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Juvenile hormone control of development of selected tissues in the migratory grasshopper, Melanoplus.. 1981

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JUVENILE HORMONE CONTROL OF DEVELOPMENT OF SELECTED TISSUES IN THE MIGRATORY GRASSHOPPER, Melanoplus sanguinipes (Fabr.) (ORTHOPTERA:ACRIDIDAE) by ELNORA PALMER B.Sc. A g r i c , University of B r i t i s h Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE DEPARTMENT OF PLANT SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1981 (c) Elnora A. Palmer, 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 Date ftpfi( / i i ABSTRACT Changes i n t o t a l body weight and i n dry weights of the internal organs indicated that male and female adults of Melanoplus sanguinipes undergo a biphasic growth pattern. Regression analyses indicated that the overall growth rates were comparable i n the two sexes during the somatic growth phase but dif f e r e d markedly during the reproductive phase. Reasons for these differences are discussed in l i g h t of the behavior and physiology of the sexes. Head width, t i b i a length, tegmina length, and dry weights of gonads, fat body, and f l i g h t muscles were highly correlated i n normal adults. This indicated that growth patterns were highly coordinated within individual insects. Fluctuations i n the dry weights of the fat body and f l i g h t muscles during reproductive development indicated that these tissues were a source of protein for the developing ovaries or accessory glands. The s i m i l a r i t y i n the pattern of changes i n males and females indicated that development may be synchronized between the two sexes. Factors contributing to t h i s apparent synchrony are discussed. Depending upon the time of application, both the a n t i - a l l a t o t r o p i n , precocene II and the juvenile hormone analog (JHA), R-20458 have been shown to d r a s t i c a l l y a l t e r the development of various tissues i n M. sanguinipes. The present studies substantiate previous reports that JH regulates the development of the fat body and gonads. In addition, JH has been shown to regulate metamorphosis, / i i i somatic growth, coloration, wing length, and development of the f l i g h t muscles and fat body during the f i f t h i n s tar. Precocene applied to fourth instars caused precocious metamorphosis and the production of diminutive adults. However, nearly normal development was produced i n pxecocene-treated insects when R-20458 was applied 4 days l a t e r . Later JHA treatments resulted i n the production of nymphal-adult intermediates. Intermediates were also produced when JHA alone was applied to f i f t h instars. However, s p e c i f i c effects depended upon precise application time. Supernumerary molting occurred only i n insects treated with JH during the middle of the f i f t h i n star stadium. Therefore, absence of JH at t h i s precise time seems to be necessary to permit the imaginal molt. Green-colored and short-winged adults char a c t e r i s t i c of locust solitarious phase, were produced when JHA was applied at certain times within the f i f t h i n s tar. JHA application to f i f t h instars resulted i n a s i g n i f i c a n t reduction i n wing length and i n dry weight of the f l i g h t muscles. Flight muscles were sensitive to JHA throughout the f i f t h stadium whereas wing length was only s i g n i f i c a n t l y affected by JHA during certain periods of the stadium. Therefore, the f l i g h t muscles must be developing separately from the wings. JHA applications to precocious adultoids were too late to change the commitment of f l i g h t muscles i n either sex, or of the male gonads. However, the fat body of males and females, and the female gonads were s t i l l susceptible to JHA at this time, and the precocene-induced s t e r i l i t y of females was reversed. / i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS. i v LIST OF TABLES v i LIST OF FIGURES i x LIST OF PLATES x i i ACKNOWLEDGEMENTS x i v INTRODUCTION 1 A. Pest Status 1 B. M. sanguinipes - Grasshopper or Locust? 1 C. Phase Polymorphism in Locusts 3 D. Environmental Factors Influencing Locust Phase Determination ° E. Juvenile Hormone Control of Selected Aspects of Insect Development ^ (a) Reproduction 10 (h) Wing Length 1 1 (c) F l i g h t Muscle Development 1 1 F. Mode of Action of JH on Fli g h t Muscles 1 3 (a) Effect on Protein Content... 1 3 (b) C r i t i c a l Timing 1 4 G. JH Effects on Migration 1 5 H. A l t e r i n g JH Levels with Precocene 1 6 I. Summary of Major Objectives ^ MATERIALS AND METHODS 2 0 A. Rearing Techniques 2 0 B. Growth Measurements 2^ C. Protein Determinations 2 2 /v Page D. Chemical Treatments 23 (a) JHA Studies 23 (b) A n t i - a l l a t o t r o p i n Studies. 25 E. S t a t i s t i c a l Analysis 25 RESULTS 26 A. Normal Development 26 B . Normal Fl i g h t Muscle Protein Content 40 C. JHA Studies 46 (a) Solvent T r i a l s 46 (b) JHA Dose-response T r i a l s 46 D. S e n s i t i v i t y of F i f t h Instars to R-20458 50 E. Effects of Adult Aging on JHA-treated Insects 57 F. S e n s i t i v i t y of M. sanguinipes to Precocene II 64 G. JHA Effects on Precocene-treated Insects 72 (a) Precocene Effects 72 (b) JHA Applied to Adultoids 72 (c) JHA Applied after Precocene but Prior to the Next Molt 74 DISCUSSION 83 A. Normal Development 83 B. Role of JH i n Development 88 C. Reversing Precocious Metamorphosis with JHA 90 D. JHA Studies on Molting and Metamorphosis .92 E. JH Effects on the Development of Gonads, Fat Body, and Fli g h t Muscles 98 F. Grasshopper Control 102 LITERATURE CITED 104 APPENDIX v 121 / v i LIST OF TABLES Table , Page I Phase characteristics of locusts 4 II Environmental factors influencing locust phase determination 8 III Changes i n various growth parameters during early adulthood i n normal M. sanguinipes 27 IV Correlation between the dry weight of the f l i g h t muscles and other body measurements i n normal M. sanguinipes 35 V Linear regression analyses showing relationship between fresh weight (Y) and age (X) i n young adult M. sanguinipes 37 VI Analysis of variance for f l i g h t muscle dry weight and protein content during the f i r s t 9 days of adulthood 41 VII Linear regression equations showing relationship between protein content (Y) and dry weight (X) of the f l i g h t muscles during the f i r s t 9 days of adulthood 44 VIII Effects of t o p i c a l application of three solvents to f i f t h i n star nymphs 3 days after treatment 47 / v i i Table Page IXa S e n s i t i v i t y of different stages of M. sanguinipes to high dosages of the JHA, R-20458 48 IXb S e n s i t i v i t y of different stages of M.. sanguinipes to low dosages of the JHA, R-20458 51 Xa Comparison of various body parameters of normal 5-day-old male adults and those treated with 0.05 yg R-20458 at various intervals during the f i f t h stadium 55 Xb Comparison of various body parameters of normal - 5-day-old female adults and those treated with 0.05 yg R-20458 at various intervals during the f i f t h stadium 56 XIa Overall effects of R-20458 on adult male body measurements when applied at various times during the f i f t h stadium 58 Xlb Overall effects of R-20458 on adult female body measurements when applied at various times during the f i f t h stadium 59 XIla Mean body measurements (± S.D.) i n untreated and . JHA-treated males dissected as 5- or 6-day-old adults, or 14-day-old adults 62 XIlb Mean body measurements (± S.D.) i n untreated and JHA-treated females dissected as 5- or 6-day-old adults, or 14-day-old adults 63 / v i i i Table Page XIII S e n s i t i v i t y of various stages of M. sanguinipes to varying dosages of precocene II 69 XIV Mean body measurements (± S.D.) of 6- to 10-day-old normal and precocene-treated adults. Precocene (300 yg) was applied to newly emerged fourth instars 73 XV Effect of 0.05 yg JHA applied after the f i n a l molt to precocene-treated adultoids 75 XVI Overall effects of 0.05 ug R-20458 applied to precocene-treated insects at various intervals p r i o r to the next molt 78 XVII Effects of timed JHA applications to precocene- treated fourth i n s t a r s . Measurements were taken 4-5 days a f t e r adult emergence 82 / i x LIST OF FIGURES Figure Page 1 Changes in t o t a l body weight of normal males and females during early adulthood. Arrow indicates approximate oviposition time 28 2 Changes i n (a) tegmina and wing length, (b) t i b i a length, and (c) head width in normal males and females during early adulthood 29 3 Changes in gonad dry weight of normal males and females during early adulthood. Arrow indicates approximate oviposition time 30 4 Changes i n fat body dry weight of normal males and females during early adulthood 32 5 Changes in f l i g h t muscle dry weight of normal males and females during early adulthood 33 6 Correlations among body measurements in normal adult M. sanguinipes 34 7 Regression lines showing relationship between fresh body weight and age in normal males and females during early adulthood. Mean fresh weights of males and females with standard errors ( v e r t i c a l lines) are also shown 38 /x Figure Page 8 Regression lines showing relationships between age and body fresh weight in normal males and females during the f i r s t 3 days of adulthood, and from 4 to 9 days after emergence 39 9 Changes i n dry weight and protein content of f l i g h t muscles in normal males during early adulthood 42 10 Changes in dry weight and protein content of f l i g h t muscles in normal females during early adulthood.... 