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Amino acid requirements of Schistocerca gregaria (Forskal) Williams, David Colin 1980

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el AMINO ACID REQUIREMENTS OF SCHISTOCERCA GREGABIA (FORSKAL) DAVID COLIN WILLIAMS B.Sc., The U n i v e r s i t y C o l l e g e of Wales, Aberystwyth, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES (Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY CF BRITISH COLUMBIA May 1980 0 David C o l i n W i l l i a m s , 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date i i ABSTRACT Ihe development of a c h e m i c a l l y d e f i n e d a r t i f i c i a l d i e t f o r S c h i s t o c e r c a g r e q a r i a (Forsk.) i s described,. The d i e t that permitted the best growth of S. a r e n a r i a was used to determine the amino a c i d requirements of t h i s animal. F u r t h e r s t u d i e s were performed to determine whether amino a c i d s were r e q u i r e d as n u t r i e n t per se or as phagostimulants. The moist a r t i f i c i a l d i e t s i n i t i a l l y used i n growth t r i a l s shewed sign s of d e t e r i o r a t i o n a f t e r 20-days storage at - i 1 5 ° C . T h i s d e t e r i o r a t i o n was evidenced by reduced growth of animals on 20-day-old d i e t s , and by the l o s s of a s c o r b i c a c i d from such d i e t s . Freeze-or oven-drying d i e t s i n c r e a s e d t h e i r storage l i f e and t h e i r s t a b i l i t y under experimental c o n d i t i o n s . Growth t r i a l s showed that d r i e d d i e t s were s t a b l e f o r at l e a s t 2 months when stored i n vacuum d e s i c c a t o r s . L i t t l e a s c o r b i c a c i d was degraded i n d i e t s kept under experimental c o n d i t i o n s (30°C, 55% E.H.) f o r 2 days, sugge s t i n g t h a t such d i e t s could be kept under these c o n d i t i o n s f o r at l e a s t 2 days without being r e p l a c e d . Jj» g r e q a r i a showed poor growth on a r t i f i c i a l d i e t s c c n t a i n i n g e i t h e r an ad hoc mixture of amino a c i d s or a mixture of amino a c i d s based on a n a l y s i s of l e t t u c e p r o t e i n . However, growth of animals was improved by using an amino a c i d mixture based on a n a l y s i s o f cabbage p r o t e i n s . D i e t s could be prepared mere r a p i d l y i f the vitamin s o l u t i o n s used i n compounding the d i e t s were r e p l a c e d by vitamins t r i t u r a t e d i n sucrose..The i i i p h y s i c a l p r o p e r t i e s of the d i e t were important, and fine-powder d i e t s caused heavy m o r t a l i t y of S. q r e q a r i a hoppers.. Powder d i e t s had to be formed i n t o granules or t a b l e t s before they could be u t i l i z e d by S. q r e q a r i a . Although few animals reached the a d u l t stage on a r t i f i c i a l d i e t s , the best d i e t d i d allow J» .g r e q a r i a hoppers to develop to the 5th i n s t a r (with a mean weight of approximately 550 mg) a f t e r 33-days growth.. lo determine which amino a c i d s were e s s e n t i a l f o r S. . q r e q a r i a , the growth of animals reared on t e s t - d i e t s l a c k i n g an i n d i v i d u a l amino a c i d was compared with growth of animals on c o n t r o l d i e t s c o n t a i n i n g the f u l l complement of amino a c i d s . I f the removal of an amino a c i d had no e f f e c t on the growth of animals i t was c l a s s e d as an i n e s s e n t i a l amino a c i d ; i f i t had an marked d e t r i m e n t a l e f f e c t i t was c l a s s e d as an e s s e n t i a l amino a c i d , and i f i t only had a m a r g i n a l l y d e t r i m e n t a l e f f e c t i t was termed a s e m i - e s s e n t i a l amino a c i d . . The r e s u l t s of growth t r i a l s i n d i c a t e d t h a t t y r o s i n e , a l a n i n e , a s p a r t a t e , glutamate, c y s t i n e , s e r i n e and p r o l i n e were i n e s s e n t i a l amino a c i d s f o r S. q r e q a r i a , whereas l y s i n e , p h e n y l a l a n i n e , i s o l e u c i n e , v a l i n e , t h r e o n i n e , tryptophan, l e u c i n e , h i s t i d i n e , methionine and a r g i n i n e were e s s e n t i a l , and g l y c i n e was semi-e s s e n t i a l . Although the growth of animals on a r g i n i n e d e f i c i e n t d i e t s was poor enough to warrant a r g i n i n e being c l a s s e d as an e s s e n t i a l amino a c i d , S. . q r e q a r i a ' s requirements f o r t h i s amino a c i d d i d not appear to be as s t r i n g e n t as i t s reguirements f o r the ether e s s e n t i a l amino a c i d s . Ihe s e m i - e s s e n t i a l nature of g l y c i n e , and the s u p r i s i n g l y good growth of animals on a r g i n i n e - d e f i c i e n t d i e t s are d i s c u s s e d i n r e l a t i o n to S. q r e q a r i a 1 s amino-acid metabolism. i v I t i s suggested that the poor growth of animals on g l y c i n e -d e f i c i e n t d i e t s i s a r e s u l t of g l y c i n e not being s y n t h e s i z e d r a p i d l y enough t o meet S. . g r e g a r i a 1 s metabolic requirements, and that the growth of animals on a r g i n i n e - d e f i c i e n t d i e t s i s due t c l i m i t e d s y n t h e s i s of t h i s amino a c i d v i a the o r n i t h i n e c y c l e . Feeding t r i a l s i n d i c a t e d that the f a i l u r e of animals to show good growth on d i e t s l a c k i n g any of the 10 e s s e n t i a l amino a c i d s was due to the reduced feeding a c t i v i t y of animals reared cn these d i e t s . I n d i v i d u a l removal of any of the other 8 amino a c i d s ( i . e . the 7 i n e s s e n t i a l amino a c i d s and g l y c i n e ) had no e f f e c t on food consumption. The r o l e of amino a c i d s as phagostimulants i s d i s c u s s e d i n r e l a t i o n s h i p to c u r r e n t t h e o r i e s concerning h o s t - p l a n t s e l e c t i o n by phytophagous i n s e c t s , and i t i s suggested that food s e l e c t i o n i s based on a l e a r n e d - a v e r s i o n response to the metabolic e f f e c t s a s s o c i a t e d with the i n g e s t i o n of an imbalanced r a t i o of n u t r i e n t s . V Acknowledgements Thanks t o: Tom Carefoot f o r h e l p f u l comments d u r i n g the course of t h i s work, and pa t i e n c e during the pr o d u c t i o n of the t h e s i s ; Jchn P h i l l i p s , Bob E l l i o t t and B i l l N e i l l f o r reading and commenting on the manuscript; Jeneen Weekes f o r t y p i n g the t h e s i s and C a r o l Henry f o r d r a f t i n g the f i g u r e s ; Fergus, Don and A u s t i n f o r b u i l d i n g equipment necessary f o r the study; Mary Chamberlin f o r advice concerning amino a c i d a n a l y s i s ; and to a l l the people a t 3573 West 15th Ave., e s p e c i a l l y C a r o l i n e and Rcdney who provided space f o r me t o work when I could no longer pay r e n t . v i Table of Contents st r act i i Acknowledgements v L i s t Of Tables i x L i s t Of F i g u r e s ....................................... x General I n t r o d u c t i o n 2 S e c t i o n A. P r e l i m i n a r y I n v e s t i g a t i o n s Concerning The Production Of A Chemically Defined A r t i f i c i a l D i e t For S c h i s t p c s r c a g r e q a r i a ............................ . . . . . . 3 I n t r o d u c t i o n ....................................... 3 M a t e r i a l s And Methods .............................. 5 O b j e c t i v e Of Study .............................. 5 Experimental Design And Rearing C o n d i t i o n s . . . . . . 6 Diet Composition And P r e p a r a t i o n . . . . . . . . . . . 8 Experimental Animals . . . . 8 Development Of A r t i f i c i a l D i e t s . . . 1 0 Experiment 1.. Storage P r o p e r t i e s Of Moist And F r e e z e - d r i e d D i e t s .......... .................... 10 Experiment 2. A s c o r b i c Acid Degradation In Moist And F r e e z e - d r i e d D i e t s , Under Storage And Experimental C o n d i t i o n s . . . 1 4 Experiment 3..Growth Of S c h i s t o c e r c a G r e q a r i a On Cabbage, L e t t u c e , Or A Lettuce Plus Bran-mix v i i C o n t r o l D i e t ....... 18 Experiment 4.. Growth Of S c h i s t o c e r c a G r e g a r i a Hoppers On D i e t s C o n t a i n i n g An Amino Acid R a t i o Based On A n a l y s i s Of Cabbage Or Lettuc e .........20 Experiment 5. . S i m p l i f y i n g The P r e p a r a t i o n Of A r t i f i c i a l D i e t s , And E f f e c t Of D i e t C o n s i s t e n c y On Growth Of S c h i s t o c e r c a Greqaria ....25 Experiment 6. S t a b i l i t y Of Improved D i e t s .......29 Experiment 7..Growth Of S c h i s t o c e r c a G r e q a r i a On Improved A r t i f i c i a l D i e t s , And On A L e t t u c e Plus Bran-mix C o n t r o l Diet ......31 D i s c u s s i o n .........................................34 S e c t i o n E. Amino Ac i d Requirements, And Phagostimulant P r o p e r t i e s Of Amino Acids For S c h i s t o c e r c a q r e q a r i a ...42 I n t r o d u c t i o n 42 M a t e r i a l s And Methods .............................. 43 Experimental O u t l i n e ............................ 43 Diet Production .......44 Growth T r i a l s 44 Feeding T r i a l s .................................. 45 Results ...... ...................................... 46 Removal Of " I n e s s e n t i a l " Amino Acids ...46 Removal Of " E s s e n t i a l " Amino Acids .............. 53 Feeding T r i a l s 60 v i i i D i s c u s s i o n .........................................62 Requirement For G l y c i n e ......................... 63 Requirement For Arginine ........................ 66 Amino Acids As Phagostimulants ..........73 References ...........................................'80 i x l i s t of Tables Table 1. Composition Of A r t i f i c i a l D i e t s .............. 9 Table 2. Adult Weight And Time Taken To Develop To The Adult Stage For S c h i s t o c e r c a G r e q a r i a Beared On A L e t t u c e Plus Bran-mix C o n t r o l D i e t , Or On Cabbage, Le t t u c e Or Grass 19 Table 3. Amino A c i d R a t i o s Based On A n a l y s i s Of Cabbage And Lettuce 22 T a t l e 4. Weight And I n s t a r Reared By S c h i s t o c e r c a G r e q a r i a Reared For 32 Days On Fresh Or Old A r t i f i c i a l D i e t s ....30 T a t l e 5..Weight And Developmental Stage Reached By S c h i s t o c e r c a G r e q a r i a Reared For 33 Days On A L e t t u c e Plus Bran-mix C o n t r o l Diet Or A Chemically Defined D i e t 33 Table 6. Amount Of Diet Consumed By S c h i s t o c e r c a G r e q a r i a Hoppers Feedinq On C o n t r o l D i e t s Containing The F u l l Complement Of Amino A c i d s , Or On Test D i e t s L a c k i n q A S i n q l e Amino Aci d . ..........................61 X L i s t of F i g u r e s F i g u r e 1. Storage L i f e Of Moist And F r e e z e - d r i e d D i e t s As I n d i c a t e d By The E f f e c t Of Diet-age On Growth Of S c h i s t o c e r c a G r e q a r i a Hoppers . . . . . . . . . . . . . . . . . . . . . . . . . 13 F i c u r e 2. E f f e c t Of F r e e z e - d r i e d Moist D i e t s On Degradation Of A s c o r b i c Acid During Storage And Under Experimental C o n d i t i o n s (upper Graph), And Water Content Of D i e t s Under Experimental C o n d i t i o n s (lower Graph) 17 F i g u r e 3. Growth Of S c h i s t o c e r c a G r e q a r i a Hoppers On A r t i f i c i a l D i e t s C o n t a i n i n g E i t h e r The Amino Acid R a t i o Developed By Dadd, An Amino Acid R a t i o Based On A n a l y s i s Of Cabbage Or An Amino Ac i d Ratio Based On A n a l y s i s Of Lettuc e . . . . . . . . 2 4 F i g u r e 4. Growth Of S c h i s t o c e r c a G r e q a r i a Hoppers On Granulated, T a b l e t e d , Or Powdered A r t i f i c i a l D i e t s . . . . 2 8 F i g u r e 5. Growth Of S c h i s t o c e r c a l G r e q a r i a Hoppers Reared On Complete A r t i f i c i a l D i e t s Or On D i e t s L a c k i n g E i t h e r T y r o s i n e , Alanine Or Aspartate . 4 8 F i g u r e 6. Growth Of S c h i s t o c e r c a G r e q a r i a Hoppers Reared On Complete A r t i f i c i a l D i e t s Or D i e t s L a c k i n g E i t h e r Glutamate, Cystine Or S e r i n e 50 F i g u r e 7. Growth Of S c h i s t o c e r c a G r e q a r i a Hoppers Reared On Complete A r t i f i c i a l D i e t s Or D i e t s L a c k i n g G l y c i n e Or P r o l i n e . . .- 52 F i g u r e 8. Growth Of S c h i s t o c e r c a G r e q a r i a Hoppers Reared On Complete A r t i f i c i a l D i e t s Or D i e t s Lacking E i t h e r H i s t i d i n e , Leucine Or Methionine 55 F i g u r e 9. Growth Of S c h i s t o c e r c a G r e q a r i a Hoppers Reared On Complete A r t i f i c i a l D i e t s Or D i e t s Lacking E i t h e r Tryptophan, Threonine, V a l i n e Or I s o l e u c i n e . . . . 5 7 F i g u r e 10. Growth Of S c h i s t o c e r c a G r e q a r i a Hoppers On Complete A r t i f i c i a l D i e t s Or D i e t s Lacking E i t h e r X I A r g i n i n e , Phenylalanine Or L y s i n e ..................... 59 F i g u r e 11. Diagram Of The O r n i t h i n e Cycle ...»......... 71 2 General I n t r o d u c t i o n A r t i f i c i a l d i e t s provide a means of examining the n u t r i t i o n a l requirements of i n s e c t s . Thus, by comparing the growth of i n s e c t s on d i e t s l a c k i n g i n d i v i d u a l n u t r i e n t s with the growth of i n s e c t s on complete d i e t s , i t i s p o s s i b l e to determine whether the d e l e t e d n u t r i e n t can be s y n t h e s i z e d by the i n s e c t . Since the production of the f i r s t a r t i f i c i a l d i e t s f o r i n s e c t s (reviewed by F r a e n k e l , 1959; F r i e n d , 1958; Uvarov, 1928) a l a r g e number of d i e t s have become a v a i l a b l e (see l i s t s of i r s e c t d i e t s by House, e t a l . , 1971; Singh, 1972), e n a b l i n g the determination of the n u t r i t i o n a l requirements of a wide ranqe of i n s e c t s p e c i e s (reviewed by Dadd, 1973; House, 1974).. Despite the l a r q e body of i n f o r m a t i o n p e r t a i n i n g to the n u t r i t i o n a l requirements of i n s e c t s , nothinq i s known of the amine a c i d requirements of any l o c u s t or grasshopper s p e c i e s . The present study was undertaken t o develop an a r t i f i c i a l d i e t that would support s u f f i c i e n t growth of S. g r e q a r i a to allow d i e t a r y - d e l e t i c n techniques t o be used to determine t h i s animal's amino a c i d requirements. The study i s d i v i d e d i n two s e c t i o n s : s e c t i o n A i s concerned with i n c r e a s i n g the storage l i f e of d i e t s , d e c r e a s i n g t h e i r p r e p a r a t i o n time and improving t h e i r composition; s e c t i o n B c o n t a i n s the growth and feeding t r i a l s conducted to determine which amino a c i d s are e s s e n t i a l f o r £. g r e q a r i a and whether growth on amino a c i d d e f i c i e n t d i e t s c c u l d be r e l a t e d to d i e t consumption. 3 Sect i o n A. P r e l i m i n a r y I n v e s t i g a t i o n s Concerning the Production of a Chemically, Defined A r t i f i c i a l D i e t f o r S c h i s t o c e r c a g r e q a r i a I n t r o d u c t i o n Dadd (1960 a-e; 1961a,b,c) o u t l i n e d the development of an a r t i f i c i a l d i e t f o r the l o c u s t s S c h i s t o c e r c a g r e q a r i a and Locusta m i q r a t o r i a . The d i e t allowed both s p e c i e s to develop from the hopper to the a d u l t stage, and enabled the determination of the v i t a m i n , carbohydrate and f a t t y a c i d requirements cf these animals. Commercially a v a i l a b l e animal and/or veqetable p r o t e i n s are o f t e n used t c supply amino-nitroqen i n d i e t s developed f o r grasshoppers, l o c u s t s and c r i c k e t s ( Borisova, 1966; Dadd, 1960c; Kreasky, 1962; LeBerre and T i r a , 1976; Luckey and Stone, 1968; McFarlane, 1964; McGinnis and K a s t i n q , 1967; Mulkern and Toczek, 1970; Nayar, 1964a,b; Patton, 1967),. In the only study of t h i s economically important group of animals concerned with the replacement of p r o t e i n s by a mixture of amino a c i d s , Dadd (196 1c) found t h a t an ad hoc mixture of 19 amino a c i d s could r e p l a c e the mix of p r o t e i n s u s u a l l y used i n a r t i f i c i a l d i e t s f c r S. g r e q a r i a . Although animals reared on t h i s amino aci d d i e t did not grow as w e l l as those on d i e t s c o n t a i n i n g p r o t e i n s ( c a s e i n , egg albumin and peptone), they d i d develop to the a d u l t stage. 