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Reproductive dynamics of the parasitoid, Spalangi endius (Walker) (Hymenoptera: Pteromalidae), with particular… Jones, James William 1982

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R E P R O D U C T I V E D Y N A M I C S OF T H E P A R A S I T O I D , S P A L A N G I A E N D I U S ( W A L K E R ) ( H Y M E N O P T E R A : P T E R O M A L I D A E ) , W I T H P A R T I C U L A R R E F E R E N C E T O H O S T - S E A R C H I N G A N D H O S T - A C C E P T A N C E B E H A V I O U R b y J A M E S W I L L I A M J O N E S B . S c , B r o c k U n i v e r s i t y , S t . C a t h a r i n e s O n t a r i o , 1 9 7 7 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 O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( D e p a r t m e n t o f Z o o l o g y ) We 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 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 A p r i l 1 9 8 2 (c) J a m e s W i l l i a m J o n e s , 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date /o y/f^.  DE-6 (.3/81) i i ABSTRACT O v i p o s i t i o n and r e l a t e d behaviour i n the host s e a r c h i n g and host acceptance r e p e r t o i r e of Spalangia endius (Walker) were examined to determine how such behaviour might be c o n t r o l l e d by the presence of mature eggs in t h i s p a r a s i t i c i n s e c t . In a l l experiments, Musca domestica L. pupae were the h o s t s . Host s e a r c h i n g was observed i n g l a s s p e t r i - d i s h arenas c o n t a i n i n g a sawdust s u b s t r a t e , h a l f of which was contaminated with the odour of mature host l a r v a e . The e f f e c t s on s e a r c h i n g behaviour of a s e r i e s of o v i p o s i t i o n s by one set of p a r a s i t o i d females c o u l d not be d i s t i n g u i s h e d from a c o n t r o l group that d i d not o v i p o s i t . A study of burrowing i n d i c a t e d that one-quarter of the p o p u l a t i o n sampled c o u l d not e x p l o i t hosts b u r i e d i n the sawdust s u b s t r a t e . The p o r t i o n of search time that was spent i n antennal i n v e s t i g a t i o n of the s u b s t r a t e and burrowing i n the s u b s t r a t e was found to i n c r e a s e with f e c u n d i t y . The p r o b a b i l i t y that given hosts would produce p a r a s i t e progeny was p r e d i c t a b l e f o r one- and two-day-old females that were given a s e r i e s of hosts to a t t a c k i n s u c c e s s i o n ; the second and seventh hosts always produced progeny. Mated females were more fecund than v i r g i n s ; the p a t t e r n s of o v i p o s i t i o n observed i n mated and v i r g i n females suggested that mating r e l e a s e s o v i p o s i t i o n i n h i b i t i o n . TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES . . . ix ACKNOWLEDGEMENTS x I. I n t r o d u c t i o n 1 I I . Host Searching S t u d i e s 4 Host Searching i n the Pteromalidae 6 F a c t o r s That Increase Search Time 9 F a c t o r s That Decrease Search Time 11 GENERAL METHODS AND MATERIALS FOR HOST SEARCHING STUDIES - 15 Expt.1: V a r i a b i l i t y of Search Behaviour with the Time of Last O v i p o s i t i o n Known—Set 1 17 Expt.2: V a r i a b i l i t y of Search Behaviour with the Time of Last O v i p o s i t i o n Known--Set 2 20 Expt.3: C o r r e l a t i n g Age, Reproductive Dynamics and Search Behaviour 25 Expt.4: P r o p o r t i o n of Burrowers i n the P o p u l a t i o n ... 30 Expt.5: Short-term E f f e c t s of O v i p o s i t i o n on Search Behaviour 39 I I I . Host Acceptance S t u d i e s 50 Host Acceptance i n the Pteromalidae 50 Information C o l l e c t e d P r i o r to I n s e r t i o n 51 Information C o l l e c t e d A f t e r I n s e r t i o n 54 i v GENERAL METHODS AND MATERIALS FOR HOST ACCEPTANCE STUDIES 58 Expt.6: Reproductive Dynamics 60 Expt.7: Host Acceptance and Host Death 66 IV. D i s c u s s i o n 82 V. Co n c l u s i o n s 91 Appendices 93 Appendix 1. L i f e H i s t o r y Data f o r Some Common Pteromalid P a r a s i t o i d s of Muscoid D i p t e r a 93 Appendix 2. S t a n d a r d i z a t i o n P r a c t i c e s 99 Appendix 3. . Notes on Emergence and Mating Behaviour ...101 References C i t e d 105 V LIST OF TABLES Table 1. Behaviour observed d u r i n g Expt.1 19 Table 2. Time spent by females on arena s u b s t r a t e s 28 Table 3. Anova for t o t a l time spent on the host-contaminated s u b s t r a t e 33 Table 4. Anova for the per cent time spent i n v e s t i g a t i n g and burrowing 33 Table 5. E f f e c t i v e n e s s of d i s t i n g u i s h i n g between females that burrowed 38 Table 6. D i s t r i b u t i o n s of p a r a s i t e progeny and emerging hosts . 39 Table.7. S p l i t - p l o t anova f o r Expt. 5 45 Table 8. P a i r e d comparisons of the mean d u r a t i o n of search time 45 Table 9. The data of Table 8, m o d i f i e d 46 Table 10. S p l i t - p l o t anova f o r m o d i f i e d data set 46 Table 11. Search 1 times grouped a c c o r d i n g to number of eggs l a i d 48 Table 12. Anova for search times of the o v i p o s i t i o n groups 48 Table 13. Rank scores f o r Search 1 times 49 Table 14. Host acceptance r e s u l t s f o r the p r e - t e s t c o n d i t i o n i n g p e r i o d s 50 Table 15. Mean number of o b s e r v a t i o n s of host-acceptance behaviour i n one- and two-day-old females 67-Table 16. C o n d i t i o n of hosts a f t e r being stung by S.endius. 69 Table 17. P a i r e d comparisons of the d u r a t i o n of host-acceptance a c t s separated by 24 hours 72 Table 18. P a i r e d comparisons of the l a t e n c y of host acceptance 73 Table 19. Dur a t i o n s f o r the f i r s t TA bout performed by one-day-old females 73 Table 20. Phases of host acceptance of one- and two-day-old S.endius females 74 Table 21. Number of d r i l l i n g bouts performed by two S.endius females on f i v e s u c c e s s i v e l y accepted h o s t s . .. 74 Table 22. Mean number of d r i l l s i t e s u t i l i z e d i n s u c c e s s i v e a t t a c k s on M. domest i c a p u p a r i a 75 Table 23. Occurrence of d r i l l i n g on M.domestica p u p a r i a . .. 75 Table 24. D i s t r i b u t i o n of a t t a c k s on M.domestica p u p a r i a . . 77 Table 25. D i s t r i b u t i o n of a t t a c k s and per cent l e a d i n g to i n s e r t i o n 80 Table 26. Heterogeneity G-test of the p r o p o r t i o n of females l a y i n g eggs i n c o n s e c u t i v e a t t a c k s 80 Table 27. D i s t r i b u t i o n of progeny by sex in the f i r s t e i g h t hosts a t t a c k e d 81 Table 28. Unproductive host a t t a c k s compared f o r three experimental groups 81 Appendix Table 1. Examples of s t a n d a r d i z a t i o n regimens 100 LIST OF FIGURES F i g u r e 1. The d i s t r i b u t i o n of mean burrowing time during Expt. 1 21 F i g u r e 2. Search d u r a t i o n vs. the number of hosts k i l l e d by females of Expt.2 26 F i g u r e 3. Number of eggs l a i d d a i l y by Expt.3 females 31 F i g u r e 4. P r o p o r t i o n of t e s t time spent i n d i f f e r e n t p a r t s of the arena 31 F i g u r e 5. Time spent burrowing and i n v e s t i g a t i n g by females d u r i n g Expt.3 35 F i g u r e 6. C o r r e l a t i o n of ranks f o r cumulative egg p r o d u c t i o n 35 F i g u r e 7. Search times f o r Expt.5 females 43 F i g u r e 8. Longevity of mated and v i r g i n S.endius females. . 62 F i g u r e 9. Mean number of progeny (emerged + f a i l e d ) per mated and v i r g i n female cohorts 62 F i g u r e 10. Unproductive host d e s t r u c t i o n d u r i n g the f i r s t f i v e days a f t e r emergence 64 F i g u r e 11. Mean d a i l y p r o d u c t i o n of progeny 64 F i g u r e 12. P r o p o r t i o n of females l a y i n g eggs i n a s e r i e s of a t t a c k s 77 Appendix F i g u r e 1. Emergence schedule of Spalangia endius 102 Appendix F i g u r e 2. Emergence schedule of M u s c i d i f u r a x z a r a p t o r (K. & L.) 102 v i i i ACKNOWLEDGEMENTS Many people were of a s s i s t a n c e d u r i n g t h i s p r o j e c t . I am g r a t e f u l to A g r i c u l t u r e Canada and B.D. F r a z e r , p a r t i c u l a r l y , f o r p r o v i d i n g l a b o r a t o r y space and equipment, and to many others who a l s o p r o v i d e d equipment. G.W. Eaton's a d v i c e on s t a t i s t i c s was i n v a l u a b l e . Thank you, B i l l N e i l l , f o r showing me by your example something p r e c i o u s . 1 I. INTRODUCTION Spalanqia endius (Walker), a small (3 mm) c h a l c i d p a r a s i t o i d , i s one of s e v e r a l s p e c i e s which l a y s i t s eggs on the pupae ( w i t h i n the puparia) of muscoid D i p t e r a . Many such s p e c i e s have been used to c o n t r o l f l y i n f e s t a t i o n s i n North American domestic l i v e s t o c k i n s t a l l a t i o n s d u r i n g the l a s t twenty years (Legner,1966,1974; Legner and Olton,1971; Morgan et al.,1975). These a p p l i c a t i o n s have r e q u i r e d d e t a i l e d knowledge of t h e i r o v i p o s i t i o n - r e l a t e d behaviour (e.g., Wylie,l965, 1966a, 1967, 1970; Legner and Gerling,1967; Legner,1976, 1977, 1979a). The p h y s i o l o g y of r e p r o d u c t i o n has been s t u d i e d f o r only one s p e c i e s in t h i s group of p t e r o m a l i d p a r a s i t o i d s , Nasonia (Mormoniella) v i t r i p e n n i s (Walker) (Edwards,1954a; King and R a t c l i f f e , 1 9 6 9 ; King and Richards,1969). Consequently, r e p r o d u c t i v e behaviour i n t h i s s p e c i e s i s the model with which other p a r a s i t o i d s are compared (Edwards,1954b; Wylie,l958; Whiting,1967). The b e h a v i o u r a l framework w i t h i n which the r e p r o d u c t i v e behaviour of S.endius w i l l be d i s c u s s e d here, was e s t a b l i s h e d by Laing (1937,1938) and Doutt (1959,1964), and f u r t h e r m o d i f i e d i n t o the h o s t - s e l e c t i o n h i e r a r c h y (Vinson,1976), a c o n c e p t u a l i z a t i o n of the processes which b r i n g the female p a r a s i t o i d i n t o c o n t a c t with the host (host searching) and allow her to make a d e c i s i o n about e g g - l a y i n g (host a c c e p t a n c e ) . S.endius i s unusual i n that i t burrows i n t o s u i t a b l e s u b s t r a t e s and i s p r o f i c i e n t at p a r a s i t i z i n g hosts that pupate in the s o i l (Legner,1967,1977), q u a l i t i e s not recognized as 2 e s s e n t i a l i n an enemy of such f l i e s d u r i n g e a r l y c o n t r o l attempts. Host s e a r c h i n g i s examined here, not only with respect to burrowing, but a l s o to the p a r t i t i o n i n g of the t o t a l search time amongst the other kinds of behaviour i n the searc h i n g r e p e r t o i r . An a d a p t a t i o n of Waage's (1978,1979) apparatus f o r t e s t i n g behaviour of the ichneumonid, N e m e r i t i s canescens Grav., has been employed i n my examination, an important part of which depends on answers to the q u e s t i o n , "What b e h a v i o u r a l changes occur d u r i n g repeated s e a r c h i n g and how might these changes be a s s o c i a t e d with o v i p o s i t i o n ? " To i n v e s t i g a t e t h i s q u e s t i o n , I have t e s t e d S.endius females f o r the e f f e c t s of o v i p o s i t i o n on host s e a r c h i n g and host acceptance over a s e r i e s of days. P r i o r t o t e s t i n g , females had t o be s t a n d a r d i z e d , that i s , brought i n t o experimental " r e a d i n e s s " . The i m p l i c a t i o n s of and procedures f o r induci n g o v i p o s i t i o n readiness (Flanders,1942; Edwards,1954a; King and Rate 1if f e , 1 9 6 9 ) have r e s u l t e d i n the adoption of v a r i o u s s t a n d a r d i z a t i o n regimens (e.g., W y l i e , l 9 7 l ; Legner,1979b) which tend to conc e n t r a t e on i n d i v i d u a l s o l d e r than three days. I found i t d i f f i c u l t to o b t a i n 'standardized' behaviour w i t h i n such a d i f f u s e c l a s s , however, and so examined d i f f e r e n t age groups i n order to c h a r a c t e r i z e t h e i r o v i p o s i t i o n responses to b u r i e d hosts, t h e i r h o s t - k i l l i n g responses, and t h e i r host s e a r c h i n g i n the t e s t apparatus. O v i p o s i t i o n d u r i n g these short-term s t u d i e s was compared with data c o l l e c t e d over the l i f e t i m e s of females. E a r l y i n the re s e a r c h programme, the o v i p o s i t i o n response 3 of v i r g i n females l e s s than twelve hours o l d was noted to be d i f f e r e n t from that seen when they were o l d e r . Such a d i f f e r e n c e might be caused by t h e i r v i r g i n s t a t e , although c o n t r o l of o v i p o s i t i o n may be e f f e c t e d through s e v e r a l mechanisms (Engelmann,1970). The p o s s i b l e e f f e c t s of mating on o v i p o s i t i o n response, coupled with the l a c k of i n f o r m a t i o n on the behaviour of very young females d u r i n g s t a n d a r d i z a t i o n , r a i s e d q u e s t i o n s of the e f f e c t s of such m a n i p u l a t i o n p r i o r to t e s t i n g . These q u e s t i o n s prompted f u r t h e r examination of o v i p o s i t i o n - r e l a t e d behaviour i n females l e s s than four days o l d . 4 I I . HOST SEARCHING STUDIES Laing (1937) suggested that a p a r a s i t o i d f i r s t seeks the h a b i t a t of the host and then the host i t s e l f . T h i s s e q u e n t i a l a t t r a c t i o n i s e x e m p l i f i e d by the behaviour of braconids that p a r a s i t i z e t e p h r i t i d l a r v a e ; e.g., B i o s t e r e s (Opius) l o n g i c a u d a t u s Ashm. i s f i r s t a t t r a c t e d to fungal fermentation products i r r e s p e c t i v e of i n f e s t a t i o n by i t s host (Greany _e_t a l . , 1976,1977). Pt e r o m a l i d p a r a s i t e s of synanthropic D i p t e r a , on the other hand, a p p a r e n t l y do not f o l l o w t h i s p a t t e r n (Varley and Edwards,1953; Edwards,1954; W y l i e , l 9 5 8 ) . Instead, they are s a i d to home in on t h e i r hosts much as the ichneumonid, Nemeritis  canescens , l o c a t e s i t s host, the Mediterranean f l o u r moth. In N.canescens , h o s t - h a b i t a t f i n d i n g and host f i n d i n g are both mediated by compounds i n t i m a t e l y a s s o c i a t e d with host presence (Matsumoto and Huffaker,1973); y et, the s e q u e n t i a l nature of the " f i n d i n g " process--arrestment, f o l l o w e d by the tapping and t u r n i n g responses (Waage,1978,1979)--remains. Decaying organic-matter h a b i t a t s support a v a s t complex of d i p t e r a n s p e c i e s (Legner and Olton,1971). That a wide v a r i e t y of these s p e c i e s are a t t a c k e d by the Sp a l a n g i a , i s perhaps due more to the hosts' a v a i l a b i l i t y to the p a r a s i t e s than to innate c h a r a c t e r i s t i c s of the hosts (Olton and Legner,1973). The degree of h a b i t a t s p e c i f i c i t y e x h i b i t e d by searc h i n g p a r a s i t o i d s would become a l l the more important i f there was l i t t l e or no host s p e c i f i c i t y . I t has been suggested that Spalangia spp. a s s o c i a t e d with synanthropic D i p t e r a are a t t r a c t e d to the host's h a b i t a t by humidity g r a d i e n t s and chemical s t i m u l i 5 a s s o c i a t e d with the h a b i t a t (Simmonds,1954) and that Spalangia  endius i s "more a t t r a c t e d to a mixture of organic m a t e r i a l and s i l a g e than to s i l a g e a l o n e " (Morgan and Patterson,1977). But, s i n c e the h a b i t a t (manure p a r t i c u l a r l y ) can be changed r a p i d l y by weather and by d i f f e r e n c e s i n the d i e t , age and breed of animals (Legner and O l t o n , l 9 7 l ) , i t i s d o u b t f u l that one set of chemical a t t r a c t a n t s would be common to a l l such h a b i t a t s . Some c l u e s r e l a t e d to the presence of the host must a l s o be i n v o l v e d . In f a c t , o l f a c t o m e t e r experiments using odours of the 'h a b i t a t ' with and without i n f e s t i n g f l y l a r v a e showed that s e a r c h i n g N . v i t r i p e n n i s females were more a t t r a c t e d to sources c o n t a i n i n g host l a r v a e (Edwards,1954). Edwards' study a l s o demonstrated that i t was the a c t i o n of C a l l i p h o r a l a r v a e on l i v e r , not b a c t e r i a l decomposition, that was r e s p o n s i b l e f o r the inc r e a s e d a t t r a c t i v e n e s s . Using an ol f a c t o m e t e r resembling one designed by V a r l e y and Edwards (1953), Wylie (1958) obtained s i m i l a r r e s u l t s with N . v i t r i p e n n i s . In a d d i t i o n , he showed that s e a r c h i n g N . v i t r i p e n n i s females p r e f e r r e d dry, uncontaminated a i r to moist a i r , even a f t e r s t a r v a t i o n f o r 24 hours. This l a t t e r f i n d i n g at f i r s t appears i n c o n s i s t e n t with the idea that the decaying o r g a n i c matter i n the h a b i t a t should promote • a high humidity preferendum i n se a r c h i n g p a r a s i t o i d s . However, s i n c e Nasonia v i t r i p e n n i s has been recovered from h a b i t a t s as d i v e r s e as bovine feces and b i r d ' s nests (Whiting,1967), the idea that high moisture l e v e l s i n v a r i a b l y must be i n v o l v e d (Legner and Olton,1971) i s perhaps too broad a g e n e r a l i z a t i o n . Indeed, s i n c e the a t t r a c t i v e n e s s of the h a b i t a t 6 appears to be s y n e r g i s t i c a l l y mediated by host presence, a p a r a s i t o i d female in the h o s t - h a b i t a t f i n d i n g phase of f o r a g i n g i s probably a l s o r e c e i v i n g i n f o r m a t i o n on