43 11 Regression lines showing the relationship between the protein content and the dry weight of f l i g h t muscles i n normal males and females during early adulthood 45 12a Correlations among body measurements i n male adults after R-20458 was applied during the f i f t h stadium 60 12b Correlations among body measurements i n female adults a f t e r R-20458 was applied during the f i f t h stadium. 61 13a The effect of aging and JHA treatment on correlations among body measurements i n 5- or 6-day-old adult males 65 / x i Figure Page 13b The effect of aging and JHA treatment on correlations among body measurements i n 14-day-old adult males 66 13c The effect of aging and JHA treatment on correlations among body measurements i n 5- or 6-day-old adult females 67 13d The effect of aging and JHA treatment on correlations among body measurements i n 14-day-old adult females ~r 68 14a Correlations among body measurements in male adultoids treated with precocene II as fourth instars and with R-20458 after t h e i r precocious molt 76 14b Correlations among body measurements in female adultoids treated with precocene II as fourth instars and with R-20458 after t h e i r precocious molt 77 / x i i LIST OF PLATES Plate Page 1 Cages used to rear stock colonies of M. sanguinipes 21 2 Cages used to determine the growth patterns of normal, precocene- and juvenile-hormone-treated grasshoppers 21 3a Abnormally large female in supernumerary stadium r e s u l t i n g from JHA application (0.375 yg) to f i f t h i n s tar M. sanguinipes . For comparison, a normal untreated female i s also shown 49 3b Normal, untreated male and large, green, short-winged supernumerary male r e s u l t i n g from JHA treatment described above. The treated insects had d i f f i c u l t y casting t h e i r exuvia 49 4a External morphology of 2-day-old adult females that were treated with 0.05 ug R-20458 as newly emerged f i f t h i n star nymphs..1 52 4b Two-day-old adult males showing the effect of a single application of 0.05 yg R-20458 to 4-, 5-, and 6-day-old f i f t h i n star nymphs 52 5 Two-day-old adult males, showing the effect of a single application of 0.05 yg R-20458 to (a) 4-, (b) 5-, and (c) 6-day-old f i f t h instar nymphs. An untreated control (d) i s also indicated 54 / x i i i Plate Page 6a Dorsal view of untreated male adult and precocious male adultoid r e s u l t i n g from precocene application (200 yg) to newly emerged fourth instars 71 6b Side view of untreated female adult and precocious female adultoid r e s u l t i n g from precocene application (200 yg) to newly emerged fourth instars 71 7a F i f t h instar nymph and precocious adultoids r e s u l t i n g from precocene (300 yg) application to 1-day-old fourth instar nymphs '. 79 7b Normal-looking f i f t h instar nymphs which received a single precocene application (300 yg) as 1-day-old fourth i n s t a r s , followed by 0.05 yg R-20458 on day 4 of the same stadium 79 7c Two semi-adultoids which late r died attempting another molt and two f i f t h instar nymphs which became non-reproducing adults. The insects were treated as mentioned previously, except that the JHA was applied on- day 5 of the fourth stadium 80 7d Two true adultoids and two f i f t h instar nymphs which la t e r became reproductive adults. The insects were treated as above, except that the JHA was applied on day 6 of the fourth stadium 80 /xiv ACKNOWLEDGEMENTS I wish to thank my research- supervisor, Dr.R. H. Elliott, and committee members, Dr. V. C. Runeckles and Dr. J. A. McLean, for their suggestions and criticisms. Special thanks go to Ms. E. Iyer for providing invaluable practical information, advice, and encouragement. Her efficient maintenance of the insectary made this study possible. Thanks also goes to Mr. D. Johnson for his insightful advice on statistical analysis, and to my fellow-student, Mr. K. Verma, for providing information on precocene and lively discussions. I am also grateful to Mr. B. McMillan, faculty photographer, who produced Plates Z to 7, and to Ms. J. Hollands for her care in typing this manuscript. Finally, my thanks go to my husband, John, to friends, Ms. D. Henderson, Ms. A. Stammers, and to fellow-student, Ms. S. Barnaby, for their suggestions and moral support. /I INTRODUCTION A. Pest Status The migratory grasshopper, Melanoplus sanguinipes (Fabr.), i s generally regarded as the most widespread and destructive grasshopper species i n North America (Pickford and Mukerji, 1974; Hewitt, 1977; Uvarov, 1977). M. sanguinipes damages cereal grains, vegetables, forages, and even the leaves and bark of f r u i t trees (Metcalf and F l i n t , 1962; Parker and Connin, 1964). Locusts, close r e l a t i v e s of M. sanguinipes, are a major world pest that have caused periodic crop decimation and famine for centuries (Baron, 1972; H i l l , 1975). Improved methods of grasshopper and locust control are currently needed (Uvarov, 1977). The International Study Conference on the Current and Future Problems of Acridology recommended the study of insect hormones as possible potent, environmentally-compatible insecticides and p a r t i c u l a r l y emphasized the potential of locust control through phase manipulation with juvenile hormone (Anonymous, 1970). B. M. sanguinipes - Grasshopper or Locust? The migratory grasshopper may actually be a locust. Grasshoppers are distinguished from the locusts i n the same family by their i n a b i l i t y to transform into a gregarious, highly mobile phase during t h e i r l i f e cycle. While locusts migrate i n huge swarms over 12 vast distances and eat most of the plants i n t h e i r path, grasshoppers remain r e l a t i v e l y l o c a l i z e d and s o l i t a r y . The name "migratory grasshopper" shows the ambivalence of s c i e n t i s t s regarding the status of M. sanguinipes. The species does not show the two extreme morphological forms common i n locusts, but i s capable of migrations of 10 to 50 miles per day ( W i l l i s , 1939) for distances of up to 575 miles (Riegert, 1962). Mass migrations of M. sanguinipes have also been reported by Bethune (1874), Gurney (1952), and Parker et al. (1955). In addition, the genus Melanoplus i s categorized by Rowell (1967) as containing locust species. Members of the genus exhibit the locust c h a r a c t e r i s t i c of green/brown polymorphism, at least i n the haemolymph of individuals with high corpora a l l a t a a c t i v i t y ( P f e i f f e r , 1945). A closely related species, the Rocky mountain locust, Melanoplus spretus (Walsh), was the predominant species during the early 1900's but appears to have become extinct. However, some authors (Buckell, 1972; Faure, 1933; Huffaker and Messenger, 1976) believe i t may be a rarely-occurring phase of M. sanguinipes. M. spretus i s very s i m i l a r to M. sanguinipes but has longer, broader wings and a darker coloring (Heifer, 1953). Perhaps M. sanguinipes i s a locust whose extreme gregarious phase i s not triggered by present North American conditions. /3 C. Phase Polymorphism in Locusts Locust phase theory, f i r s t expounded by Uvarov (1921, cited by Ordish, 1976) explains the natural polymorphism of many ac r i d i d populations and forms the foundation of modern locust control. Uvarov observed two extreme physiological states or phases, a s o l i t a r y and a gregarious phase. Although not common to a l l locust species, the two phases exhibit a variety of characteristics (Table I ) . Locusts i n the solitaria phase display grasshopper-like behavior and morphology including a more arched pronotum, shorter wings, and larger femur/head capsule r a t i o than gregaria adults. Solitaria are often light-colored or even green, feed and develop as isolated individuals and make only short f l i g h t s . Rates of feeding, and development i n solitaria are slower than i n gregaria, and solitaria locusts sometimes have an extra nymphal instar. Egg-laying i s delayed i n solitaria, but the females are larger and t h e i r fecundity i s higher than i n gregaria locusts (Kennedy, 1961). Phase gregaria exhibits long-distance migrations. These f l i g h t s begin under suitable weather conditions when the morphology and behavior of the new locust generation have begun to change from the s o l i t a r y to the gregarious form. Dark-colored gregarious nymphs begin marching and are joined by s i m i l a r individuals u n t i l a large hopper band has formed. After the nymphs molt into long-winged adults, they m i l l around for several days, taking short practice f l i g h t s u n t i l they are a l l ready for f l i g h t (Chapman, 1969). Swarms form and the TABLE I: Phase characteristics of locusts Genus or Phase character Effect species References behavior color s o l i t a r i a limited f l i g h t ; no grouping or marching individuals avoid each other gregaria mass f l i g h t ; march i n groups individuals stay together s o l i t a r i a green, yellow, or l i g h t beige gregaria tan to dark brown or black may have pinkish background color or yellowing general Loousta Schistocerca Loaustana grasshopper spp. Kennedy (1961) G i l l e t t (1978) Cassier (1966) Kennedy (1961) G i l l e t t (1978) Rowel1 (1967) morphology 1. femur/head capsule 2. wing length; length of elytron/hind femur r a t i o 3. pronotum compound eyes and. time of f l i g h t larger in solitaria; smaller in gregaria solitaria shorter wings s l i g h t l y lower E/F r a t i o arched in solitaria gregaria longer wings s l i g h t l y higher E/F r a t i o solitaria eyes have lig h t - colored spots or bands f l y at night gregaria eyes are dark