4 Ihe replacement of p r o t e i n s by amino a c i d s i s an important step i n the development of a r t i f i c i a l d i e t s as i t allows d i e t a r y d e l e t i o n techniques to be used to determine the amino a c i d requirements of the animal being s t u d i e d . Although a seemingly simple step, r e p l a c i n g p r o t e i n s by an amino a c i d mixture has not always proved f e a s i b l e . Thus d e s p i t e almost 20 years of work, the p r o t e i n used i n a r t i f i c i a l d i e t s f o r the mealworm Tenebrio m o l i t o r , can s t i l l not be adequately r e p l a c e d by an amino a c i d mixture (Davis, 1971, 1974, 1975, 1978; L e c l e r c q and Lopez-Francos, 1964, 1966, 1967).. With t h i s i n mind I hoped to use the amino a c i d r a t i o and d i e t developed by Dadd (196 1c) f c r S. q r e q a r i a to determine the amino a c i d requirements of t h i s animal. However, p r e l i m i n a r y growth t r i a l s showed that S. q r e q a r i a hoppers r e a r e d f o r 30 days on t h i s d i e t only reached a mean weight of about 130 mg, i . e . . one tenth the mean weight of about 1300 mg achieved by S. q r e q a r i a hoppers r e a r e d cn the same d i e t by Dadd (1961c). In a d d i t i o n , I found t h a t d i e t s prepared a c c o r d i n g to Dadd's r e c i p e showed signs cf d e t e r i o r a t i o n a f t e r 20 days sto r a g e . Ihe present study was undertaken t o determine whether the p r e p a r a t i o n and storage p r o p e r t i e s of Dadd's d i e t c o u l d be improved, and t o examine the e f f e c t of r e p l a c i n g Dadd's ad hoc mixture of amino a c i d s with amino a c i d r a t i o s based on a n a l y s i s of p l a n t m a t e r i a l s . 5 M a t e r i a l s and Methods Ofc-jective of Study When attempting to d e f i n e or improve a r t i f i c i a l d i e t s f o r i n s e c t s s e v e r a l c r i t e r i a should te s a t i s f i e d . Thus i t i s important t h a t d i e t s r e t a i n t h e i r o r i g i n a l composition under storage or experimental c o n d i t i o n s , c o n t a i n n u t r i e n t s in s u f f i c i e n t g u a n t i t y and balance to s a t i s f y the animal's metabolic needs, and are c h e m i c a l l y and p h y s i c a l l y a c c e p t a b l e tc the animal. In a d d i t i o n , from a pr o c e d u r a l p o i n t of view, i t i s d e s i r a b l e that production of d i e t s i n v o l v e s as few o p e r a t i o n s (weighings, volume measurements) as p o s s i b l e . With the above p o i n t s i n mind the present study was undertaken to determine: (a) the storage l i f e of moist a r t i f i c i a l d i e t s prepared according to Dadd's r e c i p e (Dadd, 1961c), and whether f r e e z e - d r y i n g d i e t s i n c r e a s e d t h e i r s t a b i l i t y (Experiment 1) ; (b) i f the storage p r o p e r t i e s of moist and f r e e z e - d r i e d d i e t s c o u l d be r e l a t e d to the degradation of n u t r i e n t s , as i n d i c a t e d by the l o s s of a s c o r b i c a c i d i n s t o r e d d i e t s , and whether a s c o r b i c a c i d was more r e a d i l y degraded under experimental than storage c o n d i t i o n s (Experiment 2); (c) whether the chemical adequacy of d i e t s could be improved by basing the amino a c i d content of d i e t s on the r a t i o of amino a c i d s present i n p l a n t foods shown to support good growth i n S. q r e q a r i a (Experiments 3 and 4 ) ; (d) i f d i e t p r e p a r a t i o n c o u l d be s i m p l i f i e d by s u p p l y i n g vitamins 6 t r i t u r a t e d i n s u g a r i n s t e a d o f i n s o l u t i o n s , a n d w h e t h e r t h e p h y s i c a l p r o p e r t i e s o f t h e d i e t c o u l d b e i m p r o v e d by m a k i n g i t i n t o g r a n u l e s o r t a b l e t s ( E x p e r i m e n t 5 ) ; (e) i f i m p r o v e d d i e t s c c u l d b e s t c r e d f o r p e r i o d s i n e x c e s s o f a m o n t h ( E x p e r i m e n t 6 ) ; a r d ( f ) t h e g r o w t h o f a n i m a l s o n c h e m i c a l l y d e f i n e d d i e t s r e l a t i v e t o g r o w t h o f a n i m a l s o n a l e t t u c e p l u s b r a n - m i x d i e t u s e d t o m a i n t a i n s t o c k s o f S . . g r e q a r i a a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a ( E x p e r i m e n t 7) . S i n c e t h e m o d i f i c a t i o n s made t o a n y a r t i f i c i a l d i e t w e r e n e c e s s a r i l y d e p e n d e n t o n t h e r e s u l t s o f p r e v i o u s e x p e r i m e n t s , a f u l l d e s c r i p t i o n o f t h e r a t i o n a l e a n d m e t h o d s a s s o c i a t e d w i t h p a r t i c u l a r e x p e r i m e n t s w i l l be p r e s e n t e d , t o g e t h e r w i t h t h e r e s u l t s , i n t h e s e c t i o n e n t i t l e d " D e v e l o p m e n t o f A r t i f i c i a l D i e t s " . F o l l o w i n g i s a d e s c r i p t i o n o f m e t h o d s c o m m o n t c , o r m o d i f i e d o n l y s l i g h t l y i n , e a c h e x p e r i m e n t . E x p e r i m e n t a l D e s i g n a n d B e a r i n g C o n d i t i o n s T h e a d e q u a c y o f d i e t s p r e s e n t e d t o S . . g r e q a r i a was d e t e r m i n e d b y f o l l o w i n q t h e q r o w t h r a t e a n d d e v e l o p m e n t o f n e w l y h a t c h e d a n i m a l s r e a r e d o n t h e s e d i e t s . T h e c h e m i c a l l y d e f i n e d d i e t t h a t a l l o w e d f o r t h e b e s t q r o w t h o f S . . g r e q a r i a i n a p a r t i c u l a r e x p e r i m e n t was u s e d a s t h e c o n t r o l d i e t i n s u b s e q u e n t e x p e r i m e n t s . . I n c a s e s w h e r e t w o o r m o r e d i e t s p e r m i t t e d e q u a l l y g o o d g r o w t h , t h e d i e t t h a t was e a s i e s t t o p r e p a r e was u s e d a s t h e c c n t r c l i n s u b s e q u e n t e x p e r i m e n t s . When l a r g e d i f f e r e n c e s i n g r o w t h o n d i e t s w e r e e x p e c t e d , 7 three groups of 10 hoppers each were started on each diet type, and animals were weighed every 7-8 days over a period of 28-32 days. Results from these experiments are presented graphically. When smaller differences i n growth were expected, larger numbers cf animals were started on each diet type (either 3 repli c a t e s of 50 animals each or 2 repl i c a t e s of 200 animals). Results from these experiments are presented either as weight and instar reached after 32 days growth, or as mean adult weight and time taken to develop to the adult stage. _ When replicates of 10 experimental animals were started on each di e t , they were kept i n c y l i n d r i c a l (22 cm-dia.). 2 - l i t r e ice-cream p a i l s . The inside of each p a i l was lined with zinc mesh to provide a rough surface suitable for locomotion and moulting, and the top was replaced by p l a s t i c mesh to allow a i r tc c i r c u l a t e through the container. For experiments involving larger numbers of hoppers (>50), wooden containers of greater volume (64-litres) were used. A l l experiments were conducted i n a constant environment chamber at 30°C and 55% R.H. under a 12-hr light:12-hr dark l i g h t i n g regime. Water was provided in the form of moist cotton pads with wicks projecting into a water reservoir.. Such an arrangement kept the pads moist for a minimum of 3 days. Chemically defined diets were presented on 9 cm-dia. . pet r i dishes placed on the flo o r of the container. Diet and water were replaced every 2-3 days, excess food and water always being present at the end of this time..When fresh lettuce or cabbace formed part of the diet, i t was replaced d a i l y . 8 D i e t C o m p o s i t i o n a n d P r e p a r a t i o n I h e g r o s s c o m p o s i t i o n o f t h e c h e m i c a l l y d e f i n e d d i e t s u s e d w a s t h e s a m e a s t h a t p r e s e n t e d b y D a d d ( 1 9 6 1 c ) a n d i s g i v e n i n T a b l e 1 . V i t a m i n s o l u t i o n s w e r e m a d e u p i n 2 0 % e t h a n o l a n d s t o r e d i n d i v i d u a l l y i n d a r k e n e d c o n t a i n e r s a t - 6 0 ° C . R i b o f l a v i n , t h i a f f i n e - H C l a n d p y r i d o x i n e w e r e a d j u s t e d t o p H 3 . 5 , m e s o -i n o s i t o l a n d c h o l i n e c h l o r i d e w e r e l e f t u n a d j u s t e d , a n d t h e r e m a i n i n g B - v i t a m i n s w e r e a d j u s t e d t o p H 7 . 0 . A l l p H a d j u s t m e n t s w e r e m a d e u s i n g H C l o r N a O H . T o p r e p a r e t h e d i e t t h e c h o l e s t e r o l , l i n o l e i c a c i d a n d b e t a - c a r o t e n e w e r e m i x e d w i t h 10 m l o f c h l o r o f o r m a n d a d d e d t o t h e c e l l u l o s e p o w d e r . T h i s m i x t u r e w a s h o m e g e n i z e d u s i n g a m o r t a r a n d p e s t l e a n d o v e n - d r i e d f o r 2 - 3 h r s a t 6 0 ° C . T h e s a l t s , a m i n o a c i d s , s u c r o s e a n d a s c o r b i c a c i d w e r e g r o u n d i n a t a l l - m i l l o v e r n i g h t , a n d a d d e d t o t h e c e l l u l o s e m i x t u r e t o p r o v i d e a h o m o g e n o u s d i s p e r s a l o f d r y - d i e t c o m p o n e n t s . „ T h e v i t a m i n s o l u t i o n s w e r e a d d e d a n d t h e w h o l e g r o u n d t o f o r m a n e v e n l y c o n s i s t e n t m o i s t d i e t . E x p e r i m e n t a l A n i m a l s A n i m a l s f c r g r o w t h t r i a l s w e r e o b t a i n e d a s h a t c h l i n g s (< 1 d a y - o l d ) f r o m a t r e e d i n g s t o c k k e p t a t t h e U n i v e r s i t y o f B r i t i s h C c l u m t i a . T h e s t o c k w a s e s t a b l i s h e d a t U . B . C . i n 1 9 6 4 , f r o m a n i m a l s o r i g i n a l l y o b t a i n e d f r o m t h e A n t i - L o c u s t R e s e a r c h 9 T a b l e 1. C o m p o s i t i o n of a r t i f i c i a l d i e t s . * c e l l u l o s e ( s i g m a , C-8002) c h o l e s t e r o l (sigma) l i n o l e i c a c i d (BDH, 29820) B - c a r o t e n e ( s i g m a , C-9750) a s c o r b i c a c i d ( s i g m a , A-7506) v i t a m i n m i x t u r e ** s u c r o s e ( s i g m a , S-850) amino a c i d mix *** s a l t mix **** wt of d i e t ( n o t i n c l u d i n g v i t a m i n s o l u t i o n ) * A f t e r Dadd 1961c, T a b l e 11, column B 1 5 grm 50 mg 0.3 ml 25 mg 0.2 grm 1 0 ml 1 0 grm 1 0 grm 1 . 5 grm 37.0 gm ** C o n c e n t r a t i o n of i n d i v i d u a l v i t a m i n s e x p r e s s e d below as w e i g h t of each v i t a m i n (mg) r e q u i r e d i n 100 ml o f 20% e t h a n o l to p r o v i d e s u f f i c i e n t of t h a t v i t a m i n f o r 37 grm of d i e t i n a 1 ml a l i q u o t of v i t a m i n s o l u t i o n . T h i a m i n e H C l 92 . 5 M e s o - i n o s i t o l 1850 R i b o f l a v i n , 92.5 Ca. p a n t o t h e n a t e 185 N i c o t i n i c a c i d 370 p - a m i n o b e n z o i c a c i d 92 . 5 P y r i d o x i n e 92.5 C h o l i n e C h l o r i d e 4625 F o l i c a c i d 92 . 5 B i o t i n 3 . 7 *** R a t i o of amino a c i d s g i v e n by Dadd 1961c, p r e s e n t e d below p a r t s p e r 10 0. L - a l a n i n e 5.4 L - a r g i n i n e 11.5 L - a s p a r t a t e 3.9 L - c y s t i n e 2 . 3 L - g l u t a m a t e 14.0 g l y c i n e 5 . 4 L - h i s t i d i n e 2.3 L - h y d r o x y p r o l i n e 1 .6 L - i s o l e u c i n e 4.7 L - l e u c i n e 7.8 L - l y s i n e HCl 4 .,7 L - m e t h i o n i n e 2 . 3 L - p h e n y l a l a n i n e 6 . 2 L - p r o l i n e 5.4 D l - s e r i n e 6.2 L - t h r e o n i n e 2 . 3 L - t r y p t o p h a n 5.4 L - t y r o s i n e 3.4 L - v a l i n e 4.7 **** O r i g i n of s a l t mix g i v e n by Dadd 1961c, T a b l e 5, column Q u a n t i t i e s below e x p r e s s e d as p a r t s by w e i g h t . Na CO 60 K SO 175 C a C l 100 K CO 165 CaHPO 125 MgSO .7H 0 100 KH P0 1 05 FeSO .7H 0 20 K HPO 125 MnSO . H 0 25 10 Centre, London. Stock animals were maintained on .bran-mix d i e t (60% bran, 20% d r i e d grass, 10% yeast, and 10% skim milk powder, % by volume) supplemented with f r e s h l e t t u c e . Male and female animals from t h i s stock had F/C r a t i o s (femur length/head width) of 3.34± 0.09 (SD) and 3.21± 0.05 r e s p e c t i v e l y , i n d i c a t i n g they were morphometrically s i m i l a r to the phase t r a s i e n s d e s c r i b e d by D i r s h (1953) as having F/C r a t i o s cf 3.15-3.75 (males) and 3.15-3.85 (females). Development of A r t i f i c i a l D i e t s Experiment 1. Storage P r o p e r t i e s of Moist and Freeze- d r i e d D i e t s P r e l i m i n a r y growth t r i a l s i n d i c a t e d t h a t d i e t s prepared and s t o r e d a c c o r d i n g to methods o u t l i n e d by Dadd (1960c, 1961c) r a p i d l y d e t e r i o r a t e d i n q u a l i t y . I f e l t t h a t the i n s t a b i l i t y of d i e t s prepared a c c o r d i n q to Dadd's r e c i p e may have been due to t h e i r high moisture content (approx. 25%). To determine whether moist d i e t s were unstable, the growth of animals r e a r e d and d i e t s prepared a f r e s h each week, was compared with growth of animals cn d i e t s t hat were not r e p l a c e d during the 32-day experimental p e r i o d . S i m i l a r experiments were conducted using f r e e z e - d r i e d d i e t s to determine whether moisture content was a f a c t o r governing the s t a b i l i t y of d i e t s . : Moist d i e t s were prepared according to Dadd's r e c i p e (Table 1,), f r e e z e - d r i e d 11 d i e t s were p r e p a r e d by f r e e z i n g t h e m o i s t d i e t o v e r l i g u i d n i t r o g e n and e v a p o r a t i n g u n d e r vacuum. M o i s t d i e t s were s t o r e d a t -15°C, and f r e e z e - d r i e d d i e t s were s t o r e d i n d e s i c c a t o r s a t room t e m p e r a t u r e . The r e s u l t s c f t h i s e x p e r i m e n t a r e shown i n F i g . 1. By t h e 2 0 t h day o f t h e e x p e r i m e n t , a n i m a l s f e d on t h e u n r e p l a c e d m o i s t d i e t h a d s t o p p e d g r o w i n g . B e t ween t h e 2 0 t h and 2 4 t h day t h e s e a n i m a l s l e s t w e i g h t , and by t h e 2 6 t h day a l l a n i m a l s cn t h i s d i e t were d e a d . No a n i m a l s on t h e u n r e p l a c e d m o i s t d i e t d e v e l o p e d p a s t t h e 2nd i n s t a r . M o i s t d i e t s had t o be r e p l a c e d w e e k l y t c a l l c w f u r t h e r g r o w t h . . F r e e z e - d r y i n g t h e d i e t i n c r e a s e d t h e d i e t ' s s t a b i l i t y s u c h t h a t w e e k l y r e p l a c e m e n t was no l e n g e r n e c e s s a r y . These d i e t s e n a b l e d some a n i m a l s t o d e v e l o p t o t h e 4 t h i n s t a r by t h e end o f 32 d a y s . G r o w t h cn t h e f r e e z e - d r i e d d i e t s , and t h e m o i s t d i e t s r e p l a c e d w e e k l y , was v e r y p o o r . Under t h e same c o n d i t i o n s , a n i m a l s r e a r e d on a l e t t u c e p l u s b r a n - m i x c o n t r o l d i e t ( n o t shown i n F i g . 1) had r e a c h e d t h e a d u l t s t a g e w i t h a mean w e i g h t of 1842 ± 143 mg (95% c o n f i d e n c e l i m i t s ) a t t h e end o f 32 d a y s , w h e r e a s t h e h i g h e s t mean w e i g h t a c h i e v e d by a n i m a l s on a c h e m i c a l l y d e f i n e d d i e t was o n l y 137 + 35 mg. . 