the d e n s i t y and the d i s p e r s i o n of the hosts i n that h a b i t a t . Host Searching i n the Pteromalidae D i p t e r a n s f e e d i n g i n decaying organic matter are not a l l subj e c t to the same p a r a s i t i z a t i o n p r e s s u r e because of d i f f e r e n c e s i n host pupation h a b i t s and the a b i l i t i e s of p a r a s i t o i d females to f i n d these pupae. C l a s s i f y i n g f l y s p e c i e s a c c o r d i n g to t h e i r "degree of exposure" to p a r a s i t e s ( U l l y e t t , 1 9 5 0 ) or p r o x i m i t y to the l a r v a l h a b i t a t at pupation (Whiting,1967) has shown how t h i s v a r i a b i l i t y may be advantageous f o r the host or p a r a s i t e , but no study has e x p l i c i t l y examined p o s s i b l e hide-and-seek s t r a t e g i e s of s o i l -pupating f l i e s . Although i t i s customary to assume that hosts are aggregated to some degree, i t has a l s o been suggested that " i s o l a t e d hosts are common i n nature because f l y l a r v a e o f t e n crawl long d i s t a n c e s (up to 15 m) from a c a r c a s s before pupating" (Werren,1980). The type of f l y h a b i t a t c l e a r l y has a bearing on whether pupation i s near or remote from the l a r v a l s i t e . For example, ex t e n s i v e manure d e p o s i t s from penned l i v e s t o c k p r o h i b i t the seg r e g a t i o n of host from h a b i t a t , and sampling has shown that f l i e s pupate at v a r i o u s depths i n such d e p o s i t s (Legner,1966). There, they are p a r a s i t i z e d to a degree dependent on t h e i r depth and the p a r a s i t e s p e c i e s 7 ( e . g . , M . r a p t o r G . & S . p a r a s i t i z e d m o r e h o s t s a t t h e s u r f a c e o f m a n u r e d e p o s i t s w h i l e S p a l a n g i a s p p . w e r e m o r e a c t i v e a t g r e a t e r d e p t h s - - L e g n e r , 1 9 6 7 ) . I n t h e l a b o r a t o r y , b e h a v i o u r a l o b s e r v a t i o n s o f p a r a s i t i z a t i o n o f M . d o m e s t i c a p u p a e a r r a n g e d i n w h e a t f l a k e s a t t h r e e s t r a t a ( s u r f a c e , 2 cm a n d 4 c m d e p t h s ) s h o w e d S p a l a n g i a s p p . ( S . c a m e r o n i P e r k . , S . e n d i u s , S . l o n q e p e t i o l a t a B o u c e k a n d S . n i g r a L a t r . ) t o b e m o r e e f f e c t i v e a t g r e a t e r d e p t h t h a n M . r a p t o r , M . z a r a p t o r o r S p h e g i g a s t e r s p . ( L e g n e r , 1 9 7 7 ) . T h i s d i f f e r e n t i a l p r o p e n s i t y t o b u r r o w i n t o t h e s u b s t r a t e v a r i e d w i t h m o i s t u r e c o n t e n t o f t h e w h e a t f l a k e s , e v e n w i t h i n g e n e r a a n d s p e c i e s ; f o r i n s t a n c e , a d a p t a t i o n s o f g e o g r a p h i c r a c e s o f S . e n d i u s t o d i f f e r e n t r a n g e s o f t e m p e r a t u r e , r e l a t i v e h u m i d i t y , a n d h a b i t a t m o i s t u r e c o n t e n t a f f e c t e d m a n y a s p e c t s o f s e a r c h b e h a v i o u r a n d s e a s o n a l a b u n d a n c e ( L e g n e r , 1 9 7 7 , w i t h c i t a t i o n s o f M a r k w i c k ' s ( 1 9 7 4 ) w o r k o n t h e B a r b a d o s s t r a i n o f S . e n d i u s ) . T h e m o r e s t a b l e c h a r a c t e r i s t i c s o f s p e c i e s , n o t a b l y i n t r i n s i c r a t e o f n a t u r a l i n c r e a s e ( b u t s e e A p p e n d i x 1 ) , c o m p e t i t i v e a b i l i t i e s o f a d u l t s a n d l a r v a e a n d , t o s o m e e x t e n t , e m e r g e n c e - a n d o v i p o s i t i o n r e s p o n s e s t o t e m p e r a t u r e , a l l o w s o m e g e n e r a l i z a t i o n s a b o u t a c t i v i t y l e v e l s a n d n u m e r i c a l r e s p o n s e s ( e . g . , M u s e i d i f u r a x s p p . a r e " c o o l - w e a t h e r p a r a s i t e s " ; S p a l a n g i a s p p . a n d S p h e g i g a s t e r s p . a r e " c o m p e t i t i v e l y i n f e r i o r " ) . H o w e v e r , t h e p e r f o r m a n c e o f a n y p a r t i c u l a r s p e c i e s w i t h i n a g i v e n h a b i t a t m u s t b e e x p e r i m e n t a l l y d e t e r m i n e d . A d d i t i o n a l r e s p o n s e s e x h i b i t e d b y a f e m a l e p a r a s i t o i d w h i l e s e a r c h i n g o n t h e h o s t h a b i t a t h a v e b e e n e x a m i n e d m o r e i n t e n s i v e l y f o r i n s e c t s o t h e r t h a n t h e m u s c o i d f l y p a r a s i t e s . 8 Since the concepts are fundamental to t h i s t h e s i s , however, and more bro a d l y , to b e h a v i o u r a l ecology, they are i n t r o d u c e d here, then developed more f u l l y at l a t e r p o i n t s . S p e c i e s - s p e c i f i c cues guide the s e a r c h i n g female p a r a s i t o i d to i n c r e a s i n g l y s m a l l e r area l i k e l y to c o n t a i n the host. When she e n t e r s that area, the female's behaviour changes to "area-c o n c e n t r a t e d search" (Curio,1976; Fantino and Logan,1979) or "success-motivated s e a r c h i n g " (Vinson,1976). The s t i m u l i e l i c i t i n g such changes may be v i s u a l or t a c t i l e , and operate at c l o s e range (reviewed by Waage,l978; host-produced "contact chemicals" by Vinson,1976). The response e l i c i t e d on f i r s t c o n t a c t i s "arrestment", an o r t h o k i n e t i c response, f o l l o w e d by ( i n N e m e r i t i s canescens ,see Waage,1978,1979) r a p i d drumming of the s u b s t r a t e , proceeding across the patch at reduced speed with o c c a s i o n a l - stops, probing with the o v i p o s i t o r , and o c c a s i o n a l sharp turns ( k l i n o k i n e s i s ) . S t u d i e s of N . v i t r i p e n n i s showed that a t t r a c t i o n to the host h a b i t a t was accomplished through the combined odours of l i v e r and i n f e s t i n g C a l l i p h o r a l a r v a e (Edwards,1954; W y l i e , l 9 5 8 ) . From these same s t u d i e s , i t was noted that females d i d not remain in the " c r i t i c a l a rea" of the o l f a c t o m e t e r ( d e f i n e d by the odour plume) i f " c l e a n " pupae were used as the odour source (these hosts were prepared by washing prepupal l a r v a e and a l l o w i n g them to pupate i n paper). In c o n t r a s t , puparia of unwashed l a r v a e e l i c i t e d t u r n i n g motions at the patch edge. In a l l such s t u d i e s , search time i s c o n s i d e r e d to s t a r t when p e r c e p t i o n of a stimulus f i r s t a r r e s t s motion (or causes some other d i s t i n c t b e h a v i o u r a l 9 c h a nge) a t t h e p a t c h edge. The f a c t o r s w h i c h t h e n a c t t o m a i n t a i n s e a r c h i n t e r e s t a r e o f i m p o r t a n c e i n b e h a v i o u r a l c o n t r o l t h e o r y . F a c t o r s T h a t I n c r e a s e S e a r c h Time The most i m p o r t a n t f a c t o r a c t i n g t o i n c r e a s e s e a r c h t i m e on t h e p a t c h i s h o s t d e n s i t y or i t s e q u i v a l e n t r e p r e s e n t a t i o n . In t h e c a s e of O p i u s l e c t u s Gahan. ( P r o k o p y and W e b s t e r , 1 9 7 8 ) , s t i m u l i o t h e r t h a n t h o s e p r o d u c e d by t h e p o t e n t i a l h o s t can i n t e r v e n e a t any p o i n t i n t h e h o s t s e l e c t i o n p r o c e s s and s e r v e t o augment t h e p e r c e p t u a l s p e c t r u m of t h e s e a r c h i n g p a r a s i t o i d f e m a l e ; e.g.,. 0. l e c t u s c u e s i n on, among o t h e r f a c t o r s , t h e o v i p o s i t i o n - d e t e r r i n g pheromone o f t h e h o s t p a r e n t a l g e n e r a t i o n (and see a l s o , t h e r e v i e w s by L e w i s e t a l . I975a,b on t h e r o l e s o f k a i r o m o n e s ) . S i n c e s t i m u l a n t s and a t t r a c t a n t s c a n be b r o a d l y c a t e g o r i z e d as b e i n g c a p a b l e or i n c a p a b l e of c a r r y i n g i n f o r m a t i o n a b o u t h o s t d e n s i t y ( H a s s e l l and Southwood,1978), i t i s o f t e n d i f f i c u l t t o a s s e s s how a f e m a l e p e r c e i v e s " h a b i t a t r i c h n e s s " . B e h a v i o u r a l i n d i c a t o r s o f a p e r c e i v e d i n c r e a s e i n r i c h n e s s may i n c l u d e : ( i ) more r a p i d and p r o l o n g e d a n t e n n a l p a l p a t i o n o f t h e c o n t a m i n a t e d a r e a ; ( i i ) p r o l o n g e d o r t h o k i n e t i c r e s p o n s e upon f i r s t c o n t a c t ; ( i i i ) i n c r e a s e d r a t e o f t u r n i n g o r p r o l o n g a t i o n o f k l i n o k i n e s i s ; ( i v ) an o v e r a l l i n c r e a s e i n t i m e s p e n t on a p a t c h . In g e n e r a l , i t has been shown t h a t an i n c r e a s e i n h o s t d e n s i t y p r o d u c e s an i n c r e a s e i n s e a r c h t i m e . C o m p a r i s o n s o f S p a l a n g i a and M u s e i d i f u r a x s p e c i e s d e m o n s t r a t e d t h a t S p a l a n g i a 1 0 females from Chile and S.cameroni from Puerto Rico moved between patches more often and spent more time on the patches with higher densities of hosts than did Muse i d i furax spp. (Legner,1967). Abies and Shepard (1974b) corroborated this finding and went on to describe the functional responses of S.endius and M.raptor as being linear and quadratic, respectively, at house-fly densities of up to 80 per female per 72-hour period. That i s , a straight l i n e described the rela t i o n s h i p between the number of hosts attacked and host density for S.endius, and a curved l i n e described the response for M.raptor. The functional response i s dependent on temperature and the ef f e c t s of temperature on oviposition are often reported (Burnett,1951,1954, for Dahlbominus fuse ipennis (Zett.); Podoler and Mendel,1979, for M.raptor; Abies and Shepard,1976, for S.endius (maximum 17 progeny emerging/24-hour oviposition period at 32.2°C) and M.raptor). In addition, age of the ovipositing female may l i m i t effectiveness at high host densities, as may the related factors of female siz e , n u t r i t i o n a l history (Wylie,1965,1966) and the oviposition/host feeding experience of the adult (Flanders,1935; Edwards,1954) . The relationships among patch stimulus strength (chemotactile), host density and behavioural response have not been developed for this group of parasitoids as they have for N.canescens (Corbet,1973--concentration of mandibular gland secretions of i t s host, Ephestia kuehniella Z e l l . ; Waage,l979— Plodia interpunctella Hubn. secretions are l i n e a r l y related to 11 search time), or Apanteles melanoscelus (Ratz.) (Weseloh,1980— examination time of 10 or 50 strands of host Lymantria dispar L. s i l k ) . Theoretical speculations on the effects of host dispersion on parasite response form too large a body of l i t e r a t u r e to review adaquately here, but some points can be made about the u t i l i t y of dispersion in the habitat. In laboratory studies, hosts are generally placed in convenient, regular arrays or in a r b i t r a r i l y sized groups, usually without acknowledgement of the implications of such designs or of the lack of information about real host dispersion. Even so, Chabora (1967) was able to select two strains of N.vitripennis on the basis of oviposition response to a host dispersion pattern; one str a i n had an ..only s l i g h t l y higher reproductive rate than the other, yet par a s i t i z e d a s i g n i f i c a n t l y greater number of adjacent Phaenicia  sericata (Meig.) host pupae (20%) that were arranged 1 cm apart in a square 6 X 6 pattern. Since host density i s a determinant of patch time, so too must be patch area (or volume) and shape. Factors That Decrease Search Time In some species, an increased proportion of parasitized hosts in a patch may represent a perceived decrease in host density and thus produce lower search times. Whether a par a s i t i z e d host i s 'available' can depend on what Wylie (1972) described as a species' " i n t r i n s i c competitive a b i l i t y " , that i s , "those factors giving the parasitoid larva a competitive edge"; these are, (i) developmental time of the egg and larva, 12 and ( i i ) p r e d a t o r y a g g r e s s i v e n e s s of the f i r s t - i n s t a r l a r v a . S . e n d i u s , f o r i n s t a n c e , has been d e s c r i b e d as h a v i n g " c o m p e t i t i v e l y i n f e r i o r " l a r v a e (Markwick,1974 i n Legner,1977) compared w i t h M . z a r a p t o r , M . u n i r a p t o r K. & L. and P a c h y c r e p o i d e u s vindemiae ( R o n d a n i ) , meaning t h a t the S p a l a n g i a l a r v a e d i d not s u r v i v e i n s u p e r p a r a s i t i z e d h o s t s . Legner (1977) s t a t e s f u r t h e r t h a t Muse i d i f u r a x s p e c i e s have an " i n n a t e c o m p e t i t i v e s u p e r i o r i t y " t o S p a l a n g i a s p e c i e s , a l t h o u g h h a b i t a t p a r t i t i o n i n g and temperature e f f e c t s on b e h a v i o u r may make such a g e n e r a l i z a t i o n u n t e n a b l e . E l i m i n a t i o n of c o m p e t i t o r s t h r o u g h a t t a c k by f i r s t - i n s t a r l a r v a e has been r e p o r t e d f o r many Hymenoptera ( i n c l u d i n g ichneumonids, b r a c o n i d s , e u l o p h i d s , c y n i p i d s , c h a l c i d i d s , e n c y r t i d s and one s p e c i e s of p a r a s i t i c D i p t e r a — V i n s o n and Iwan t s c h , 1 9 8 0 ) , and i s one o p t i o n taken by c o m p e t i t i v e l y s u p e r i o r l a r v a e . Developmental s u p p r e s s i o n of a p r e p u p a l s t a g e of the c o m p e t i t o r i s another ( r e p o r t e d f o r S . c a m e r o n i — G e r l i n g and Legner,1968). An edge i n de v e l o p m e n t a l time can determine the outcome i n the case of n e a r l y s i m u l t a n e o u s a t t a c k s by d i f f e r e n t s p e c i e s . For i n s t a n c e , e i t h e r M.raptor . or N . v i t r i p e n n i s may win i n a s i m u l a t a n e o u s a t t a c k , and any s i g n i f i c a n t ( e . g . , 24- or 48-h) l e a d by one or the o t h e r w i l l e nsure v i c t o r y ( W y l i e , 1 9 7 2 ) . I t can thus be seen t h a t the q u e s t i o n of a v a i l a b i l i t y of a p a r a s i t i z e d h o s t becomes a c o m p l i c a t e d i s s u e i n which one must a l s o c o n s i d e r a s p e c i e s ' d i s c r i m i n a t o r y a b i l i t y - - t h e a b i l i t y of the female t o det e r m i n e whether a hos t i s p a r a s i t i z e d - - a n d the a b i l i t y t o e x e r c i s e 13 o v i p o s i t i o n r e s t r a i n t in those cases l i k e l y to 'waste' eggs. Three o ther responses that tend to decrease s earch time a r e : ( i ) P h y s i c a l i n t e r a c t i o n s w i th males . T h i s i s p r o b a b l y a minor p o i n t s i n c e , in g e n e r a l , hymenopteran females mate on ly once ( B a r r a s s , 1 9 7 6 ; P r i c e , 1 9 8 0 ; Appendix 3 ) . However, m u l t i p l e matings i n N . v i t r i p e n n i s may occur under c e r t a i n c o n d i t i o n s (van den Assem and V i s s e r , 1 9 7 6 ) , r e s u l t i n g in time wastage for the female (Walker ,1980) and a concomitant i n c r e a s e in male progeny (van den Assem,1977; van den Assem and d e . B r u i j n , 1 9 7 7 ) . ( i i ) P h y s i c a l i n t e r a c t i o n s w i t h o t h e r f emales . A c l a s s i c experiment by Debach (1944), u s i n g 1 0 - g a l l o n t i n s f i l l e d wi th b a r l e y g r a i n s and v a r i o u s d e n s i t i e s of Musca p u p a r i a and s e a r c h i n g N . v i t r i p e n n i s females , demonstrated the e x i s t e n c e of a f emale -produced s e a r c h - a r e a contaminant tha t decreased l e v e l s of p a r a s i t i s m . ( i i i ) P h y s i o l o g i c a l response decrement . I t i s supposed tha t some decrementa l p r o c e s s ( e s ) , o b s e r v a b l e a t the b e h a v i o u r a l l e v e l and o p e r a t i n g on the n e u r o p h y s i o l o g i c a l sys tem, may be u l t i m a t e l y r e s p o n s i b l e for ending a s e a r c h . Such proces se s are e n v i s i o n e d as hav ing c e n t r a l ( i . e . , i n the sensory c o r t e x ) or p e r i p h e r a l (at the r e c e p t o r or e f f e c t o r ) changes which , in the case of h a b i t u a t i o n , cause "the p r o g r e s s i v e decrease in magnitude a n d / o r p r o b a b i l i t y of a response r e s u l t i n g from i t s repeated e l i c i t a t i o n s by an i n t e r m i t t e n t s t i m u l u s " ( E i s e n s t e i n , 1 9 7 2 ) . In e l u c i d a t i n g another p r o c e s s , C o r b e t (1973) g i v e s an " i n t e r p r e t i v e h y p o t h e s i s " of sensory a d a p t a t i o n based on the 1 4 assumption that changes i n responsiveness r e l a t e to changes at the chemosensory r e c e p t o r s . Her work with N e m e r i t i s canescens demonstrated the e f f e c t s of exposing p a r a s i t o i d females to a range of c o n c e n t r a t i o n s of mandibular gland m a t e r i a l s of t h e i r host, E p h e s t i a k u e h n i e l l a . The data suggested that the responses of grooming, and jabbing the s u b s t r a t e with the o v i p o s i t o r , c o u l d be i m p l i c a t e d i n r e l a t i n g patch time, o v i p o s i t i o n e f f i c i e n c y and host d e n s i t y . A f o r a g i n g model f o r N e m e r i t i s canescens has been suggested (Waage,l979) in which host d e n s i t y i s an incremental v a r i a b l e and host odour s t r e n g t h (the p e r c e i v e d host d e n s i t y ) s p e c i f i e s the female's response l e v e l i n a d i r e c t , l i n e a r f a s h i o n . A second incremental v a r i a b l e , o v i p o s i t i o n , adds one u n i t per o v i p o s i t i o n to the response l e v e l . T h i s l e v e l , a measure of the i