i e s o f d e f i n i t i v e h o s t s w e r e u s e d . M a t e r i a l s a n d M e t h o d s M e t a c e r c a r i a e , o b t a i n e d b y t h e m e t h o d d e s c r i b e d f o r " D e v e l o p m e n t i n N a t u r a l H o s t s " (p.33T) , w e r e f e d t o t h e f o l l o w i n g d e f i n i t i v e h o s t s : B u f o b o r g a s B a i r d a n d G i r a r d , 1852; RllH r e c j i l l a B a i r d a n d G i r a r d , 1852; R a n a a u r o r a B a i r d a n d G i r a r d , 1852; R a n a c l a m i t a n s L a t r e i l l e , 1802; R a n a p i p i . e n s S c h r e b e r , 1782; a n d R a n a p r e t i o s a . A l l e x p e r i m e n t a l h o s t s w e r e c a u g h t i n t h e w i l d f r o m n o n — i n f e c t e d p o p u l a t i o n s ; c o l l e c t i n g l o c a l i t i e s f o r h o s t s a r e g i v e n i n T a b l e 5. W i l d - t r a p p e d h o s t s w e r e h e l d i n t h e l a b o r a t o r y f o r 21 d a y s , a n d t h e f e c e s w e r e c h e c k e d p e r i o d i c a l l y t o v e r i f y t h a t n a t u r a l i n f e c t i o n s o f l u n g f l u k e s w e r e n o t p r e s e n t . F i f t e e n f r o g s o f e a c h s p e c i e s w e r e i n f e c t e d i n t h e m a n n e r d e s c r i b e d o n p a g e j i * . A l l f r o g s w e r e m a i n t a i n e d a t 20°C on a d i e t o f b e e f h e a r t s u p p l e m e n t e d w i t h e a r t h w o r m s d u r i n g t h e d e v e l o p m e n t a l p e r i o d o f t h e p a r a s i t e s . 58 Samples of t h r e e f r o g s of each s p e c i e s were examined a t 5, 14,: 21, 28 and 60 days. Specimens of H. b u t t e n s i s from the v a r i o u s hosts were then compared with specimens of s i m i l a r age from R. p r e t i o s a as w e l l as with younger and o l d e r worms. 59 Table 5. Collecting sites for uninfected definitive hosts. Host Collecting locality Bufo boreas Haney, B. C. Hyla regilla U. B . C . Endowment Lands Rana aurora Chemainus Lake, near Duncan, B .C. Rana clamitans Blaine, Washington Rana pipir.ns Foxborough, Ontario Rana pretiosa 15 miles east of Lumby, B. C. 60 R e s u l t s ..: Ccmparative measurements of H. b u t t e n s i s from experimental h o s t s are presented i n appendices 17 to 20. Host r a p i d development took p l a c e i n the n a t u r a l host B. p r e t i o s a and i n B.; boreas, which i s i n f r e q u e n t l y found n a t u r a l l y i n f e c t e d . Development i n R. aurora was the same as t h a t found f o r R. c l a m i t a n s ; i n both, the f l u k e s took about one week longer to produce eggs than d i d f l u k e s developing i n B. boreas or R. p r e t i o s a . So worms were found i n R. p i p i e n s or H. r e g i l l a a t any , t i i e a i i r i n g the s a i p l i n g p e r i o d (Table 6 ) ; they were judged t o be u n s u i t a b l e hosts. Pigures 29 t o 34 and F i g u r e s 36 to 38 i l l u s t r a t e some y a r i a t i o n s o c c u r r i n g ^ i n 60-day o l d worms from R. p r e t i o s a . :- H. a u r o r a . R. c l a m i t a n s and B. boreas. „ Body s i z e : Adult H. b u t t e n s i s recovered from B. p r e t i o s a were u s u a l l y l a r g e r than those obtained from 8. box.eag, R. aurora or R. c l a m i t a n s - f o r a l l ages sampled. T h e r e f o r e , the former h o s t , of those t e s t e d , appears to be the optimal one a t s i x t y days, i -•H.;,;.;,/b,uttensis • from R. p r e t i o s a had an average l e n g t h 6.41 ( F i g . 27). Sixty—day o l d f l u k e s developed i n B. aurora or i n B. c l a m i t a n s were 4.95 and 4.88 l o n g , r e s p e c t i v e l y and i n jB. boreas 5.35 {Fig. 27) . 61 Table 6. Variations in rate of development of H. buttensis maturing in various amphibian hosts. Host First appearance of gravid worms (days) Bufo boreas 21 Rana ajjrora 28 Rana clamitans 28 Rana p.voiens No worms present Rana p i etiosa 14. Hyla re gilla No worms present Determined by dissecting frogs, removing H. buttensis and preparing slides of the worms. 62 F i g . 27 The r e l a t i o n s h i p between body l e n q t h , body width, and 0/A r a t i o of H. b u t t e n s i s developed i n d i f f e r e n t f r o g hosts., R. a. fiiM aurora R. c. Rana cl§mitans R. p. Rana p r e t i o s a B. b. Bufo boreas Body Length (mm) Body Width (mm) 0/A Ratio 63 F l u k e s recovered from R.;.clamitans 28 and 60 days p o s t - i n f e c t i o n were the s m a l l e s t of comparably aged worms recovered d u r i n g t h i s study {Fig. 27). Horms recovered from B. boreas were in t e r m e d i a t e i n s i z e . Average body widths were g r e a t e r f o r f l u k e s from R. p r e t i o s a at a l l ages examined. Average body widths f o r 60-day-old f l u k e s from B. p r e t i o s a were 1.40; from B. boreas. 1.37 and from R. aurora and R. cl a m i t a n s 1.20 and 1.17 r e s p e c t i v e l y ( F i g . 27). Worms, r e g a r d l e s s o f host, showed an i n c r e a s e i n l e n g t h and width with i n c r e a s e d age of f l u k e . Sucker s i z e : The o r a l sucker was c o n s i s t e n t l y l a r g e r than the acetabulum i n a l l f l u k e s observed. O r a l sucker width v a r i e d i n f l u k e s harboured by d i f f e r e n t host s p e c i e s . The maximum width (0.372) of the o r a l sucker was a t t a i n e d by a 60-day-old f l u k e developed i n B. boreas. The maximum diameter of the acetabulum was 0.123 which occurred i n a 6 0—day-old worm developed i n R. c l a m i t a n s . The s m a l l e s t diameter i n a 60—day-old worm occurred i n B. boreas (0.082) . The 0/A r a t i o o f mature worms developed i n B. boreas was g r e a t e r (2.9—3.1) than f o r f l u k e s developed i n the ether host s p e c i e s (2.4-3.1). The l a r g e r 0/A r a t i o i n f l u k e s from B.,boreas i s a r e s u l t of s m a l l e r acetabulum diameter i n those worms. , 6 4 Fig. 28 the e f f e c t of d i f f e r e n t amphibian hosts on oral sucker length, o r a l sucker width, and acetabulum diameter of H. buttensis. 0.12 H 0.10-1 E J: E o 5 E ojas-l *5 i 0.06-1 w < 0.04-1 E JL 0 . 4 H x a 0 . 3 H e a 0 . 2 el D R.aurora A R.clamitans • R.pretiosa O B.boreas O O 0 . 1 - | E X I 1 0.2-fl O 0.1-Bassaomi , „_ -Q _o Q • • 20 40 50 60 Age (Days) 65 Ovary s i z e and shape: V a r i a t i o n s i n gonad s i z e i n a d u l t H. b u t t e n s i s recovered from d i f f e r e n t host s p e c i e s are even more pronounced than v a r i a t i o n s i n t h i s organ i n f l u k e s obtained from a s i n g l e host s p e c i e s . Flukes p o s s e s s i n g u n u s u a l l y l a r g e o v a r i e s s e r e obtained from experimental i n f e c t i o n s i n B, c l a m i t a n s ( F i g s . 31, 33 and 35 ) . The ovary of one a d u l t s i x t y - d a y - o l d fl. b u t t e n s i s from R. c l a m i t a n s measured 0.85 long and 0.73 wide. The ovary d i d not become apparent u n t i l 21 days a f t e r i n f e c t i n g f r o g s . T h i s was a l s o t r u e f o r f l u k e s developed i n R, auro r a . I n f e c t i o n s i n R. aurora y i e l d e d H. b u t t e n s i s a d u l t s with s m a l l o v a r i e s . The s m a l l e s t ovary i n a 60-day-old f l u k e from R. au r o r a was 0.392 long by 0.113 wide. The l a r g e s t t e s t e s were found i n one a d u l t 60—day-old worm from R. c l a m i t a n s . the a n t e r i o r t e s t i s of which measured 1.32 long by 0.58 wide and t h e p o s t e r i o r t e s t i s measured 1.46 long by 0.65 wide. The s m a l l e s t t e s t i s was found i n a sixty^-day-old worm from R. aurora: the a n t e r i o r t e s t i s measured 0,62 long by 0.31 wide, and the p o s t e r i o r t e s t i s 1.25 lo n g by 0.28 wide ( F i g . 35). 66 Gonad s i z e and u t e r i n e loop v a r i a t i o n i n f l u k e s developed i n d i f f e r e n t d e f i n i t i v e hosts. F i g . 29 F l u k e developed i n Rana p r e t i o s a . F i g . 3 0 Fluke developed i n Rana aurora.; F i g . 31 Fl u k e developed i n Rana c l a m i t a n s . F i g . 32 Fluke developed i n jgufo boreas. F i g . 33 Fluke developed i n Ban a) c l a m i t a n s . F i g . ,34 Fluke developed i n Rana aurora. The e f f e c t of d i f f e r e n t amphibian hosts t e s t e s and ovary l e n q t h of H. b u t t e n s i s . 67A Age (Days) 6 8 V i t e l l a r i a : V i t e l l a r i a became evi d e n t 14 days p o s t - i n f e c t i o n i n B. boreas and R. p r e t i o s a but only a f t e r 21 days i n R. clamitans and B. anfgr.i,. ,The l e n g t h of t h e v i t e l i a r i a on the r i g h t s i d e of th e worm was - c o n s i s t e n t l y s h o r t e r than t h a t on the l e f t ( F i g s . 36 t o ; 3 f j i . The a n t e r i o r l i m i t o f v i t e l l i n e f o l l i c l e s o c c u r r e d between the acetabulum and pharynx i n 60—day-old worms recovered from a l l hosts. V i t e l l i n e f o l l i c l e s i n f l u k e s from R. aurora and R. c l a m i t a n s reached a l e v e l j u s t a n t e r i o r to the acetabulum ( F i g s . 37 and 38). However, i n worms from R. p r e t i o s a and B. ..boreas, v i t e l l i n e f o l l i c l e s extended f u r t h e r a n t e r i o r to the r e g i o n between the acetabulum and pharynx ( F i g . 36). Specimens from R. p r e t i o s a and B. boreas had a s o l i d band of v i t e l l a r i a a c r o s s the body between the pharynx and acetabulum. In specimens from R. aurora and R. cl a m i t a n s the v i t e l l i n e band o f t e n l a y beneath the acetabulum. Incomplete bands were sometimes observed, and i n three cases a t r a n s v e r s e band was absent ( F i g . 38). Much g r e a t e r v a r i a b i l i t y c h a r a c t e r i z e s the p o s t e r i o r l i m i t s of the v i t e l l i n e f o l l i c l e s i n specimens recovered from a l l amphibian hosts. A band o f v i t e l l i n e f o l l i c l e s u s u a l l y extended m e d i a l l y half-way a c r o s s the worm from the p o s t e r i o r p a r t o f the l e f t v i t e l l i n e band p a s s i n g p b s t e r i a d o f t h e p o s t e r i o r t e s t i s . The experimental s t u d i e s demonstrate t h a t the presence or absence o f a v i t e l l i n e band, or t h e extent of the a n t e r i o r o r p o s t e r i o r V i t e l l a r i a , a r e u n r e l i a b l e c r i t e r i a f o r d i s t i n g u i s h i n g s p e c i e s w i t h i n t h i s genus. Fig.,36 D i s t r i b u t i o n developed i n F i g . 3? D i s t r i b u t i o n developed i n F i g . 38 D i s t r i b u t i o n developed i n of v i t e l l i n e a l l f l u k e s drawn i n v e n t r a l o f v i t e l l a r i a i n worms Sana p r e t i o s a . ; o f v i t e l l a r i a i n worms Sana aurora. of v i t e l l a r i a i n worms Sana c l a m i t a n s showing absence band. , view. , 69A 70 E x t r a c a e c a l f o l d s of the uterus: E x t r a c a e c a l l o n g i t u d i n a l f o l d s of the uterus were f i r s t e v i d ent at 14 days p o s t - i n f e c t i o n i n R. p r e t i o s a and B. boreas but d i d not become e v i d e n t u n t i l the twenty-eighth day i n B. c l a m i t a n s or B. auro r a . The r i g h t l o n g i t u d i n a l f o l d was long e r than the l e f t i n specimens developed i n B. p r e t i o s a . The reverse was t r u e f o r specimens developed i n B. boreas (compare F i g s . 29 with 32). The r i g h t and l e f t l o o p s were of about the same le n g t h i n specimens from fi. aurora and B. c l a m i t a n s . The r i g h t u t e r i n e f o l d s of 60-day o ld worms developed i n •B^ypretiosa reached a d i s t a n c e half—way along the p o s t e r i o r t e s t e s . The l e f t loop j u s t reached the p o s t e r i o r part of the p o s t e r i o r t e s t i s ( F i g . 29). E x t r a c a e c a l l o o p s reached about half—way t o the p o s t e r i o r t e s t i s i n worms developed i n B. boreas. The l e f t loop extended somewhat beyond the r i g h t ( F i g . 32). These u t e r i n e l o o p s extended o n l y 1/3 the way to the p o s t e r i o r t e s t i s i n 60—day-old worms developed i n R. aurora and R. c l a m i t a n s ( F i g s . 30,31,33 and 34). Development i n d i f f e r e n t d e f i n i t i v e hosts a l t e r e d the a n t e r i o r extent o f the u t e r i n e l o o p s . The u t e r i n e loops extended the f a r t h e s t i n the n a t u r a l - host>• S.-• v p r e t i e s a, and the l e a s t i n B . a u r o r a and R. c l a m i t a n s . ^ S p i n a t i o n : Spines were u n i f o r m l y d i s t r i b u t e d over the e n t i r e body s u r f a c e o f f l a k e s of a l l ages from a l l hosts. 71 Egg s i z e : Eggs were not present i n the uterus u n t i l 14 days p o s t — i n f e c t i O n i n R. p r e t i o s a ; 21 days i n B. boreas; and 28 days i n B . a u r o r a and R. c l a m i t a n s . Eggs were o f approximately the same l e n g t h and width i n 60-day o l d worms developed i n R. p r e t i o s a . 8. c l a m i t a n s and -fi. boreas. Eggs were s m a l l e s t i n worms from R. augora (appendices 17—20). 7 2 8. V A R I A T I O N DOE TO DEVELOPMENT I N D I F F E R E N T I N S I C T I N T E R M E D I A T E HOSTS. I n t r o d u c t i o n H a v i n g d e m o n s t r a t e d t h a t some t a x o n o m i c c h a r a c t e r s a r e a l t e r e d when f l u k e s d e v e l o p i n d i f f e r e n t d e f i n i t i v e h o s t s , we m u s t now e s t a b l i s h w h e t h e r c h a n q e s o c c u r when worms d e v e l o p i n d i f f e r e n t i n t e r m e d i a t e h o s t s . M a t e r i a l s a n d M e t h o d s I n f e c t e d s n a i l s , P. n u t t a l l i , m a i n t a i n e d a t 2 0 ° C o n a d i e t o f b o i l e d l e t t u c e , s h e d x i p h i d i o c e r c a r i a e ( o f t h e " o r n a t a g r o u p " ) o f H. b u t t e n s i s a p p r o x i m a t e l y 2 8 d a y s a f t e r i n g e s t i o n o f t h e e g g s . I n s e c t n y m p h s w e r e c o l l e c t e d f r o m a s m a l l p o n d i n H a n e y , B.C. T h i s p o n d d o e s n o t c o n t a i n a n y f r o g s ; n a t u r a l l y - o c c u r r i n g i n f e c t i o n s o f H a e m a t o l o e c h u s s p . a r e t h e r e f o r e a b s e n t . T e n c e r c a r i a e r e c o v e r e d f r o m e x p e r i m e n t a l l y i n f e c t e d s n a i l s w e r e p l a c e d i n e a c h o f s i x t y 60-mm p l a s t i c p e t r i d i s h e s . T w e n t y d i s h e s c o n t a i n e d o n e n a i a d e a c h o f t h e d a m s e l f l y , I s c h n u r a o e r p _ a r v a , t w e n t y c o n t a i n e d I . c e r y u l a a n d t w e n t y c o n t a i n e d o n e l a r v a e a c h o f t h e d r a g o n f l y A e s c h n a £ a l m a t a . A l l l a r v a e w e r e a p p r o x i m a t e l y 16 mm l o n g . C e r c a r i a e a n d i n s e c t s w e r e l e f t t o g e t h e r f o r f i v e h o u r s , t h e n n a i a d s w e r e r e m o v e d a n d w a s h e d , a n d a l l n a i a d s o f a s p e c i e s w e r e p l a c e d i n a 100-mm p l a s t i c p e t r i d i s h . F i v e d a y s l a t e r , m e t a c e r c a r i a e f r o m t h e t w o s p e c i e s o f d a m s e l f l i e s a n d t h e d r a g o n f l y w e r e j u d g e d t o b e i n f e c t i v e t o f r o g s . G r o u p s o f f i f t e e n R. E . r e t i o s a , 5 0 - 5 5 mm 73 l o n g , were i n f e c t e d with metacercariae from one of the i n s e c t hosts* Frogs were maintained at 20°C. Samples of three f r o g s were examined from each group 5, 14, 21, 28 and 60 days p o s t - i n f e c t i o n . R e s u l t s Flukes recovered from f r o g s f e d i n f e c t e d d a m s e l f l i e s d i f f e r e d from those from f r o g s f e d i n f e c t e d d r a g o n f l i e s i n having a s m a l l e r body s i z e (length and width), s m a l l e r gonad s i z e , and s h o r t e r extent o f the e x t r a c a e c a l u t e r i n e loops (Table 7). The mean and range f o r body le n g t h and width, and gonad l e n g t h s are shown i n Fi g u r e 39. The average l e n g t h of worms with I. c e r v u l a or I. perparva i n t e r m e d i a t e hosts i s 6.6 3 and 6.39, r e s p e c t i v e l y . Worms averaged 7.33 long when Aegchna palmata was the i n s e c t host. Body width measured 1.52, 1.46 and 1.69, r e s p e c t i v e l y . L a r g e r average gonad s i z e was a t t a i n e d i n worms that had ft. palmata as t h e i r I n s e c t host and i n these worms both e x t r a c a e c a l loops extended the f u l l l e n g t h o f the p o s t e r i o r t e s t i s . The l e f t e x t r a c a e c a l loop extended 1/4 to 1/3 the l e n g t h of , the p o s t e r i o r t e s t i s and the r i g h t loop extended 1/2 to 3/4 the l e n g t h o f the p o s t e r i o r t e s t i s when the i n s e c t host i s e i t h e r I. c e r v u l a or I . perparva* The r a t i o o f the o r a l sucker diameter t o acetabulum diameter was the same (2.4 t o 2.7) r e g a r d l e s s o f i n s e c t host. Table 7. Morphological variation in 60-day old H. buttensis reared in Ischnufa cervula. Ischnura perparva. or Aeschna palmata as the Second intermediate host. Character I. cervula Intermediate host , I. perparva A. palmata Body length 6.63(6. 27-7. 24) 6. 39(5. 88-6.93) 7. 33(7.08-8. 35) Body width 1. 52( 1. 33-1. 60) 1. 46( 1. 37- 1. 68) 1.69(1.61-2.01) O/A ratio 2. 5 (2.4 -2. 6 ) 2. 5 (2.4 -2. 7 ) 2. 6 (2.4 - 2.7 ) Ovary length 0.79(0.74-0. 82) 0. 73(0. 69-0.79) 0. 86(0. 81-0.94) Ovary width 0.41(0.37-0.45) 0. 38(0.34-0.42) 0.45(0.42-0. 53) Ant. testis length i.2:-{ 1.18- 1. ?2) 1.16(1.14-1.30) 1.36(1.28- 1.47) Ar>t. testis width 0. 52(0.49-0. 61 ) 0. 46(0.40-0. 57) 0. 54(0.47-0. 69) Post, testis length 1. 33( 1. 29- 1. 42) 1. 24( 1. 19- 1. 36) 1.45( 1. 33- 1. 56) Post, testis width 0. 57(0. 50-0. 65) 0. 52(0.49-0. 67) 0.61(0. 53-0. 76) Ext r a c a e c a l loops *L. 1/4 to 1/3 P T . 1/4 to 1/3 P. T. ful l length E x t r a c a e c a l loops R. 1/2 to 3/4 P.T. 1/2 to 3/4 P.T. fu l l length Extent of extracaecal uterine loop along the posterior testis. V a r i a t i o n s i n some body measurements of H. b u t t e n s i s r e a r e d i n d i f f e r e n t i n s e c t i n t e r m e d i a t e hosts, , 9.0 8.0-in O 5.0-1 E 4.0-3.0-2.0-t i "T"° f—r* a b c Body Length i — ! — T a b c Body Width 1.0 —5 0.5-a m A., palmata b > I. cervula e = I. perparva 4 a b c Post. Testis Length Character Pleasured r b A n t . Test is Length s f s s a f m b c Ovary Length a > 76 9. .MORPHOLOGICAL VARIATION IN H.-„BUTTSHSIS BHEN CEBCABIAE ABE DEVELOPED IB VARIOUS SNAIL HOSTS. I n t r o d u c t i o n Underwood and Dronen (1977) noted t h a t measurements of c e r c a r i a e shed from a h c y l i d s n a i l s { F e r r i s s i a sp.) e x p e r i m e n t a l l y i n f e c t e d with H. b r e v i p l e x u s . d i f f e r e d from those of t h e same lung f l u k e given by S c h e l l {1965) / who used Gvraulus sp. as molluscan host, underwood and Dronen d i d not compare the a d u l t stages. However, they noted that the d i f f e r e n c e s may i n d i c a t e t h a t these f l u k e s , i d e n t i f i e d as H,. b r e v i p l e x u s i n Idaho and Texas, are not the same s p e c i e s . They s t a t e d t h a t c r o s s — i n f e c t i o n s t u d i e s , u t i l i z i n g hosts and p a r a s i t e s from Idaho and Texas, are necessary t o determine t h a t the d i f f e r e n c e s are not a r e s u l t of host—induced v a r i a t i o n . M a t e r i a l s and Methods To determine the p o s s i b l e e f f e c t s o f v a r i o u s f i r s t i n t e r m e d i a t e hosts on the morphology of a d u l t H. b u t t e n s i s . experimental i n f e c t i o n s o f Physa n u t t a l l i . S t a g n i c o l a e l o des and H e 11 s p ma t r i v o l v i s were attempted. Eggs o f p. b u t t e n s i s used f o r experimental feedings were o r i g i n a l l y o b tained from worms i n n a t u r a l l y i n f e c t e d B. p r e t i o s a c o l l e c t e d i n Manning Park. Flukes removed from the lungs were pla c e d i n d e c h l o r i n a t e d tapwater. A f t e r one hour, mature eggs r e l e a s e d by the worms were f e d t o 30 each of the t h r e e s p e c i e s of s n a i l s . C e r c a r i a e which developed i n and were shed from s n a i l s were 77 placed i n 100-mm p l a s t i c p e t r i d i s h e s . Each d i s h contained ten c e r c a r i a e and one I . perparva. Twenty naiads i n a l l were i n f e c t e d i n t h i s way. F i v e days l a t e r the metacercariae were f e d t o R. pr.et.iosa 5 0 - 5 5 nm i n snout-vent l e n g t h . F i f t e e n f r o g s were exposed and maintained at 20<>C. Samples of th r e e f r o g s were examined at 5 , 1 4 , 2 1 , 2 8 and 6 0 days. . R e s u l t s Only Ph£sa n u t t a l l i became i n f e c t e d . I n f e c t e d s n a i l s shed c e r c a r i a e approximately 2 8 days a f t e r i n g e s t i o n o f the eggs. Flukes recovered from R. p r e t i o s a which were p r e v i o u s l y developed i n I. perparva and P. n u t t a l l i showed no morphological v a r i a t i o n s d i f f e r e n t from those i n experiments on "Development i n Rana p r e t i o s a " (Appendix 2 1 ) . 78 10. DISCUSSION AND CONCLUSIONS FBOH EXPEBIMENTAL RESULTS Body S i z e : The l e n g t h and width of the body of H. b u t t e n s i s i n c r e a s e d by as much as 78f and 140% r e s p e c t i v e l y when weight was added to the c o v e r s l i p d u r i n g the d r y i n g process of s l i d e p r e p a r a t i o n . In t h i s study, reduced growth r a t e i n H. buttepis-is o c c u r r e d i n f l u k e s maintained a t 12°c. Worms maintained i n h o s t s a t higher temperatures (20°C and 27<>C) were l a r g e r than f l u k e s of a comparable age maintained i n hosts kept a t lower temperatures. S t u d i e s on the e f f e c t s o f temperature on trematode development are l i m i t e d . W i l l e y (194 1) observed t h a t the trematode z y g o c o t y l e l u n a t a ( D i e s i n g . 1836} matured more r a p i d l y i n ducks than i n r a t s . He a t t r i b u t e d t h i s t o the higher body temperature of b i r d s . Wohlgemuth (1920) and l a t e r Izyumova (1956) r e p o r t e d t h a t v a r i a t i o n s In water temperature a f f e c t development of the monogenetic trematode, Dactyloqyrus v a s t a t o r N y b e l i n , 1924, a p a r a s i t e on the g i l l s of f i s h . Watertor (1965) found t h a t t h e growth and development o f a d u l t T e l o r c h i s bonnerensis Waitz„ 1960 were g r e a t l y a l t e r e d when e x p e r i m e n t a l l y i n f e c t e d d e f i n i t i v e hosts ( a d u l t Ambystoma t i g r i n n m Green. 1825) were maintained a t v a r i o u s co n s t a n t temperatures of 10°, 22°, 30° and 34°C. S t u d i e s on other p a r a s i t i c organisms have demonstrated s i m i l a r responses to temperature. Smaller H. but tens i s were recovered from B. p r e t i o s a e x p e r i m e n t a l l y i n f e c t e d with l a r g e r numbers o f metacercariae.,. 79 The body s i z e of f l u k e s decreased as ouch as t w o - t h i r d s i n h e a v i l y i n f e c t e d f r o g s (Table 4). The number of worms present i n a h o s t has been shown to be an important s i z e — c o n t r o l l i n g f a c t o r i n trematodes ( L y u b i n s k i and Kulakovskaya, 1940; W i l l e y , 1941; Dawes* 1962; Watertor, 1965, and o t h e r s ) . T h i s has a l s o been demonstrated f o r nematodes (Chitwood, 1957; Haley and Parker, 1961) and tapeworms ( P a v l o v s k i and G n e z d i l o v , 1949, 1953; Brooks and Hayes, 1976; and o t h e r s ) . Bankin (1937) observed t h a t specimens were un u s u a l l y s m a l l when l a r g e numbers o f worms were present i n n a t u r a l i n f e c t i o n s of the salamander trematodes, Brachycoelium. Plagituray.and Megalod i s c u s § p. Body l e n g t h s were on an average 18^-30% g r e a t e r i n 60-day-o l d H. b u t t e n s i s r e a r e d i n R. p r e t i o s a than i n those r e a r e d i n amphibian hosts which are seldom n a t u r a l l y i n f e c t e d . S i m i l a r l y , 60-day-old f l u k e s were as much as 15% longer when the d r a g o n f l y naiad was used as second i n t e r m e d i a t e host than when a dams e l f l y naiad was used. Experimental s t u d i e s by Beaver (1937) on Echinostomum reVolutum F r o e l i c h , 1802 developing i n a v i a n and mammalian h o s t s , the work o f Bankin (1937) on Brachycoelium c o l l e c t e d from v a r i o u s s p e c i e s of n a t u r a l l y i n f e c t e d salamanders, and s t u d i e s by Wharton (1940) and watertor (1967, 1968) on s e v e r a l s p e c i e s o f Telorchisjp. f ro a c h e l o n i a n and amphibian hosts, present f u r t h e r evidence t h a t m o r p h o l o g i c a l changes may occur when a d u l t trematodes mature i n d i f f e r e n t s p e c i e s of d e f i n i t i v e h o s t S i Boddeke (1960b) s t u d i e d experimental i n f e c t i o n s of P r os t ho go n j mu s cvatus and observed great morphological v a r i a t i o n s among these f l u k e s from d i f f e r e n t 80 a v i a n hosts. Blankespoor's 1974 s t u d i e s on P l a q i o r c h i s n o b l e i i n a v i a n hosts demonstrate t h f l u k e s recovered from p a s s e r i n e b i r d s were u s u a l l y l a r g e r ( i n l e n g t h and width) than those o b t a i n e d from g a l l i n a c e o u s b i r d s . Development of • j3. btsltensis i n four s i z e c l a s s e s of !• p r e t i o s a d i d not g r e a t l y a f f e c t the morphology of t h i s f l u k e . As w e l l , f l u k e morphology was not a f f e c t e d by the sex of the host. flerrick (1925) and Ackert (1935) observed marked d i f f e r e n c e s i n l e n g t h of A s c a r i d i a g a l l i • (Shrank, 1788) from ch i c k e n s of v a r y i n g ages. Worms from o l d e r b i r d s were s i g n i f i c a n t l y s m a l l e r than those from younger b i r d s . Bouchard (1951) found the p a r a s i t e s i n l a r g e r hosts (frogs of v a r i o u s s p e c i e s ) t o be l a r g e r r e g a r d l e s s of the p a r a s i t e s p e c i e s . According t o Bead and Bothman (1958), the s i z e of Hymenolgpjs diminuta (Budolphi. 1819) was a f u n c t i o n of the age of the r a t host. Bourns (1966), working with n a t u r a l i n f e c t i o n s of P l a s i o r c h i s n o b l e i i n n e s t l i n g red-winged b l a c k b i r d s , found a h i g h e r percentage of g r a v i d a d u l t s i n o l d e r h o s t s . A l o n g e r time was r e q u i r e d to become g r a v i d i n domestic c h i c k e n s . Blankespoor (1974) i n f e c t e d 1-, 7- and 20-day o l d l a b o r a t o r y - r e a r e d house sparrows and noted t h a t age of sparrows di d not g r e a t l y a f f e c t the body s i z e of a d u l t P. n o b l e i * Gonad S i z e and Shape: The l e n g t h and width of ovary i n c r e a s e d as much as 271 and 64% r e s p e c t i v e l y when weight was added t o the cover s l i p d u r i n g s l i d e p r e p a r a t i o n . S i m i l a r l y , a n t e r i o r and p o s t e r i o r t e s t e s 81 i n c r e a s e d as much as 32% and 50% i n len g t h and width, r e s p e c t i v e l y . A major problem i n i d e n t i f y i n g s p e c i e s of the genus Haematoloechus has been t h e l a c k of experimental data on c h a r a c t e r changes with i n c r e a s i n g s i z e of worms. My experimental s t u d i e s on H. b u t t e n s i s reared i n a d u l t R. p r e t i o s a provided evidence of pronounced a g e - r e l a t e d v a r i a t i o n i n gonad s i z e and shape. Both the ovary and t e s t e s continued to i n c r e a s e i n l e n g t h and width d u r i n g the study p e r i o d . O v a r i e s changed from smooth t o h i g h l y lobed, and t e s t e s e i t h e r remained smooth or became hi g h l y lobed. S e v e r a l authors have used the degree o f l o b i n g i n o v a r i e s and : i n t e s t e s as a s p e c i e s - s p e c i f i c c h a r a c t e r . I n g l e s (1936) used i t t o he l p him d i s t i n g u i s h H. b u t t e n s i s (ovary never lobed) from H. p a r v i p l e x u s (ovary deeply l o b e d ) . I n g l e s (1932) a l s o used, i t to se p a r a t e H. confusus (ovary and t e s t e s lobed) from H.: c o l o r a d e n s i s (ovary and t e s t e s unlobed). Manter (1938) i n h i s key t o North american s p e c i e s o f f r o g lung f l u k e s made e x t e n s i v e use of degree of l o b a t i o n o f t e s t e s and ovary t o separate s p e c i e s . The ovary and t e s t e s were s m a l l e r i n worms maintained i n hosts a t 27°C compared with worms developed at 20°C. T h i s response to temperature produced longer and wider worms with s m a l l e r gonads a t 27°C than a t 20°C. T h i s i s an important o b s e r v a t i o n because f r o g s are o f t e n c o l l e c t e d from d i f f e r e n t areas with v a s t l y d i f f e r e n t environmental c o n d i t i o n s The s i z e of the ovary and t e s t e s and degree of l o b a t i o n o f these organs were a l s o a f f e c t e d when worms were reared i n 8 2 d i f f e r e n t d e f i n i t i v e hosts. F l u k e s possessing u n u s u a l l y l a r g e o v a r i e s were obtained from experimental i n f e c t i o n i n R. c l a m i t a n s . I n f e c t i o n s i n R, aurora y i e l d e d a d u l t worms with s m a l l o v a r i e s . The l o n g e s t a n t e r i o r t e s t e s were found i n worms reared i n R. c l a m i t a n s . the s m a l l e s t i n worms from R. au r o r a . However, the longest p o s t e r i o r t e s t e s were found i n worms reared i n B.r boreas and the s m a l l e s t i n R. p r e t i o s a . Sucker S i z e and 0/A R a t i o : The l e n g t h and width o f the o r a l sucker and ace t a b u l u s of H*i b u t t e n s i s i n c r e a s e d when weight was added to the c o v e r s l l p s d u r i n g s l i d e p r e p a r a t i o n . Sucker s i z e a l s o v a r i e s with age o f worm and host s p e c i e s . An i n c r e a s e i n s i z e of the suckers o c c u r r e d d u r i n g the 60-day study p e r i o d . However, the 0/A r a t i o remained f a i r l y constant r e g a r d l e s s of age of worm or method of PREPARATION. Blankespoor (1970) determined the e f f e c t s of f l a t t e n i n g and f i x a t i o n on morphology o f ad u l t P l a g i o r c h i s n o b l e i Park, 1936 developed i n a f i v e - d a y o l d c h i c k . He found t h a t v a r i o u s degrees of f l a t t e n i n g had l i t t l e e f f e c t on the s i z e — r a t i o o f muscular s t r u c t u r e s such as the o r a l sucker and acetabulum. S i m i l a r r e s u l t s were found by Boddeke (1960a) i n h i s s t u d i e s with the f l u k e Prosthogonimus ovatus Rudolphi, 1803. He concluded that the .,; only taxonoraic c h a r a c t e r u s e f u l i n d i s t i n g u i s h i n g s p e c i e s w i t h i n t h a t genus i s the r a t i o between the diameter of the o r a l and v e n t r a l suckers. Blankespoor (1974) repo r t e d the u n r e l i a b i l i t y of sucker s i z e i n P l a g i o r c h i s n o b l e i r e a r e d i n a v a r i e t y of avian and 83 mammalian hosts. However, he noted t h a t the O/A r a t i o was a s t a b l e taxonomic c h a r a c t e r i n a d u l t s o f t h i s s p e c i e s . B e r r i e {1960). Boddeke {1960a) and B a t e r t o r (1967) have a l s o found s i g n i f i c a n t d i f f e r e n c e s i n sucker s i z e between a d u l t f l u k e s recovered from d i f f e r e n t host s p e c i e s . S p i n a t i o n : Hy experiments demonstrated t h a t d i s t i l l e d water caused l i v e H. b u t t e n s i s to l o s e s p i n e s ; the warmer the water the sore r a p i d t h e l o s s . T h i s f i n d i n g i s important because some workers {Cort, 1915a) assembled t h e i r specimens i n water before f i x a t i o n . The presence or absence o f sp i n e s has been used t o sep a r a t e the f o l l o w i n g s p e c i e s : H. c o l o r a d e n s i s (Cort. 1915a) (spined) from H, complexus (Seely, 1906) (no spines) fl. £onfusus I n g l e s , 1932 (spines) from H. o x y o r c h i s I n g l e s 1932 (no spines) H. tumidus I n g l e s , 1932 (spined) from H . k e r n e n s i s I n g l e s . 