12 F i g u r e 1. Storage l i f e of moist and f r e e z e - d r i e d d i e t s as i n d i c a t e d by the e f f e c t of di e t - a g e on growth of S c h i s t o c e r c a g r e q a r i a hoppers T h i r t y animals (3 r e p l i c a t e s of 10 animals each) were s t a r t e d cn each d i e t . Each p o i n t r e p r e s e n t s the mean weight of s u r v i v i n g animals, bars represent 95% confidence l i m i t s f o r the number cf animals i n d i c a t e d . Open c i r c l e s , moist d i e t not r e p l a c e d during the course of the experiment; c l o s e d c i r c l e s , moist d i e t r e p l a c e d weekly during the course of the experiment; open squares, f r e e z e - d r i e d d i e t s not r e p l a c e d during the course cf the experiment; c l o s e d squares, f r e e z e - d r i e d d i e t s r e p l a c e d weekly dur i n g the course of the experiment; //, a l l animals dead. Growth t r i a l s conducted at 30°C and 55% R.H. LIVE WEIGHT (mg) 14 Experiment 2. A s c o r b i c A c i d Degradation i n Moist and F r e e z e -d r i e d D i e t s , Under Storage and Experimental C o n d i t i o n s The p r e v i o u s l y presented growth t r i a l s demonstrated that f r e e z e - d r i e d d i e t s were mere s t a b l e than moist d i e t s under t h e i r r e s p e c t i v e storage c o n d i t i o n s . However, growth t r i a l s c c u l d not be used to judge the s t a b i l t i y of d i e t s under experimental c o n d i t i o n s , s i n c e d i e t s were only kept under these c o n d i t i o n s f o r 2 days before being r e p l a c e d . This being so, I f e l t that the s t a b i l i t y of d i e t s under experimental c o n d i t i o n s could best be determined by f o l l o w i n g the degradation of a p a r t i c u l a r d i e t a r y n u t r i e n t . . A s c o r b i c a c i d , which i s i n i t i a l l y present i n the d i e t at a high l e v e l (0.5% by dry weight), i s knewn to be very l a b i l e , e s p e c i a l l y i n s o l u t i o n s (Altman and D i t t i r e r , 1972) . The present experiment was performed to determine i f the s t a b i l i t y of moist and f r e e z e - d r i e d d i e t s under storage c e n d i t i o n s c c u l d b'e r e l a t e d to the degradation of .ascorbic a c i d , and i f t h i s was so, to-use the same c r i t e r i a to judge the s t a b i l i t y of d i e t s under experimental c o n d i t i o n s . To t h i s . end, the a s c o r b i c a c i d content of moist and f r e e z e - d r i e d d i e t s kept under storage c o n d i t i o n s was determined every week over a one-month p e r i o d . In a d d i t i o n , the moisture and a s c o r b i c a c i d content of d i e t s kept under experimental c o n d i t i o n s was determined every 12 hr over a 48 hr period..Moist and f r e e z e -d r i e d d i e t s were prepared and s t o r e d as i n Experiment I.The a s c o r b i c a c i d content of a r t i f i c i a l d i e t s was determined by t i t r a t i o n a g a i n s t 2,6-dichloroindophenol according to methods d e s c r i b e d by the AOAC (1975) f o r dry m a t e r i a l s c o n t a i n i n g 15 a p p r e c i a b l e amounts of b a s i c substance, although t h i s method i s s u b j e c t to i n t e r f e r e n c e by other reducing compounds (Roe, 1961) no d i f f i c u l t y was experienced i n o b t a i n i n g a l a s t i n g end point (> 5 s e e s ) , and the method c o r r e c t l y estimated the a s c o r b i c a c i d content of f r e s h d i e t s c o n t a i n i n g known amounts of a s c o r b i c a c i d , and d i e t s prepared without a s c o r b i c a c i d . r i g . 2 shows t h a t only 40% of the a s c o r b i c a c i d i n i t i a l l y present i n the moist d i e t remains a f t e r 14 days st o r a g e at -15°C. However, f r e e z e - d r i e d d i e t s , s t o r e d i n a d e s i c c a t o r at room temperature, showed l i t t l e l o s s of a s c o r b i c a c i d over the 28-day experimental p e r i o d . These r e s u l t s are i n accordance with the s t a b i l i t y o f the d i e t s as determined i n growth t r i a l s (Experiment 1) and suggest that a s c o r b i c a c i d can be used as an i n d i c a t o r of general d i e t s t a b i l i t y . Under experimental c o n d i t i o n s (30°C, 55% R.H.) both moist and f r e e z e - d r i e d d i e t s showed a l o s s of a s c o r b i c a c i d (see F i g . 2). However the l o s s was g r e a t e r i n moist d i e t s , and appeared to be a s s o c i a t e d with the water content of the d i e t (lower graph, F i g . 2 ) . Thus i t took approximately 14 hr f o r the r a t e of a s c o r b i c a c i d l o s s i n the moist d i e t to l e v e l o f f , and about the same time f o r the water content of the moist d i e t to decrease, and reach e q u i l i b r i u m with the a i r i n the experimental chamber. 16 F i g u r e 2.. E f f e c t of f r e e z e - d r y i n g moist d i e t s on degradation of a s c o r b i c a c i d d u r i n g storage and under experimental c o n d i t i o n s (upper graph), and water content of d i e t s under experimental c o n d i t i o n s (lower graph) A s c o r b i c a c i d or water ccntent of d i e t s under experimental c o n d i t i o n s were measured every 12 hours. Ascorbic a c i d content of d i e t s under storage c o n d i t i o n s was measured every 7 days. Experimental c o n d i t i o n s : 30°C and 55% R.H. .Storage c o n d i t i o n s : mcist d i e t s s t o r e d at -15°C; f r e e z e - d r i e d d i e t s s t o r e d i n d e s i c c a t o r s a t room temperature. Each point r e p r e s e n t s the mean of 5 d e t e r m i n a t i o n s ; bars r e p r e s e n t 95% confidence l i m i t s . 1 / FREEZE-DRIED MOIST DIET (STORAGE CONDITIONS) 0 DAYS 7 H 21 28 HOURS 12 24 36 48 TIME 18 Experiment 3. Growth of S c h i s t o c e r c a q r e q a r i a on Cabbage,  L e t t u c e , Grass, or a Lettuce plus,, bran-mix C o n t r o l D i e t Dadd (196 1c) d i d not study the e f f e c t of d i f f e r i n g amino a c i d r a t i o s on the growth of S. g r e q a r i a , and I f e l t t h a t the poor growth of animals i n the present study may have been due to an imbalance i n the ad hoc mixture of amino a c i d s developed by Dadd (1961c) and used i n the f o r e g o i n g experiments. With t h i s i n mind I decided to t e s t amino a c i d r a t i o s based on a n a l y s i s of p l a n t s t h a t could be shown to support good growth of S. q r e q a r i a . Three p l a n t m a t e r i a l s were i n i t i a l l y choosen f o r growth experiments and subsequent amino a c i d a n a l y s i s : (1) a l o c a l l y a v a i l a b l e lawn qrass, sin c e qrass has been r o u t i n e l y used f o r the l a b o r a t o r y r e a r i n q of S. q r e q a r i a (Davey, 1954; Howden and Hunter-Jones, 1958); (2) cabbage (Brassicae o l e r a c e a c a p i t a t a ) , s i n c e cabbage has a l s o been used i n l a b o r a t o r y r e a r i n g of S. jgregaria (Mehrctra and Eao, 1966; Mehrotra et a l , 1 972) ; (3) l e t t u c e (Lactuca s a t i v a c a p i t a t a ) , s i n c e t h i s i s r e a d i l y a cceptable t o S. q r e q a r i a and i s r e g u l a r l y used as an adjunct i n bran-mix a r t i f i c i a l d i e t s . Cabbage and l e t t u c e were obtained from supermarkets, and the grass from lawns on the U n i v e r s i t y Campus. The bran-mix c o n s i s t e d cf 60% bran, 20% d r i e d g r a s s , 10% yeast and 10% skim milk powder (% ty volume). . The r e s u l t s of growth t r i a l s on " n a t u r a l " foods are shown i n Table 2. Animals reared on grass f a i l e d t o reach the adult stage by the time the experiment was terminated ( a f t e r 45 19 T a b l e 2. A d u l t w e i g h t and tim e t a k e n t o d e v e l o p t o the a d u l t s t a g e f o r S c h i s t o c e r c a g r e g a r i a r e a r e d on a l e t t u c e p l u s b ran-mix c o n t r o l d i e t , or on cabbage, l e t t u c e or g r a s s d i e t s . Growth t r i a l s c o n d u c t e d a t 30°C and 55% R.H. T h r e e r e p l i c a t e s o f 50 a n i m a l s were s t a r t e d on each d i e t , p r o v i d i n g an i n i t i a l N-v a l u e of 150 f o r each d i e t . R e p l i c a t e s were combined f o r s u b s e q u e n t a n a l y s i s . Sexes a r e t r e a t e d s e p a r a t e l y s i n c e p r e l i m i n a r y a n a l y s i s showed t h a t , on any g i v e n d i e t , males d e v e l o p e d f a s t e r and had a lower a d u l t w e i g h t t h a n f e m a l e s (ANOVA, p < 0.05). To r e a d t h e t a b l e : f o r each sex, f i g u r e s i n columns f o l l o w e d by t h e same l e t t e r a r e s t a t i s t i c a l l y s i m i l a r (p > 0.05, S c h e f e ' s t e s t ) , i . e . f e m a l e s r e a r e d on l e t t u c e (ab) had s t a t i s t i c a l l y s i m i l a r mean w e i g h t s t o t h o s e r e a r e d on cabbage (b) and l e t t u c e p l u s b r a n - m i x ( a ) . D i e t Type and Sex Fema1es l e t t u c e + b r a n - m i x cabbage l e t t u c e g r a s s M ales l e t t u c e + b r a n - m i x cabbage l e t t u c e g r a s s Number of A d u l t s O b t a i n e d 2 1 15 15 41 18 17 Mean Wt. (mg) + S.D. 1679(a) + 87 1503(b) + 116 1601(ab) + 120 1397(a) + 117 1274(b) + 105 1249(b) + 89 Time t o D e v e l o p t o A d u l t S t a g e (Days) + S.D. 31.5(a) + 0.6 37.0(b) + 0 . 7 3 7 . 6 ( c ) + 0.6 30.9(a) + 0.8 34.9(b) + 0.9 35.0(b) + 0.7 20 d a y s ) . Although the reason f o r t h i s f a i l u r e i s not c l e a r , the grass used was c o l l e c t e d i n mid-summer, when grass q u a l i t y might be expected t o be poor..On the three other d i e t s , females took s i g n i f i c a n t l y l onger than the males to reach the ad u l t stage and were s i g n i f i c a n t l y heavier than males (p<0.05,ANOVA), so the sexes were t r e a t e d s e p a r a t e l y i n subsequent s t a t i s t i c a l a n a l y s i s . Females reared on the l e t t u c e p l u s bran-mix c o n t r o l d i e t achieved a higher a d u l t weight than those reared on cabbage, and a s i m i l a r a d u l t weight to those reared on l e t t u c e . In a d d i t i o n , females cn the c o n t r o l d i e t reached the ad u l t stage some 6 days sooner than females reared on e i t h e r cabbage or l e t t u c e . Males reared on the c o n t r o l d i e t achieved a higher a d u l t weight than those reared on cabbage or l e t t u c e , and reached the a d u l t stage 4 days sooner than animals on the other two d i e t s . Experiment 4. Growth of S c h i s t o c e r c a g r e q a r i a Hoppers,,on D i e t s C o n t a i n i n g an Amino Acid Eatio, Eased on A n a l y s i s of Cabbage or l e t t u c e Althouqh S. g r e q a r i a d i d not develop as f a s t on cabbage or l e t t u c e as on a l e t t u c e plus bran-mix c o n t r o l d i e t , animals on both these d i e t s d i d reach the a d u l t stage with a mean a d u l t -weight only s l i g h t l y l e s s than t h a t of animals reared on the c o n t r o l d i e t . T h i s suggests that S. g r e q a r i a i s ab l e to o b t a i n a balanced r a t i o c f amino acids from e i t h e r cabbage or l e t t u c e p r o t e i n s . Thus samples of cabbage and l e t t u c e were analysed f o r 21 amine a c i d content, and the growth of animals on d i e t s c o n t a i n i n g amino a c i d r a t i o s based on t h i s a n a l y s i s was compared with growth of animals on d i e t s c o n t a i n i n g the amino a c i d r a t i o developed by Dadd (1961c). For amino a c i d d e t e r m i n a t i o n , samples of cabbage or l e t t u c e (15 mg dry wt.) were hydrolysed by a d d i t i o n of 6K HCl and by heating under vacuum f o r 24 hrs a t 110°C. The r e s u l t i n g h y d r o l y s a t e was f r e e z e - d r i e d and d i s s o l v e d . i n 2 ml of l i t h i u m c i t r a t e b u f f e r (pH 2.2). T h i s s o l u t i o n was c e n t r i f u g e d to remove humin and 150 u l of the supernatent was analysed using a Beckman 119C amino a c i d a n a l y s e r . The r e s u l t s of the amino a c i d a n a l y s i s and the t e s t amino a c i d r a t i o s based on t h i s a n a l y s i s are shown i n Table 3..Growth experiments conducted using these amino a c i d r a t i o s , and the amino a c i d mixture developed by Dadd (see Table 1) are presented i n F i g . 3. From the r e s u l t s of the growth t r i a l s i t can be seen t h a t a f t e r 32 days, animals reared on the d i e t c o n t a i n i n g the amino a c i d r a t i o based on cabbage were over twice the weight c f those reared using Dadd's amino a c i d r a t i o . At the end of 32 days, 8 animals had reached the 4th i n s t a r and 6 animals had reached the 5th i n s t a r on d i e t s c o n t a i n i n g the amine a c i d r a t i o based on a n a l y s i s of cabbage, compared with 9 animals reaching the 3rd, and 4 animals the 4th i n s t a r on d i e t s c o n t a i n i n g Dadd's amino a c i d r a t i o . Animals reared on the d i e t i n c o r p o r a t i n g the amino a c i d r a t i o based on l e t t u c e showed l i t t l e change i n weight over the 32-day p e r i o d , although a t the end of t h i s time they had developed t o the 2nd i n s t a r . These 2nd-instar animals were unusual as they e x h i b i t e d white patches of c u t i c l e cn and around the pronotum, i n c o n t r a s t to the 22 T a b l e 3. Amino a c i d r a t i o s b a s e d on a n a l y s i s of cabbage and l e t t u c e . R e s u l t s o f amino a c i d a n a l y s i s a r e t h e means o f 3 d e t e r m i n a t i o n s and and e x p r e s s e d as % (+S.D.) of t o t a l amino a c i d s p r e s e n t . Amino a c i d r a t i o s used i n d i e t s were d e r i v e d from t h e r e s u l t s of amino a c i d a n a l y s i s by (a) i n c l u d i n g t r y p t o p h a n a t an a r b i t r a r y 1%, (b) r e p l a c i n g h y d r o x y p r o l i n e w i t h p r o l i n e and (c) a d j u s t i n g g l u t a m a t e l e v e l s to p r o v i d e f o r a t o t a l of 100 p a r t s . A l l d i e t a r y amino a c i d s were s u p p l i e d as L - i s o m e r s . Amino a c i d a n a l y s i s Amino a c i d r a t i o u s e d i n D i e t s cabbage l e t t u c e cabbage l e t t u c e mean(%)+ S.D. mean(%)+ S.D. % % a l a n i n e 5.03 + 0.15 3.87 + 0.11 5 . 0 3.9 a r g i n i n e 6 . 08 + 0.92 5.96 + 0 . 72 6. 1 6 . 0 a s p a r t a t e 9.86 + 0 .42 14.63 + 0.85 10.0 14.6 c y s t i n e o. 69 + 0 . 09 0 . 09 + 0.01 0.7 0 . 1 g l u t a m a t e 2 1.80 + 0 .26 3 1.34 + 0.33 19.7 29 . 2 g l y c i n e 4.71 + 0.21 4 .66 + 0.17 4.7 4.7 h i s t i d i n e 1 . 58 + 0.11 1 .58 + 0.09 1 .6 1 . 6 h y d r o x y p r o l i n e 2.11 + 0.14 - - - -i s o l e u c i n e 3.43 + 0.17 3.91 + 0.16 3.4 3.9 l e u c i n e 7.01 + 0 .95 5 . 60 + 0.48 7 . 0 5.6 l y s i n e 6 . 92 + 0.19 4.57 + 0.13 6.9 4.6 m e t h i o n i n e 1 . 78 + 0.14 0.10 + 0.00 1 .8 0 . 1 p h e n y l a l a n i n e 3 . 84 + 0.13 3 .49 + 0.18 3.8 3.5 p r o l i n e 6.21 + 1.33 4. 02 + 0.61 8 . 0 4.0 s e r i n e 7 .28 + 0.05 6.57 + 0.10 7 . 3 6.6 t h r e o n i n e 5 . 06 + 0.21 5.61 + 0 .25 5 . 1 5.6 t r y p t o p h a n - - - - 1 . 0 1 . 0 t y r o s i n e 2 . 48 + 0.23 0.19 + 0 .03 2.5 0 . 