n s e c t ' s d r i v e , decrements l i n e a r l y to the l e a v i n g t h r e s h o l d , at which p o i n t , "turning-back" i s no longer e l i c i t e d when she c r o s s e s the patch boundary. F u r t h e r , the model p r o v i d e s f o r two d e c i s i o n s by the s e a r c h i n g f e m a l e - - ( i ) the d e c i s i o n to stop and i n v e s t i g a t e ; ( i i ) the d e c i s i o n encompassing host a c c e p t a n c e — both of which a c t to prolong search time i n a stepped f a s h i o n . A t h i r d d e c i s i o n , to leave, g e n e r a l l y i n c o r p o r a t e d i n t o the search-time concept (e.g., Charnov,1976; Cook and Hubbard,1977), i s e l i m i n a t e d . Of concern to t h i s t h e s i s , i s the N e m e r i t i s model's p r e d i c t i o n that o v i p o s i t i o n while on the patch produces an i n c r e a s e i n the female's search time. 15 GENERAL METHODS AND MATERIALS FOR HOST SEARCHING STUDIES Test Arena The bottoms of glass p e t r i dishes (diam. 13.7 cm, height 2.0 cm) were used as arenas for searching behaviour. Female Spalangia were introduced into an arena by placing them at the centre of a clear plexiglass sheet that was then inverted to serve as a cover for the arena. A s t r i p of acetate (2.0 X 50.0 cm) wrapped around the inside perimeter of the dish provided a square edge for the cover to rest on and prevented escape of the insects. Substrate Sawdust co l l e c t e d at a hardwood lumber m i l l was sieved twice (mesh sizes: 2/cm, 6.4/cm) to recover p a r t i c l e s of uniform size. (Sawdust of cedar, a softwood, was used in the f i r s t three experiments because a vigorous search response could be e l i c i t e d with sawdust that had been used as the host pupation medium in the colony maintenance regimen.) When tamped down l i g h t l y , these p a r t i c l e s presented an unobstructed walking surface for the females. The arena fl o o r was divided into semi-circular areas and covered to a depth of 0.5 cm with l i g h t l y tamped sawdust treated in two ways: (i) 'uncontaminated'—moistened only; ( i i ) 'host-contaminated'--moistened, and prepared by leaving unwashed, mature host larvae to pupate in the sawdust. Hosts were removed from the sawdust by sieving the batch to be used 16 that day. Experimental Animals Hosts and p a r a s i t e s were reared at 25°C f 50-60 % R.H. And a 14:10 L:D photoperiod. Hosts: Musca domestica l a r v a e were reared i n 4 1 g l a s s j a r s on a mixture of wheat bran (400 g), y e a s t ( l 5 g), molasses (15 ml) and water (700 ml) (a formula used f o r h o u s e f l y r e a r i n g at the Simon Fr a s e r U n i v e r s i t y i n s e c t a r y ) . Time to maturity depended on the d e n s i t y of l a r v a e p a r t i c u l a r l y of- the t h i r d i n s t a r s ; too few ( s e v e r a l hundred) or too many ( s e v e r a l thousand) lengthened the l a r v a l stage. G e n e r a l l y , the developmental p e r i o d ranged between 7 and 10 days. Prepupal l a r v a e and r e a r i n g medium were dumped onto a screen, the holes of which were j u s t l a r g e enough to permit the l a r v a e t o d r o p i n t o a t r a y below that c o n t a i n e d moistened sawdust. T h i s s e p a r a t i o n of l a r v a e from the medium was f a c i l i t a t e d by a 100— watt lamp s h i n i n g on the screen. The sawdust was s i e v e d to o b t a i n pupae of known age and s i z e . Age of pupae was determined from the time of e v e r s i o n of the pupal head. P a r a s i t e s : Host pupae 6-48 h o l d were p a r a s i t i z e d by p l a c i n g them in the Spalangia endius colony cages f o r 24 h. Females s t a r t e d to emerge 24 days a f t e r p a r a s i t i z a t i o n . F u r t h e r d e t a i l s are given i n the s t a n d a r d i z a t i o n procedures f o r experiments. Recording Behaviour Two types of r e c o r d e r s were used: a s i x t e e n - c h a n n e l 1 7 behaviour r e c o r d e r designed by the E n g i n e e r i n g and S t a t i s t i c a l Research I n s t i t u t e ( A g r i c u l t u r e Canada) and a twenty-channel E s t e r l i n e Angus chart r e c o r d e r . Behaviours recorded were: walk; burrow ( d i g g i n g i n t o the s u b s t r a t e ) ; f l y ; r e s t ; and antennate (waving the antennae i n the a i r while s t r e t c h e d up on the l e g s ) . Avoidance behaviour, that i s , t u r n i n g back upon encountering the edge of e i t h e r s u b s t r a t e type with antennae and/or f o r e t a r s i , without r e s t i n g or e n t e r i n g the new patch, was a l s o recorded. Events were recorded f o r four p a r t s of the arena: top, s i d e , and contaminated (C) and uncontaminated (U) s u r f a c e s . L a t e n c i e s ( i . e . , the p e r i o d s before f i r s t occurrence) of behaviours or events were noted as: l a t e n c y of search (time from the beginning of the t e s t to f i r s t c o n t a c t with the s u b s t r a t e ) ; and l a t e n c y of burrowing (time from f i r s t s u b s t r a t e contact to f i r s t burrowing e v e n t ) . Expt. 1: V a r i a b i l i t y of Search Behaviour with the Time of Last  O v i p o s i t i o n Known--Set J_ Object i v e : to estimate v a r i a b i l i t y in search behaviour among females when the time of t h e i r l a s t o v i p o s i t i o n was known. Methods -and M a t e r i a l s ( 1 ) S t a n d a r d i z a t i o n of Females A group of eight females was s e l e c t e d from those amongst f r e s h host puparia on the f l o o r of a colony cage. The cage contained a cohort of mated females that were 6 - 8 days o l d . Each 18 female was i n t r o d u c e d i n t o a p e t r i d i s h (4.0 cm diam. by 1.2 cm depth) c o n t a i n i n g only one host puparium (24-56 h old) and a small square of f i l t e r paper moistened with honey s o l u t i o n . The f i r s t f i v e females to complete host acceptance were used to t e s t search behaviour in order that the c o n s e c u t i v e searches of i n d i v i d u a l s c o u l d be separated by one hour. Hosts were removed immediately a f t e r the females l e f t them. (2) S u b s t r a t e The t e s t arena was set up as d e s c r i b e d e a r l i e r , with f i n e cedar sawdust, contaminated and uncontaminated. (3) T e s t i n g Each female was observed f o r three ten-minute p e r i o d s at h o u r l y i n t e r v a l s commencing one hour a f t e r completion of the -..host . acceptance t r i a l . Females were t e s t e d c o n s e c u t i v e l y a c c o r d i n g to the order of t h e i r completion of host acceptance and each was c o n f i n e d to her own i n d i v i d u a l p e t r i d i s h a f t e r the s t a n d a r d i z a t i o n procedure was completed and a l s o between o b s e r v a t i o n p e r i o d s . Each female was t r a n s f e r r e d i n a separate v i a l . F i v e arenas, one f o r each female, were prepared before the f i r s t o b s e r v a t i o n p e r i o d as d e s c r i b e d above under "General Methods and 'M a t e r i a l s " and were kept covered between search p e r i o d s . R e s u l t s The contaminated s u b s t r a t e was touched f i r s t i n 80 per cent of the searches (Table 1). Time spent on both host-contaminated (C) and uncontaminated (U) s u b s t r a t e s decreased i n s u c c e s s i v e searches but t h i s decrease was s i g n i f i c a n t f o r • C only 19 (P < 0.02). More of the t o t a l time was spent on C than on U dur i n g Search 1 (P < 0.05). A change i n the p r o p o r t i o n of females a v o i d i n g the C and U patches occurred i n the f i r s t three minutes of the t e s t s ; t u r n i n g back i n t o the uncontaminated patch i n c r e a s e d d u r i n g Search 2. No s e a r c h i n g was observed during the l a s t ( t h i r d ) t e s t p e r i o d , d e s p i t e lengthening the r e s t time from 1 h'( female #3) to 2 h (#2,5) and 3 h (#1,4). Table 1. Behaviour observed f o r females s e a r c h i n g host-contaminated (C) and uncontaminated (U) s u b s t r a t e s i n c l u d e d the s u b s t r a t e t y p e , f i r s t c o n tacted by the females, the number of times females avoided each s u b s t r a t e type f o r the ten-minute o b s e r v a t i o n p e r i o d and f o r the f i r s t three minutes of that p e r i o d , and the mean t o t a l s u b s t r a t e time (times i n minutes). Search 1 Search 2 . Substrate type U 'C U C F i r s t c o n t a c t (# females) 1 4 1 4 # Avoidance events 20 9 14 15 # Avoidance events o c c u r r i n g in f i r s t three minutes 9 1 7 6 Mean of t o t a l time spent 0.4 3.3 0.2 0.9 on the s u b s t r a t e +/- s.d. 0.47 1.13 0.14 0.42 A l l females burrowed but only d u r i n g the f i r s t search and only on the host-contaminated s u b s t r a t e . Latency of burrowing was b r i e f , v a r y i n g from 0.1 to 0.7 minutes. The mean number of burrowing bouts per female was 8 and these had a mean d u r a t i o n of 0.1 minutes. The d i s t r i b u t i o n of burrowing throughout the experiment i s 20 shown i n F i g u r e 1 as the mean per cent time spent burrowing d u r i n g each minute. T h i s p r o p o r t i o n was g r e a t e s t d u r i n g minute 2 of the ten-minute t e s t p e r i o d (44%), and l e s s than 5% i n minutes 4-10. F l i g h t occurred only from s u b s t r a t e s u r f a c e s (not from s i d e s or top of the arena) and occ u r r e d sooner i n s u c c e s s i v e searches (at a mean time of 4.0 minutes a f t e r the s t a r t of Search 1 and at 1.0 minutes a f t e r the s t a r t of Search 2). P r e - t e s t host acceptance r e s u l t s were v a r i a b l e : 2 f l i e s emerged and 2 p a r a s i t e s were produced. I f the r e s u l t s of the p r e - t e s t host-acceptance (and hence any e f f e c t on searc h i n g due to time s i n c e the , l a s t o v i p o s i t i o n ) can be used to p r e d i c t search response, then at l e a s t two of the v a r i a b l e s of t h i s experiment had to be re-examined i n an attempt to o b t a i n a r e p e t i t i o n * of searching performance by i n d i v i d u a l females, and a c o n s i s t e n t response to the host. Expt.2: V a r i a b i l i t y of Search Behaviour with the Time of Last  O v i p o s i t i o n Known—Set 2 O b j e c t i v e : to improve s e a r c h i n g performance by removing the pungent cedar odour from the arena s u b s t r a t e m a t e r i a l s and by s e l e c t i n g a more uniform group of s u b j e c t s . Methods and M a t e r i a l s (1) S t a n d a r d i z a t i o n of Females Seven t h r e e - d a y - o l d females were s e l e c t e d from a cohort t h a t had emerged w i t h i n a 20-h p e r i o d . A l l had mated and fed on honey and h o s t s . I n d i v i d u a l s were chosen from those that were 21 F i g u r e 1. The d i s t r i b u t i o n of mean b u r r o w i n g t i m e d u r i n g E x p t . 1 22 BURROWING 4 0 - 1 2 0 . 1 1 5 - 1 H 1 1 1 1 1-B> 9 - 1 0 . SEARCH INTERVAL CMIIM-) 23 o v i p o s i t i n g i n host puparia on the f l o o r of the cage. Each female was given a p r e - t e s t host at 1530 h. The f i r s t f i v e females completing the host acceptance procedure were s e l e c t e d for study. A f t e r t e s t i n g , females were placed i n host exposure dishes c o n t a i n i n g 15 f r e s h hosts, honey and water. The next day, they were removed at 1430 h, at which time each female was given a p r e - t e s t host, 24-36 h o l d , that was recovered immediately a f t e r she l e f t i t . They were then t e s t e d f o r Search 2. (2) T e s t i n g The f i v e females were observed f o r two ten-minute p e r i o d s , separated by about 22.5 h, that commenced w i t h i n an hour of host acceptance. Arena set-up and t r a n s f e r c o n d i t i o n s were as d e s c r i b e d f o r Set 1. (3) S u b s t r a t e F i n e uncontaminated cedar sawdust was washed with 95% e t h a n o l , then tap water, and d r i e d . One hundred mature l a r v a e were put i n t o a 500 cc p o r t i o n of sawdust f o r 24 h. A c o n t r o l batch of the washed sawdust was l e f t uncontaminated. R e s u l t s The ethanol washes removed most of the cedar odour and some of the brown c o l o u r from the sawdust. Twenty-six per cent of the l a r v a e had pupated by the f i r s t day of the experiment, 76% by the second day. A sample of the pupation sawdust was used i n p r e p a r i n g the contaminated h a l f of the arena f l o o r . Females spent very l i t t l e time on the s u b s t r a t e (Table 2). That three i n d i v i d u a l s d i d burrow i n d i c a t e d that s u f f i c i e n t s timulus was a v a i l a b l e i n the contaminated s u b s t r a t e . There was 24 no s e a r c h i n g on the second day; females spent most of the ten-minute p e r i o d on the c e i l i n g p l a t e . Grooming behaviours recorded were: grooming antennae with the f o r e l e g s ; wings and abdomen with the h i n d l e g s ; both f o r e l e g s and h i n d l e g s grooming si m u l t a n e o u s l y . No s i g n i f i c a n t d i f f e r e n c e c o u l d be found i n grooming performance d u r i n g the f i r s t search p e r i o d s (pooled mean t o t a l s / f e m a l e : E x p t . 1 , 83 ± 69; Expt.2, 33 ± 24) to i n d i c a t e that washing the s u b s t r a t e had a f f e c t e d search behaviour. Table 2. Length of time ( i n min.) spent by females on host-contaminated (C) and uncontaminated (U) arena s e c t i o n s . In the f i r s t , search, the C s e c t i o n was the f i r s t c o n t a c t e d by four i n d i v i d u a l s ; i n the second search, a l l had to be p l a c e d on the s u b s t r a t e to s t a r t the t e s t . . -.. . The p r e - t e s t host-acceptance r e s u l t s show whether an egg was l a i d or not (y or n ) . Female 1 Search 2 3 1 4 5 1 Search 2 2 3 4 5 F i r s t touched C U c c c had to be pla c e d on C T o t a l C time 0.29 0 1 .02 0.67 1 .39 0.14 0.17 0.76 0.81 T o t a l U time 0.40 0.17 0.64 0.49 0.96 0.48 0.41 0.09 0.09 Burrow time 0.16 0 0.19 0 0.12 0 0 0 0 performed on C - C - C - - - -P r e - t e s t Egg y y y y y n y y n n A l l females o v i p o s i t e d i n the f i r s t p r e - t e s t host, while only two d i d so i n the second. During the 22.5 hours s e p a r a t i n g the search p e r i o d s , host a t t a c k was q u i t e v a r i a b l e , with 3 to 11 progeny being produced from the 15 a v a i l a b l e h o s t s . Female 3 25 produced the fewest progeny and had the highest burrowing score i n the f i r s t search (Table 2). Female 4 had been r e l u c t a n t to accept the second p r e - t e s t host, d i d not complete the acceptance f o r s e v e r a l hours, and was not t e s t e d . Female 1 was t e s t e d f o r Search 2 but had shown no i n t e r e s t i n the host. Another i n d i c a t o r of the frequency of p a r a s i t o i d - h o s t i n t e r a c t i o n , the number of hosts k i l l e d , suggested a negative a s s o c i a t i o n of search time and k i l l i n g a c t i v i t y ( F i g . 2 ) , although too few degrees of freedom were a v a i l a b l e to d i s p r o v e the n u l l h y p o t h e s i s . Expt.3: C o r r e l a t i n g Age, Reproductive Dynamics and Search  Behaviour O b j e c t i v e : to assess changes in search behaviour o c c u r r i n g over a p e r i o d of 1 week from emergence i n terms of the cumulative r e p r o d u c t i v e output of the i n d i v i d u a l . Methods and M a t e r i a l s (1) P a r a s i t e Females F i f t e e n females, emerged d u r i n g a 19 h p e r i o d , were mated and a r b i t r a r i l y a ssigned to 2 groups. The t e s t group of f i v e i n d i v i d u a l s was t e s t e d f o r search behaviour over a one-week p e r i o d from emergence. (2) Hosts D a i l y c o l l e c t i o n s of host pupae were made; any e x h i b i t i n g p h y s i c a l a b n o r m a l i t i e s were d i s c a r d e d . Pupae were s i e v e d to remove a l l i n d i v i d u a l s smaller than 2.5 mm diam; these were 26 F i g u r e 2. Search d u r a t i o n vs. the number of hosts k i l l e d by females of Expt.2 1.5 I 1-0 1 0-5 I 0 , 0 H 1 1 1 1 1 1 1 1 1 1 1 1—3 1. E« 3- 4- 5- E« 7. B- 9«10*11-1H-13«14 # HOSTS KILLED 28 a l s o d i s c a r d e d . Maximum age of the pupae was determined by the l a s t c o l l e c t i o n date; minimum age was set by removing a l l white to l i g h t - t a n pupae, g i v i n g an experimental group 4-30 h o l d . (3) Substrate Ethanol-washed s u b s t r a t e was arranged i n contaminated and uncontaminated arena s e c t i o n s as b e f o r e . (4) Host Exposure Dishes Each female was t r a n s f e r r e d to a c l e a n p e t r i d i s h every 24 h f o r 28 days or f o r as long as she remained a l i v e . The dishes c o n t a i n e d hosts (20/day f o r the f i r s t 4 days, 15/day t h e r e a f t e r ) on 1 sheet of moistened f i l t e r paper s p r i n k l e d with contaminated sawdust and a f i l t e r paper square moistened with honey s o l u t i o n on the cover p l a t e . (5) . T e s t i n g The f o l l o w i n g exceptions to the standard t e s t format were implemented: ( i ) Latency of Search--a maximum of f i v e minutes was allowed f o r the female to contact the s u b s t r a t e . I f con t a c t was not made, she was t r a n s f e r r e d by v i a l to the s u b s t r a t e s u r f a c e . ( i i ) A l l behaviours were recorded over a ten-minute p e r i o d commencing with f i r s t s u b s t r a t e c o n t a c t . ( i i i ) I n v e s t i g a t i o n - - a new behaviour, d i s t i n c t from 'walk' r e g a r d l e s s of pace, by v i r t u e of the antennae being h e l d together or p a r a l l e l , as opposed to being splayed, while i n v e s t i g a t i n g a p o i n t source or hole i n the s u b s t r a t e . ( i v ) Latency of I n v e s t i g a t i o n — t i m e from f i r s t s u b s t r a t e contact to f i r s t i n v e s t i g a t i o n . 