1932 (no spines) S p i n a t i o n was not a f f e c t e d by: f l u k e age; s p e c i e s , sex or s i z e o f h o s t ; o r temperature a t which the i n f e c t e d host was ,maintained. Egg S i z e : Egg s i z e d i d not d i f f e r i n f l u k e s o l d e r than 21 days.. Eggs averaging 0.024 by 0.016 i n s i z e were found i n the a n t e r i o r p o r t i o n of the uterus i n 21- to 60-day o l d worms. Egg s i z e was not a f f e c t e d by any o f the other v a r i a b l e s i n v e s t i g a t e d . 84 E x t r a c a e c a l L o o p s : T h e l e n g t h s o f t h e e x t r a c a e c a l l o o p s d i f f e r e d i n f l u k e s f r o m d i f f e r e n t f r o g h o s t s a s w e l l a s f r o m d i f f e r e n t i n s e c t h o s t s ( F i g s . 2 9 - 3 5 , T a b l e 7 ) . . T h e v a r i a t i o n s f o u n d e x p e r i m e n t a l l y f o r l i . E i J t i S S s i s o v e r l a p t h o s e r e p o r t e d a s b e i n g a d i s t i n g u i s h i n g f e a t u r e f o r H. f l o e d a e a n d H. p a r v i p l e x u s . M e a s u r e m e n t s f r o m t h r e e o t h e r d e s c r i b e d s p e c i e s t h a t h a v e e x t r a c a e c a l u t e r i n e l o o p s ( H. s i ID i i i p l e x u s , H. u n i _ p l _ e x u s a n d H. y a r i c p l e x u s ) a l s o f a l l w i t h i n t h e r a n g e o f t h o s e d e s c r i b e d h e r e . T h i s s u g g e s t s t h a t a s i n g l e h i g h l y v a r i a b l e s p e c i e s may b e p r e s e n t a n d n o t s i x d i s t i n c t s p e c i e s . D i s t r i b u t i o n o f t h e V i t e l l a r i a : T h i s t h e s i s h a s d e m o n s t r a t e d t h a t v i t e l l i n e d i s t r i b u t i o n was a f f e c t e d b y d e v e l o p i n g i n d i f f e r e n t d e f i n i t i v e h o s t s . V i t e l l i n e f o l l i c l e s e x t e n d e d f u r t h e r a n t e r i o r i n w o r m s r e a r e d i n R. p r e t i o s a o r B. b o r e a s t h a n i n w orms r e a r e d i n R. a u r o r a o r !• c l a m i t a n s . T h e b a n d o f v i t e l l a r i a e x t e n d i n g a c r o s s t h e worms d e v e l o p e d i n R. E i e t i o s a a n d B. b o r e a s was i n c o m p l e t e o r a b s e n t i n w o r m s d e v e l o p e d i n R. a u r o r a a n d R. c l a m i t a n s . R a n k i n ( 1 9 3 7 ) , W h a r t o n ( 1 9 4 0 ) , W a t e r t o r ( 1 9 6 7 ) a n d B l a n k e s p o o r ( 1 9 7 4 ) h a v e a l s o s h o w n t h e u n r e l i a b i l i t y o f v i t e l l i n e d i s t r i b u t i o n a s a t a x o n c m i c c h a r a c t e r . S u m m a r y : S e v e r a l t r e m a t o d e s p e c i e s h a v e b e e n s h o w n t c u n d e r g o m o r p h o l o g i c a l c h a n g e s when s u b j e c t e d t o v a r i o u s m e t h o d s o f f i x a t i o n o r f l a t t e n i n g . T h e s e c h a n g e s make i t v e r y d i f f i c u l t t o 85 compare the a b s o l u t e s i z e and shape of va r i o u s f l u k e s when d i f f e r e n t h a n d l i n g procedures are used. Thus, statements such as " I t r H. v a r i o p l e x u s S t a f f o r d , 1902] r i v a l s No.2 I H. b r e y i p l e x u s ] i n s i z e " ( S t a f f o r d , 1902, p.906); "the pharynx i was ,somewhat s m a l l e r than i s u s u a l " f H. f l o e d a e Harwood. 19321 i (Banter, 1938, p. 31) and "The specimens of H. l o n g i p l e x u s from R. p i p i e n s are on the average s m a l l e r than those d e s c r i b e d by S t a f f o r d from the b u l l f r o g " (Cort, 1915a, p. 213), are d i f f i c u l t to i n t e r p r e t . Changes i n f l u k e morphology r e s u l t i n g from d i f f e r e n t methods of f i x a t i o n and f l a t t e n i n g have a l s o been demonstrated by fJlmer (1950), working w i t h the trematode Postharmostomum h e l i c i s (Leidy,1847) ; G i l f o r d (1955), working with Allassoqonoporus v e s p e r t i l i o n i s Hacy, 1940 from b a t s ; and by Angel (1959)working with P l a g i p r c h i s maculosus (Rudolphi. 1802). Kavelaars and Bourns (1968) i n d i c a t e d t h a t many of the d i f f e r e n c e s seen i n a d u l t P l a g i g r c h i s p e t e r b o r e n s i s Kavelaars and Bourns, 1968 reared i n a l a b o r a t o r y mouse ( Mus musculus Linnaeus, 1758), r e s u l t e d from v a r i o u s k i l l i n g methods and degrees of f l a t t e n i n g . The v a l i d i t y of a s p e c i e s , i n a group, should be c a r e f u l l y c o n s i d e r e d when c h a r a c t e r s used f o r s p e c i e s determination are a l t e r e d by changing h a n d l i n g t e c h n i g u e s . The degree of v a r i a t i o n caused by v a r i o u s procedures should be s t u d i e d f o r each group, and an e f f o r t be made to s t a n d a r d i z e these procedures. A major problem i n i d e n t i f y i n g s p e c i e s of the genus flaglg£olpech us has been t h e l a c k of experimental data on 86 host-Induced m o r p h o l o g i c a l m o d i f i c a t i o n s . , In the present study I have demonstrated t h a t a d u l t H.- b u t t e n s i s can be e x p e r i m e n t a l l y e s t a b l i s h e d i n f o u r s p e c i e s of d e f i n i t i v e hosts and t h r e e s p e c i e s of i n s e c t second i n t e r m e d i a t e hosts. J 3 . b u t t e n s i s has p r e v i o u s l y been d e s c r i b e d from two other f r o g hosts f B. b o y l e l . and B. catesbeIan a Sha w, 1802) (appendix 23). The apparent l a c k of host s p e c i f i c i t y i n l a b o r a t o r y i n f e c t i o n s , as w e l l as t h e morphological v a r i a t i o n s which occurred i n f l u k e s developed i n d i f f e r e n t f r o g and i n s e c t h o s t s , c a s t s doubt on the use of host s p e c i f i c i t y alone f o r d e l i n e a t i n g s p e c i e s w i t h i n the genus Haematol^chus, Host s p e c i f i c i t y of a d u l t trematodes has been used i n s e p a r a t i n g one s p e c i e s from another; y e t morphological d i f f e r e n c e s t h a t c o n s t i t u t e the c r i t e r i a f o r the d e f i n i t i o n o f new s p e c i e s may be the r e s u l t o f the i n f l u e n c e of the p a r t i c u l a r host s p e c i e s (Stunkard, 1957). Rankin (1938) noted c o n s i d e r a b l e v a r i a t i o n i n c h a r a c t e r s i n s p e c i e s of Brachvcoelium c o l l e c t e d from twenty d i f f e r e n t s p e c i e s of salamanders. T h i s v a r i a t i o n rendered i d e n t i f i c a t i o n of the f l u k e s with d e s c r i b e d s p e c i e s impossible, as a r e s u l t of h i s study, Bankin (1938) reduced twelve s p e c i e s t o synonymy, s i a i l a r c o n c l u s i o n s from s t u d i e s on other f l u k e s have been a r r i v e d at by Beaver (1937) , Boddeke (1960a), K i n s e l l a (1971) and o t h e r s . The c h a r a c t e r s which appear t o be the most s t a b l e i n H. b u t t e n s i s and consequently may have the g r e a t e s t value f o r s e p a r a t i n g s p e c i e s of Haematoloechus a r e : the 0 / A r a t i o , egg s i z e of 21-day o l d and o l d e r worms, p o s i t i o n of the e x t r a c a e c a l loops r e l a t i v e the the t e s t e s and ovary, s p i n a t i o n o f the tegument and p o s i t i o n of the t e s t e s . 8 8 PAST I I . TAXONOMY AND MORPHOLOGICAL VARIATION I n t r o d u c t i o n Our present knowledge of s p e c i e s of Heamatoloechus from Canada and the United S t a t e s i s s p o t t y . Most published r e p o r t s of new s p e c i e s d e a l o n l y with one or two l o c a l i t i e s which are u s u a l l y c l o s e together ( S t a f f o r d , 1902; C o r t , 1915a; I n g l e s , 1932,1936; and o t h e r s ) . Brooks (1976) has noted t h a t a l a r g e number o f amphibian p a r a s i t e surveys have been p u b l i s h e d i n North America, but i n most c a s e s , u n l e s s new ta x a were d e s c r i b e d , no morphological data have been presented or specimens made a v a i l a b l e by d e p o s i t i o n i n a museum c o l l e c t i o n . To date no attempt has been made to c o l l e c t over a broad g e o g r a p h i c a l area and to make i n t r a s p e c i f i c and i n t e r s p e c i f i c comparisons o f c h a r a c t e r s regarded as important i n s e p a r a t i n g s p e c i e s of t h i s group. A study was undertaken t o assess the v a r i a t i o n i n c e r t a i n c h a r a c t e r s from f l u k e s c o l l e c t e d from s e v e r a l l o c a l i t i e s i n Canada and the United S t a t e s . , Emphasis i s placed on those c h a r a c t e r s p r e v i o u s l y used t o d e l i n e a t e s p e c i e s i n t h i s group. M a t e r i a l s and Methods C o l l e c t i o n s of trematodes made from B r i t i s h Columbia, A l b e r t a , Saskatchewan, Washington and Oregon by the author, were examined f o r v a r i a t i o n . Shipments of l i v e f r o g s were sent by a i r by s e v e r a l Canadian and American c o l l e c t o r s t o the U n i v e r s i t y o f B r i t i s h Columbia. F r e s h m a t e r i a l was d i s s e c t e d on a r r i v a l and f l u k e s 89 r e c o v e r e d f r o m t h e l u n g s w e r e f i x e d i n h o t 7 0 % a l c o h o l a n d s t a i n e d w i t h H a r r i s ' s H a e r a a t o x y l i n ( s e e A p p e n d i x 3 f o r p r o c e d u r e ) . H o s t s p e c i e s , s e x , s i z e a n d p r e s e n c e o f o t h e r worm p a r a s i t e s w e r e a l s o r e c o r d e d . Two t h o u s a n d c n e h u n d r e d a n d s i x t y - t w o l u n g f l u k e s c o l l e c t e d i n t h i s m a n n e r w e r e e x a m i n e d . S l i d e s o f s p e c i m e n s b o r r o w e d f r o m p r i v a t e a n d i n s t i t u t i o n a l c o l l e c t i o n s w e r e a l s o e x a m i n e d f o r v a r i a t i o n . T h e t o t a l n u m b e r o f s p e c i m e n s e x a m i n e d f r o m t h e s e s o u r c e s w a s 4 2 2 . R e s u l t s I n t h e p r e s e n t s t u d y , 2 8 5 1 a m p h i b i a n s , r e p r e s e n t i n g 14 s p e c i e s a n d f i v e f a m i l i e s , w e r e c o l l e c t e d a n d e x a m i n e d f o r n a t u r a l i n f e c t i o n s o f H a g m a t o l o e c h u s b y t h e a u t h o r ( T a b l e 8 ) . N a t u r a l i n f e c t i o n s w e r e f o u n d i n s e v e n s p e c i e s r e p r e s e n t i n g t w o f a m i l i e s . W i t h i n t h e f a m i l y R a n i d a e , f r o g s p e c i e s 2« c a t e s b e i a n a , R. c l a m i t a n s , R, p i p i e j g s , R. p r e t i o s a a n d !• s y l v a t i c a L e C o n t e , 1 8 2 5 c o n t a i n e d n a t u r a l i n f e c t i o n s o f H a e m a t o l o e c h u s , a s d i d t o a d s B u f g a m e r i c a n u s H c l b r o c k , 1 8 3 6 a n d B. b o r e a s o f t h e f a m i l y B u f o n i d a e . T h e h i g h e s t p e r c e n t a g e o f n a t u r a l i n f e c t i o n s w a s f o u n d i n m e m b e r s o f t h e R a n i d a e . O t h e r k n o w n d e f i n i t i v e h o s t s o f H a e m a t o l o e c h u s i n C a n a d a a n d t h e U n i t e d S t a t e s a r e g i v e n i n A p p e n d i x 2 3 . Of t h e 2 8 5 1 f r o g s e x a m i n e d i n t h i s i n v e s t i g a t i o n , 1 0 9 0 ( 3 8 . 2 % ) h a r b o r e d i n f e c t i o n s o f H a e m a t o l o e c h u s . P r e v a l e n c e 2 o f n a t u r a l i n f e c t i o n s a m ong t h e s e h o s t s r a n g e d f r o m 2.9 t o 6 4 . 0 p e r c e n t . T h e h i g h e s t p r e v a l e n c e o f i n f e c t i o n w a s i n R a n a p i p i e n s P r e v a l e n c e i s d e f i n e d h e r e a s t h e p e r c e n t o f a n i m a l s i n a p o p u l a t i o n w h i c h a r e i n f e c t e d . 9 0 ( 6 4 . 0?>) a n d i n B. g r e t i o g a (46.5%) . 91 Table 8. Summary of amphibian hosts examined for natural infections of H a e m a t o l o e c h u s spp. Host # adults examined Ranidae Rana aurora R. catesbeiana  R. clamitans _R. pipiens R. pretiosa R. sylvatica R. grylio S t e j n e g e r , 1901 Hylidae Hyla rc gill a Pseudacris triseriata Bufonidae Bufo americanus B. boreas B. marinus Scaphiopodidae Scaphiopus hammondi Ascaphidae Ascaphus truei _ 65 81 112 602 1405 77 32 196 12 8 243 3 10 # infected 0 14 11 385 653 18 0 0 "0 2 7 0 Total 2851 0 1090 Lung fluke present H. longiplexus H . long'tplexus H . breviplexus H. medioplexas H. medioplexus H. buttensis H . parviplexus H. medioplexus H. buttensis Identifications are based on type descriptions and type specimens when available. ' 92 The maximum number of f l u k e s recovered from a s i n g l e f r o g (R. clamitans) was 358. The mean3 number o f f l u k e s per i n f e c t e d •B.,vpr.e^Aosa- was 3.8 and f o r R. c l a m i t a n s i t was 64. The high mean found i n 8 . c l a m i t a n s was a r e s u l t of l a r g e numbers of f l u k e s i n two of the i n f e c t e d f r o g s . The specimens of • R^iiyClagl^ans • c o l l e c t e d from Maple Ridge, BsG. , c o n t a i n e d both -H origj plexu s a n d H. b r e v j p l e x u s . T h e remaining n i n e i n f e c t e d •rR.%cl;fmitan-s c o n t a i n e d o n l y • H.•• j b r e v l p l e y u s J - A l l . other f r o g s c o l l e c t e d from any one l o c a l i t y contained o n l y a s i n g l e s p e c i e s of vHaematolqechus.-. •., Examination of prepared s l i d e s o f Haematoloechus a nd worm specimens f i x e d and sent i n v i a l s s u p p l i e d an a d d i t i o n a l source of f l u k e s f o r examination. T h i s m a t e r i a l r e p r e s e n t s c o l l e c t i o n s by many people from numerous l o c a t i o n s throughout Canada and the United S t a t e s . A t o t a l o f 2584 specimens was examined. V a r i a t i o n s i n c h a r a c t e r s p r e v i o u s l y c o n s i d e r e d as important f o r s e p a r a t i n g s p e c i e s i n t h i s genus w i l l be d i s c u s s e d f o r the above m a t e r i a l under each s p e c i e s heading. 'The mean number of p a r a s i t e s per i n f e c t e d host i s c a l l e d the i n t e n s i t y of i n f e c t i o n by some authors. 93 Generic Diagnosis Haematoloechus Looss. 1899 syn. Pneumgnoeces Looss, 1902 Ostipium P r a t t , 1903 Pneupobites War d, 1917 The genus Haematoloechns Looss, 1899 i s here d e f i n e d as f o l l o w s : ( m o d i f i e d a f t e r Yamaguti, 1 9 7 1 ) Haematoloechidae. Body e l o n g a t e , spinose or not. acetabulum i n a n t e r i o r or middle t h i r d o f body, and u s u a l l y s m a l l e r than the o r a l sucker. T e s t e s o b l i g u e or symmetrical, i n p o s t e r i o r h a l f o f body. C i r r u s and c i r r u s pouch present, c o n t a i n i n g s e a i n a l v e s i c l e . G e n i t a l pore v e n t r a l t o pharynx. Ovary lobed or not, c l o s e to acetabulum. Seminal r e c e p t a c l e l a r g e , o f t e n l a r g e r than ovary* Laurer's canal absent. V i t e l l a r i a f o l l i c u l a r , d o r s a l and l a t e r a l , e xtending f o r v a r i a b l e l e n g t h . Uterus c o i l e d , occupying roost of hindbody, with or without e x t r a c a e c a l u t e r i n e l o o p s . The ascending limb passes i n t e r c a e c a l l y through the forebody. Eggs, numerous, embryonated, do not hatch u n t i l i n g e s t e d by a p p r o p r i a t e s n a i l . E x c r e t o r y v e s i c l e Y-shaped. P a r a s i t i c i n the lungs of Anura. Type s p e c i e s Haematoloechus v a r i e g a t u s (Pudolphi, 1819) Looss, 1899. Species of t h i s genus co n s i d e r e d v a l i d from North America may be d i v i d e d i n t o two groups: Group I : E x t r a c a e c a l u t e r i n e loops are present and extend a n t e r i a d f o r v a r y i n g l e n g t h s . T h i s group i n c l u d e s H. l o n g i p l e x u s , H. b r e v i p l e x u s . £. v a r i c p l e x u s . and fl« K§rngnsis. Group I I : e x t r a c a e c a l u t e r i n e loops are absent. T h i s group 94 i n c l u d e s H. m e d i o p l e x u s a n d H. c o m p l e x u s . 95 V a l i d Species Group I Haematoloechus l o n q i p l e x u s S t a f f o r d Haematoloechus l o n g i p l e x u s S t a f f o r d , 1902: 90-1. ( P i g . 4Z) Pneumonoeces lon g ! p l e x u s S t a f f o r d , 1905: 687. Pneunobiteg l o n q i p l e x u s Hard, 1917: 5. H e - d e s c r i p t i o n (measurements based on 20 specimens): Body el o n g a t e , 4 .90-8.64 (5.86) long by 1.50-2.27 (1.77) wide. Tegument spined or not. O r a l sucker t e r m i n a l , 0.286—0.469 (0.380) l o n g by 0.330-0.512 (0.149) wide. Acetabulum medial, s l i g h t l y p o s t e r i o r o f ovary, 0 .18-0.260 <0.197) wide.,0/A r a t i o 1.6:1.0 to 2 .6 :1 .0 ( 2 .2 :1 .0 ) . Pharynx muscular, 0 .165-0.336 (0.243) long by 0 .154-0.276 (0.26) wide. O/P r a t i o 1.9:1.0 t o 2.5:1 .0 ( 2 . 2 : 1 . 0 ) . I n t e s t i n a l caeca : narrow tubes, extending t o near p o s t e r i o r extremity, T e s t e s p a r a l l e l , e l o n g a t e , l o b e d or not. Ovary lobed or not, a n t e r i o r t o acetabulum, p r e t e s t i c u l a r . E x t r a c a e c a l u t e r i n e l o o p s present, e x t e n d i n g a n t e r i o r beyond the ovary, o f t e n extending t o the l e v e l of the pharynx. G e n i t a l pore v e n t r a l to pharynx. V i t e l l a r i a f o l l i c u l a r , s y m m e t r i c a l l y p l a c e d on each s i d e of the body. Extent ranging from about l e v e l of i n t e s t i n a l b i f u r c a t i o n , and t e r m i n a t i n g p p s t e r i o r t o the t e s t e s . Eggs o p e r c u l a t e , 0 .022-0.025 (0.023) long by 0;015-0.018 (0.016) wide. Host: Sana catesbeiana*„ S i t e o f i n f e c t i o n : l u n g s . 96 Specimens examined; T h i r t y - o n e specimens. Nova S c o t i a * H a l i f a x ex c a t e s b e i a n a , (Mr. and Mrs. Eonnyraan), author's c o l l . ; 2 specimens, Iowa, ex R. c a t e s b e i a n a . Dr. R, Campbell c o l l , ; 110 specimens, Nebraska, ex R. c a t e s b e i a n a * H.8. Manter L a b o r a t o r i e s c o l l . ; 23 specimens. Hew York, ex R. c a t e s b e i a n a . author's c o l l . ; 17 specimens, O n t a r i o * B e l l e v i l l e , ex R. c a t e s b e i a n a , author's c o l l . ; 20 specimens, B r i t i s h Columbia, Maple Bidge* ex R. c a t e s b e i a n a , author's c o l l . Type specimens were not a v a i l a b l e to the author. 97 Fig.40 Haefflatbloechus p a r y i p l e x u s ,• r,.r;, fgneumoBoeces p a r v i p l e x u s o f Irw i n * 1929} : . r e d r a w n f r o a the type specimen. Fig.41 H. -previplexus redrawn from S t a f f o r d (1902). Fig.,42 H. l i l o n g i p l e x u s redrawn from S t a f f o r d (1902J;. ,-• S e v e r a l s t r u c t u r e s have been o m i t t e d f o r c l a r i t y . A l l specimens are d e p i c t e d i n v e n t r a l view. S t a f f o r d (1902) d i d not give a s c a l e t o h i s drawings and so ab s o l u t e s i z e i s unknown f o r h i s type drawings. 41 98 D i s c u s s i o n : S t a f f o r d (1902) d e s c r i b e d l i . l o n g i plexus as being s p i n e l e s s . H is type m a t e r i a l f o r t h i s s p e c i e s was not a v a i l a b l e f o r examination. However, o f the 203 specimens examined by me, from s i x l o c a l i t i e s (Map 1), 170 had s p i n e s . A l l f l u k e s from Iowa; B e l l e v i l l e , O n t a r i o ; Haple Ridge, B.C.; and New York had s p i n e s ; 12.9% (4/31) o f t h e worms from H a l i f a x and 26.4% (29/110) from Nebraska had none. The e x t r a c a e c a l loops i n H. l o n q i p l e x u s commonly l i e between the p o s t e r i o r border o f t h e pharynx at t h e i r g r e a t e s t extent and 1/2 the d i s t a n c e between t h e ovary and pharynx a t t h e i r l e a s t e x t e n t ( F i g s . 43 and 44) . The p o s i t i o n of these l o o p s , d i d not vary i n worms from d i f f e r e n t c o l l e c t i o n s i t e s . The f o l l o w i n g r e s u l t s are based on 203 worms examined: 69% o f the worms had u t e r i n e l o o p s o f egual o r near eg u a l l e n g t h . Of these, 55% had both loops r e a c h i n g the l e v e l o f the pharynx or 90% of the d i s t a n c e from the ovary t o the pharynx. Twenty-eight percent had the l o o p s extending 75-85% o f t h i s d i s t a n c e and s i x t e e n percent had loops extending about one-half the d i s t a n c e . T h i r t y — o n e percent o f the specimens had u t e r i n e loops of unegual l e n g t h . Of these, 72.7% had s h o r t e r l e f t u t e r i n e l o o p s . Extreme v a r i a t i o n i n e x t r a c a e c a l loop extent occurred i n 2% of the specimens examined from Nebraska ( F i g . 45 and 47). Elongate, p a r a l l e l or near p a r a l l e l t e s t e s ( F i g . 44) occurred i n 98% of the specimens^ Abnormal t e s t e s p o s i t i o n i n v o l v e d e i t h e r the l e f t or r i g h t t e s t i s ( F i g , 45 and 47). These specimens were a l s o from Nebraska; Ei g h t y percent o f the a n t e r i o r and p o s t e r i o r t e s t e s were 99 unlobed. Twenty percent of the a n t e r i o r and p o s t e r i o r t e s t e s were lobed and 51.5% of the o v a r i e s were l o b e d / 48.5% unlobed. The 0/A r a t i o ranged from 1i 5:1* 0 to 21.8:1,0 with a mean o f 2.1;1.0. V a r i a t i o n s i n the 0/A r a t i o s between p o p u l a t i o n s o f H. l c n g i p l e x u s are given i n Map 1, There was no s i g n i f i c a n t d i f f e r e n c e i n the 0/A r a t i o when t h e means o f f i v e l o c a l i t i e s were compared u s i n g a one-way a n a l y s i s of va r i a n c e , , The Iowa sample was too s m a l l t o be compared with those from e t h e r l o c a l i t i e s . / The r a t i o between t h e t r a n s v e r s e diameters of the o r a l sucker and pharynx ;{0/P r a t i o ) d i d mrt d i f f e r i n :flukes from Jthe "TrXve d i f f e r e n t l o c a l i t i e s . The o/P r a t i o had a range of 1.9:1.0 • t o ^ i i ^ : ^ ^ 2. 2:1. 0. Egg l e n g t h s and widths d i d not d i f f e r i n f l u k e s from d i f f e r e n t l o c a l i t i e s ( F i g . 48). Variation in 0/A r a t i o between populations of H. lonqiplexus. Each l o c a l i t y shows the mean (horizontal l i n e ) , standard deviation (open rectangle), and range ( v e r t i c a l l i n e ) . The samples s i z e i s given below each range. 101 Fig.43 H. l o n q i p l e x u s showing v a r i a t i o n i n e x t r a c a e c a l u t e r i n e l o o p s . Fig.44 B. I o n g i p l e x u s showing v a r i a t i o n i n e x t r a c a e c a l u t e r i n e l o o p s . Fig.45 abnormal extent of e x t r a c a e c a l u t e r i n e loops i n H. lonqiplexus.. : Fig.,46 abnormal extent of e x t r a c a e c a l u t e r i n e loops i n H. l o n q i p l e x u s . Fig.47 Abnormal t e s t e s p o s i t i o n i n H. l o n q i p l e x u s . , 101A V a r i a t i o n i n egg measurements of a . I o n g i p l e x u s from d i f f e r e n t l o c a l i t i e s , i n d i c a t i n g the mean, range, and standard d e v i a t i o n (open r e c t a n g l e ) , with the sample s i z e i n d i c a t e d f o r each l o c a l i t y . Ma pi* Ridge B.C. New York Belleville Ont. Halifax MS. • AtHrtson Neb. ' Sprlngvlew Neb. Humboldt Neb. Bouchard Neb, EGG LENGTH MM. t CO _1_ g g o o o ' CJ Ui CO o 10 o o o o 3 = 6 VI Maple Ridge B.C. Hew York Belleville Ont. Halifax Atkinson Sprlngvlew Humboldt Bouchard EGG WIDTH MM. 103 Haematoloechus br e v i p l e x us St a f f o r d Haematplpec feu's- b r e y i p l e x u s S t a f f o r d , 1902: 904. ( F i g . 41) Pneu aonoecus b r e v i p l e x u s S t a f f o r d . 1905: 687. Pneumobites b r e v i p l e x u s Hard. 1917: 5. R e - d e s c r i p t i o n (based on 20 specimens): Body el o n g a t e 7. 13-10.99 (8.86) long by 1.80-2.27 (2.06) wide. Tegument spined or not. O r a l sucker terminal,0.46 5-0.573 (0.526) 40 l o n g by 0;462-0.558 (0.489) wide. Acetabulum medial, may o v e r l i e the ovary or be a n t e r i o r t o i t , 0.165-0.198 (0.176) wide. O/A r a t i o 2.3: 1.0 to 3. 4: 1.0 (2.8:1.0) . Pharynx muscular, 0. 198-0.264 (0.225) l o n g by 0.209-0.252 (0.236) wide. 0/P r a t i o 1.8:1.0 t o 2.4:1.0 (2.1:1.0). I n t e s t i n a l caecaL narrow tubes, extending t o near p o s t e r i o r e x t r e m i t y . T e s t e s , e l o n g a t e , o v e r l a p from 35% to 60% of t h e i r l e n g t h s , lobed or not. ovary lobed o r not, p r e t e s t i c u l a r . E x t r a c a e c a l u t e r i n e l o o p s present, extend from the a n t e r i o r border of the p o s t e r i o r t e s t i s , t o t h e p o s t e r i o r border of the ovary. G e n i t a l pore v e n t r a l t o pharynx. V i t e l l a r i a , f o l l i c u l a r , s y m m e t r i c a l l y placed on each s i d e of the body. Extent ranging from about l e v e l o f i n t e s t i n a l b i f u r c a t i o n , and t e r m i n a t i n g p o s t e r i o r t o the t e s t e s . Eggs o p e r c u l a t e , 0.OT9-0.025 (0.023) l o n g by 0.015-0.019 (0.017), wide.; Host: R. c a t e s b e i a n a , R. c l a m i t a n s . R. g r y l i o - R . utricularia..-./ 104 S i t e c f i n f e c t i o n : Lungs Specimens* examined: Seventeen specimens, B r i t i s h Columbia, Maple Ridge, ex. 1 . c a t e s b e i a n a . author's c o l l . ; 1 specimen, F l o r i d a , J a c k s o n v i l l e , ex R. u t r i c u l a r i a . Dr. G.J. Bowers c o l l . ; 3 specimens, L o u i s i a n a , Avery I s l a n d , ex R. q r v l i o . Dr. M.L. Eberhard c o l l . ; 3 specimens, Massachusetts, North Dartmouth, ex I - : £Si£sjbeiaaa, Dr. , R. a. Campbell c o l l . ; 1 specimen, M i s s i s s i p p i , ex R. c a t e s b e i a n a . Dr. J.D. Lynch c o l l i ; 10 specimens, Nevada, LasVegas, ex R. c a t e s b e j a n a . Dr. B.B. Sabero c o l l . ; 10 specimens. Nova S c o t i a , H a l i f a x , ex R. c a t e s b e i a n a . (Mr. and Mrs. Bonnyman), author's c o l l . ; 12 specimens, O n t a r i o , B e l l e v i l l e , ex R. c a t e s b e i a n a . (Dr. R.J. G. L e s t e r ) , author* c o l l . ; 5 specimens, V i r g i n i a , Richmond, ex R. c l a m i t a n s . Dr., R. A. ;«:Campbell c o l l . Type specimens were not a v a i l a b l e to the author. D i s c u s s i o n : S i x t y - s i x H. b r e v i p l e x u s from ten l o c a l i t i e s i n Canada and the Onited S t a t e s were examined f o r v a r i a t i o n i n t a x o n o m i c a l l y important c h a r a c t e r s (Map 2 ) . , Hp s i g n i f i c a n t d i f f e r e n c e i n c h a r a c t e r s o f f l u k e s from the d i f f e r e n t l o c a l i t i e s o ccurred. Too few samples of R. g r v l i o . R. u t r i c u l a r i a Harlan, 1826 and fi. c l a m i t a n s were obtained t o t e s t f o r d i f f e r e n c e s i n f l u k e s from d i f f e r e n t hosts. The f o l l o w i n g r e s u l t s r epresent pooled data from a l l l o c a l i t i e s . Spines were l o c a t e d on the a n t e r i o r o n e - t h i r d of the tegument o f f l u k e s from Nevada and L o u i s i a n a . Two specimens from 105 V i r g i n i a d i d not have spines, a l l other specimens examined had spin e s over t h e i r e n t i r e s u r f a c e . The ovary was lobed i n 81.51 of the f l u k e s and smooth i n 18.5%. Testes were lob e d i n 76.951 of the worms and unlobed i n 23.1%. N e i t h e r t e s t i s was lobed more f r e q u e n t l y than the oth e r . Both t e s t e s were el o n g a t e and overlapped f o r at l e a s t 1/3 t h e i r l e n g t h . S t a f f o r d , 1902 used the arrangement o f the uterus as a u s e f u l c h a r a c t e r f o r s e p a r a t i n g H. l o n q i p l e x u s from H. b r e y i p l e x u s . He noted t h a t the e x t r a c a e c a l u t e r i n e l o o p s i n H. b r e y i p l e x u s do not extend beyond t h e p o s t e r i o r t e s t i s , whereas, i n H. l o n q i p l e x u s they extend nearly t o the pharynx. I found the e x t r a c a e c a l loops i n H. b r e y i p l e x u s t o extend to t h e l e v e l of the ovary. The r i g h t loop i s most f r e q u e n t l y t h e longer (71.451 of the cases) and ranged from 1/2 the d i s t a n c e along the l e n g t h o f the ovary t o the l e v e l of the ovary. The l e f t loop ranged from the l e v e l of the p o s t e r i o r t e s t i s to 1/2 the d i s t a n c e along t h e l e n g t h o f the p o s t e r i o r t e s t i s . Thus d e s p i t e the c l o s e morphological resemblance t o H.l:pnqiiplexus. the two s p e c i e s do d i f f e r i n the exte n t of t h e i r e x t r a c a e c a l u t e r i n e l o o p s . The O/a r a t i o ranged from 2.1:1.0 to 3.4:1.0 with a mean o f 2.8:1.0. The o/P r a t i o d i d not d i f f e r i n f l u k e s from d i f f e r e n t l o c a l i t i e s . T h i s r a t i o ranged from 1.4:1.0 to 2.4:1.0 (2.0:1.0). Egg l e n g t h s ranged from 0.019 t o 0. 