2 v a l i n e 5.44 + 0.24 4.75 + 0.27 5.4 4.8 T o t a l amino a c i d n i t r o g e n i n 100 p a r t s of amino a c i d 13.5 13.3 mix 23 Figure 3. Growth of S c h i s t o c e r c a q r e q a r i a hoppers on a r t i f i c i a l d i e t s c o n t a i n i n g e i t h e r the amino a c i d r a t i o developed by Dadd (a.a Dadd), an amino a c i d r a t i o based on a n a l y s i s of cabbage (a.a cabb) or an amino a c i d r a t i o based on a n a l y s i s cf l e t t u c e (a.a l e t t ) T h i r t y animals (3 r e p l i c a t e s of 10 animals each) were s t a r t e d cn each d i e t . Each p o i n t r epresents the mean weight of s u r v i v i n g animals; bars r e p r e s e n t 95% c onfidence l i m i t s f o r the number cf animals i n d i c a t e d . Growth t r i a l s were conducted at 30°C and 55% E.H. . \ Z 4 25 o v e r a l l black c o l o u r cf 2nd-instar animals on the other d i e t s . Experiment 5. S i m p l i f y i n g the P r e p a r a t i o n of A r t i f i c i a l D i e t s , and the. E f f e c t c f D i e t Consistency cn Growth of S c h i s t o c e r c a g r e q a r i a S e v e r a l commercial vitamin p r e p a r a t i o n s are s u p p l i e d t r i t u r a t e d with sugar (see ICN catalogue, 1978), and I f e l t t hat addinq vitamins to a r t i f i c i a l d i e t s as a suqar-vitamin mix, i n s t e a d of i n s o l u t i o n s , would s i m p l i f y the p r e p a r a t i o n of d i e t s . .Thus, mixinq the vitamins with sugar would mean t h a t the time consuming volume measurements and pH adjustments r e q u i r e d f o r the production of vitamin s o l u t i o n s would no longer be necessary. In a d d i t i o n , I hoped t h a t the dry d i e t s produced using a suqar-vitamin mix would not have to be processed f u r t h e r ( i . e . f r e e z e - d r i e d ) to i n c r e a s e t h e i r s t a b i l i t y . However, the fine-powder d i e t produced when sug a r - v i t a m i n mixtures were used made i t u n l i k e l y t h a t such d i e t s would be accepta b l e to S. g r e q a r i a , and attempts were made t o improve the p h y s i c a l c h a r a c t e r i s t i c s of the d i e t by producing t a b l e t s or granules from the f i n e powder. The growth of animals on powder, gran u l a t e d , or t a b l e t e d d i e t s , produced using a sugar-vitamin mix, was compared with growth on a granulated c o n t r o l d i e t produced i n the usual way by f r e e z e - d r y i n g a moist d i e t c o n t a i n i n g vitamins added i n s o l u t i o n . The sugar-vitamin mixture was prepared by adding s u f f i c i e n t v i tamins t o sucrose to provide the standard v i t a m i n 26 c o n c e n t r a t i o n (see Table 1) i n the 10 grm of sucrose used as a carbohydrate source i n the a r t i f i c i a l d i e t , and g r i n d i n g i n a b a l l - m i l l f o r 5 hr. Powder d i e t s were produced by adding the vitamins and sucrcse to the other d i e t i n g r e d i e n t s as a sugar-vitamin mix, i n s t e a d of the separate a d d i t i o n of sucrose and vitamin s o l u t i o n s . Tableted d i e t s were produced from pcwder d i e t s using a "Stokes" single-punch t a b l e t i n g machine, which formed biconvex t a b l e t s , 8 mm i n diameter, weighing about 0.5 grm. . Granulated d i e t s were produced by mixing 37 grm of the powder d i e t with 10 ml of water, f r e e z e or oven-drying (60°C f o r 5 h r ) , and then p u l v e r i z i n g i n a mortar and p e s t l e . A l l d i e t s contained the amino a c i d r a t i o based on a n a l y s i s of cabbage, and were s t o r e d i n vacuum d e s i c c a t o r s (at room temperature) i n an e f f o r t to prevent p o s s i b l e o x i d a t i o n of n u t r i e n t s . The r e s u l t s of growth t r i a l s on d i e t s c o n t a i n i n g a sugar-vitamin mixture i n place of tiie u s u al v i t a m i n s o l u t i o n s are shown i n F i g . 4. As expected, the f i n e powder d i e t was unacceptable. A l l animals cn t h i s d i e t d i e d between the 7th and 14th day of the experiment, and none developed past the 1st i n s t a r . However, growth cf animals on powder d i e t s formed i n t o granules or t a b l e t s was as good as growth on c o n t r o l d i e t s produced using vitamin s o l u t i o n s . The r e s u l t s show t h a t the p h y s i c a l p r o p e r t i e s of the d i e t are important, and t h a t sugar-vitamin mixtures can only be used i f d i e t s are l a t e r t a b l e t e d , cr moistened and d r i e d to form g r a n u l e s . Since the granulated d i e t formed by a d d i t i o n of water t c pcwder d i e t s , and subsequent oven-drying, was as gocd or b e t t e r than the other d i e t s t e s t e d , and took the l e a s t time to 27 F i g u r e 4. Growth of S c h i s t o c e r c a g r e q a r i a hoppers on g r a n u l a t e d , t a h l e t e d , cr powdered a r t i f i c i a l d i e t s T h i r t y animals (3 r e p l i c a t e s of 10 animals each) were s t a r t e d on each d i e t . Each p o i n t r e p r e s e n t s the mean weight of s u r v i v i n g animals; bars re p r e s e n t 95% confidence l i m i t s f o r the number cf animals i n d i c a t e d . . Open c i r c l e s , moist d i e t g r a nulated by f r e e z e - d r y i n g ; c l o s e d c i r c l e s , powder d i e t g r a nulated by a d d i t i o n of water and f r e e z e - d r y i n g ; open sguares, powder d i e t formed i n t o t a b l e t s ; c l o s e d squares, powder d i e t granulated by a d d i t i o n of water and oven-drying; c l o s e d t r i a n g l e s , unmodified powder d i e t ; --//, a l l animals dead. Grcwth t r i a l s were conducted at 30°C and 55% E.H. zo TIME (days) 29 produce, a l l f u r t h e r a r t i f i c i a l d i e t s were made i n t h i s way. Experiment 6. S t a b i l i t y of Improved D i e t s P r e l i m i n a r y experiments on the storage p r o p e r t i e s of c h e m i c a l l y d e f i n e d d i e t s i n d i c a t e d t h a t f r e e z e - d r i e d d i e t s were s t a b l e f o r at l e a s t a month (Experiment 1 ) . - However, these f i r s t experimental d i e t s had a poorly balanced amino a c i d r a t i o and i t was p o s s i b l e t h a t any d e l e t e r i o u s e f f e c t s due to prolonged storage were being masked by the o v e r a l l poor growth of animals on these d i e t s . The present experiment was undertaken to evaluate the s t a b i l i t y of oven-dried d i e t s c o n t a i n i n g the improved amino a c i d r a t i o based on a n a l y s i s of cabbage. 2o determine whether improved d i e t s were s t a b l e , the growth of animals on a d i e t that had p r e v i o u s l y been s t o r e d f o r one month i n a vacuum d e s i c c a t o r was compared with growth of animals r e a r e d on d i e t s prepared a f r e s h each week. Both d i e t s contained the amine a c i d r a t i o based on the a n a l y s i s of cabbage, were prepared using a v i tamin-sucrose mix, and were formed i n t o granules by a d d i t i o n of water and subsequent oven-dr y i n g (for f u r t h e r d e t a i l s r e l a t i n g to d i e t p r o duction see Experiment 5). Ihe r e s u l t s of growth t r i a l s using " f r e s h " and " o l d " a r t i f i c i a l d i e t s are shown i n Table 4. There was no s i g n i f i c a n t d i f f e r e n c e i n the weight of male or female animals r e a r e d on 30 T a b l e 4. Weight and i n s t a r r e a c h e d by S c h i s t o c e r c a g r e g a r i a r e a r e d f o r 32 days on f r e s h or o l d a r t i f i c i a l d i e t s . F r e s h d i e t s are t h o s e r e p l a c e d w e e k l y d u r i n g the c o u r s e of t h e e x p e r i m e n t , o l d d i e t s a r e t h o s e s t o r e d i n vacuum d e s i c c a t o r s f o r 1 month p r i o r to the e x p e r i m e n t , and not r e p l a c e d d u r i n g the c o u r s e of the e x p e r i m e n t . Growth t r i a l s were c o n d u c t e d a t 30 °C and 55% R.H. Two r e p l i c a t e s of 200 a n i m a l s were s t a r t e d on each d i e t , p r o v i d i n g an i n i t i a l N - v a l u e of 400 f o r each d i e t . R e p l i c a t e s were combined f o r s u b s e q u e n t a n a l y s i s ; s e x e s were not s e p a r a t e d s i n c e t h e mean w e i g h t of t h e males and f e m a l e s were not s i g n i f i c a n t l y d i f f e r e n t (p > 0.05, ANOVA). Mean w e i g h t s f o l l o w e d by the same l e t t e r a r e s t a t i s t i c a l l y s i m i l a r (p > 0.05, ANOVA). D i e t Type Number of A n i m a l s Mean wt. Number of A n i m a l s S u r v i v i n g ( o f 400) (mg) A f t e r 33 days + S . D. and I n s t a r ( i n b r a c k e t s ) A f t e r 32 Days f r e s h d i e t 56 560(a) +78 1 8 ( 4 t h ) , 3 8 ( 5 t h ) o l d d i e t 52 572(a) +95 1 6 ( 4 t h ) , 3 6 ( 5 t h ) 31 e i t h e r of the d i e t s (p>0.05, ANOVA) so sexes were not t r e a t e d s e p a r a t e l y . At the end of 32 days, most animals on both d i e t s were in the 5th i n s t a r and there was no s i g n i f i c a n t d i f f e r e n c e between the weight of animals reared on d i e t s p r e v i o u s l y s t o r e d f o r 1 mcnth and those reared on d i e t s r e p l a c e d weekly. Ihese r e s u l t s confirm t h a t dry d i e t s are s t a b l e , and j u s t i f i e s the experimental procedure of making up s u f f i c i e n t d i e t at the beginning of a growth experiment t o l a s t f o r 4-5 weeks. Experiment 7. Growth of S c h i s t o c e r c a q r e q a r i a on Improved  A r t i f i c i a l , Diets,,and on a Lettuce plus bran-mix C o n t r o l Di e t I h u s f a r , the adequacy of improved a r t i f i c i a l d i e t s ( c o n t a i n i n g the amino a c i d r a t i o based on the a n a l y s i s of cabbage) has net been t e s t e d r e l a t i v e t o the l e t t u c e p l u s bran-mix d i e t used to maintain l o c u s t s t o c k s at 0.B.C. In a d d i t i o n , although i t has been shown t h a t animals reared on the improved a r t i f i c i a l d i e t can reach the 5th i n s t a r a f t e r 32-days growth (Experiment 6), no experiments have been conducted f o r longer periods of time t o determine i f the d i e t i s good enough to allow animals to develop to the a d u l t stage..The present experiment was conducted to compare the weight and developmental stage of animals reared on c h e m i c a l l y d e f i n e d d i e t s f o r 32 days, with the weight and developmental stage reached by animals reared on a l e t t u c e p l u s bran-mix c o n t r o l d i e t , and to determine i f animals could reach the a d u l t stage on a c h e m i c a l l y d e f i n e d d i e t . The c h e m i c a l l y d e f i n e d d i e t 32 contained the amino a c i d r a t i o based on a n a l y s i s of cabbage, and was prepared using a vitamin-sucrose mix, and formed i n t o granules by a d d i t i o n of water and subsequent oven-drying (for f u r t h e r d e t a i l s see Experiment 5)..The composition of the bran-mix used i s given i n Experiment 3. The r e s u l t s cf the growth t r i a l s on c h e m i c a l l y d e f i n e d and l e t t u c e plus bran-mix (control) d i e t s are presented i n Table 5. Females reared on the c o n t r o l d i e t were s i g n i f i c a n t l y heavier than aales (p<0.05, ANOJfA) (no such d i f f e r e n c e e x i s t e d f o r animals on the c h e m i c a l l y d e f i n e d d i e t s ) and thus sexes were t r e a t e d s e p a r a t e l y i n subsequent a n a l y s i s . Comparisons of mean weiqht were made using the K r u s k a l l W a l l i s non-parametric t e s t s i n c e the var i a n c e s i n weight of animals on the two d i e t s were net homogenous (p<0.05, B a r t l e t t ' s t e s t ) . . At the end of 32 days, a l l animals on the c o n t r o l d i e t had reached the adult stage whereas, at t h i s time, the ma j o r i t y of animals r e a r e d on the c h e m i c a l l y d e f i n e d d i e t were only i n the 5th i n s t a r . . Male and female animals reared on the c o n t r o l d i e t were some 2-3 times heavier than those reared on the c h e m i c a l l y d e f i n e d d i e t . Animals were kept on the c h e m i c a l l y d e f i n e d d i e t f o r a f u r t h e r 30 days, by the end of which time they had a l l d i e d . During t h i s a d d i t i o n a l time, 4 male animals moulted to the a d u l t stage with a mean weight of 808 ± 37 mg (+SD), and 5 female animals moulted to the a d u l t stage with a mean weight of 752 ± 45 mg. The males took 45±6 days to develop to the a d u l t stage and the females took 5 1+4 days. Only 9 of the 400 animals s t a r t e d on the c h e m i c a l l y d e f i n e d d i e t reached the a d u l t stage, whereas 158 cf 400 reached the a d u l t stage on the lettuce+bran-mix d i e t , showing t h a t the c h e m i c a l l y d e f i n e d d i e t i s not adequate 33 T a b l e 5. Weight and d e v e l o p m e n t a l s t a g e r e a c h e d by S c h i s t o c e r c a g r e g a r i a r e a r e d f o r 33 days on a l e t t u c e p l u s b ran-mix c o n t r o l d i e t or a c h e m i c a l l y d e f i n e d d i e t . Growth t r i a l s were c o n d u c t e d a t 30°C and 55% R.H. Two r e p l i c a t e s of 200 a n i m a l s were s t a r t e d on each d i e t , p r o v i d i n g an i n i t i a l N - v a l u e o f 400 a n i m a l s f o r each d i e t . R e p l i c a t e s were combined f o r s u b s e q u e n t a n a l y s i s . Sexes a r e t r e a t e d s e p a r a t e l y s i n c e p r e l i m i n a r y a n a l y s i s showed t h a t f e m a l e s r e a r e d on the l e t t u c e + b r a n - m i x d i e t were s i g n i f i c a n t l y h e a v i e r a f t e r 33 days t h a n t h e males (p < 0.05, ANOVA). F o r each sex, mean w e i g h t s f o l l o w e d by the same l e t t e r a r e s t a t i s t i c a l l y s i m i l a r (p > 0.05, K r u s k a l l - W a l l i s t e s t ) . D i e t Type Number o f Mean Wt. Number o f and sex A n i m a l s (mg) +S.D. A n i m a l s i n S u r v i v i n g Each I n s t a r ( i n A f t e r 33 b r a c k e t s ) A f t e r 33 days 33 Days Females l e t t u c e + b r a n - m i x c h e m i c a l l y d e f i n e d 48 1656(a) +51 4 8 ( a d u l t s ) 26 552(b) +131 1 0 ( 4 t h ) , 1 6 ( 5 t h ) Males l e t t u c e + b r a n - m i x 110 1349(a) +43 c h e m i c a l l y d e f i n e d 18 564(b) +157 1 1 0 ( a d u l t s ) 4 ( 4 t h ) , 1 4 ( 5 t h ) 34 f o r continuous r e a r i n g of S. q r e q a r i a . D i s c u s s i o n Ihe i n s t a b i l i t y of d i e t s prepared a c c o r d i n g to Dadd's r e c i p e (Dadd, 1960c, 1961c) was i m p l i c i t i n h i s weekly replacement of such d i e t s , and noted when he blamed poor growth of animals on d i e t age (Dadd, 1960e). In the growth s t u d i e s presented here, i t was shewn that such d i e t s were u n s t a b l e , and caused reduced growth r a t e s and heavy m o r t a l i t y a f t e r 20 days of storage at -15°C. However, the i n s t a b i l i t y of these d i e t s could be overcome by f r e e z e - d r y i n g and s t o r i n g i n a d e s i c c a t o r . Many i n s e c t s are i n c a p a b l e of s y n t h e s i z i n g a s c o r b i c acid (Gupta e_t a l . , 1972), and i t i s known to be an e s s e n t i a l n u t r i e n t f o r some phytophagous i n s e c t s (see reviews by Dadd, 1970, 1973; House, 1974 and Vanderzant, 1966), i n c l u d i n g S» q r e q a r i a (Dadd, 1957, 1960b,c). A s c o r b i c a c i d i s a n o t o r i o u s l y unstable compound, o x i d i z i n g r e a d i l y i n s o l u t i o n s (Altman and Dittmer, 1972a), and has been shown to degrade i n s e v e r a l i n s e c t a r t i f i c i a l d i e t s (Bounias and Bonnot, 1976; Chipendale and Beck, 1964; Dadd e t a l . , 1967; I t o and A r a i , 1965; Navon, 1978; Vanderzant and Richardson, 1963). Dadd et a l . , (1967) found t h a t l o s s of a s c o r b i c a c i d i n l i g u i d d i e t s prepared f o r aphids was l a r g e l y prevented by continuous storage a t -20°C, although much more was l o s t i f the d i e t s were thawed f o r as l i t t l e as 1 hr a day. The r e s u l t s 35 presented i n F i g . 2 show that a s c o r b i c a c i d i s r e a d i l y degraded i n mcist d i e t s prepared according to Cadd's r e c i p e and s t o r e d at -15°C. The r a t e of a s c o r b i c a c i d l o s s was i n i t i a l l y high .