29 R e s u l t s Randomized complete block analyses were performed on the data. Females of the search group l i v e d as long as, and l a i d as many eggs as, the c o n t r o l group; the d a i l y egg p r o d u c t i o n of the two groups was a l s o s i m i l a r ( F i g . 3 ) . (Comparison of the d a i l y progeny p r o d u c t i o n of the c o n t r o l group with that of v i r g i n females i s d e l a y e d u n t i l S e c t i o n I I I ( F i g . 11) f o r a more complete treatment.) T o t a l time spent on the contaminated s u b s t r a t e (C) v a r i e d s i g n i f i c a n t l y among p e r i o d s (Table 3). The t e s t group was not provided with hosts on days 5 and 6, and was not t e s t e d f o r s e a r c h i n g in P e r i o d 6. By the t e s t time of P e r i o d 7, t h i s group had been without hosts f o r about 51 hours; time spent on C was longer- (P < 0.01) d u r i n g t h i s t e s t ( F i g . 4 ) . .Although burrowing c o n t r i b u t e d to t h i s i n c r e a s e i n patch time ( F i g . 5 ) , i t appeared i r r e g u l a r l y throughout the week. Burrowing and i n v e s t i g a t i o n times, combined to give the p r o p o r t i o n of s e a r c h i n g on E, suggested that females and p e r i o d s were d i f f e r e n t ; t h i s was not supported s t a t i s t i c a l l y at the 95% l e v e l i n the o v e r a l l anova (Table 4). Females of the search group were ranked a c c o r d i n g to t h e i r d a i l y cumulative egg p r o d u c t i o n . T h i s ranking, when compared with t h e i r rank f o r p r o p o r t i o n of i n t e n s i v e search behaviour (burrowing + i n v e s t i g a t i n g ) d u r i n g t h e i r t o t a l C time, showed p o s i t i v e c o r r e l a t i o n of s e a r c h i n g i n t e n s i t y with egg p r o d u c t i o n ( F i g . 6 ) . D e l e t i n g the maximum p r o d u c t i v i t y day f o r each female from the c a l c u l a t i o n s of Spearman's rho y i e l d e d : day 1, 0.88; day 2, 0.65; day 3, 0.63; day 4, 0.75; day 6, 0.80. F u r t h e r experimentation on the nature 30 of burrowing behaviour was i n d i c a t e d . Expt.4: P r o p o r t i o n of Burrowers i n the P o p u l a t i o n O b j e c t i v e : to devise a method f o r s e l e c t i n g animals p r o f i c i e n t at e x p l o i t i n g hosts b u r i e d i n a sawdust s u b s t r a t e . Hypothesis : that there e x i s t s a segment of the Spalangia endius p o p u l a t i o n that does not e x p l o i t b u r i e d h o s t s . M a t e r i a l s and Methods Two methods were used to assess burrowing i n t h i s experiment. Both i n v o l v e d using the post-emergence p e r i o d f o r three purposes: (1) to c o n d i t i o n the i n s e c t s to the s t i m u l i presented ( i . e . , host odour and sawdust); (2) to use the i n i t i a l r e s u l t , number of hosts not emerging, to i d e n t i f y experimental i n d i v i d u a l s f o r f u r t h e r t e s t s ; (3) to develop an index f o r burrowing and host e x p l o i t a t i o n . Two methods were used f o r c o n d i t i o n i n g females f o r study; both were employed in t h i s experiment, but only the second was used i n the next. C o n d i t i o n i n g Method 1 i n v o l v e d c o v e r i n g 16 hosts which were evenly spaced on a p e t r i d i s h bottom with 0.5 cm of contaminated sawdust. Females newly emerged and mated were intr o d u c e d i n t o the d i s h e s with water, honey d r o p l e t s and s h e l t e r f o r a p e r i o d of 3 d. During t h i s time, females were scored f o r burrowing ( i . e . , being out of s i g h t ) . L a t e r counts of p a r a s i t i z e d hosts were used to c o n f i r m the v a l i d i t y of these s c o r e s . C o n d i t i o n i n g Method 2 employed a s h o r t e r c o n d i t i o n i n g 31 F i g u r e 3. Number of eggs l a i d d a i l y by Expt.3 females of the Search ( V ) and No Search ( A ) groups. Hosts were not given to the Search group on days 5 and 6. F i g u r e 4. P r o p o r t i o n of t e s t time spent i n d i f f e r e n t p a r t s of the arena (U=uncontaminated, C=host-contaminated arena s u b s t r a t e s ) . Standard d e v i a t i o n given f o r C. 32 Table 3. T o t a l time spent by females on the host-contaminated s u b s t r a t e (C) f o r each of the s i x pe r i o d s i n which s e a r c h i n g was t e s t e d (days 1-7). Source d. f . S.S. M.S. F- r a t i o Prob. Female 4 18.606 4.6515 2. 4546 0.08097 Per iod 5 84.990 16.998 8. 9698 0.00017 E r r o r 19 36.005 1.8950 T o t a l 28 1,44.85 Table 4. Anova f o r the per cent time spent i n v e s t i g a t i n g and burrowing by the females of Expt, 3 duri n g searches performed over a one-week p e r i o d from emergence. Source d . f . S.S. M.S. F - r a t i o Prob. Female 4 2608.6 652.15 2.6525 0.06507 P e r i o d 5 3028.0 605.59 2.4631 0.07000 E r r o r 19 4671.4 245.86 T o t a l 28 10499. 3V F i g u r e 5. Time spent burrowing and i n v e s t i g a t i n g by females d u r i n g Expt.3 i n c r e a s e d with time spent on the contaminated (C) s u b s t r a t e . F i g u r e 6 . C o r r e l a t i o n of ranks f o r cumulative egg p r o d u c t i o n and the p r o p o r t i o n of i n t e n s i v e s e a r c h i n g (burrowing + i n v e s t i g a t i n g time) on the host-contaminated s u b s t r a t e (C). Days of maximum egg p r o d u c t i o n f o r each female are shown as A Y = -0'S844 * 0-6135 »X N = 29 f 1 0 . 9 - . . Z s 0 . 1 - 3 - 5 - 7 . 9 - 1 0 . TOTAL G TIME (MIN -) 0- !• S« 3» 4. RANK CUM- # EGGS 3* p e r i o d (1 day), fewer hosts (4) and u t i l i z e d the arrestment response at the patch edge to cue the female i n to hosts b u r i e d i n small patches. These four c i r c u l a r patches (1.5 cm diam.) of contaminated sawdust covered one host each and were formed i n a 0.5 cm deep l a y e r of uncontaminated sawdust. R e s u l t s C o n d i t i o n i n g Method 1 was deemed i n e f f e c t i v e f o r the purposes s t a t e d . I t s s i t u a t i o n d i d not c l o s e l y approximate the t e s t s i t u a t i o n ; a uniform l a y e r of sawdust contaminated by pupating l a r v a e was not p r o v i d i n g the "patch edge" stimulus that the females would encounter i n the t e s t arena. Attempts to d e v i s e a d i f f e r e n t c o n d i t i o n i n g method produced the f o l l o w i n g r e s u l t , when newly emerged, mated females were in t r o d u c e d i n t o - . p e t r i .dishes c o n t a i n i n g a l a y e r of uncontaminated sawdust with a small (1.5 cm diam.) patch of contaminated sawdust embedded at i t s c e n t r e . Such females t y p i c a l l y would walk at a f a s t r a t e a c r o s s the s u b s t r a t e , enter the c i r c u l a r contaminated area without slowing down, and proceed d i r e c t l y a c r o s s i t without s t o p p i n g . Immediately upon l e a v i n g the c i r c u l a r area, however, they would stop and take f l i g h t . A few hours l a t e r , they responded to the same arrangement by stopping when they f i r s t c o n t a c t e d the c o n d i t i o n e d s u b s t r a t e . C o n d i t i o n i n g Method 2 a l s o attempted to i d e n t i f y burrowing i n d i v i d u a l s by i n t e r m i t t e n t l y r e c o r d i n g any disappearance i n t o the sawdust. Table 5 shows that while t h i s method c o r r e c t l y d e signated "burrowers" (those d i s a p p e a r i n g at l e a s t once) as the group r e s p o n s i b l e f o r k i l l i n g a l l f l i e s (see #0), and "non-37 burrowers" as the group k i l l i n g none (see #4), the intermediate 77% of the sample c o u l d not be a c c u r a t e l y c l a s s i f i e d . S e l e c t i o n of t e s t i n d i v i d u a l s on t h i s basis'was i m p r a c t i c a l , t h e r e f o r e . Table 5. E f f e c t i v e n e s s of d i s t i n g u i s h i n g between females that burrowed f o r hosts and those that d i d not by i n s p e c t i n g dishes d u r i n g a 24-h p e r i o d i s evident i n 10/44 cases. Dishes each contained four h o s t s . # F l i e s Emerging per Dish 0 1 2 3 4 # Females c a l l e d "Non-burrowers" 0 3 6 8 1 # Females c a l l e d "Burrowers" 9 5 4 8 0 # Cases 9 8 10 16 1 A c o n s e r v a t i v e estimate of the s i z e of the non-burrowing segment of the p a r a s i t e p o p u l a t i o n i s obtained r e a d i l y from Table 6—20.3%, or 27/133 d i s h e s that produced 4 a d u l t f l i e s . Host m o r t a l i t y due to causes other than s t i n g i n g had been found to be 11 ± 5%. Of 532 hosts used i n t h i s study, however, 23.9% produced n e i t h e r hosts nor p a r a s i t e s . N a t u r a l m o r t a l i t y c o u l d thus add 6 females (from the "0 p a r a s i t e s " row) and enlarge the estimate of the non-burrowing segment of t h i s age group to 24.8%. 3$ Table 6. D i s t r i b u t i o n s of p a r a s i t e progeny and emerging hosts among d i s h e s c o n t a i n i n g four b u r i e d host p u p a r i a . Females were exposed to hosts as d e s c r i b e d i n " C o n d i t i o n i n g Method 2" # F l i e s Emerging per D i s h 0 1 2 3 4 Sum % # 0 3 5 1 2 32 27 79 59.4 P a r a s i t e s 1 7 2 8 1 2 29 21.8 Emerging 2 1 7 6 14 10.5 per 3 2 4 6 4.5 D i s h 4 5 5 3.8 Sum 18 18 26 44 27 1 33 Expt.5: Short-term E f f e c t s of O v i p o s i t i o n on Search Behaviour Hypothesis: that o v i p o s i t i o n w i l l i n c r e a s e search d u r a t i o n f o r a s e r i e s of c o n s e c u t i v e searches t e r m i n a t i n g i n o v i p o s i t i o n s . M a t e r i a l s and Methods (.1 ) S t a n d a r d i z a t i o n of Females Females were c o l l e c t e d from emergence d i s h e s at 1600 h each day,, checked fo r mating s t a t u s — m a l e s showed no i n t e r e s t i n mated females (q.v. Appendix 3)--and put i n d i v i d u a l l y i n t o prepared host exposure d i s h e s (see " P r o p o r t i o n of Burrowers" above) f o r p e r i o d 1 ( d u r a t i o n 24 h) . Dishes and hosts were r e p l a c e d the next day f o r p e r i o d 2. A l l hosts exposed to females were r e t a i n e d f o r the p r e - t e s t o v i p o s i t i o n r e c o r d . T e s t i n g was done on the t h i r d day a f t e r emergence. (2) S u b s t r a t e P r e p a r a t i o n One-half of the arena and the four patches c o v e r i n g hosts in the host exposure dishes c o n s i s t e d of hardwood sawdust that had been contaminated with host odour in the f o l l o w i n g way. Mature host l a r v a e were added to a c o n t a i n e r of s i f t e d , -59 moistened, hardwood sawdust and l e f t to pupate. D a i l y c o l l e c t i o n s of pupae were made and mature maggots were added as needed to maintain a supply of pupae. A f t e r s i f t i n g the sawdust to remove ho s t s , a sample was set a s i d e f o r the day's d i s h e s and t e s t arenas. O c c a s i o n a l l y , small a d d i t i o n s of uncontaminated sawdust were made to the pupation c o n t a i n e r . (3) Experimental Design The exact source of p o t e n t i a l v a r i a b i l i t y i n batches of contaminated sawdust gathered d a i l y , whether due to d i f f e r i n g numbers of l a r v a e pupating or to other causes, was not of d i r e c t concern. The "days", confounded with the "searches" as p l o t s , were thus - r e p l i c a t i o n s and served to reduce experimental e r r o r . "Treatments", the s p l i t - p l o t e f f e c t , was arranged by s e l e c t i n g •_.pai.rs , ..of ,' females .from a group, of four s t a n d a r d i z e d per .day f o r the with-host (H+) and without-host (H-) treatments. S e l e c t i o n was based on those v i s i b l e at t e s t time and females were randomized among treatments. (4) T e s t i n g T r a n s f e r of each female from host exposure d i s h to arena, from arena to host pupa and back, was e f f e c t e d by p l a c i n g a v i a l over the i n s e c t and w a i t i n g f o r her to walk i n t o i t . The up- or down-orientation of the walking i n s e c t was used to complete her t r a n s f e r to the new s u r f a c e . With a female at the ce n t r e of the arena l i d , the t e s t began when the l i d was put i n p l a c e . T e s t i n g stopped when any of the f o l l o w i n g c r i t e r i a was met: ( i ) the i n s e c t remained o f f the t e s t s u b s t r a t e f o r 15 c o n s e c u t i v e seconds; ( i i ) the i n s e c t 'io walked onto the cover of the arena; ( i i i ) the i n s e c t flew. At t h i s p o i n t , the H+ i n s e c t was t r a n s f e r r e d to the host by i n v e r t i n g the t r a n s f e r v i a l over the pupa; the female i n v a r i a b l y o r i e n t e d downward and found the host immediately. The H- i n s e c t was t r a n s f e r r e d a f t e r the search to a c l e a n , empty p e t r i d i s h and was l e f t there f o r a p e r i o d approximating the mean d u r a t i o n of host acceptance (30 min.). Upon completion of t h i s p e r i o d , i n s e c t s were p l a c e d at the c e n t r e of the cover f o r the s t a r t of Search #2. T h i s p a t t e r n was repeated f o r f i v e searches i n t e r s p e r s e d with treatment p e r i o d s . R e s u l t s (J_) Search Times "Search time" was that p e r i o d between the time that females touched the sawdust s u b s t r a t e and the time that they.met-one of the . l e a v i n g c r i t e r i a ; i t c o n s i s t e d of walking, antennal i n v e s t i g a t i o n , and burrowing. Antennating and grooming were never performed on the s u b s t r a t e . Search times v a r i e d between 21.51 and 0 minutes, the l a t t e r recorded f o r c o n t a c t i n g the s u b s t r a t e and immediately walking away ( F i g . 7 ) . The t r a n s f o r m a t i o n ln(Y + 10) was a p p l i e d to normalize the data which had an o v e r a l l mean of 1.99 minutes (with 95% confidence l i m i t s of 2.57 'and 1.44 minutes). The anova i n d i c a t e d s i g n i f i c a n t treatment e f f e c t (Table 7). Examining the i n t e r a c t i o n of search with treatment, I found that both Duncan's and Newman-Keul's m u l t i p l e range t e s t s put the H+ and H-treatments f o r Search 1 i n t o separate subgroups. P a i r e d comparisons of treatment means for the f i v e search groups i n d i c a t e d no treatment e f f e c t f o r Searches 1, 3 and 4 4J V (.05 < P < .10, Table 8), although t h i s d e c i s i o n regarding Search 1 appeared q u e s t i o n a b l e . Searches 2 and 5 were s i g n i f i c a n t l y d i f f e r e n t . A number of p o s s i b l e c o r r e c t i o n s produced u n i f o r m i t y i n the Search 1 times. One example i s the d e l e t i o n of data f o r the t h i r d (day 3) p a i r of females that c o n t r i b u t e d the lowest v a l u e s in the H+ group (Table 9). D e l e t i o n of data f o r the p a i r c o n t a i n i n g the highest S1 value i n the H- group gave the same r e s u l t s : a high p r o b a b i l i t y of s i m i l a r i t y of treatment groups i n S1, S3 and S4, and a low p r o b a b i l i t y of s i m i l a r i t y i n S2 and S5. Although i t i s not e n t i r e l y a p p r o p r i a t e to d e a l with the problem of non-homogeneous S1 groups in t h i s manner, the s e n s i t i v i t y of the data set i s demonstrated. I t i s noteworthy, too, that the modified, data s e t . o f Table 9 y i e l d e d an i n s i g n i f i c a n t treatment e f f e c t (Table 10). The v a l i d i t y of assuming a treatment e f f e c t ( i . e . of a c c e p t i n g homogeneity of S1 groups) w i l l be examined again l a t e r . (2) O v i p o s i t i o n Experience Did o v i p o s i t i o n p r i o r to t e s t i n g have an e f f e c t on (S1) search times? I t was noted i n the " P r o p o r t i o n of Burrowers" study t h a t 24.8% of females d i d not burrow or k i l l b u r i e d hosts; c o u l d t h i s e f f e c t be detected i n the search or burrowing times? The number of p r e - t e s t eggs l a i d was used to group the log-transformed S1 times of both groups (Table 11). No evidence f o r an added component of v a r i a n c e due to o v i p o s i t i o n experience c o u l d be found (Table 12), although comparing the "no eggs l a i d " group with a l l o t h e r s combined produced a l a r g e r F value (F=2.03, .75 < P < .90) than d i d the gure 7. Search times f o r Expt.5 females searching an area contaminated with odour of i t s host M.domestica. H+ females ( A ) were given a host pupa to p a r a s i t i z e between searches, H- females ( X ) were not. A b s c i s s a shows mean s t a r t times f o r the two treatment groups. . SEARCH TIME 4' -5' 20 - 45. 70- EE- ISO- 145 MINUTES 4« Table 7. S p l i t - p l o t anova f o r Expt.5. Y i e l d was search times of Spalangia endius i n host- and no-host treatments. Source d.f. S.S. M.S. E r r o r Prob. Day (D) 9 0.37195 0.04133 Search (S) 4 2.4294 0.60736 DS 0.1918E-7 DS . 36 1 . 1584 0.03218 Treatment (T) 1 0.25385 0.25385 DT 0.0304 DT 9 0.34729 0.03859 ST 4 0. 