and a s o l i d color day f l i g h t Schistocerca Locusta Zonocerus variegatus Schistocerca Locusta Schistocerca Locus tana Locusta G i l l e t t (1978) Nolte (1976) McCaffery S Page (1978) Kennedy (1961) Fuzeau-Braesch § Nicholas (1970) Nolte (1978) Cassier (1965) Davey (1959) (continued). TABLE I: (continued). Genus or Phase Character Effect species References development 1. egg hatching 2. maturation rate 3. young hoppers 4. early adulthood 5. sexual dimorphism 6. maturity 7. fecundity solitaria slower than gregaria solitaria slower than gregaria solitaria sometimes has an extra instar gregaria nymphs are heavier than solitaria, and contain more dry matter gregaria feed more rapidly than solitaria and have a higher metabolic rate solitaria have bigger ? and smaller <f than gregaria Therefore in gregaria there i s a smaller difference between the sexes. a) gregaria begin oviposit ion later than solitaria b) gregaria begin oviposition e a r l i e r than solitaria lower i n gregaria than in solitaria Schistocerca Locustana Schistocerca Locusta Nomadacris Schistocerca Nomadacris Locusta Schistocerca Nomadacris Schistocerca Nomadacris Locusta Schistocerca Nomadacris Locusta Nomadacris Schistocerca cf. Kennedy (1961) /6 locusts f l y together for hundreds of miles. During the migration, the female grasshoppers or locusts contain undeveloped oocytes, whereas the males are usually older and more sexually mature (Davey, 1959; Johnson, 1969; Chapman et al., 1978). Migrations usually continue u n t i l the females begin to form eggs and leave the swarm in search of oviposition s i t e s , when the band disperses. D. Environmental Factors Influencing Locust Phase Determination Locusts benefit substantially by th e i r a b i l i t y to change state i n response to t h e i r fluctuating environment. In wet weather, when food i s p l e n t i f u l , they are dispersed amongst the vegetation producing large numbers of eggs and growing slowly into large, pale-colored or green adults which are well camoflaged i n t h e i r surroundings. When the climate becomes hot and dry, the locusts numerous, and food plants scarce, locusts migrate to more favorable areas. The long wings, rapid development, and organized behavior of gregaria are i d e a l l y suited to large-scale dispersal. Within a few generations locusts adapt to t h e i r new conditions. Locust phases are determined by both genetics and environment. Although selective breeding can bring about d i s t i n c t solitaria and gregaria l i n e s within four generations, the same v a r i a t i o n i n phase character can be induced within one generation by environmental s t i m u l i such as rearing density. The altered state i s then maternally inherited ( c f . Kennedy, 1961). p Enviromental conditions triggering phase change are l i s t e d i n Table I I . Not su r p r i s i n g l y , form gregaria develops when the insects are densely populated. High temperatures, low r e l a t i v e humidity, and long days also favor phase gregaria, while solitaria i s encouraged by the opposite conditions. Even the presence of faeces from certain other locust stages has been found to influence phase determination i n caged nymphs ( G i l l e t t and P h i l l i p s , 1977). In addition, locusts are phase-sensitive to carbon dioxide levels (Fuzeau-Braesh and Nicolas, 1970; Doane, 1973) and to diet. Brett (1947) produced brachypterous M. sanguinipes by rearing the ~ grasshoppers on lucerne at low temperatures and high humidity (Uvarov, 1966). Perhaps M. spretus was just an extreme phase of M. sanguinipes that was no longer favored by the new food plant communities resulting from the human settlement of North America. Changes i n diet can a l t e r the hormonal balance within insects and may increase or decrease the l i k e l i h o o d of migration. According to Staal (1967), "the discovery that particular plant substances simulate the effects of juvenile hormone and molting hormone of the insects may throw new light on the migratory behavior of many phytophagous insects". E. Juvenile Hormone Control of Selected Aspects of Insect Development Environmental factors appear to trigge r hormonal responses that i n i t i a t e phase change. The solitaria phase i s thought to be a TABLE I I : Environmental factors influencing locust phase determination Phase character Effect Genus or species References crowding status of parents humidity- temperature photoperiod food plants extreme s o l i t a r i u s phase at very low density extreme gregarious phase at very high density intermediate forms at intermediate densities solitaria parents produce a higher proportion of solitaria offspring than do the intermediates gregaria parents produce a higher proportion of gregaria offspring low humidity favors gregaria high humidity favors solitaria low temperatures favor solitaria high temperatures favor gregaria short days favor solitaria long days favor gregaria various plants favor one phase Zonocerus Schistocerca Locusta Nomadacris Locusta Zonocerus variegatus Locusta Schistocerca Schistocerca Schistocerca M. sanguinipes McCaffery § Page (1978) Chapman et al. (1978) Kennedy (1961) Kennedy (1961) Hunter-Jones (1958) Nolte (1976) Chapman et al. (1978) Davey (1959) Doane (1973) G i l l e t t (1978) Albrecht et al. (1978) G i l l e t t (1978) Albrecht et al. (1978) Doane (1973) Staal (1961) McCaffery $ Page (1978) Uvarov (1966) Brett (1947) (continued). TABLE I I : (continued). Genus or Phase character Effect species References carbon dioxide faeces high levels of carbon dioxide favor solitaria faeces from crowded adults increase solitaria characters in nymphs •faeces from crowded numphs increase gregaria characters in numphs Locusta Schistocerca Doane (1973) Fuzeau-Braesch § Nicholas (1970) G i l l e t t § and P h i l l i p s (1977) /io permanently neotenized form a r i s i n g from high juvenile hormone (JH) t i t r e s during the l a s t nymphal stage (Kennedy, 1961; Doane, 1973; Cassier and Delorme-Joulie, 1976). When locust nymphs receive corpora a l l a t a (CA) implants or are treated with juvenile hormone analogs (JHA), solitaria-like adults are produced (Joly, 1960; Staal, 1961; Doane, 1973). Conversely, s o l i t a r y Loousta females with one CA removed produce progeny which show gregarious ch a r a c t e r i s t i c s (Cassier, 1966; Doane, 1973). Phase coloration changes can also be induced by allatectomy (Kennedy, 1961), CA implants, and external application of JHA (Joly and Meyer, 1970; Nemec, 1970; Kruse Pedersen, 1978). Rowell (1967) reports that color change seems to be a general char a c t e r i s t i c of the Acridoidea, and can be induced with high JH levels even i n species where green individuals i n the wild are rarely found ( P f e i f f e r , 1945). (a) Reproduction In many a c r i d i d species including M. sanguinipes, the CA have been shown to stimulate ovarian development ( G i l l o t t and E l l i o t t , 1976; E l l i o t and G i l l o t t , 1976, 1977, 1978, 1979). Since JH accelerates reproductive growth low JH levels i n early adult migrants could explain the slow sexual maturation i n the females of some locust species. S i m i l a r l y , JH i s also involved i n the reproductive functions of male locusts. In S. gregaria, allatectomies performed on young males completely abolished male / I I sexual behavior (Loher, 1961; Pener, 1967). Evidence for CA control of the development and functioning of the male accessory glands has been demonstrated i n M. sanguinipes ( G i l l o t t and F r i e d e l , 1976a,b). (b) Wing Length "It is well-known that JHA applied to insects at the pveimaginal stage disturb the imaginisation of integuments and wing formation" (Chudakova et al., 1976). In the t r o p i c a l pest grasshopper, Zonocerus variegatus, crowding or low JH levels during the l a s t nymphal in s t a r produced long-winged adults capable of long f l i g h t s (McCaffery and Page, 1978). Allatectomy performed on 3-day-old f i f t h instars of Z. variegatus produced only long-winged adults (McCaffery and Page, 1978). In contrast, JHA applied to f i n a l i nstar Locusta and Schistocerca caused curly and/or shortened wings, the effects being dependent on precise timing of JHA application (Nemec, 1970). (c) Flight Muscle Development The role of the CA i n insect f l i g h t muscle development remains uncertain (Gilbert and King, 1973). However, researchers have observed that "maturation of flight muscles differs between species and sexes and may be under the control of the CA" (Rockstein and Miguel, 1973). In the majority of species investigated, JH i n h i b i t s f l i g h t muscle development i n nymphs / 1 2 and encourages f l i g h t muscle degeneration i n adults. In H. cecrovia, formation of many adult tissues occurs during the f i r s t 2 days of pupal development, when JH levels are low. However, when CA are implanted during early pupation, f l i g h t muscle formation i s suppressed (Williams, 1961). In in vitro experiments on various insects, JH suppressed imaginal wing disc development (Patel and Madhaven, 1969; Chihara and Fristrom, 1973; Benson and Oberlander, 1974; Oberlander and Silhacek, 1976). Some insects normally exhibit wing-casting and/or f l i g h t muscle degeneration after the i n i t i a l active adult f l i g h t s give way to reproductive a c t i v i t y . However, i n some of these insects f l i g h t loss can be prevented by removal of the CA. In the house c r i c k e t , Aoheta domestical, allatectomies performed during the f i n a l nymphal instar prolonged f l i g h t muscle persistance i n the adults (Chudakova and Bocharova-Messner, 1968b) and resulted i n a permanent retention of f l i g h t a b i l i t y (Chudakova and Gutmann, 1978). Conversely, JHA application caused rapid degeneration of the f l i g h t muscles i n both nymphal and adult Aoheta (Chudakova and Bocharova-Messner, 1968a, 1968b). Adult f i r e ants, Solenopsis inviota, f a i l e d to cast t h e i r wings or undergo normal f l i g h t muscle h i s t o l y s i s a f ter allatectomy (Barker, 1979) but subsequent JHA application to the adults resulted i n rapid f l i g h t muscle degeneration. Implantation of CA into adult female, Dysdercus intermedins (Edwards, 1970), also e l i c i t e d f l i g h t muscle h i s t o l y s i s . /13 In various bark beetle species, JHA induce degeneration of the adult f l i g h t muscles (Borden and Slat e r , 1968; Unnithan and Nair, 1977). F. Mode of Action of JH on Flight Muscles (a) Effect on Protein Content The diameter and protein content of the f l i g h t muscles of some insects mirror t h e i r increasing f l i g h t c a p a b i l i t y during early adulthood (Poels and Bennakkers, 1969; B u r s e l l , 1973; Panar and Nair, 1975; Baker, 1976). Protein, the major "component of muscle (Panar and Nair, 1975), i s also affected by JH lev e l s . In Poels and Beenakkers* experiments (1969), protein accumulation i n adult locust f l i g h t muscles was greatly reduced, but not t o t a l l y blocked, by implanting active CA during the early f i f t h i n s t ar. The l i k e l i h o o d of uneven release from the CA throughout adult development makes i t impossible to determine from t h e i r experiment whether or not there i s a c r i t i c a l stage during which JH influences f l i g h t muscle development. Perhaps protein synthesis i n the f l i g h t muscles would not be affected i f JH were applied at the l a t e f i f t h i n star stage. However, since the CA caused the greatest i n h i b i t i o n of f l i g h t muscle protein during the d i f f e r e n t i a t i o n stage (2 to 5-day-old adults), JH applications to newly-emerged adults should be effec t i v e i n reducing f l i g h t muscle protein content unless the tissues are already committed at t h i s time. The /14 s e n s i t i v i t y of the f l i g h t muscles of f i f t h instar nymphs to CA implantation led Poels and Beenakkers (1969) to suggest that i n normal Locusta, f l i g h t muscle development only begins when the JH t i t r e i s very low. The authors presume that JH t i t r e s i n Loausta are n e g l i g i b l e throughout the f i f t h nymphal stadium. C a p i l l a r y gas chromatography with electron capture (Blight and Wenham, 1976a,b; Huibregetse-Minderhoud et al., 1980) and Gallevia bioassays (Johnson and H i l l , 1973b) on Schistoeerca and Loausta confirmed that the high JH levels i n the early fourth instar stadium decreased at the end of the stadium and were almost undetectable during much of the f i f t h i n star stadium and early adulthood. (b) Critical Timing The s e n s i t i v i t y of various a c r i d i d tissues to JH-induced effects appears to vary according to the development stage. In Locusta and Schistoeerca, exogenous JHA applied i n small doses at the beginning of the last l a r v a l i n s t a r cause changes in morphology whereas the same doses applied l a t e r i n the same instar result i n phase change effects (Nemec, 1970). In terms of the effects of JH on wing and f l i g h t muscle development, several authors (Staal, 1975; McCaffery and Page, 1978; Chapman et al., 1978) believe the l a s t nymphal instar to be the c r i t i c a l period. Doane (1973), however, states that i n locusts "...the sensitive period for hormonal action in the case of...wing response was /15 during the -post-molt period of rapid mitosis". In further experiments by Beenakkers (1973), allatectomy f a i l e d to affect locust f l i g h t muscle development. However, since the operation was performed one day after adult ecdysis, the procedure may have been too late to reverse a commitment and increase f l i g h t muscle dry weight and protein content. The JH s e n s i t i v i t y of the system during adult development and the length of the sensitive period have not been extensively investigated. Rankin (1980) agrees that "...more work needs to be done to produce a dose-response curve of JH to flight as well as JH titre determinations on reproductive males and females". G. JH Effects on Migration In the t r o p i c a l grasshopper, Z. variegatus, wing length i s highly correlated with migratory capacity (McCaffery and Page, 1978), and short-winged insects have poorly developed f l i g h t muscles (Chapman et al., 1978). JHA applications to f i n a l nymphal in s t a r grasshoppers resulted i n progressively shorter wings as JH dosage increased (McCaffery and Page, 1978). Chapman et al. (1978) state that i n determining the migratory status of these grasshoppers, "the important dimorphism is in the development of the wing muscles". Perhaps the high JH and ecdysone levels at the c r i t i c a l f i n a l nymphal instar stage i n h i b i t f l i g h t muscle as well as wing development i n solitaria locusts. Unfavorable JH levels for maximum f l i g h t muscle /16 development could be a major cause of non-migration i n many insects. I f migrating insects possessed low JH levels i n early adulthood, f l i g h t muscle development would be favored and reproductive development delayed. These char a c t e r i s t i c s are indeed seen i n gregaria locust females. There may be a d i f f e r e n t response to JH of f l i g h t muscles and gonads i n the two sexes during development, but t h i s p o s s i b i l i t y has not yet been thoroughly investigated. H. Altering JH Levels with Precocene Recently, a new insect growth regulator precocene II has been discovered which has an a n t i - a l l a t o t r o p i c effect (Bowers et al., 1976). Actions of the compound on various insects include a reduction of CA a c t i v i t y , CA degeneration, precocious metamorphosis, and s t e r i l i t y (Bowers and Martinez-Pardo, 1977; Kruse Pedersen, 1978; Pener et al., 1978; Schooneveld, 1979; Chenevert et al., 1979). In vitro experiments have shown that precocene II i s capable of d i r e c t l y i n a c t i v a t i n g the CA (Pratt and Bowers, 1977; Muller et al., 1979). CA of Oncopeltus, excised and incubated in vitro with precocene, lost the a b i l i t y to induce supernumerary molting which normally results i n many CA-implanted insects (Muller et al., 1979). This work has been supported by further in vitro experiments on Schistocerca. Precocene caused CA parenchyma c e l l degeneration i n second, t h i r d , and fourth instar Schistocerca nymphs (Unnithan et al., 1980). Bowers /17 and Aldrich (1980) confirmed that the brain and neurosecretion are not involved i n precocene in a c t i v a t i o n of the CA. No side effects of precocene have yet been demonstrated. Non-target tissues such as the fat body and gut rapidly metabolize precocene (Pratt et al., 1980). The authors suggest that t h i s process may form the basis of precocene resistance i n non-target tissues. Precocene s e n s i t i v i t y of the CA also varies from species to species. Of nine insect species tested, Ohta et al. (1977) found "a variation of at least 37-fold in metabolic rate" of precocene. The authors proposed that i n insects i n which precocene i s rapidly degraded, the compound i s less bioactive, and that t h i s may be a basis for i t s s e l e c t i v i t y . Precocene applications are only eff e c t i v e at certain developmental stages. Kelly and Fuchs (1978) found no antigonadotropic effects on the adult female mosquito, Aedes aegypti, when precocene was applied one hour after adult emergence, while l a t e r applications after a blood meal slowed ovarian maturation and produced abnormal oviposition. Unnithan and Nair (1979) applied precocene II to fourth and f i f t h i n s t a r Oncopeltus fasciatus and found a n t i a l l a t o t r o p i c effects i n the e a r l i e r instars but not during the f i n a l i n s t a r . They proposed that "apparently precocene is effective only when the insect's CA is active or it is free from any inhibitory control". Timing of precocene application i s c r i t i c a l i n Locusta as well (Kruse Pedersen, 1978). /18 Although the degeneration of precocene-treated CA i s permanent, some precocene effects seem to be short-lived. Sahota and Fa r r i s (1980) temporarily arrested the normal degeneration of f l i g h t muscles i n log-colonizing female spruce bark bettles (Dendroctonus rufipennis) by applications of precocene to adults. The normal increase i n f l i g h t muscle DNA/protein r a t i o was delayed. According to Sahota and Farris (1980), "...it would appear that with JH-induaed muscle degeneration3 precocene II interferes with JH (production or effects) and indirectly sustains t r a n s c r i p t i o n at a normal level". The authors were not able to explain why muscle degeneration-later proceeded after a delay of 8 days. Comparable effects have been observed i n Oncopeltus (Masner et al., 1979). I. Summary of Major Objectives The main purpose of th i s study was to investigate the role of JH on the development of selected tissues i n M. sanguinipes from the following aspects: 1. What i s the normal pattern of growth during early adulthood i n male and female M. sanguinipes? What i s the normal change i n f l i g h t muscle protein content during t h i s period? 