025 (0.021) and egg width from 0.015 to 0.018 (0.016). areas p r o v i d i n g samples of H. b r e v i p l e x u s . Sample s i z e i s r e p r e s e n t e d i n the c i r c l e s . 106A 107 Haematoloechus v a r i o p l e x u s S t a f f o r d Haematplpechus v a r i o p l e x u s S t a f f o r d , 1902: 906. ( F i g . 65) Haematoloechus s i a i i i p l e x u s S t a f f o r d . 1902: 907 ( F i g . 64) Haegatoldechus p a r v i p l e x u s (Irwin, 1929) : 74. s ^ j a . nov. (Fi g . 40) Haematoloechus f l o e d a e Harwood, 1932: 16. sjrn. nov. (F i g . 66) Haematoloechus u n i p l e x u s Harwood, 1932: 18. sy.n. A• nov. , (Fi g . 67) Haematoloechus b u t t e n s i s I n g l e s . 1936: 78., syn. nov. ( F i g . 63) H e — d e s c r i p t i o n (based on 78 specimens): Body elongate, 3.57-11.75 <6.19) long by 0.73- 4.02 (1.31) wide. Tegument spined. O r a l sucker t e r m i n a l , 0.039-0.589 (0.340)long by 0.187-0.589 (0.317) wide, acetabulum medial, o v e r l i e s the ovary, 0.Q66-0.363 (0.156) wide. 0/A r a t i o 1.1:1.0 to 3.4:1.0 (2.3:1.0:. Pharynx muscular, 0.110^0.297 (0. 175) long by 0.110-43.314 (0.165) wide. ; O/P r a t i o 1.2:1.0 t o 2.3:1.0 (1.7: 1.0) . I n t e s t i n a l caecao narrow tubes, e x t e n d i n g t o near the p o s t e r i o r e x t r e m i t y . T e s t e s tandem or o b l i q u e , round t o e l l i p t i c a l , smooth or lob e d . Ovary lbbed o r not, p r e t e s t i c u l a r . E x t r a c a e c a l u t e r i n e l o o p s p r e s e n t , extending a n t e r i o r to a n t e r i o r border c f the p o s t e r i o r t e s t i s , o c c a s i o n a l l y extending to mid-region of a n t e r i o r t e s t i s . . G e n i t a l pore v e n t r a l t o pharynx, v i t e l l a r i a f o l l i c u l a r , v a r i a b l e i n d i s t r i b u t i o n . 1 0 8 Extent r a n g i n g from midway between ovary and pharynx t o p o s t e r i o r to the p o s t e r i o r t e s t i s on the s i d e o p p o s i t e the ovary or t o , the middle of the p o s t e r i o r t e s t i s on the o v a r i a n s i d e . Eggs o p e r c u l a t e , 0.018-0.043 (0.028) long by 0.013-0.031 (0.017) wide. . . . ,.,: .Hostsr;;\E.v:,ca"teg:b^iana;> R. cl a m i t a n s ; • R. p i p i e n s ; - R b l a i r i Hecham* t i t t l e J o h n , Oldham, Brown and Brown, 1973; R. p r e t i o s a ; ' R. s y l y a t i c a ; .E..ri?spBenocephal;a ;•« B. .woodhonsei •• G i r ar d,. 1854. S i t e o f i n f e c t i o n : l u n g s . Specimens examined: A. H. p a r v i p l e x u s : • • , 71 specimens, B r i t i s h Columbia, ex '• 8. s v l v a t i c a , author's c o l l . ; 83 specimens, Nebraska, ex > R.- c a t e s b e i a n a . H. B. .-Banter L a b o r a t o r i e s c o l l . ; 1 • , r, . •'specimen UVS. N.B. Helm. C o l l . So. 8085. •!*&.-•.- • "; H. v a r i o p l e x u s - / H. s i m i l i p l e x u s : 12 specimens, Ontario,; ex R. c l a m i t a n s . Hr., B. Baker's c o l l . ; 10 specimens, V i r g i n i a , ex , • :v£2t§s.||ei.ana, .-.Dr.. R.A. Campbell c o l l . ; 23 specimens, Nebraska, ex R. p i p i e n s . R. b l a i r i . B. wpodhousei, H.W. Banter L a b o r a t o r i e s c o l l . Type specimens were not a v a i l a b l e to the author. C. . H. b u t t e n s i s : One thousand f i v e hundred s i x t y - t w o specimens, B r i t i s h Columbia, ex R. p r e t i o s a . a u thor's c o l l . ; 1 type specimen, O.S.N.B. Helm. C o l l . No.,8926. D. H. f l o e d a e : 1 type specimen, 0. S.N. B. Helm. .Col 1. , No. .30879., 109 E. H. u n i p l e x u s : 1 type specimen, U.S.N.M. Helm. C o l l . No. 30880. D i s c u s s i o n : H. p a r v i p l e x u s . one hundred and f i f t y — f o u r , specimens of H. p a r v i p l e x u s . from f i v e l o c a l i t i e s , were examined f o r v a r i a t i o n i n c h a r a c t e r s used to d i s t i n g u i s h t h i s s p e c i e s from other North American members of Haematoloechus. The o v a r i e s were lobed i n 87.1% and unlobed i i i 12.9% o f the specimens examined. The a n t e r i o r t e s t i s was lobed i n 17.9$ and unlobed i n 82.1$ while the p o s t e r i o r t e s t i s was lobed i n 11.3% and unlobed i n 85.7% of t h e specimens. The G/A r a t i o c o u l d be determined f o r 139 of the f l u k e s . The acetabulum could not be l o c a t e d i n the other 15 f l u k e s so a r a t i o c o u l d not be c a l c u l a t e d . A one-way a n a l y s i s o f var i a n c e f o l l o w e d by a Newman—Kxuls M u l t i p l e Range Tes t was used to t e s t f o r s i g n i f i c a n c e between the means of the f i v e l o c a l i t i e s sampled. There was no s i g n i f i c a n t d i f f e r e n c e between the iseans of f l u k e s from Vanderhoof, S l i m Lake, or McBride ( F i g . 49) . A l l of these f l u k e s o c c u r r e d i n Rana s y 1 v a t i c a . The At k i n s o n , Nebraska, sample had a mean s i g n i f i c a n t l y d i f f e r e n t from t h a t of the B r i t i s h Columbia samples (<* <0.001) . The Humboldt sample was a l s o s i g n i f i c a n t l y d i f f e r e n t frem the B r i t i s h Columbia sample { < K < 0.001) and from the Atkinson sample (<* <0.001) . The samples from Nebraska were from R. c a t e s b e i a n a . / The o/P r a t i o ranged from 1.2:1.0 to 2.1:1.0 (1.5: 1.0) . T h i s r a t i o d i d not d i f f e r s i g n i f i c a n t l y i n f l u k e s from d i f f e r e n t 110 l o c a l i t i e s or h o s t s . V a r i a t i o n s i n the extent of the e x t r a c a e c a l l o o p , between l o c a l i t i e s sampled, d i d not d i f f e r . A d e s c r i p t i o n of the v a r i a t i o n i n e x t r a c a e c a l l o o p s i s given from pooled r e s u l t s . The tegument of a l l specimens had s p i n e s . 111 F i g . 49 O/ft r a t i o s of H. pary,iplexus from c o l l e c t i o n s made i n B r i t i s h Columbia and Nebraska. The range, mean and standard d e v i a t i o n (open r e c t a n g l e ) are gi v e n . Sample s i z e i s i l l u s t r a t e d a t the lower l i m i t of each range. 3.8 3.4 R. sylvatica R . .catesbeiana © "5" 3.0-2-6-2.2-1.8-14 -28 27 16 39 29 o a o > u ca E 55 d ca BQ u « 2 e o V) c 2 o E LOCALITY 112 E x t r a c a e c a l u t e r i n e Icops were egua l or near egual i n le n g t h i n 31.9% o f t h e f l u k e s examined; the r i g h t loop l o n g e r than the l e f t i n HI.7% and the l e f t longer than the r i g h t i n 26.4% o f the f l u k e s . The l e f t e x t r a c a e c a l l o o p was absent i n 4.211 of the cases. When both loops are present and of egual or near egu a l l e n g t h , the v a r i a t i o n i n extent i s from 1/2 the d i s t a n c e from the p o s t e r i o r end t o the p o s t e r i o r border of the p o s t e r i o r t e s t i s to 3/4 the d i s t a n c e along the l e n g t h o f the p o s t e r i o r t e s t i s . The average e x t e n t i s 1/3 the d i s t a n c e along the l e n g t h of the p o s t e r i o r t e s t i s . When the r i g h t l o o p i s longer than the l e f t i t extends between 1/5 and 9/10 the d i s t a n c e along the l e n g t h o f the p o s t e r i o r t e s t i s , with a mean of 52.6%. The l e f t l o op ranges from j u s t r e a c h i n g the p o s t e r i o r border , o f the p o s t e r i o r t e s t i s ; t o 25. 7% the d i s t a n c e along the p o s t e r i o r t e s t i s . When t h e l e f t l o op i s l o n g e s t i t extends from 20% to 80% t h i s d i s t a n c e , with a mean o f 54.5%. The r i g h t loop extends from 18% to 50%, with a mean of 30.4%. Some v a r i a t i o n s i n ovary and t e s t i s shape and i n u t e r i n e l o o p s are given i n Fi g u r e s 50 t o 52. There i s no s i g n i f i c a n t d i f f e r e n c e i n widths of eggs from d i f f e r e n t l o c a l i t i e s or i n the len g t h of eggs from Vanderhoof, Slim Lake, McBride and Humboldt ( F i g . 53). Fl u k e s from Vanderhoof, S l i m Lake and McBride had JR. s y l y a t i c a as t h e i r def i n i t i v e h ost. Fluked from Nevada had as t h e i r d e f i n i t i v e host R. c a t e s b e i a n a . The egg l e n g t h s i n f l u k e s from R. s y l v a t i c a d i f f e r e d s i g n i f i c a n t l y frcm f l u k e s from R . c a t e s b e i a n a from Atkinson (<*. <0.005). Egg l e n g t h s of f l u k e s from Humboldt 113 d i f f e r e d s i g n i f i c a n t l y from those of worms from Atkinson (°< <0.001). The tegument of a l l specimens had sp i n e s . 114 F i g . 50 t o 52. Some v a r i a t i o n s i n shape of o v a r i e s and t e s t e s and i n e x t e n t of e x t r a c a e c a l u t e r i n e l o o p s i n H . p a r v i p l e x u s , 114A Length and width of eggs of H. p a r v i p l e x n s from d i f f e r e n t l o c a l i t i e s , i n d i c a t i n g mean, standard d e v i a t i o n ( v e r t i c a l r e c t a n g l e ) , and the range ( v e r t i c a l l i n e ) , with t h e sample s i z e and f r o g host i n d i c a t e d f o r each l o c a t i o n . 115A ft.C«Usbeiana O.030-4 0.028 H R.sylvatica s C5 z ui 0.026 -1 28 L3 0.024 Ui OJ022-1 0020-4 u aa e o « > 27 16 51 u to E •5 "T o a a u S 31 O Z T « z •o 1 E R»sylvatica R. catesbeiana 28 .27 16 31 o •5 o c O CS 0 a E 5 51 I I T B.C. A m Xi B.C. o z Z • 2 c o n c 2 "o J> c Met Atkl c 3 X h 0.020 r-0.019 h-0.018 h0.017 Q 1-0.016 — C3 h 0.015 C5 Ui - 0 . 0 1 4 -0 .013 10CALITY 116 B. v a r i o p l e x u s and H. s i m i l i p l e x u s S e v e n t y — f i v e specimens o f f l u k e s l a b e l l e d e i t h e r H., v a r i o p l e x u s or H. s i a i 1 i p 1 ex us were examined. The range i n v a r i a t i o n of c h a r a c t e r s used t o separate H. s i m i l i p l e x u s from fi. y a r i o p l e x n s was too g r e a t to be c e r t a i n of p o s i t i v e i d e n t i f i c a t i o n . T h e r e f o r e specimens l a b e l l e d H. v a r i o p l e x u s and H. s i m i l i p l e x u s were t r e a t e d a c c o r d i n g to host. The 0/A r a t i o of f l u k e s from d i f f e r e n t l o c a l i t i e s and d i f f e r e n t hosts were c a l c u l a t e d and are presented i n F i g u r e 54. A Hewman-Keuls M u l t i p l e Range Test was used to t e s t f o r s i g n i f i c a n c e of means between samples from d i f f e r e n t l o c a l i t i e s . There i s no s i g n i f i c a n t d i f f e r e n c e between f l u k e s c o l l e c t e d from Genoa and L i n c o l n , Nebraska when the host i s R. p i p i e n s . However, th e r e i s a s i g n i f i c a n t d i f f e r e n c e between these f l u k e s and worms from Genoa recovered from B. woodhousei (<K =0.02). A s i g n i f i c a n t d i f f e r e n c e between f l u k e s from B. wobdhousei and R. p i p i e n s a l s o occurs when the data f o r R. p i p i e n s a r e pooled (<=*<0.001) . The o/A r a t i o of f l u k e s recovered from JR. c a t e s b e i a n a from Richmond, V i r g i n i a d i f f e r e d from those i n .S^VSiauiiiaJBS • from Long P o i n t , O n t a r i o <°< <0.05). The means, standard d e v i a t i o n s and ranges are given f o r samples egual to or g r e a t e r than f o u r and f o r pooled data ( F i g . 54). The O/P r a t i o d i d not d i f f e r s i g n i f i c a n t l y i n f l u k e s from d i f f e r e n t l o c a l i t i e s . , The r a t i o ranged from 1.3:1.0 to 2.3:1.0 (1.7:1.0). Q/ft r a t i o s of B> v a r i o p l e x u s and H. s i m i l i p l e x u s from d i f f e r e n t l o c a l i t i e s , i n d i c a t i n g the mean , s t a n d a r d d e v i a t i o n ( v e r t i c a l r e c t a n g l e ) and the range ( v e r t i c a l l i n e ) , with sample s i z e and f r o g host i n d i c a t e d f o r each l o c a t i o n . O/A RATIO o o > Oenoa N«b. — Oenoa Nab, — Richmond Va . — L i n c o l n Nab. — Long Point Ont* — Pooled Data — Pooled Data — Pooled Data —J S S c b to CD i t I l I . t .. i R. plplena B, woodhousel R. catesbeiana 3 "t=i=> R. plplena R. clamitans R. Djjpjena ( H . varloplexua) S l | T R.blalr l R. plplena (H. var l 0 plexua«f -H. slmliiplexua^ > 118 Fgg measurements from f l u k e s from Nebraska d i d not d i f f e r s i g n i f i c a n t l y , Egg measurements of f l u k e s from Long P o i n t , O n t a r i o and Richmond, V i r g i n i a d i f f e r e d s i g n i f i c a n t l y from each other as we l l as from Nebraska (<* <0.001) ( F i g , 55). No d i f f e r e n c e i n e x t r a c a e c a l loops i n f l u k e s from d i f f e r e n t hosts o r l o c a l i t i e s o c c u r r e d . A d e s c r i p t i o n of these l o o p s i s gi v e n t o i n d i c a t e t h e i r v a r i a b i l i t y . When both e x t r a c a e c a l loops reached the p o s t e r i o r t e s t i s (88.9% o f the time) the loops were of approximately the same leng t h i n 13.3% of the f l u k e s . In 35.1% of the f l u k e s the r i g h t loop was l o n g e s t and i n 21,6% the l e f t . When u t e r i n e loops were of near-egual l e n g t h they extend 40-65% the d i s t a n c e along the p o s t e r i o r t e s t i s . When the r i g h t loop was l o n g e s t i t reached at l e a s t 50% the d i s t a n c e along the p o s t e r i o r t e s t i s , when the l e f t loop was longest i t a l s o reached at l e a s t 50% the d i s t a n c e along the p o s t e r i o r t e s t i s . In 11.1% of the cases examined one or both u t e r i n e Icops f a i l e d t o reach the p o s t e r i o r t e s t i s . Of these c a s e s , 16.7% had loo p s the same l e n g t h and 66.7% had the r i g h t loop l o n g e r than the l e f t , 8.3% had the l e f t loop l o n g e s t and 8.3% had the l e f t loop missing. Some of the v a r i a t i o n s i n extent of e x t r a c a e c a l l o o p s are given i n F i g u r e s 56 to 59. A n t e r i o r and p o s t e r i o r t e s t e s and o v a r i e s are commonly unlobed. Only 2.6% o f t h e a n t e r i o r t e s t i s examined and 5.7% of the p o s t e r i o r t e s t i s examined were lobed, and 5.6% o f a l l o v a r i e s examined were lobed. A l l specimens had s p i n e s d i s t r i b u t e d over the tegument. 119 F i g . 5 5 Length and width o f eggs o f H. varioplexus- ;-and • -'H.:-siMlir#exns - from • d i f f e r e n t -l o c a l i t i e s , i n d i c a t i n g t h e ; mean, standard d e v i a t i o n ( v e r t i c a l r e c t a n g l e ) and the range ( v e r t i c a l l i n e ) , with the sample s i z e and amphibian host i n d i c a t e d f o r each l o c a l i t y . 119A 0.074 -OJ070-0.066-OJ062 — 0.058— s S 0.054-1 O 0.050—\ z u i "*J 0.046 H o W 0.042 H 0.038-0D34— 0j030— K I 12 o «« 3 E Kl 10 KI a c 12 C0| 17 11 3 K | 10 - 0.048 -0.044 -0.040 •0.036 S 5 J- 0.032 X O f- 0.028 ^ h-0B24 « a | L 17 26 26 11 h- 0.020 h- 0.016 i — r 4 > x w ae z z • e c • Q • TJ e A. o e e a. t> c o 2 c e E z LOCALITY 120 F i g u r e s 56 to 59. V a r i a t i o n s i n extent o f e x t r a c a e c a l u t e r i n e loops.- in^Hv-B/va^ic'ple?cus.-- .,> 58 120A 59 ' 121 H. c a t ten s i s , one t h o u s a n d f i v e hundred s i x t y - t w o H. buttensj.s c o l l e c t e d from . s e v e n t y — f o u r l o c a t i o n s i n B r i t i s h Columbia were examined. A l l specimens were from B. p r e t i o s a and were prepared by the author. Lobed o v a r i e s were found i n 93,8$ of the specimens examined ( F i g . 60). A n t e r i o r and p o s t e r i o r t e s t e s were lobed i n 18.7% of the f l u k e s examined. Tes t e s overlapped 15 51 t o 50% of t h e i r l e n g t h , averaging 32.5% ( F i g . 61). E x t r a c a e c a l u t e r i n e loops are equal or near-equal i n l e n g t h 72.71 of the time ( F i g . 62). In these c a s e s the r i g h t loop ranged: a d i s t a n c e o f 1/4 the l e n g t h o f the p o s t e r i o r t e s t i s to 3/1 the d i s t a n c e . At the same time the l e f t l o o p extends 1/2 the d i s t a n c e to the p o s t e r i o r t e s t i s from the end of the worm. When the r i g h t loop i s l o n g e s t the a n t e r i o r extent of the r i g h t l oop reached a d i s t a n c e 1/3 the way along the p o s t e r i o r t e s t i s . When the. l e f t e x t r a c a e c a l loop i s l o n g e s t the l e f t l o op extends, a t most, to the p o s t e r i o r margin of the p o s t e r i o r t e s t i s . In 9S o f the f l u k e s examined the r i g h t l o o p was absent. The 0/A r a t i o ranged from 2.2:1.0 to 2.9:1.0 with an average of 2.6:1.0. The O/P r a t i o d i d not d i f f e r i n f l u k e s c o l l e c t e d from d i f f e r e n t l o c a l i t i e s . T h i s r a t i o had a range of 0.9:1.0 to 1.9:1.0 and a mean o f 1.4:1.0. So s i g n i f i c a n t d i f f e r e n c e o c c u r r e d i n egg l e n g t h s or i n egg widths when fukes from d i f f e r e n t l o c a l i t i e s were compared. Eggs measured 0.020 t o 0.025 (0.023) l o n g by 0.016 to 0.018 (0.017) wide. Spines covered the e n t i r e body s u r f a c e on a l l f l u k e s . F i g . 60 Lobed o v a r i e s and o v e r l a p o f t e s t e s i n H. b u t t e n s i s . Fig.61 Lobed o v a r i e s and o v e r l a p o f t e s t e s i n H. b u t t e n s i s . Fig.6 2 Extent of e x t r a c a e c a l u t e r i n e loops i n H•.,-.buttensjs.. 123 Examination of the type specimen of H. b u t t e n s i s (U.S. N.M. Helm, C o l l . No. 8926) showed c e r t a i n i n c o n s i s t e n c i e s between d e s c r i p t i o n and observed morphology. In g l e s * drawing of the type specimen i s i n d o r s a l view, not v e n t r a l view as s t a t e d by him i n t h e c a p t i o n to h i s F i g u r e 3. T h i s view d e p i c t e d the l e f t e x t r a c a e c a l loop extending 1/3 along the l e n g t h of the p o s t e r i o r t e s t i s , and the r i g h t loop d i d not r e a c h the p o s t e r i o r t e s t i s . Examination of the type specimen i n d i c a t e d t h a t both e x t r a c a e c a l l o o p s j u s t reach the l e v e l of the p o s t e r i o r t e s t i s ( F i g . 63). " C u t i c l e armed with s p i n e s p o s t e r i o r l y t o the acetabulum" ( I n g l e s , 1936) . T h e r e are s p i n e s over the e n t i r e body, but they become s m a l l e r and l e s s numerous p o s t e r i o r of the acetabulum. H. f l o e d a e and H. uniplexus , Only the type specimens of H. f l o q d a e and H. uniplexus ( F i g s i 66 and 67) were a v a i l a b l e f o r examination. These specimens showed some i n c o n s i s t e n c i e s between d e s c r i p t i o n and observed, morphology. H. f l o e d a e (U.S.N.M. Helm. C o l l . No. 30879): Harwood d e s c r i b e d H.. f l o e d a e as being " e n t i r e l y without spines"/ 1 and t h a t "the o r a l sucker measured 3.6 to 4.4 mm i n diameter," p. 16. My examination of the type specimen showed s p i n e s t o be present. There a r e l a r g e s p i n e s on the o r a l sucker and a n t e r i o r 20% of the worm. Spines become s m a l l e r p o s t e r i o r l y . The o r a l sucker o f the type specimen has a t r a n s v e r s e diameter of 0.432mm. Therefore I f e e l Harwood's d e s c r i p t i o n 124 should be amended to r e a d : o r a l sucker measures 0.36 t o 0.44 mm in diameter. N, The absence of spines i n H.%floedae was one character used by Garwood t o d i s t i n g u i s h t h i s species from H.-/parviplexus•/' I - l S i ^ i e 2 « s (O.S. H.«. Helm. C o l l . No. , 30880) : Harwood (1932: 18) reported that the «cuticula i s smooth and i ; without s p i n e s .* However, I found small spines over most of the body surface and l a r g e r spines i n the region of the o r a l sucker. No emphasis i s placed on the supposed smooth nature of the tegument f o r species s e p a r a t i o n . The main feature used f o r recognizing t h i s species i s the s i n g l e uterine l o o p . The v a l i d i t y of t h i s character for I d e n t i f y i n g species i n t h i s genus w i l l be discussed i n the next s e c t i o n . 125 F i g . 63 By b u t t e n s i s • r e d r a a n from the type m a t e r i a l . Fig., ,64 H. s i m i l i p l e x a s redrawn from S t a f f o r d < 1902). Fig.,,,65 v a r i o p l e x u s redrawn from S t a f f o r d (1902). F i g . 66 H. f l o e d a e redrawn from the type specimen. Fi g . • 67; H. uniplexus redrawn from the type specimen. , S e v e r a l s t r u c t u r e s have been omitted f o r c l a r i t y . A l l specimens are d e p i c t e d i n the v e n t r a l view. S t a f f o r d (1902) d i d not give a s c a l e to h i s drawings and so a b s o l u t e s i z e i s unknown f o r h i s type drawings. 125A 126 \, Haematoloechus kernensis Ingles Haematoloechus kernensis Ingles, 1932: 19As (Fig. 69) Haem a toloec hus tti midu s Ingles, 1932: 199. syn. no v. (Fig. 70) Description (based on 2 specimens): Body elongate, 5.51-8.57 (7.04) long by 0.98-2.93 (1.95) wide. Tegument s pined. Oral sucker terminal, 0. 369-0.614 (0.492) long by 0.351-0.693 (0.522) wide, acetabulum medial, o v e r l i e s the ovary or anterior to i t , 0.292-0.759 (0.526) wide.; O/a r a t i o 0.9: 1.0 to 1.2: 1.0 (1.1:1.0) . Pharynx muscular* 0.27-0.498 (0.353) long by 0.243-0.473 (0.358) wide. O/P r a t i o 1.4:1.0 to 1.5:1.0 (1.5:1.0). I n t e s t i n a l caecai narrow tubes, extending to near posterior extremity. Testes obligue, round, lobed or not. Ovary not lobed, p r e t e s t i c u l a r . Extracaecal uterine loops present, extending anterior to the posterior border of the anterior t e s t i s . Genital pore ventral to pharynx. V i t e l l a r i a , f o l l i c u l a r , symmetrically placed on each side of the body. Extent ranging from midway between acetabulum and pharynx and terminating posterior to the posterior t e s t i s on the side opposite to the ovary and extending midway along the posterior t e s t i s on the side of the ovary. Eggs operculate, 0.023-0.036 (0.030) long by 0.016-0.020 (0.018) wide., Host: Rasa aurora dravtoni Camp. 1917 Site of i n f e c t i o n : Lungs. 127 Specimens examined: One type specimen, U.S.N.M. Helm. C o l l . No. 8654; 1 type specimen, U.S.N. H. Helm. C o l l . No. >8657. 128 F i g . 68 H a em at o l o e c hus coaplexus fPneuaonoeces complexus of Seely, 1906) , redrawn from Seely (1906). No s c a l e g i v e n . F i g . 69 H. k e r n e n s i s redrawn from the type specimen. F i g . 70 H. tumidus redrawn from the type specimen. 70 69 129 D i s c u s s i o n : Examination of the type specimen of H. k e r n e n s i s (O.S.N.M. Helm.,, C o l l . „ No . v 8 6 5 » ) and H. tumidus (O.S.N.M. Helm. C o l l . No. 8 6 5 7 ) , showed c e r t a i n i n c o n s i s t e n c i e s between d e s c r i p t i o n and observed morphology. j i n g l e s (1932) s t a t e s t h a t "The c u t i c u l a of j Haematoloechus k e r n e n s i s i s e n t i r e l y smooth, there being no i n d i c a t i o n of s p i n e s at any p l a c e , " p. 191. The absence o f spi n e s i s one of the main f e a t u r e s d i s t i n g u i s h i n g t h i s f l u k e frem H. tumidus, which i s s p i n e d . ; Examination o f t h e type m a t e r i a l r e v e a l e d the presence o f s p i n e s . Spines are s p a r s e l y d i s t r i b u t e d a n t e r i o r of the acetabulum, more concentrated a t the l e v e l of the acetabulum and p o s t e r i o r to i t . The tegument appears to be sloughed o f f i n s e v e r a l p l a c e s a n t e r i o r t o the acetabulum, s p i n e s a r e present around the middle p o r t i o n o f the worm where the tegument i s i n t a c t . I n g l e s (1932) s t a t e s t h a t " Haematoloechus tumidus d i f f e r s from a l l the p r e v i o u s l y d e s c r i b e d s p e c i e s o f t h i s genus i n being l a r g e r , and i n having the acetabulum l a r g e r than the o r a l sucker," p.200. However, I n g l e s measured the length and width o f l i v i n g animals. When he measured mounted specimens he found them to be s m a l l e r , but even then he f e l t t h e i r s i z e t o be g e n e r a l l y l a r g e r than t h a t o f oth e r s p e c i e s o f Haematoloechus p r e v i o u s l y d e s c r i b e d . S i z e alone i s a poor c r i t e r i o n to use i n s p e c i e s s e p a r a t i o n o f lu n g f l u k e s . H. tumidus d i f f e r s from H. ke r n e n s i s mainly i n having s p i n e s . However, I have demonstrated t h a t S* k e r n e n s i s i s 130 spi n e d . T h e r e f o r e I f e e l t h a t the two s p e c i e s should be synonymized and that H. k e r n e n s i s has page p r i o r i t y and i s the v a l i d name. The above two s p e c i e s have been r e p o r t e d o n l y one other time, t h a t by I n g l e s (1936) . More specimens are needed to assess t h e r e l a t i o n s h i p of the above two s p e c i e s t o other Haematoloechus sp. ^'hich a l s o c o n t a i n e x t r a c a e c a l l o o p s . 131 Group I I Haematoloechus medicplexus Stafford, Haematploechus medioplexus S t a f f o r d , 1902: 908. ( F i g . 75) Pneumonoeces medidplexns Stafford« 1905: 5. r O g M ' O l w f o r mo sum P r a t t , 1903; S t a f f o r d , 1905: 5. ^  syn. nov. R e - d e s c r i p t i o n (based on 25 specimens): Body e l o n g a t e , 8.43^9.15 (8.71) long by 0.93-1.53 11.05) wide. Tegument s p i n e d . O r a l s u c k e r t e r m i n a l , 0.275-0.365 (0.327) long by 0.275-0.352 (0.314) wide. Acetabulum medial, o v e r l i e s ovary, 0.083-0.099 (0.083) wide. 0/A r a t i o 3.0:1.0 t o 3.9:1.0 (3.4 (1.0) . Pharynx muscular, 0. 176-0.231 (0.204) l o n g by 0. 192-0. 242 (0. 203) wide. 0/P r a t i o 1.3:1.0 to 1.6:1.0 (1.5:1.0). I n t e s t i n a l caecae narrow tubes, extending to near p o s t e r i o r extremity. Testes tandem, round to e l l i p t i c a l , lobed or not. Ovary lobed or not, p r e t e s t i c u l a r . E x t r a c a e c a l u t e r i n e loops absent. G e n i t a l pore v e n t r a l t o pharynx. V i t e l l a r i a f o l l i c u l a r , v a r i a b l e i n y d i s t r i b u t i o n . Extent ranging from midway between ovary and pharynx t o p o s t e r i o r t o the p o s t e r i o r t e s t i s on the s i d e o p p o s i t e the ovary or half-way along the p o s t e r i o r t e s t i s on the o v a r i a n s i d e . Eggs o p e r c u l a t e , 0.025-0.030 (0.026) l o n g by 0; 015-0*021 (0.018) wide. Hosts: R. p i p i ens. R. p r e t i o s a , B. americanus. S i t e o f i n f e c t i o n : Lungs. Specimens examined: 19 specimens. A l b e r t a , ex R. p i p i e n s and B. americanus, 132 author's c o l l . ; 54 specimens, B r i t i s h Columbia, ex B. p i p i e n s and B. p r e t i o s a . author's c o l l . ; 1 specimen, Iowa, ex E* p i p i e n s . Dr. R.A. Campbell c o l l . ; 87 specimens, O n t a r i o , ex fi.pipiens (from Dr. B.J.G. L e s t e r and Hrvfl. Baker), author's c o l l . ; 14 specimens, Nebraska, ex B. p i p i e n s . H.H.; Banter L a b o r a t o r i e s c o l l . ; 18 specimens, Saskatchewan, ex B. p i p i e n s . author's c o l l . ; 1 specimen, W i s c o n s i n / ex B. p i p i e n s . Dr. J.D. Lynch c o l l . ; 232 specimens, S i s c o n s i n , ex fi. p i p j e n s (Bio1oaica1 Supply) , author's c o l l . D i s c u s s i o n : H. medioplexus . Four hundred and twenty—six specimens o f H. medioplexus from 27 c o l l e c t i n g l o c l i t i e s i n Canada and the United S t a t e s were examined. The f o l l o w i n g v a r i a t i o n s i n c h a r a c t e r s o f major taxonomic importance are presented below. The r e s u l t s given f o r o v a r i e s , t e s t e s and u t e r i n e l o ops are f o r pooled data from a l l l o c a l i t i e s . E x t r a c a e c a l u t e r i n e loops were absent i n a l l specimens examined. Seventy-seven p o i n t e i g h t percent of the o v a r i e s were unlobed and 22.2% lobed. The a n t e r i o r t e s t i s were lobed i n 82.5% of t h e specimens, unlobed i n 17.5%. P o s t e r i o r t e s t e s were lobed i n 88>9% of the specimens and unlobed i n 11.131 of the specimens. The 0/A r a t i o c o u l d be determined f o r onl y 371 f l u k e s . The range,, mean and standard d e v i a t i o n f o r ten c o l l e c t i n g s i t e s a re given i n F i g u r e 71. Nebraska r e p r e s e n t s pooled data from s i x l o c a l i t i e s i n the s t a t e . . No s i g n i f i c a n t d i f f e r e n c e i n 0/A r a t i o occurred a mo 0 . ^ 3 . 