(0-14 days), with only 50% of the o r i g i n a l a s c o r b i c a c i d remaining a f t e r 14 days, but then the r a t e of l o s s decreased (14-28 days). I f thawing of the f r o z e n moist d i e t s was r e s p o n s i b l e f o r the majority of a s c o r b i c a c i d l o s s , as determined f o r aphid d i e t s (Dadd e t a l . , 1967) , then the r e s u l t s presented here w i l l be an underestimate of the amount of a s c o r b i c a c i d l o s t i n the moist d i e t s used i n growth experiments, as d i e t s used i n these experiments were thawed every 2-3 days, whereas those used i n a s c o r b i c a c i d degradation experiments were only thawed every 7 days. This may e x p l a i n why 40% of the o r i g i n a l a s c o r b i c a c i d s t i l l remained i n one month-old d i e t s , w h i l s t Dadd (1960e), using s i m i l a r d i e t s and storage c o n d i t i o n s , but f e e d i n g animals (and presumably thawing d i e t s ) every 2 days c o u l d f i n d " l i t t l e t r a c e " of a s c o r b i c a c i d i n 1 month o l d d i e t s . . A l t h o u g h r e a d i l y degraded i n moist d i e t s , a s c o r b i c a c i d seemed to remain s t a b l e i n f r e e z e - d r i e d d i e t s s t o r e d i n d e s i c c a t o r s . Several workers have shown that a s c o r b i c a c i d i s l o s t more r a p i d l y under experimental c o n d i t i o n s than under storage c o n d i t i o n s (Dadd et a l . , 1967; I t o and A r a i , 1965; Vanderzant and Bichardson, 1963).. Under the present experimental c o n d i t i o n s (30°C, 50% B.H.) the f r e e z e - d r i e d d i e t showed l i t t l e a s c o r b i c a c i d l e s s over the p e r i o d of 48 hrs, whereas by the end c f t h i s time the non-dried d i e t contained only 80% of the a s c o r b i c a c i d i n i t i a l l y present.. Both d i e t s were hygroscopic, t a k i n g up cr l o s i n g moisture to come i n t o e q u i l i b r i u m with the 36 moisture content cf the surrounding a i r . I n t e r e s t i n g l y , the l o s s cf a s c o r b i c a c i d i n non-dried d i e t s l e v e l l e d o f f at approximately the same time t h a t the water content of the d i e t s t a b i l i z e d at 6%, suggesting t h a t the d r y i n g of moist d i e t s under experimental c o n d i t i o n s may serve to preserve a s c o r b i c a c i d a c t i v i t y . Although a s c o r b i c a c i d degradation seemed to be c o r r e l a t e d with the poor growth and heavy m o r t a l i t y of hoppers r e a r e d on unreplaced moist d i e t s , there i s no evidence to show a c a u s a l r e l a t i o n s h i p . However, the r e s u l t s of these experiments do show that water content of d i e t s i s a major f a c t o r governing the r a t e c f a s c c r b i c a c i d l o s s under both experimental and storage c o n d i t i o n s , and suggests t h a t n u t r i e n t s w i l l be more s t a b l e i n dry d i e t s . Dadd (1960c) f r e q u e n t l y r e f e r s to h i s d i e t ( e s s e n t i a l l y the same d i e t as used i n the experiments d i s c u s s e d above) as being dry, however examination of the composition of t h i s d i e t shows t h a t i t c o n t a i n s approximately 25% water. W h i l s t such d i e t s are d r i e r than the agar d i e t s they superceded ( c o n t a i n i n g approximately 84% water) (Dadd, 1960c) they c e r t a i n l y cannot be considered to be dry. S i m i l a r l y , Nayar (1964a) d e s c r i b e s a d i e t developed f o r the grasshopper Melanoplus b i v i t t a t u s as being dry, «hen i t a l s o contained approximately 25% water.. In the present study i t has been shown t h a t there are lar g e d i f f e r e n c e s i n storage p r o p e r t i e s between t r u l y dry d i e t s (<1% water) and moist d i e t s (25% water), and with t h i s i n mind i t i s t c he recommended t h a t more care be taken i n d e s c r i b i n g the moisture content of d i e t s . . In the present study, S. q r e q a r i a hoppers reared on an 37 a r t i f i c i a l d i e t c o n t a i n i n g the amino a c i d r a t i o developed by DaddiJ1961c) only reached a mean weight of 130 mg a f t e r 30 days growth. . Dadd< 1961c) , using the same d i e t s and s i m i l a r experimental c o n d i t i o n s , found that a f t e r 30 days growth S. g r e g a r i a hoppers had reached a mean weight of approximately 1300 mg. Thus the animals used by Dadd were almost 10 times h e a v i e r , a f t e r 30 days growth, than the animals used i n the present study. T h i s d i f f e r e n c e i n growth of animals on the same a r t i f i c i a l d i e t c o u l d be e x p l a i n e d by assuming t h a t the experimental animals used by Dadd were g e n e t i c a l l y d i f f e r e n t frcm those used here..Sang(1959) emphasized t h a t n u t r i t i o n a l requirements could be expected to d i f f e r w i t h i n animal s p e c i e s , and s t u d i e s have shown that w i l d p o p u l a t i o n s or l a b o r a t o r y s t r a i n s of the same i n s e c t s p e c i e s can d i f f e r i n t h e i r performance cn a r t i f i c i a l d i e t s ( A u c l a i r and S r i v a s t a v a , 1978; H i n t c r , 1959; 0'meara and Evans, 1973; S r i v a s t a v a and A u c l a i r , 1S78). In a d d i t i o n i t has been shown t h a t amino a c i d balances p e r f e c t e d f o r one s t r a i n of i n s e c t s p e c i e s do not n e c e s s a r i l y allow f c r qood growth of another s t r a i n ( G r i f f i t h s e t a l . , 1375)..With these s t u d i e s i n mind, i t seems u n r e a l i s t i c to expect that S. g r e q a r i a obtained from s t o c k s at U.B.C.. would respond i n the same way to an amino a c i d r a t i o and a r t i f i c i a l d i e t developed by Dadd (1960c) f o r S. g r e q a r i a obtained from stocks at the A n t i - L o c u s t Research Centre i n London. Ihe d e t r i m e n t a l e f f e c t of amino a c i d imbalance on growth of i n s e c t s i s w e l l known (Davis, 1961a,b, 1968a,b,c, 1969a #b, 1972, 1974, 1978; Gordon, 1959; House, 1963; Pausch and F r a e n k e l , 1966; P r a t t et a l . , 1972; Rock and King, ,1967; Thompson, 1976; Vanderzant, 1973), and s e v e r a l s t u d i e s of 38 phytophagous i n s e c t s have demonstrated that such i n s e c t s grow b e t t e r when reared on a r t i f i c i a l d i e t s c o n t a i n i n g an amino a c i d r a t i o e q u i v a l e n t t c that present i n p l a n t s t h a t support good growth (DeGroot, 1953; Fukada et a l . , 1962; M i t t l e r and Dadd, 1962; Vanderzant, 1958). In the present study i t was found t h a t both cabbage and l e t t u c e allowed S. .qreqaria t o develop to the a d u l t s t a g e , and presumably both s u p p l i e d an adeguate amount and balance of amino a c i d s . However, although the amino a c i d r a t i o based on a n a l y s i s cf cabbage p r o t e i n s allowed animals to develop to the 5th i n s t a r and was s u p e r i o r t o the amino a c i d r a t i o developed by Dadd, the amino a c i d r a t i o based on a n a l y s i s of l e t t u c e p r o t e i n did not support good growth when i n c o r p o r a t e d i n an a r t i f i c i a l d i e t . . Since both the a r t i f i c i a l d i e t based on a n a l y s i s of l e t t u c e and th a t based on a n a l y s i s of cabbage contained s i m i l a r amounts of amino n i t r o g e n , the f a i l u r e of the amino a c i d r a t i o based on l e t t u c e must be r e l a t e d t o an amino a c i d imbalance. There c o u l d be s e v e r a l p o s s i b l e reasons f o r t h i s imbalance and conseguent f a i l u r e of the a r t i f i c i a l d i e t : (a.) the l e t t u c e p r o t e i n a c t u a l l y contained an imbalanced r a t i o of amino a c i d but the animals consumed s u f f i c i e n t l e t t u c e to compensate f o r t h i s imbalance, w h i l s t not consuming s u f f i c i e n t a r t i f i c i a l d i e t t c do the same; (b) the r a t i o of amino a c i d r e l e a s e d during the v i o l e n t h y d r o l y s i s procedure a s s o c i a t e d with amino a c i d a n a l y s i s d i d not r e f l e c t the r a t i o made a v a i l a b l e to the animal by i t s d i g e s t i v e enzymes; (c) the r a t i o of amino a c i d s a v a i l a b l e when feed i n g on i n t a c t p r o t e i n i s not the same as that a v a i l a b l e when f e e d i n g on amino a c i d mixes. Thus, when feed i n g on the a r t i f i c i a l d i e t , the simultaneous 39 presence of many f r e e amino a c i d s i n the gut may l e a d to competition f o r amino a c i d uptake s i t e s , r e d u c i n g the a v a i l a b i l i t y of p a r t i c u l a r amino a c i d s (Elvehjem, 1956) ; (d) the animals used and r e g u i r e d p a r t i c u l a r p e p t i d e s or polyp e p t i d e s a v a i l a b l e from the l e t t u c e p r o t e i n but not a v a i l a b l e i n the a r t i f i c i a l d i e t (such a reason has been suggested f o r the f a i l u r e o f other amino a c i d r a t i o s based on p r o t e i n a n a l y s i s , Kasting et a l . , 1962; L e c l e r c q and Lopez-Francos, 1964; Lipke and Fr a e n k e l , 1956); (e) the amino a c i d a n a l y s i s procedure did net a c c u r a t e l y determine the amounts of the anino a c i d s present i n the l e t t u c e p r o t e i n . although the f a i l u r e of the d i e t based on l e t t u c e p r c t e i n could be due to any or a l l of the above reasons there i s some evidence to suggest that i t was due to the known i n a c c u r a c i e s a s s o c i a t e d with q u a n t i t a t i v e amino a c i d a n a l y s i s . . T h u s the three amino a c i d s : methionine, c y s t i n e , and t y r o s i n e , which are considered to be p a r t i a l l y or completely destroyed d u r i n g the a c i d h y d r o l y s i s procedure a s s o c i a t e d with amino a c i d a n a l y s i s (Blackburn, 1978) were only found i n low amounts i n l e t t u c e p r o t e i n . Methionine i s an e s s e n t i a l amino a c i d f o r S. . g r e q a r i a , whereas c y s t i n e and t y r o s i n e are not (see s e c t i o n B)..Although not themselves e s s e n t i a l , both c y s t i n e and t y r o s i n e are considered to be produced i n i n s e c t s from the e s s e n t i a l amino a c i d s methionine and phenylalanine (Dadd, 1970, 1973; House, 1974). I t would thus be expected that any l a c k o f c y s t i n e or t y r o s i n e could only be compensated f o r by a corres p o n d i n g r e d u c t i o n i n the amount of the methionine and phen y l a l a n i n e a v a i l a b l e f c r p r o t e i n s y n t h e s i s . Thus the low l e v e l s of each or a l l cf the three amino a c i d s , methionine, c y s t i n e and t y r o s i n e , 40 could e x e r t a profound e f f e c t on growth.. fls well as f a i l i n g to grow on the d i e t based on l e t t u c e p r o t e i n , animals a l s o developed areas of white c u t i c l e e s p e c i a l l y around the head and pronotum. These areas could represent i n c o m p l e t e l y melanized c u t i c l e , which would suggest that i n s u f f i c i e n t t y r o s i n e was present, s i n c e t h i s amino a c i d i s known tc play a r o l e i n the formation of melanin i n i n s e c t s ( C o t r e l l , 1964; Furzeau-braesch, 1966; Schlossberger-Raecke and K a r l s c n , 1964). However, i t i s a l s o p o s s i b l e t h a t these areas represent white d e p o s i t s of u r i c a c i d , as s i m i l a r d e p o s i t s have been noted i n cockroaches fed on high p r o t e i n d i e t s (Haydak, 1953) , and could presumably a l s o be caused by a gen e r a l imbalance i n the r a t i o o f amino a c i d s . Many authors have emphasized the importance of the p h y s i c a l p r o p e r t i e s c f a r t i f i c i a l d i e t s developed f o r i n s e c t s (Beck e t a l . , 1949; Chippendale, 1970; Hagen and Tassan, 1965; Pausch and F r a e n k e l , 1966; Rodriguez et a l . , 1976; S i n o i r , 1968; S n e l l e r and Dadd, 1977; I s i t s i p i s , 1977; Vanderzant, 1969) . The f a i l u r e of Kreasky (1962) to produce a s u c c e s s f u l a r t i f i c i a l d i e t f o r the grasshopper Melanoplus b i v i t t a t u s was l a t e r suggested as being due to the p h y s i c a l p r o p e r t i e s of the d i e t (Nayar, 1964a).. In a recent review, Davis (1972) s t a t e s that Mulkern and Toczek (1970) "have r e c e n t l y s o l v e d the p h y s i c a l d i e t a r y reguirements of the grasshopper Melanoplus femurrubrum, by producing p e l l e t s of d i e t on which t h i s i n s e c t a c t i v e l y feeds." In f a c t Mulkern and Toczek (1970) made no such c l a i m s f o r t h e i r d i e t , and p e l l e t i n g was c a r r i e d out as the powdered d i e t was s u b j e c t to s p i l l a g e and blowing away. Ihe p r e p a r a t i o n of d i e t s used i n t h i s study was made l e s s 4 1 time-consuming by using a sugar-vitamin mixture i n s t e a d of vitamin s o l u t i o n s . However, the f i n e dry powder d i e t formed by the use cf sugar-vitamins was p h y s i c a l l y unacceptable to q r e q a r i a , and such d i e t s had to be p e l l e t e d or made into granules by a d d i t i o n of water and subsequent f r e e z e - o r even-d r y i n g . The f a i l u r e of the powder d i e t to support growth was not unexpected, given (a) the i n s e c t i c i d a l a c t i o n of f i n e powders (Banks, 1976) and (b) t h a t S. q r e q a r i a has chewing mouth-parts adapted f o r b i t i n g at the edges of leaves (Uvarov, 1966, 1977) and not adapted f o r t a k i n g up f i n e powders.. Cven-d r y i n g moist d i e t s produced an apparently s u i t a b l e t e x t u r e , and s i n c e t h i s procedure was l e s s time-consuming than e i t h e r p e l l e t i n g dry d i e t s or f r e e z e - d r y i n g moist d i e t s , i t was used i n the production of a l l f u r t h e r d i e t s . . The improved a r t i f i c i a l d i e t s ( c o n t a i n i n g an amino a c i d r a t i c based on a n a l y s i s of cabbage p r o t e i n , vitamins added as a vitamin-sucrose mixture, moistened, and oven-dried to produce granules) were s t a b l e f o r at l e a s t 2 months when s t o r e d i n vacuum d e s i c c a t o r s . Such d i e t s enabled S..qreqaria hoppers to develop to the 5th i n s t a r , with a mean wt of approximately 550 mg, a f t e r 33 days growth. However, very few animals on these d i e t s s u r v i v e d to become a d u l t s , and growth was s i g n i f i c a n t l y poorer than on a l e t t u c e plus bran-mix c o n t r o l d i e t . . Despite these shortcomings the d i e t was good enough to allow i n v e s t i g a t i o n of the importance of amino a c i d s i n governing food consumption and growth of S. q r e q a r i a (see s e c t i o n B ) . 42 Sectio n B. Amino Acid Requirements, and Phaqpstimulant P r o p e r t i e s cf Amino Acids f o r S c h i s t o c e r c a g r e q a r i a I n t r o d u c t i o n A l l animals r e q u i r e an exoqenous supply of d i e t a r y n i t r o g e n i n the form of p r o t e i n s or amino a c i d . Of the 20 commcn amino a c i d s only 10 (the e s s e n t i a l amino acids) u s u a l l y have to be s u p p l i e d by the d i e t , the other (the i n e s s e n t i a l amine acids) being r e a d i l y synthesized by the animal. D i e t a r y -d e l e t i o n techniques ( i . e . removal of i n d i v i d u a l amino acids from a r t i f i c i a l d i e t s ) have shown t h a t , i n gen e r a l , the CIO amine a c i d s found to be e s s e n t i a l f o r the r a t (Rose, 1938) and other v e r t e b r a t e s (Altman and Dittmer, 1972b) are a l s o e s s e n t i a l f o r i n s e c t s (see general reviews of i n s e c t n u t r i t i o n by: Altman and Dittmer, 1972; Dadd, 1970, 1973; House, 1974; Lipke and F r a e n k e l , 1956; Trager, 1953). Un f o r t u n a t e l y , only r a r e l y are amino a c i d d e l e t i o n s t u d i e s accompanied by i n v e s t i g a t i o n of the e f f e c t s of such d e l e t i o n on the consumption of a r t i f i c i a l d i e t s . T h i s makes i t i m p o s s i b l e to determine whether an amino a c i d i s e s s e n t i a l f o r p r o t e i n formation ( i . e. cannot be s y n t h e s i z e d by the animal) or whether i t i s a phagostimulant necessary f o r the i n g e s t i o n of the d i e t . However, some s t u d i e s have shown that the f a i l u r e of an i n s e c t to grew on an a r t i f i c i a l d i e t l a c k i n g a p a r t i c u l a r amino a c i d can be r e l a t e d to reduced d i e t intake..Thus, 43 L e c k s t e i n and L l e w e l l y n (1974) showed that poor growth of the aphid A£his f abae on d i e t s l a c k i n g a l a n i n e or p r o l i n e c o u l d be a t t r i t u t e d to reduced food i n t a k e , and M i t t l e r (1970) showed the same f o r the aphid Myzus p e r s i c a e feeding on d i e t s l a c k i n g h i s t i d i n e , methionine or c y s t e i n e . . Despite the p l e n i t u d e of s t u d i e s on amino a c i d reguirements of i n s e c t s (see reviews c i t e d e a r l i e r ) , to my knowledge no i n f o r m a t i o n i s a v a i l a b l e on t h i s aspect of the n u t r i t i o n cf any l o c u s t or grasshopper s p e c i e s . The development of a c h e m i c a l l y d e f i n e d a r t i f i c i a l d i e t f o r S c h i s t o c e r c a q r e q a r i a (see s e c t i o n A) provides a method of i n v e s t i g a t i n g the amine a c i d reguirements of t h i s s p e c i e s . The present study was undertaken to determine the amino a c i d requirements of S. g r e q a r i a , and whether amino ac i d s were r e q u i r e d as n u t r i e n t s per se or as phaqcstimulants necessary f o r the i n g e s t i o n of d i e t s . M a t e r i a l s and Methods Experimental O u t l i n e 1c determine •which amino a c i d s were e s s e n t i a l f o r j3. q r e q a r i a the qrowth of hoppers reared on t e s t d i e t s l a c k i n g i n d i v i d u a l amine a c i d s was compared with growth of animals on c o n t r o l d i e t s c o n t a i n i n g the f u l l complement of 18 