14880 0.03720 DST 0.1785 DST 36 0.80224 0.02228 E r r o r -T o t a l 99 5.5120 Table 8. P r o b a b i l i t i e s f o r p a i r e d compari-sons of the mean d u r a t i o n of search time f o r Searches 1-5 i n Expt.5. H+ (with hosts) and H- (without hosts) were the treatment groups. Data have been transformed by Y'=ln(Y+10). S1 S2 S3 S4 S5 n 10 10 10 9 8 H+ x 2.673 2.384 2.390 2.403 2.337 s* 0.051 0.002 0.011 0.011 0.001 n 10 10 10 9 6 H- x 2.900 2.525 2.409 2.505 2.391 s*- 0.081 0.008 0.009 0.131 0.002 P 0.064 0.4E-3 0.674 0.430 0.033 Table 9. The data of Table 8, mo d i f i e d by the d e l e t i o n of search times f o r one H+ / H- p a i r of i n s e c t s . See t e x t f o r f u r t h e r d e t a i l s . SI S2 S3 S4 S5 n 9 9 9 8 7 H+ x 2.7123 2.3832 2.3953 2.4132 2.3261 s* 0.0400 0.0027 0.0117 0.0115 0.0004 n 9 9 9 8 6 H- x 2.8572 2.5262 2.3828 2.5127 2.4066 s* 0.0707 0.0090 0.0026 0.1491 0.0014 P 0.2098 0.0011 0.7567 0.4942 0.0006 Table 10. S p l i t - p l o t anova f o r the m o d i f i e d data s e t — m i s s i n g Day 3. Treatment e f f e c t i s not s i g n i f i c a n t . See text f o r f u r t h e r d e t a i l s . Source d.f. S.S. M.S. E r r o r Prob. Day (D) 8 0.37026 0 .04628 Search (S) 4 2.2031 0 .55078 DS 0.3577E-6 DS 32 1.1316 0 .03536 Treatment (T) 1 0.14633 0 .14633 DT 0.0650 DT 8 0.25617 0 .03202 ST 4 0.08306 0 .02077 DST 0.3191 DST 32 0.54173 0 .01693 E r r o r -T o t a l 89 4.7323 44 comparison of 5 groups, or "low" and "high" groups (0,1,2 vs. 3,4 eggs l a i d ) . Spearman rank c o r r e l a t i o n t e s t s were performed on the data f o r number of p r e - t e s t progeny produced per female vs. t o t a l burrowing time i n the f i v e searches (rho = 0.180), and p r e - t e s t progeny vs. the sum of burrowing and i n v e s t i g a t i o n times in S1 (rho = 0.137). Number of p r e - t e s t hosts not emerging was a l s o t e s t e d a g a i n s t burrowing and search times. The r e s u l t was the same: There was no a s s o c i a t i o n between these t e s t behaviours and the s t i n g i n g or o v i p o s i t i o n behaviour i n the p r e - t e s t p e r i o d s when these p e r i o d s were lumped together. However, the number of progeny from the f i r s t p r e - t e s t p e r i o d was w e l l c o r r e l a t e d with t o t a l burrowing time throughout the experimental s e r i e s (rho = 0.629, P<0.01); the second p r e - t e s t .. p e r i o d d i d not. show c o r r e l a t i o n with experimental v a r i a t e s (Table 13). The trend d u r i n g these two c o n d i t i o n i n g p e r i o d s was that of i n c r e a s e d host k i l l and progeny p r o d u c t i o n (both P<0.005; Wilcoxon matched-pairs, signed ranks t e s t - - T a b l e 14). Table 11. Search 1 times grouped a c c o r d i n g to number of eggs l a i d by the females p r i o r to t e s t i n g i n Expt.5. Times i n l n (min. + 10). # Eggs L a i d P r i o r to T e s t i n g 0 1 2 3 4 2.3214 2.5848 2.9796 2.6561 2.7441 H+ 2.3437 2.9663 2.6497 2.6383 2.8484 3.2802 2.6872 3.4207 2.9962 3.0096 H- 2.6912 2.8190 2.6145 2.5649 2.9118 Table 12. Anova f o r search times of the o v i p o s i t i o n groups shown i n Table 11. Source d . f . S.S. M.S. F O v i p o s i t i o n Groups 4 0.2350 0.0588 0.7318 E r r o r 15 1.2042 0.0803 T o t a l 19 1.4392 4<9 Table 13. Rank scores f o r Search 1 times (A and B) and t o t a l burrowing times (C and D) fo r the twenty Expt.5 females (ranked from 20 , h i g h ) . # PROGENY # HOSTS KILLED A B P e r i o d 1 Pe r i o d 1 0 1 2 0 1 2 3 4 P 0 1,2,3, 4,9 1 2 2 3,4, e 10,19 10,19 r 1 15 7,16, 1,9 12,20 i 20 o 2 13 5,14, 6 13,15 7 5,6, 16 d o 1 7 14 3 8 18 •17 18 2 4 1 1 8,11 C D P 0 2,2,7, 6,14 1 7 7 2,13, e 13,16 14,16 r 1 11 8,9, 2,6 17,18 i 18 o 2 5 12,19, 1 5 5,11 9 12,15, 8 d 20 20 3 4 1 0 19 10 2 4 2 2,4 Table 14. Host acceptance r e s u l t s f o r the p r e - t e s t c o n d i t i o n i n g p e r i o d s . Twenty females e x p l o i t e d four b u r i e d Musca domestica pupae per 24-h p e r i o d . P e r i o d 1 P e r i o d 2 P Host K i l l x 1.30 1.60 <0.005 s* 1.17 1.27 # Progeny x 0.65 1.20 <0.005 s* 0.67 1.24 I I I . HOST ACCEPTANCE STUDIES Host Acceptance i n the Pteromalidae Edwards (1954) d i v i d e d the host-acceptance behaviour of Nason i a v i t r i p e n n i s i n t o d r u m m i n g - d r i l l i n g and o v i p o s i t i o n -f e e d i n g phases, a - d i v i s i o n which separated h o s t - e x t e r n a l and h o s t - i n t e r n a l s t i m u l i and produced c l a s s e s having d i s t i n c t l y d i f f e r e n t t i m e - e f f o r t requirements. In t h i s group of pupal p a r a s i t e s , the phase p r i o r to i n s e r t i n g the o v i p o s i t o r may be c o n s i d e r e d time-extensive and energy-expensive, whereas the p o s t - i n s e r t ion phase may be c o n s i d e r a b l y s h o r t e r , with a minimal o u t l a y of metabolic energy. The i m p l i c a t i o n s of such a d i v i s i o n can be v i s u a l i z e d by t r e a t i n g the separate acceptance behaviours as a h e i r a r c h y of " d e c i s i o n p o i n t s " (Dawkins and Dawkins,1973). Information-bearing s t i m u l i are r e c e i v e d and i n t e g r a t e d , then motor responses are r e l e a s e d to accomplish a succession of i n t e r m e d i a r y d e c i s i o n s . Consequently, the success or f a i l u r e of host acceptance i s not merely the r e s u l t of a s i n g l e g o a l -o r i e n t e d behaviour, o v i p o s i t i o n , but i s determined i n s t e a d by 5 0 the economic c o n s t r a i n t s of time and energy a r i s i n g from the organism's responses to i n f o r m a t i o n r e c e i v e d at each intermediary step. In t h i s s e c t i o n r e f e r e n c e w i l l be made to resear c h showing that s p e c i f i c types of i n f o r m a t i o n are c o l l e c t e d by the female engaged i n host acceptance. The host-e x t e r n a l / h o s t - i n t e r n a l s t r u c t u r e i s again r e l e v a n t i n d i s c u s s i n g the t i m i n g of the i n s e c t ' s e v a l u a t i o n of t h i s i n f o r m a t i o n . Information C o l l e c t e d P r i o r to I n s e r t i o n At f i r s t contact with the host, a female may not stop to i n v e s t i g a t e , or may p a l p a t e the host only b r i e f l y before l e a v i n g i t . The techniques of s t a n d a r d i z a t i o n of females promote readiness to o v i p o s i t and e l i m i n a t e or reduce incomplete host-acceptance responses (Edwards,1954; . Wylie,1971). The s t a t e of 'readiness' and i t s i m p l i c a t i o n s f o r stimulus relevance a s i d e , some a t t r i b u t e s of the host are ap p a r e n t l y more important than others f o r a p a r t i c u l a r p a r a s i t o i d s p e c i e s . Consequently, the a t t r i b u t e s presented below may not be r e l e v a n t f o r every spec i e s . Movement of the host may be a necessary p r e r e q u i s i t e f o r att a c k and o v i p o s i t i o n f o r some p a r a s i t o i d s (Arthur et a l . 1972, c i t i n g F i s h e r , 1 9 5 9 ) , Such movements may s t i m u l a t e ( r e l e a s e ) o v i p o s i t i o n or increase excitement ( r e f s . i n Vinson,1976), or may p r o h i b i t o v i p o s i t i o n i n egg p a r a s i t o i d s ( i n d i c a t i n g advanced development of the host--Jackson,1966). Age of the host i s o f t e n c o r r e l a t e d with s i z e , although these two parameters may be p e r c e i v e d independently by t h i s 5:i: group of pupal p a r a s i t o i d s . Since a space between pupa and puparium i s necessary f o r o v i p o s i t i o n i n these e c t o p a r a s i t i c p a r a s i t o i d s , and such a space i s not formed for a few hours f o l l o w i n g pupation, Edwards (1954) i n v e s t i g a t e d the exudate found on newly formed hosts ( " i n d i c a t i n g the presence of a space in the puparium") for i t s p o s s i b l e f u n c t i o n as a host acceptance r e l e a s e r . No such property c o u l d be shown f o r these droplet.s. Comparable a g e - r e l a t e d e x t e r n a l s t i m u l i have not been suggested. L i t t l e work has been, done ( d e s p i t e i t s p o p u l a t i o n consequences—Charnov e_t a l . 1981) on the a b i l i t y of the female to determine host s i z e , per se. C o r r e l a t i o n s between host s i z e and progeny sex r a t i o or number (e.g., Wylie,1966c) have been made, u s u a l l y without r e v e a l i n g p o s s i b l e mechanisms. In a set of elegant experiments, Klomp and Teerink (1962) demonstrated the p o s s i b l e r o l e of drumming in determining host s i z e by the g r e g a r i o u s egg p a r a s i t o i d , Trichogramma embryophagum Htg. They f i r s t e s t a b l i s h e d that l a r g e r s p e c i e s of hosts e l i c i t e d longer mean times f o r drumming and more eggs per o v i p o s i t i o n . Eggs of the s m a l l e s t host s p e c i e s ( E p h e s t i a ) were then clumped together using g e l a t i n and agar to form a "super-egg" that approximated that of the intermediate s i z e d host, E l l o p i a . The number of eggs l a i d i n the a r t i f i c i a l host (x" =4.1, range 3-5) was more than that l a i d i n E p h e s t i a (x~ = 1.1, range 1-2). The d e c i s i v e r o l e played by s i z e ( r a t h e r than by a h o s t - i n t e r n a l r e c o g n i t i o n of s p e c i e s ) was f u r t h e r supported when a small host was o f f e r e d f o r acceptance and, at the moment d r i l l i n g was to commence, an egg of a l a r g e r host s p e c i e s (or the super-egg), 51 was s l i p p e d between the l e g s of the a c c e p t i n g female. T h i s experiment and i t s converse c o n v i n c i n g l y demonstrated that host s i z e was p e r c e i v e d before o v i p o s i t o r i n s e r t i o n , probably by p a l p a t i o n of the antennae on the host's s u r f a c e . I t has been suggested that other g r e g a r i o u s p a r a s i t o i d s p e c i e s are, i n some f a s h i o n , a l s o able to determine host s i z e before a l l o t i n g t h e i r progeny; e.g., Caraphractus c i n c t u s Walker (myrmarid egg p a r a s i t e , suggested but not demonstrated i n Jackson,1966 ) ; N . v i t r i p e n n i s and M.raptor (Wylie,1 958 ,1967); Hyssopus thymus (P u r r i n g t o n and Uleman,1972). Host c o l o u r has a l s o been i m p l i c a t e d as a host-acceptance c r i t e r i o n by Takahashi and Pimentel (1967) . Although not supported by any b e h a v i o u r a l data, they suggested that N . . v i t r i p e n n i s females: " p r e f e r r e d " (as evidenced by the r e l a t i v e number of f l i e s emerging) black puparia over brown or the h y b r i d which was i n d i s t i n g u i s h a b l e from brown. A f t e r some p e r i o d of antennal drumming and abdomen tapping, the female o b t a i n s a f o o t - h o l d and begins to d r i l l . T h i s d r i l l i n g p e r i o d may l a s t from 10 to 60 minutes f o r N . v i t r i p e n n i s (5-45 m i n u t e s — W y l i e , l 9 7 0 ) d u r i n g which time hosts are seldom r e j e c t e d or abandoned, although the d r i l l i n g process may be i n t e r r u p t e d (Whiting,1967). A study by J a c o b i (1939, c i t e d i n Edwards,1954) rep o r t e d 80% of the d r i l l s i t e s f o r N . v i t r i p e n n i s to be at an intersegmental notch where the puparium, though t h i c k e r , has a v e r t i c a l arrangement of i t s c h i t i n . The p r e f e r r e d d r i l l i n g s i t e s f o r N . v i t r i p e n n i s , r e p o r t s Wylie (1970) , are the v e n t r a l t h o r a c i c and abdominal areas over the l e g s and wings of 5^ the pupa. V a r i o u s aberrant s u r f a c e f e a t u r e s of the puparium w i l l cause the female-to r e j e c t a host; a broken puparium w i l l not e l i c i t d r i l l i n g , although a puparium with a concealed break w i l l (Edwards,1954), as w i l l a p a r t i a l l y exposed puparium (Wylie,1971). A naked pupa i s not i n v e s t i g a t e d (Whiting,1967). Given that such a s i g n i f i c a n t p r o p o r t i o n of the host acceptance time and energy expenditure i s taken up by the pre-i n s e r t i o n behaviours, one would expect that these pupal p a r a s i t o i d s should possess a system f o r d i s c r i m i n a t i n g h e althy from p a r a s i t i z e d hosts p r i o r to d r i l l i n g . Simmonds (1954) r e p o r t s that Spalangia d r o s o p h i l a e Ashm. o f t e n r e j e c t s p a r a s i t i z e d hosts before attempting to d r i l l (and l a y s only one egg per o v i p o s i t i o n - - v a n - Lenteren,1976). The g r e g a r i o u s -N..--vitr ipennis- .does- n61. d i s c r i m i n a t e p r i o r to d r i l l i n g , however (Wylie,1970). I t i s o f t e n impossible to determine whether an o l d . - d r i l l h o l e , remnants of female-deposited odour, or some e x t e r n a l chemical change i n the host i s r e s p o n s i b l e f o r d i s c r i m i n a t i o n . Old d r i l l h o les can be de t e c t e d by females of some s p e c i e s and may be re-used by the same female or by another female of the same or d i f f e r e n t s p e c i e s , thus l e a d i n g to super- or m u l t i -p a r a s i t i s m (e.g., M.raptor--Wylie,1971). Information C o l l e c t e d A f t e r I n s e r t i o n A f t e r i n s e r t i n g the o v i p o s i t o r , one or more of the f o l l o w i n g b e h a v i o u r a l responses may be e l i c i t e d by s t i m u l i from the pupa: o v i p o s i t i o n (or i t s r e s t r a i n t ) ; egg f e r t i l i z a t i o n (or i t s r e s t r a i n t ) ; venom i n j e c t i o n ; host f e e d i n g . Wylie's (1970) 5% work with Nasonia v i t r i p e n n i s , showed that " a f t e r p i e r c i n g the enclosed pupa with the o v i p o s i t o r , each female u s u a l l y l a y s fewer eggs on a p a r a s i t i z e d pupa than on an u n p a r a s i t i z e d one and leaves the former much sooner." D i f f e r e n t r e s u l t s were obtained when m u l t i p a r a s i t i z e d hosts were p a r t i a l l y wrapped i n aluminum f o i l and presented f i r s t to M.raptor or N . v i t r i p e n n i s , and then presented to another N . v i t r i p e n n i s female f o r r e d r i l l i n g and p i e r c i n g . These l a t t e r N . v i t r i p e n n i s females r e j e c t e d 50% of the hosts p r e v i o u s l y p a r a s i t i z e d by c o n s p e c i f i c s , but only 14% of those p a r a s i t i z e d by M.raptor. D i s c r i m i n a t i o n , i t was suggested, was based on d e t e c t i o n of g e n e r a l i z e d changes i n the host haemolymph ra t h e r than d e t e c t i o n of the wound or d r i l l s i t e or other l o c a l i z e d f a c t o r s a s s o c i a t e d .with_ - p i e r c i n g . - Holmes': ••(-•19-72-) work on s u p e r p a r a s i t i s m i n N . v i t r i p e n n i s a l s o i n d i c a t e d that fewer and fewer eggs, with an i n c r e a s i n g p r o p o r t i o n of these being male, were l a i d by second and t h i r d females p a r a s t i z i n g Sarcophaga b u l l a t a Parker pupae. Among pte r o m a l i d s , there i s a comparatively long time l a g between p a r a s i t i z a t i o n and the onset of i n t r a s p e c i f i c d i s c r i m i n a t i o n and r e s t r a i n t . A f i v e - h o u r i n t e r v a l has been r e p o r t e d f o r M.raptor on M.domestica, depending on d i s t a n c e between d r i l l s i t e s (Wylie,1971). A much s h o r t e r i n t e r v a l has been r e p o r t e d f o r Pseudeucoila bochei Weld, a c y n i p i d e ndoparasite of D r o s o p h i l a melanoqaster Meig. l a r v a e : 60 seconds fo r 80% r e j e c t i o n (van Lenteren,1976). King and R a f a i (1970), working with N . v i t r i p e n n i s (on an u n s p e c i f i e d host of unstated age), showed a gradual d e c l i n e i n percent s u p e r p a r a s i t i s m (85-5 5 66) over a 60-hour i n t e r v a l from f i r s t to second p r e s e n t a t i o n of the host to a female, f o l l o w e d by a p r e c i p i t o u s drop in acceptance at 72 hours. Although t e s t c o n d i t i o n s d i d not allow the discernment of i n d i v i d u a l s ' responses (and group v a r i a n c e s were not r e p o r t e d ) , i t was hypothesized that c e s s a t i o n of host heartbeat between 60 and 72 hours and changes in host haemolymph were r e s p o n s i b l e f o r d i s c r i m i n a t i o n and r e s t r a i n t . That s p e c i f i c changes occur i n the haemolymph of hosts as a r e s u l t of p a r a s i t i z a t i o n has been demonstrated f o r other p a r a s i t o i d groups (e.g., host E p h e s t i a l o s e s p h e n y l a l a n i n e and l e u c i n e and gains two u n i d e n t i f i e d amino a c i d s d u r i n g p a r a s i t i s m by N e m e r i t i s — F i s h e r and Ganesalingam,1970). Questions concerning host death are more r e l e v a n t to the present study. Ratcli.f fe and-King (1 967 ) demonstrated that C a l l iphora pupae were k i l l e d by m a t e r i a l s from the a c i d gland and a s s o c i a t e d r e s e r v o i r .of N . v i t r i p e n n i s , r a t h e r than by o v i p o s i t o r i n s e r t i o n , egg or l a r v a l e f f e c t s , or m a t e r i a l s from other (e.g., a l k a l i n e ) glands a s s o c i a t e d with the p a r a s i t o i d ' s r e p r o d u c t i v e system. Host death, caused by o v i p o s i t o r p i e r c i n g , with or without o v i p o s i t i o n , was found to occur w i t h i n 4 to 5 hours of s t i n g i n g by N . v i t r i p e n n i s and M.raptor, and w i t h i n 24 hours of s t i n g i n g by S.cameroni (Wylie-,1970) and Eupteromalus dubius (Ashm.) (Wylie,1976). The r e l a t i o n s h i p between host f e e d i n g and venom pr o d u c t i o n was d e l i n e a t e d by R a t c l i f f e and King (1967) f o r N . v i t r i p e n n i s on C a l l i p h o r a ; c o n t r o l f l y emergence, 91.83%; emergence of f l i e s p a r a s i t i z e d by females h o s t - s t a r v e d f o r 4 days p r i o r , 55.67%; or by females h o s t - f e d for 4 days p r i o r , 556 19.78% ( a l l s i g n i f i c a n t at P < 0.001). These f i n d i n g s have i m p l i c a t i o n s f o r s t a n d a r d i z a t i o n procedures ( d i s c u s s e d l a t e r ) and f o r estimates of the h o s t - d e s t r u c t i v e p o t e n t i a l of p a r a s i t o i d s p e c i e s . In most, i n s t a n c e s , a p p a r e n t l y , o v i p o s i t o r probing causes host death. But i s host death necessary f o r p a r a s i t i z a t i o n ? Both o v i p o s i t i o n and p a r a s i t e development occur when M.domestica pupae that have been k i l l e d by f r e e z i n g are o f f e r e d to female p a r a s i t o i d s (e.g., Pachycrepoideus vindemiae --Pickens and M i l l e r , 1 9 7 8 ) . S i m i l a r r e s u l t s have been obtained with "coddled" ( i . e . h e a t - k i l l e d ) pupae with N . v i t r i p e n n i s (Wylie,1963), although Edwards (1954) re p o r t e d that t h i s s p e c i e s w i l l d r i l l and probe, but not o v i p o s i t i n , froze n or 1 i g a t i o n - k i l i e d h o s t s . Legner , ( 1 979) showed, that r e f r i g e r a t e d hosts ( 1 0°C f o r up to 21 days) were a l s o s u i t a b l e f o r r e a r i n g M.raptor, M.zaraptor and S.endius. (In a l l of the above i n s t a n c e s , c o n t r o l progeny p r o d u c t i o n r a t e s on h e a l t h y , untreated hosts were as much as 50% higher.) The o v i p o s i t i o n response can a l s o be a f f e c t e d by host age (Wylie,1963). N . v i t r i p e n n i s females o f f e r e d M.domestica puparia of o v e r l a p p i n g 48-h age c l a s s e s , l a i d more eggs per female on hosts that were between 24- and 72-h o l d , than on o l d e r h o s t s . Coats (1976) suggested that M.zaraptor a l s o e x h i b i t e d host-age b i a s e d o v i p o s i t i o n p r e f e r e n c e s ("females s e l e c t e d fewer one-day-o l d than 2- or 3-day-old hosts f o r o v i p o s i t i o n " ) as evidenced by a general decrease i n female progeny p r o d u c t i o n c o r r e l a t e d with i n c r e a s i n g host age (5, 1-day c l a s s e s ) . 