1 t h e means o f the ten l o c a l i t i e s ( A n a l y s i s o f 133 Variance p > 0.05). R. p i p i e n s . B. boreas, and B. p r e t i o s a were the d e f i n i t i v e hosts f o r a l l f l u k e s . When a l l data are pooled, the r a t i o ranged f r o a 2.6:1.0 to 4.3:1.0 (3.5:1.0). The O/P r a t i o d i d not d i f f e r i n f l u k e s from d i f f e r e n t l o c a l i t i e s . The range of the O/P r a t i o was from 1.1 t o 1.6:1.0 (1.4:1.0). No s i g n i f i c a n t d i f f e r e n c e occurred i n egg l e n g t h or i n width when worms from d i f f e r e n t l o c a l i t i e s were compared. Egg le n g t h ranged from 0.024 to 0.035 (0.028) and width from 0.015 t o 0.021 (0.018) . all specimens were s p i n e d . 0/A r a t i o of H. medioplexus frcm d i f f e r e n t l o c a l i t i e s , i n d i c a t i n g the mean, standard d e v i a t i o n ( v e r t i c a l r e c t a n g l e ) and the range ( v e r t i c a l l i n e ) , with the sample s i z e i n d i c a t e d f o r each l o c a t i o n . Nebraska r e p r e s e n t s pooled data from s i x l o c a l i t i e s i n t h a t S t a t e . 134A 4.2 -4 . 0 -3 . 8 -3 . 6 -5 3.4 -5.3.2-1 3.0-J 2 . 8 -» 2 . 6 -* 2 . 4 -30 40 11 1 2 1 3 1 2 33 e O v c O c O o 39 z • Q 2 198 i — r » •3 e e U a a e E a S m U m JC o o it a to e B LOCALITY 135 Haematoloechus complex us (Seely) Haematplpechus complexus (Seely, 1906): 249. ( F i g . 68) Haematoipechus c o l o r a d e n s i s C o r t , 1915a: 213. sy_n. nov. ( F i g . 74) Haematoloechus o x y o r c h i s I n g l e s , 1932: 193. sy,n. nov. ( F i g . 12.) Haematglpechus c p n f u s u s I n g l e s , 1932: 195. s y n . nov. ( F i g . 73) R e — d e s c r i p t i o n (based on 50 specimens): Body el o n g a t e , 2. 73-7.72 15.82) long by 0.35-1.95 (1.23) wide. Tegument spined or not. O r a l sucker t e r m i n a l , 0.253-0.513 (0.379) l o n g by 0.242-0.492 (0.375) wide, acetabulum medial, o v e r l i e s the ovary, o r a n t e r i o r t o i t , 0.120-0.450 (0.271) wide. 0/A r a t i o 1.0:1.0 to 3.5:1.0 (1.5:1.0). Pharynx muscular, 0. 127-0.432 (0.228) long by 0,122-0.369 (0.208) wide. ,O/P r a t i o 1.2:1.0 t o 2.4:1.0 (1.9:1.0). I n t e s t i n a l caeca'- narrow tubes extending t o near p o s t e r i o r e x t r e m i t y . T e s t e s tandem, round, lobed or not. Ovary l o b e d or not, p r e t e s t i c u l a r . ; E x t r a c a e c a l u t e r i n e loops absent. V i t e l l a r i a f o l l i c u l a r , s y m m e t r i c a l l y placed on each s i d e o f the bodyj extent ranging from midway between ovary and pharynx t o p o s t e r i o r t o p o s t e r i o r t e s t i s on the s i d e o p p o s i t e the ovary, or half-way along the p o s t e r i o r t e s t i s on the o v a r i a n s i d e . Eggs o p e r c u l a t e , 0.023-0.043 (0.034) long by 0.014-0.024 (0.019) wide. Host: 5. b l a i r i . R. p i p i e n s . §. c a t e s b e i a n a . S i t e o f i n f e c t i o n : Lungs. 136 F i g . 72 H. o x y o r c h i s redrawn from the type m a t e r i a l . , F i g . 73 H. confusus redrawn from the type specimen. F i g . 74 H. c o l o r a d e n s i s ( Pneumonogees c o l o r a d e n s i s of c o r t , 1915a) redrawn from C o r t (1915a). F i g . 75 H. medioplexus redrawn from S t a f f o r d (1902). S e v e r a l s t r u c t u r e s have been omitted f o r c l a r i t y . A l l specimens are d e p i c t e d i n the v e n t r a l view. S t a f f o r d (1902) ) d i d not give a s c a l e t o h i s drawings and so a b s o l u t e s i z e i s unknown f o r 136A 74 75 1 3 7 Specimens examined: Av; H. complex us: 105 specimens, Nebraska, ex B. b l a i r i . H.H. Manter L a b o r a t o r i e s c o l l . ; 5 specimens, Nevada, ex B. p i p i e n s . Dr. B.B. Babero c o l l . , The t y p e specimen was not a v a i l a b l e to the author. , B. v,.,. H. c o l o r a d e n s i s : 10 specimens, Nebraska, ex 1» •••pijpigas* H. 8. Manter l a b o r a t o r i e s c o l l . ; 5 specimens, F l o r i d a , ex B. c a t e s b e i a n a . Dr. G.J. Bowers c o l l . The type specimen was not a v a i l a b l e to the author. C. fl. o x y o r c h i s : 1 type specimen, U.S.N.M. Helm. C o l l . No. 8655, ex Sana aurora d r a y t o n i . D. .. H. confusus: 1 type specimen, U.S.N.M. Helm. C o l l n c 8656, ex Sana aurora dra y t o p i . D i s c u s s i o n : H. coaplexus H. complexus ( pneumonoeces .gcmplexus of S e e l v . 1906) was d e s c r i b e d by Seely (1906) as without s p i n e s . However, he s t a t e s t h a t t h i s may have been due t o maceration o f the worm. Only patches of i n t a c t tegument remained and these d i d not have s p i n e s . U n f o r t u n a t e l y Seely d i d not mention the p o s i t i o n o f these patches. Type m a t e r i a l was not a v a i l a b l e f o r examination. 138 Of the 105 H. complexas examined from Nebraska and f i v e from Nevada, 90 had s p i n e s over t h e e n t i r e body s u r f a c e . , S p i n e s are s m a l l e r and l e s s numerous p o s t e r i o r of the acetabulum. •>i-.•it"V;^ -.ij'fi'..'f;i'|;'^  E x t r a c a e c a l l o o p s are absent i n t h i s s p e c i e s . The a n t e r i o r t e s t i s was lobed i n 6% of the specimens, the p o s t e r i o r t e s t i s i n 11$. A l l o v a r i e s were unlobed. The 0/A r a t i o ranged from 1.0:1.0 to 2.4:1.0 with a mean of 1.4:1.0. The worm with the 2.4:1.0 r a t i o had a very s m a l l acetabulum. The O/P r a t i o o f f l u k e s from d i f f e r e n t l o c a l i t i e s i -: d i d not d i f f e r ;'sig*Tiif ic;a»ti'y^ a ; range of 1.6:1.0 to 2.0:1.0. The widths of eggs d i d not d i f f e r s i g n i f i c a n t l y i n f l u k e s from d i f f e r e n t l o c a l i t i e s ( F i g . 76). Egg l e n g t h s from the four l o c a l i t i e s i n Nebraska d i d not d i f f e r s i g n i f i c a n t l y ( F i g . 76). The egg lengths of f l u k e s from Nevada d i f f e r e d s i g n i f i c a n t l y (Newman-Keuls,^> 0.05) from the egg l e n g t h s of f l u k e s of pooled data from Prague, Davey and Head (Nebraska) (<<< 0.05) , however, the sample s i z e from Nevada i s very s m a l l (n=5) . Hore samples from t h i s l o c a l i t y a re needed. Egg l e n g t h had a range of 0. 030 to 0.043 (0.036). Egg width had a range of 0.017 to 0.025 (0.020). H. c o l o r a d e n s i s when a l l a n t e r i o r t e s t e s were compared. 86.7$ were observed to be l o b e d . This a l s o h o l d s f o r the p o s t e r i o r t e s t e s . A l l o v a r i e s examined were lobed., Only 15 specimens from the uni t e d s t a t e s and Canada were examined. E x t r a c a e c a l loops were absent i n a l l of these 1 3 9 specimens. However, s h o r t loops do occur o c c a s i o n a l l y (21.0% o f the t i n e ) but do not extend e x t r a c a e c a l l y ( F i g s . 79 and 8 0 ) . T h e form of c o i l i n g of the p o s t e r i o r u terus depended on the host. When the host was R. p i p i e n s , the c o i l s formed t i g h t s p r i n g — l i k e l o o p s ( F i g . 77). When the host was R. b l a i r i , the c o i l s formed l o o s e t w i s t s ( F i g . 78). The 0/A r a t i o ranged from 1.0:1.0 to 1.4:1.0 with a mean of 1.2:1.0. The O/P r a t i o d i d not d i f f e r s i g n i f i c a n t l y i n f l u k e s from d i f f e r e n t l o c a l i t i e s or hosts. T h i s r a t i o ranged from 1.2: 1.0 t o 2.0:1.0 (1.4:1.0). No d i f f e r e n c e o c c u r r e d i n egg length or width i n f l u k e s from d i f f e r e n t hosts or l o c a l i t i e s . Egg l e n g t h ranged from 0.031 to 0.038 (0.035) and width from 0.017 to 0. 024 (0.019) . , A l l specimens were sp i n e d . 1 4 0 F i g . 76 Length and width o f eggs of H. complexes from d i f f e r e n t l o c a l i t i e s , i n d i c a t i n g the mean, standard d e v i a t i o n ( v e r t i c a l r e c t a n g l e ) and range ( v e r t i c a l l i n e ) , with the samples s i z e i n d i c a t e d f o r each l o c a l i t y . Data f o r Verdon and Humboldt were pooled as are data f o r Prague, Davey and Mead. R. b l a i r i was the host f o r a l l f l u k e s from Nebraska and R. p j p i e n s f o r f l u k e s from Nevada. 140A 0.044 ~\ 0.042 H 0.040-J 0.038 H 0.036 O C3 Ul 0.034 0032-0.030-17 25 30 12 "I I I I I — e 1 I i c e "8 c o « Z & a o h 0.026 17 25 5 12 30 h-0.024 g -0.022 jE Q -0.020 ^ u -0.018 w -0.016 I I I I I •2 -S •*> * z z J3 « Z 5 o & i 4 1 * s 9 0 e e "S , a t LOCALITY 141 F i g . 77 The shape of p o s t e r i o r u t e r i n e c o i l s of H. c o l o r a d e n s i s when the f l u k e i s recovered from R. p i p i e n s . F i g . 78 The shape of p o s t e r i o r u t e r i n e c o i l s of H. c o l o r a d e n s i s when the f l u k e i s recovered from a .volair1. Fig.79-80 Some v a r i a t i o n s i n u t e r i n e loops of H. coloradensis.•„•• 141A 1 4 2 H. o x y o r c h i s and H. c o n f u s u s E x a m i n a t i o n o f t h e t y p e s p e c i m e n s o f H. o x y o r c h i s ( U . S . N . B . H e l m . C o l l . N O . 8655) a n d H. c o n f a s i s ( O . S . N . B . H e l m . C o l l . Ho . 8656) r e v e a l e d some d i s c r e p a n c i e s b e t w e e n d e s c r i p t i o n and o b s e r v e d m o r p h o l o g y . H. o x y o r c h i s : i n g l e s ( 1 9 3 2 : 193) d e s c r i b e d H. o x y o r c h i s a s h a v i n g " c u t i c u l a f r e e f r o m s p i n e s . " I f o u n d t h a t t h e t e g u m e n t a p p e a r e d t o be l a c k i n g a n t e r i o r o f t h e a c e t a b u l u m . S m a l l s p i n e s a r e p r e s e n t on t h e a n t e r i o r and m i d - b o d y where t h e t e g u m e n t i s i n t a c t . T h e s e s p i n e s a r e v e r y s m a l l , b a r e l y p r o t r u d i n g t h r o u g h t h e t e g u m e n t . S p i n e s a r e a b s e n t p o s t e r i o r o f t h e p o s t e r i o r t e s t e s . The main f e a t u r e used t o d i s t i n g u i s h H. o x y o r c h i s f r o m H. c o n f u s u s h a s b e e n t h e s u p p o s e d a b s e n c e o f s p i n e s f r om t h e f o r m e r s p e c i e s . The p r e s e n c e o f s p i n e s i n t h e t y p e s p e c i m e n o f H . , o x y o r c h i s s u g g e s t s t h a t t h e s e two s p e c i e s s h o u l d be s y n o n y m i z e d . ; .." H. c o n f u s u s : :. " e g g s a v e r a g e 26 m i c r o n s i n l e n g t h and 15 m i c r o n s i n w i d t h " ( I n g l e s , 1932) . T h e a v e r a g e measurement o f 25 e g g s measured f r o m l o c a t i o n A ( s e e A p p e n d i x 2 2 , F i g . 81) was , 0 . 0 3 1 mm l o n g by 0 . 0 2 3 mm w i d e . T h e d i s c r e p a n c y between I n g l e s * measu rements and mine may be due t o m e a s u r i n g e g g s f r o m a d i f f e r e n t p a r t o f t h e u t e r u s . Egg s i z e and t h e p r e s e n c e o f s p i n e s were u s e d by I n g l e s t o 143 d i s t i n g u i s h t h i s s p e c i e s from H. complexes, d e s c r i b e d by Seely t^9p6)v,The type specimens o f fl. ccmplexus i s not a v a i l a b l e f o r examination. The d i f f e r e n c e i n egg s i z e b et »ee n H. a co ap l e x tt s (0.029 mm by 0.0 14 mm) and H. confusus i s not s u f f i c i e n t f o r s p e c i e s s e p a r a t i o n . 144 DISCUSSION AND-CONCLUSIONS A. D i f f e r e n t i a t i o n o f Species HV l o n q i p l e x u s and H. b r e v i p l e x us: • -H.v,-Aongi^lex/us - can only be confused with one other North American haematoloechid^: B. b r e y i p l e x u s . They are s i m i l a r i n t h e i r 0/A r a t i o s , 2.0:1.0 f o r both s p e c i e s ( S t a f f o r d , 1902; Co r t , 1915a). My o b s e r v a t i o n s show a range of 2.1:1.0 to 3.4:1.0 (2.8:1.0) f o r g. JbreyJ:pJLexus and 1,5:1.0 t o 2.8M..0 (2.2:1.0) f o r , H. l o n g i p l e x u s . These r a t i o s d i d not vary g e o g r a p h i c a l l y (Map 1). These s p e c i e s a re a l s o s i m i l a r i n shape of t e s t e s and ovary and i n g e n e r a l body s i z e . Two f e a t u r e s serve t o se p a r a t e these s p e c i e s : t he exte n t o f t h e e x t r a c a e c a l loops and the o r i e n t a t i o n of the t e s t e s . The e x t r a c a e c a l l o o p s i n H. l o n g i p l e x u s are the l o n g e s t i n any North American s p e c i e s , and extend a n t e r i o r l y to near t h e pharynx ( S t a f f o r d , 1902; C o r t , 1915a)., Some v a r i a t i o n does e x i s t , but the l o o p s are never found l e s s than 1/2 the d i s t a n c e between the ovary and the pharynx, U t e r i n e l o o p s i n H. b re vi. plexus a re s h o r t e r and extend from the a n t e r i o r border of the p o s t e r i o r t e s t i s t o the p o s t e r i o r border of the ovary (Cort, 1915a). My o b s e r v a t i o n s i n d i c a t e a s l i g h t l y g r e a t e r v a r i a t i o n f o r the l e f t u t e r i n e loop. T h i s l o o p may extend from 1/2 the le n g t h of the p o s t e r i o r t e s t i s t o the l e v e l of the a n t e r i o r border o f the a n t e r i o r t e s t i s . However, i n H. b r e y i p l e x u s t h e lo o p s never extend a n t e r i o r beyond the l e v e l of the ovary. / The elongate t e s t e s are p a r a l l e l i n H. 1enqiplexus. t h a t i s 145 they : overlap for more than 70% of t h e i r length. The testes are unequal in s i z e , the smaller t e s t i s occasionally extending 20% of i t s length anterior of the larger t e s t i s . The testes of • H. hreviglexus are also elongate, but they overlap for only 1/2 t h e i r length {Stafford, 1902) . T h i s overlap ranged from 35% to 60% in my c o l l e c t i o n s . The testes were never p a r a l l e l . The extent of the extracaecal loops and the shape and orientation of testes serve to separate these two species from other North American representatives of Haematoloechus and from one another. H. varioplexus and H. s i m i l i p l e x u s : The extent of the extracaecal loops, size and shape of testes and ovaries, and O/P r a t i o are s i m i l a r for these two species. The uterine loops i n both species commonly extend a distance 40 to 60% along the posterior t e s t i s . The ovary and testes are usually unlobed, but lobing in some specimens i n both species was noted. The O/P r a t i o did not d i f f e r between these two species. This r a t i o ranged from 1.3:1.0 to 2.3: 1.0 (1.7:1.0) for both species. The 0/A r a t i o was not reported by Stafford (1902) for either species. Cort (1915a) reported the 0/A r a t i o for H. s i s i i i p l e x u s as averaging 4:3 (1.33:1.0). He did not examine specimens of H. varioplexus. Brooks (1976) considered H. similiplexus and H. yarioplexus synonyms and reported the 0/A r a t i o as having a range of 1.0:0.69 to 0.84 (1.5:1 to 1.2:1). However, when the data for 0/A r a t i o are plotted according to 146 host, and not f l u k e s p e c i e s , the G/ft r a t i o v a r i e s with host. The same i s t r u e f o r egg s i z e . fl. v a r i o p l e x u s has an egg length of 0.029, whereas H« g i m i l i p l e x u s i s r e p o r t e d as having an egg l e n g t h of 0.039 ( S t a f f o r d , 1902) . C o r t (1915a) recorded an egg length range o f 0.034 t o 0.040 f o r H. s i m i l i p l e x u s . L a t e r , while examining specimens of H. s i m i l i p l e x u s from R. p i p i e n s from Oshkosh, i i s c o n s i n , C o r t (1915b) noted that the eggs were s m a l l e r than p r e v i o u s l y r e p o r t e d f o r t h i s s p e c i e s . These i n d i v i d u a l s had an , average egg l e n g t h of only 0.034, and t h e l i m i t s of v a r i a t i o n were from 0.030 t o 0.037. Brooks (1976) c i t e d an egg l e n g t h range f o r H. v a r i o p l e x u s o f 0.032 to 0.037. Bouchard (1951) noted v a r i a t i o n s i n H. s i m i l i p l e x u s from the lungs o f J * c l a m i t a n s and R. s e p t e n t r i o n a l l s c o l l e c t e d i n Maine., He reported t h a t some specimens had an egg l e n g t h average of 0.029. He a l s o noted t h a t some o f these f l u k e s had l o n g e r e x t r a - c r u r a l u t e r i n e f o l d s than r e p o r t e d by Cort(1915a) and S t a f f o r d {1902). F i g u r e 55 demonstrates the great v a r i a t i o n o c c u r r i n g i n egg measurements. T h i s v a r i a t i o n appears t o be r e l a t e d to host and not g e o g r a p h i c a l i n f l u e n c e s . Thus B. v a r i o p l e x u s and • H s i m i l i p l e x u s cannot be s e p a r a t e d . H. p a r v i p l e x u s : Irwin (1929) and l a t e r Brooks (1976) d e s c r i b e d the ovary o f •:- Hy|::.^ ;pariViiglexus as being deeply l o b e d . Hy experimental r e s u l t s u s i n g H. b u t t e n s i s show t h a t g r e a t v a r i a t i o n can occur i n the degree of l o b i n g of the ovary and t e s t e s , even i n worms c f the same age. The ovary may be unlobed ( F i g . 50) or deeply lobed 147 ( f i g s i 51 and 52). C o l l e c t i o n s o f H. p a r v i p l e x u s from f i v e l o c a l i t i e s show t h a t l o b i n g i n o v a r i e s and t e s t e s i s h i g h l y v a r i a b l e and may or may not o c c u r . There i s t h e r e f o r e too much v a r i a b i l i t y i n t h i s c h a r a c t e r to be used i n s p e c i e s s e p a r a t i o n . U t e r i n e f o l d s i n fl. p a r v i p l e x u s a r e r e p o r t e d by Irwin (1929) as never r e a c h i n g the a n t e r i o r border of the p o s t e r i o r t e s t i s ; s c a r c e l y r e a c h i n g the p o s t e r i o r t e s t i s i n some,; while i n others the loops may extend along 3/4 the l e n g t h of the p o s t e r i o r t e s t i s , ft s i m i l a r degree o f v a r i a t i o n was found i n the 154 specimens examined by me. I have shown, i n t h i s t h e s i s , t h a t the 0/A r a t i o f o r t h i s s p e c i e s i s v a r i a b l e , and may be h o s t - r e l a t e d ( F i g . 49) as w e l l as v a r y i n g g e o g r a p h i c a l l y . , T h i s s p e c i e s cannot t h e r e f o r e be separated from H. b u t t e n s i s . . H. b u t t e n s i s : I n g l e s (1936) used a combination o f c h a r a c t e r s t o separate h i s newly-described s p e c i e s , H. b u t t e n s i s ( F i g . 63) from ether members of the genus with which i t may be confused. H. b u t t e n s i s d i f f e r e d from H. p a r v i p l e x u s (Irwin, 1929) not a b l y i n the 0/A r a t i o , the shape of the ovary, and the extent of the e x t r a c a e c a l u t e r i n e f o l d s . V a r i a t i o n s i n the shape of the ovary and the exte n t of the e x t r a c a e c a l u t e r i n e f o l d s have been d i s c u s s e d i n the p r e v i o u s s e c t i o n . The main f e a t u r e d i s t i n g u i s h i n g H. b u t t e n s i s from H. p a r v i p l e x u s was s t a t e d to be the 0/A r a t i o , H. b u t t e n s i s having an average r a t i o of 1.0:0.7 (1.4:1.0) ( I n g l e s , 1936). Flukes c o l l e c t e d from B. p r e t i o s a from 148 B r i t i s h Columbia had an average O/A r a t i o of 2.6:1.0 (2.2:1.0 t o 2.9:1.0). H. p a r v i p l e x u s has a r a t i o o f 4:1 (Irwin, 1929). However, Brooks (1976) found the Nebraska c o l l e c t i o n t o range from 2.3:1.0 to 2.6:1.0, and my c o l l e c t i o n s from B r i t i s h Columbia averaged 3.0:1.0 (2.6 t o 3.4:1.0). T h e r e f o r e , the 0/A r a t i o i n these two s p e c i e s i s too s i m i l a r to be used as a d i s t i n g u i s h i n g c h a r a c t e r . H. b u t t e n s i s i s s e p a r a t e d from fi. . s d m i l i p l e x u s S t a f f o r d , 1902 ( F i g . 64) by having s m a l l e r eggs, average 0.027 by 0.014 (0.0 25 t o 0.030 and 0.022 to 0.017 f o r length and width r e s p e c t i v e l y ) , and l a r g e r t e s t e s . T e s t e s measured 0.82 long by 0.64 wide (0.45 t o 1.03 by 0.48 t o 0.87). Both t e s t e s were d e s c r i b e d as being n e a r l y the same s i z e and shape. V i t e l l a r i a were a l s o c o n s i d e r e d as d i f f e r e n t l y d i s t r i b u t e d . However, specimens examined by me d i d not d i f f e r i n v i t e l l i n e d i s t r i b u t i o n . S t a f f o r d (1902) gave the egg measurements of H.:; s l a i i i pie x us as averaging 0.039 by 0. 019, but s t a t e d t h a t " v a r i a t i o n s a l i t t l e above and below occurred. 1' H. , s i m i l i p l e x us was d e s c r i b e d from the lungs of jg. v i r e s c e n s (= B. p i p i e n s ) and !• l e n t i g i n o s u s (= B. americanus ) . He d e s c r i b e d H. v a r i o p l e x u s from the lungs of B. c l a m j t a p s . The type d e s c r i p t i o n was very incomplete, and S t a f f o r d t r u s t e d h i s drawings t o i l l u s t r a t e i t s c h i e f c h a r a c t e r i s t i c s . No s c a l e was g i v e n f o r h i s drawings. As shown above, H. b u t t e n s i s cannot be separated from H. p a r v i p l e x u s or from H. s i m i l i p l e x u s and H. v a r i o p l e x u s . 149 - R. uiuniplexus: ' The type d e s c r i p t i o n of B. u n i p l e x u s Harwood, 1932 ( F i g . 67) i s based on a s i n g l e specimen. T h i s f l u k e i s d i s t i n g u i s h e d from; a l l other worms o f the genus (con s i d e r e d here) by p o s s e s s i n g a s i n g l e s h o r t e x t r a c a e c a l u t e r i n e loop. The v a r i a t i o n s i n e x t r a c a e c a l u t e r i n e l o o p s have a l r e a d y been d i s c u s s e d f o r s e v e r a l s p e c i e s . The freguency of occurrence of a s i n g l e l o o p i n samples c o l l e c t e d from numerous l o c a l i t i e s i n d i c a t e s t h a t t h i s alone i s not a good c h a r a c t e r f o r s e p a r a t i n g s p e c i e s i n t h i s genus. T h i s s p e c i e s has not been r e p o r t e d s i n c e i t s o r i g i n a l d e s c r i p t i o n . H. f l o e d a e : Harwood (1932) separated H. floedae from H. t, par v i plexus. which i t most c l o s e l y resembles, by the s m a l l e r pharynx, l a r g e r acetabulum, the smooth tegument, s m a l l e r eggs and the l o n g e r l o n g i t u d i n a l u t e r i n e f o l d s ( F i g s . 66 and 40 r e s p e c t i v e l y ) . The G/P r a t i o i n H. f l o e d a e i s n e a r l y 1:2; (0.5:1) the r a t i o does not f a l l below 2:5 (0.4:1) (Harwood, 1932)*, In • H. p a r y j p l e x u s t h i s r a t i o i s 3:2 (Irwin, 1929)*, The O/ft r a t i o i n H. f l o e d a e i s 3:1 (Harwood, 1932). I n >• H. p a r v i p l e x u s • t h i s r a t i o i s 4:1 (Irwin, 1929).; Eggs o f ! • l o e d a e vary from 0.021 by 0.017 t o 0.017 by 0.013.,Eggs of H. p a r v i p l e x u s range i n s i z e from 0.023 t o 0.029 i n l e n g t h and from 0.016 to 0.019 i n width, averaging 0.025 by 0.017. The l e n g t h s of the e x t r a c a e c a l f o l d s of the uterus i n H. f l o e d a e have been shown, by examining the type specimen, not to extend beyond 1/2 the d i s t a n c e along the a n t e r i o r t e s t i s . 150 U t e r i n e f o l d s on the o v a r i a n s i d e of the body are o f t e n s h o r t e r than i t s mate {Harwood, 1932). V a r i a t i o n s i n O/k r a t i o , egg measurements, and extent of e x t r a c a e c a l loops have p r e v i o u s l y been d i s c u s s e d f o r H. p a r v i p l e x u s . Because of the v a r i a t i o n i n egg measurements, O/a r a t i o s , and extent of e x t r a c a e c a l l o o p s , i t i s i m p o s s i b l e to s e p a r a t e H. y a r i o p l e x u s . H. s i m i l i p l e x n s . H. p a r v i p l e x u s . H. b u t t e n s i s . H. f l o e d a e and H. unjplexus from one another. However, the e x t r a c a e c a l l o o p s never extend beyond the a n t e r i o r t e s t i s . T h e r e f o r e , t h i s group can be separated from fl. l o n q i p l e x u s and 5» b r e y i p l e x u s . H.complexus, H. conf usus, and H. o x y o r c h i s : I n g l e s (1932) s t a t e s that " H. conf usus more c l o s e l y resembles H. complexus than any other d e s c r i b e d lung f l u k e . I t agrees f a v o u r a b l y with t h i s l a t t e r s p e c i e s i n s i z e , type o f u t e r u s , the lobed t e s t e s and ovary, and i n r a t i o of the o r a l sucker t o the acetabulum. I t d i f f e r s from H. complexus. however, i n having s p i n e s , i n having s m a l l e r eggs, i n the arrangement o f the v i t e l l a r i a , and i n the t e s t e s and ovary always being lobed" £sicj. H. o x y o r c h i s d i f f e r s from H. complexus i n having s m a l l e r eggs. Egg s i z e was the main f e a t u r e d i s t i n g u i s h i n g H. o x y o r c h i s - • from --fl ..•s.jcpmplexas. Type specimens of fl. complexus were not a v a i l a b l e f o r i n s p e c t i o n . However, Seely (1906) s t a t e s that t h i s s p e c i e s i s "without s p i n e s " but notes t h a t " t h i s may have been due t o 151 maceration". Eighty-two percent o f t h e specimens examined by me had spines. The egg s i z e s of these two s p e c i e s o v e r l a p the low egg s i z e range of H. complexus. I f e e l t h a t there i s not enough d i f f e r e n c e i n these measurements t o warrant s e p a r a t i o n of these two s p e c i e s from H. complexus. I have shown t h a t i n c o l l e c t i o n s o f H. complexus s i x percent of the a n t e r i o r and el e v e n percent of the p o s t e r i o r t e s t i s were lob e d . H. c o l o r a d e n s i s : Cort (1915a) reviewed the North American lung f l u k e s and d e s c r i b e d H. c o l o r a d e n s i s n. sp. i n the same paper. He s t a t e d t h a t H. c o l o r a d e n s i s ( F i g . 74) i s most c l o s e l y r e l a t e d t o H. complexus (Seely, 1906) ( F i g . 68) i n having the same body shape and g e n e r a l arrangement of the uterus as that s p e c i e s . D i f f e r e n c e s between t h e s e two s p e c i e s were noted i n the 0/A and O/P r a t i o s and i n s i z e of eggs. The 0/A r a t i o f o r H. c o l o r a d e n s i s i s 5:4 (=1.3: 1.0) (C o r t , 1915a) and 1.1:1.0 f o r H. complexus (Seely, 1906). fly c o l l e c t i o n s show t h a t the r a t i o s o v e r l a p completely f o r the two s p e c i e s . The r a t i o s ranged from 1.0 t o 1.4:1.0 (1.2:1.0) f o r H. c o l o r a d e n s i s and 1.0 to 2.4:1.0 (1.4:1.0) f o r H. complexus. The O/P r a t i o f o r H. c o l o r a d e n s i s i s 10:7 (= 1.4:1.0) (Cort, 1915a) and was not given f o r fl. complexus by Seely (1906). Uy data show a great d e a l o f o v e r l a p i n t h i s r a t i o f o r the two s p e c i e s , H. c o l o r a d e n s i s has an O/P r a t i o range of 1.2 t o 2.4:1.0 (1.4:1.0) and ]f. complexus has a range of 1.6 to 2.0:1.0 152 (1.9:1.0). The average egg measurement f o r H. complexas i s 0.029 by 0.014 (Seely, 1906) and 0.034 by 0.020 f o r fl.coloradensis as give n by C o r t , 1915a. Specimens examined by me i n d i c a t e d almost a complete o v e r l a p i n these measurements f o r the two s p e c i e s . The average measurement f o r H. c o l o r a d e n s i s was 0.035 by 0.019 and f o r H. complexus 0.036 by 0.020. , Because of the o v e r l a p i n measurements used i n attempting to s e p a r a t e these two s p e c i e s the s e p a r a t i o n i s not v a l i d . 153 B. Examining Type Specimens, ft re-examination of a v a i l a b l e type specimens of North American Haematoloechus spp. i n d i c a t e d some e r r o r s i n e a r l i e r o b s e r v a t i o n s . The absence of s p i n e s reported f o r s i x s p e c i e s o f Haematoloechus has been used t o he l p separate these s p e c i e s from those f l u k e s which bear s p i n e s . I n g l e s (1932) re p o r t e d t h a t H. o x y o r c h i s d i f f e r e d from H. confusus i n being s p i n e l e s s . S i m i l a r l y , the absence o f s p i n e s i n H . f l o e d a e was one c h a r a c t e r used by Harwood (1932) t o d i s t i n g u i s h t h i s s p e c i e s from H. p a r v i p l e x u s . Examination of the type specimens of a l l the above f o u r s p e c i e s demonstrated that a l l had s p i n e s . • • H. k e r n e n s i s - • was d e s c r i b e d by I n g l e s (1932) as having no s p i n e s . T h i s f e a t u r e was used t o help separate that s p e c i e s from H. tumidus (described i n the same paper), which had s p i n e s . , H.; iuniplexus was a l s o d e s c r i b e d as being s p i n e l e s s (Harwood, 1932), but Harwood d i d not use t h i s c h a r a c t e r t o separate i t from other Haematoloechus spp. Be—examining the type specimens of if. k e r n e n s i s . fl. tumidus and fl. f l o e d a e de mo ns t r a t e d t h a t a l l were spined. The type specimen of H. complexus was not a v a i l a b l e f o r examination. T h i s s p e c i e s was d e s c r i b e d by Seely (1906) as being without s p i n e s . Seely s t a t e d t h a t " t h i s may have been due, however, to maceration," p. 249. I have shown e x p e r i m e n t a l l y t h a t f l u k e s l o s e t h e i r s p i n e s when t r e a t e d i n d i s t i l l e d water. Furthermore, I have shown that a c e r t a i n p r o p o r t i o n of f l u k e s of each s p e c i e s c o l l e c t e d i n the f i e l d , i n c l u d i n g H. complexus r do not have s p i n e s . ; 154 Because of the e r r o r s made i n r e p o r t i n g s p i n e s , the experimental r e s u l t s demonstrating the l o s s of s p i n e s d u r i n g p r e — f i x i n g treatment, and the v a r i a t i o n found i n f i e l d o b s e r v a t i o n s , t h i s c h a r a c t e r cannot be c o n s i d e r e d r e l i a b l e f o r s p e c i e s s e p a r a t i o n i n t h i s genus. The shape of t e s t e s and o v a r i e s has a l s o been used t o separate s p e c i e s i n t h i s genus. I n g l e s (1932) used these f e a t u r e s to help s e p a r a t e H.ykernensis from H. tumidns. The ovary i s lobed and the t e s t e s are unlobed i n fi. tumidus I n g l e s , 1932. The shape of the t e s t e s was a l s o used by I n g l e s (1932) t o separate H. confusus from H. o x y o r c h i s . Examination of t h e type specimens of B. confusus and comparison with H. o x y o r c h i s i n d i c a t e d t h a t the shape of the t e s t e s o f the two s p e c i e s was not d i f f e r e n t ( F i g s . 