amino a c i d s . Feeding t r i a l s were conducted to see i f growth of animals on 4 4 d i e t s l a c k i n g i n d i v i d u a l amino a c i d s c o u l d be r e l a t e d t o t h e amount o f d i e t consumed. D i e t I r o d u c t i o n D i e t c o n s t i t u e n t s ( o t h e r t h a n t h e t e s t a m i n o a c i d m i x t u r e s ) were a s p r e s e n t e d i n t h e M a t e r i a l s and M e t h o d s o f t h e p r e v i o u s s e c t i o n . a l l d i e t s c o n t a i n e d a s u g a r - v i t a m i n m i x t u r e and were m o i s t e n e d a n d o v e n - d r i e d t o p r o d u c e g r a n u l e s a c c e p t a b l e t o S. q r e g a r i a . . C o n t r o l d i e t s c o n t a i n e d an amino a c i d m i x t u r e b a s e d on a n a l y s i s o f c a b b a g e ; amino a c i d m i x e s f o r t e s t d i e t s were p r o d u c e d by o m i t t i n g an i n d i v i d u a l amino a c i d f r o m c c n t r c l m i x t u r e s , w i t h no a t t e m p t b e i n g made t o k e e p t e s t d i e t s i s o n i t r o g e n o u s w i t h c o n t r o l d i e t s . S u f f i c i e n t d i e t f o r t h e e x p e r i m e n t a l p e r i o d was made up i n a d v a n c e and s t o r e d i n vacuum d e s i c c a t o r s a t room t e m p e r a t u r e . G r o w t h T r i a l s A n i m a l s f o r g r o w t h t r i a l s were o b t a i n e d a s h a t c h l i n g s (< 1 day e l d ) , and were r e a r e d i n m o d i f i e d i c e - c r e a m p a i l s u nder c o n d i t i o n s p r e v i o u s l y d e s c r i b e d (see M a t e r i a l s and M e t h o d s , p r e v i o u s s e c t i o n ) . T h r e e r e p l i c a t e s o f 1 5 a n i m a l s e a c h were s t a r t e d on e a c h d i e t t y p e ( p r o v i d i n g a s t a r t i n g t o t a l c f 4 5 a n i m a l s on e a c h d i e t ) , r e p l i c a t e s b e i n g c o m b i n e d f o r s u b s e g u e n t s t a t i s t i c a l a n a l y s i s , a n i m a l s were i n d i v i d u a l l y w e i g h e d e v e r y 45 week ever a p e r i o d of 5-weeks. The r e s u l t s of growth t r i a l s are presented g r a p h i c a l l y , with 95% c o n f i d e n c e l i m i t s given f o r the means on days 21 and 35 i f the number of animals s u r v i v i n g at these times was g r e a t e r than 2.. Feeding T r i a l s The a c c e p t a b i l i t y of d i e t s was assessed by determining how much d i e t was consumed by groups (3 animals) of 2nd i n s t a r animals over a p e r i o d of 3 days. Weighed amounts of dry d i e t (< 1% water) were presented to animals and the dry weight of food consumed was g r a v i m e t r i c a l l y determined by s u b t r a c t i n g the dry weight of d i e t l e f t at the end of the experimental p e r i o d ( d i e t remnants oven-dried f o r 5 hrs at 60°C) from the dry weight of d i e t i n i t i a l l y presented. At the end of 3 days the groups of animals reared on the d i e t s were k i l l e d and oven-dried (60°C f o r 5 h r s ) , and the amount of food consumed was c o r r e c t e d f o r animal weight by using a consumption index ( i . e . dry weight of food consumed/mean dry weight of animals on the d i e t ) . . The mean dry weight of animals on the d i e t was determined by averaging the i n i t i a l and f i n a l dry weights of the 3 animals. The i n i t i a l dry weight of the animals placed on d i e t s was approximated by oven-drying (60°C f o r 5 hrs) r e p r e s e n t a t i v e samples of animals of known l i v e weight, and a p p l y i n g the l i v e weight/dry weight c o n v e r s i o n f a c t o r to the l i v e weight of animals placed on d i e t s . . Each experiment i n v o l v e d a comparison between a t e s t d i e t 46 ( i . e . a d i e t l a c k i n g an amino acid) and a c o n t r o l d i e t ( c o n t a i n i n g a l l 18 amino a c i d s ) . Ninety animals, 1-2 days i n t o the 2nd i n s t a r , were c o l l e c t e d from the st o c k s maintained at U.B.C., and 45 animals were randomly assigned t o e i t h e r the t e s t cr c o n t r o l d i e t . These 45 animals were s u b d i v i d e d i n t o groups of 3 animals each, p r o v i d i n g 15 r e p l i c a t e s of 3 animals each f o r each d i e t t e s t e d . I f any animals i n a r e p l i c a t e died during the course of an experiment, the r e s u l t s of that r e p l i c a t e were d i s c a r d e d . Consumption i n d i c e s were c a l c u l a t e d (as o u t l i n e d above) f o r each r e p l i c a t e of 3 animals on a p a r t i c u l a r d i e t , and comparisons between i n d i c e s were made using the Mann-Whitney U t e s t . R e s u l t s Removal.of " I n e s s e n t i a l " Amino Acids J i g s . 5, 6 and 7 show the e f f e c t on growth of i n d i v i d u a l d e l e t i o n of those amino a c i d s c o n s i d e r e d to be i n e s s e n t i a l f o r the r a t ( t y r o s i n e , a l a n i n e , a s p a r t a t e , glutamate, c y s t i n e , s e r i n e , g l y c i n e , and p r o l i n e ) . The r e s u l t s show that c n l y the removal of g l y c i n e ( F i g . .7) caused a s i g n i f i c a n t l y reduced r a t e of growth r e l a t i v e to the c o n t r o l d i e t . On the g l y c i n e d e f i c i e n t d i e t , animals had only reached a mean weight of 320 mg a f t e r 35 days, compared to the mean weight of 450 mg achieved by animals on the c o n t r o l d i e t . In «7 F i g u r e 5. Growth of S c h i s t o c e r c a g r e q a r i a hoppers reared on complete a r t i f i c i a l d i e t s or on d i e t s l a c k i n g e i t h e r t y r o s i n e , a l a n i n e or a s p a r t a t e F o r t y - f i v e animals (3 r e p l i c a t e s of 15 animals each) were s t a r t e d on each d i e t . Each p o i n t r e p r e s e n t s the mean weight of s u r v i v i n g animals, bars r e p r e s e n t 95% confidence l i m i t s f o r the number cf animals i n d i c a t e d . S o l i d squares, complete d i e t c o n t a i n i n q the f u l l complement of 18 amino a c i d s ; open squares, d i e t l a c k i n q a s p a r t a t e ; c l o s e d c i r c l e s , d i e t l a c k i n g t y r o s i n e ; c l o s e d t r i a n g l e s , d i e t l a c k i n g a l a n i n e . Growth t r i a l s were conducted at 30°C and 55% R.H. . 4 b 49 F i g u r e 6. Growth of S c h i s t o c e r c a g r e q a r i a hoppers reared on complete a r t i f i c i a l d i e t s or d i e t s l a c k i n g e i t h e r glutamate, c y s t i n e or s e r i n e F o r t y - f i v e animals (3 r e p l i c a t e s of 15 animals each) were s t a r t e d cn each d i e t . Each p o i n t r e p r e s e n t s the mean weight of s u r v i v i n g animals, bars represent 95% confidence l i m i t s f o r the number of animals i n d i c a t e d . S o l i d squares, complete d i e t c o n t a i n i n g the f u l l complement of 18 amino a c i d s ; open squares, d i e t l a c k i n g s e r i n e ; s o l i d c i r c l e s , d i e t l a c k i n g glutamate; s o l i d t r i a n g l e s , d i e t l a c k i n g c y s t i n e . Growth t r i a l s were conducted at 30°C and 55% R.H. 5 1 F i g u r e 7. Growth of S c h i s t o c e r a g r e q a r i a hoppers reared on complete a r t i f i c i a l d i e t s or d i e t s l a c k i n g g l y c i n e or p r o l i n e F o r t y - f i v e animals (3 r e p l i c a t e s of 15 animals each) were s t a r t e d on each d i e t . Each p o i n t r e p r e s e n t s the mean weight of s u r v i v i n g animals, bars represent 95% c o n f i d e n c e l i m i t s f o r the number c f animals i n d i c a t e d . . S o l i d squares, complete d i e t c o n t a i n i n g the f u l l complement of 18 amino a c i d s ; s o l i d c i r c l e s , d i e t l a c k i n q q l y c i n e ; s o l i d t r i a n q l e s , d i e t l a c k i n q p r o l i n e . . Growth t r i a l s were conducted at 30°C and 55% R.H... 53 a d d i t i o n , development of animals on the g l y c i n e - d e f i c i e n t d i e t was r e t a r d e d , with 8 animals reach i n g the 4th i n s t a r and only 5 r e a c h i n g the 5th i n s t a r a f t e r 35 days, as compared with 2 animals reaching the 4th and 10 animals the 5th i n s t a r on the c o n t r o l d i e t . These r e s u l t s suggest t h a t g l y c i n e should be c o n s i d e r e d a s e m i - e s s e n t i a l amino a c i d f o r S. q r e q a r i a . The removal of any other " r a t - i n e s s e n t i a l s " had no apparent e f f e c t on the growth of S. .qreqaria (see F i g s . .5,6 & 7). . Removal of " E s s e n t i a l " Amino Acids In c o n t r a s t to the r e s u l t s presented above, the removal of any of the amino a c i d s c o n s i d e r e d e s s e n t i a l f o r the r a t ( h i s t i d i n e , l e u c i n e , methionine, tryptophan, t h r e o n i n e , v a l i n e , i s o l e u c i n e , a r g i n i n e , p henylalanine and l y s i n e ) had an extremely d e t r i m e n t a l e f f e c t on growth and development (Figs. 8, 9 and 10). The absence of l e u c i n e ( F i g . 8), i s o l e u c i n e , or v a l i n e ( F i g . 9) exerted the most profound e f f e c t s on growth and development. A l l animals reared on d i e t s d e f i c i e n t i n any of these 3 amino a c i d s had d i e d by the 14th day of the experiment, f a i l e d to develop past the 1st i n s t a r and showed no i n c r e a s e i n weight. Animals reared on d i e t s l a c k i n g threonine ( F i g . 9) or l y s i n e ( F i g . .10) were a l l dead by the end cf 21 days. Although animals showed l i t t l e growth on d i e t s l a c k i n g t h r e o n i n e cr l y s i n e , 2 animals on each d i e t d i d reach the 2nd i n s t a r . Cn d i e t s d e f i c i e n t i n h i s t i d i n e , methionine ( F i g . . 8), cr phenylalanine ( F i g . 10), a l l animals died by the end of 28 54 F i g u r e 8, Growth of S c h i s t o c e r c a g r e q a r i a hoppers reared on complete a r t i f i c i a l d i e t s or d i e t s l a c k i n g e i t h e r h i s t i d i n e , l e u c i n e or methionine F o r t y - f i v e animals (3 r e p l i c a t e s of 15 animals each) were s t a r t e d cn each d i e t . Each p o i n t r e p r e s e n t s the mean weight of s u r v i v i n g animals; 95% confidence l i m i t s are presented f o r weights cn days 21 and 35 i f more than 2 animals were s t i l l s u r v i v i n g at these times. Growth t r i a l s were conducted at 3 0 ° C and 55% E . H . . 56 F i g u r e S. . Growth of S c h i s t o c e r c a g r e q a r i a hoppers reared on complete a r t i f i c i a l d i e t s or d i e t s l a c k i n q e i t h e r tryptophan, t h r e o n i n e , v a l i n e or i s o l e u c i n e F o r t y - f i v e animals (3 r e p l i c a t e s of 15 animals each) were s t a r t e d cn each d i e t . Each point r e p r e s e n t s the mean weight of s u r v i v i n q animals; 955! confidence l i m i t s are presented f o r weights cn days 21 and 35 i f more than 2 animals were s t i l l s u r v i v i n g at these times. Growth t r i a l s were conducted at 30°C and 55% E. H. 58 Fi g u r e 10.. Growth of S c h i s t o c e r a g r e g a r i a hoppers on complete a r t i f i c i a l d i e t s or on d i e t s l a c k i n g e i t h e r a r g i n i n e , phenylalanine or l y s i n e F o r t y - f i v e animals (3 r e p l i c a t e s of 15 animals each) were s t a r t e d cn each d i e t . Each p o i n t r e p r e s e n t s the mean weight of s u r v i v i n g animals; 95% confidence l i m i t s are presented f o r weights cn days 21 and 35 i f more than 2 animals were s t i l l s u r v i v i n g at these times. Growth t r i a l s were conducted at 30°C and 55% J.H. . 3 3 60 days. Only 2 animals reached the 2nd i n s t a r on the methionine-or h i s t i d i n e - d e f i c i e n t d i e t s , w h i l s t 4 reached t h i s i n s t a r on the d i e t l a c k i n g p h e n y l a l a n i n e . Animals reared on the d i e t l a c k i n g a r g i n i n e d i d s u r p r i s i n g l y w e l l ( F i g . 1 0 ) , showing the best growth and s u r v i v a l of any animals reared on d i e t s l a c k i n g any ere of the 10 " e s s e n t i a l " amino a c i d s . Six animals (mean wt..65 mg), a l l i n the 2nd i n s t a r , s u r v i v e d on the a r g i n i n e -d e f i c i e n t d i e t u n t i l the experiment was terminated a f t e r 35 days. The r e s u l t s from these, experiments show that the 10 amino a c i d s found to be e s s e n t i a l f o r the r a t , are a l s o e s s e n t i a l f o r S. g r e g a r i a . F e e d i n g _ T r i a l s Table 6 shows the r e s u l t s of experiments conducted to compare S. g r e g a r i a 1 s consumption of c o n t r o l d i e t s , c o n t a i n i n g a l l 18 amino a c i d s , with t e s t d i e t s , l a c k i n g one of the 18 amino a c i d s . The r e s u l t s show that the absence of any of the amino a c i d s found to be e s s e n t i a l f c r the growth of S . . g r e g a r i a ( i . e . h i s t i d i n e , l e u c i n e , methionine, tryptophan, t h r e o n i n e , v a l i n e , i s c l e u c i n e , a r g i n i n e , phenylalanine or l y s i n e ) caused a c o n s i d e r a b l e r e d u c t i o n i n the amount of d i e t consumed. These amino a c i d s can thus be c l a s s e d as phagostimulants f o r S. g r e g a r i a . In a d d i t i o n , the r e s u l t s demonstrate t h a t the absence of amino a c i d s found to be i n e s s e n t i a l f o r S . . g r e q a r i a ( i . e . t y r o s i n e , a l a n i n e , a s p a r t a t e , qlutamate, c y s t i n e , s e r i n e and p r o l i n e ) or only s e m i - e s s e n t i a l ( i . e . g l y c i n e ) , d i d not 61 Table 6. Amount of d i e t consumed by S c h i s t o c e r c a g r e g a r i a hoppers feed i n g on c o n t r o l d i e t s c o n t a i n i n g the f u l l complement of amino a c i d s , or on t e s t d i e t s l a c k i n g a s i n g l e amino a c i d . Consumption index d e f i n e d as: the amount of d i e t consumed by 3-2nd i n s t e r animals/mean weight of animals. A more d e t a i l e d d e s c r i p t i o n of the consumption index used i s presented in m a t e r i a l s and methods. Results presented here are the mean consumption i n d i c e s of the i n d i c a t e d number of r e p l i c a t e s . Comparison of t e s t d i e t s with c o n t r o l d i e t s : * = p < 0.001 (Mann Whitney U t e s t ) . Amino Acid Mann missing from Whitney test diet Control Diet Test Diet U test Number of mean groups of consumption 3-animals index + S.D. number of mean groups of consumption 3-animals index + S.D. alanine 11 1.59 + 0.41 9 1.58 + 0.38 arginine 10 1.81 + 0.50 11 0.40 + 0.15 * aspartate 11 1.58 + 0.41 12 1.60 + 0.35 cystine 12 1.62 + 0.40 10 1.56 + 0.30 glutamate 12 1.70 + 0.33 9 1.70 + 0.42 glycine 11 1.63 + 0.32 10 1.72 + 0.30 histidine 13 1.57 + 0.36 11 0.46 + 0.18 * isoleucine 11 1.75 + 0.39 8 0.23 + 0.16 * leucine 10 1.64 + 0.41 7 0.28 + 0.21 * lysine 12 1.54 + 0.39 12 0.56 + 0.22 * methionine 11 1.63 + 0.40 10 0.30 + 0.19 * phenylalanine 12 1.61 +• 0.31 11 0.31 + 0.18 * proline 11 1.59 + 0.27 12 1.52 + 0.23 serine 12 1.60 + 0.29 11 1.44 + 0.22 threonine 12 1.67 + 0.44 7 0.54 + 0.13 * tryptophan 9 1.74 + 0.52 9 0.31 + 0.23 * tyrosine 12 1.56 + 0.39 12 1.65 + 0.40 valine 9 1.42 + 0.38 10 0.56 + 0.22 * 62 a f f e c t d i e t consumption.. D i s c u s s i o n The present study demonstrated t h a t S. g r e g a r i a has s i m i l a r amino a c i d requirements to that of other i n s e c t s . . Thus the 10 amine a c i d s ( i . e. l y s i n e , phenylalanine, i s o l e u c i n e , v a l i n e , t h r e o n i n e , tryptophan, l e u c i n e , h i s t i d i n e , methionine and a r q i n i n e ) found to be e s s e n t i a l f o r most i n s e c t s (Dadd, 1973; House, 1974) were a l s o r e q u i r e d f o r qood growth of S. g r e q a r i a . In a d d i t i o n , 7 of the amino acids ( i . e. t y r o s i n e , a l a n i n e , a s p a r t a t e , qlutamate, c y s t i n e , s e r i n e and p r o l i n e ) c o n s i d e r e d i n e s s e n t i a l f o r other i n s e c t s (reviews, op c i t ) were a l s o i n e s s e n t i a l f o r S. g r e q a r i a . However, g l y c i n e , a u s u a l l y i n e s s e n t i a l amino a c i d , appeared to be necessary to improve the growth of S. . g r e g a r i a , and S. g r e q a r i a 1 s requirements f o r a r q i n i n e d i d not appear to be as s t r i n g e n t as