2. How i s the normal developmental pattern changed by to p i c a l JHA treatments? Is timing of JHA application c r i t i c a l even within an instar? /19 3. How is normal development changed when the activity of the CA is impaired by precocene II? 4. Can the developmental changes caused by the application of precocene II be reversed by subsequent JHA treatment? /20 MATERIALS AND METHODS A. Rearing Techniques The insects used i n th i s study were of the non-diapause st r a i n of M. sanguinipes(Pickford and Randell, 1969). Stock colonies were reared under crowded conditions (ca. 30 to .100 grasshoppers) i n cages described by Pickford (1958) (Plate 1). Photoperiod was maintained at 12L:12D and temperatures at 30 ± 5°C. The stock colonies and experimental insects were fed an a l f a l f a meal mixture (100 g a l f a l f a meal:100 g bran:10 g brewer's yeast:12 ml corn o i l ) supplemented by d a i l y additions of fresh lettuce ( G i l l o t t and Dogra, 1972). B. Growth Measurements The i n i t i a l experiments were aimed, at establishing the normal growth patterns of adult males and females. Within 3 to 4 hrs after the imaginal molt, insects i n the stock colony were transferred into glass j a r cages (Plate 2). At various intervals after ecdysis, the grasshoppers were decapitated and weighed on a Mettler microbalance. The wing length, t i b i a length, and head width were measured with vernier c a l i p e r s . Dissections were performed under a stereo- microscope. Using the methods described by H i l l et at. (1968), the fat body, gonads, and f l i g h t muscles were removed, dried to constant weight at 35°C, and weighed on a microbalance. F l i g h t muscles were l a t e r used for protein determinations. Analysis of variance was performed to determine the effects of age on adult body measurements. PLATE 2: Cages used to determine the growth patterns of normal, precocene- and juvenile-hormone-treated grasshoppers Ill The significance of correlations between body parameters was also determined. To obtain more precise estimates of d a i l y changes i n t o t a l body weight, the fresh weight was measured i n the same insects during the f i r s t 9 days of adulthood. Since the presence of mature males affects the maturation rate of other female acridids . (Uvarov, 1966), both sexes (2 males and 2 females) were placed i n glass jars (Plate 2), and reared under conditions i d e n t i c a l to those i n the stock colony. Each day after the imaginal molt, the insects were anaesthetized with. C0 2 and the fresh weight determined. The data were subjected to regression analysis to determine the ov e r a l l relationship between age and fresh weight i n both sexes. As d i s t i n c t phases of somatic and reproductive development were evident i n both sexes, additional regression analyses were performed on 1- to 3-day-old and 4- to 8-day-old insects. C. Protein Determinations The procedure employed to determine the protein content of the f l i g h t muscles was modified after Schacterle and Pollack (1973) and Lowry et al. (1951). Three pre-weighed portions of each sample (0.2-0.3 nig) were s o l u b i l i z e d i n 1.0 ml 0.5 N NaOH for 7 min at 100°C (Lowry et al., 1951). After adding 10 ml of copper reagent (10% sodium carbonate, 0.1% potassium t a r t r a t e , and 0.05% copper sulfate, but lacking NaOH), 4.0 ml of phenol reagent (Fisher 723 S c i e n t i f i c Co.) was added. Heating was omitted because the mixture was observed to be more stable at room temperature. After 10 min, transmittance readings were taken at 620 nm i n a Spectronic 20. By knowing the t o t a l dry weight of the f l i g h t muscles and the amount used in each protein determination, the t o t a l protein content of the f l i g h t muscle was calculated, D. Chemical Treatments The role of JH i n somatic and reproductive development was investigated using a juvenile hormone analogue (JHA) and an a n t i - a l l a t o t r o p i n . Newly ecdysed fourth i n s t a r s , f i f t h i n s t a r s , and adults were removed from the stock colony. They were isolated i n glass jars i n groups of 6-10 grasshoppers/jar and exposed to conditions described previously (Plate 2). Adult emergence data were recorded for each insect. When adult emergence was asynchronous, the insects were marked i n d i v i d u a l l y with dots of Testor's PLA enamel paint (Testor Corp.). (a) JHA Studies At selected i n t e r v a l s , the insects were anaesthetized with nitrogen or carbon dioxide. As the JHA, R-20458 (6,7-epoxy-l- (p-ethylphenoxyl)-3,7-dimethyl-2-octene; Stauffer Chem. Co.), has been shown to be b i o l o g i c a l l y active i n M. sanguinipes ( E l l i o t t and G i l l o t t , 1978), the compound was diluted appropriately. To determine the best solvent for the JHA t r i a l s , 1.0 u l samples /24 of acetone, o l i v e o i l , or an acetone/olive o i l mixture (1:1) were applied using a micropipette to the abdomenal terga of s i x insects. The o l i v e o i l and acetone-oil mixture inh i b i t e d molting whereas acetone alone was non-inhibitory. To determine the l e t h a l and morphological effects of R-20458, the JHA was s e r i a l l y diluted i n acetone. The JHA doses examined ranged from 0.375-0.0375 yg. One y l samples were applied t o p i c a l l y with a micropipette to fourth and f i f t h i n star nymphs. Treated insects were kept i n glass j a r cages u n t i l 4 to 5 days after adult emergence, when mortality and external appearance were noted. The s e n s i t i v i t y of d i f f e r e n t stages of f i f t h i n star nymphs to R-20458 was investigated by treating 1- to 6-day-old f i f t h instars with single applications of 0.05 yg JHA/insect. Five days after molting, the effects of the treatment on external morphology and growth of the various body tissues were assessed. Analysis of variance was performed on the data to see i f there were any s i g n i f i c a n t differences between various body measurements with d i f f e r e n t JHA application times. Correlations between body measurements were also determined. In order to establish whether the effects of the JHA treatments on the various tissues were temporary or permanent, 0.05 yg R-20458 was applied to newly emerged f i f t h instar nymphs. The treated insects were dissected 5 and 14 days after adult emergence and t h e i r body measurements compared. /25 (b) Anti-allatotropin Studies The insect a n t i - a l l a t o t r o p i n , precocene II (6,7-dimethoxy- 2,2-dimethyl chromene; Aldr i c h Chem. Co.) was stored under nitrogen at 6°C. Stock solutions were prepared by d i l u t i o n i n dimethyl sulfoxide (Pound and Olive r , 1979; Verma, 1981). Since 1 y l of the solvent exhibited no l e t h a l or other deleterious properties (see Appendix 8), solvent-treated controls were not used i n a l l the precocene experiments. Instead, the normal growth patterns established i n the preceeding studies were used. In order to determine a b i o l o g i c a l l y - a c t i v e but non-lethal dosage of precocene, fourth i n s t a r , f i f t h i n s t a r , and adult grasshoppers were treated with doses ranging from 25 to 1000 yg/insect. Effects on mortality and external appearance were recorded. To determine the effects of precocene II on adult body measurements, 1-day-old, fourth i n s t a r nymphs were treated with 300 yg precocene I I . The insects were dissected 6-16 days after molting. To reverse the effects of precocene on molting, metamorphosis, and/or the development of the internal organs, insects which had been treated with precocene as newly ecdysed fourth instars were also treated with 0.05 yg R-20458. The JHA was applied at various intervals p r i o r to the molt and 6 to 10 days after the molt. E. Statistical Analyses Analysis of variance, correlation matrices, and linea r regression analysis were performed using the MIDAS computer package. 1 (Michigan Interactive Data Analysis System); Fox and Guire, 1976. 726 RESULTS A. Normal Development The t o t a l body weight of males and females increased s i g n i f i c a n t l y during the f i r s t 9 days of adulthood (Table I I I ) . The da i l y increments are shown i n Fig. 1. In both sexes, the fresh weight increased markedly during the f i r s t 3 days. However, after t h i s , growth patterns were d i s t i n c t l y d i f f e r e n t . In males, the t o t a l body weight increased marginally to reach a maximum on day 5 when mating normally begins. Concurrent with t h i s , the fresh weight declined and remained r e l a t i v e l y stable for the remainder of the assessment period. In contrast, the t o t a l body weight of females increased s i g n i f i c a n t l y u n t i l day 8 when oviposition occurred. In both sexes, no signficant change i n tegmina and wing length, t i b i a length, or head width occurred after emergence (Table I I I , Fig. 2). However, s i g n i f i c a n t changes i n the dry weight of various internal organs were evident (Table I I I ) . In newly-emerged females, the ovary was poorly developed and weighed less than 2 mg (Fig. 3). The dry weight remained r e l a t i v e l y constant u n t i l day 3, after which a pronounced increase occurred u n t i l day 8. At t h i s point, the ovary contained mature eggs i n the oviducts and weighed nearly 46 mg. The sharp decrease i n ovarian dry weight reflected the deposition of these eggs. In newly emerged males, the testes were well developed. The mean combined dry weight of the testes-accessory gland complex was Table I I I : A n a l y s i s of d a i l y Changes i n various growth parameters d u r i n g e a r l y adulthood i n normal M. sanguinipes Males Females Growth parameter D.F. F value S i g n i f i c a n c e D.F. F value S i g n i f i c a n c e T o t a l body f r e s h weight (mg) 8,44 5.49 .0034 7,39 5.92 .0001 T i b i a length (mm) 8,44 1.65 . 