73 and 72). The, presence or absence of l o b i n g of o v a r i e s and t e s t e s has been shown, i n t h i s t h e s i s , t o be h i g h l y v a r i a b l e f o r s e v e r a l other members o f t h i s genus. I n g l e s ' (1936) drawing o f H. b u t t e n s i s showed t h a t the l e f t e x t r a c a e c a l u t e r i n e l o o p extended 1/3 the way along the l e n g t h of the p o s t e r i o r t e s t i s , and the r i g h t loop d i d not reach the p o s t e r i o r t e s t i s . Examination of the type specimen i n d i c a t e d t h a t both e x t r a c a e c a l loops j u s t r e a c h the l e v e l of the p o s t e r i o r t e s t i s . Examination of the type specimen of fi. f l o e d a e demonstrated t h a t the l e f t e x t r a c a e c a l l o o p extended o n l y a d i s t a n c e 1/2 way along the a n t e r i o r t e s t i s . The r i g h t loop extended half-way along the p o s t e r i o r t e s t i s . Harwood's drawing of the type specimen shows both l o o p s extending a d i s t a n c e 1/2 way along the ovary. I t i s very important to be c o r r e c t i n 155 r e p o r t i n g the v a r i a t i o n i n t h i s s e parate s e v e r a l s p e c i e s of v a r i a t i o n i n t h i s c h a r a c t e r Haematoloechus sp. c h a r a c t e r , as i t i s used to help Haematoloechus. The range o f has not been repo r t e d f o r many 156 PABT I I I . TAXONOHIC DISCUSSION Haematoloechus Looss, 1899, one of the most commonly encountered genera of f r o g trematodes, has been repo r t e d from p r a c t i c a l l y every c o n t i n e n t . Although Looss named the genus i n 1899, he renamed i t Pneumonoeces i n 1902. T h i s was done because the genus Haematoloecha had been given to an hemipteran by St'al i n 1874. Harwood(1932), and I n g l e s (1932) independently p o i n t e d out t h a t the name f i r s t chosen by Looss f o r t h i s genus was not i n v a l i d a t e d , and should stand i n accordance with t h e I n t e r n a t i o n a l Code o f Z o o l o g i c a l N omenclature.However, some authors, notably S k r j a b i n and A n t i p i n (1962), r e t a i n e d FjJSJlsnc-eces. A d d i t i o n a l g e n e r i c names have been a p p l i e d t o trematodes o f t h i s group. Ward (1917) accepted the genus Pneumonoeces but s t a t e d that one group iof f l u k e s , i n t h i s genus, should be separated as a new genus, to which he a p p l i e d the name Pneumobiteg and gave Pneumobftes l o n g i g l e x us ( Haematoloechus l o n g i p l e x u s of S t a f f o r d , 1902) as the type of the genus. Ward c h a r a c t e r i z e d t h i s genus as having elongate l a t e r a l and n e a r l y symmetrical t e s t e s , and lobed ovary - i n c o n t r a s t to the round, median t e s t e s and e n t i r e ovary o f Pneumonoeces. E x t r a c a e c a l l o n g i t u d i n a l f o l d s of the uterus are more pronounced i n Pneumpbites. r e a c h i n g n e a r l y the length of the body. Ward i n c l u d e d Pngumobjtes b r e v i p l e x u s { H. b r e v i p l e x u s of S t a f f o r d , 1902) i n t h i s new genus. In my t h e s i s I have demonstrated that the g e o g r a p h i c a l v a r i a t i o n i n extent o f e x t r a c a e c a l l o o p s , and o r i e n t a t i o n o f the t e s t e s i s co n s t a n t f o r t h e above two s p e c i e s , r e g a r d l e s s of 157 c o l l e c t i n g l o c a l i t y . However, the lobed nature of the ovary and t e s t e s i s h i g h l y v a r i a b l e . Mayr (1969) s t a t e s t h a t **a genus i s a taxonomic c a t e g o r y c o n t a i n i n g a s i n g l e s p e c i e s , or a monophyletic group of s p e c i e s , which i s separated frem other t a x a of the same rank (other genera) by a decided gap". Mayr (1969) recommended t h a t the s i z e of the gap be i n i n v e r s e r a t i o to the s i z e o f the taxon., The soundest genera are based on an o v e r a l l a p p r e c i a t i o n of the members of the taxon under c o n s i d e r a t i o n (Mitcherier, 1957) and i d e a l l y should be based on the occurrence of c o r r e l a t e d c h a r a c t e r complexes (Mayr, 1969) To e r e c t new genera based on incomplete knowledge of a few r e p r e s e n t a t i v e s of the world fauna c u r r e n t l y r e c o g n i z e d under the genus Haematoloechus i s c l e a r l y unwise. V a r i a t i o n s i n e x t e n t of e x t r a c a e c a l l o o p s , and o r i e n t a t i o n of t e s t e s are not known f o r many European s p e c i e s . . The c h a r a c t e r s proposed by Ward (1917) to c h a r a c t e r i z e the genus Pneumobites are present i n d i f f e r e n t combinations i n some European s p e c i e s . For example. H. s i b e r i c u s has round t e s t e s but a l s o elongate e x t r a c a e c a l l o o p s , which extend a t l e a s t as f a r as i s found i n H. b r e y i p l e x u s . which Ward i n c l u d e d i n - Pneumobites; S i m i l a r l y , there are s p e c i e s i n which the t e s t e s are p a r a l l e l , but round and smooth { H. rnanchanqensis r e p o r t e d from Japan).» T h e r e f o r e , I f e e l t h a t t h e genus Pneumobites sh c u i d be r e j e c t e d , and Haematoloechus r e t a i n e d . The extent o f the e x t r a c a e c a l loops and the shape and o r i e n t a t i o n of t e s t e s serve to s e p a r a t e H. l o n g i p l e x u s and H . i b r e y i p l e x u s from one-another as w e l l as from a l l other North 158 American r e p r e s e n t a t i v e s of Haemtaoloechus. Because of the c o n s i s t e n t c h a r a c t e r d i f f e r e n c e s , I propose that these two s p e c i e s remain as v a l i d s p e c i e s . ; N a t u r a l i n f e c t i o n s of H. l c n g i p l e x u s have most f r e g u e n t l y been d e s c r i b e d from a. c a t e s b e i a n a . but have been recorded from s i x s p e c i e s of Ranidae and one o f Bufonidae. B. .breviplexus has a l s o been r e p o r t e d from R. c a t e s b e i a n a . but occurs i n two other r a n i d s and one b u f o n i d . The above f l u k e s have been r e p o r t e d only from North America and have not so f a r been r e p o r t e d north of the 50ON p a r a l l e l or south of the 28°N p a r a l l e l . The geographic d i s t r i b u t i o n of these s p e c i e s f o l l o w s c l o s e l y t h a t d e s c r i b e d f o r R. c a t e s b e i a n a . The occurrence of these f l u k e s i n R. .catesbeiana and o c c a s i o n a l l y i n 8. c l a m i t a n s may be r e l a t e d t o d i f f e r e n c e s i n s e a s o n a l r e p r o d u c t i v e timing of the host, or food p r e f e r e n c e s at the time when i n f e c t e d odonates are a v a i l a b l e to f r o g s . T h e r e f o r e , host s p e c i f i c i t y of lj^3ifiiS3La§ f°r i . c a t e s b e i a n a , as suggested by r e p o r t e d f i e l d o b s e r v a t i o n s , may be a r e s u l t of e c o l o g i c a l f a c t o r s . H a b i t a t p r e f e r e n c e s of R.i c a t e s b e i a n a and R. c l a m i t a n s were demonstrated by Stewart and Sanderson (1972). The d i f f e r e n t p r e f e r e n c e s r e s u l t e d i n 8. c a t e s b e i a n a t a k i n g Coenagrionidae (Odonata) as a food item, whereas R. c l a m i t a n s d i d not consume any odonates. Cstiolum P r a t t , 1903 was d i f f e r e n t i a t e d from Haematoloechus Looss, 1899 only on the absence of e x t r a c a e c a l l o o p s i n the former and the presence of one or two loops i n the l a t e r . Odening (1960a) and S k r j a b i n (1964) have a l s o accepted t h i s genus as being v a l i d . I f t h i s genus i s accepted, on the b a s i s o f t h i s c h a r a c t e r d e s i g n a t i o n , two genera would be r e c o g n i z e d : 159 Ostlolum P r a t t , 1903, and Haematoloechus Looss. 1899. .The genus Q§ticlui would i n c l u d e f i v e Hcrth American s p e c i e s p r e s e n t l y r e c o g n i z e d i n the genus Baematolbechus : H. medioplexus, H. complexus. H. :Colgradfe:nsis,- ]|.v,pxiyo-r<;fais. and H. conf usus. A s i n g l e c h a r a c t e r may j u s t i f y the c r e a t i o n Of a new s p e c i e s but i t cannot be the s o l e c r i t e r i o n f o r the e r e c t i o n o f a new genus ( V e r s t e r , 1969}.,If the above c r i t e r i o n was f o l l o w e d i t would be necessary t o e r e c t a t l e a s t f o u r new genera: one to accommodate H. u n i p l e x u s . i n which t h e r e i s a s i n g l e e x t r a c a e c a l u t e r i n e loop; one t o accommodate s p e c i e s with egg lengths g r e a t e r than 0.058 mm; one t o accommodate s p e c i e s with v e n t r a l suckers egual to o r l a r g e r than t h e o r a l sucker; and one to accommodate s p e c i e s with round or o v a l t e s t e s . I have shown, e x p e r i m e n t a l l y , and by a n a l y s i s of f i e l d c o l l e c t i o n s , t h a t v a r i a t i o n s w i t h i n the genus may r e s u l t from d i f f e r e n c e s i n age and degree o f maturity, extent of crowding, s p e c i e s of host, and other f a c t o r s . Because of these v a r i a t i o n s , I f e e l t h a t the genus O s t i g l n a should not be accepted, and t h a t the genus Haematoloechus should c o n t a i n the above f i v e s p e c i e s . I r e c o g n i z e o n l y two of the above f i v e s p e c i e s as being v a l i d . I c o n s i d e r H. medioplexus v a l i d . T h i s s p e c i e s can be d i s t i n g u i s h e d from the other f o u r s p e c i e s of the group by i t s 0/A r a t i o , which remained constant r e g a r d l e s s o f c o l l e c t i n g l o c a l i t y . T h i s r a t i o can serve to separate H. medioplexus from a l l other Haematoloechus sp. which do not c o n t a i n e x t r a c a e c a l u t e r i n e l o o p s . fl. flgdjoplexus occurs p r i m a r i l y in---R^vpi:piens, but has been recorded i n f r e g u e n t l y from seven other s p e c i e s o f Ranidae and 160 onespecies of Bufonidae. My r e c o r d s show that H. medioplexus has not been recorded above the 50°N p a r a l l e l or below the 40<>H p a r a l l e l , or west of the Cascade mountains. The l i f e - c y c l e of 8. ;,medioplexus may have developed by a d a p t a t i o n to the e a r l y breeding season of R« p i p i e n s . T h e r e f o r e , metacercariae o f H.medioplexus may be a v a i l a b l e t o j | . : v i i 2 i e j s a t a time when i t i s u s i n g the pond most h e a v i l y and may not be a v a i l a b l e t o other f r o g s using t h e pond l a t e r i n t h e season. R. p i p i e n s may be d i s p l a c e d from the ponds when other f r o g s are using the area. T h i s displacement may r e s u l t i n a s h i f t i n food items taken by i * t ^ E i B t o s . , , >-fUvcom plexus/-'is the other s p e c i e s i n t h i s group which I c o n s i d e r v a l i d . 1 . o x y o r c h i s , g. con,fUsus and H. c o l o r a d e n s i s are here c o n s i d e r e d synonyms of H.. cqmplexus. Egg s i z e , and l o b a t i o n o f t e s t e s and ovary, as w e l l as absence o f s p i n e s i n H. , complexus a nd H. o x y o r c h i s were the main f e a t u r e s p r e v i o u s l y used to s e p a r a t e these s p e c i e s from one another. The range o f egg s i z e s of H. c o l o r a d e n s i s and H. complexus d i d not vary g e o g r a p h i c a l l y . However, egg l e n g t h and width of these two s p e c i e s o v e r l a p completely, and are t h e r e f o r e u n r e l i a b l e f o r s e p a r a t i n g them. The egg s i z e s of H. o x y o r c h i s and B. confusus o v e r l a p completely with each other and with the lower l i m i t o f egg l e n g t h of H. complexus. I have shown t h a t egg s i z e v a r i e s i n fl. b u t t e n s i s when t h i s f l u k e i s developed i n d i f f e r e n t f r o g h o s t s . I have a l s o shown t h a t egg l e n g t h v a r i e s g e o g r a p h i c a l l y f o r s e v e r a l other members of t h i s genus. S e p a r a t i n g s p e c i e s on the b a s i s of egg s i z e alone i s t h e r e f o r e very u n r e l i a b l e . 1.61 Lo b a t i o n of ovary and t e s t e s i s a l s o an u n r e l i a b l e c h a r a c t e r to use f o r s e p a r a t i n g s p e c i e s i n t h i s genus. I have demonstrated e x p e r i m e n t a l l y t h a t l o b i n g i n these s t r u c t u r e s i s dependent, i n p a r t , on the age of the worm. Furthermore, f i e l d c o l l e c t i o n s Of H. complexus and H. c o l o r a d e n s i s ha ve shown that l o b a t i o n may or may not be p r e s e n t . T h i s c h a r a c t e r i s t h e r e f o r e unusable f o r s e p a r a t i n g s p e c i e s . For the above reasons, H. complexus, H. o x y o r c h i s . H. confusus and H. c o l o r a d e n s i s are con s i d e r e d synonyms, fly,-jCOg'PleMS (Seely, 1906) i s the v a l i d name. ;H. complexus uses •• R. p j p j e n s .• most f r e q u e n t l y as i t s d e f i n i t i v e h o s t , as does •H.\,'Medioplex:us^!^!fl.";.,cofflpiex^s may even have, a r i s e n from H. medioplexus. A l l f l u k e s designated by me as H. complexus occur between the 30° N and 40° N p a r a l l e l s . §• 9.9%QMadensis- has been r e p o r t e d from v a r i o u s l o c a l i t i e s i n Utah, Colorado, Idaho and Nebraska. Two sp e c i e s (H. o x y o r c h i s and H. complexus) reco r d e d from the west c o a s t by I n g l e s (1932a) may have been d e r i v e d from what Co r t (1915) has c a l l e d H.; c o l o r a d e n s i s . "A L i t t l e I c e Age" f o l l o w e d some time a f t e r the c l o s e of the Wisconsin. Dumas (1966)believed the c o o l e r , moister c l i m a t i c c o n d i t i o n s o f t h i s age permitted an i n v a s i o n a c r o s s the low passes of the Rockies of the e a s t e r n pond f r o g R. p j p j e n s . I t i s du r i n g t h i s time t h a t R. Pijsiens may have passed on t o R. aurora the lung f l u k e , • -H'..-;celoradensi;s, and what I n g l e s (1932) has c a l l e d fl. .oxyor-c-fcis; - and H. cpnfu^us. These l a t t e r lung f l u k e s are m o r p h o l o g i c a l l y very s i m i l a r t o H. c o l o r a d e n s i s . and the d i s t r i b u t i o n of H. c o l o r a d e n s i s i n R. p i p i e n s suggests t h i s may be the stock which s u p p l i e d I n g l e s with h i s f l u k e s . 162 I n g l e s (1936) s t a t e s t h a t H. tumidas i s always found i n f r o g s which i n h a b i t streams and never i n f r o g s from ponds. The p o n d - i n h a b i t i n g f r o g s are always i n f e c t e d with H. o x y o r c h i s . I f my t r a n s f e r theory i s c o r r e c t , then fi. o x y o r c h i s may have been t r a n s f e r r e d from R. p i p i e n s t o Sana aurora d r a y t o n i and adapted t o a pond-type l i f e c y c l e . H. tumidus may be the o r i g i n a l lung f l u k e Rana aurora d r a y t o n i of the stream type. What was o r i g i n a l l y d e s c r i b e d as H. complexus (Seely, 1906) may a l s o have a r i s e n from fi. c o l o r a d e n s i s but i s now using R. , b l a i r i • and R. u t r i c u l a r i a (both p r e v i o u s l y c o n s i d e r e d !• E i i i l i s ) as i t s amphibian host., Odening (1960a) i n c l u d e d i n the genus Haematoloechus a l l those f r o g lung f l u k e s which contained e x t r a c a e c a l l o o p s . . F l u k e s without e x t r a c a e c a l l o o p s were i n c l u d e d i n t h e genus Ostjclum. Odening d i d not accept Pn en mg b i t e s Ward, 1917 as being v a l i d , i n a p r e v i o u s paper odening (1958) i n c l u d e d a l l f r o g l u n g - f l u k e s i n ; t h e genus Haematoloechus. The v a l i d i t y of t h e s p e c i e s which have e x t r a c a e c a l loops i s d i s c u s s e d • below. r *•• R . v l o n f r i p i e f f i s * and I . b r e v i p l e x u s have been d i s c u s s e d e a r l i e r and w i l l not be i n c l u d e d here. i B« JS^rnensis and §. t u i i d j j s are separated cn the b a s i s of absence of s p i n e s i n H. k e r n e a s i s * - d i f f e r e n c e s i n O/P r a t i o , and shape of ovary and t e s t e s . I have pointed out e a r l i e r , i n the d i s c u s s i o n on examining type specimens, t h a t the O/P r a t i o s are the same f o r both s p e c i e s , and t h a t spines are present on H. k e r n e n s i s . The presence or absence of l o b i n g of o v a r i e s and t e s t e s has been shown, i n t h i s t h e s i s , to be h i g h l y v a r i a b l e f o r s e v e r a l other members o f t h i s genus. T h e r e f o r e , I recommend t h a t 163 H. tumidus be c o n s i d e r e d a synonym of H. k e r n e n s i s . The type d e s c r i p t i o n of H. k e r n e n s i s should be amended t o read "tegument with s p i n e s " . Some of the R. aurora c o l l e c t e d from Kern County, C a l i f o r n i a by I n g l e s (1932, 1936) contained H. k e r n e n s i s . T h i s f l u k e has never been c o l l e c t e d o u t s i d e t h a t v i c i n i t y nor h a s i t ever been r e p o r t e d from any other amphibian host. H. tumidus. a l s o from R. aurora was r e p o r t e d by I n g l e s (1932, 1936) from Kern County north to the San F r a n c i s c o Bay r e g i o n . I t i s always found i n f r o g s which i n h a b i t streams and never i n f r o g s which i n h a b i t ponds ( I n g l e s , 1936). The f o l l o w i n g f i v e s p e c i e s , which a l s o c o n t a i n e x t r a c a e c a l l o o p s , are c o n s i d e r e d synonyms of H. v a r i o p l e x u s . They are: - H s i f f i i i i plexus r< H. ,: p a r v i p l e x u s • • • H. b u t t e n s i s . ft. ioed.ae - an d B. u n i p l e x n s . These s p e c i e s are c h a r a c t e r i z e d as having e x t r a c a e c a l loops t h a t do not extend to the a n t e r i o r margin of the, a n t e r i o r t e s t i s . T h i s group d i f f e r s from H. k e r n e n s i s i n having O/fi r a t i o s u s u a l l y g r e a t e r than 1 . 1 : KO. r :H nni plexa y • was • p r e v i o u s l y separated from the other f i v e s p e c i e s by having only one e x t r a c a e c a l loop. I have demonstrated that as much as 8 . 5 % of the specimens i n t h r e e of the s p e c i e s i n the H. v a r i o p l e x u s group a l s o had only one e x t r a c a e c a l loop. The extent of the e x t r a c a e c a l u t e r i n e loops has been important i n s e p a r a t i n g H. p a r v i p l e x u s , •• -•H.-'U'but-t-enslsr and H. f l o e d a e from one another as w e l l as f r o w fl. v a r i o p l e x u s . I have demonstrated, e x p e r i m e n t a l l y , that the e x t r a c a e c a l u t e r i n e f o l d s were i n p a r t host-dependent f o r H. b u t t e n s i s . Examination of H. b u t t e n s i s from s e v e r a l l o c a l i t i e s i n B.C., has shown 164 f u r t h e r t h a t the u t e r i n e f o l d s may extend as f a r as the a n t e r i o r border of the p o s t e r i o r t e s t i s or as l i t t l e as 1/3 the d i s t a n c e from the end of the worm t o the p o s t e r i o r t e s t i s . I have a l s o shown a c o n s i d e r a b l e v a r i a t i o n i n t h i s c h a r a c t e r f o r H. v a r i o p l e x u s and H. p a r v i p l e x u s . The d e s c r i p t i o n s of the extent of e x t r a c a e c a l loops i n H. s i m i l i p l e x u s and H. f l o e d a e l i e iftithin t h i s range of v a r i a t i o n . I t thus becomes i m p o s s i b l e to separate these s p e c i e s u s i n g t h i s c h a r a c t e r . . Egg s i z e has been used t o s e p a r a t e s i m i l i p l e x u s from H« v a r i o p l e x u s ; H. b u t t e n s i s from H. s i m i l i p l e x u s and H. f l o e d a e from H. p a r v i p l e x u s . Egg s i z e i n some s p e c i e s can vary c o n s i d e r a b l y . The egg s i z e> of H * p a r v i piexu s v a r i e d with host as well as v a r y i n g geog ra p h l e a l 1 y . The egg l e n g t h s of H. v a r i o p l e x u s and H. s i m i l i p l e x u s v a r i e d with host. Species of Haematoloechus occur, under n a t u r a l c o n d i t i o n s , i n a v a r i e t y o f amphibian hosts (see Appendix 23 f o r a l i s t o f known d e f i n i t i v e h o s t s ) . A wider range of p o t e n t i a l hosts has been demonstrated e x p e r i m e n t a l l y f o r R. b u t t e n s i s i n t h i s t h e s i s than has been found under n a t u r a l c o n d i t i o n s i n B r i t i s h Columbia, y I n d i v i d u a l s p e c i e s may d i f f e r from each ot h e r through s l i g h t d i f f e r e n c e s i n s i z e , arrangement and l o c a t i o n of v a r i o u s s t r u c t u r e s . Stunkard (1965) s t a t e s t h a t : "the problem of the taxonoraist then i s t o d i s t i n g u i s h between v a r i a t i o n s w i t h i n a p a r t i c u l a r design and between d i f f e r e n t d e s i g n s . " He went on t o s t a t e t h a t " c o n s i d e r a t i o n must be given to the p o s s i b i l i t y t h a t r e p r e s e n t a t i v e s of a s i n g l e s p e c i e s may complete t h e i r 165 development i n d i f f e r e n t h o s t - s p e c i e s and furthermore t h a t , as a r e s u l t of development i n d i f f e r e n t hosts, i n d i v i d u a l s of the same s p e c i e s may manifest d i f f e r e n c e s i n s i z e and shape, i n r a t e of growth and s e x u a l m a t u r i t y , and i n extent of development of v a r i o u s t i s s u e s and organs." Dronen (1975, 1977) noted t h a t a l l odonates tested,-- ftnax sp. ,. ; L l b e l l a - sp. , Tr amea sp., and Enallagma sp. , became i n f e c t e d with metacercariae o f H. c o l o r a d e n s i s , and c o u l d serve as second i n t e r m e d i a t e h o s t s . S c h e l l (1965) used the d r a g o n f l y , fteschna m u l t i c o l o r flagen, 1861 as an experimental host f o r fl.breviplexus; Dronen (1977) used L j b e l l u l a - sp. In d i f f e r e n t areas d i f f e r e n t odonate s p e c i e s may be p r e f e r r e d as second i n t e r m e d i a t e hosts. Dronen (Personal Communications) noted t h a t the c e r c a r i a e of ^ H* b r e v i p l e x u s were l a r g e r i n the s n a i l , F e r r i s s i a . which he used as f i r s t i n t e r m e d i a t e host than i n Gvraulus s i m i l a r i s (Baker, 1919) used by S c h e l l (196 5). Species of ga^§atolqechus can and do u t i l i z e a wide range of h o s t s . T h e r e f o r e , i t i s not a d v i s a b l e t o use h o s t - s p e c i f i c i t y as the s o l e c r i t e r i o n f o r d i a g n o s i n g s p e c i e s . Stuhkard (1957) s t a t e s " i t i s abundantly c l e a r t h a t flatworm p a r a s i t e s are a b l e to a c q u i r e ne» h o s t s , and t h a t they change hosts with d i f f e r i n g e c o l o g i c a l s i t u a t i o n s . I t i s e g u a l l y c l e a r t h a t development i n d i f f e r e n t h o s t - s p e c i e s and under d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s of the i n d i v i d u a l host may profoundly a l t e r the p a r a s i t e . In many i n s t a n c e s , v a r i e t i e s o r r a c e s p e c u l i a r to c e r t a i n h o s t s , h o s t a l v a r i e t i e s , may be r e c o g n i z e d , but t h e r e i s no sound reason to b e l i e v e t h a t they 166 r e p r e s e n t d i f f e r e n t s p e c i e s " . Forms showing marked host s p e c i f i c i t y c o u l d be c o n s i d e r e d s u b s p e c i e s r a t h e r than s p e c i e s . In view of the demonstrated v a r i a b i l i t y i n c h a r a c t e r s used to separate the above s i x s p e c i e s I recommend t h a t they become synonyms, and t h a t H. v a r i o p l e x u s be the v a l i d name. In t h i s t h e s i s I have recommended r e c o g n i z i n g o n l y s i x s p e c i e s of Haematoloechus from Canada and the United S t a t e s as being v a l i d . They are as f o l l o w s : a..,^-l-onqlpl¥xqis' S t a f f o r d , 1902 H. b r e v i p i e x u s S t a f f o r d , 1902 H. v a r i o p l e x u s S t a f f o r d , 1902 (= H. ..parviplexys. = H. b u t t e n s i s , - H. s i p i l i p l e x u s , = H. f l o e d a e . - H. u n i p l e x u s I H.^kernensis i n g l e s , 193 2 (= H. tumidus ) H. medioplexus S t a f f o r d , 1902 H. complex us (Seely, 1906) (= H. cq3.Qradensi§, ~ H. conf usus. = !• o x y o r c h i s ). The above f i n d i n g s suggest s e v e r a l p o s s i b i l i t i e s i n regard t o r e l a t i o n s h i p s between s p e c i a t i o n and d i s p e r s a l of the host, and the lung f l u k e s they c o n t a i n . These i d e a s are s t i l l h i g h l y s p e c u l a t i v e and incomplete. However, they do add support t o c u r r e n t t h e o r i e s of s p e c i a t i o n i n some North American Ranidae, and help to c l a r i f y some as p e c t s of haematoloechid s y s t e m a t i c s i n North America. For these reasons I have o u t l i n e d my ideas below. , (' \ • Current s p e c i e s d i s t r i b u t i o n i n North America i s a t t r i b u t e d to P l e i s t o c e n e and Recent c l i m a t i c changes ( P o r t e r , 1972). The southeastern United S t a t e s has been the najor c e n t e r of d i s t r i b u t i o n , for^) North American Ranidae ( P o r t e r , 1972) . 167 Leopard f r o g s from North America are comprised of numerous separate s p e c i e s (Pace, 1974). f o u r o f these (Rana p i p i e n s . B. u t r i c u l a r i a , £. b e r l a n d i e r i . and R . b l a i r i ) were at one time c o n s i d e r e d a s i n g l e s p e c i e s , b e l o n g i n g t c the R. p i p i e n s Complex. Pace (1974) d i s t i n g u i s h e d the above four s p e c i e s by c e r t a i n m o r p h o l o g i c a l , b i o c h e m i c a l , and v o c a l c h a r a c t e r i s t i c s . The g e o g r a p h i c a l ranges o f these s p e c i e s are i n g e n e r a l mutually e x c l u s i v e with c o n t i g u o u s boundaries, and are d e p i c t e d i n Pace (1974). Pace (1974) suggested that F l o r i d a p o p u l a t i o n s of R. u t r i c u l a r i a . and Texas p o p u l a t i o n s of J« b e r l a n d i e r i are d e r i v e d from the same a n c e s t r a l s t o c k . The Texas p o p u l a t i o n (R. b e r l a n d i e r i ) , s p e c i a t e d from R. u t r i c u l a r i a but the F l o r i d a p o p u l a t i o n d i d • not.../ R. u t r i c u l a r i a may have invaded from the south, meeting R. p i p i e n s north of t h e i r present zone o f i n t e r a c t i o n (Pace, 1974) (Map 3). , : My concept of the H. complexus group (=H.coloradensis. =H. confusus, and -H. oxyorchis) f i t s , and supports t h i s i n t e r p r e t a t i o n . Lung f l u k e s from B. b e r l a n d i e r i are m o r p h o l o g i c a l l y , more s i m i l a r t o those found i n R. u t r i c u l a r i a than those recovered from R. b l a i r i or R. p i p i e n s ; ( F i g . 81). T h i s suggests a c l o s e r r e l a t i o n s h i p to f l u k e s from R. u t r i c u l a r i a . • I t i s not known where R . b l a i r i s u r v i v e d g l a c i a t i o n , i f i t was a d i s t i n c t s p e c i e s at t h a t time (Pace, 1974). I t s spread i n t o the midwest f o l l o w e d the post-Wisconsin expansion of the P r a i r i e P e n i n s u l a (Smith, 1957). The morphology o f lung f l u k e s from R. b l a i r i i s i n t e r m e d i a t e between those from the 1 6 8 southwestern R. p i p i e n s and those from R. u t r i c u l a r i a . S i m i l a r i t i e s between t h e i r l u n g f l u k e s suggest a southwestern o r i g i n f o r B . , b l a i r i - . perhaps around A r i z o n a and New Mexico, a t which time R. b l a i r i may not have been d i s t i n c t . T h i s h y p o t h e s i s , while extremely tenuous, n e v e r t h e l e s s may account f o r the g r e a t e r s i m i l a r i t y of H. complexus from M B . - j - b l a i r i - t o H. complexus from R. p i p i e n s than t o lung f l u k e s from J • u t r i c u 1 a r i a. Where d i d the H. complexus <=H. o x y o r c h i s . =H. conf usus) > found i n R. aurora o r i g i n a t e ? I suggested e a r l i e r t h a t they arose from what was p r e v i o u s l y considered H. c o l o r a d e n s i s . , In many r e s p e c t s the f l u k e s from R. au r o r a , i n no r t h e r n C a l i f o r n i a , are s i m i l a r to f l u k e s (~ H. c o l o r a d e n s i s ) found i n southwestern R. p i p i e n s . T h i s suggests to me a l a t e r t r a n s f e r o f f l u k e s t o R. aurora from R. p i p i e n s . The a n c e s t r a l lung f l u k e was probably what was p r e v i o u s l y c o n s i d e r e d fl. c o l o r a d e n s i s . B. c l a m i t a n s and B. c a t e s b e i a n a are n a t i v e t o e a s t e r n United S t a t e s . The n a t u r a l western l i m i t s are u n c e r t a i n because of t h e i r i n t r o d u c t i o n i n t o many l o c a l i t i e s (Conant, 1958} (Map 4) . The above two s p e c i e s form a n a t u r a l group d i s t i n c t from other North American Ranidae {Wallace, e t . a l . , 1973).wThese f r o g s c o n t a i n fl.