i t s requirements f o r the other 9 e s s e n t i a l amino a c i d s . Feedinq t r i a l s i n d i c a t e d t h a t removal of any of the 7 i n e s s e n t i a l amino a c i d s had no e f f e c t on d i e t consumption. These r e s u l t s , together with the r e s u l t s of the growth t r i a l s , show that these 7 amino ac i d s are not phagostimulants for ,S. qreqaria, and t h a t they can be s y n t h e s i z e d by t h i s animal. The f a i l u r e of animals to qrow well on d i e t s l a c k i n q each of the 10 e s s e n t i a l amino a c i d s was found to be due to the reduced 63 feeding a c t i v i t y of animals reared on these d i e t s . . T h u s , these amino a c i d s are a c t i n g as phagostimulants, and i t i s not p o s s i b l e t c say whether or not they can be s y n t h e s i z e d by S- q r e q a r i a . Only the r e l a t i v e l y poor qrowth of animals on g l y c i n e - d e f i c i e n t d i e t s , and the s u r p r i s i n g l y good growth of animals on a r g i n i n e - d e f i c i e n t d i e t s c o u l d not be f u l l y e x p l a i n e d i n terms of the amount of food consumed by animals.. Thus feeding t r i a l s i n d i c a t e d t h a t the d i e t s l a c k i n g g l y c i n e were consumed i n the same g u a n t i t y as c o n t r o l d i e t s c o n t a i n i n g the f u l l conplement of amino a c i d s , and d i e t s l a c k i n g a r g i n i n e d i d not appear to be consumed i n l a r g e r amounts than d i e t s l a c k i n g any of the other e s s e n t i a l amino a c i d s . . T h i s means that the growth of S. q r e q a r i a on g l y c i n e - o r a r g i n i n e - d e f i c i e n t d i e t s must be e x p l a i n e d i n terms of the animal's o v e r a l l metabolism. . Ihe f o l l o w i n g i s a more d e t a i l e d d i s c u s s i o n of S. q r e q a r i a ' s reguirements f o r g l y c i n e and a r g i n i n e , and an assessment of the r o l e played by phagostimulant amino a c i d s i n the n u t r i t i o n of i n s e c t s . Jeguirement f o r G l y c i n e As well as being r e g u i r e d f o r good growth of S . , q r e q a r i a , q l y c i n e has a l s o been found t o be r e q u i r e d f o r optimal development of the mosquitoes Culex p i p i e n s (Dadd, 1978) and Aedes a e g y p t i (Lea and DeLonq, 1958; Sinqh and Brown, 1957), other d i p t e r a n s ( F r i e n d , 1968; Hinton e t a l , 1951), screwworm 64 l a r v a e ( G i n g r i c h , 1964) , the g r a i n b e e t l e Oryzaephilus  surinamensis (Davis, 1956, 1969a, 1972b), and the f l o u r b e e t l e Te.ne.trio roolitor (Davis, 1978). In these and the present study, an a b s o l u t e reguirement f o r g l y c i n e could not be demonstrated, sugges t i n g t h a t i n c e r t a i n i n s e c t s , i n c l u d i n g S. g r e g a r i a , i n s u f f i c i e n t g l y c i n e can be s y n t h e s i z e d to meet the animal's metabolic needs. Although g l y c i n e can be s y n t h e s i z e d using b i o c h e m i c a l pathways i n v o l v i n g t h r e o n i n e or c e r t a i n metabolic i n t e r m e d i a t e s of carbohydrate metabolism (Karlson, 1970), i n mammals at l e a s t , the major pathway of g l y c i n e s y n t h e s i s i s v i a the d e c a r b o x y l a t i o n of s e r i n e (Lehninger, 1970) (see Egn..1).. Egn.1 S e r i n e + T e t r a h y d r o f o l a t e G l y c i n e * M e t h y l e n e - T e t r a h y d r o f o l a t e Evidence f c r the e x i s t e n c e of s e r i n e - g l y c i n e i n t e r c o n v e r t i b i l i t y i n i n s e c t s comes from r a d i o - i s o t o p e s t u d i e s (Chefurka, 1965) and n u t r i t i o n a l s t u d i e s which have shown that s e r i n e can p a r t i a l l y r e p l a c e g l y c i n e i n some i n s e c t d i e t s (Davis, 1969a; Seede, 1961). In mammals, the s e r i n e f o r the r e a c t i o n shown i n Egn..1 i s e i t h e r derived from the d i e t , cr i s u l t i m a t e l y s y n t h e s i z e d from 3-phosphoglyceric a c i d ( l e h n i n g e r , 1970). T e t r a h y d r c f o l a t e i s d e r i v e d from the vitamin f o l i c a c i d s u p p l i e d i n the d i e t . . 65 I f s e r i n e i s the major pre c u r s o r of g l y c i n e S- i t e q ^ a r i a , then there are three p o s s i b l e reasons f o r the f a i l u r e of the g l y c i n e - f r e e d i e t to support good growth: (1) i n s u f f i c i e n t s e r i n e i n the d i e t to meet g l y c i n e requirements; (2) i n s u f f i c i e n t t e t r a h y d r o f o l a t e ( f o l i c acid) present to c a t a l y s e c o n v e r s i o n of s e r i n e to g l y c i n e , and (3) c o n v e r s i o n of s e r i r e t c g l y c i n e proceeds at too slow a r a t e to meet the animal's g l y c i n e requirements. Since the d e l e t i o n of s e r i n e from a r t i f i c i a l d i e t s had no d i s c e r n a b l e e f f e c t on qrowth of S. g r e q a r i a , s e r i n e must ne r e a d i l y s y n t h e s i z e d i n t h i s animal, and i t thus i s u n l i k e l y that low l e v e l s of s e r i n e are l i m i t i n g tne rate of g l y c i n e formation. F o l i c a c i d i s present i n the d i e t a t some 10 times the minimal l e v e l found necessary to support optimal growth of J . g r e g a r i a (Dadd, 1961b). In a d d i t i o n , para-aminox>enzoic a c i d , which i s not a n u t r i t i o n a l requirement of S. g r e q a r i a (Dadd, 196 1b), but i s known to be converted to f o l i c a c i d r»y many organisms (Lehninger, 1970), i s a l s o present i n the d i e t . Ihe presence of such high l e v e l s of f o l i c a c i d , and i t s p r e c u r s o r para-aminobenzoic a c i d , make i t u n l i k e l y that low l e v e l s of t h i s vitamin are l i m i t i n g g l y c i n e s y n t h e s i s . Since i t i s u n l i k e l y t h a t a l a c k of s e r i n e or t e t r a h y d r o f o l a t e i s r e s p o n s i b l e f o r the s e m i - e s s e n t i a l nature of g l y c i n e , i t seems probable t h a t i t i s the k i n e t i c s of the r e a c t i o n c o n v e r t i n g s e r i n e to q l y c i n e t h a t i s l i m i t i n g g l y c i n e s y n t h e s i s . Although there i s no d i r e c t evidence f o r t h i s , g l y c i n e i s known t c be a requirement f o r the growing chick (Meister, 1965), suqqesting t h a t i t i s l e s s r e a d i l y s y n t h e s i z e d than ether u s u a l l y i n e s s e n t i a l amino a c i d s . 66 Kequirgment f o r , A r g i n i n e In the present study i t was found t h a t animals r e a r e d on d i e t s l a c k i n g a r g i n i n e showed b e t t e r growth and s u r v i v e d longer than did animals reared on any other d i e t l a c k i n g an e s s e n t i a l amino a c i d . There could be s e v e r a l e x p l a n a t i o n s f o r the f a c t that requirements f o r a r g i n i n e appeared to be l e s s c r i t i c a l than requirements f o r other e s s e n t i a l amino a c i d s : (1) animals obtained a r q i n i n e throuqh c a n n i b a l i s m ; (2) s u f f i c i e n t maternally d e r i v e d a r g i n i n e was present i n h a t c h l i n g s to allow f o r seme f u r t h e r growth and development; (3) the animal's a r g i n i n e reguirements were p a r t i a l l y met by symbiotes; and (4) S» _ q r e q a r i a i s capable of l i m i t e d a r g i n i n e s y n t h e s i s . Each of these p o s s i b i l i t i e s w i l l be c o n s i d e r e d i n t u r n . Although attempts were made to remove dead animals from r e a r i n g - c o n t a i n e r s as o f t e n as p o s s i b l e , the o c c a s i o n a l f i n d i n g of p a r t i a l l y eaten animals provided evidence that c a n n i b a l i s m s t i l l o c c u r r e d . However, two o b s e r v a t i o n s m i t i g a t e a g a i n s t c a n n i t a l i s m being the reason f o r the weight gain e v i d e n t i n animals reared cn the a r g i n i n e d e f i c i e n t d i e t . F i r s t , l i t t l e a r g i n i n e would be expected to be a v a i l a b l e i n the dead animals, unless they contained maternally derived a r g i n i n e , or were s y n t h e s i z i n g , or o b t a i n i n g a r g i n i n e from symbiotes. _Secondly, animals reared on d i e t s l a c k i n g the other e s s e n t i a l amino acids di d net appear to b e n e f i t from such c a n n i b a l i s m as o c c u r r e d . . S e v e r a l s t u d i e s have i n d i c a t e d that n u t r i e n t s r e g u i r e d i n s m a l l amounts, such as c e r t a i n vitamins and minerals, can be s u p p l i e d i n s u f f i c i e n t g u a n t i t y i n i n s e c t eggs to allow f o r 67 p o s t - h a t c h i n g growth on d i e t s l a c k i n g these n u t r i e n t s . . T h u s s e v e r a l g e n e r a t i o n s of the l e a f hopper Macrosteles f a s c i f r o n s had to be reared on d i e t s l a c k i n g copper and z i n c before reguirements f o r these minerals c o u l d be demonstrated (Hou and Brooks, 1978). Other s t u d i e s have shown or suggested the importance of m a t e r n a l l y d e r i v e d v i t a m i n s , such as c a r o t e n e i n the l o c u s t L. m i q r a t o r i a (Dadd, 1961a), i n o s i t o l i n the roach B l a t e l l a qermanica (Gordon, 1959), a s c o r b i c a c i d i n the aphid Myzus p e r s i c a e ( M i t t l e r , 1976), and the B-vitamins i n the r a t -f l e a Xenopsylla cheppis (Pausch and F r a e n k e l , 19 66) and Myzus  p e r s i c a e (Dadd and M i t t l e r , 1 965; Dadd et a l , 1967).. No s t u d i e s have been performed on the e f f e c t of m a t e r n a l l y d e r i v e d amino a c i d s on the growth of i n s e c t s reared on d i e t s l a c k i n g those amino acids..However, i n r e f e r e n c e to the present study, i t i s known t h a t f r e e a r g i n i n e i s only present i n small amounts ( r e l a t i v e to other amino acids) i n h a t c h l i n g s of S. g r e q a r i a (Colombo et a l . , 1962), making i t u n l i k e l y that s u f f i c i e n t a r g i n i n e was present to allow f o r the weight i n c r e a s e observed in h a t c h l i n g s r e a r e d on a r g i n i n e - d e f i c i e n t d i e t s . Symbiotes have been shown to supply normally e s s e n t i a l amine a c i d s to t h e i r i n s e c t h o s t s . Thus, f o r the aphid J - p e r s i c a e , the d i s p a r i t y between the number of amino a c i d s determined as being e s s e n t i a l using r a d i o - i s o t o p e techniques (Strong and Sakamoto, 1963) and the l e s s e r number determined as being e s s e n t i a l using d i e t a r y - d e l e t i o n techniques (Dadd and K r i e g e r , 1968), was suggested as being due to the presence of i n t r a - c e l l u l a r symbiotes (Dadd and K r i e g e r , 1968)..The r o l e of aphid-symtiotes i n s u p p l y i n g e s s e n t i a l amino-acids was l a t e r 68 confirmed by M i t t l e r (1971) who showed that aposymbiotic aphids had more s t r i n g e n t amino a c i d requirements than aphids with the ncrmal complement cf symbiotes.. Other s t u d i e s usinq s e p s i s / a s e p s i s techniques have demonstrated t h a t symbiotes can supply normally e s s e n t i a l amino a c i d s t o the b e e t l e Steqcbium paniceum (Pant e t a l . , 19 60), and the roach B l a t e l l a germanica (Henry and Block, 1960,1962).. In a d d i t i o n , symbiotes are thouqht t c supply amino a c i d s f o r the f l i e s Dacus o l e a (Haqen, 1966) and fihaqoletus pomanella (Miguzaki et a l . , 1968), and the tug Cysdercus s i m i l i s (Singh, 1 976). , Although the gut of S. g r e g a r i a i s r i c h l y endowed with micro-orqanisms (Morgan, 1976), the l a c k of i n f o r m a t i o n on the e f f e c t c f a s e p s i s on the n u t r i t i o n a l requirements of these, and other l o c u s t s and qrasshoppers, makes i t d i f f i c u l t to assess the p o s s i b l e r o l e of symbiotes i n s u p p l y i n q nutrients..However, with reqard t o the present study, i t seems u n l i k e l y that a r q i n i n e was s u p p l i e d by i n t e s t i n a l micro-orqanisms s i n c e : (a) Dadd (1961b), usinq d i e t a r y d e l e t i o n techniques, was a b l e to demonstrate t h a t s e p t i c S. g r e g a r i a had the usual B-vitamin requirements, suggesting that micro-organisms could not supply s u f f i c i e n t of these vitamins to meet the animal's needs, and (b) i n cases where symbiotes have been shown t o provide e s s e n t i a l amino a c i d s (Henry and Block, 1960,1962; M i t t l e r , 1971; Pant et a l . , 1960) they have always s u p p l i e d s e v e r a l amine a c i d s , and not j u s t one as suggested by the present study. Since there i s l i t t l e evidence to support the t h e s i s that exogenous s u p p l i e s of a r g i n i n e were being obtained by animals reared on a r g i n i n e - f r e e d i e t s , i t i s p o s s i b l e t h a t t h i s amino 69 a c i d can be s y n t h e s i z e d by S. q r e q a r i a . L i m i t e d s y n t h e s i s of a r g i n i n e has been shown to occur i n other animals.. Thus, although a r g i n i n e i s r e g u i r e d f o r optimal growth of the young r a t (Eose, 1938; Eorman et a l . , 1946) , i t s absence from d i e t s reduces growth to a l e s s e r degree than the absence of the ether e s s e n t i a l amino a c i d s (Milner and Visek, 1975; P r i o r e t a l . , 1975) , and s u f f i c i e n t may be s y n t h e s i z e d to meet the reguirements of the a d u l t r a t ( S c u l l and Eose, 1 9 3 0 ) a r g i n i n e b i o s y n t h e s i s has a l s o been shown to occur i n r a b b i t s (Adamson and l i s h e r , 1976) and pigs (Easter and Baker, 1976).. In r a t s the semi-dispensable nature of a r g i n i n e i s due to i t s production as an i n t e r m e d i a t e i n the o r n i t h i n e (Krebs-H e n s e l e i t ) c y c l e (see f i g . .11). The end product of t h i s c y c l e i s a r g i n i n e and urea i n u r e o t e l i c animals, and a r g i n i n e i n n c n - u r e c t e l e s (Jones, 1971). The e x i s t e n c e of a complete o r n i t h i n e c y c l e i n i n s e c t s has yet to be proved (Cochran, H975; Pant and Kumar, 1978), and Dadd (1973) has questioned the r a t i o n a l e behind e x p e c t i n g to f i n d t h i s c y c l e i n the predominately u r i c o t e l i c t e r r e s t r i a l i n s e c t s . . S u c h evidence as does e x i s t f o r the presence of an o r n i t h i n e c y c l e i n i n s e c t s r e l i e s on the two o b s e r v a t i o n s t h a t urea i s excreted i n small amounts by some i n s e c t s ( B u r s e l l , 1967; Chefurka, 1965; Gilmcur, 1961, 1965; S c h o f f e n i e i l s and G i l l e s , 1970) and that the three amine a c i d s found i n the c y c l e ( a r g i n i n e , o r n i t h i n e and c i t r u l l i n e ) are found i n some i n s e c t t i s s u e s ( K i l b y and N e v i l l e , 1957; Beddy and Campbell, 1969). A d d i t i o n a l evidence f o r the presence of at l e a s t a p o r t i o n of the c y c l e comes from n u t r i t i o n a l s t u d i e s i n which i t has teen found that d i e t a r y c i t r u l l i n e can "spare" the a r g i n i n e requirements of some 7 0 Figure 1 1 . Diagram of the Ornithine Cycle Adapted from Karlson ( 1 9 7 0 ) . Enzymes refered to i n text are presented in brackets. FUMARATE ASPARTATE ARGININE CITRULLINE ORNITHINE (arginase) UREA CARBON DIOXIDE CARBAMYL PHOSPHATE (carbamyl phosphate synthetase) AMMONIA 72 ; i n s e c t s (Davis, 1962; Hinton e t a l . , 1951; InoXuchi et a l . , 1969 ; Bock, 1969; Vanderzant and Chremos, 1 97 1)._ However, these same s t u d i e s have a l s o shown th a t w h i l s t c i t r u l l i n e can spare a r g i n i n e reguirements, o r n i t h i n e cannot, and t h i s has been construed to mean th a t a complete o r n i t h i n e c y c l e does not e x i s t i n the i n s e c t s s t u d i e d . The reasoning behind t h i s c o n c l u s i o n i s not c l e a r , s i n c e n u t r i t i o n a l s t u d i e s with r a t s (aniicals kncwn to possess a complete o r n i t h i n e cycle) have a l s o shown o r n i t h i n e cannot spare a r g i n i n e reguirements._ Thus the i n a b i l i t y cf o r n i t h i n e to r e p l a c e a r g i n i n e cannot be used as a c r i t e r i o n t c determine whether an animal possesses a complete o r n i t h i n e c y c l e . Few s t u d i e s have been performed to determine whether the enzymes of the o r n i t h i n e c y c l e are present i n i n s e c t t i s s u e s , although Pant and Kumar (1978) found a l l the c o n s t i t u e n t enzymes (except carbamyl phosphate synthetase) i n the f l y Sarcophaqa r u f i c o r n i s , and K i l b y and N e v i l l e (1957) found evidence of arginase a c t i v i t y i n the t i s s u e s of S. g r e q a r i a . In the absence of more d e f i n i t i v e s t u d i e s , the e x i s t e n c e of the o r n i t h i n e c y c l e i n i n s e c t s cannot be r u l e d out^ The present f i n d i n g s that S. q r e q a r i a 1 s reguirements f o r a r g i n i n e was net as c r i t i c a l as i t s requirements f o r the other u s u a l l y e s s e n t i a l amine a c i d s could be due to a l i m i t e d s y n t h e s i s of a r g i n i n e v i a the o r n i t h i n e c y c l e . 