1373 NS 7,39 1.39 .2328 NS Tegmina length (mm)* 8,44 0.31 .9581 NS 7,39 0.67 .6923 NS Head width (mm) 8,44 1.05 .4178 NS 7,39 0.83 .5654 NS Gonad dry weight (mg) 8,44 14.18 .0000 7,40 24.25 .0000 Fat body dry weight (mg) 8,44 2.41 .0298 7,40 6.87 .0000 F l i g h t muscle dry weight (mg) 8,44 6.88 .0000 7,40 4.89 .0005 NS = not s i g n i f i c a n t (P = 0.05) * wing length comparable /28 100 H i i i i i t I . I i i 1 2 3 4 5 6 7 G 9 AGE (days) FIGURE 1: Changes in t o t a l body weight of normal males (A) and females (o) during early adulthood. Arrow indicates approximate oviposition time. In this and remaining figures, the mean ± S.F. are indicated. /29 20 15 M 10 CO 3 t J 5 3 4 4 t 5 (A 5 A 5 5 o A 5 A A (B i 1 1 5 I 5 o (C 3 4 5 8 AGE (days) FIGURE 2; Changes i n (a) tegmina and wing length, (b) t i b i a length, and (c) head width i n normal males (A) and females (o) during early adulthood. /30 40 30 e H I—I 20 10 S FIGURE 3: 8 0 AGE (days) Changes i n gonad dry weight of normal males (A) and females (o) during early adulthood. Arrow indicates approximate oviposition time. /31 4.2 mg (Fig. 3). Pronounced weight increases occurred between days 3 and 5 when the dry weight of the complex s t a b i l i z e d at approximately 10 mg. Highly s i g n i f i c a n t (P = 0.01) changes i n both fat body and f l i g h t muscle dry weight took place i n males and females during early adulthood (Table I I I ) . Although the fat body was larger i n females than i n males, the pattern of d a i l y changes in the dry weight of the tissue was s i m i l a r i n the two sexes (Fig. 4). In both, the fat body dry weight increased markedly after adult emergence and peaked on day 5, when mating usually begins. Then the dry weight decreased rapidly in both sexes, plateauing around days 8-9. S i m i l a r l y , f l i g h t muscle dry weight was greater i n females than i n males (Fig. 5). However, i n both sexes, the dry weight of the f l i g h t muscles followed a pattern s i m i l a r to that of the fat body v i z . r i s i n g sharply after adult emergence, peaking on days 5-6, then declining. A l l body measurements were highly correlated with each other in normal M. sanguinipes (Fig. 6). The exception was the gonads, which were not correlated with t i b i a length or head width i n either sex, nor with tegmina and wing length i n females. F l i g h t muscle dry weight was highly correlated with the other body measurements i n normal M. sanguinipes (Table IV). Although the former fluctuated after imaginal ecdysis, i t was s t i l l correlated with tegmina and wing length which are of fixed size after adult emergence. More detailed fresh weight analyses were performed on a second group of newly emerged adults. The same insects, reared under 25 20 h e 15 e - 1 10 o A 4 T A i I i J I L i 111 \ T t 3 4 5 6 7 8 9 AGE (days) FIGURE 4: Changes i n fat body dry weight of normal males (A) and females (o) during early adulthood. /33 20 R 15 00 '6 V / E- 10 I—I J 1 3 J 1 1 I 1 I I t 1 5 6 AGE (days) FIGURE 5: Changes in f l i g h t muscle dry weight of normal males (A) and females (o) during early adulthood. Fresh Weight T i b i a Length Tegmina Length /34 Wing Length Fl i g h t Muscle Dry Weight Fat Body Dry Weight Head Width Gonad Dry Weight a) Males FIGURE 6: Correlations among body measurements i n normal adult M. sanguinipes. In t h i s and remaining figures, a double l i n e joining two points denotes a s i g n i f i c a n t correlation at the 1% l e v e l ; a single l i n e denotes significance at the 5% l e v e l . When the growth of two body parameters was not correlated, they were not joined with a l i n e . There were on negative correlations. TABLE IV: Correlation between the dry weight of the f l i g h t muscles and other body measurements in normal M. sanguinipes Growth parameter correlated with f l i g h t muscle dry weight D.F. Males R Significance D.F. Females R Significance Total body fresh weight (mg) 54 .86 1% 54 .86 1% T i b i a length (mm) 54 .48 1% 54 .50 1% Tegmina length (mm) 54 .55 1% 54 .56 1% Wing length (mm) 54 .55 1% 54 .56 1% Head width (mm) 54 .42 1% 54 .56 1% Gonad dry weight (mg) 54 .85 1% 54 .56 1% Fat body dry weight (mg) 54 .71 1% 54 .77 1% — /36 density-controlled conditions, were used throughout t h i s study. Analysis of variance revealed a s i g n i f i c a n t variation i n fresh body weight of both sexes during early adulthood (Table V). The increments i n fresh weight were highly dependent upon age, yie l d i n g s i g n i f i c a n t regressions i n males and females for the 9-day period (Table V; Fig. 7). Analysis of the residuals confirmed the normal d i s t r i b u t i o n of residual sizes, and the random d i s t r i b u t i o n of the residuals with respect to age. Regression equations of Y f e m a l e = 275.7 + 21.IX and v m a ^ e = 242.4 + 9.8X show a faster growth rate for females than for males. Examination of changes i n the fresh weight and dry weight of the internal organs i n normal adults indicated that there were two growth periods occurring i n grasshoppers during the f i r s t 9 days of adulthood (Figs. 1-5). During the f i r s t 3 days, limited reproductive growth occurred i n both sexes so the increase i n fresh weight was due primarily to growth of somatic tissues including the fat body, f l i g h t muscles and c u t i c l e . However, after t h i s the converse was true so that the increase i n t o t a l body weight was largely due to the growth of the reproductive organs. Fig. 8 shows the dependence of fresh body weight on age during the two periods of early adult growth. During the f i r s t 3 days, the slopes of the regression lines for males and females were s i m i l a r , being 28.8 and 26.3, respectively (Fig. 8). Dependency of fresh body weight on age was s i g n i f i c a n t i n both sexes at t h i s time (Table V). However, after day 3, the regression equations TABLE V: Linear regression analyses showing relationship between fresh weight (Y) and age (X) i n young adult M. sanguinipes Sex Growth period D.F. F value Significance Regression equation Males Days 1 - 9 Days 1 - 3 Days 4 - 9 6,21 1,10 1,14 4.6 6.3 1.3 .0041 .0305 .2777 NS Y = 242.4 + 9.8 X Y = 224.0 + 23.8 X Y = 273.9 + 4.6 X Females Days 1 - 9 Days 1 - 3 Days 4 - 9 6,21 1,10 1,14 22.3 29.5 16.2 .0000 .0003 .0013 Y = 275.7 + 21.1 X Y = 264.4 + 26.3 X Y = 310.4 + 15.6 X 738 500 r 400 6 X i—i UJ 300 h 200 h 100 (- 9.8 X AGE (days) (X) FIGURE 7: Regression lines showing relationship between fresh body weight and age i n normal males and females during early adulthood. Mean fresh weights of males (A) and females (o) with standard errors ( v e r t i c a l lines) are also shown. /39 500 >- 400 Y = 310.4 + 15.6X 60 s H X 300 I—I Y - 264.4 + 26.3 Y_ 273.9 +4.6X .0 +23.8X 200 100 AGE (days) (X) FIGURE 8: Regression lines showing relationships between age and body fresh weight i n normal males and females during the f i r s t 3 days of adulthood, and from 4 to 9 days after emergence. /4 0 for both sexes were quite d i f f e r e n t . In males, there was no s i g n i f i c a n t change i n fresh body weight with age (Table V); the slope of the regression l i n e being only 4.6 (Fig. 8). However, i n females, fresh body weight increased s i g n i f i c a n t l y with age (Table V) and yielded a regression slope of 15.6. B. Normal Flight Muscle Protein Content With the exception of T r i a l 1, f l i g h t muscle dry weight and protein content usually varied s i g n i f i c a n t l y (P = 0.05) with age in normal males and females (Table VI). In males, changes i n the protein content and dry weight of the f l i g h t muscles paralleled each other (Fig. 9). In both t r i a l s , these two parameters increased rapidly immediately a f t e r ecdysis u n t i l days 5-7, when they decreased. Similar results were also observed i n females (Fig. 10). Although the protein content and dry weight of the f l i g h t muscles were highly correlated, i t i s apparent that smaller muscles contained more protein per mg dry weight than the larger muscles (Table VII; Fig. 11). The slopes of the regression lines of these two parameters were sim i l a r for the two sexes i n both t r i a l s . In t r i a l 1, the regression slopes for males and females were 0.54 and 0.46, respectively, whereas i n t r i a l 2 they were 0.62 and 0.58. Analysis of the residuals confirmed the normal d i s t r i b u t i o n of residuals with respect to age. TABLE VI: Analysis of variance for f l i g h t muscle dry weight and protein content during the f i r s t 9 days of adulthood Males Females Tissue parameter T r i a l D.F. F value Significance T r i a l D.F. F value Significance Flight muscle dry weight 1 7,15 2.34 2 8,18 11.96 .0793 NS .0000 7,16 8,18 2,58 7.06 .0554 NS .0003 Flight muscle protein content 1 7,14 2.32 2 8,18 13.36 .0858 NS .0000 7,15 8,18 4.38 6.01 .0079 .0008 NS = not si g n i f i c a n t (P = 0.05) /4'2 T r i a l 1 18 M H 10 c3 UJ I i i 2 3 4 5 6 AGE (days) 8 9 T r i a l 2 J T t 5 $ I $ 2 J !