\longiplexus and H. b r e v i p l e x u s which I have shown form a group g u i t e d i s t i n c t from other North American haematoloechids. H. l o n q i p l e x u s and H. b r e v i p l e x u s e x h i b i t very l i t t l e m o rphological v a r i a t i o n r e g a r d l e s s o f c o l l e c t i n g l o c a l i t y . I have i n s u f f i c i e n t data t o warrant a good hypothesis concerning the spread of the H. v a r i o p l e x u s group recognized i n 169 t h i s t h e s i s . Hore specimens are needed from many l o c a l i t i e s before any p r e d i c t i o n s can be made. 170 Hap, 3. The d i s t r i b u t i o n of the Sana p i p i e n s Complex ( a f t e r Pace, 1974) and t h e i r lung f l u k e s i n North america. R. blairi 171 Hap 4. The d i s t r i b u t i o n o f R. catesbeiana and •R. c l a m i t a n s ( a f t e r Conant, 1958) and t h e i r lung f l u k e s i n North america. 172 F i g . 81 A proposed d e r i v a t i o n o f the body types which c o n s t i t u t e H. complexus i - H * o x y o r c h i s , =H.^  c o n f usus. =H. c o l o r a d e n s i s ) . H. o x y o r c h l a H. conf U I U I R. aurora R. aurora 173 ft KEY TO THE H ft E l f ftTOL 0 ECHO S 5P. IX CANADA AND THE ONITED STATES 1 E x t r a c a e c a l u t e r i n e l o o p s present ........2 1' E x t r a c a e c a l u t e r i n e loops--absent--"..?f:Vvw-*VV'5 2(1) E x t r a c a e c a l u t e r i n e loops extending beyond a n t e r i o r t e s t i s ........................ .3 2* E x t r a c a e c a l u t e r i n e loops do not reach 3(2) T e s t e s p a r a l l e l or ne a r l y p a r a l l e l ; e x t r a c a e c a l u t e r i n e l o o p s extending beyond ovary 3* Testes o v e r l a p 1/3 to 1/2 t h e i r l e n g t h ; e x t r a c a e c a l u t e r i n e loops extend to ovary H« :breviplexus 4(2») T e s t e s e l l i p t i c a l ; 0 / A r a t i o u s u a l l y g r e a t e r than 1.4:1.0; i f l e s s than 1.4:1.0 t e s t e s not round ....H. v a r i o p l e x u s 4» Testes round; 0/A r a t i o 0.8:1.0 to 1.1:1.0 5(1*) 0/A r a t i o g r e a t e r than 2.0r1.0 (2.6 t o 4.3:1.0) 5« 0/A r a t i o 2.0:1.0 or l e s s (1.2 to 2.o:1.0) 174 SUSMSB? 1. F l a t t e n i n g of worms durin g mounting caused great v a r i a t i o n i n s i z e and shape of the body, gonads, and suckers. Body s i z e i n c r e a s e d with i n c r e a s e d temperature of f i x a t i v e , and when d i s t i l l e d water was used t o assemble l i v e f l u k e s b e f o r e f i x a t i o n . Tegumental s p i n e s were l o s t d u r i n g t h i s p r e — f i x a t i v e treatment. The 0/A r a t i o was t h e onl y c h a r a c t e r measured t h a t d i d not change. 2. V a r i a t i o n i n fl. b u t t e n s i s r e s u l t e d from d i f f e r e n c e s i n age and degree of matu r i t y ; from extent of "crowding," from type of host and temperature a t which i t was maintained..The s i z e and shape of B. b u t t e n s i s was not a f f e c t e d by host s i z e or sex. 3. A d u l t fl. b u t t e n s i s were r e a r e d e x p e r i m e n t a l l y i n fo u r d i f f e r e n t s p e c i e s of f r o g s and t h r e e s p e c i e s of i n s e c t s . S i z e , shape, and p o s i t i o n of ovary and t e s t e s , body s i z e , and extent of v i t e l l a r i a were so v a r i a b l e t h a t they were o b v i o u s l y u n r e l i a b l e f o r s e p a r a t i n g s p e c i e s . S t a b l e c h a r a c t e r s i n c l u d e d the 0/A r a t i o , s p i n e d tegument, and t o some degree, extent of the:, e x t r a c a e c a l u t e r i n e l o o p s . a. H.> l o n q i p l e x u s and H. b r e v i p l e x u s c o l l e c t e d from v a r i o u s l o c a l i t i e s had two c h a r a c t e r s s u i t a b l e f o r s p e c i e s i d e n t i f i c a t i o n : e x t e n t o f e x t r a c a e c a l u t e r i n e l o o p s , and degree of o v e r l a p o f the a n t e r i o r and p o s t e r i o r t e s t e s . There was was no o v e r l a p i n these c h a r a c t e r s , which are t h e r e f o r e , c o n s i d e r e d good taxonomic f e a t u r e s . 5. Examination of specimens l a b e l l e d H. v a r i o p l e x u s or H s i f l i l i p l e x u s i n d i c a t e d t h a t c h a r a c t e r s p r e v i o u s l y used t o d i s t i n g u i s h these s p e c i e s overlapped so much t h a t i t was 175 i m p o s s i b l e t o l a k e a p o s i t i v e i d e n t i f i c a t i o n . , S p e c i m e n s sere t h e r e f o r e t r e a t e d a c c o r d i n g t o host. When t h i s was done I t was noted t h a t the 0/A r a t i o and egg s i z e v a r i e d with h o s t . 6. A n a l y s i s of the o/A r a t i o s f o r fi. p a r v i p l e x u s showed a s i g n i f i c a n t v a r i a t i o n between specimens from B.G. and those from Nebraska. Both a geographic and a host e f f e c t was i n d i c a t e d . As w e l l , egg length of worms from A t k i n s o n , Nebraska d i f f e r e d from those o f worms from B.C. or Humboldt, Nebraska. Other c h a r a c t e r s measured d i d not show s i g n i f i c a n t host or geographic v a r i a t i o n . 7. , Nine s p e c i e s have p r e v i o u s l y been d e s c r i b e d as c o n t a i n i n g p a i r e d e x t r a c a e c a l u t e r i n e l o o p s . However, as much as 9% of the specimens examined from f i e l d c o l l e c t i o n s c o n t a i n e d a s i n g l e e x t r a c a e c a l loop. T h i s suggested t h a t H« u n i p l e x u s , d e s c r i b e d from a s i n g l e specimens as having one e x t r a c a e c a l l o o p , i s not a v a l i d s p e c i e s . 8. Examination o f type specimens r e v e a l e d that some e r r o r s i n p u b l i s h e d d e s c r i p t i o n s had been made f o r fl. f l o e d a e . H.,^ernensis, H . : o x y o r c h i s . H. un i p l e x u s and fi. b u t t e n s i s . These f i n d i n g s , t o g e t h e r with c o n c l u s i o n s drawn from experiments on H. b u t t e n s i s and examination of c o l l e c t i o n s of other lung f l u k e s , l e d to t h e c o n c l u s i o n t h a t H. f l o e d a e , H. o x y o r c h i s . I . vSSiPiSJSS and H. b u t t e n s i s were not v a l i d s p e c i e s . 9. Harked m o r p h o l o g i c a l v a r i a t i o n and l a c k o f host s p e c i f i c i t y documented duri n g t h i s study i n d i c a t e d t h a t the f o l l o w i n g s i x s p e c i e s are v a l i d : H. l o n g i p l e x u s S t a f f o r d , 1902 H. b r e v i p l e x u s S t a f f o r d , 1902 176 H. v a r i o p l e x u s S t a f f o r d , 1902 (= H. s i m i l i p l e x u s . = H. b u t t e n s i s , • = H. p a r v i p l e x u s . => H. f j o e d a e . = H. gnipl e x u s ). , H. medioplexus S t a f f o r d . 1902 H. complexus (Seely, 1906) (= H. c o l o r a d e n s i s . = ••!!.••>/ conf usus-, =H. o x y o r c h i s ). 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I n t r a s p e c i f i c v a r i a t i o n of a d u l t T e l o r c h i s bonnerensis {Treaatoda: T e l c r c h i i d a e ) i n amphibian and r e p t i l i a n hosts. J . P a r a s i t o l . 53: 962-968. .Watertor,- J.L. 1968. E f f e c t s o f temperature s t r e s s on growth and development of l a r v a l and a d u l t T e l o r c h i s bonnerensis (Trematoda: T e l o r c h i i d a e ) . J. P a r a s i t o l . 54: 506-509. Wharton, G.W. 1940. The genera T e l o r c h i s . Protenes. and Auridistornum (Trematoda: R e n e f e r i d a e ) . J . P a r a s i t o l . 26: 497-518. Wharton, G.W. 1957. I n t r a s p e c i f i c v a r i a t i o n i n the p a r a s i t i c A c a r i n a . , Syst. Z o o l . 6: 24-28. W i l l e y , G.H. 1941. The l i f e h i s t o r y and bionomics o f the trematode, Zygocotyle l u n a t a IParamphistbrnidae). Z o o l o g i c a (N.Y.). 26: 65-887 Wohlgemuth, 1920. Die Witkung von Temperature Schwankungen auf d i e P a r a s i t e n der Pishkaut. C i t e d In B u l l . S t a t e S c i . Res. In s t y , Lake and R i v e r F i s h . 49: 124. Wrightj C.a. 1960. R e l a t i o n s h i p s between trematodes and molluscs. ann. Trop. Med. P a r a s i t o l . 5*1: 1-7. Yamaguti, S. 1971. Synopsis of Digenetic Trematodes of Vertebrates. Vol.1. Keigaku Pub. Co. Ltd. Tokyo, Japan. 1074 pp. Addendum Odening, K. 1964. Zur Taxionomie der Trematodenunterordnung Plagiorchiata. Monatsber Deut. Akad. Wess. B e r l i n . 6:191-193. (in German) 185 Appendix 1. Summary of measurements used. AC Diameter of Acetabulum: The g r e a t e s t a c e t a b u l a r diameter measured orthogonal t o the long a x i s of the worm. ATE A n t e r i o r T e s t i s from P o s t e r i o r End. The d i s t a n c e between the p o s t e r i o r end of the a n t e r i o r t e s t i s and the p o s t e r i o r end o f the worm. ATI, A n t e r i o r T e s t i s Length. G r e a t e s t length o f the a n t e r i o r t e s t i s measured along the lo n g a x i s of the worm. ATS A n t e r i o r T e s t i s Shape, Designated as e i t h e r smooth, m i l d l y l o b e d , o r h i g h l y l o b e d . ATW A n t e r i o r T e s t i s Width. G r e a t e s t width o f the a n t e r i o r t e s t i s measured ort h o g o n a l t o the l o n g a x i s of the worm.'. BL Body Length. G r e a t e s t body l e n g t h , BW Body Width. Measured a t the widest p a r t o f the worm. EL , Egg Length. The g r e a t e s t l e n g t h o f an egg. EW Egg Width. The g r e a t e s t width o f an egg. Egg measurements were made cn eggs found i n the most a n t e r i o r p o r t i o n o f the ute r u s ( F i g . 8«i). LVL Length of L e f t v i t e l l a r i a . Distance between the a n t e r i o r and p o s t e r i o r e x t e n t o f the v i t e l l a r i a , e x c l u s i v e of the c e n t r a l band o f v i t e l l a r i a which extends between the l e f t and r i g h t v i t e l l i n e a r e a s . 0/A O r a l sucker width d i v i d e d by acetabulum width. OAC Ovary from Acetabulum. The d i s t a n c e between the p o s t e r i o r border of the acetabulum and the a n t e r i o r border of the ovary. 186 OAT Ovary from A n t e r i o r T e s t i s . The d i s t a n c e between the p o s t e r i o r border o f t h e ovary and t h e a n t e r i o r margin j o f the a n t e r i o r t e s t i s ( F i g . 82). OL Ovary Length. Greatest ovary l e n g t h measured along the long a x i s o f the worm. OSH Ovary Shape. Designated as e i t h e r smooth, m i l d l y lobed or h i g h l y l o b e d . / OSL The Length o f the Or a l Sucker. The area a n t e r i o r to the o r a l sucker, when the sucker i s su b t e r m i n a l , i s hot i n c l u d e d ( F i g . 82). OSW Width of the O r a l Sucker. T h i s measurement i n c l u d e s only the o r a l sucker proper, and does not i n c l u d e the area l a t e r a l to the o r a l sucker ( F i g . 82) * OW; v Ovary Width. G r e a t e s t ovary width measured orthogonal to the lo n g a x i s o f the worm. PL Pharynx Length. I n c l u d e s the e n t i r e l e n g t h of the pharynx, even when the pharynx extends some d i s t a n c e i n t o t h e o r a l sucker ( F i g . 82). PTE P o s t e r i o r T e s t i s from P o s t e r i o r end. The d i s t a n c e between the p o s t e r i o r margin o f the p o s t e r i o r t e s t i s and the end , ., .,. of-;the worm. / PTL • P o s t e r i o r T e s t i s Length. Greatest l e n g t h o f the p o s t e r i o r t e s t i s ( F i g . 82). PTS P o s t e r i o r T e s t i s Shapes,Designated as e i t h e r smooth, m i l d l y lobed, o r h i g h l y l o b e d . PTW P o s t e r i o r t e s t i s width. G r e a t e s t width of the p o s t e r i o r t e s t i s measured o r t h o g o n a l t o the lo n g a x i s of the worm. PW . Pharynx Width. G r e a t e s t pharynx width. 187 BOP l e n g t h of Bight L o n g i t u d i n a l F o l d o f Uterus. The a n t e r i o r extent o f t h a t p a r t of the uterus which extends t o the o u t s i d e o f the l e f t i n t e s t i n a l caecum. The f o l d i s measured from i t s most p o s t e r i o r extent, BVL Length of Bight V i t e l l a r i a , Distance between the a n t e r i o r and p o s t e r i o r e x t e n t of the v i t e l l a r i a , e x c l u s i v e of ,the c e n t r a l band o f v i t e l l a r i a which extends between the r i g h t and l e f t v i t e l l i n e areas, TES S e p a r a t i o n of Te s t e s , T h i s i s t h e d i s t a n c e between the p o s t e r i o r end of the most a n t e r i o r t e s t i s i and the a n t e r i o r end o f the most p o s t e r i o r t e s t i s . TO Overlap of Testes, The v e r t i c a l d i s t a n c e between the te s t e s . , f t p o s i t i v e value i s giv e n when t e s t e s a c t u a l l y o v e r l a p with one another, ft negative v a l u e denotes t e s t e s separated by some d i s t a n c e . TT Tegument Thickness, . VA A n t e r i o r Extent of V i t e l l a r i a . Measured between the a n t e r i o r extent o f the v i t e l l a r i a and the p o s t e r i o r end of the worm, VE Distance o f V i t e l l a r i a from end o f worm. The d i s t a n c e between the p o s t e r i o r v i t e l l i n e l e v e l and the p o s t e r i o r end of the f l u k e . 188 F i g . 82. .Delineation of some worm measurements. The uterus has been-excluded f o r c l a r i t y . F i g . 83 D e l i n e a t i o n of some worm measurements. The t e s t i s , ovary and acetabulum have been omitted f o r c l a r i t y . 188A 189 Appendix 2. Formulae f o r f i x a t i v e s and s o l u t i o n s used i n experiments. AEA ( a l c o h o l - f o r m o l - a c e t i c ) f i x a t i v e A l c o h o l , B5*v**VwiUvv**W m l Formalin, commercial . 10 ml A c e t i c a c i d , g l a c i a l 5 ml Bouin's ( P i c r o - f o r m o l - a c e t i c ) f i x a t i v e P i c r i c a c i d , s a t u r a t e d aqueous s o l u t i o n ........75 ml • Formalin, commercial •.'vv'^r:r;,;vv.4:*..=.;..y-vv**vv*. 25 ml ... A c e t i c a c i d , g l a c i a l - . V v v * u ^ v v v ^ ml' For m a l i n , 10% Water, d i s t i l l e d .......90 ml Formalin, commercial 10 ml Frog S i n g e r ' s S o l u t i o n - KCl 0 . 14 gm • NaCl--.-*>v*V?«W*r*^**^ • 0.5O' gm . .CaCl2 -ii;-...v.v,vvvv^.vVW. . ,vy.v.;.>;r.^.;i^ 1 2 9m NaHC03 .. 0.20 gm T>istilUJ- <c/*«r |o0 ml Scbaudinn's f i x a t i v e Mercuric c h l o r i d e , s a t u r a t e d aqueous s o l . ......66 ml i • • • . . A l c o h o l , 9 5 1 T v v : * v » . y , r y « > ^ A c e t i c a c i d , g l a c i a l 3 ml Appendix 3. Flow sheet for preparing whole mounts. Fixative 1 35% \ Water I Harris' H a e m a t o x y l i n 1 Water 1 35% 1 50% 1 70% Acid Alcohol I 85% 1 95% I Methyl Benzoate Permount Standardized Procedure 191 Appendix 4 . Effect of flattening on character measurements of H . buttens is . Unflattened W e i g h t u s e d 5 grams 10 grams Body length 3. 05(2. 86--3. 60) 4.67(3. 85--4.91) 5.43(4.71--5. 88) Body width 0. 63(0. 55--0. 74) 0.95(0. 88--1.13 ) 1.51 (1.27--1. 69 ) Acetabulum diameter 0. 09(0. 08-•0. 09) 0.11 (0.10--0.12) 0.13(0.12- 0. 14) Oral sucker length 0. 21(0.19-•0. 23) 0. 24(0. 21-0. 27) 0. 27(0. 24-0. 30) Oral sucker width 0. 23(0. 20-•0. 25) 0.29(0. 26-•0. 31 ) 0. 35(0. 32-o. 38) O/A ratio 2. 6 (2. 6 -•2. 8 ) 2.6 (2.5 -•2.6 ) 2.7 Pharynx length 0. 13(0.12-•0. 15) 0.15(0.12--0.16 ) 0.18(0.15- •o. 20) Pharynx width 0. 15(0.13- 0. 16) 0. 22(0.19 -•0. 24) 0. 31(0. 27-•0. 33) Ovary length 0. 52(0.40-•0. 61) 0. 63(0. 59-0. 67) 0. 66(0. 61-•e. 76) Ovary width 0. 25(0.19- 0. 31) 0.35(0. 28-•0.44) 0.41(0.34-•o. 53) Ant. Testis length 0. 63(0. 57- 0. 74) 0.79(0. 68- 0. 85) 0. 83(0.75-•o. 93) Ant. testis width 0. 32(0. 26- 0. 40) 0.43(0.39- 0. 53) 0. 48(0.42-•o. 61) Post, testis length 0. 67(0.59- 0. 78) 0.84(0.79-• 1. 01) 0. 89(0.85- l . 07) Post, testis width. 0. 41(0. 35- 0. 55) 0.51(0. 45- 0. 66) 0. 57(0.50- 0. 75) A l l measurements i n m i l l i m e t e r s . Appendix 5. Average measurements of Haematoloechus buttensis of varying ages developed in Rana pretiosa. Metacercarla 5 days 14 days 21 days 2 8 days 60 days # recovered 10 24 18 20 21 17 Body length 0 . 6 8 ( 0 . 5 7 - 0 . 80) 0 . 9 9 ( 0 . 9 2 - 1.11 ) 1.75( 1 . 6 1 - 1 .98) 3 . 1 0 ( 2 . 4 3 - 3 . 6 1 ) 3 . 9 0 ( 3 . 5 0 - 4 . 59 ) 6 . 01 ( 5 . 5 8 - 6 . 6 7 ) Body width 0 . 2 9 ( 0 . 2 4 - 0 . 35) 0 . 3 4 (0 . 3 3 - 0 . 36) 0 . 5 1 ( 0 . 4 7 - 0 . 59) 0 . 6 8 ( 0 . 5 3 - 0 . 8 5 ) 0 . 8 2 ( 0 . 7 8 - 0 . 8 7 ) 1. 2 6 ( 1 . 1 2 - 1. 4 6 ) Acetabulum 0 . 0 9 ( 0 . 0 8 - 0 . 1 2 ) 0 . 0 5 ( 0 . 0 5 - 0 . 0 6 ) 0 . 0 7 ( 0 . 0 6 - 0 . 0 7 ) 0 . 0 8 ( 0 . 0 7 - 0 . 0 9 ) 0 . 1 0 ( 0 . 0 9 - 0 . 1 1 ) 0 .11 ( 0 . 0 9 - 0 . 1 2 ) Oral sucker 0 . 11 ( 0 . 1 0 - 0 . 1 3 ) 0 . 12 (0 .11 - 0 . 1 3 ) 0 . 1 6 ( 0 . 1 4 - 0 . 1 8 ) 0 . 2 0 ( 0 . 1 9 - 0 . 2 2 ) 0 . 2 4 ( 0 . 2 3 - 0 . 2 5 ) 0 . 2 5 ( 0 . 2 1 - 0 . 29) length Oral sucker 0 . 1 2 ( 0 . 0 9 - 0 . 1 5 ) 0 . 1 3 ( 0 . 1 2 - 0 . 1 3 ) 0 . 1 8 ( 0 . 1 7 - 0 . 2 0 ) 0 . 2 2 ( 0 . 1 9 - 0 . 24 ) 0 . 2 5 ( 0 . 2 4 - 0 . 26 ) 0 . 2 9 ( 0 . 2 5 - 0 . 32 ) width O/A ratio 1 .2 ( 1 . 0 - t. 3 ) 2 . 4 ( 2 . 2 - 2 . 4 ) 2 . 6 ( 2 . 5 - 2 . 7 ) 2 . 5 ( 2 . 1 - 2 . 7 ) 2 . 5 ( 2 . 4 - 2 . 6 ) 2 . 6 ( 2 . 4 - 2 . 8 ) Pharynx 0 . 0 6 ( 0 . 0 4 - 0 . 0 7 ) 0 . 0 8 ( 0 . 0 7 - 0 . 0 9 ) 0 . 1 2 ( 0 . 1 1 - 0 . 1 3 ) 0 . 1 3 ( 0 . 1 2 - 0 . 1 5 ) 0 . 1 5 ( 0 . 1 3 - 0 . 2 0 ) 0 . 1 9 ( 0 . 1 7 - 0 . 2 3 ) length Pharynx 0 . 0 6 ( 0 . 0 4 - 0 . 0 7 ) 0 . 1 0 ( 0 . 0 8 - 0 . 1 1 ) 0 . 1 3 ( 0 . 1 2 - 0 . 1 4 ) 0 . 1 5 ( 0 . 1 4 - 0 . 1 8 ) 0 . 1 7 ( 0 . 1 5 - 0 . 1 8 ) 0 . 2 0 ( 0 . 1 8 - 0 . 2 3 ) width Ovary length Absent 0.'j£;0.06-0.10 } 0 . 2 4 ( 0 . 2 0 - 0 . 3 2 ) 0 . 4 8 ( 0 . 3 3 - 0 . 5 3 ) 0 . 5 5 ( 0 . 5 0 - 0 . 6 2 ) 0 . 7 1 ( 0 . 7 1 - 0 . 7 4 ) Ovary width Absent 0 . 0 8 ( 0 . 0 7 - 0 . 0 9 ) 0 . 1 7 ( 0 . o 4 - 0 . 2 0 ) 0 . 2 3 ( 0 . 1 1 - 0 . 3 4 ) 0 . 2 8(0. 2 4 - 0 . 3 5 ) 0 . 3 6 ( 0 . 3 1 - 0 . 4 0 ) Ant. testis 0 . 0 3 ( 0 . 0 2 - 0 . 05) 0 . 13(0 .11 - 0 . 21) 0 . 3 4 ( 0 . 2 9 - 0 . 3 7 ) 0 . 6 2 ( 0 . 5 1 - 0 . 7 4 ) 0 . 6 0 ( 0 . 5 4 - 0 . 67 ) 1.12 ( 1 . 0 5 - 1. 21 ) length Ant. testis 0 . 0 3 ( 0 . 0 2 - 0 . 0 4 ) 0 . 0 9 ( 0 . 0 6 - 0 . 1 1 ) 0 . 2 2 ( 0 . 1 9 - 0 . 29) 0 . 3 4(0. 2 5 - 0 . 4 0 ) 0 . 4 2 ( 0 . 3 9 - 0 . 4 8 ) 0 . 4 8 ( 0 . 3 1 - 0 . 5 9 ) w idth Post, testis 0 . 0 4 ( 0 . 0 2 - 0 . 0 6 ) 0 . 1 3 ( 0 . 1 1 - 0 . 1 7 ) 0 . 3 1 ( 0 . 2 6 - 0 . 33) 0 . 6 5(0. 5 3 - 0 . 7 8 ) 0 . 7 5 ( 0 . 6 5 - 0 . 9 0 ) 1. 22(1 .15 - 1 . 33 ) length Post, testis 0 . 0 3 ( 0 . 0 2 - 0 . 0 4 ) 0 . 0 8 ( 0 . 0 5 - 0 . 11 ) 0 . 2 2 ( 0 . 1 8 - 0 . 2 7 ) 0 . 3 8(0. 2 5 - 0 . 5 4 ) 0 . 4 5 ( 0 . 3 5 - 0 . 53 ) 0 . 5 2 ( 0 . 4 0 - 0 . 65 ) width All measurements ln millimeters. 193 Appendix 6. Measurements and s t a n d a r d i z e d measurement system. T h i r t y - f i v e c h a r a c t e r s were measured f o r each i n d i v i d u a l , the number being determined on the b a s i s of those c h a r a c t e r s most commonly used t o i d e n t i f y members of the genus. Before the measurements were taken, a l l the s l i d e s of the f l u k e samples were mixed without i d e n t i f i c a t i o n of the f l u k e s p e c i e s . A l l the measurements were recorded, the s l i d e s then r e -i d e n t i f i e d , and the data re-grouped a c c o r d i n g t o the f l u k e s p e c i e s . In t h i s way b i a s e s due t o changes i n measurement technigue and p r o g r e s s i v e and p e r s o n a l e r r o r were minimized.. Egg s i z e s vary i n d i f f e r e n t p a r t s of the u t e r u s {Fig. / 84). For. t h i s reason i t i s important to choose a s i t e i n the uterus from which a l l egg measurements are done. The most a n t e r i o r p a r t Of t h e uterus {Site A, F i g . 84) was chosen. The eggs are embrycnated when they reach t h i s s i t e and; hence are the most f u l l y developed and b e s t r e p r e s e n t the true egg s i z e and shape of the s p e c i e s being measured. A l l measurements were made over a one-year p e r i o d (1S76). A l l 35 c h a r a c t e r s were measured on one specimen£, then a second specimen was measured, and so on u n t i l a l l the specimens had been measured. A l l measurements were made by means of a L e i t z compound microscope eguipped with a l i n e a r - g r a d u a t e d o c u l a r micrometer t h a t had been c a l i b r a t e d from a s t a n d a r d stage micrometer t o the nearest 0.1 micrometer. Before measuring a c h a r a c t e r on each specimen, the c o r r e c t m a g n i f i c a t i o n f o r t h a t c h a r a c t e r was s e t . The specimen was focused on the v e n t r a l s i d e and the c h a r a c t e r was always i n 194 focus. In this study a l l measurements were f irst recorded as micrometer units and later converted to millimeter units. 195 F i g . 84 V a r i a t i o n i n s i z e and shape of eggs taken from d i f f e r e n t p a r t s of the uterus of H. b u t t e n s i s . 195A 196 E r r o r s and b i a s e s i n measurement may a r i s e from p e r s o n a l experience, p e r s o n a l v i s u a l a b e r r a t i o n s , measuring h a b i t s , and inadequacies of the measurements system (Kim, Brown, and Cook, 1966) . The r e l i a b i l i t y of measurements i n t h i s study was assessed f o r each of 28 c h a r a c t e r s . One i n d i v i d u a l of each s p e c i e s of Haematoloechus s t u d i e d was measured on a l l 28 c h a r a c t e r s at thr e e widely spaced times (Tables A t o 0). Before measuring a c h a r a c t e r on each specimen, the c o r r e c t m a g n i f i c a t i o n f o r t h a t c h a r a c t e r was s e t . The specimen was focused on the c o r r e c t s i d e and t h e c h a r a c t e r was always i n focus. The r e l i a b i l i t y of measurements f o r each s p e c i e s was t e s t e d and the r e s u l t s are presented i n T a b l e s A to 0 i n c l u s i v e . The most u n r e l i a b l e measurement i s tegument t h i c k n e s s (TT), with c o e f f i c i e n t s of v a r i a t i o n 33.3$ f o r H. c o l o r a d e n s i s and H. complexus; 24.5$ f o r H. l o n g i p l e x u s ; 20.0$ f o r H. v a r i o p l e x u s ; 19.6% f o r - H. p a r v i p l e x f l s ; • 17.9$ f o r H. o x y o r c h i s ; 15.9$ f o r H. confusus; 15.8% f o r fl, k e r n e n s i s ; 13.3$ f o r H. b r e v i p l e x u s and H. b u t t e n s i s : 17.6$ f o r H. medioplexus; 10.8$ f o r H. s i m i l i p l e x u s : 10.6$: f o r fl .; yuniplexux; 10.2% f o r H. f l o e d a e : and 6. 0$ • for- H. turn i d us. , H, c o l o r a d e n s i s had a C.v..Of -10.1$ f o r the c h a r a c t e r , o v e r l a p o f t e s t e s . H. medioplexus had a -10.2$ C.v. For the same c h a r a c t e r . , r Table A. R E L I A B I L I T Y F C R 2 8 C H A R A C T E R S M E A S U R E D O F C H A R A C T E R MEASUREMENTS 1 2 3 ( J A N 1 6 / 7 6 M A P P . 1 3 / 7 6 X S E P 1 0 / 7 6 ) H . Q R E V I P L E X U S AC l s e . o 2 0 3 . 0 2 0 1 . 0 0 5 L 511 .0 557 . r. 5 0 6 . 0 OSW 51 - 2 1 i i K O 5 1 6 . 0 PL .2 2 09 . 0 2 1 2 . 0 Pw 2 5 1 . 9 2 5 7 .C 2 5 5 . 5 0-v. 961 .0 9 5 0 . 0 9 6 3 . 0 OW 6 5 1 . 0 6 5 2 . 0 6 5 4 . 0 OAT - 6 2 . 0 - 5 9 . 0 - 6 . 5 OAC - 3 7 2 . J - 3 7 0 . C - 3 7 7 . 0 ATL 1 3 J 2 . 0 1 3 0 7 . C 1 3 4 0 . 0 AI W 5i;v .o 5 7 6 . C • 5 V 1 . 0 ATE 20 7 7 . 0 2 0 5 5 . 0 2 0 B 5 . 0 P T L 1 5 5 0 . 0 1 5 6 3 . C 15 4 2 .5 PTW 7 7 3 . 0 7 ^ 9 . 0 7 7 5 . 0 P T E 1 3 6 4 . 0 1 3 5 5 . 0 . 1 3 6 9 . 0 ro 9 C 1 . 0 9 0 0 . C , 9 5 2 . 0 IHS - ' . C O - A O . 0 - 4 1 . 0 RUF 4601 .0 4 c 4 0 . C 4 6 8 5 . 0 LUF 4 2 U . 0 4 2 0 U .0 4 2 3 2 . 5 RVL 5 5 b 0 . 0 5 5 4 5 . C 5 5 9 0 . 0 LVL 4 S 0 2 . 0 4 4 5 0 . C 4 4 6 5 . 0 VA 5 8 4 0 . 0 5 6 5 0 . C 5 9 0 0 . 0 VE 6 5 1 . 0 6 4 5 . C 6 5 5 . 0 bL oes 7.0 8 9 5 0 . 0 8 8 9 5 . 0 Bw 1 7 9 8 . 0 1 7 8 0 . 0 1 8 0 0 . 0 TT 12 .5 1 0 . C 1 0 . 0 EL 2 0 . 0 . 2 2 . 0 20 .0 tw I t . 3 1 6 . C 1 5 . 5 MEAN MAX < l l « l » * « t * < . « r i « i ^ < i « * « < * « . # i « * 2 0 0 . 7 203 .0 . 5 6 4 . 7 5 6 7 . 0 5 1 3 . 6 5 1 6 . 0 2 1 0 . 7 2 1 2 . 0 2 5 4 . 6 2 5 7 . 0 9 5 U . 0 9 6 3 . 0 6 5 2 . 3 6 5 4 . 0 - 6 1 . f i - 5 9 . 0 - 3 7 3 . 0 - 3 7 0 . 0 1 3 2 6 . 7 1 2 4 0 . 0 5 6 5 . 3 5 9 1 . 0 2 0 7 2 . 3 2 0 U 5 . 0 1 5 3 2 . 5 1 5 6 5 . 0 7 6 ^ . 7 7 7 5 . 0 l i t 2 .7 1 3 6 9 . 0 95 7.7 y61 . 0 - 4 4 . 3 - 4 1 . 0 4 6 6 8 . 7 4 6 6 5 . 0 4 2 1 6 . 2 4 2 3 2 . 5 5571 . 7 S59 -J .0 4 4 3 9 . 0 4 4 6 5 . 0 5 8 U 0 . 0 5 9 0 0 . 0 6 5 0 . 3 055 . J 8 9 1 4 . 0 O9>50.0 1 7 9 2 . 7 1 8 0 0 . 0 1 0 . 0 1 2 . 5 2 0 . 7 2 2 . 0 1 5 . 9 1 6 . 3 M IN 1 9 6 . 0 561 . 0 5 1 0 . 0 209 .0 25 1.9-9 5 0 . 0 651 .0 -64 . 5 - 3 7 7 ;6-1 3 0 7 . 0 5 7 6 . 0 2055 1542 74 9 135 5 952 -46 4 64 0 5 5 f 5 . 0 4 4 0 2 . 0 5 8 - J O . 0 64 5 .0 0C95 . 0 1 7 8 0 . 0 1 0 . 0 2 0 . 0 1 5 . 5 .0 .5 .0 .0 .0 .0 .0 S T O O H V V A ^ t 2 . 5 6. 3.2 1 0 . 3.2 1 0 . 1.6 2. 2 . 6 6. 7 .0 4 9 . 1.6 2. 2 .8 7. 3 . 6 13. 1 7 . 4 3 0 2 . 8.1 6 6 . 1 5 . 5 2 4 1 . 1 1 . 5 131 . 1 3 . 8 19 0. 7.1 5 0. 4 . 9 2 4 . 2 .9 0. 2 4 . 8 6 1 6 . 1 6 . 2 2 6 4 . 2 4 . 5 6 0 0 . 3 2 . 9 1 0 3 0 . 26 .7 71 2. 5 . 0 2 5 . 3 1 . 5 9 9 2 . 1 1 . 0 1 2 2 . 1.4 2. 1.2 1. 0 . 4 0. ASCE C V 3 1 .3 3 0 . 6 2 0 . 6 4 0 . 7 9 1.0 0 0 . 7 5 0 . 2 6 - 4 . 5 0 - - 1 . 3 5 1.3 3 1.4 5 0 . 7 5 0 . 7 5 1.8 5 0 . 5 5 0 . 5 3 - 6 . 5 0 0 . 5 0 0 . 4 0 0 . 4 0 0 . 7 0 0 . 5 5 0 . 8 0 0 . 4 3 3 . 6 1 1 3 . 3 3 5 . 6 2 2 . 5 . Table B. RELIABILITY FCR 28 CHARACTERS MEASUKEO OF W. BUTTENSI S V_/* * * ****** *** *** * MEASUREMENTS I 2 3 CHARACTER (JAN 17/7fc)IAPR 1 1 / 7 6 X S E P 10/76) MEAN MAX M IN STD OEV VARIANCE CV V * * * * * * * * * * * * * * * * * * * * * * * * * * * ^ . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ^ AC 1 0 1 . 0 9 9 . 5 102 .0 100.8 1 0 2 . 0 . 9 9 . 5 1.3 1. 6 1.2 OSL 2 5 0 . 8 2 4 5 . C 2 5 3 . 0 2^9.6 253 . 0 245 . 0 4 .1 1 7 . 1 1.7 CSW 3 J 0 . 0 325 . 0 3 3 0 . 0 3 2 8 . 3 3 3 0 . 0 32 5. 0 2 . 9 8. 3 0.9 PL 209 .0 2 0 4 . C 2 0 7 . 0 2 0 6 . 7 209 .0 20-.. 0 2 .5 6. 3 1.2 Fw 24 7.5 2 4 5 . C 2 4 5 . 0 24 5.8 247 . 5 245 . 0 1 .4 2. 1 0 .6 CL 5 5U.0 547 . 0 5 6 3 . 0 5 5 6 . 0 5 6 3 . J 5 4 7 . 0 8.2 6 7 . 3 1.5 Uw 2 4 8 .0 2 5 0 . 0 2 5 9 . 0 2 5 2 . 3 259 .0 24a . 0 5 . 9 34. 3 2 .3 CAT - 5 5 i l . O - 5 4 0 . C - 5 4 0 . 0 - 5 4 6 . 0 - 5 4 0 . 0 - 5 5 S . O 1 0 . 4 10 0. G - 1 . 9 OAC - 5 3 . 0 -55 .0 - 5 0 . 0 - 5 2 . 7 - 5 0 . 0 -55 . 0 2 .5 6. 3 -4.8 AT L 1 9 5 3 . 0 1 9 2 3 . C 1 9 5 5 . 0 1 9 4 3 . 7 1 9 5 5 . 0 192 3 . 0 1 7 . 9 32 1. 5 0.9 A T W 4 6 5 . 0 455 . 0 4 5 5 . 0 4 5 0 . 3 4 6 5 . 0 4 5 5 . 0 5 .0 3 3 . 3 U 3 ATE 2 1 7 0 . 0 2 1 4 0 . C 2 1 5 0 . 0 2 1 5 3 . 3 2 1 7 0 . 0 2 1 4 0 . 0 1 5 . 3 23 3. 5 3. 7 P T L 1 3 0 2 . 0 1 3 3 5 . 0 1 3 3 5 . 0 1 3 2 4 . 0 1 3 3 5 . 0 1 3 0 2 . 0 19 .1 3 6 3 . 0 1.4 PTW 558 .0 5 4 5 . 0 5 6 0 . 0 5 5 4 . 3 560 . 0 5 4 5 . 0 8.1 6 6 . 3 1.5 PTE 119 3.5 1 1 7 5 . C 1 1 9 5 . 0 118 7.8 1 1 9 5 . 0 1 1 7 5 . 0 11 .1 124 . 0 0.9 TO 4 U 3 . 0 4 15.C 4 1 5 . 0 411. 0 4 1 5 . 0 403 . 0 6 .9 4 0 . 0 1.7 THS - 3 4 1 . 0 - 3 4 5 . 0 - 3 5 0 . 0 - 3 4 5 . 3 - 3 4 1 .0 - 3 5 0 . 0 4 . 5 2 0. 3 - 1 . 3 RUF 1U91 .0 l t - 8 0 . 0 1 9 C 0 . 0 109 0 .3 1 9 0 0 . 0 1 8 8 0 . 0 1 0 . 0 1 0 0 . 5 0.5 LUF 12 4 0 . 0 1 2 4 0 . 0 1 2 4 5 . 0 1 2 4 1 . 7 1 2 4 5 . 0 1 2 4 J . 0 2 . 9 C. 5 0 .2 RVL 3 0 3 8 . 0 3 0 4 0 . C 3 0 4 5 . 0 3*>4l.O 3045 .0 3 0 J 8 . 0 2.8 0.' 3 0. 1 LVL 4 2 / 8 . 0 4 2 7 5 . C 4 2 6 0 . 0 42 7 7. 7 4 2 6 0 . 0 4 2 7 5 . 0 0 . 0 0. 0 C O V A 5 1 6 1 . 5 5 1 6 5 . 0 5 1 7 5 . 0 5167.2 5 1 7 5 . 0 5 1 o l . 5 6 . 3 4 0. 3 3.1 VE 1 C 2 3 . 0 1 0 1 5 . 0 1 C l 5 .0 101 7. 7 1023 .0 1 0 1 5 . 0 4 .6 2 1 . 5 3.5 BL 6 1 5 3 . 5 6 1 0 0 . 0 6 1 2 5 . 0 6 126.2 6 1 5 3 . 5 6 1 0 0 .0 26.8 . 7 2 0 . 0 3.4 Bw 1 3 0 2 . 0 1 3 0 0 . 0 1 3 1 5 . 0 1 3 0 5 . 7 1 3 1 5 . 0 1 3 0 0 . 0 •• 6 .2 6 6. 5 3.6 TT 1 2 . 5 1 0 . 0 1 0 . 0 1 O.U 1 2 . 5 1 0 . 0 1.4 2. 1 1 3 . 3 EL 2 5 . 0 2 5 . 0 2 5 . 5 25.2 2 5 . 5 2 5 . 0 0 . 3 0. 1 1.1 EW 1 2 . 5 1 2 . 5 1 2 . 0 1 2 . 3 1 2 . 5 1 2 . 0 3.3 3. 1 2 . 3 Tab le C . R E L I A B I L I T Y FCR 28 CHARACTERS MEASURED OP MEASUREMENTS 1 2 CHARACTER (KAY 14 /76 ) (MAY 2 1 / 7 0 ) ( JUN 3 /76 ) AC 3 5 2 . 0 3 5 0 . 0 3 5 3 . 0 OSL 3 9 c . 0 3 9 fc; C 2 9 9 . 0 OSw 3 35 S 3 3 3 . 0 3 3 2 . 0 PL 3 0 7 . 0 303 .0 ? H 2 5 7 . 4 2 5 7 . C 2 5 9 . 0 OL 5 C 6 . 0 5 U . C 51 I .0 CW 4 2 3 . 5 12 J . C 1 2 o . O OAT 5 73.5 5 6 5 . C 5 6 9 . 0 OAC 1 3 2 . J 1 2 5 . 0 1 3 1 . 0 ATI. 5 2 7 . J S i b . C 5 2 3 . 0 AT W 5 5 U . 0 563 . C ' 5 6 5 . 0 ATE 2 2 9 i . o 2 2 4 3 . 0 22713 .0 P T L 1 9 0 . 0 4 9 0 . 0 1 9 1 . 0 PTW 6 2 0 . J 6 2 0 . 0 6 l a .0 P T E 192 7.0 195r l . 0 1 9 5 8 . 0 TO - 9 <i. 0 - 9 0 .0 - 1 1 0 . 0 THS 3 1 2 . 0 3'. 3 .0 3 2 7 . 0 RUF 2 7 5.0 2 u 4 . 0 2 8 0 . 0 LUF 2 75.0 2 75 . 0 201 . 0 R VL 2 2 1 : .0 3 2 0 J . 0 3 2 1 2 . 0 L V L 4 l 2 d . O 4 1 5 9 . 0 1 1 2 6 . 0 VA 5 0 4 6 . 0 5 0 5 7 . 0 5 0 3 5 . 0 VE 1 1 1 7 . 0 1 1 1 6 . C 1 1 1 7 . 0 bL 6 1 7 3 . 0 6 13 1.0 621 3.0 Bw 13 0 5.0 1 3 6 1 . C 13b0 .0 TT 7.5 5 .C 1 0 . 0 EL 3 7.5 3 7 . 0 3 5 . 0 E« 2 1 . 3 2 0 . 0 . 2 0 . 0 . 8 . 0 .2 . 2 .3 , 0 Mt AN 3 5 1 . 7 3 9 7 . 0 3 3 3 . 5 3 0 5 . 3 257 . 511 . 123 . 5b V.1 3 J . 522 . 5 b 2 . 0 2 2 7 d . 3 1 9 3 . 3 6 l 9 . 3 1 9 1 7 . 7 - 9 9 . 327 . 2 73, 27 7. 321 7. 413U. 5 J l o . 1136 , 6 1 7 9 . 1379, 7. 36. 20, 7 .3 . 0 .0 . 7 .3 0 , 7 . 0 .7 . 5 , 5 .1 . 0 . J .0 . 5 .0 .0 MAX. 3 5 3 . 0 3 9 9 . 0 3 3 5 . 5 307 .0 259 51b 126 573 131 52 7 5 6 5 . 0 2 2 9 1 . J 1 9 6 . 0 6 2 0 . 0 195 0 . 0 - 9 0 . 0 3 1 3 . 0 2 8 0 . 0 2 (> 1 . 0 3 2 1 2 . 0 1 1 5 9 . 0 5 0 5 7 . 0 1 1 1 7 . 0 6 2 1 3 . 0 1 3 9 i , . 0 1 0 . 0 3 7 . 5 2 1 . 3 H.COLOP.AUENSIS MIN STO OEV VARIANCE CV 350 . 0 1.5 2. 3 0 . 4 396 . 0 1.7 3. 0 0 . 4 3 3 2 . 0 1 .8 3. 3 0 .5 303 . 0 2.1 1. 2 2=7 25 7.0 l . l 1. 1 0 . 4 5 J O . 0 5 . 0 25 . 0 1.0 12 J . 0 3 . 0 9. ' 0 . 7 565 . 0 1 . 3 18. 1 0 . 7 1 2 5 . J 1 . 7 22 . 3 3 .6 51 6 . 0 5.6 3 1 . 0 1.1 5 5 6 . 0 3 . 6 13. 0 0 . 6 22o3 . 0 1 5 . 6 2 1 2 . 0 0. 7 1 9 U . 0 3.1 9. 3 0 . 6 6 U . 0 1.2 1. 5 0 . 2 l 9 2 7 . 0 1 7 . 9 3 2 2 . 0 0 . 9 - I I O . O 1 0 . 