73 amino acids as Phagostimulants The present study showed t h a t the poor growth of S. g r e q a r i a cn d i e t s l a c k i n g any one of the 10 " e s s e n t i a l " amine a c i d s : h i s t i d i n e , l e u c i n e , methionine, tryptophan, t h r e o n i n e , v a l i n e , i s o l e u c i n e , a r g i n i n e , p h e n y l a l a n i n e or l y s i n e ; c c u l d be e x p l a i n e d by reduced d i e t consumption. The removal of amino a c i d s u s u a l l y considered to be i n e s s e n t i a l { t y r o s i n e , a l a n i n e , a s p a r t a t e , glutamate, c y s t i n e , s e r i n e , g l y c i n e and p r o l i n e ) had no e f f e c t on d i e t uptake._These r e s u l t s argue f o r S. g r e g a r i a b e i n q able to determine the presence or absence of the 10 e s s e n t i a l amino a c i d s , e i t h e r on the b a s i s of i n i t i a l c o n t a c t with the d i e t , or by a s s e s s i n g the p o s t - i n g e s t i v e e f f e c t s of consuming such d i e t s . S e v e r a l s t u d i e s have shown that the m a x i l l a r y and l a b i a l palps of S. g r e q a r i a are r i c h l y endowed with c o n t a c t chemcreceptcrs (Blaney and Chapmen, 1970; H a s k e l l and Mordue, 1969; H a s k e l l and Schccnhoven, 1969; Mordue, 1979; Thomas, 1966; Winstanley and Blaney, 1S78). although none of these r e c e p t o r s have been t e s t e d f o r response to amino a c i d s , e l e c t r o p h y s i o l o g i c a l s t u d i e s have demonstrated the presence of amine a c i d - s e n s i t i v e chemoreceptors on the mouthparts of l e p i d e p t e r c u s l a r v a e (Dethier, 197 1, 1978; Dethier and Kuch, 1971; Schoonhoven, 1969). Although there i s no d i r e c t evidence f o r amino a c i d - s e n s i t i v e chemoreceptors i n l o c u s t s or grasshoppers, s e v e r a l b e h a v i o r a l s t u d i e s have shown t h a t these animals w i l l consume more of an i n e r t m a t e r i a l ( f i l t e r paper or wcod pith) when i t i s impregnated with amino a c i d ( s ) (Bernays 74 and Chapman, 1978; Cook, 1977; T h o r s t e i n s o n , 1960). S i m i l a r s t u d i e s have shown t h a t c e r t a i n amino a c i d s w i l l i n c r e a s e the amount of an i n e r t m a t e r i a l consumed by the Colorado potato b e e t l e , L e p t i n o t a r s a decemlineata (Hsiao and F r a e n k e l , 1968); the b e e t l e , S e r c e s t h i s geminata (Wensler and Du d z i n s k i , 1972) ; the aphids. Myzus p e r s i c a e ( M i t t l e r , 1967a, b, c; M i t t l e r and Dadd, 1964) and Acyrothosiphon pisum ( S r i v a s t a v a and A u c l a i r , 1975). Thus both e l e c t r o p h y s i o l o g i c a l and r e h a v i o r a l s t u d i e s have demonstrated that i n s e c t s are capable of d e t e c t i n g the presence or absence of amino a c i d s and a d j u s t i n g t h e i r f e e d i n g behavior a c c o r d i n g l y . For S. g r e g a r i a to reduce f e e d i n g i n response to the post-i n g e s t i v e e f f e c t s of d i e t s l a c k i n g an e s s e n t i a l amino a c i d , three c r i t e r i a must be met. There must be some p e r t u r b a t i o n of the animal's metabolism when such d i e t s are i n g e s t e d , and the animal must be able to detect t h i s p e r t u r b a t i o n , and to a s s o c i a t e the " f l a v o u r " of the d i e t with the metabolic d i s t u r b a n c e . In the f o l l o w i n g d i s c u s s i o n of these three c r i t e r i a i t w i l l be assumed t h a t animals cannot s y n t h e s i z e the 10 amino a c i d s found e s s e n t i a l f o r growth. Although d i r e c t evidence f o r t h i s i s l a c k i n g s i n c e animals did not consume s u f f i c i e n t d i e t to determine whether the amino a c i d s c o u l d be syn t h e s i z e d or not, the f a c t t h a t the 10 amino a c i d s found to be e s s e n t i a l f o r S. g r e g a r i a are the same as those which cannot be sy n t h e s i z e d by most other animals suggests t h a t the assumption i s a reasonable one. The i n g e s t i o n of d i e t s l a c k i n g an amino a c i d would be expected to e x e r t a profound e f f e c t on an animal's metabolism as no p r o t e i n s c o n t a i n i n g t h a t amino a c i d c o u l d be s y n t h e s i z e d , 75 and as a c o r o l l a r y of t h i s , the excess amino acids i n g e s t e d i n the d i e t ( i . e . those no longer r e g u i r e d f o r p r o t e i n s y n t h e s i s ) would have to be excreted. That these d e t r i m e n t a l e f f e c t s occur i n i n s e c t s was demonstrated by Inokuchi (1970) who showed that when one e s s e n t i a l amino a c i d was missing from the d i e t of the silkwcrm Bombyx mpri, the haemolymph l e v e l s of most other amino a c i d s were i n c r e a s e d . In a d d i t i o n Horie and Inokuchi (1978) shewed, f c r the same s p e c i e s , that removal of an e s s e n t i a l amino a c i d from the d i e t r e s u l t e d i n a decrease i n p r o t e i n s y n t h e s i s and an i n c r e a s e i n u r i c a c i d e x c r e t i o n . Consumption of d i e t s l a c k i n g an e s s e n t i a l amino a c i d would te expected to cause (a) a decrease i n the l e v e l of t h a t amino a c i d i n the blood; (b) an i n c r e a s e i n the l e v e l of other amino a c i d s , and (c) an i n c r e a s e i n the l e v e l of nitrogenous e x c r e t o r y products or t h e i r i n t e r m e d i a t e s . . although no experiments have been performed to determine whether any of the above e f f e c t s could be r e s p o n s i b l e f o r reduced d i e t i n t a k e i n i n s e c t s feeding on d i e t s l a c k i n g an e s s e n t i a l amino a c i d , s t u d i e s with the r a t have shown t h a t depressed food i n t a k e on d i e t s c o n t a i n i n g an imbalanced r a t i o of amino a c i d s can be caused by i n c r e a s e d l e v e l s of ammonia (an in t e r m e d i a t e i n the formation cf urea) i n the blood (Noda, 1975), or by decreased l e v e l s of a l i m i t i n g amino a c i d (Leung and Rogers, 1969).. I f S. g r e g a r i a i s capable of r e l a t i n g the tast e of a d i e t l a c k i r g an i n d i v i d u a l amino a c i d to the d e t r i m e n t a l e f f e c t s of i n g e s t i n g t h a t d i e t , i t must be able to assess the f l a v o u r of that d i e t and "remember" that d i e t s e x h i b i t i n g that f l a v o u r are t c be avoided. I n s e c t s are known to be capable of d e t e c t i n g a wide range of n u t r i e n t s other than amino a c i d s and both 76 b e h a v i o r a l and e l e c t r o p h y s i o l o g i c a l s t u d i e s have shown that s a l t s , f a t t y a c i d s and carbohydrates can a l s o i n f l u e n c e f e e d i n g behavior (Dadd, 1960a; Davis, 1968; D e t h i e r , 1978; Hsiao, 1972; Hsiao and F r a e n k e l , 1968; Schoonhoven, 1969)..In a d d i t i o n , i t i s known that combinations of n u t r i e n t s can have s y n e r g i s t i c or a d d i t i v e e f f e c t s on the feeding behavior of i n s e c t s (Bernays and Chapman, 1978; Hsiao, 1972; Hsiao and F r a e n k e l , 1968), and t h a t i n s e c t s are capable of d i s t i n g u i s h i n g between a r t i f i c i a l d i e t s c o n t a i n i n g n u t r i e n t s i n d i f f e r i n g r a t i o s (House, 1970, 1971a,b; 1972). These f i n d i n g s suggest that i n s e c t s are capable of a s s e s s i n g the n u t r i t i o n a l f l a v o u r of d i e t s . Jermy e t a l . , (1966) showed t h a t l a r v a e o f Maduca sexta and rieliothis zea develop c l e a r preferences f o r p l a n t s on which they have been re a r e d , and Gordon (1968,1972) has shown t h a t p r i o r experience with an a r t i f i c i a l d i e t can a f f e c t an i n s e c t ' s response to n o v e l d i e t s . Thus i n s e c t s are capable of responding to d i e t s on the b a s i s of past experience and appear to possess both p r e r e q u i s i t e s ( i . e . a b i l i t y t o r e c o g n i z e d i e t f l a v o u r , and memory f o r that f l a v o u r ) necessary to demonstrate a l e a r n e d a v e r s i o n response. . No s t u d i e s have been performed to determine whether i n s e c t s depress or e l e v a t e food i n t a k e on the b a s i s of i n i t i a l c o n t a c t with the d i e t , or on the b a s i s of p o s t - i n g e s t i v e metabolic e f f e c t s . That such s t u d i e s have not been conducted i s probably a f u n c t i o n of the emphasis that b e h a v i o r a l and e l e c t r o p h y s i o l o g i c a l s t u d i e s have placed on the importance of i n i t i a l c o n t a c t with d i e t s . Thus b e h a v i o r a l s t u d i e s on the phagcstimulant p r o p e r t i e s of n u t r i e n t s , almost always i n v o l v e determining whether the a d d i t i o n of a n u t r i e n t to an i n e r t 77 m a t e r i a l ( i . e . one of l i t t l e or no n u t r i t i o n a l value to the i n s e c t ) i n c r e a s e s the amount of m a t e r i a l consumed. Experiments such as these cannot provide an i n s i g h t i n t o the e f f e c t of i n g e s t i n g d i e t s with imbalanced r a t i o s of n u t r i e n t s , and serve only to i n d i c a t e whether i n s e c t s have chemoreceptors capable of d e t e c t i n g the presence or absence of p a r t i c u l a r n u t r i e n t s . . S i m i l a r l y , e l e c t r o p h y s i o l o g i c a l s t u d i e s demonstrate the range of chemoreceptors present i n i n s e c t s but do not i n d i c a t e whether such r e c e p t o r s are used to provide an i n i t i a l assessment of the d i e t , or whether they supply " f l a v o u r " i n f o r m a t i o n necessary f o r a l e a r n e d a v e r s i o n response.. I t i s i n t e r e s t i n g t c note t h a t , i n the r a t , where t h e r e i s a great deal of i n f o r m a t i o n r e g a r d i n g the e f f e c t s of i n g e s t i n g imbalanced d i e t s , i t i s b e l i e v e d that "... the major mechanism whereby an animal l e a r n s to s e l e c t proper d i e t s , at l e a s t with regard to amino a c i d balance, i s v i a " l e a r n e d a v e r s i o n s " (to p o s t - i n g e s t i v e e f f e c t s cn metabolism)" (Rogers and Leung, 1976) ; whereas i n i n s e c t s , where such i n f o r m a t i o n i s l a c k i n g , i t i s b e l i e v e d that "... i n s o f a r as the mechanism of i n d i v i d u a l choice (of f o o d - p l a n t s by i n s e c t s ) i s concerned, the post-i n g e s t i v e e f f e c t s of the s t i m u l a n t s are l e s s important than the i n i t i a l and immediate e f f e c t s . " (Dethier, 1970).. In the absence of f u r t h e r experimental work, i t i s d i f f i c u l t to determine which of the two mechanisms ( i . e . i n i t i a l c o n t a c t with the d i e t , or a l e a r n e d a v e r s i o n response) plays the most important r o l e i n a s s e s s i n g d i e t s u i t a b i l i t y . However, the present study demonstrated t h a t the absence of a n u t r i e n t that would be expected t o e x e r t a profound e f f e c t on S. q r e q a r i a 1 s metabolism ( i . e . a u s u a l l y 78 e s s e n t i a l amino a c i d ) r e s u l t e d i n a decrease i n d i e t consumption. That animals were not a s s e s s i n g the d i e t on the fcasis of the absence of a p a r t i c u l a r amino a c i d i s suggested by the f a c t t h at e l e c t r o p h y s i o l o g i c a l s t u d i e s have shown that i n s e c t chemoreceptors respond i n a g u a n t i t a t i v e l y s i m i l a r way to a wide range of amino ac i d s (Dethier, 1971; Schoonhoven, 1969) , and do net j u s t respond to p a r t i c u l a r or e s s e n t i a l amino a c i d s . I t i s thus u n l i k e l y t h a t i n s e c t s would be capable of d i s t i n g u i s h i n g (on the b a s i s of sensory information) the absence of a p a r t i c u l a r amino a c i d i n s i t u a t i o n s where many other amine a c i d s are present, and t h i s suggests t h a t i n s e c t s may te capable of demonstrating a learned a v e r s i o n response to the p o s t - i n g e s t i v e e f f e c t s of d i e t s l a c k i n g i n d i v i d u a l amino a c i d s . . Brues (1920) suggested that t a s t e played a major r o l e i n h c s t - p l a n t s e l e c t i o n by phytophagous i n s e c t s , and P a i n t e r (1953) proposed the theory that the absence of a p a r t i c u l a r n u t r i e n t from a p l a n t might make t h a t pl a n t r e s i s t a n t to attack by i n s e c t s r e g u i r i n g t h a t n u t r i e n t . F r a e n k e l (1953) re c o g n i z e d that . i n s e c t s appear to have g u a n t i t a t i v e l y s i m i l a r n u t r i t i o n a l reguirements and that a l l p l a n t s appeared to c o n t a i n these n u t r i e n t s , and he l a t e r suggested that the p a l a t a b i l i t y of p l a n t s was based cn the presence or absence of secondary plant compounds ( i . e . chemicals present i n the p l a n t having no known f u n c t i o n i n p l a n t metabolism; F r a e n k e l , 1959, 1 969)..Since the e a r l y wcrk cf F r a e n k e l , a l a r g e number of secondary plant compounds ( a l l e l c c h e m i c s ) have been i s o l a t e d from p l a n t s and have been shown to a c t e i t h e r as d e t e r r e n t s or a t t r a c t a n t s f o r i n s e c t s (Adams and Bernays, 1978; Chapman, 1974; Rhoades and 79 Cates, 1976; Schoonhoven, 1972; Schoonhoven and Derksen-Kcppers, 1976; Schoonhoven and Jermy, 1977). The l a r g e number of secondary p l a n t compounds found to have an i n h i b i t i n g e f f e c t on i n s e c t f e e d i n g , coupled with F r a e n k e l ' s o b s e r v a t i o n that p l a n t s appear to c o n t a i n a l l the n u t r i e n t s r e g u i r e d by i n s e c t s , seems t o have r e s u l t e d i n a b e l i e f t h a t n u t r i e n t s do not play an important p a r t i n host-p l a n t s e l e c t i o n by i n s e c t s . Thus Beck and Eeese (1976) i n a recent review of i n s e c t - p l a n t i n t e r a c t i o n s s t a t e t h a t " i t does not seem at a l l l i k e l y t h a t the i n s e c t ' s n u t r i t i o n a l requirements play more than a minor r o l e i n determining host-p l a n t s p e c i f i c i t y " . However, i f i n s e c t s are capable of demonstrating a learned a v e r s i o n response based on the d e t r i m e n t a l e f f e c t s of i n g e s t i n g an imbalanced d i e t , as suggested i n the present study, then i t would be the balance of n u t r i e n t s present i n the p l a n t t h a t would govern i t s p a l a t a b i l i t y , and not the presence or absence of n u t r i e n t s . I f the f i e l d of i n s e c t - p l a n t i n t e r a c t i o n s i s t o produce a comprehensive t h e c r y concerning the s e l e c t i o n of h o s t - p l a n t s by phytcphagcus i n s e c t s , f u r t h e r s t u d i e s must be performed to e l u c i d a t e the mechanisms whereby i n s e c t s are able t o a s s e s s the n u t r i t i o n a l composition of foods. 80 References Adams CM. and Bernays E.A. (1978). The e f f e c t of combinations of d e t e r r e n t s on the f e e d i n g behavior of Locusta m i g r a t o r i a . Entcmologia Exp. Appl 23, 101-109. 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