5-7 GENERAL METHODS AND MATERIALS FOR HOST ACCEPTANCE STUDIES (A) Behaviours Recorded The beginning and end of host acceptance are marked by the i n i t i a l antennal and t a r s a l c o n t a c t s with the host and departure from the host. I n t e r i m behaviours, accounting f o r the observed d u r a t i o n of host acceptance, were recorded as: ( 1 ) Antennal t a p — t h e p a l p a t i o n of the surface of the host . (2) Abdomen tap--the touching of the abdomen t i p to the s u r f a c e of the puparium. (3) Walk--antennal tap accompanied by locomotion. (4) D r i l l i n g i n i t i a t e d - - a p o i n t event c h a r a c t e r i z e d by the female's assuming a d r i l l i n g posture ( f e e t clamped f o r purchase and the o v i p o s i t o r t i p i n con t a c t with the puparium). (5) . Ovipositor.. I.nsert-ion--a p o i n t event preceded by r a p i d v i b r a t i o n of the abdomen while the female i s i n the d r i l l i n g p osture, and seen as: ( i ) the simultaneous extension of the abdomen t i p away from the puparium s u r f a c e and ( i i ) the d i s t e n s i o n of the v e n t r a l s u r f a c e of the abdomen around the po i n t of attachment of the o v i p o s i t o r to the female. These movements i n v o l v e the pronounced s t r e t c h i n g of the abdominal t e r g i t e s as the o v i p o s i t o r i s extended to i t s f u l l l e n g t h . ( 6 ) O v i p o s i t o r withdrawal--a p o i n t event c l e a r l y v i s i b l e as the o v i p o s i t o r t i p comes away from the host. The i n t e r v a l between i n s e r t i o n and withdrawal i n c l u d e s events not v i s i b l e to the observer: ( i ) e x p l o r a t i o n of the pupal s u r f a c e ; ( i i ) puncture of the pupal integument; ( i i i ) o v i p o s i t i o n . (7) R e - i n s e r t i o n - - t h e o v i p o s i t o r i s i n s e r t e d i n t o the o r i g i n a l d r i l l h o l e . (This event was i n f e r r e d . Although 'holes' were never seen even a f t e r p r e c i s e l y n o t i n g d r i l l s i t e l o c a t i o n s , the much s h o r t e r d u r a t i o n of the second ' d r i l l i n g ' r u l e d out the p o s s i b i l i t y that the females had i n i t i a t e d new h o l e s . ) (8) Grooming—of any body p a r t ; occurred o c c a s i o n a l l y d u r i n g host acceptance but was not necessary f o r completion of the sequence. (9) Host feeding--the a c t of d r i n k i n g or l a p p i n g a d r o p l e t of host blood exuded at the d r i l l s i t e . (10) Rest--the absence of v i s i b l e a c t i v i t y . (B) Method of Underwater I n s p e c t i o n of Hosts Throughout pupal l i f e , the f l y ' s t h i r d l a r v a l i n s t a r .integument --(the. puparium) changes from t r a n s p a r e n t to opaque, f i n a l l y assuming a reddish-brown or black c o l o u r . At f i r s t the pupa .is c l e a r l y v i s i b l e , the d o r s a l a o r t a s t anding out a g a i n s t the white of the f a t body. By the time the host i s s u i t a b l e f o r o v i p o s i t i o n , that i s , a f t e r the a i r space has formed (about three to s i x hours a f t e r e v e r s i o n of the pupal head), pigmentation has u s u a l l y proceeded s u f f i c i e n t l y to obscure any d e t a i l s of subsequent events w i t h i n the puparium. A f t e r 24 hours, the puparium has become opaque. I n t e r n a l events can s t i l l be monitored, however, i f the puparium i s viewed through a l a y e r of water. Attached to a p e t r i d i s h bottom by a dab of v a s e l i n e , a puparium immersed i n water w i l l u s u a l l y , depending on c u t i c l e t h i c k n e s s and age of the pupa, render v i s i b l e the pupa and any s u r f a c e f e a t u r e s , i n c l u d i n g the a o r t a or a p a r a s i t o i d egg or l a r v a . Expt.6: Reproductive Dynamics O b j e c t i v e : to monitor the e f f e c t s of a t t a c k s on hosts by a group of v i r g i n females, with p a r t i c u l a r a t t e n t i o n given to the f i r s t four days of adulthood. Methods and M a t e r i a l s (1) P a r a s i t e Females Ten v i r g i n females were c o l l e c t e d at emergence. Time of emergence was noted. (2) Hosts F i f t e e n pupae about 48 h o l d were used / f e m a l e / p e r i o d . (.3 ).. Procedure - -Females were provided with water d r o p l e t s but no honey; -host-contaminated sawdust was s p r i n k l e d on the bottom of the d i s h to about 1 mm depth to h o l d hosts i n an evenly spaced p a t t e r n . Females were t r a n s f e r r e d by v i a l to a c l e a n d i s h with f r e s h hosts at the s t a r t of each p e r i o d . During the f i r s t 4 days, experimental p e r i o d s were 12 h i n l e n g t h . T h e r e a f t e r ( f o r the l i f e of the female), they were 24 h. Dishes were changed at 1400 and 0200 for the f i r s t e i g h t p e r i o d s and at 1400 f o r the 24 h p e r i o d s . R e s u l t s Mated and v i r g i n females were found to d i f f e r c o n s i d e r a b l y . Length of l i f e was s h o r t e r f o r the cohort of ten v i r g i n s (7.1 ± 2.13 days) than f o r the cohort of ten mated females 60 (22.8 ± 4.92 days, F i g . 8 — a l t h o u g h t h i s experiment was terminated at 28 days with 3 unproductive females s t i l l a l i v e ) . F e c u n d i t y was enhanced by mating. Net r e p r o d u c t i v e e f f o r t was estimated at where mx i s the number of e f f e c t i v e ( i . e . emerged) female progeny per mother, and 1 K i s the number of s u r v i v i n g mothers times 0.9, an estimate of the p r o b a b i l i t y of s u r v i v a l to adulthood p u b l i s h e d by Legner (1976). T o t a l r e p r o d u c t i v e output was estimated as the number emerged p l u s the number f a i l e d and i s thus c o n s e r v a t i v e — f a i l e d a d u l t s , pupae and mature l a r v a e ,w.er.e-..Gonspicuous but the rema-ins of sma l l e r l a r v a e were r a r e l y seen. Mated females l a i d 133 ± 23.36 eggs while v i r g i n s l a i d 31.0 ± 11.55. Peak pr o d u c t i o n occurred on day 2 f o r mated females, day 3 f o r v i r g i n females ( F i g . 9 ) . V i r g i n s k i l l e d about 9.5 times as many hosts as produced progeny d u r i n g the f i r s t twelve hours a f t e r emergence ( F i g . 1 0 ) . T h e r e a f t e r , the r a t i o of number k i l l e d to number p a r a s i t i z e d d e c l i n e d but remained g r e a t e r than one. Percent female progeny remained c l o s e to the o v e r a l l average of 82.7 ± 6.24 throughout the f i r s t two weeks of the experiment ( F i g . 1 1 ) . A mean of 4.1 days without o v i p o s i t i o n preceded death f o r the mated females. 61 F i g u r e 8 . Longevity of mated and v i r g i n S.endius females. F i g u r e 9 . Mean number of progeny (emerged + f a i l e d ) per mated and v i r g i n female c o h o r t s . Mated ( •+- ) V i r g i n ( V ). . •AYS 63 F i g u r e 10 . Unproductive host d e s t r u c t i o n d u r i n g the f i r s t f i v e days a f t e r emergence. Means f o r ten v i r g i n females are c o r r e c t e d f o r host m o r t a l i t y not caused by s t i n g i n g . F i g u r e 1 1 . Mean d a i l y p r o d u c t i o n of progeny produced by a cohort of ten mated S.endius females. Males ( V ). Females ( A ) . LNPRDQJCTIVE HCBT DESTRUCTION iO-0 _, 65" Expt.7: Host Acceptance and Host Death In a p r e l i m i n a r y experiment, the host-acceptance behaviours of 15 mated one- and two-day-old S.endius females d i d not demonstrate an age e f f e c t (Table 15). Each day's s u b j e c t s were observed simultaneously as they walked about, each i n her own d i s h c o n t a i n i n g 20 hosts, age 20-43 h, spaced i n a 1.5 cm 2 g r i d p a t t e r n . Behaviours of a l l females were noted at one-minute i n t e r v a l s u n t i l a minimum of f i v e hosts had been a t t a c k e d per female, which produced t e s t d u r a t i o n s of 3.68 h (day 1) and 6.16 h (day 2). T h i s method of sampling, i t was decided, n e g l e c t e d the r a p i d s h i f t i n g . o f behaviours, d e t a i l that p o s s i b l y c o ntained the a g e - r e l a t e d d i f f e r e n c e s sought. A c c o r d i n g l y , these data showed only that the t e s t days produced s i g n i f i c a n t l y d i f f e r e n t behaviour (e.g., r e s t i n g means between t e s t p e r i o d s — .01 < P < .02) . O b j e c t i v e s : (1) to observe the e f f e c t s t h at s t i n g i n g had on the host (2) to determine i f d i f f e r e n c e s e x i s t i n the host-acceptance behaviours of newly emerged, host-naive females and those one day o l d e r having host experience. Methods and M a t e r i a l s (1) P a r a s i t e Females Females were c o l l e c t e d at emergence and checked f o r mating. The newly emerged group was t e s t e d about 2 h a f t e r emergence, the day-2 group about 28 h a f t e r emergence. (2) Hosts M.domestica pupae were 20-43 h o l d . 6S Table 15. Mean number of o b s e r v a t i o n s (% of t o t a l ) of host-acceptance behaviour i n one- and two-day-old S.endius females. D r i l l , groom and r e s t were s i g n i f i c a n t l y d i f f e r e n t between days (.01 < P < .05). Ant Tap Abd Tap D r i l l Ovip Ins Groom Walk Rest Between 1 9.5 3.3 25.0 11.4 9.0 7.0 30.2 Days 2 7.7 4.7 55.0 15.2 2.7 10.9 1 .6 Between 1 8.6 5.0 35.3 12.9 4.6 8.9 14.4 Ages 2 8.3 3.2 38.9 14.5 5.9 9.8 11.5 (3) Procedure . At t e s t time, each female was i n t r o d u c e d i n t o a c l e a n p e t r i d i s h c o n t a i n i n g honey and 20 hosts spaced in a 1.5 cm 2 g r i d pattern.. Host acceptance behaviours, the p o s i t i o n and number of d r i l l i n g bouts per host, and the occurrence or non-occurrence of host feeding' were recorded. The c o n d i t i o n of a group of a t t a c k e d hosts was assessed at about 2 and 26 h a f t e r a t t a c k . A f t e r a host had been a t t a c k e d , i t was removed from the d i s h , p l a c e d i n a v i a l f o r r e a r i n g , and a f r e s h host was put i n i t s p l a c e to preserve the e q u a l i t y of the p o t e n t i a l encounter r a t e . T h i s procedure was repeated u n t i l f i v e hosts had been att a c k e d by each female. For a s s e s s i n g host death, two newly emerged, mated females were each o f f e r e d f i v e hosts i n s u c c e s s i o n ; one of these females was o f f e r e d an a d d i t i o n a l four the f o l l o w i n g day. The hosts were in s p e c t e d underwater to assess t h e i r appearance and to d e t e c t heartbeat and p a r a s i t e eggs. R e s u l t s 67 Host Death Table 16 shows that the mean times of f i r s t and second i n s p e c t i o n s were 2.0 and 25.3 h a f t e r the s t a r t of host acceptance. A l l hosts i n which the a o r t a c o u l d be d e t e c t e d on the f i r s t day showed no heartbeat at times ranging from 21.6 to 31.2 h a f t e r the p a r a s i t e s ' i n i t i a l c o n t a c t with the hosts. In 10/14 cases, the aorta had changed in appearance to being dark grey and l a t e r a l l y t h i c k e n e d . One egg (of four observed) had hatched by 28.7 h a f t e r s t i n g i n g . Host Acceptance Durations of host acceptance performed by the same i n d i v i d u a l s on the two days are presented i n Table 17. Females took longer to complete host acceptance f o r the hosts presented on day 1 than f o r those presented on day 2 (.01 < P < .025). Since the time spent between h o s t s , that i s , the l a t e n c y of host acceptance, d i d not d i f f e r from period' 1 to p e r i o d 2 (Table 20), p a r a s i t i z a t i o n r a t e i n c r e a s e d concomitantly with host acceptance d u r a t i o n on day 2. T y p i c a l l y , a female would c o n t a c t a host, examine i t with her antennae while walking slowly over the puparium, tap her abdomen t i p ( o f t e n s e v e r a l times) onto the puparium s u r f a c e , and then resume antennal t a p p i n g . A f t e r r e p e a t i n g t h i s process a number of times, she would s t a r t to d r i l l but o f t e n would not f i n i s h ; t h i s i n t e r r u p t i o n of d r i l l i n g might be followed by antennal tapping (the o b l i g a t o r y f i r s t behaviour of the sequence) or by abdomen t a p p i n g . I t was of i n t e r e s t to know whether naive females were l e s s e f f i c i e n t at l o c a t i n g the f i n a l d r i l l s i t e . For a n a l y s i s , host acceptance was d i v i d e d i n t o four p a r t s : Table 16. C o n d i t i o n of host M.domestica a f t e r being stung by S.endius Times are given i n hours a f t e r the s t a r t of host acceptance. Appearance of the host a o r t a was graded from 0. (not v i s i b l e ) through 3 (prominent). Host heartbeat was Present, Absent, or not observable. R e s u l t s of the s t i n g i n g were Male and Female a d u l t p a r a s i t e s , a dead p a r a s i t e Larva and PUpa, and a dead host bearing no evidence of p a r a s i t i s m . Host Observation Aorta Heart # Time Appearance Beat Res u l t 1 2 1 2 1 2 1 3.3 21 .6 2 2 P A F 2 2.5 20.7 0 0 - F 3 1.9 20. 1 0 0 - - PU 4 1.4 19.7 0 3 - A F 5 1.0 19.3 0 0 - - F 6 3.3 31 .2 1 3 P A F 7 2.9 30.8 2 3 P A M 8 2.4 30.3 1 3 P A F 9 1 .2 29.0 2 3 P A -10 0.8 28.7 2 3 P A L 1 1 2.4 26.2 1 3 P A M 12 2. 1 25.8 1 3 P A F 1 3 1 .8 25.6 1 3 P A F 14 1 .3 25.2 1 3 P A F 4>S Table 17. P a i r e d comparison of the d u r a t i o n ( i n min.) of two host-acceptance a c t s separated by 24 hours and performed by the same female. The d i f f e r e n c e i s s i g n i f i c a n t (0.05 < P < 0.02). Age P a i r Day 1 Day 2 D i f f . 1 39.3 27.4 + 11.9 2 23.8 26. 1 - 2.3 3 36.7 14.7 + 22.0 4 21.0 10.4 + 10.6 5 24.7 29.3 - 4.6 6 54.5 18.8 + 35.7 7 32.7 35.3 - 2.6 8 57.2 8.3 + 49.0 9 16.6 10.5 + 6.1 1 0 21.6 17.4 + 4.2 ~x 32.8 19.8 + 13.0 p r e - d r i 1 1 i n g , d r i l l i n g , i n s e r t i o n , and post-withdrawal. At f i r s t c o n t a c t with the host (the beginning of the p r e - d r i l l i n g phase), the f i r s t bout of antennal tapping (T) followed by abdomen tapping (A) was always longer than the second, and i n f a c t , the mean d u r a t i o n of the f i r s t TA bout was longer than the mean of a l l other TA bouts (P < .001, Table 19). For these groupings of the TA bouts there was no . s i g n i f i c a n t treatment effeet,.however. The l e n g t h of the p e r i o d from f i r s t touching the host to the beginning of the f i r s t d r i l l i n g bout (that i s , the p r e - d r i l l i n g phase d u r a t i o n ) was compared between age groups; the means d i d not d i f f e r (Table 20). In the d r i l l i n g phase, the number of d r i l l i n g bouts per host suggested a gre a t e r e f f e c t of 7<? d i f f e r e n c e s between females than between age groups (Table 21). The d u r a t i o n of the s u c c e s s f u l d r i l l i n g bouts had a s i g n i f i c a n t l y g r e a t e r mean time on the f i r s t day (15.3 min.) than on the second. (8.7 min.). Once d r i l l i n g had been achieved, there was l i t t l e change in the female's behaviour which might have i n d i c a t e d events o c c u r r i n g while the o v i p o s i t o r was i n s e r t e d . T h i s i n s e r t i o n p e r i o d and the p e r i o d from withdrawal to departure from the host were found to be s i m i l a r f o r the treatment groups (Table 20). D r i l l i n g performance was measured by s c o r i n g the number of s i t e s u t i l i z e d per host. " S i t e s " were: the host's anal s p i r a c l e s ; and the p u p a r i a l segments ( e i t h e r "on" or "between" segments) d i v i d e d i n t o s i d e , d o r s a l and v e n t r a l areas. Table 22 i n c l u d e s data f o r females of the p r e l i m i n a r y , host acceptance experiment; there was no p a t t e r n evident i n number of d r i l l s i t e s u t i l i z e d by the two age groups. P a r t i c u l a r d r i l l s i t e s were p r e f e r r e d , however. Of 164 a t t a c k s observed ("attack" being the commencement of d r i l l i n g ) , 61.0% l e d to i n s e r t i o n of the o v i p o s i t o r . The d i s t r i b u t i o n of t o t a l number and p r o p o r t i o n of s u c c e s s f u l a t t a c k s i s shown in Tables 23 and 24. Ranking s i t e p r e f e r e n c e by t o t a l number of a t t a c k s and number of s u c c e s s f u l a t t a c k s f o r both treatment groups pooled and f o r day-one females y i e l d e d very s i m i l a r scores f o r the most p r e f e r r e d d r i l l s i t e s (Table 23). The p o s t e r i o r two p u p a r i a l segments r e c e i v e d 57.3% of a t t a c k s , the a n t e r i o r two 24.4%. Att a c k s were d i s t r i b u t e d evenly on v e n t r a l , d o r s a l and l a t e r a l areas of the p u p a r i a (Table 25). Table 18. P a i r e d comparison of the l a t e n c y of host acceptance (min.) f o r two a c t s performed by the same females 24 h a p a r t . The d i f f e r e n c e i s not s i g n i f i c a n t . Age P a i r Day 1 Day 2 Di f f. 1 12.9 4.3 + 8.6 2 2.5 2.6 - 0.1 3 2.0 0.3 + 1.7 4 1 .7 6.4 - 4.7 5 11.6 22.6 -11.1 6 53.3 4.0 + 49.3 7 7.4 0.1 + 7.4 8 0.1 3.9 - 3.8 9 18.6 3.0 + 15.6 10 10.4 1 .9 + 8.5 X 12.1 4.9 + 7.2 Table 19. Bout d u r a t i o n s (min.) f o r the f i r s t antennal tap to the f i r s t abdomen tap ( f i r s t TA) compared with a l l other TA bouts performed by one-day-old and two-day-old females. The means are s i g n i f i c a n t l y d i f f e r e n t only w i t h i n age groups. Age F i r s t TA Bout a l l other TA Bouts X 0.8 0.3 1 a S 0.4 0.1 n 1 2 48 X 0.7 0.3 2 s* 0.1 0.1 n 16 45 Table 20. Phases of host acceptance compared f o r d i f f e r e n c e in mean d u r a t i o n s ( i n min.) between one- and two-day-old S.endius females. Age n x s* P / 95% C.L. P r e - d r i l l i n g 1 10 3.5 4.89 n.s. 2 10 3.6 12.32 D r i l l i n g 1 9 15.3 46. 