0 100 . 3 - 1 0 . 1 3 1 2 . 0 1 5 . 5 2 1 0 . 3 4 . 7 2b1 . U 0 . 2 6 7. 0 3 .0 2 7 5 . 0 3 . 5 • 12. 0 1.3 3 2 0 0 . 0 2 1 . 7 4 7.2. 0 0 . 7 4 1 2 3 . 0 1 7 . 9 3 2 0 . 0 0 . 4 5 0 3 5 . 0 1 1 . 0 12 0. 0 0 .2 1 116 . 0 1 7 . 9 3 2 0 . 5 1.6 6 1 3 1 . 0 1 2 . 7 1 3 2 1 . 0 0 . 7 1 3 b * . 0 1 5 . o 2 4 2 . 0 1.1 5 . 0 2 .3 6. 3 3 3 . 3 3y . 0 1.3 1. S 3 .6 2 0 . 0 0 . 8 0. 6 3 .7 Tab le D. R E L I A B I L I T Y FOR 28 CHAPACTEKS MEASURED OF H.COMPLEXUS **« * * » ? * » **** * MEASUREMENTS 1 2 3 CHARACTER (MAY 14/76)(MAY 21 /76) (JUN 3/76) MEAN MAX MIN STO DEV VARIANCE CV AC 2 8 6 . 9 2 8 0 . C 2 8 9 . 0 2 8 5 . 0 2 8 V . O 2 U 0 . 0 4 . 6 2 1 . 0 1.6 CSL 4 C 5 . 9 4 0 J . 0 405 .0 4 0 3 . 6 4 0 5 . 9 4 0 0 . 0 3 .2 10. 2 3 . 0 OSW 4 0 1 . 5 4 0 0 . 0 4 0 0 . 0 4 0 0 . 5 401.5 4 0 0 . 0 0 . 9 0. 8 0 . 2 PL 1 9 0 . 0 1 9 5 . 0 2 0 0 . 0 19 7. r 2 0 0 . 0 1 9 5 . 0 2 .5 6. 3 1.3 Prf 2 31 .0 2 2 8 . 0 2 3 5 .0 2 3 1 . 3 2 3 5 . 0 2 2 0 . 0 3 .5 1 2. 3 1.5 CL 4 5 o . 5 4 5 0 . C 4 6 3 . 0 4 5 U . 5 4o3 . 0 456 . 0 3 .9 1 5. 3 0 . 9 Cw 3 L9 .0 . 3 1 5 . 0 3 2 2 . 0 31 n. r 32 2 .0 3 1 5 . 0 3.5 12. 3 1.1 OAT 6 1 3 . 8 61 J . C 6 1 6 . 0 6 1 3 . 3 61 6 .0 6 1 0 . 0 3 .0 9. 3' 0. 5 OAC 3 3 . 0 3 3 . 0 ' 2 1 . 0 3 2 . 3 33.0 3 1 . 0 1.2 I. 3 3 .6 A T L 7 4 4 . 0 74 J . O 731 . 0 7 3 8 .3 7 4 4 . 0 7 3 1 . 0 6 .7 4 4 . 5 0 . 9 AT A 6 75.8 6 8 0 . 0 6 8 3 .0 6 7 9 . 6 6 8 3 . 0 675 .6 3.6 13. 0 0.5 ATE 1 5 5 5 . 0 152 1.0 1 5 3 C . 0 1 5 3 0 . 0 1 5 5 5 . 0 1 5 2 1 . 0 1 7 . 0 2 9 0 . 0 1.1 PTL 899 .0 6 6 8 . 0 9 0 0 . 0 609.0 9 0 0 . 0 floO. 0 1 6 . 2 3 3 1 . 3 2 . 3 PTW 7 4 4 . 0 732 . C 7 3 7 . 0 73 7. 7 7<-H.O 732 . 0 6 . 0 36 . 5 3 . 6 PTE 96<< .0 S 7 0 . 0 S C O . O 97 7.3 9 0 2 . 0 S7 0. 0 6 .4 4 1 . 5 3.7 TO 1 3 7 . 0 1 3 5 . 0 1 4 0 . 0 1 3 7 . 5 1 4 0 . 0 1 3 5 . 0 2.5 6. 3 l . b IMS - 3 0 7 . 0 - 2 6 0 . C - 3 6 0 . 0 - 3 6 2 . 3 - J O 0.0 - 3 6 7.0 4 . 0 16. 3 - 1 . 1 RUF 7 1 3 . 0 7 1 5 . C 7 1 9 . 0 7 1 5 . 7 7 1 9 . 0 713 . 0 3.1 9. 5 3 . 4 LUF 06H . 0 8 6 0 . 0 8 6 9 . 0 8 6 5 . 7 0 6 9 . 0 8 6 0 . 0 4 . 9 2 4 . 5 0 . 6 PVL 3391.0 3372 . 0 3 3 9 6 . 0 3 3 8 0 . 0 3 3 9 8 . J 3372.0 1 3 . 9 19 ?. 0 0.4 LVL 4 3 1 2 . 0 4 3 0 0 . 0 4 2 9 0 . 0 4 3 0 0 . 7 4312 . 0 4 2 9 0 . 0 1 1 . 0 1 2 0 . 0 0 . 3 VA 5 3 2 1 . 0 5300 .0 5 2 0 0 . 0 530 7.0 5 3 2 1 . 0 5 300.0 1 2 . 3 152 . 0 3 . 2 VE 5C4 .0 5 0 0 . 0 4 9 5 .0 4 V 9 . 9 504 .6 4 9 5 . 0 4 . 8 2 3. 1 1.3 BL 6 1 9 3.0 6 1 0 5 . C 6 1 8 2 . 0 . 6 1 b 7 . 0 6 1 9 3 . 0 6 1 6 3 . 0 5.7 3 2 . 0 0 . 1 Bw 1 2 7 1 . 0 1 2 6 5 . 0 1 2 7 5 . 0 1 2 7 0 . 3 1 2 7 5 . 0 1 2 o 5 . 0 5.0 2 5. 5 3.4 TT 7.5 5 . 0 1 0 . 0 7.5 1 0 . 0 5 . 0 2.5 6. 3 33 .3 EL 32.5 32.5 3 5 . 0 3 3 . 3 3 5 . 0 3 2 . 5 1 .4 2. 1 4 . 3 EH 17.5 17.5 1 7 . 0 1 7 . 3 17.5 1 7 . 0 0 . 3 0. 1' 1.7 Table E . R E L I A B I L I T Y FOR 28 CHARACTERS MEASURED OF H.CONFUSUS **************** MEASUREMENTS I 2 3 C H A R A C T E R (NOV 9/76)(NCV 13/7 6)(NOV 11/76) MEAN MAX MIN STO OEV VARIANCE CV AC 2S7.9 3 0 0 . 0 2 9 6 . 0 2 9 8.0 3 0 0 . 3 . 2 9 6 . 0 2 . 3 4 . 0 0. 7 OSL 4 95 .9 4 9 3 . 2 4 9 6 . 0 4 9 5 . 0 4 9 0 .0 4 9 3 . 2 1.6 2.6 0.3 OSW 4 3 6 .5 4 3 5 . 6 4 3 7 . 5 4 3 6 . 5 4 3 7 . 5 4 3 5 . 6 1.3 1. 3 3.2 PL 3 7 U . J 3 7 5 . 6 3 8 0 . 0 3 7 7 . 9 360.0 3 7 5 . 6 2.2 4 . 9 3.6 Pw 22a.5 3 2 7 . 5 2 3 0 . 2 32 0 . 7 3 3 0 . 2 32 7.5 1.4 I. 9 0 . 4 OL 3 3 3 . 0 3 3 5 . 0 3 3 5 . 0 334,3 3 3 5 . 0 333,0 1.2 I . 3 0.3 Ow 3C6.3 3 0 5 . 0 3 0 7 . 3 3 0 6 . 1 3 0 7 .3 3 o J . o 1.2 1. 3 0 . 4 O A T 4b.0 4 7 . 0 4 5 . 9 46.0 4 7.0 4 5 . 0 1.0 1. 0 2.2 OAC 36.0 3 4 . 2 3 5 . 3 3 5 . 1 3 6 . 0 34.2 0.9 3. 8 2.6 A T L 5 1 3 . 0 5 1 2 . C 5 1 0 . 0 5 1 1 . 7 • 5 1 3 . 0 5 1 0 . 0 1.5 2. 3 0.3 A T H 3 5 1 . 0 3 4 7 .0 • 3 4 9 . 5 3 4 9 . 2 3 5 1 . 0 3 4 7. 0 2.3' 4 . 1 3.6 A T E 12 7 0 . 3 1 2 7 9 . 3 12 7 5.8 1 2 7 7 . 7 1 2 7 9 . 3 1 2 7 5 . 6 1.6 2. 5 3.1 P T L 466.3 4e8.C 4 8 5 . 7 46o.6 4 a 6 . 0 4 6 5 . 7 1.3 1. 7 0. 3 P T * 4 6 8 . 3 4 7 1 . 3 4 7 0 . 0 46 V . 8 4 7 1 . 3 4 6 6.0 1.7 2. 8 0.4 P T E 6 9 1 . 3 8 6 7 . 5 0 9 0 . 5 8 6 9 . 7 8 9 1 . 0 8 6 7 .5 1.9 3. 5 0. 2 T O 4 C 5 . 0 4 0 0 . 5 4 0 3 . 4 4 0 J . 0 4 0 5 . 0 4 0 0 . 5 2.3 5. 3 0.6 THS - S 9 . 0 - 9 7 . 6 - 1 0 J .0 - 9 8 . 9 - 9 7 . 6 - 1 0 0 . 3 1.2 1. 5 -1.2 RUF A B S E N T A B S E N T A 8 S L N T LUF AbSENT A U S C A ' T A B S E N T RVL 2 1 6 7 . 3 2 1 9 ? . ? 2 1 8 5 . 3 2 1 6 8 . 4 2 1 9 3 . 3 2 1 8 5 . 3 4.9 24. 0 3.2 LVL 1 6 4 7 . J 1 6 'j'l .1 i 6 i u . 5 1 6 4 6 . 9 1 6 5 1 . 3 1 6 4 7.0 3 .5 12. 0 0.2 VA 2 ; i . o 2 9 5 6 . 7 2 9 5 9 . 0 2 9 5 8 . 9 2 9 6 1 . 0 2 9 5 6 . 7 2 . 8 v- 3 0.1 V E 7 t 5 . 3 7 6 3 . 3 7 6 6 . 7 7 6 4 . 9 7 6 6 . 7 ' 7 6 3 . 0 1.9 3. 5 BL 393 7.5 2 9 3 5 . 0 393e.5 3 9 3 7 . 0 3 9 3 8 . 5 3 9 3 5 . 0 0.0 0. 0 0.0 BW 0 02.3 fcbO. 1 6 8 3.0 681.7 663 .3 8 0 0 . 1 • 1.6 2. 5 3.2 T T 1 3 . 8 1 0 . 0 12.0 1 1 . 9 1 3 . 8 1 0 . 0 1.9 3. 6 15.9 EL 3 0 . 9 3 0 . 0 3 1 . 0 3 0 . 6 3 1 . 3 3 0 . 0 3.6 0. 3 i.e E« 22.6 22.5 22.3 2 2 . 4 22.6 22.0 0.3 3. 1 1 . 4 T a b l e F . R E L I A B I L I T Y F O R 28 C H A R A C T E R S M E A S U R E D O F H . F L O E D A E M E A S U R E M E N T S 1 2 3 CHARACTER ( N C V 9 / 7 6 U N 0 V 1 0 / 7 6 1 ( N C V 1 1 / 7 6 ) MEAN MAX" M IN S T D D E V V A P I A N C E CV AC 153.0 155.0 154.5 154.2 155.0 153.0 1.3 1. 1 3. 7 CSL 450.0 44 7 .5 449.0 44 8. 6 450.0 4-.7.S 1.3 1. 6 3.3 OS* 432 .0 430.0 4 3 1.3 431.1 432.0 430.0 1.3 1. 3 3.2 PL 162. J 160.0 159.3 160.4 162.0 159.3 1 .4 2. 3 3.9 PW 197.1 198.7 196.0 19 7.3 196.7 196 . 0 1 .4 I. 9 0.7 OL C69.4 871.0 867.7 869.4 671.0 86 7. 7 1.7 3. 0 3.2 OH 5 3 5.5 537. C 533.0 535.2 537 .0 533 .0 2.3 4. i 3.4 CAT 1 H9 .0 168.0 166.8 16 7.9 1 H 9 . 0 166. 6 1.1 1. 3 0.6 OAC -53 1.0 -526.0 -533.0 -529.7 -526.0 -531.0 1.5 2. 3 -3.3 ATL l C C b . O 10C5.0 1011.3 1006. 1 1011.3 1005.0 3.2 10. 0 0.3 A Trl 535.5 53 7 .1 536.6 5 3 7 .1 538.6 535 . 5 1.6 2. 4 3.3 ATE 14 17.5 1422.0 1425.0 1421.5 1425.0 1417.5 3.9 15. 5 3.3 PTL 1023.8 1025.C 1C21.8 1023.5 102 5.0 1021.8 1.6 2. 5 0.2 PT« 5 10.3 512.0 5 06 .7 510.3 512.0 508. 7 1.6 2. 7 3.3 PTE 75c.0 (50.0 755 .0 756.3 75U. 0 755 .0 1 .0 2. 5 0. 2 TO 4 C 9 . 5 407. 1 4 10.0 4U6. 9 410.0 40 7.1 1 .6 2. 5 0.4 THS -126.0 -125.0 -122.0 -124.3 -122.0 - 12u.O 2.1 4. 3 -1.7 RUF 1772.0 27 70.C 2 7 6 5.0 2 769.0 2772.0 2 7o5.0 2 .0 e. o 0. 1 LUF 2929.5 2 93 1 .C 2933.4 293 1 .3 2933.4 2929.5 2.0 a^o 3. 1 RVL 2 1 L C. 5 ctto.Z 2 t o 1 .3 2665.3 2666 . 0 2 S o l .3 4 .9 24.0 3.2 LVL 39 J17. 5 2935.0 3944.0 3938.8 . 3944.0 3 93 5.0 4.9 24. 0 0.1 VA 40c3.5 4065.0 4 Zu'j. 0 406 2.6 4 0 6 5 . J 4 Oo 0 . 0 2.8 6. 3 3. 1 VE 4 9 0.5 486 .0 4 92 .2 49 0. 2 492 . 2 466 .0 2.1 4. 5 0.4 BL 5C96.7 5093 .C 5100.0 5096.6 51uu.0 5 0.3.0 2.0 e. o 3.1 Bw 1354.5 1 3 5 J . C 1356.3 1353.6 1356.3 1350.0 3.2 13. 3 3.2 TT 10.1 6.5 6.5 9.0 10.1 8.5 3.9 0. 9 10.2 EL 22.1 22.0 • 22.5 22.2 22.5 22. *J 0.3 3. ii 1.2 EW 1-.3 14.3 12.5 13.9 14.3 13.5 0.4 3. 2 2.9 Table G . R E L I A B I L I T Y F C R 28 C H A R A C T E R S M E A S U R E D OF H . K E R N E N S I S M E A S U R E M E N T S 1 2 3 CHARACTER ( N O V 9 / 7 6 1 ( N O V 1 3 / 7 6 M N O V 1 1 / 7 6 J MEAN MAX M I N S T O DEV AC O S L OSW P L Pn C L O H C A T C A C A r L A T W ATE P T L P T W PTE T U T H S R U F L U F R V L L V L VA V E B L bW T T EL EW VARIANCE CV 432. 0 430.0 433 . 0 . 431.7 433 . 0 430 .0 1.5 2.3 3.4 459 . 0 460.-- 4.'. C O 459. 7 460 . 0 459.0 0.6 0. 3 C . 1 4 5u:'« 450.0 453 .0 451. 3 4 5 3 . 0 4 5 J . 0 1.5 2, 3 3.3 266 .0 290. C 293.5 289. 5 290.5 288.0 1.3 1. 8 3-5 294.3 295. 0 29 3.0 294.1 295 .0 293 . 0 1 .0 1. 0 0.3 630.0 <-35 . 0 632.5 632.5 635.0 630.0 2.5 6. 5 3.4 427.5 426. 5 428.0 42 7.3 420 .3 42o .5 0.6 3. 6 0.2 459.0 460.0 4 4 7. 5 455.5 460.0 4<. 7. 5 7.0 4 0. 3 1 .5 -52.2 -52.0 -50.0 -51.4 -50.0 -52.2 1.2 1. 5 -2.4 9 13.5 915.0 912.0 913.5 915.0 912.0 1.6 2. 5 0.2 9 7o.5 9 73 .0 '9 75 .0 9 74.0 9 76.5 973.0 1.7 3. 0 0.2 21C1.1 2100.0 210 5.0 2102.0 2105.0 2100.0 3.5 12. 5 0.2 1020.0 1023.C 1C18.0 1019.5 1020.6 1013.0 1 .4 2. 0 0. 1 1C39.5 1037.0 1035 .3 103 7.3 1039 . 5 1035.3 2.1 4. 5 0.2 122 1.7 1234.0 1237.5 12J4.4 1237.5 1231.7 2 .9 8. 5 0.2 157.5 155 . 0 155.0 155.8 157.5 155.0 1.4 2. 1 0.9 -535.5 -537.0 -533.0 -535.2 - 5 3 3 . 0 -537.0 2.3 4. 1 -3.4 Ab SCNT A8SEKT ABSENT ABSENT ABSENT AllSeNT 3C43 . 0 3041.0 364 1.0 3841.7 3043 .3 3841.0 3.3 3..; 3 3 .0 21.66 .5 2u6fl.C 2865.0 2866.5 2Uo0 .0 2065 . 0 0.0 o.'o 0.0 5166.6 5155.0 5158.0 5159.9 5166.6 5155 . 0 4 .9 24. 3 3.1 14C6. 1 l<O0. 0 1410.0 1406.0 1410.3 1400.0 5.8 33. 5 0.4 719 4.6 7 190. 1 719 7.5 7 194. 1 7197.5 7 190. 1 2.8 3. 0 0.0 1638 . 0 1640.5 1637.3 1638.6 1640.5 1637.3 • 2 .9 8. 5 3.2 22.1 17. C 19 . 0 19.7 23.1 17 . 0 3.1 9. 7 15.8 36 . 0 36 . 0 3 5 .0 35.7 36 . 0 35.0 3.6 0.3 1.6 . 23.1 22. 5 22.5 22.7 2 3 . 1 22.5 3.3 0. 1 1.5 . T a b l e H. R E L I A B I L I T Y FCR 28 CHARACTERS MEASURED OF H.LONGIPLEXUS MEASUREMENTS **** CHARACTER * • » * » • • • • « AC OSL OSW PL Pw OL Ow OAT . OAC ATL ATM ATE P TL PTW PTE TO TMS RUF LUF RVL LVL VA VE BL Bw T T EL EM • I 2 3 ( J A N 1 5 / 7 6 M A P R 9/76) (SEP 10/7) MEAN 187.0 189. C 192.0 573.5 563.C 568.0 558.0 563. 0 555.0 264.0 263 .0 262.0 221.0 236. 0 231.0 ICC 5 . J 1126.0 1069.0 620.0 604 .0 626.0 124.0 113.0 110.0 -3 56.0 -2 2 3.0 -'234.0 12<. o.O 1271.0 1249.0 5 5 H. 0 5 70. 0 570.0 2 7 5V.0 269H.0 2 72 7.0 1550.0 1581.0 1555.0 620.0 631.0 620.0 1091.0 1860.C 1VC 7.0 402.0 414.0 414.0 -240.0 -212.C - 2 3 7 . 0 4 4 9 5 . 0 4526 . C 454 1 .0 42 78.0 42S4.0 4 2 9 0 . 0 5 733 .0 5*0-'; . C 5723.0 42 4', 425V.0 42 7 7 . 0 6^-. 1 . 0 65 72. C 6 5 3 1 . 0 651.0 640.0 656.0 7U12.0 7800.0 7662.0 2129.0 2122.C 21 5 0 . 0 16.3 10.0 12.5 2 2 . 5 22.5 • 25.0 17 . 5 16.C 17.0 189 568 556 263 232 10V3 616 118.3 -337.7 1253 5o6 2 728 1562 623 1836 410.3 - 2 3 2 . 3 4520.7 4 26 7.3 5 720.0 4 26 0.7 654 0.0 649.0 7624 . 7 2137.0 12.9 23.3 16.8 .3 . 0 . 0 .0 . 7 . 0 . 0 .0 . J .0 MAX 192.0 573.5 563 .0 264. 236. 1 1 2 6 . 626 , 124.0 - 3 2 3 . 0 1271.0 570.0 2 759.0 1561.0 6 3 1 . 0 1907.0 4 1 4 . 0 M IN -212 454 1 4294 5733 4277 6572 656 78O2.0 2 1 5 0 . 0 16.3 25.0 1 7.5 1 8 7 563 555 262 231 1 06 9 604 113 -356 1240.0 55 8 .0 2 6 9 6 . 0 1550 .0 620.0 16o 0. 0 403 • 0 -246 4495 • 42/6 5 704 4 24 6 6531 64 0.0 7800.0 2 1 2 2 . 0 10.0 22.5 16.0 .0 .0 .0 .0 .0 . 0 STO OEV VAPIAMCE CV 2.5 6. 3 1.3 5.3 2 7. 6 3.9 4.0 16. 3 0. 7 1 .0 1. 0 0.4 2.9 8. 3 1.2 29.4 8 6 4 . 5 2.7 11.4 129. 5 1.8 5.5 33 . 3 4.7 16.3 2 8 2 . 3 -5.0 16 . J' 254. 5 1.3 6.9 4f>. 0 1.2 30.5 9 2 8 . 0 1.1 16.7 • 27C. 3 1. 1 6.4 40. 5 1.3 23.9 572. 3 1.3 6.4 43. 3 1.5 18.4 340. 3 - 7 . 9 23.3 544. 0 0.5 8.3 64. 0 3.2 15.0 2 2 4 . 0 0.3 15.2 •?- 3 3.4 21.4 4 5 6. 3 6.2 6 7 . 3 1.3 33.3 1112. 3 3.4 14.2 20 0 . 5 0. 7 3.2 13. 1 24 . 5 1 .4 2. 1 6.2 0.8 0. 6 4 . 5 o •o « tn « r> * x * ui a _i » o. « o * * o * Ui • £ « « ft X * o U J cf. Z3 v> •d Ul ac «/> or ui »-o < or < x o « « > ft o • « « • U J • o * z « < « CC • < ft > ft • « > ft 111« o « ft o ft »- ft tn « • « » * Z ft * 2: ft « « ft ft ft « ft X ft < <• x ft * « « « z ft < * UJ ft Z. « ft ft « ft m i f t i o « « « i N N ^ o^-.rMco-.OfM-.f. r> o- 0 4 J> n • • • • I - "> «M - -< co _ - • O ^ ' ^ f v i j - ^ r o ^ o - . r . ^ c o r t j J - r , ™ —< o- —' IN <^ m co r\j <N —« o ~« M ^ ^ M r t o ^ n ^ u ^ O - r v ^ J o ^ r n ^ O r J ^ ^ ^ o ^ o "~* «-< lO ~< . -« —I _ l fVJ — « • - < - . ° ^ * ^ ' l ^ ^ ° ^ ° 3 ^ ° ^ « o ^ o o o o o o o . r , o . n f - O 1 . t 5 > M O rj- -O 0 r 0 v ) - O , 0 ~ - < r \ i o O t - - . r N t - _ . , r i r - > - i . _ ro cvj w - r- ro w « £ o ^ ^ S ^ S ^ o S S S ™ -M -< I I st vj- J\ -< r-" " " ^ ° ^ ™ ^ ° ^ ° ° o o n o o n o o o a o i n » .0 r v :M .M r > m O ^ O O S O H I N H ^ a " * -« I I -4- J\ -I N . m w IM-c 1 - r o o m m o ^ o w r N - ^ o ^ j ^ ^ - " - I I >r vj- IA r-— ft O ft co CM Of u u. ro < V* ft O ft CO ~ l « ft a ft U l ft v> • — ft tn ~ » »- O f t f ~ ft Ul «s. ft a : 0 0 u i CM « t t « 3 c i ' < tn o . • ui a; < » — » « » »>- • V. ft tn « 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ,nr,^ m rv rv, cv r- ro *^OOLOo2^'rs12-0^2S3S ^ CM —< I 1 -T <r m —« r-0 0 0 0 0 0 0 » 0 0 0 0 0 0 0 ^ 0 w o « T i C i n o H v > r j H . ^ n » j . M < i i I I vr ^- IT. ^  r-co o t o n o o o o o o o o o o o o o o o o o o o o m o c o tt) H —< I I -r j> ui - 1 r-Z « < ft -> ft —• • « or • U l • ft- • 0 • < ft 01 * - 1 3 . ^ O J J i u J l i u ifl u. u. J j < i o K w J i j j < < < > - f c « - M - i - c : j D > > < u j j j > - j j 1 * < o o i a o o o o < i < < a . ( x ( ) . h i - r j ; j u J > > i i S f - i u u i Table J . R E L I A O I L I T Y FCR 28 CHARACTERS MEASURED OF H.OXYORCHIS CHARACTER AC OSL OSW PL P-i OL Ow OAT OAC A r L ATW ATE PTL PTW , PTE TO THS RUF LUF R VL LVL VA VE OL BW TT EL EH MEASUREMENTS I 2 3 (NOV 9/76MNOV 10/76)(NOV 11/76) # * * * * f « * * * * * t> * »<« 291.6 290.0 289 .1 369.3 3 7.?, C " L . 5 3 5 1-0 349 .0 350. 7 c w 7 • 0 205.0 206.3 243.0 245. C 241.2 5 U5 . 0 587. 0 503.5 3 23.3 335. C 333.7 153.0 150.5 155.0 I 80.0 170.6 102.0 729.0 735. C 730. 5 612.0 610.0 6 13.4 1002.0 16C7. C 1 6 0 J . 0 6 75.0 6 73 . 5 6/5.3 648.0 650.3 6 4 7 . 1 963.0 961. <. 960.5 3 6.0 • 34.5 33.6 -117.0 -115.0 -115.0 A H S C N T A B S E N T A B S E N T A B S E N T A B S E N T A B S E N T 39C6.0 3910.0 3904.0 2740.5 2 742 . 5 2744.0 43 78.5 4300.C 4385.0 540.3 542.3 545 .0 5512.5 5516.C 5515.6 974.5 979.C 975 .0 6.9 7. C 5.0 23.1 22.5 22.5 15.7 15.C 15.5 MEAN « p <• !> I 290.2 371.2 350.2 2J6.0 243.1 585.2 333.9 15 2.8 100.3 , 5 , 8 0 5 5 6 7 7 731 611 1603 674, 64b, 961. 34. -115. 3906.7 2742.3 4381.2 542.3 5514.7 976.8 6.3 22. 7 15.4 .0 . 0 .0 . 4 .0 .0 MAX 291 .6 373 .0 351.0 200.3 245.0 567.0 335 .0 155, 182, 735, 613. 1607 , 67 5, 650.3 963.0 3o. 0 -115.0 3910.0 2744.0 4385.0 545.0 5516.0 979.0 7.0 23.1 15.7 MIN 11> «> «i *<.»:«> < 289.1 369 .0 319.0 205 .0 24 1. i 583, 333. 150. 17o. 72 9, 610, 1600.0 673 .5 647. 1 9t>0.5 33.6 - 1 1 7 . 0 STD DEV VARIANCE .5 .0 .5 . 6 . 0 .0 3904.0 2740.5 4378.5 540.0 5512.5 975.0 5.0 22.5 15.3 1.3 2.3 1.1 1 1 1 1 2, 1 3 1. 3, 1. 1. 1. 1.2 1.2 1.6 4. 1 * • c 2. 8 3. 6 3. I 1. 0 5. I 2. 7 iO. 0 3. 3 13. 5 1. 0 2. 5 1. 5 1. 5 1. 3 CV 3.4 0.5 3.3 0.6 O . C 3.3 3. 2 1.5 3.9 0.4 0.3 0.2 0. I 0. 2 3.1 3.5 -1.3 2.8 8 . 3 3.1 0.0 0.0 0.0 2.8 e. 0 3. 1 2.5 6. 3 0.5 2.8 8 . 0 0.1 2.3 4. 3 3.2 1.1 1. 3 17.9 0.3 ?. 1 1.5 0.4 3. 1 2.3 . Table K. R E L I A B I L I T Y F C R 28 C H A R A C T E R S M E A S U R E D O F H . P A R V I P L E X U S M E A S U R E M E N T S 1 2 3 C H A R A C T E R ( KAY 1 4 / 7 6 U M A Y 2 1 / 7 6 ) ( J U N 3 / 7 6 ) MEAN MAX K I N S T O D E V V A R [ &NCE C V »*»f«*«»»»»»**e«ti>»«»»**«»«tsr.e»«4»*»*t*i>***»#*<i]»<i<i«i)«*c<i»««*«*«*<i««^ AC 87.5 90. C 85.5 87.7 90.0 85.5 2 . 3 "5. 1 2 . 6 OSL 220.0 225 .0 230.5 2 2 5 . 2 230.5 22 0. 0 5 . 3 2 7. 6 2 . 3 OSw 167 . 3 167.0 192.5 160.6 192 .5 18 7.0 3.2 10. 1 1 . 7 P-L 143.0 14 6. 0 . 140.0 143.7 148.0 140. 0 4 . 0 16. 3 2.3 PW 154.0 159.0 152.0 155.0 159 .0 152.0 3.6 1 3. 3 2 . 3 OL £ Co. 0 795. C 800.0 800.3 8 06 . 0 7^5.0 5.5 3 3. 5 0 . 7 OW 356.5 351.0 356.0 354.5 35o.5 351.0 3 . 1 . . 3 3.9 OAT 111 . 3 191 .0 187.0 166.3 191.0 106.0 2.6 7. 3 1.4 CAC -122.0 -1 30. 0 —12 8.0 -130.0 -128.0 -132.0 2.0 4 . 0 -1.5 ATL 6i:2 .0 667. 0 67b.0 662. 3 607 .0 6 7 o . 0 4 . 5 23. 5 0. 7 AT W 46C.0 4 t l . 0 476.0 400. 2 47o .0 401.0 7.5 56. 3 1 . 6 ATE 2 J 3'/. 0 2326.0 2333.0 2333.3 2339.0 232b.0 5.5 30. 5 0.2 PTL 7H«..0 730. C 74J.0 7 J 8 . 0 744.0 7 3 0 . 3 7,2 52. 0 1.3 PTW 558.0 556. C 570.0 S o l . 3 5 7 0.0 55o . 0 7.6 57. 3 1 . 3 PTE 1225.0 1200.C 1 20 7.0 1210.7 1225.0 1 2 J J . 0 12.9 166. 5 1.1 TO - l i b . O -133.C -133.0 -133.7 -133.0 -128.0 4.0 16. 2 - 3 . 3 THS -253.0 -347.0 -245.0 -340.3 -345.0 - 3 5 J . 0 4.2 17. 3 -1.2 RUF I 5 c 1.0 1550.0 1563.3 1563.7 156 1.0 1550.0 15.9 252. 0 1 . 3 LUF U 1 2 . 0 1612.0 162 6.0 l o l 7 . 3 1620.0 1612.0 9.2 85. 5 3 . 6 RVL • 3303.0 3280.0 3232.0 3305.0 33_>2.0 32JO .0 26.1 6£'0. 0 s.e LVL 4 1 7 3 . 3 4 1 <: 2. 4 1'-: "J . 0 4 l o 5 . 3 . 4160.0 4 1 4 J . 0 19.6 334. 0 3.5 VA 52 7 52 /9.0 5246.0 5267.3 5279.0 5 240 . 0 16.7 280. 0 0 . 3 VE 1 i J I . 9 1090 .0 1105.0 . 1 J9b. 7 1105.0 l 0 9 0.0 7.8 6-1.5 0 . 7 OL 6422.0 6400.0 6400.0 6 4 J 7 . 3 6422 . 0 6430 .0 14. 1 200. 0 0.2 BW 116b.0 1155.C 1170.0 1164.3 1170.0 1155.0 •• 6.2 6 6 . 5 0.7 TT 12.5 8.5 10.0 10. 3 12.5 6. 5 2.0 4. 1 1 9 . 6 EL 22.8 22.e 22. 5 23.0 23.5 22.8 0.4 0. 2 1 . 6 E» 15.0 15.0 14 .0 14. 7 15.0 14.0 0 . 6 0. 3 3 . 9 . O • • « i o o o o r > o < n - « o o o o o o o m o o r i o o o o o o ' A « M Ui • o • z » •a » o — ft. . a' « <? « > » > * 0 * 0 ft • ft • - « ft ft « r> « V ft • U l « _ l • « a * ft • - • ft Z ft _ i ft —• 5 • H 0 E ft 2T ft ft • H ft ft 1/1 ft ft • ft « X « ft ft u. • ft 0 V « < ft 0 s: » •u ft C C « to c < U i « s: it zz » i/> < s tx' U J ft U l £ ft V- » 0 ft < ft f x ft <. ft X ~ ft U 0 ft r - ft co N» « C M m ft ro •» C C » L I a « It. UJ ft «/> » J - — « t-— i- 0 «• r - ft — i>i \ « I A ft U i C M li-—• CC ft _J _ l « U i i/» r> » D. «x -> * UJ — t ac <* * •0 « f- « « 0 » V < J N " 0 - f O < ; j « i f t o j d « J ^ J cJ'rJ d o s o pi 01 r! d —' <M CM .n 11 • - o i i u i n n » i j A i r > n m o ^ n i o o o 1 o c» » 3 »i N H H O N O N N n l N r j r j ^ H ^ H / \ n N O O N O H N O o ^ s ^ o r -0 0 0 0 0 ifi j 3 in ^ r- -T 0- r» M O t~ -t x> -O s* f" ^ C 1 0 O O I A C5 0 O O O D O 0 O n O O O D --> 0 0 0 1 J-1 iA O O 0 . A -O —t r-l O IO 0 r> r - A r - .A <JV ^M 1 r> r» S> U3 Lf> -r O O lA —1 —1 •3 r - r - vr >r O —1 t"M — I • - 4 IA —1 O • r 0 fM <T O IA 0 CO 0 r - 17» i n 1 r-i CM rH CM M* .-M VA m r-( O O O O O O O O O O O O O O O O O o O O .A iA O rg N- 0 O <\ -> «-\ •NJ O lA -J- NT I f J-l O O O CM O =5 CJ T> • j iA r - .•3 IA - J rvj O- r - -O >J- J - ro r l fM O I A O CM Ti O •*» i> lA •O -3 ~) r - j> A 1 rH CM —* r-l r-l CM M- *M tn S\ — 4 m O fM r*. O r - rj CO 1— O O CO r - as • • • • • • • • • • • • • • # • a 4 n r— •J- r— O -r f\J n -0 I ' l *M X )^ cr CM 0 r-— J> -.* •— <V u'i —4 •—1 r- -r -r -J- —1 rg —i I A 0 O IvJ / 0 CT LA 0 j> .1 r - 7- J> 1 r-l CM r—i r-l rH CM vj" -vi l A i n rH o n j - < o o r > o o o o o o o o o o i A O o o o o o o i n o i A i n o ^ t ^ u - . ^ r j « r r - o o o j - > M n j a c 5 o r ~ r l ^ i n o r - i n o ' M n o i m — i r - i o i A — • t-> I A o I M m o P I j m o c j u M M H | r - | C M r—I rH H O , l A - H O O ^ I A O O o o o m o o 0 0 0 0 0 ci 0 0 c> 0 0 0 0 o 0 0 H I < I « 1 H H 0 J l - H O i A O C M - l - O n i u - l A O J v - i r - v T i / , rH fM M* CM L A i n rH r Q rt H > ft <J » i : <-V ft CC « tu • • o • •a ft < « X • O ft 0 O CO O 0 0 O 0 O O O 0 O O O O • • • « • • • • • • • • • • a a a • —* r» 0 in 0 r—i •O O 0 -r .r r - fM 1 ) r- uv u vr vr r— W i-'V —t : M IU r— r— .r «-« <l .1 *H 1 1 J rH V" rvj fM • H M* O rH Cl cr-rH LA O fM M" 30 rvj •."> o o o r> o i n 0 r r-i y o O eg si i* <J H r i nj H 1 > r - iv m l J » O l A - j 3 J 3 « I < J l - - - r - r - r - r - O X O D > > < U J < o o < m o o o o i < < o . i \ ( L h h i . j K j > > •i I r - _ l X ' 3 a~ ui Ul Tab le M. RELIABILITY FCR 28 CHARACTERS MEASURED OF H.TUMIDUS MEASUREMENTS I 2 3 CHARACTER (NOV 9/76MNCV 13/76MNOV 11/76J AC 75?.2 755. J 761 .0 OSL £ 1 0 . 0 616.0 CSW 693.0 fc\ : • L . 693 .0 PL 1 495.3 5 0 J . 0 ?w Ml. 5 466. ) 474.0 CL 1C96.2 I Z95.0 1091.0 Cw 645.0 640.5 6 J 8 . 1 OAT 15 7.5 155.C 159. 3 OAC - 4 3 4 . 7 - 4 3 7 . 0 -430 .5 ATL 1764.5 1760.0 - 1766.0 AT W 1102.5 1100.5 1105.2 ATE 2563.3 . 2590.C 2 59 2.0 PTL 1764.0 1 770.C 1760.3 PTW 1354.5 1348.6 1350.0 PTE 16 0 9.7 16C0.C 1635.0 TU 693.0 690. 0 695.5 TH S 203.5 280.0 265 .1 RUF 2110.5 21C5.2 2103.5 LUF 1056.5 l e s s . o 1C60 .1 RVL 5071.5 5065.0 5073.3 LVL 6174.0 6 17 0.0 6175.0 VA 4 3 4 C . 7 4335.0 4333.7 VF. 945.0 949 . 0 947.6 BL £ 5 6 e. 0 B579.0 856 7. 7 b r f 2929.5 2933.C 2920.0 TT 19.4 17.5 17.5 EL 3d .4 36. 5 37.0 Ew 19.3 19.5 23.3 Mb AN MAX MIN STO DEV VAPIANCE cv 7 5 6.4 761.0 755.0 3.1 9v 5 3.4 6 1 3 . 4 6 1 6 . 0 6 1 0 . 0 3.1 9. 5 0.5 69 5.0 6 9 7 . 0 69 3 . 0 2 . 0 4. 3 3.3 4 9 7. 7 5 0 0 . 0 4 9 5 . 3 2 . 3 3. 5 4 7 1.5 4 7 4 . 0 4 6 0 . 1 2 . 1 9. 5 0 . 7 1 0 9 4 . 1 1096 . 2 109 1. 0 2 . 7 7. 5 3.3 641 . 5 64 5 . 8 636 .1 3.9 15,3 3. 6 •15 7 .3 15 9 . 3 1 5 5 . 0 2 . 2 4. 7 1.* - 4 3 6 . 7 - 4 3 4 . 7 - 4 3 0 . 5 2.3 3. 8 -3.4 1 7 6 j . 5 1 766 . 0 1 7 o 0 . 0 2 . 5 1 2 . 0 0.2 1 1 0 2 . 7 11 J 5 . 2 1100.5 2 . 2 5. 3 3.2 2 5 6 0 . 3 2 5 9 2 . 0 2 5 6 3 . 0 4.9 2 4 . 3 3.2 1 7 6 7 . 4 1 7 7 0 . 0 1 7 6 4 . 0 3.9 1 5 . 5 0 . 2 1351.3 1 3 5 4 . 5 134b.6 5.1 9. 5 3. 2 1 6 0 4 . 9 1 6 0 9 . 7 1600 . 0 4 .9 2 4 . 5 0.2 6 9 2 . 8 6 9 5 . 5 6 9 0 . 0 2 . 7 7. 5 3.4 282 . 9 2 6 5 . 1 280 . 0 2 . 6 6. 8 3.9 2 1 0 6 . 4 2 1 1 0 . 5 2 1 3 3 . 5 4.6 21.0 0.2 1 C 5 7 . 9 I 8 6 0 . 1 1 0 5 5 . 0 3.7 1 3 . 5 3.2 5 3 0 9 . 6 5 0 7 3 . 0 5 06 5 . 0 4 .9 24.0 0. 1 6 1 7 3 . 0 . 6 1 7 5 . 0 6 1 7 0 . 0 5 . 7 3 2 TO 0 . 1 4 3 3 6 . 5 4 3 4 0 . 7 4 3 3 3 . 7 4.3 16. 0 3. 1 9'r 7. 2 94 9 . 0 ' 945 . 0 2.3 4. 0 0.2 6 5 7 1 . 6 85 7 V . 0 856 1. 7 6.3 4 0. 0 3. 1 2 9 3 0 . 8 2 9 3 3 . 0 2 9 2 9 . 5 • 0.3 0. 0 0.0 1 0 . 1 1 9 . 4 1 7 . 5 1.1 1. 2 6.3 3 6 . 6 37.3 3 6.4 3.3 •6.1 0.9 1 9 . 8 2 0 . 0 1 9 . 5 0.3 0. 1 1.3-Tab le N. RELIABILITY FCR 28 CHAP ALTER S MEASURED OF H.UNIPLEXUS ME ASOfEMfcNTS 1 2 3 CHARACTER (KCV 9/7OMMJV IO/7OMNCV I I / 76» Mt A N MAX MIN STD OEV VAPIANCE CV AC 63.3 83.0 81.3 82.4 63.0 ei .3 1.0 1. 0 :.2 OSL 2 3 0.5 227 .C 221.4 229.6 • 231.4 227 .0 2.3 5. 4 1.0 OSx 216.7 215.0 218.0 216.6 218.0 215.0 1.5 2. 3 0.7 PL 119.9 113.0 121.4 119.8 12 1.4 116.0 1.7 2. 9 1.4 Pw 136.3 137.5 140.0 138.6 140.0 13 7.5 1.3 1. 7 0.9 O L 432.9 4 2 1.0 429.6 431.2 432.9 429.6 1.7 2. 8 0.4 CW 121.4 130.0 133.0 131.5 133.0 130.0 1.5 2. 3 1.1 O A T -90.3 -92 .0 -89.0 -90. 3 -69.0 -92 . 0 1.5 2. 3 -1.7 OAC -164.4 -1G6.4 -107.0 -165.9 -164.4 -187.0 1.4 1. 9 - 0 . 7 A f L 441 .0 4 39. 7 . 4^3.5 •44 1 . 4 443 .5 43 9. 7 2.0 2. 8 0.4 ATW 160 .5 166.0 165 .0 165.6 166 . 5 165.0 0.8 0. 6 0.5 ATE 6 5 5.0 658.7 860.0 657.9 OoO . 0 055.0 2.5 6. 5 0. 3 P TL 4 05.0 401. 3 4 C 7 .9 4 04. 7 407 .9 401 .3 3.3 1 1. 0 0.8 PTw 189.0 187.5 193.0 109.6 193.0 l o 7 . 5 2.3 G. 1 1.5 P I E 4 96 .8 495. C . 500.0 49 7.3 500 .0 495 .0 2.5 6. 5 0.5 T O 8 1.0 79.C 61.0 60. 6 81.8- 79.0 1 .4 2. 1 1.8 T H s 27. J 26. 0 25.4 26. 1 27.0 25.4 0.8 0. 7 3.1 RUF ABSENT A IS SENT ABSENT LUF 549.0 54 7 .C 551.0 549.0 551 .0 54 7.0 2.0 4.0 0.4 RVL 232 /.9 23 3 J . 0 2 2 2 4.0 2327.3 2330.0 2324 .0 3.7 14.0 0.2 LVL l o 7 5 . e 1679.C 1673.0 167 3.9 . 1679.0 1673.0 3.9 15.0 0.2 VA 2595.6 2O00.0 2593.4 2596.3 2o00.0 2 59 3.4 4.0 16. 0 0.2 VE 10 0 4.9 1000.C 1006.6 l O O t . 5 1006.6 1000.0 4.3 18. 5 0.4 bL 3c. 66 • 6 3 259.0 3771.1 3432.c 3771.1 3259.0 292 .5 8614 4.0 8.6 BW 711.0 7C6.4 712.0 7J9. 6 712.0 7.^ -,.4 3.0 9. 0 0.4 T T 9.2 C. C 7.5 8. 2 9.2 7.5 0.9 0. 8 10.6 EL 2C.3 20. 5 2 1 .0 20. 8 21.0 20.5 0.3 0. 1 1.2 EW 16.6 16.C 15.5 16.0 16.6 15.5 0.6 0. 3 3.4 « > • K t A i o r / ' t r i ^ O i A - * o g > o • «HJ O O -H O O O •H cf o o o o o r-t r- i M M H n o o i \ i O ' ' r > i M • 1 I rg • U l « o * z * <s « O i A r O O r o r o O O O C O O O O O O rc> •-« • «o CO r o o r v ro o r~ O i - i n -o O r^ r o O ^) r v -O -o o A O "0 r o O O < « •H r-i 1A ^ r o f - l A O r o IM •o r-t • ^  i * - »-< > « r g r-t r-t r v r-t r-t I A I A —1 « gj ro cv • « > ft u i « O « CO o I A r v - A r » o r v i A - A rO ~t — i co • v as o a o O 6 rH rg r n ~H —« o gr r v r » r v CV r o r o 3* •o o r o r o o — i —< r o o o « H r l H M r i ^ I M r v ^< r o rg • «/> * * ft * z>- ft ft X * • UJ ft —1 « ft o o o o O ."> o O O O l A o O CO o o o o o o O o o o o o rs o £ L e z « O « ft r o N o m a o .—< o r s r v vO 75 D — i i n o r s CO o r s <• -g i n r v I A »-« • s: ft O H r v ^ ^ O ' ^ ' i —( •o o o —« r o A c o ~> -t i n •t o -J- r g ~ « CL' « * r-i I A A t v r v -J- m r o i c v -r co i ^ - r o r-l r-t >T r-i O 3D c A i n « r-t ~-t 1 ro r o vT • A <3 —1 > e • • « i X « * « u. m r s o o o r s o o r s o o o o r> r> o o r> rs o r> n • . - i 0 0 0 . 1 D o < * o S O H N J> rr- VA -A r - r v O r v IM A r> A o r o •0 . - i o v 1 r v ro 0 o o ^ m M i f , i r-i rsl —i .0 r v .—i O r~ i — J\ i-O O >o -JO r v r v U l » —« I A m rv r v *r x\ •o 1 r v ~r -o r-i < ^ - . M —< o O i n A o j-> or « rH r-t 1 r o r o s>- >!• I A O - i O « </> * < « U l 3E « X rO O 25 r o r o l ~ r> rO rO O r o r o r~ CO z » CO < « *r lA f - CO O l » CO IA r - CO g-O r o n co —• r v cc r v -0 rO r o o lA O CO <M A or U J ft o H N ^ ^ J* r o —I r v r> J J w-i vT O H O r~ r - A 1 O r l N H U l i . ft •H <A m r v r v c s\ rO 1 r v J - Cf r - r o r-l —< - r — i O X O r IA OJ i - ft 1 r O CO gj •j\ gj — i o ft < ft or. « <i ft X — ft o <e e r- ft CD «v ft t n O O O O D O O o o o o o n o o o o O O o r-t. A o O O i A o ro co ft to e •O r - r> — i w c- > r ^ M P N o r v ^ lA — i o v> e> r o J-i —i vj J - r o o cC ft O r v r v o* j ^ r o <-* r v " O r-i —< A r - o t n I- r o C- • O C CO —1 . M r-t U ft —^  I A I A r v r v A r o 1 <v ^ .Cf l — ro r-4 r-4 v — 1 CJ J - <3 u \ a\ UJ 0 ' u. ft r~4 —< r-t I «o r o vl -1- U l •o -< -> ft >- — ft t~ — ft O ft z r- ft U l >. r o u o c> o cs r> O O o « n o o Cl O o o o O O O t i C) O O O O C J a 3C —• ft U J r V I N C rO r v o I A -O -o A O : v r o o r> —< A o o O CO c r r s O g- r v I A r v ' A u r ft O ^ N H m p n —* M r ) o 3- .—i r o A r - O —t 4- ^» A g- r j O* rH IM rM _ i => >- ft •—t u * l A r g r v g- m r o 1 r v o- co r o •J- r-i o iA I A CO 0» Ui < ft r~i ^1 gj- l A g> -H ct < £ ft Ul «- ft — • o « o r- • v. • Ct o o o o o ~> O n o o o O O r> o rs o O O f 5 r j r. o o o L-> rs <u V" ft •HI r-t « t n r~ co r s -M gj 0- O C< j - i r v -T o r v ^ J> A u , j > ."O r v *v I A f\ •o •H r g <v *r o- r o —< -o < J o r v —I O VA J - m CJ I J Cf A r - l N I M V H >- ft r-t A I A ' V rg gr i A ro 1 c v o- U - ro — i CM CJ IT o l A I A CO U» rt <r • » 4 1 CO ro g; gr gi g> -H H x • w ft • « • • CL' « Ol • ft- • o »< t of • - 1 X 1- O _ l T Ul _ l U l »/» u» U . -I _ i < • O m m J J J J < < 1 - 1 - l — >- o X D r> > > < U J - l b J X X t < o o a o . o o o o < < a a. a i - » - CC - 1 CC - i > > 31 CD 1 - UJ Ul u • Appendix 7. Average measurements (mm)of fl. but tensis maintained at 12°C Age (days) Metacercariae No, of specimens 5 5 13 14 8 21 9 28 11 60 7 0, 69(0,59-0. 77) 0. 74(0. 63-0. 86) 0, 76(0.66-0.82) 0,77(0,64-0, 80) 0, 75(0, 68-0,82) 0, 80(0,79-0, 85) 0, 27(0,21-0, 33) 0.29(0. 23-0. 32) 0. 28(0. 22-0.32) 0. 28(0. 23-0. 31 ) 0. 29(0. 25-0.34) 0. 30(0. 28-0. 36 ) 0.09(0.08-0. 10 ) 0.08(0, 07-0.09) 0.08(0.07-0.09) 0.08(0.07-0.10 ) 0.08(0.07-0.09) 0.08(0.07-0.09) 0.11 (0.09-0.13 ) 0.10(0.09-0.14). 0.11 (0.08-0.15 ) 0.11 (0.09-0.13) 0.12(0.10-0.14) 0.12(0.11-0.15) 0. 10(0. 09-0. 11 ) 0.10(0. 09-0.11 ) 0.10(0.09-0.10 ) 0.11 (0.10-0 .12) 0.11(0.10-0.13) 0.12(0.11-0.13) 1.2 (1.1 -1.3 ) 1.5 (1.3 -1.6 ) 1.5 (1.2-1.7 ) 1.3 (1.2 -1.4 ) 1.5 (1.4 -1.6 ) 1.6 (1.5 -1.7 ) 0.05(0.04-0. 07) 0. 05(0.04-0.08) 0.06(0.05-0.08) 0. 08(0. 06-0..09) 0.08(0.08-0.11 ) 0.09(0.08-0.10 ) 0. 05(0. 04-0. 07) 0. 05(0.04-0.06) 0.06(0.04-0.08) 0. 07(0. 06-0.08) 0.07(0.07-0.09) 0.08(0.07-0.09) N . D . N . D . N .D . N . D . N . D . N . D . N . D . N . D . N . D . N . D . N . D . N . D . 0.02(0.02-0. 03) 0. 02(0.01-0.03). 0.02(0.02-0.03) 0.04(0.03-0.06) 0.07(0.05-0.09) 0.09(0.07-0. 11 ) 0. 03(0.02-0. 04) 0. 05(0.04-0.06) 0. 06(0.04-0.07) 0.08(0.06-0.09) 0.10 (0. 08-0.12 ) .0. 14 (0.11 -0. 20) A . T . from eno h 0. 02(0. 01 -0. 03) 0.04(0.02-0.07) 0.07(0.05-0.09) 0.09(0.08-0.11 ) 0.13(0.09-0.15 ) 0. 24(0.20-0. 26), i Body length Body width Acet. diam. a O.S. diam. ' 5 O. S. length0 O/A ratio Phyn. diam.** Phyn. length Ovary diam. Ovary length A. T. Diam. f A. T. length 8 P.. T. diam. T \ T. length^ 0.03(0.020.04; 0. 05(0. 04-0.07) 0. 06(0. 04-0.08) 0.06(0.05-0.08) 0.07(0.05-0.09) 0.09(0.06-0. 11 ) 0. 04(0.03-0.05) 0. 06(0.05-0.07) 0.07(0.05-0.08) 0.09(0.06-0.10 ) 0.09(0.07-0. 12 ) 0.13(0.11 -0. 17 ) P . T . from end 0. 01(0.01-0. 02) 0. 03(0.01-0.04) 0.05(0.03-0.07) 0.07(0.05-0.0?) 0.08(0.06-0.11 ) 0.11 (0.10-t). 13 ) a Diameter of acetabulum b Diameter of oral sucker c Length of oral sucker d Diameter of pharynx e Length of pharynx f Diameter of anterior testis g Length of anterior testis h Distance of anterior testis from posterior end of worm i Diameter of posterior testis j Length of posterior testis k Distance of posterior testis from posterior end of worm N . D. Not developed Appendix 8. Average measurements (mm) of i i - b u t t e n s i s m a i n t a i n e d a t 20°C Age (days) No. of specimens 5 21 14 18 21 19 28 11 60 14 Body length Body width Acet. diam. a O. S. diam.' 5 O. S. l e n g t h 0 O/A ratio Phyn. diam. Phyn. length O v a r y diam. O v a r y length A. T. diam. ^  A. T. l e n g t h 8 A. T. f r o m end^ P. T. d i a m . 1 P. T. length 5 P. T. f r o m end*1 1.06(0. 94-1. 20) 0. 35(0. 33-0. 37) 0. 05(0. 04-0. 06) 0.12(0.11 -0.13) 0. 11 (0. 10-0.13 ) 2. 3 (2. 2 -2. 6 ) 0. 09(0. 07-0. 11 ) 0. 08(0. 06-0.11 ) 0. 07(0.06-0.09) 0. 08(0. 06-0.11 ) 0. 09(0.06-0.11 ) 0. 14(0. 11 -0. 21) 0. 24(0. 21-0. 27) 0. 08(0. 06-0.11) 0.13(0.11 -0.17) 0.12(0.09-0.13) . 1. 78(1.73 0. 51(0.48-0. 07(0. 06-0.18(0.17.-0.16(0.14-2.6 (2.5 -0.14(0.12-0.12(0.10-0.18(0.14-0. 25(0. 20-0. 24(0. 20-0. 35(0.29-0. 39(0. 33-0.24( 0.18-0. 32(0. 25-0.17(0.15-.-1. 88 ) -0.53) -0.08) -0.20) -0.19 ) -2.8 ) -0.16 ) •0.14 ) -0.20) •0.31 ) •0.29) 0. 39) 0.45) 0. 28) 0. 32) 0.19) 3. 27(2. 66-0.75(0. 70-0. 08(0. 07-0.22(0.19-0.21(0.19-2.5 (2.2 -0.16(0.14-0.13(0.12-0.23(0.14-0.49(0. 33-0. 35(0. 26-0. 63(0. 52-0. 80(0. 45-0. 39(0. 27-0. 66(0. 53-0. 64(0. 39-•3. 74) 0. 84) 0.09) 0. 25) 0. 23) 2.7 ) -.18) 0.16) 0. 36) 0. 51) 0. 39) 0. 75) 1.06) 0. 52) 0. 78) 1. 26) 4.10(3 0. 81(0. 0.11 (0. 0. 26(0. 0. 24(0. 2. 5 (2. 0.17(0. 0.15(0. 0. 29(0. 0. 56(0. 