_ 1 2 3 4 5 6 7 AGE (days) 8 9 FIGURE 9: Changes i n dry weight (A) and protein content (A) of f l i g h t muscles i n normal males during early adulthood. /43 20 15 H H 10 T r i a l 1 _1_ 2 3 4 5 6 AGE (days) 5 I T r i a l 2 I P 1 JL 8 a 3 4 5 6 AGE (days) 8 9 FIGURE 10: Changes i n dry weight (®) and protein content (o) of f l i g h t muscles i n normal females during early adulthood. For significance of d a i l y changes in- dry weight see Appendix 3. TABLE VII: Linear regression equations showing relationship between protein content (Y) and dry weight (X) of the f l i g h t muscles during the f i r s t 9 days of adulthood Sex T r i a l D.F. F value Significance Regression equation Males Females 1,20 1,25 1,21 1,25 13.89 341.81 39.60 211.31 .0013 .0000 .0000 .0000 Y = 0.46 + 0.54 X Y = 0.13 + 0.62 X Y = 1.05 + 0.46 X Y = 0.88 + 0.58 X /45 Y . = 0.46 + 0.54 X male Y - . = 1.05 + 0.46 X female T r i a l 1 r 8 -r Z o " 6 w 5 E~ O U J 1 2 1 2 3 4 5 6 7 8 9 10 11 Y_ , = 0.88 + 0.58 X female Y . = 0.13 + 0.62 X male T r i a l 2 1 2 3 4 5 6 7 8 9 10 11 FLIGHT MUSCLE DRY WEIGHT (mg) FIGURE 11: Regression lines showing the relationship between the protein content and the dry weight of f l i g h t muscles i n normal males ( ) and females ( ) during early adulthood. /46 C. JHA Studies (a) Solvent Trials The s e n s i t i v i t y of f i f t h i n star nymphs to topical application of 1 u l of three solvents i s shown i n Table VIII. Three days after application, both o l i v e o i l and a 1:1 mixture of ol i v e oil/acetone resulted i n delayed nymphal development and at least 50% mortality. In a l l instances, mortality occurred during the imaginal molt as the insects appeared unable to cast t h e i r exuvium. Because acetone exhibited no deleterious e f f e c t s , i t was chosen as the appropriate solvent i n a l l subsequent JHA studies. (b) JHA Dose-Response Trials The s e n s i t i v i t y of different stages of M. sanguinipes to high dosages of R-20458 i s shown i n Table IXa. At higher doses (0.375 and 0.75 ug), the JHA k i l l e d most of the grasshoppers. Mortality was higher when R-20458 was applied to young and old f i f t h instars than when the treatment was applied during the middle of the stadium. Some of the surviving insects which had been treated with R-20458 i n the middle of the f i f t h i n star underwent a supernumerary molt to become large nymphal-adult intermediates with thick legs and varying color and wing length (Plates 3a and b). Sumpernumerary nymphs often died soon after the molt. Treated insects which retained the normal molting sequence often became adults with short and/or wrinkled wings. They were green or yellow in color, especially on the ventral portion of the abdomen. /47 TABLE VIII: Effects of t o p i c a l application of three solvents to f i f t h i n star nymphs 3 days after treatment No. insects No. surviving No. surviving No. Solvent treated nymphs adults died o l i v e o i l 6 1 2 3 oil/acetone (1:1) 6 1 1 4 acetone 6 0 6 0 TABLE IXa: S e n s i t i v i t y of different stages of M. sanguinipes to high dosages of the JHA, R-20458 JHA cone. Stage applied No. insects treated Mortality No. normal adults 0.75 yg ADULTS < 2 h-old 1 to 2 days-old 5th INSTAR late 0.375 yg 4th INSTAR early late 5th INSTAR early middle late 5th INSTAR newly ecdysed to 60-h-old 72-h-old 80-h-old to 85-h-old 95-h-old to 120-h-old 3 3 6 6 11 26 11 11 6 6 6 3 0 6 5 9 17 10 11 2 6 4 0 3 N/A 0 0 0 1 0 0 0 0 No. abnormal adults 0 0 N/A 0 1 0 4 0 2 Morphological and color effects no observable effects no observable effects no other effects observed no observable effects yellow color nymphal period prolonged; 1 very green yellow or green; some with short wings; 5 insects underwent a supernumerary molt yellow color no observable effects very green; 3 showed supernumerary molting no observable effects yellow-green color 0 0 /49 PLATE 3a: Abnormally large female in supernumerary stadium (right) r e s u l t i n g from JHA application (0.375 yg) to f i f t h i n s tar M. sanguinipes. For comparison, a normal untreated female ( l e f t ) i s also shown. PLATE 3b: Normal, untreated male (left) and large, green, short-winged supernumerary male (right) r e s u l t i n g from JHA treatment described above. The treated insects had d i f f i c u l t y casting t h e i r exuvia. /50 When a lower concentration (0.037 yg) was applied, mortality was infrequent and occurred only at the imaginal molt (Table IXb). Several normal-looking adults were produced, while others exhibited the characteristics observed at the higher dosages. An intermediate concentration of 0.075 yg JHA gave negli g i b l e mortality and a high frequency of eas i l y observable JHA effects. To further minimize l e t h a l effects, 0.05 yg R-20458 was applied i n subsequent studies. D. Sensitivity of Fifth Instars to R-20458 Single applications of 0.05 yg R-20458 to f i f t h instar insects of s l i g h t l y varying ages resulted i n dramatically different external and internal effects. JHA application to newly emerged f i f t h instar males and females produced mixed effects, ranging from insects which molted into normal-looking adults to those which became adultoids with pronounced nymphal ch a r a c t e r i s t i c s . Juvenile ch a r a c t e r i s t i c s exhibited by the l a t t e r included short, wings, reddish heads and pale cu t i c l e s marked with black (Plate 4a). A d i s t i n c t , pale s t r i p e extended the length of the pronotum. In normal adults, the pronotal stripe extends only halfway up the pronotum then blends into the beige-brown c u t i c l e . When the same dose of JHA was applied to 4-day-old f i f t h i n s tar nymphs, the insects molted into normal-looking adults with long wings, beige-brown c u t i c l e , and an i n d i s t i n c t pronotal stripe TABLE IXb: S e n s i t i v i t y of different stages of M. sanguinipes to low dosages of the JHA, R-20458 No. No. No. J H A Stage insects normal abnormal Morphological and cone. applied treated Mortality adults adults color effects 0.037 yg 5th INSTAR misc, 5-h-old 20-h-old 48-h-old 68-h-old 0.075 yg 5th INSTAR mostly middle 12 3 2 3 3 15 0 0 1 0 N/A 1 0 1 0 N/A 2 2 1 3 14 many had shortened wings and yellow or green coloring; 1 underwent a supernumerary molt short wings wrinkled wings; one with short wings green color a l l s l i g h t l y green green or yellow-green color; 4 underwent a supernumerary molt /52 PLATE 4a: External morphology of 2-day-old adult females that were treated with 0.05 yg R-20458 as newly emerged f i f t h i n star nymphs.' An untreated adult i s shown on the far r i g h t . PLATE 4b: Two-day-old adult males showing the effect of a single application of 0.05 yg R-20458 to ( l e f t to right) 4-, 5-, and 6-day-old f i f t h i n s t a r nymphs. An untreated grasshopper of comparable age i s shown on the far r i g h t . /53 (Plate 4a). Similar JH treatments applied to f i f t h instars from late day 4 to early day 5 usually resulted i n adults with green-brown to bright green c u t i c l e s and s l i g h t l y shortened to very short tegmina and wings (Plates 4b, 5). Maximum green color was usually seen around day 5 of adulthood. The d i s t i n c t pale pronotal s t r i p e observed i n grasshoppers which received JHA at f i f t h i n star emergence was also present i n the short-winged, green insects. Although the tegmina and wings are of equal length i n normal M. sanguinipes, treated insects often had shorter wings than tegmina. Furthermore, i n some insects, the tegmen and wing were of normal length on one side of the- body but shorter on the other. The same JHA dose applied to 5- or 6-day-old f i f t h - i n s t a r s produced l i t t l e e f f e c t , although a fa i n t green coloration and s l i g h t wing shortening occurred i n some of the adults. Tables Xa and b compare the body measurements of normal 5-day-old adults (average of two previous t r i a l s ) and those that had been treated with 0.05 yg JHA during the f i f t h i n s t ar. In males, JHA application resulted i n s i g n i f i c a n t (P = 0.05) reductions i n t o t a l fresh body weight, tegmina length, wing length, and f l i g h t muscle dry weight. Except i n t r i a l 2, the tegmina-wing length and f l i g h t muscle dry weight were s i g n i f i c a n t l y reduced i n JHA-treated females. Both sexes experienced s i g n i f i c a n t changes i n gonad dry weight after JHA application i n t r i a l 1 but not i n t r i a l 2. Ti b i a lengths, head widths, and fat body dry weights were not s i g n i f i c a n t l y d i f f e r e n t from those of untreated insects. /54 PLATE 5: Two-day-old adult males, showing the effect of a single application of 0.05 yg R-20458 to (a) 5-, (b) 5-, and (c) 6-day-old f i f t h instar nymphs. An untreated control (d) i s also indicated. TABLE Xa: Comparison of various body parameters of normal 5-day-old male adults and those treated with 0.05 yg R-20458 at various intervals during the f i f t h stadium* Body parameter Untreated JHA T r i a l 1 JHA T r i a l 2 Mean 1 S.D. Mean ± S.D. D.F. F value Significance Mean 1 S.D. D.F. F value Significance Total body fresh weight (og) 387.4±66.8 323.6138.5 1,20 7.70 .0121 327.8131.7 1,22 8.62 .0079 T i b i a length (mm) 10.8±0.6 10.710.5 1,20 0.00 .9494 NS 10.610.5 1,20 0.15 .6988 NS Tegmina length (mm) 19.3±1.6 15.213.5 1,20 7.72 .0120 IS.113.9 . 