1 4 .02 <P< .05 2 9 8.7. 52.31 6.63 + 5.86 I n s e r t i o n 1 9 5.5 4.00 n.s. 2 9 4.2 0.96 Post- 1 10 0.8 0.12 n.s. withdrawal 2 1 0 1 .0 1 .59 Table 21. Number of d r i l l i n g bouts performed by two S.endius females on f i v e s u c c e s s i v e l y accepted h o s t s . Female 18 Female 19 Host # 1 2 3 4 5 1 2 3 4 5 Day 1 4 1 2 1 1 3 2 6 3 1 Day 2 1 1 1 2 1 5 2 2 8 1 T o t a l 16 33 Table 22. Mean number of d r i l l s i t e s u t i l i z e d by S.endius females i n s u c c e s s i v e a t t a c k s on M.domestica p u p a r i a . Host # 1 2 3 4 5 6 7 8 Day 1 1 .9 1 .3 1 .7 1 .6 1 . 1 1 .2 1.0 1.3 # 10 10 10 10 9 5 4 3 Day 2 1 .6 1 .4 1 .8 1 .8 1 .3 1.0 # 10 9 9 9 6 2 Table 23. Occurrence of d r i l l i n g on the a n a l s p i r a c l e s (AS), on a segment, or on an inter-segment l o c a t i o n (e.g.,11/12) of Musca domestica puparia by Spalangia endius Host segments are numbered from a n t e r i o r (1) to p o s t e r i o r (12) . D r i l l S i t e AS 1 2 11/12 2 11 1 2/3 1/2 1 0 3/4 Pooled Treatment Groups # bouts 33 33 17 13 11 10 9 8 7 6 t s u c c e s s f u l 30 1 4 10 9 6 6 3 2 3 0 Day-one Females # bouts 24 1 7 13 3 6 5 5 5 3 1 t s u c c e s s f u l 22 9 7 3 4 4 2 2 1 . 0 71 Table 24. D i s t r i b u t i o n of a t t a c k s on M_;_ domestica p u p a r i a . S u c c e s s f u l a t t a c k s l e a d to i n s e r t i o n (shown as % of t o t a l number of a t t a c k s ) . Puparium Segment # A n t e r i o r 1 2 3 4 5 Middle 6 7 8 9 P o s t e r i o r 10 11 12 AS "on" 10 13 5 0 3 1 0 0 3 7 11 33 33 "between" 8 9 6 3 2 1 0 3 3 2 17 % t o t a l 31.1 9.8 59. 1 % s u c c e s s f u l 23 1 2 65 Blood exuded from 5 of 97 hosts r e c e i v i n g o v i p o s i t i o n punctures. Four females were i n v o l v e d i n these i n s t a n c e s , and four bouts of h o s t - f e e d i n g o c c u r r e d , one per female. Each bout of h o s t - f e e d i n g was fo l l o w e d by r e - i n s e r t i o n . The o v i p o s i t i o n r e s u l t s f o r these a t t a c k s are shown i n F i g u r e 12 as the p r o p o r t i o n of females l a y i n g eggs f o r the s e r i e s of hosts presented. Since the p a t t e r n s of o v i p o s i t i o n for the two days were remarkably s i m i l a r ( c f . day 1 i n Table 27), both days were combined f o r a n a l y s i s . The data of F i g u r e 12 were compared to the estimate of mean number of eggs l a i d per host a t t a c k (378/474=0.797) f o r days one and two of the mated females r e p r o d u c t i o n dynamics study; day 3 was found to be s i g n i f i c a n t l y d i f f e r e n t from the estimated mean (P < .05) with the h e t e r o g e n e i t y G-test (Sokal and Rolf,1969) (Table 26). Day 4, on the other s i d e of the mean, was s l i g h t l y under the 95% s i g n i f i c a n c e l e v e l . The r a t i o of hosts k i l l e d to hosts c o n t a i n i n g p a r a s i t e s - - t h e unproductive' host a t t a c k — f o r the females of t h i s experiment was s i m i l a r t o t h a t obtained f o r the 15 F i g u r e 1 2 . P r o p o r t i o n of females l a y i n g eggs in a s e r i e s o a t t a c k s on M.domestica p u p a r i a . Mean p r o p o r t i o n was estimated from pooled day-one and day-two females (n=1 from E x p t . 3 ) . . n PROPORTION DF F E M A L E 5 LAYING E G G 5 o-al o-al 0.7J. o.si o-sl 0.4J. 0.3. ATTACK # 177 mated females of Expt.3 but s i g n i f i c a n t l y l e s s than that f o r v i r g i n females shown in F i g u r e 10 (Table 28). Table 25. D i s t r i b u t i o n of a t t a c k s and per cent l e a d i n g to i n s e r t i o n . # % a t t a c k s s u c c e s s f u l "on" 77 61 "between" 54 43 i n AS 33 91 v e n t r a l 33 42 d o r s a l 35 49 l a t e r a l 45 64 Table 26. Heterogeneity G-test of the p r o p o r t i o n of females (one- to two-days-old) l a y i n g eggs i n c o n s e c u t i v e a t t a c k s . Day 3 i s s i g n i f i c a n t l y d i f f e r e n t (P < .05) from the mean p r o p o r t i o n of one- and two-day Expt.6. o l d females Attack l a y i n g eggs d.f. G 1 1 0.060 2 1 0.504 3 1 4.206 4 1 3.524 5 1 0.109 Te s t s d . f . G 6 1 0.840 Pooled , 1 0.057 7 1 0.050 Heterogeneity 7 9.794 8 1 0.560 T o t a l 8 9.851 8 9.853 Table 27. D i s t r i b u t i o n of progeny by sex i n the f i r s t e i g h t hosts a t t a c k e d by S.endius females that had mated and had been presented with hosts w i t h i n two hours of emergence. Two dead p a r a s i t e l a r v a e c o u l d not be sexed. Sequence of Host Attack 1 2 3 4 5 6 7 8 Female 8 7 7 4 5 3 3 1 Male 1 2 1 3 2 1 1 1 ? sex 0 0 2 0- 0 0 0 0 n 10 10 10 10 9 5 4 3 Table 28. Unproductive host a t t a c k s (the r a t i o of hosts k i l l e d to hosts with p a r a s i t e s ) compared f o r females of three experimental groups (age i n h a f t e r emergence). In bracke t s are the mean number of eggs l a i d f o r the females of each group. Age Expt.3 Expt.7 Expt.6 1-12 1 .18 9 .50 ( 0. 6) 1 .22 (11 .7) 1 3-24 3 .10 ( 2. 1 ) 25-48 1 .30 (13 .4) 1 .65 ( 6. 4) 49-72 1 .32 (11 .6) 1 .26 (13. 9) 73-96 1 .26 (11 .8) 2 .20 ( 5. 4) n mated 10 mated 10 v i r g i n 10 86 IV. DISCUSSION ... In t h i s t h e s i s , v a r i o u s methods were used to b r i n g females i n t o experimental r e a d i n e s s . Much of the focus has been on i n d u c i n g o v i p o s i t i o n - r e l a t e d behaviours to occur with s u f f i c i e n t i n t e n s i t y to i l l u s t r a t e r e l a t i o n s h i p s . I n d i v i d u a l s i n l a t e r experiments were monitored from emergence to o b t a i n an o v i p o s i t i o n h i s t o r y that might s u b s t a n t i a t e hypotheses r e l a t i n g number of eggs l a i d to the performance of host acceptance or host s e a r c h i n g . F u r t h e r , a more r i g o r o u s s t a n d a r d i z a t i o n regimen was imposed on females of Experiments 3, 5 and 7 to gain data that might r e l a t e changes in behaviour to day-to-day and w i t h i n -day o v a r i a n p r o d u c t i v i t y . A fundamental h y p o t h e s i s - - t h a t the presence of mature eggs c r e a t e s c o n d i t i o n s necessary f o r the performance of o v i p o s i t i o n -r e l a t e d a c t i v i t i e s — w a s t e s t a b l e . An u n d e r l y i n g assumption that a female with mature eggs w i l l n e c e s s a r i l y l a y them was examined. O v i p o s i t i o n records f o r the study of v i r g i n females (Expt.6) showed that during the f i r s t twelve hours a f t e r emergence, these females r e a d i l y a t t a c k e d and k i l l e d h osts, but produced progeny i n only a small p r o p o r t i o n (10.5%) of the hosts a t t a c k e d ( F i g . 1 0 ) . Data f o r F i g u r e 10 were c o r r e c t e d by 7.3 ± 3.2% (n=853) f o r host m o r t a l i t y due to causes other than s t i n g i n g d u r i n g the experiment as a whole and 3.3% (n=30) f o r the f i r s t twelve-hour p e r i o d . Assuming s i m i l a r s u r v i v o r s h i p of u n f e r t i l i z e d eggs from v i r g i n and mated females, the v i r g i n females a p p a r e n t l y were s t i n g i n g and k i l l i n g , but not o v i p o s i t i n g . That newly emerged females c o n t a i n e d more mature 81 eggs than were evident i n the o v i p o s i t i o n p a t t e r n s of the v i r g i n s was demonstrated with females that had emerged and mated w i t h i n the two hours p r i o r to p r e s e n t a t i o n of hosts (Expt.7). These females continued to o v i p o s i t throughout the d u r a t i o n of the experiment (a maximum of 6 hours) as shown i n Table 27. The r a t i o of k i l l e d hosts to hosts c o n t a i n i n g progeny was s i m i l a r to that f o r the mated females of Experiment 3 (Table 28). Since i t i s u n l i k e l y that an a c c e l e r a t i o n of o v a r i a n maturation r a t e due to mating would be observable w i t h i n minutes of mating, I suggest that these data show that mating f a c i l i t a t e s the r e l e a s e of eggs and that newly emerged females have mature eggs but l a y only some of them i f unmated. While the o v i p o s i t i o n and h o s t - s t i n g i n g responses of . v i r g i n s were attenuated on both s i d e s of the peak laying-day (Table 27), the mean number of hosts stung during the peak day of e g g - l a y i n g was s i m i l a r f o r mated females (day 2—17.4) and v i r g i n females (day 3—17.5) as was the mean number of eggs l a i d on these days (mated f e m a l e s — 1 3 . 4 ; v i r g i n f e m a l e s — 1 3 . 9 ) . T h i s s i m i l a r i t y i s s t r i k i n g i n view of the many d i f f e r e n c e s noted f o r the host responses of mated and v i r g i n females. C l e a r l y , o v i p o s i t i o n and i t s concomitant response, h o s t - k i l l i n g , are dependent on mating although to what degree mating c o n t i n u e s to a f f e c t each of these beyond the i n i t i a l p e r i o d j u s t mentioned i s u n c e r t a i n ; that i s , mating may a l s o a f f e c t , f o r i n s t a n c e , the r a t e of egg maturation or the rate of oocyte formation (Englemann,1970). It i s customary with some kinds of experimentation to 8 2. s t a n d a r d i z e i n s e c t s p r i o r to t e s t i n g , the intended purpose being to reduce v a r i a b i l i t y i n the data and to l i m i t treatment to a group having s p e c i f i c and i d e n t i f i a b l e c h a r a c t e r i s t i c s . Thus, Wylie (1971) s t a t e s , "Females s t a n d a r d i z e d i n t h i s way contained mature eggs and u s u a l l y a t t a c k e d hosts r e a d i l y . " A survey of s t a n d a r d i z a t i o n p r a c t i c e s f o r p t e r o m a l i d p a r a s i t o i d s showed the f o l l o w i n g t h i n g s to be v a r i e d most o f t e n : the d u r a t i o n of the p e r i o d p r i o r to experimentation--and hence the age of the i n s e c t s ; the t i m i n g of mating; the p r o v i s i o n of hosts and number pro v i d e d . As can be seen in Appendix 2, these data are o f t e n omitted from r e p o r t s . R e s u l t s of s i m i l a r experiments r e p l i c a t e d i n d i f f e r e n t l a b o r a t o r i e s may be c o n t r a d i c t o r y d e s p i t e e f f o r t s to o b t a i n the ..same stock and maintain c u l t u r e s under i d e n t i c a l c o n d i t i o n s . For i n s t a n c e , i n t h i s study I have found S.endius to have an a d u l t l i f e s p a n of 22.8 ± 4.92 days ( F i g . 8 ) ; other p u b l i s h e d r e p o r t s i n d i c a t e 10.7 to 14.6 days (Appendix 1). Net r e p r o d u c t i v e r a t e , r e p o r t e d here to be 87.3, has p u b l i s h e d values ranging from 14.58 to 62.28 daughters per female (Appendix 1). If such c h a r a c t e r i s t i c s can be so d i f f e r e n t among s t u d i e s of one s p e c i e s , other aspects of behaviour that are i n t r i c a t e l y l i n k e d to r e p r o d u c t i o n a l s o can be s u b j e c t to v a r i e d i n t e r p r e t a t i o n s . Information about a s p e c i e s ' b i o l o g y might be u s e f u l l y a p p l i e d to s t a n d a r d i z a t i o n design yet i s o f t e n not a v a i l a b l e . O c c a s i o n a l l y , i t may be r e p o r t e d that a s p e c i e s has or does not have a p r e - o v i p o s i t i o n p e r i o d , f o r i n s t a n c e , and that t h i s age-s p e c i f i c e f f e c t has been c o n s i d e r e d i n choosing an a p p r o p r i a t e 8 1 regimen: "During these two days [ N . v i t r i p e n n i s females] were fed [ o n ly] honey, as they u s u a l l y begin to p a r a s i t i z e when 24-48 hours o l d " (Wylie,1966). A s p e c i e s with a p r e - o v i p o s i t i o n p e r i o d w i l l u s u a l l y not a t t a c k and feed on hosts u n t i l ready to o v i p o s i t (Edwards,1954; Doutt,l964; Arthur,1967); thus, not p r o v i d i n g hosts i s b i o l o g i c a l l y sound. M u s e i d i f u r a x z a r a p t o r , on the other hand, w i l l l a y eggs on the f i r s t day of a d u l t l i f e (Coats,1976), so' why deny i t hosts u n t i l day two (q.v. Wylie,1971)? In the l i t e r a t u r e , c o ntinued r e f e r e n c e to e a r l i e r works that should c o n t a i n the r a t i o n a l e f o r s t a n d a r d i z a t i o n procedure i s commonplace. Legner (e.g., 1976,1979) o f t e n r e p o r t s r e p r o d u c t i v e p o t e n t i a l without i n c l u d i n g data f o r the f i r s t three d a y s - a f t e r emergence and s t a n d a r d i z e s females by p r o v i d i n g them with hosts and honey for three days p r i o r to experimental treatment. For the r a t i o n a l e , the reader i s r e f e r r e d to Legner and G e r l i n g (1967), a paper that concludes: "The present i n v e s t i g a t i o n f u r t h e r emphasized the importance of i n i t i a l [ host] f e e d i n g on the whole l i f e span." No i n f o r m a t i o n on host f e e d i n g ( i . e . feeding by the p a r a s i t e on the host) was given, however. That d i f f e r e n c e s e x i s t between s p e c i e s ' b e h a v i o u r a l and r e p r o d u c t i v e p a t t e r n s i s understood; the e f f e c t s of s t a n d a r d i z i n g are not. In Experiment 1, I used females that probably v a r i e d c o n s i d e r a b l y i n age and o v i p o s i t i o n experience. The s t a n d a r d i z e d v a r i a b l e was the time s i n c e the l a s t o v i p o s i t i o n . High v a r i a b i l i t y i n most of the search v a r i a b l e s 81 and s p o r a d i c o v i p o s i t i o n i n the p r e - t e s t host, i n d i c a t e d a need for t i g h t e r c o n t r o l over the p h y s i o l o g i c a l c o n d i t i o n of the s u b j e c t s . In the next experiment, a cohort of 3-day ± 5-hour-o l d females wajs used f o r which there was again no o v i p o s i t i o n h i s t o r y . No searching was e l i c i t e d from females on the second day of search t e s t i n g ; t h i s , and the even g r e a t e r v a r i a b i l i t y i n acceptance of the second p r e - t e s t host (Table 2) suggested that females had somehow " l o s t the m o t i v a t i o n " to search and accept hosts. In Experiment 3, the marked in c r e a s e i n time spent s e a r c h i n g i n p e r i o d 7 f o l l o w i n g 51 hours without hosts (Fig.4) was due p r i m a r i l y to i n c r e a s e d burrowing and i n v e s t i g a t i o n ( F i g . 5 ) . There was p o s i t i v e c o r r e l a t i o n between cumulative ....number, of. eggs l a i d and the p r o p o r t i o n of t o t a l time on the contaminated s u b s t r a t e spent i n burrowing and i n v e s t i g a t i o n a c t i v i t i e s ( F i g . 6 ) . The experimental group of females spent l e s s time exposed to hosts than d i d those not t e s t e d f o r s e a r c h i n g , and l a i d s l i g h t l y more eggs, though in a manner which suggested that the search group may have been more p r o d u c t i v e to begin with ( F i g . 3 ) . A l s o , the search group l a i d more eggs in p e r i o d 7 which f o l l o w e d the t e s t i n g than d i d the c o n t r o l group (.02 < P <.05), suggesting that the l a c k of hosts f o r the two days p r i o r to that p e r i o d had i n c r e a s e d the supply of mature eggs. Latency of search showed no r e l a t i o n to day-to-day changes in p r o d u c t i v i t y . That cumulative e g g - l a y i n g d i d so ( F i g . 6 ) suggests that the more p r o d u c t i v e females are vigorous s e a r c h e r s , although t h i s alone does not suggest a mechanism. 8 5 The q u e s t i o n remained: How would the presence of mature eggs i n f l u e n c e the h o s t - s e l e c t i o n behaviours, i f at a l l ? The h y p o t h e s i s that o v i p o s i t i o n would increase patch time (a query of Waage's (1979) p r e d i c t i o n f o r the Nemeritis model) was t e s t e d using a s e r i e s of contiguous and s u c c e s s i v e searches and host acceptances which was intended to s t r e s s the r e p r o d u c t i v e "dynamic" and induce b e h a v i o u r a l changes. If the treatment e f f e c t of Experiment 5 (Table 7) i s accepted, then females o v i p o s i t i n g between searches had s h o r t e r search times--as d e f i n e d by the l e a v i n g c r i t e r i a - - f o r searches 2 and 5 ( F i g . 