0. 44(0. 0. 62(0. 1.12(0. 0. 46(0. 0. 77(0. 0. 51(0. . 55-4.43) 77-0.84) 09-0.12 ) 24-0.28) 23-0. 26) 3 -2.7 ) 16-0.19 ) 13-0.21 ) 26-0. 34) 50-0.63) 41-0.49) 57-0.68) 83-1.39 ) 3.6-0. 54) 66-0.91) 26-0. 63) 6. 30(5. 78 1.40(1. 22 0.11 (0. 09 0.29(0.25 0. 25(0. 22 2.6 (2. 5 -0. 21(0.18 0.20(0.17 0. 38(0. 33 0. 72(0. 70 0.49(0. 33 1.15 (1.07' 1.79(1.48 0. 52(0.41-1. 23(1.17 • 1.01 (0.92-7.01) -1.52 ) -0.13 ) -0.33) -0.29) 2.7 ) -0.24) -0.24) -0. 42) •0.75) •0.61) • 1. 31) •2.25) •0. 66) 1.35) 1.33) a D i a m e t e r of acetabulum b Diameter of o r a l sucker c Length of o r a l sucker d Diameter of pharynx e Length of pharynx f D i a m e t e r of anterior testis g Length of anterior testis h Distance of anterior testis f r o m p o s t e r i o r end of worm i Diameter of p o s t e r i o r testis j Length of p o s t e r i o r testis k Distance of po s t e r i o r testis f r o m p o s t e r i o r end of worm A p p e n d i x 9.Average measurements (mm) of H . b u t t e n s i s m a i n t a i n e d a t 27°C. Age (days) No. of specimens 14 21 28 60 17 19 13 14 10 -Body length Body width a O. S. diam. ^  O.S. l e n g t h 0 O/A ratio Phyn. diam. ^  Phyn. length Ovar y diam. O v a r y length A. T. diam. * A. T. l e n g t h g A. T. f r o m end P. T. diam. * P.T. length J P. T. f r o m end 1.98(1.85-2 .12) 3.71(3. 69-3.83) 3.96(3.88-4.05) 4. 63(4. 27-4. 88) 7.40(6.83-7.94) 0. 37(0.32-0.41 ) 0. 49(0. 44-0. 52) 0. 75(0. 69-0.87) 0. 79-0.76-0. 83) i . 4?-(1. 25-1. 59 ) 0.06(0.04-0.07) 0.07(0.05-0.08) 0.08(0.07-0.09) 0.10(0.08-0.13 ) 0.12(0.08-0.14 ) 0.13(0.11 -0.14 ) 0.17(0.15-0. 19 ) 0.23(0.18-0. 26) 0. 27(0.25-0. 29) 0. 31(0.26-0. 33) 0.12(0.09-0.14 ) 0.17(0.14-0.18 ) 0. 22(0. 20-0. 24) 0. 25(0. 23-0.27) 0. 26(0.23-0. 28) 2.6 (2.1 -2.8 ) 2.7 (2.4 -3.0 ) 2.7 (2.6 -2.9 ) 2.8 (2.2 -3.0 ) 2.8 (2.4 -3.1 ) 0. 08(0.07-0.10 ) 0.12(0.10-0.15 ) 0.14(0.13 -0. 17 ) 0. 16(0.14-0.21 ) 0. 19(0.17 -0. 23) 0. 07(0.06-0.10 ) 0.10(0.09-0. 13 ) 0.12 (0.11-0. 16 ) 0. 13(0.11 -0.18 ) 0.17(0.15-0. 21 ) 0. 06(0.05-0.08) 0.16(0.13-0.18 ) 0.19(0.14-0. 24) 0. 23(0. 20-0. 31 ) 0. 32(0 . 29-0. 37) 0.07(0.06-0.10 ) 0.22(0.19-0.27) 0.44(0.31-0.50) 0.50(0.43-0.59) 0.65(0.61-0.70) 0.07(0.05-0.08) 0. 20(0.18-0.27) 0.29(0. 25-0. 33) 0.37(0. 34-0.42) 0.40(0. 31-0. 53) 0.12(0.09-0.18 ) 0. 29(0. 26-0. 35) 0.54(0.49-0.61 ) 0. 51(0.47-0. 59) .0.99(0.93-1. 17 ) 0. 68(0.63-0.74) 1. 14(1.11 -1.20) 2.35(2.29-2.41 ) 3. 29(3.01-3.42) 5. 31(5.17 -5.42) 0. 07(0.05-0.09) 0.19(0.16-0. 23) 0.32(0. 24-0. 47) 0. 38(0.27-0.43) 0. 44(0.39-0. 54) 0. 11(0.08-0.15 ) 0. 27(0. 21-0.29) 0.54(0. 48-0.63) 0. 65(0.56-0.79) 1.04(0.96- 1.17 ) 0. 35(0.29-0.41 ) 0.48(0. 39-0.53) 1.90(1.78-2.11 ) 1.52(0.79-1. 88 ) 3.10 (2.95-3. 22) a Diameter of acetabulum b Diameter of o r a l sucker c Length of o r a l sucker d Diameter of pharynx e Length of pharynx f Diameter of anterior testis g Length of anterior testis h Distance of anterior testis f r o m p o s t e r i o r end of worm i Diameter of p o s t e r i o r testis j Length of p o s t e r i o r testis k Distance of p o s t e r i o r testis f r o m p o s t e r i o r end of worm Appendix 10. Average measurements of H. buttensis of varying ages developed ~ r i n Rana pretiosa having a snout-vent length of 30-35 ram. Age (days) 5 14 21 • 28 60 No. of specimens 2 6 2 1 23 19 18 Body length 1.25(1.21-1.33) 1.81(1.78-1.98) 3.33(3.17-3.63) 3.88(3.77-3-95) 6.46(6.28-6.77) Body width 0.38(0.35-0.52) 0.50(0.57-0.57) 0.73(0.63-0.86) 0.83(0.80-0.94) 1.43(1.34-1.57) Acetabulum diam. 0.06(0.06-0.07) 0.07(0.07-0.08) 0.09(0.08-0.09.) 0.11(0.10-0.12) 0.11(0.10-1.13) Oral Sucker diam. 0.13(0.12-0.14) 0.18(0.17-0.19) 0 . 23(0 . 23-0.27) 0.26(0.23-0.27) 0.28(0.26-0.30) Oral Sucker length 0.12(0.09-0.13) 0.17(0.16-0.18) 0.22(0.19-0.23) 0 . 24(0 . 23-0.26) 0.25(0.23-0.26) 0/A Ratio 2.4 (2.3 -2 . 5 ) 2.5 (2.4 -2.7 ) 2.5 (2.4 -2.7 ) 2.6 (2.3 -2.7 ) 2.6 (2.4 -2.8 ) Ovary width 0.08(0.07-0.09) 0.17(0.15-0.18) 0.26(0.24-0.28) 0.30(0.28-0.33) '0.39(0.37-0.41) Ovary length 0.08(0.06-0.09) 0.24(0.22-0.25) 0.48(0.46-0.49) 0.56(0.55-0.59) 0.73(0.68-0.74) Ant. Testis width 0.10(0.09-0.11) 0.25(0.22-0.28) 0.33(0.28-0.35) 0.39(0.38-0.42) 0.46(0.43-0.49) Ant. Testis length 0.14(0.13-0.18) 0.35(0.32-0.37) 0.58(0.54-0.60) 0.62(0.58-0.63) 1.17(1.11-1.19) Post. Testis width 0.09(0.08-0.10) 0.24(0.21-0.26) 0.42(0.39-0.43) 0.47(0.46-0.49) .0.53(0.49-0.54) Post. Testis length 0.14(0.13-0.16) 0.33(0.30-0.34) 0.67(0.63-0.69) 0.74(0.72-0.77) 1.29(1.24-1.33) A l l measurements in millimeters. Appendix 11. Average measurements of fl. buttenoi8 of varying ages developed i n Rana pretiosa having a snout-vent length of 45-50 mm. Age (days) No. of specimens 5 22 14 23 21 2 i 28 26 60 25 Body Length l : l l ( l . C ? - i . i y ; Body Width 0.35(0.33-0.38 Acetabulum diam. 0.05(0.05-0.06 Oral Sucker diam. 0.14(0.13-0.15 Oral Sucker length 0.12(0.11-0.14 0/A Ratio 2.4 (2.3 -2.5 Ovary width 0.09(0.08-0.10 Ovary length 0.83(0.07-0.10 Ant. Testis width 0.09(0.08-0.10 Ant. Testis length 0.13(0.13-0.17 Post. Testis width 0.09(0.07-0.10 Post. Testis length 0.13(0.12-0.14 1*77(1.62-1.79 0 . 5 4 ( 0 . 4 6 - 0 . 5 7 0.07(0.07-0.08 0.19(0.17-6.20 0.16(0.15-0.17 2 . 5 ( 2 . 4 - 2 . 7 0 . 1 8 ( 0 . 1 7 - 0 . 2 0 0 . 2 4 ( 0 . 2 3 - 0 . 2 6 0 . 2 2 ( 0 . 2 1 - 0 . 2 5 0 . 3 6 ( 0 . 3 5 - 0 . 4 0 0 . 2 2 ( 0 . 2 1 - 0 . 2 5 0 . 3 2 ( 0 . 3 2 - 0 . 3 5 3.12(2.88-3.37 0.70(0.58-0.87 0.09(0.0'8-0.10 0.22(0.20-0.24 0.21(0.18-0.21 2.5 (2.3 -2.6 0.24(0.23-0.27 0.48(0.45-0.50 0.35(0.32-0.38 0.63(0.61-0.66 0.38(0.35-0.39 0.66(0.63-0.67 3.9K3.84-4.05) 0.85(0.77-0.92) 0.11(0.09-0.12) 0 .26(0 .25-0 .27) 0 . 2 4(0 . 2 4-0 . 2 6 ) 2 .5 {2-4 - 2 . 6 ) 0.29(0.28-0.31) 0.56(0.54-0.57) 0.42(0.41-0.44) 0.64(0.62-0.67) 0 .44(0 .42-0.45) 0 . 74(0 . 72-0 . 75) 6.11(5.83-1.35(1.06-0.12(0.10-0.28(0.26-0.26(0.23-2.6 (2.4 -0.36(0.34-0.71(0.68-0.51(0.48-1.14(1.06-0.53(0.49-1.24(1.20-6.72) 1 .48) 0 .13) 0 . 3 0 ) 0 .27) 2 .7 ) 0 . 3 8 ) 0 . 7 4 ) 0 . 5 3 ) 1 .19) O.54) 1.25) A l l measurements i n millimeters. Appendix 12. Average measurements of H. buttensis of varying ages developed in Rana pretiosa having a snout-vent length of 55-60 mm. Age (days) N o . of specimens 5 18 14 16 21 24 28 17 60 17 Body length Body width Acetabulum diam. Oral Sucker diam. Oral Sucker length 0/A Ratio Ovary width Ovary length Ant. Testis width Ant. Testis length Post. Testis width Post. Testis length 1.26(1.24-0.37(0.34-0.06(0.06-0.12(0.12-0.12(0.11-2.5i(2.4 -0.08(0.08-0.09(0.08-0.12(0.11-0.14(0.12-0.10(0.10-0.15(0.13-1.32) 0.38) 0.07) 0.13) 0.12) 2.6 ) 0.09) 0.10) 0.14) 0.16) 0.13) 0.16) 1.76(1.75-0.47(0.45-0.08(0.07-0.17(0.15-0.18(0.16-2.5 (2.3 • 0.18(0.16-0.25(0.23-0.25(0.23-0.34(0.32-0.25(0.22-0.34(0.33-1.84) 0.50) •0.08) 0.18) 0.18) •2.7 ) •0.20) •0.26) •0.26) 0.38) 0.26) •0.36) 3-31(3.29-0.69(0.63-0.09(0.08-0.24(0.23-0.21(0.20-2.6 (2.3 • 0.25(0.21-0.47(0.45-0.33(0.31-0.57(0.55-0.41(0.40-0.67(0.64-•3.47 0.71 0.10 0.25 0.22 2.6 0.27 0.48 0.36 0.60 •0.44 0.67 3.79(3.63-3.99) 6.44(5.87-6.75) 0.80(0.73-0.91) 1.37(1.22-1.43) 0.10(0.09-0.11) 0.11(0.10-0.12) 0.25(0.25-0.27) 0.28(0.26-0.29) 0.24(0.23-0.26) 0.25(0.24-0.28) 2.7 (2.4 -2.8 ) 2.6 (2.5 -2.7 ) 0.32(0.29-0.33) .0.38(0.37-0.40) 0.56(0.54-0.57) 0.71(0.66-0.72) 0.39(0.37-0-/,!) 0.46(0.41-0.49) 0.63(0.61-0.64) 1.16(1.10.-1.21) 0.46(0.43-0.47) 0.52(0.49-0.53) 0.74(0.71-0.77) 1.26(1.21-1.26) A l l measurements i n millimeters. Appendix 13. Average measurements of H. buttensis of varying ages developed i n Rana pretiosa having a snout-vent length of 65-70 mm. Age (days) 5 14 21 28 60 No. of specimens 17 26 15 !9 21 Body length 1.25(1.15-1.30) 1.78(1.69-1.80) 3.30(2.97-3.51) 3.86(3.72-4.10) 6.44(6.00-6.99) Body width 0.36(0.35-0.37) 0.49(0.42-0.52) 0.69(0.52-0.83) 0.82(0.71-0.93) 1.38(1.19-1.52) Acetabulum diam. 0.06(0.06-0.07) 0.07(0.06-0.08) 0.09(0.07-0.09) 0.10(0.09-0.10) 0.10(0.09-0.11) Oral Sucker diam. 0.13(0.12-0.13) 0.17(0.16-0.18) 0.23(0.20-0.24) 0.25(0.23-0.26) 0.27(0.25-0.28) Oral Sucker length 0.11(0.10-0.15) 0.17(0.17-0.19) 0.21(0.20-0.22) 0.24(0.22-0.25) 0.25(0.22-0.27) 0/A Ratio 2.3 (2.1 -2.4 ) 2.4.(2.3 *2.6 ) 2.5 (2.3 -2.7 ) 2.6 (2.5 -2.7 ) 2.5 (2.2 -2.6 ) Ovary width 0.08(0.07-0.09) 0.17(0.15-0.19) 0.25(0.21-0.28) 0.32(0.30-0.35) • 0.38(0.35-0.42) Ovary length 0.09(0.07-0.10) 0.24(0.22-0.26) 0.46(0.45-0.48) 0.55(0.54-0.58) 0.70(0.65-0.72) Ant. Testis width 0.11(0.09-0.12) 0.24(0.22-0.26) 0.32(0.31-0.36) 0.38(0.35-0.40) 0.45(0.43-0.49) Ant. Testis length 0.13(0.12-0.17) 0.34(0.31-0.37) 0.57(0.52-0.58) 0.62(0.59-0.65) 1.15(1.04-1.18) Post. Testis width 0.09(0.08-0.10) 0.24(0.20-0.26) 0.40(0.37-0.42) 0.47(0.44-0.49) 0.52(0.50-0.55) Post. Testis length 0.14(0.12-0.17) 0.33(0.31-0.35) 0.66(0.64-0.68) 0.74(0.72-0.76) 1.27(1.21-1.28) A l l measurements in millimeters. Appendix 14* Average measurements of H. buttensis of varying ages developed i n male Rana pretiosa having a snout-vent length of 50 mm. Age (days) 5 14 21. 28 60 No. of specimens 22 23 26 19 25 Body length 1.06(0.954-1.18) 1.78(1.59-1.88) 3.00(2.66-3.57) 3.96(3.78-4.61) 6.48(5.83-6.78) Body width 0.33(0.32-0.36) 0.55(0.48-0.62) 0.66(0.51-0.83) 0.82(0.78-0.87) 1.26(1.18-1.49) Acetabulum diam. 0.06(0.06-0.07) 0.08(0.07-0.08) 0.08(0.07-0.08) 0.12(0.11-0.13) 0.13(0.13-0.15) Oral Sucker diam. 0.12(0.11-0.12) 0.18(0.15-0.18) 0.23(0.22-0.25) 0.27(0.26-0.28) 0.29(0.27-0.32) Oral Sucker length 0.10(0.09-0.11) 0.17(0.16-0.18) 0.23(0.21-0.24) 0.25(0.24-0.27) 0.27(0.26-0.29) 0/A Ratio 2.4 (2.3 -2.6 ) 2.4 (2.3 -2.7 ) 2.6 (2.4 -2.7 ) 2.6 (2.5 -2.8 ) 2.6 (2.5 -2.7 ) Ovary width 0.09(0.08-0.10) 0.18(0.13-0.26) 0.24(0.15-0.36) 0.28(0.21-0.33)- 0.37(0.30-0.40) Ovary length 0.10(0.09-0.11) 0.26(0.25-0.36) 0.52(0.48-0.65) 0.54(0.48-0.61) 0.74(0.72-0.76) Ant. Testis width 0.12(0.09-0.13) 0.24(0.22-0.28) 0.35(0.29-0.51) 0.43(0.35-0.47) 0.50(0.46-0.61) Ant. Testis length 0.12(0.11-0.18) 0.35(0.28-0.37) 0.65(0.55-0.72) 0.63(0.58-0.70) 1.16(1.10-1.37) Post. Testis width 0.09(0.07-0.11) 0.23(0.20-0.30) 0.38(0.28-0.47) 0.45(0.36-0.54) 0.54(0.48-0.62) Post. Testis length 0.14(0.12-0.17) 0.32(0.29-0.56) 0.66(0.52-0.69) 0.79(0.68-0.93) 1.36(1.20-1.46) A l l measurements i n millimeters. Appendix 15* Average measurements of H. buttensis of varying ages developed in female Rana pretiosa having a snout-vent length of 50 mm. Age (days) .5 14 21 28 6 0 No. of specimena 27 24 19 21 23 Body length 1.09(1.04--1.22) 1.83(1.71-1.94) 3.09(2.75--3.49) 4.08(3.63--4.45) 6.51(5.93-6.81) Body width 0.35(0.32--0.36) 0.57(0.50-0.60) 0.67(0.53--0.83) 0.84(0.76--0.88) 1.33(1.27-1.51) Acetabulum diam. 0.07(0.06--0.08) 0.08(0.07-0.09) 0.09(0.08--0.09) 0.12(0.12--0.13) 0.13(0.12-0.14) Oral Sucker diam. 0.12(0.12--0.13) 0.18(0.16-0.19) 0.23(0.22--0.25) 0.26(0.26--0.28) 0.29(0.26-0.30) Oral Sucker length 0.10(0.09-•0.11) 0.17(0.16-0.18) 0.23(0.20--0.24) 0.26(0.24--0.27) 0.27(0.26-0.29) 0/A Ratio 2.3 (2.2 -•2.5 ) 2.5 (2.3 -2.7 ) 2.5 (2.4 -•2.7 ) 2.6 (2.4 -•2.7 ) 2.6 (2.5 -2.8 ) Ovary width 0.09(0.08-•0.10) 0.18(0.14-0.27) 0.24(0.16-•0.34) 0.27(0.22-•0.32) 0.37(0.28-0.39) Ovary length 0.10(0.09-•0.11) 0.25(0.25-0.34) 0.50(0.47-•0.63) 0.54(0.46-•0.59) 0.72(0.68-0.73) Ant. Testis width 0.12(0.10-•0.13) 0.24(0.21-0.26) 0.36(0.31-•0.50) 0.44(0.37-•0.47) 0,52(0.48-0.59) Ant. Testis ieug-h 0.12(0.12-0.17) 0.37(0.29-0.38) 0.64(0.54-•0.74) 0.62(0.57-•0.68) 1.15(1.13-1.36) Post. Testis width 0.09(0.07-0.11) 0.24(0.22-0.32) 0.40(0.29-•0.48) 0.46(0.38-•0.52) 0.55(0.45-0.58) Post. Testis length 0.15(0.13-0.17) 0.33(0.31-0.39) 0.67(0.55-0.67) 0.80(0.69-0.89) 1.40(1.25-1.53) A l l measurements in millimeters. Appendix 16 Effects of crowding on 60-day-old Haematoloechus buttensis in Rana pretiosa. # worms # gravid worms % gravid worms Frog Number of # worms % per lung per lung per lung * Average body • no. m e t a c e r c a r i a e recovered survival Right Left Right Left Right Left size (LxW)/2 1 10 7 70.0 3 4 3 4 100 100 3. 72(2. 38-5. 29) 2 10 10 100 7 3 7 3 100 100 3.92(3. 26-5.07) 3 10 6 60.0 4 2 4 2 100 100 3. 65(3. 62-4. 37) 4 20 16 80.0 8 8 6 8 75. 0 100 3. 29(2. 74-3.91) 5 • 20 15 75.0 9 6 7 6 77. 8 100 3. 31(2.13-4. 73) 6 20 15 75.0 8 7 8 7 100 100 3.47(2. 86-4. 55) 7 40 25 62. 5 14 11 10 7 71.4 63.6 2.80(2.25-3. 33) 8 40 27 67. 5 9 18 8 11 88.9 61.1 3;07(2. 60-3.75) 9 40 32 80.0 18 14 10 9 55. 6 64. 3 2. 35.1. 63-3. 53) ! J*i 80 43 53. 8 20 23 12 12 60.0 52.2 1.99(1. 73-2. 43) 11 80 27 33. 8 11 16 9 7 81. 8 43.8 l .8C(l. 31-2. 50) 12 80 52 65.0 31 21 11 15 35. 5 71.4 2.05(1. 66-2. 63) 13 160 85 53.1 50 35 23 19 46. 6 54. 3 1.20(0.89-1.47) 14 160 101 63.1 49 52 17 21 34.7 40.4 1.27(0198-1. 67) 15 160 120 75.0 49 71 i  19 12 38. 8 16.9 1.32(1.00-1. 80) Measurements ln square millimeters. A p p e n d i x 17. Average measurements of H. buttensis of varying ages developed in Rana pretiosa. Age (days) 5 14 21 28 60 No. of specimens 21 19 18 16 18 Acetabulum 0.054(0.053-0.056) 0.069(0.065-0.075) 0.088(0.076-0. 096) 0.098(0.090-0.109 ) 0.104(0.096-0. 110 ) Oral sucker 0.119 (0.115 -0.123 ) 0.169(0.160-0.181 ) 0. 211 (0.199-0. 227) 0. 237(0.229-0. 243) 0.241(0.211 -0. 263) length Oral sucker 0.129(0.120-0.135 ) 0.180(0.175-0.190 ) 0. 230(0.198-0. 253) 0.251(0. 236-0. 267) 0. 273(0. 250-0. 286) width OI A ratio 2.4 (2.2 -2.6 ) 2.6 (2.4 -2.7 ) 2.6 (2.5 -2.8 ) 2.6 (2.5 -2.7 ) 2.6 (2.5 -2.7 ) Ovary length 0.082(0.067-0.097) 0.243(0.211 -0. 301 ) 0. 501 (;0=. 438-0. 529) 0. 553(0. 522-0. 603) 0. 723(0. 691-0. 730) Ovary width 0.079(0.071-0.089) 0.171 (0.153.-0.190 ) 0. 246(0.163-0. 307) 0. 285(0.231-0. 337) 0. 3.72(0. 337-0. 396) Ant. testis 0.136(0.117 -0.168) 0. 338(0.299-0. 365) 0.611 (0. 526-0.730) 0. 608(0. 567-0. 667) 1.147 (1.069-1. 237 ) length Ant. testis 0. 08f.<0. 065-0.101 ) 0.235(0. 201-0. 273) 0. 339(0.253-0.401 ) 0.411 (0. 359-0. 467/ 0. 174(0. 380-0. width 540) Post:, tcatis 0.138(0.127-0.151 ) 0. 331(0. 288-0. 350) 0.669(0.597-0. 745) 0.745(0. 680-0.837) 1.263(1. 216 -1. 300 ) length Post, testis 0.079(0.059-0.101 ) 0.233(0.200-0. 279) 0.402(0. 277-0. 513 ) 0.474(0. 400-0. 521 ) 0. 528(0.440-0. 637) width Body length 1.061(0.979-1. 200 ) 1.777(1.726-1. 821 ) 3.265(2.760-3. 715 ) 3.918(3. 650-4. 300) 6.413(5. 780-7.005) Body width 0. 352(0. 329-0. 757) 0.507(0.480-0. 587) 0. 746(0.689-0.987) 0.809(0. 771-0. 850) 1. 399(1. 220-1. 518 ) Egg length — - 0.017(0.016-0. 018 ) 0.024(0.020-0. 02 9) 0.023(0.022-0. 024) 0.023(0.022-0. 025) Egg width --- 0.016(0.015-0.017) 0.017(0.016-0.018) 0.018(0.017-0.019) 0.018(0.0/7-0.018) A l l measurements in millimeters. Appendix 18. Average measurements of H . buttensis of varying ages developed in Rana aurora, Age (days) No. of specimens 1 4 2 1 2 8 60 17 1 3 1 4 1 5 11 Acetabulum Oral sucker length Oral sucker width O/A ratio Ovary length Ovary width Ant. testis length Ant. testis width Post testis length Poet, testis width Body length Body width Egg length Egg width 0 . 0 6 1 ( 0 . 0 5 5 - 9 . 0 6 3 ) 0 . 1 3 3 ( 0 . 1 2 6 - 0 . 1 4 2 ) 0 . 1 4 5 ( 0 . 1 3 7 - 0 . 1 5 8 ) 2 . 4 Absent Absent 0 . 0 8 4 ( 0 . 0 5 2 - 0 . 1 1 9 ) 0 . 0 6 5 ( 0 . 0 4 4 - 0 . 0 8 2 ) 0 . 1 3 3 ( 0 , 1 1 9 - 0 . :Si; 0 . 0 4 8 ( 0 . 0 3 3 - 0 . 0 7 3 ) 0 . 8 0 2 ( 0 . 7 5 3 - 0 . 9 4 8 ) 0 . 2 6 5 ( 0 . 2 5 7 - 0 . 2 8 8 ) Absent Absent 0 . 0 7 7 ( 0 . 0 6 9 - 0 . 0 8 1 ) 0 . 1 8 9 ( 0 . 1 7 0 - 0 . 2 0 4 ) 0 . 2 0 1 ( 0 . 1 8 8 - 0 . 2 0 6 ) 2 . 6 Absent Absent 0.212 ( 0 . 1 8 8 - 0 . 2 5 3 ) 0 . 1 6 5 ( 0 . 1 2 8 - 0 . 2 0 3 ) 0 . 3 - 4 ( 0 . 2 6 ^ - 0 . 3 5 2 ) 0 . 1 3 3 ( 0 . 0 9 6 - 0 . 1 7 5 ) 1 . 4 3 9 ( 1 . 2 6 7 - 1 . 6 8 0 ) 0 . 3 7 8 ( 0 . 3 6 4 - 0 . 3 9 7 ) Absent Absent 0 . 0 9 9 ( 0 . 0 8 7 - 0 . 1 0 3 ) 0 . 2 3 7 ( 0 . 2 1 2 - 0 . 2 4 3 ) 0 . 2 5 8 ( 0 . 2 2 5 - 0 . 2 7 9 ? 2 . 6 0 . 2 0 3 ( 0 . 1 4 8 - 0 . 2 1 3 ) 0 . 0 7 5 ( 0 . 0 6 6 - 0 . 0 8 2 ) 0 . 3 9 0 ( 0 . 2 8 8 - 0 . 4 2 7 ) 0 . 2 5 4 ( 0 . 2 1 3 - 0 . 3 0 4 ) 0 . 6 7 5 ( 0 . 5 8 3 - 0 . 7 9 7 ) 0 . 2 2 5 ( 0 . 1 8 8 - 0 . 2 6 4 ) 2 . 3 1 9 ( 1 . 9 2 6 - 2 . 5 5 1 ) 0 . 5 5 0 ( 0 . 4 9 7 - 0 . 6 2 2 ) Absent Absent 0 . 1 1 0 ( 0 . 1 0 1 - 0 . 1 2 1 ) 0 . 2 6 5 ( 0 . 2 8 7 - 0 . 2 8 0 ) 0 . 2 8 1 ( 0 . 2 5 0 - 0 . 2 8 9 ) 2 . 6 0 . 3 8 8 ( 0 . 3 2 0 - 0 . 4 1 6 ) 0 . 1 1 3 ( 0 . 0 8 7 - O . 1 2 1 ) 0 . 4 1 2 ( 0 . 3 3 9 - 0 . 4 8 0 ) 0 . 2 8 1 ( 0 . 2 6 5 - 0 . 3 1 3 ) 0 . 7 7 8 ( 0 . 7 1 9 - 0 . 8 5 0 ) 0 . 2 4 0 ( 0 . 2 0 3 - 0 . 2 8 8 ) 3 . 2 7 8 ( 2 . 9 8 1 - 3 . 4 6 4 ) 0 . 6 3 4 ( 0 . 5 8 1 - 0 . 7 1 4 ) 0 .019(0. 0 1 7 - 0 . 0 2 0 ) 0 . 0 1 6 ( 0 . 0 1 5 - 0 . 0 1 8 ) 0 . 111 ( 0 . 0 9 9 -0 . 2 7 0 ( 0 . 2 5 0 -0 . 3 0 6 ( 0 . 2 6 7 -2 . 8 0 . 4 4 8 ( 0 . 3 9 2 -0 . 1 2 6 ( 0 . 1 1 3 • 0 . 7 0 6 ( 0 . 6 1 9 6. 3 6 1 ( 0 . 3 0 5 1. 3 2 5 ( 1 . 2 4 7 -0 . 3 1 2 ( 0 . 2 7 6 4 . 9 5 1 ( 4 . 3 2 0 1 . 2 0 2 ( 0 . 9 9 5 0 . 0 2 0 ( 0 . 0 1 8 0 . 0 1 7 ( 0 . 0 1 6 0.122) 0.291) 0. 322) 0. 506) •0.136) -0.785) -0. 389) •1.381) •0.370) -5.219 ) -1. 367 ) -0.022) -0.017 . A l l measurements In millimeters. Appendix 19.A - -t . „ ' Average measurements of tJ. buttensis of varying ages developed in Rana clamitans. Age (days) 5 14 21 28 60_ No, of specimena 14 12 15 13 9 Acetabulum 0.062(0.057-0.065) 0.080(0.071-0.087) 0.101 (0.093-0. 105) 0 . 112 ( 0 . 1 0 5 -•0 .117 ) 0.119 ( 0 . 1 0 9 -0.123) Oral sucker 0 . 1 3 6 ( 0 . 1 2 8 - 0 . 1 4 2 ) 0 . 1 9 3 ( 0 . 1 5 7 - 0 . 201 ) 0 . 2 4 1 ( 0 . 2 3 5 - 0 . 2 4 1 ) 0 . 2 7 0 ( 0 . 2 6 2 - 0 . 2 9 1 ) 0. 2 7 5 ( 0 . 2 7 0 -0.273) length Oral sucker 0 . 1 4 7 ( 0 . 1 3 9 - 0 . 1 5 2 ) 0 . 2 0 5 ( 0 . 1 9 0 - 0 . 211 ) 0 . 2 6 2 ( 0 . 2 5 7 - 0 . 2 6 6 ) 0 . 2 8 6 ( 0 . 2 7 0 - 0 . 2 9 5 ) 0 . 3 1 2 ( 0 . 3 0 0 - 0 . 3 2 4 ) width O/A ratio 2 .4 2. 6 2 . 6 2 . 6 2 . 6 Ovary length Absent Absent 0 . 5 2 1 ( 0 . 4 7 3 - 0 . 5 6 2 ) 0 . 6 0 1 ( 0 . 5 1 2 - 0 . 6 8 2 ) 0. 8 7 0 ( 0 . 689--0.851) Ovary width Absent Absent 0 . 4 5 3 ( 0 . 4 0 1 - 0 . 5 2 8 ) 0 . 4 6 7 ( 0 . 4 0 9 - 0 . 521) 0 . 6 8 2 ( 0 . 6 1 4 ' -0.737) Ant. testis 0.152(0.117 -0.167) 0.378(0.348-0.417 ) 0 . 6 7 9 ( 0 . 6 1 3 - 0 . 7 0 5 ) 0 . 6 9 3 ( 0 . 6 4 9 - 0 . 7 6 1 ) 1. 2 8 4 ( 1 . 1 7 2 • -1. 3 2 2 ) length Ant. testis 0.095(0.077-0.117 ) 0.252(0.175-0. 2 7 7 ) 0 . 3 6 3 ( 0 . 311 - 0 . 4 2 7 ) 0 . 4 4 0 ( 0 . 4 1 6 - 0.49D 0 . 5 0 7 ( 0 . 4 6 6 -- 0 . 5 8 2 ) width Post, testis 0 . 1 6 r . ? o . 1 5 6 - 0 . 182 ) 0. 3 9 7 ( 0 . 3 3 5 - 0 . 4 2 7 ) 0 . 7 4 3 ( 0 . 6 6 9 - 0 . 8 7 9 ) 0 . 8 2 7 ( 0 . 7 7 4 - 0 . 86 c - ) 1, 4 0 2 ( 1 . 3 2 5 - 1 .457 ) length Pool. L c o t i s 0 . 1 4 0 ( 0 . 0 7 2 - 0 . 1 1 2 ) 0 . 2 7 7 ( 0 . 2 5 0 - 0 . 2 8 9 ) 0 . 4 2 7 ( 0 . 4 0 1 - 0 . 518 ) 0 . 5 0 2 ( 0 . 4 6 0 - 0 . 4 6 3 ) 0 . 5 6 0 ( 0 . 537-- 0 . 6 4 8 ) width Body length 0 . 8 2 3 ( 0 . 7 6 4 - 0 . 9 8 9 ) 1. 367(1 . 217 - 1 . 592 ) 2 . 2 4 7 ( 2 . 0 3 2 - 2 . 574) 3 . 1 8 6 ( 2 . 9 5 1 - 3 . 3 2 4 ) 4 . 8 7 7 ( 4 . 4 2 9 -- 5 . 2 4 3 ) Body width 0 . 2 5 9 ( 0 . 2 4 5 - 0 , 2 7 7 ) 0 . 3 7 0 ( 0 . 3 4 8 - 0 . 391 ) 0 . 5 3 8 ( 0 . 4 5 0 - 0 . 5 5 2 ) 0 . 6 3 6 ( 0 . 5 9 9 - 0 . 6 8 2 ) 1.167 ( 0 . 9 2 8 -- 1 . 2 4 5 ) Egg length Absent Absent Absent 0 . 0 1 8 ( 0 . 0 1 7 - 0 . 0 1 9 ) 0 . 0 2 3 ( 0 . 0 2 2 - 0 . 0 2 5 ) Egg width Absent Absent Absent 0 . 0 1 6 ( 0 . 0 1 5 - 0 . 017) 0 . 0 1 7 ( 0 . 0 1 6 - 0 . 0 1 8 ) A l l measurements in millimeters. Appendix 20.Average measurements of H. buttensis of varying ages developed in Bufo boreas. Age (days) __5 14 21 28 60 No. of specimens 19 17 17 21 20 Acetabulum 0.049(0.047-0. 051 ) 0.061 (0.057-0.065) 0.076(0.063-0. 083) 0.091(0.083-0.099) 0.100(0.082-0. 120 ) O r a l sucker 0.137(0.128-0. 145 ) 0.188(0.167-0. 212 ) 0.235(0.215-0.257) 0. 270(0.255-0.282) 0.291(0.242-0. 346) length O r a l sucker 0.135(0.124-0. 141 ) 0.185(0.171 -0.206) 0.237(0.195-0.258) 0. 264(0.253-0. 280) 0. 305(0. 244-0. 372) width OI A ratio 2.7 (2.6 -2.8 ) 3.1 (2.9 -3.2 ) 3.1 (2.9 -3.3 ) 2.9 (2.8 -3.1 ) 3.0 (2.9 -3.1 ) Ovary length Absent 0.231(0.190-0. 304) 0.457(0. 313-0. 514 ) 0. 527(0.475-0. 595) 0. 684(0. 552-0. 825) Ovary width Absent 0.085(0.066-0.098) 0.133 (0.130-0. 194 ) 0. 097(0. 086-0.107 ) 0.132(0.105-0.159 ) Ant. testis 6.096(0.080-0.128) 0.244(0.210-0. 271 ) 0.448(0.367-0. 539) 0.473(0.431-0. 504) 0.€11 (0. 652-0. 983) length Ant. testis 0.073(0.052-0. 085) 0.186(0.148-0. 266) 0.285(0.208-0. 333) 0. 316(0. 256-0.389) 0.406(0.326-0.490) width Post, testis 0.153(0.127-0. 201 ) 0.373(0. 314-0. 397) 0.776(0. 628-0.901 ) 0. 895(0.775-1.070 ) .1.523(1. 227-1. 835) length Post, testis 0.053(0.036-0. 069) 0.146(0.117 -0.179 ) 0.248(0.166-0. 345) 0.216(0.169-0. 252) 0. 343(0. 271-0.416 ) width Body length 0. 879(0. 824-0.962) 1.534(1.429-1.771 ) 2.695(2.112 -3.224) 3. 431(3.083-4.037) 5. 353(4. 325-6.450) Body width 0. 309(0.290-0. 321 ) 0.429(0.422-0. 542) 0. 625(0.575-0. 786) 0. 743(0.707-0. 784) 1. 366(1.104 -1. 638) E g g length Absent Absent 0.023(0.019-0.025) 0.023(0.023-0.026) 0. 023(0.023-0.025) E g g width Absent Absent 0.016(0.014-0.018) 0.018(0.017-0.018) 0.017(0.017-0.018) A l l measurements In m i l l i m e t e r s . Appendix 21. Average measurements of H. buttensis of varying ages developed ln Rana pretiosa when Physa nuttalll was the -first : Intermediate host. Age (days) 5 14 21 28 60 No. of specimens j. 23 20 19 Body length 1.11(0.97-1.19) 1.81(1.77-1.88) 3.17(2.74-3.59) 4.11(3.58-4.47) 6.64(5.89-6.96) Body width 0.34(0.32-0.36) 0.57(0.51^ 0.59) 0.68(0.66-0.79) 0.83(0.80-0.83) 1.32(1.27-1.61) Acetabulum diam. 0.07(0.05-0.07) 0.08(0.07-0.09) 0.08(0.08-0.09) 0.12(0.10-0.14) 0.14(0.12-0.15) Oral Sucker diam. 0.12(0.10-0.13) 0.18(0.17-0.21) 0.24(0.22-0.27) 0.27(0.24-0.29) 0.29(0.27-0.33) Oral Sucker length 0.11(0.10-0.12) 0.17(0.15-0.19) 0.23(0.20-0.24) 0.25(0.24-0.28) 0.28(0.26-0.30) 0/A Ratio 2.5 (2.3 -2.6 ) 2.5 (2.4 -2.7 ) 2.6 (2.4 -2.8 ) 2.5 (2.3 -2.6 ) 2.6 (2.5 -2.7 ) Ovary width 0.10(0.09-0.10) 0.19(0.16-0.21) 0.24(0.17-0.33) 0.29(0.28-0.34) 0.38(0.34-0.42) Ovary length 0.11(0.10-0.12) 0.27(0.22-0.32) 0.52(0.39-0.54) 0.56(0.53-0.63) 0.74(0.73-0.77) Ant. Testis width 0.12(0.07-0.14) 0.25(0.22-0.32) 0.36(0.28-0.39) 0.44(0.43-0.52) 0.55(0.36-0.61) Ant. Testis length 0.12(0.11-0.16) 0.36(0.30-0.39) 0.65(0.55-0.74) 0.65(0.60-0.71) 1.18(1.00-1.36) Post. Testis width 0.09(0.08-0.10) 0.24(0.20-0.26) 0.39(0.29-0.47) 0.46(0.38-0.57) 0.55(0.47-0.69) Post. Testis length 0.15(0.12-0.17) 0.33(0.27-0.34) 0.67(0.55-0.77) 0.80(0.76-0.92) 1.38(1.27-1.48) All measurements in millimeters. Appendix 2 2 Summary of some measurements given in type descriptions. H. buttensis Ingles (1936) H. similiolexus Stafford (1902) * H, similiolexus H. varioplexus >•'•••• Stafford (1902) H. floedae Harwood (1932) Acetabulum 0. 31 (0. 24-0.37) (0. 38) (0. 41) Oral sucker length 0. 33(0.18 -0.14 ) (0.44) (0. 51) Oral sucker width 0. 46 (0. 29-0.47) (0.41)(0. 57) (3. 6-4.4) O/A ratio 1:0. 7 (1:0. 6-1. 08) (1.1:1. 0).(1. 4:1. 0) 4:3 3:1 Pharynx length 0. 22 Pharynx width 0. 21 Ovary length 0. 44 (0. 26-0. 55) (0.29) (0. 46) (0. 65-0. 83) Ovary width 0. 32 (0. 20-0. 37) (0. 21) (0. 43) (0. 32-0.45) Ant. testis length (0. 34) (0. 48) (0. 7 - I. 1 ) Ant. testis width (0.34) (0.45) (0.32-0. 65) Post, testis length 0. 82 (0.45-1.03) (0.46) (0. 56) (0. 8-1.2) Post, testis width 0. 64 (0. 43-0. 87) (0.34)(0.58) (0.34-0.7) Body length 7.4 (3.2 -10.0 ) (3 - 8) 1.9(1.8-5.8) 10.5 (4.4 -10. 0) Body width 1. 3 (0. 7 -2. 2 ) (0.67-1.96) 2.0 (1.2-1.6 ) Egg length 0. 027(0. 025-0. 030) 0.039 0.038(0. 034-0.04) 0.029 (0.017-0.021) Egg width 0.014(0.011 -0.017) 0.019 0.018(0.017-0.021) 0.018 (0. 013-0. 018) *A redescription •from C<ni, H/J"*-.. Appendix 2 2 continued H. uniplexus H. parviplexus H. breviplexus H. breviplexus H. longiplexus Harwood 1932 (Irwin 1929) Stafford (1902) Stafford (1902) Acetabulum 0.08 0.16 0. 23 O r a l sucker length (0.46)(0.38) O r a l sucker width 0. 23 0. 32 (0. 4) (0. 28) (0.46) (0.49) O/A ratio 1:3 4:1 1:2 2:1 P h a r y n x width 0.14 (0.19) (0.195) Ovary length (1.16) (0.79) 1. 57 O v a r y width 0.495 (0. 314) 0. 92 Ant. testis length 0. 5 1.023 (1.04) 1. 71 Ant. testis width 0.16 0.578 (0.495) 1. 03 Post, testis length 0. 48 (1. 469) (1.02) 2. 31 Post, testis width 0.16 (0. 528) (0. 512) 1-1 Body length 4. 25 (3.9-8.49 12. 0 (5. 8) (9. 4). 7. 0 o r 8. 0 (15) Body width 0.7 (0.85-1.6) (2.0-2. 5) (1.8) (2.74) About 2. 0 (3. 0) E g g length (0.021-0. 017)0. 025(0.023-0.029) 0. 022 0. 023(0. 021-0. 026) 0. 022 E g g width (0.017-0. 013)0.017(0.016-0.019) 0. 017 0. 014(0. 013-0.016) 0.017 Appendix 22 continued _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _i ^H. longiplexus H. complexus H. kernensis H. tumidus V :y/ Seely (1906) Ingles (1932) Ingles (1932) Acetabulum 0. 7 0. 38 0.44 0. 68 O r a l sucker length 0. 36 0. 44 O r a l sucker width 0. 42 0. 4 0. 61 O/A ratio (5:2 - 2:1) 1:1 (7:6-15:16) 5:6 P h a r y n x length 0. 22 0.49 P h a r y n x width 0.18 0. 31 O v a r y length 0. 85 0.7 0. 87 O v a r y width 0. 72 0. 32 0. 37 (0. 58) Ant. testis length 1. 08 l . l Ant. testis width 0. 27 0.92 0.97 0.16 Post, testis length I. 26 1.4 Post, testis width 0. 32 0.92 9.96 0.15 Body length 4.6 (5-8) 6.3(5.5-7.0) 3.1 (6.4-9.7) Body width 2.0 1.7 1. 54 2. 5 E g g length 0.025(0.022-0.087) 0.029 0.030(0. 025-0. 039) 0. 032(0. 030-0. 036) E g g width 0.015(0.014-0.017) 0.014 0.016 0.017 Appendix 2 2 continued H. oxvorchis H. confusus T H . coloradensis H. medioolexus H. medioplexus Ingles (1932) Ingles (1932) Stafford (1902) Acetabulum 0. 32 0. 32 0. 7 (0.12)(0.15) 0. 08 Oral sucker length 0. 44 (0.22) (0.39) (0i 40) Oral sucker width 0. 41 0.46 (0. 31) (0. 24) (0. 37)(0.35) O/A ratio 5:4 (7:6-8:5) 4:3 (5:4-9:5) 5:4 (7:6-4:3) 4:1 Pharynx length 0. 34 0. 29 Pharynx width 0. 31 0. 28 0. 26 Ovary length 0.58 0. 40 (0. 30 - 0. 49) 0. 56 Ovary width 0. 51 0.19 (0. 22 - 0. 42) - 0. 28 Ant. testis length 0. 51 (0.42 - 0. 64) 0. 56 Ant. testis width 0. 76 0. 44 (0.46 - 0. 60) 0. 64 Post testis length 0. 50 (0.46 - 0. 74) 0.64 Post, testis width 0. 78 0.42 (0.44 - 0. 86) 0. 6 Body length 5. 8 (3. 8-6. 5) 3.9 (3.3-4.9) 8.1 11.0 (3.9-7. 8) Body width 0. 87 0.9 1. 55 1. 25 (0.59-1. 2) Egg length 0.027(0.026-0.030) 0.034(0.032-0.039) 0.026(0.024-0.029) Egg width 0.017 0.015 0.020(0.018-0.021) 0.028 0.018 0.026(0.022-0.029) 0.015(0.013-0. 017) Appendix «t J Known Hosts of Adult Haematoloechus sp. ln Canada and the On 1 ted States H a e n a t o l o e c h u s Host b r i t v l i ' l t f x u s b u t t e n a ! a C'lloradensls con: l*?xus eonfusua f l o e d a e kerner.sla ^ o n r l i l - j x u a Ranldae R a n i . T u r . ' r n ft.aurora d r a v t o n l R . b l i l r l R . b c v l i R . c a t e s b e i a n a a . c l a - i t a n s R.f r y l l o R . r y . °Ti:-e H.r.4lnlr.-=5 R . c a l u a t r l s R . p l r l i n s R . c r e t l o a a ft.pret1?sa R.sert«'^t-ionalis 1^ 12.30,37. 4,23,42. 13 38 32(experlcental) Hybrids R.srh>v. j j . - p h a l a BuTonlJao Sufo .'.rfrlcnnua 3 . r l c r o 3 c a r h u 9 H y l i i a e Kvl3 c h r v s p s c e l l s 41 27.31. 27,31. 41 IB 4.27.29,31, 3,4,10.12, 46,41. 16\22,J6, 41. 3,4,12. 41 41 25 13 9 J}15.28, 41 1.8,11.12 15, 29.J2.35.36, 38,41. 8,21. •« r n 32,36. 32 21 41 1.Stafford,1=02) 2 . i t i : T o r d ( 1 9 0 5 ) J . S e e l y ( l r C = ) 4.~ort( 1*515 J 5..73rtr.er!l?23) 6. : r - : r . ( l v i ? ) 7. : r j l l ( l V 3 1 ) «..-.ar.-3Si(l-,32) 9.:-t-:es(l?32) ir..:r.r»es(r,3)) 12. Brandt(1936) 13.1ngles(1936) l/..oonnctt(1938) 15. :-*anter(1938) 16. Caballero(1941) 17. Parker(1941) 13. Caballero(1942) 19.S;mkin(1945) 20."Jribe-riecrahlta(1948) 21.5 )uchara(1951) 11."..-conjee and Hefley ^2.UJlau^f 19i4) (1934) 23. NaJarian(1955) 24. Turner(1958) 25. Chenc and ProventatI960) 26. Cheng(1960) 27. rranc3en and CrundmantI960) 28. Loftin(1360) 29...aitz(1961) 3t.Kninht,Jarbay and Morrison (19o5) 31. Farry and Crundir.an(1965) 32. w c l . u l l ( l % 5 ) 33. Jacobs and Korrison(1966) 34. Campbell(1968) 35. Clark and Longeat(1970) 36. UlT.er(1970) 37. Hollis(1972) 3c!.b-ibero ana GolHng( 1974) 39. Cain ar.a Krcnch( 1975) 40. Dronen'1975) 41.3rooks(1076) 42.Hoben anu .,anis( 1976) 43 .iierineuy( unpublished) A p p e n d i x _ J COUt . Known Hoats of Adult ftaanatoloechus op. ln Canada and the Unltad -te'.tt. Kaeratoloechua Host aedloolexus oyxorchls parviplexus slrr.lllplexus tumidus unlplexus varioplexus Ranidae H<nA aut-cra draytoni R . b l a i r i 41 3. boy11 R.c »; e any l.ma 39 R.c . a-.ltans 19,21. R . - r v l i o R.£2T__J__ 13 R.cal-loes 20 R .calur.trls 19,21. ft. r-l Mens R.rr'tlosa R . r r t - t l o s a R.svlvntica R.sor-tt;n*.rlonall3 21 R. a y ' : :enoci*cha l a R.svlv.tlca B u f o n t r U r Eufo nT.erlcanus 1 B.rr.lcroscashus a.wojcihouall Hylldae Kv •n chr y f o s c e l l s 9,10. 1.4.5,18,19, 2^ ,J6,37,U. Hybrids 14,38,41. 6,14.23,29, 43. 16 U 1,34,35. 21 1.4,5,36. 24 21 1,4,36. 13 9 41 41. 23 41 1.2-.af:-3.-j;:?C2) 2 .9*3:Tord{ I9Cj) 3~5eelyU9Co) 4. :ort(1915) 5. rortr.^r! 1923) 6. :.-w:n(l?2-J) 7. Kr-.;il(1931) 2.H&rwe5-.(l'}32) 9.Ir.r:os( 1932) 10.:r.c:es( 1933) 12.3-andt(1936) 13.1n5le8(1936) 14.3*nnett(1938) 15. Minter(193ti) 16. Ciballero(1941) 17. ?arfcer{1941) 18. Ciballero(1942) 19. ?.snkin(19'.5) 20. L: rioe-^ie'irahlta(1948) 21. uoucr.ard(1951) l l . : rowbricse ani Hafley 22.0r;lauc(l^^l (193C) 23. NaJarlRn(1955) 24. Turner(195S) 25. Cheng and Provenza{1960) 26. ChenrJ(1960) 27. Frandsen and Grundman(1960) 28. Loftln(1960) 29. '.vaitz(i96l) 30. f . n i i ; h r , , 3 a r b a y and Morrison (l9o5) 31. P a r r y a n d Grundman(1965) 32 . S c h e l l(19o5) i) .C^^ozz a n d .•;orrison(19o6) 34. Ca-npbell(196a) 35. Clark and Loneest(1970), 36. Ulmer{1970) 37. Kollis(1972) 38.3abero and Golllng(1974) 39.Cain and rrench(1975) 40..L-ror.en(1975) 41.3rooits(1976) i>2.H03ian a n c ,:anls( 1°76) 43 . f . o : i n e < j y ( u t i p'jbll3hed) 

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