7 ) . The o v i p o s i t i o n records for a l l females were c o l l e c t e d as part of the s t a n d a r d i z a t i o n procedure. Since p a r a s i t i z a t i o n of b u r i e d hosts c o u l d be achieved by a segment of the p o p u l a t i o n (Table 6).., it-.was f e l t , that an, a p p r o p r i a t e procedure would provide both a r e c o r d of r e p r o d u c t i v e output and a cohort of i n d i v i d u a l s having . s i m i l a r experiences with a stimulus environment approximating that of the t e s t arena. An a n a l y s i s of the number and t i m i n g of o v i p o s i t i o n s d u r i n g s t a n d a r d i z a t i o n would i n d i c a t e whether such a procedure was warranted. The number of o v i p o s i t i o n s p r i o r to t e s t i n g had been evenly d i s t r i b u t e d among females of both treatment groups (Table 11) and so probably would not have c o n t r i b u t e d to the low P value obtained when the Search 1 means were compared (Table 9). These o v i p o s i t i o n s were not w e l l c o r r e l a t e d with the search behaviour d u r a t i o n s , the exception being that females l a y i n g no eggs in the f i r s t p r e - t e s t p e r i o d ranked lower in t o t a l burrowing time than females l a y i n g eggs (Table 13C). Approximately equal 86 numbers of females i n c r e a s e d , decreased, and remained unchanged in the numbers of eggs they l a i d (n=8,3,9) and hosts they k i l l e d (n=6,6,8) between f i r s t and second c o n d i t i o n i n g p e r i o d s (Table 13) d e s p i t e the o v e r a l l i n c r e a s e i n h o s t - r e l a t e d a c t i v i t i e s (Table 14). Day-to-day changes in the e x p l o i t a t i o n of b u r i e d hosts was, in t h i s experiment, as v a r i a b l e as the s e a r c h i n g behaviours of Experiment 3. I t i s probable that the l i m i t e d range of o v i p o s i t i o n s observed (0 - 4) i n the s t a n d a r d i z e d females c o u l d not have produced, in any d e t e c t a b l e way, a d i f f e r e n t i a l o v i p o s i t i o n experience. Given a l s o that these eggs were l a i d over a two-day p e r i o d p r i o r to t e s t i n g on the t h i r d day, the d i f f e r e n c e between o v i p o s i t i o n groups would have been f u r t h e r minimized. T e s t s designed to i m p l i c a t e o v a r i a n s t a t u s i n _.a-_-•-..mec-hanism;i . f o r behavioural- change would have to examine, more completely, the short-term e f f e c t s of o v i p o s i t i o n . S i m p l i f i c a t i o n of the Experiment 5 d e s i g n , by removal of the repeated searches of a s u b s t r a t e , p r o v i d e d a means of examining these e f f e c t s . Host acceptance in Spalangia . endius has the general appearance of that of Nasonia v i t r i p e n n i s (Edwards,1954): an e s s e n t i a l l y l i n e a r sequence of events. The age-group d i f f e r e n c e in the d u r a t i o n of the d r i l l i n g p h ase—newly emerged females took longer to perform the s u c c e s s f u l d r i l l i n g bout than day-two i n d i v i d u a l s (Table 2 0 ) — c o u l d not be a t t r i b u t e d to any of the f o l l o w i n g : number of d r i l l i n g bouts (Table 20), which was c o n s i s t e n t w i t h i n females (Table 21); number of d r i l l s i t e s u t i l i z e d by the two groups (Table 22); a change i n d r i l l s i t e 8.7 p r e f e r e n c e (Table 23). The two age groups were a l s o s i m i l a r i n the p a t t e r n of, o v i p o s i t i o n s that showed a marked c y c l i n g i n p r o p o r t i o n of females o v i p o s i t i n g ( F i g . 1 2 ) . The d i f f e r e n c e i n the number of hosts a t t a c k e d (or eggs l a i d ) between peaks i n t h i s s e r i e s of a t t a c k s was four; s i n c e the number of o v a r i o l e s i s probably even in most i n d i v i d u a l s (168/177 N . v i t r i p e n n i s had an even number of o v a r i o l e s — K i n g and R a t c l i f f e , 1 9 6 9 ) , an even number of eggs being l a i d before any sudden change i n p a t t e r n seems reasonable. What i s c u r i o u s , however, i s that the f i r s t apparent shortage of eggs should occur at host f o u r . Why are only three eggs a v a i l a b l e on both days for the s t a r t of t h i s sequence of host a t t a c k s ? I t i s p o s s i b l e that non-viable eggs in a stage of r e s o r p t i o n were l a i d i n the f o u r t h a t t a c k s . If ;S.endius i s s i m i l a r to- N . v i t r i p e n n i s -in -that -the egg nearest the o v i d u c t i s always the f i r s t to be resorbed (Richards and :• .King, 1 967) , perhaps three mature eggs i s the maximum storage c a p a c i t y f o r t h i s s p e c i e s . U n d e r l y i n g the s t a t e d purposes f o r some of the experiments of the t h e s i s were q u e s t i o n s concerning the m o t i v a t i o n of behaviour. I n t u i t i v e l y , one f e e l s that the s t i m u l i producing the search, host acceptance, or o v i p o s i t i o n response, a r i s e d i r e c t l y from the presence of mature eggs. When t h i s supply becomes t e m p o r a r i l y d e p l e t e d , what manner of b e h a v i o u r a l accommodation w i l l o f f s e t the o v i p o s i t i o n delay? I t seems reasonable that S.endius might employ a d e l a y i n g t a c t i c , such as r e p e a t i n g the d r i l l i n g - s t i n g i n g sequence or r e s t i n g with the o v i p o s i t o r i n s e r t e d , although such t a c t i c s c o u l d not be shown from the &8 d a t a . 8T V. CONCLUSIONS (1) M.domestica host pupae d i e w i t h i n 20 - 30 hours of s t i n g i n g by S.endius. From the data presented i t cannot be shown that the p a r a s i t e egg i s or i s not i n v o l v e d i n host death; however, i t has been demonstrated f o r other p t e r o m a l i d s p e c i e s that i t i s the female's venom that i s r e s p o n s i b l e f o r host death, and not the presence of an egg or any product of o v i p o s i t i o n ( R a t c l i f f e and King,1967; Wylie,1970,1976). (2) In S.endius, mating r e l e a s e s o v i p o s i t i o n r e s t r a i n t . T h i s b e h a v i o u r a l e f f e c t was most pronounced w i t h i n f o u r t e e n hours of emergence. (3) One-quarter of the S.endius p o p u l a t i o n sampled c o u l d not e x p l o i t b u r i e d hosts. Lack of such i n f o r m a t i o n about the a c c e s s i b i l i t y of hosts has been c i t e d as c o n t r i b u t i n g to the f a i l u r e of programmes f o r the b i o l o g i c a l c o n t r o l of pest f l i e s (Whiting,1967). (4) I t i s probable that an i n c r e a s e d supply of mature eggs promoted more i n t e n s i v e and longer s e a r c h i n g of the host-contaminated s u b s t r a t e a f t e r hosts had been w i t h e l d from females f o r more than two days. (5) Judgement i s w i t h e l d on the e f f e c t of w i t h o l d i n g hosts from females repeatedly s e a r c h i n g a host-contaminated s u b s t r a t e 90 (Experiment 5 ) . In view of the c y c l i c i t y of o v i p o s i t i o n noted in the host acceptance experiment, d i f f e r e n c e s i n search behaviour due to treatment, i f present, may have been masked by v a r i o u s e f f e c t s . APPENDICES Appendix 1. L i f e H i s t o r y Data f o r Some Common Pteromalid  P a r a s i t o i d s of Muscoid D i p t e r a (1) Spalangia endius (Walker) 1839 (2) Spalangia cameroni Per k i n s 1910 (3) Mu s e i d i f u r a x r a p t o r G i r a u l t and Sanders 1910 (4) Muse i d i furax z a r a p t o r Kogan and Legner 1970 (5) Nasonia v i t r i p e n n i s (Walker) 1836 Appendix 2. Examples of S t a n d a r d i z a t i o n Regimens  Appendix 3• Notes on Emergence and Mat ing Behaviour 9A Appendix 1. L i f e H i s t o r y Data f o r Common Pt e r o m a l i d P a r a s i t o i d s of Muscoid D i p t e r a S p alangia endius Time from egg to a d u l t males: 23.96 ± 2.503 d; females: 24.59 ± 1.814 d at 25 ± 2°C on Musca domestica ( t h i s study) L i fespan 22.8 ± 4.92 d ( t h i s study) 10.7 ± 6.02 d; max. 21 d (Legner,1972) 14.6 d (when pupae r e t a i n e d at 10°C f o r 180 d ) ; 11.8 d at 25°C (Legner,1976) Fecundi t y R 0 = 87.3 . ( t h i s study) R 0 =14.58; R 0 =34.94 shown for F2 progeny of S.E. A u s t r a l i a . males X S. C a l i f o r n i a females (Legner,1972) Ra =62.25; rm =0.1676 (Legner,1979b) Mating Type: B i p a r e n t a l Larvae a r e : S o l i t a r y Host Range Euxesta notata (Olton and Legner,1973); Fannia c a n i c u l a r i s , F . f e m o r a l i s (Legner and Olton,1971); Haematobia i r r i t a n s ( L . ) , Spiloqona spp. (Watts and Combs, 1977); Musca domestica (Wylie,1972a); Physiphora aenoena ( F . ) , Stomoxys c a l c i t r a n s (L.) (Morgan and Patterson,1977); C e r a t i t i s c a p i t a t a (Wied.), Dacus  d o r s a l i s Hendel, Fannia l e u c o s t i c t a (Meig.), F . s c a l a r i s ( F . ) , Muscina sp., Ophyra sp., Sarcophaqa sp. (Krombeiri et al.,1979). 93 Spalangia cameroni  Time from egg to a d u l t 24-27 d f o r 90% emergence at 26»C, 55% R.H. (Legner and Gerling,1967) L i fespan 30 d with no hosts f o r f i r s t 4 d; about 46 d with continuous host supply (Legner and Gerling,1967) Host Range Euxesta notata (Weid.) (Olton and Legner,1973); Haematobia  i r r i t a n s (L.) (Watts and Combs,1977); Musca domest i c a L. (Wy1ie,1972a); Fannia f e m o r a l i s ( S t e i n ) , F . c a n i c u l a r i s , Stomoxys  c a l c i t r a n s (L.) (Legner and Olton,1971); Anastrepha suspensa .(Loew), H i p p e l a t e s c o l l u s o r (Towns.), Muse ina spp., Ophyra sp. , Phaenic i a sp. (Krombein et a l . , 1 9 7 9 ) . 9.4-M u s e i d i f u r a x r a p t o r  L i fespan x' = 7.9 d (range 4-15); x"=15.5 (5-32) with pupae h e l d f o r 55 d at 10°C; X"=19.9 (5-33) with pupae h e l d f o r 180 d at 10°C (Legner,1976) Fe c u n d i t y R 0 =152.64; r m =0.1702 (Legner,1979); R e =26.55; r„ =0.1260 (Legner,1976) R 0 =64 (approx.;an I s r a e l i s t r a i n ; Podoler and Mendel,1979) Mating Type: Uni- / b i p a r e n t a l - Larvae a r e : S o l i t a r y - o r Gregarious Host Range Haematobia i r r i t a n s (L.) (Peck,1974); Musca autumnalis l a r v a e (Wylie,1973a); M.domestica L. (Wylie,1972a); C e r a t i t i s c a p i t a t a (Weid.) (Podoler and Mendel,1979); Cochliomyia m a c e l l a r i a ( F . ) , Fannia c a n i c u l a r i s ( L . ) , Phormia regina (Meig.), Stomoxys  c a l c i t r a n s (L.) (Krombein et al.,1979). 9 5 Muse i d i furax z a r a p t o r Time from egg to a d u l t males: x"=18.82 ± 1.400 d (range 16-23, n = l 8 l ) ; females: x"=21.50 ± 1.344 d (19-28, n=167); at 25 ± 3°C on Musca  domest i c a ( t h i s study) 34 d (Coats,1976). L i fespan of a d u l t : 19.5 d; generation time: 26 d (Coats,1976). F e c u n d i t y R 0 =170.44; rm=0.1767 (Legner,1979). R 0 -163; rm=0.195 (Goats,1976). Mating type: B i p a r e n t a l Larvae a r e : S o l i t a r y Host Range Haematobia i r r i t a n s (Peck,1974); Musca domestica (Wylie,1972a). 9S Nasonia v i t r i p e n n i s  Time from egg to a d u l t 14 d at 25°C (E: 1; L: 6-7; P: 7); 10 d at 28°C; on Sarcophaqa spp. (Whiting,1967) L i f e s p a n males: 1.62 ± 0.10 d; females: 6.96 ± 0.60 d (range 1-22) (Whiting,1967) Fecundity x- prog./female = 13.98 (range 0-802) (Whiting (1967) c i t e s Nagel and Pimentel (1963)); max. > 700 prog, per female u s i n g hosts < 48 h o. (Whiting (1967) c i t e s Wylie (1962)). Mating Type: B i p a r e n t a l Larvae a r e : Gregarious Host Range C a l l i p h o r a spp. (King et al.,1969); L u c i l i a spp. (King and R a t c l i f f e , 1 9 6 9 ) ; Musca domestica L. ( i n l a b only; r a r e l y found p a r a s i t i z i n g other h o u s e f l y types, f o r i n s t a n c e , Fannia spp. Legner and Gerling,1967); P h a e n i c i a s e r i c a t a (Meig.) (Chabora,1967); Chrysomia c h l o r o p y q i a , C . a l b i c e p s , 68 spp. of c y c l o r r a p h o u s D i p t e r a (Whiting (1967) c i t e s U l l y e t t (1950)); Sarcophaqa b u l l a t a Parker (Holmes,1972); C a l l i p h o r a v i e i n a R.D., Cochliomyia hominivorax (Coq.), C . m a c e l l a r i a ( F . ) , Fannia 9.7 c a n i c u l a r i s ( L . ) , F . f e m o r a l i s ( S t e i n ) , F . s c a l a r i s ( F . ) , Hubneria  estigmenensis ( S e l l e r s ) , Hypoderma sp., Luc i l i a i l l u s t r i s (Meig.), Musca sp., Omotoma fumi feranae ( T o t h i l l ) , Ophyra  aenescens (Wied.), 0.leucostomata (Wied.), P h a e n i c i a eximia (Wied.), P.mexicana (Macq.), Phormia regina (Meig.), P i o p h i l a  c a s e i ( L . ) , P o l i e t e s l a r d a r i a ( F . ) , P r o t o c a l l i p h o r a avium S.and D., P . m e t a l l i c a (Towns.), P . s i a l i a S.and D., Synthesiomyia  nud i s e t a (Wulp) (Krombein et al.,1979) . -Appendix 2. S t a n d a r d i z a t i o n • P r a c t i c e s Appendix T a b l e 1 Examples of treatment a f f o r d e d p a r a s i t o i d s of muscoid pupae p r i o r to e x p e r i m e n t a t i o n . Parameters of t h i s p e r i o d a r e : d u r a t i o n ; whether the females were mated; the t i m i n g of mating; the p r o v i s i o n of h o s t s ; the number of h o s t s p r o v i d e d ; whether honey was p r o v i d e d ; whether host f e e d i n g was observed. S p e c i e s Dur. Mated T imi ng of Mat i ng Hosts Prov-i ded 0 of Hosts Honey Prov — i ded Feed i ng Obs . Nature of Expt. Reference M. . r a p t o r 24 h yes ? no - yes - s e a r c h i ng,. di s c r imi nat ion P o d o l e r & Mendel(1979) S, . en d i u s 1 d ? ? ? ? ? ? r a t e of p a r a -s i t i s m Schmidt & Morgan(1978) M. S. . r a p t o r ca.36 . en d i u s h yes ? ? ? ? ? f u n c t i onal response A b i e s 5 Shepard(1974) P . v i n - < 1 d demiae yes ? ? ? ? ? r e p r o d u c t i v e p o t e n t i a 1 Morgan et a l . (1978) P . dubius 1-53d yes ? no - sugar ? r e p r o d u c t i ve NostvikC1954) M. . z a r a p t o r 72 & o t h e r s h yes day 1 day 2 onl y ? yes d1S3 . yes most e x p t s . most spp. Wyl i e (e.g.1971 ) N. . v i t r i - 4 d penni s yes d 1-2 d 3-4 ? d 1-2 yes female s i z e vs r e p r o d u c t ion Wy l i e (19S6) S M . e n d i u s 3 d . r a p t o r & o t h e r s yes p r i o r yes to host exposure 20 yes yes most e x p t s . most spp. Legner (e.g.1979) Appendix 3. Notes on Emergence and Mating Behaviour In many i n s e c t s , males emerge before females ( p r o t a n d r y ) . T h i s i s a l s o true f o r Spalangia endius (Appendix Fig.1) and Muse i d i furax z a r a p t o r (Appendix F i g . 2 ) . I t was o f t e n observed that male S.endius remained with a group of host puparia from which p a r a s i t e s were emerging i n s t e a d of f l y i n g away as females d i d . Males were s t r o n g l y a t t r a c t e d to puparia i n which females had s t a r t e d to chew t h e i r e x i t h o l e s . I f s e v e r a l males had gathered at such a s i t e , a l l would e x c i t e d l y i n v e s t i g a t e the opening by i n s e r t i n g t h e i r antennae and as much of t h e i r heads as p o s s i b l e . None, however, was ever observed to chase other males away or to e x h i b i t other t e r r i t o r i a l behaviour as noted by Whiting (1967) in Nasonia v i t r i p e n n i s . If the developing hole were too small f o r him to gain entrance, the male, a l t e r n a t i n g with the female, would chew at i t s edge to enlarge i t . To f i n d out whether mating c o u l d occur w i t h i n the c o n f i n e s of the puparium, and before the female had emerged, two puparia c o n t a i n i n g females that had j u s t s t a r t e d t h e i r e x i t h o l e s were i s o l a t e d , each with a male. The males gained entrance as d e s c r i b e d and, a f t e r a short p e r i o d , emerged and l e f t . The females then completed t h e i r e x i t h o l e s , emerged, and were p l a c e d i n d i v i d u a l l y i n p e t r i d i s h e s which each c o n t a i n e d twelve h o s t s . A f t e r a 48-h exposure p e r i o d , both s e t s of hosts subsequently produced mainly female progeny — i n a l l 14 females, 3 males. Mating had o c c u r r e d p r i o r to emergence. Females were observed to mate only once i n t h e i r l i v e s . The behaviour of males toward a female changed r a d i c a l l y w i t h i n the 1 oo Appendix F i g u r e 1. Emergence schedule of Spalangia endius eggs l a i d w i t h i n a 24-h p e r i o d at 25"BC~! Males CiAA) Females (// / ) . Appendix F i g u r e 2. Emergence schedule of M u s c i d i f u r a x z a r a p t o r (K. & L.) eggs l a i d w i t h i n a 24-h p e r i o d at 25°C. Males (Wv) Females (7~7~7) . 107 D/EREENLE !EO-ECU_E CF EPAI-ANJIA ENDILEi 5 5 - _ . LLI Hi IJ fil i 3 3D- I 1 5 -1 . 0 -DAYS AFTEH LAYLM3 DvCRGENCE 9CH-OJJE CF MLISCIOEFIRAX ZARAPTOR ui L> 6 W 1 5 -1D> - n - t - i -Q A Y 5 A F T E R L A Y I N G 101 hour a f t e r her mating: vigorous male c o u r t s h i p c o u l d be e l i c i t e d f o r only a few minutes a f t e r mating, and males were soon immediately r e p u l s e d by merely touching t h e i r antennae to a mated female's abdomen. The scent of a v i r g i n female o f t e n draws more than one s u i t o r . If more than one male i s i n c o n t a c t with the female, she e i t h e r crouches and lowers her abdomen to the s u b s t r a t e to a v o i d them, or walks away, sometimes b e a r i n g a l l s u i t o r s on her back. Both of these responses are a l s o e x h i b i t e d by u n r e c e p t i v e ( i . e . mated) females. I f the s u p e r f l u o u s males are removed, and the one remaining continues to tap h i s antennae on the female's head and eyes, w i t h i n a couple of seconds the female adopts the r e c e p t i v e posture—abdomen t i p r a i s e d to present the g e n i t a l pore between the separated mid-abdominal s t e r n i t e s . The male then walks backward and c u r l s h i s abdomen under hers. C o p u l a t i o n ensues and i s completed w i t h i n f i v e seconds. 103 REFERENCES CITED Abies, J.R. and M. Shepard. 1974. 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