1,22 6.47 .0189 Wing length (mo) 19.311.6 13.713.7 1,20 12.41 .0023 14.513.6 1,22 9.79 .0051 Head width (mm) 4.0*0.2 3.910.2 1,20 2.04 .1692 NS 3.810.1 1,22 5.78 .0255 Gonad dry weight (mg) 10.312.6 7.911.3 1,20 8.78 .0080 9.211.7 1,22 1.57 .2241 NS Fat body dry weight (mg) 11.7±4.0 8.513.5 1,20 3.23 .0883 NS 9.512.6 1,22 2.28 .1460 NS Flight muscle dry weight (mg) 14.113.7 9.812.0 1,20 12.20 .0024 9.812.5 1,22 10.41 .0040 •In Tables Xa-XIb, R-20458 was applied on days 1, 3, 4, 5 or 6 of the f i f t h stadium i n T r i a l 1 whereas in T r i a l 2, the JHA was applied on days 1, 2, 4, 4 1/2, 4 3/4 or 5 of the f i f t h stadium. TABLE Xb: Comparison of various body parameters of normal 5-day-old female adults and those treated with 0.05 vg R-20458 at various intervals during the f i f t h stadium* Body parameter Untreated JHA T r i a l 1 JHA T r i a l 2 Mean t S.D. Mean t S.D. D.F. F value Significance Mean ± S.D. D.F. F value Significance Total body fresh weight (mg) 475.8+85.2 431.8±66.8 1,20 1.59 .2222 NS 448.6+47.0 1,20 0.90 .3555 NS Ti b i a length (mm) 11.4±0.6 11.7±0.6 1,20 1.48 .2383 NS 11.7±0.4 1,19 2.01 .1734 NS Tegmina length (mm) 20.0tl.6 16.0±3.6 1,20 6.69 .0181 17.5±2.9 1,20 3.78 .0667 NS Wing length (mm) 20.0±1.6 1S.0+.3.7 1,20 9.71 .0057 16.4+3.1 1,20 7.02 .0158 Head width (mm) 4.2±0.2 4.2±0.2 1,20 0.07 .7880 NS 4.2+0.1 1,20 0.17 .6828 NS Gonad dry weight (mg) 26.6±3.9 13.5+11.8 1,20 6.82 .0172 33.9±14.7 1,20 1.41 .2496 NS Fat body dry weight (mg) 24.8+8.4 20.3+6.6 1,20 1.71 .2061 NS 18.8+6.7 1,20 2.94 .1028 NS Flight muscle dry weight (mg) 17.5±4.8 12.0±2.7 1,20 11.07 .0024 12.0+1.8 1,20 15.02 .0010 /57 Tables XIa and b show the effects of R-20458 on adult body measurements when 0.05 ug of the JHA was applied at various times during the f i f t h stadium. In general, the timing of JHA application only had a s i g n i f i c a n t effect upon the fat body, tegmina, and wings of 5-day-old adults. In some cases s i g n i f i c a n t changes i n ovarian dry weight, t i b i a length, and f l i g h t muscle dry weight were also produced. However, these changes were not consistent enough to provide a basis for accurately predicting the size of these parameters from JHA application time (Appendix 4). Correlations among body parts were much lower i n JHA-treated than i n normal grasshoppers, and varied with sex and with t r i a l (Figs. 12a and b). E. Effects of Adult Aging on JHA-Treated Insects Tables XIla and b show the various body measurements i n normal insects and those treated with R-20425 as newly emerged f i f t h instars. The measurements were taken 5 or 6 and 14 days after adult emergence. Treated insects had shorter than normal tegmina and wings i n both age groups and female gonad dry weight was variably altered. Five- to 6-day-old males and females exhibited reduced f l i g h t muscle dry weight after f i f t h i n star JHA treatment. However, no reduction i n f l i g h t muscle dry weight was seen i n 14-day-old adults after the same treatment. The main effect of aging on the body parameters was reduced fat body dry weight v i z . i n both sexes, the weight i n 14-day-old insects was about h a l f that of 5- to 6-day-old insects. TABLE XIa: Overall effects of R-20458 on adult male body measurements when applied at various times during the f i f t h stadium Body parameter T r i a l 1 T r i a l 2 D.F. F value Significance D.F. F value Significance Total body fresh weight (mg) 4,14 2.86 .0808 NS 5,16 0.63 .6833 NS Ti b i a length (mm) 4,14 5.07 . .0170 5,16 0.69 .6423 NS Tegmina length (mm) 4,14 23.12 .0000 5,16 25.23 .0000 Wing length (mm) 4,14 30.78 .0000 5,16 16.43 .0001 Head width (mm) 4,14 3.03 .0704 NS 5,16 0.37 .8561 NS Gonad dry weight (mg) 4,14 1.31 .3313 NS 5,16 2.91 .0653 NS Fat body dry weight (mg) 4,14 4.74 .0210 5,16 3,56 .0370 Fl i g h t muscle dry weight (mg) 4,14 4,65 .0222 5,16 1.14 .3974 NS On OO TABLE Xlb: Overall effects of R-20458 on adult female body measurements when applied at various times during the f i f t h stadium Body parameter T r i a l 1 T r i a l 2 D.F. F value Significance D.F. F value Significance Total body fresh weight (mg) 4,14 2.25 .1365 NS 4,14 1.19 .3742 NS Ti b i a length (mm) 4,14 6.60 .0072 4,12 3.08 .0744 NS Tegmina length (mm) 4,14 5.76 .0114 4,14 14.27 .0004 Wing length (mm) 4,14 20.84 .0001 4,14 41.52 .0000 Head width (mm) 4,14 1.86 .1945 NS 4,14 0.21 .9267 NS Gonad dry weight (mg) 4,14 111.66 .0000 4,14 1.97 . .1749 NS Fat body dry weight (mg) 4,14 5.08 .0170 4,14 16.67 .0002 Flight muscle dry weight (mg) 4,14 2.46 .1132 NS 4,14 3.77 .0403 Fresh Weight Tibia Length Tegmina Length Head Width Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight (i) T r i a l 1 Fresh Weight Tibia Length Tegmina Length Head Width ( i i ) T r i a l 2 Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight FIGURE 12a: Correlations among body measurements i n male adults after R-20458 was applied during the f i f t h stadium. Negative correlations i n t h i s and succeeding figure are denoted by dotted l i n e s . /61 Fresh Weight Tibia Length Tegmina Length Head Width Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight (i) T r i a l 1 Fresh Weight Tibia Length Tegmina Length Head Width ( i i ) T r i a l 2 Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight FIGURE 12b: Correlations among body measurements i n female adults after R-20458 was applied during the f i f t h stadium. TABLE X l l a : Mean body measurements (1S.D.) i n untreated and JHA-treated males dissected as 5- or 6-day-old adults, or 14-day-old adults, (N = 3 to 6) Body parameter 5- or 6--day-old adults 14-day-old adults Untreated JHA-treated Untreated JHA-treated Total body fresh weight (mg) 387.4166.8 361.2130.5 344.8140.1 318.6122.7 Tib i a length (mm) 10.8±0.6 11.110.5 10.711.0 10.610.0 Tegmina length (mm) 19.3±1.6 16.411.3 18.711.6 10.611.8 Wing length (mm) 19.311.6 11.811.0 18.711.6 10.611.8 Head width (mm) 4.010.2 4.010.1 3.910.2 3.910.1 Gonad dry weight (mg) 10.312.6 8.013.2 11.2+1.1 11.711.8 Fat body dry weight (mg) 11.714.0 9.710.6 5.811.0 5.711.5 Flight muscle dry weight (mg) 14.113.7 11.811.0 12.012.1 12.010.7 ON TABLE X l l b : Mean body measurements (±S.D.) i n untreated and JHA-treated females dissected as 5- or 6-day-old adults, or 14-day-old adults (N = 3 to 6) Body parameter 5- or 6-day-old adults 14-day-old adults Untreated JHA-treated Untreated JHA-treated Total body fresh weight (mg) 475.8±85.2 383.7±80.3 427.4192.0 420.1156.7 Tibia length (mm) 11.4±0.6 11.4±0.1 11.610.8 11.110.6 Tegmina length (mm) 20.0±1.6 14.6±2.4 19.911.7 13.012.8 Wing length (mm) 20.011.6 12.6±1.6 19.911.7 11.911.6 Head width (mm) 4.2±0.2 4.1±0.2 4.210.3 4.210.2 Gonad dry weight (mg) 26.6±3.9 6.7±2.4 33.311.81 51.8122.2 Fat body dry weight (mg) 24.8±8.4 19.9±7.0 9.214.6 7.112.0 Flight muscle dry weight (mg) 17.5+4.8 11.0±2.6 10.612.2 9.911.1 764 Correlations between body measurements were reduced with age and with JHA treatment i n both sexes (Fig. 13a-d). In 13-day-old, JHA-treated males and females, the only s i g n i f i c a n t correlation (P = 0.05) was between tegmina and wing length. F. Sensitivity of M. sanguinipes to Precocene II Table XIII shows the dose response of M. sanguinipes to precocene II applied at various times during the fourth, f i f t h , and adult instar s . Precocious metamorphosis was produced i n early fourth instars when doses of 200-300 ug precocene were applied (Plates 6a and b). Lower doses had no apparent effect on adult emergence while doses exceeding 400 yg produced high mortality (Table X I I I ) . Two of the survivors of these high precocene doses exhibited JHA-like effects, namely short wings and juvenile coloration. Precocene II did not resul t i n precocious metamorphosis when applied to f i f t h i n s tars, although applications of 400-500 yg caused some mortality. In two cases, the ovaries of 5-day-old adult females remained undeveloped. Newly-emerged adults experienced some mortality and growth retardation when 1000 yg precocene II was applied. At least 500 yg was needed for any effect on newly-emerged adults and.ovarian development appeared to be normal. However, more extensive tests would be necessary to confirm t h i s finding. (i) Untreated insects FIGURE 13a: The e'ffect of aging and JHA treatment on correlations among body measurements i n 5- or 6-day-old adult males. Fresh Weight Tibia Length Tegmina Length Head Width Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight (i) Untreated Fresh Weight Wing Length Tibia Length Tegmina Length Head Width ( i i ) JHA-treated Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight FIGURE 13b: The effect of aging and JHA treatment among body measurements i n 14-day-old on correlations adult males. FIGURE 13c: The effect of aging and JHA treatment on correlations among body measurements i n 5- or 6-day-old adult females. Fresh Weight Tibia Length Tegmina Length Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Head Width Gonad Dry Weight ( i ) Untreated FIGURE 13d: The effect of aging and JHA treatment on correlations among body measurements i n 14-day-old adult females. TABLE XI I I : S e n s i t i v i t y of various stages of M. sanguinipes to varying dosages of precocene II No. Stage Precocene No. insects No. normal abnormal applied dose (N) No. dead adults adults Effects 4th INSTAR early 100 yg 8 0 early 200 yg 23 7 newly emerged 300 yg 8 0 1-day-old 300 yg 7 2 4-days-old 300 yg - -late 300 yg - -early 400 yg 6 6 misc. 500 yg 6 3 5th INSTAR early 25 yg 5 0 late 25 yg 5 0 misc. 50 yg 31 0 early 100 yg 9 0 early 200 yg 27 0 newly emerged 250 yg 6 0 misc. 300 yg - -newly emerged 400 yg 8 1 late 400 yg 3 0 new 500 yg 4 0 middle 500 yg 7 2 8 0 no effect 11 5 precocious metamorphosis 7 1 long wings, no ovarian development 2 2 precocious metamorphosis - no effect _ _ II 0 0 " 1 2 short wings, juvenile coloring 5 0 no effect 5 0 " 31 0 9 0 " 27 0 " 6 0 " 7 0 " 3 0 " 3 1 undeveloped ovary 4 1 undeveloped ovary; 'tegmina longer than wings (continued). TABLE XIII: (continued). No. Stage Precocene No. insects No. normal abnormal applied dose CN) No. dead adults adults Effects ADULTS young 400 yg newly emerged 500 yg 3-4 days old 500 yg newly emerged 1000 yg 1 day old 1000 yg 1-4 days old 1000 yg 3-4 days old 1000 yg 7 5 6 6 5 5 6 0 0 0 3 0 0 0 7 5 6 0 5 5 6 0 0 0 3 0 0 0 no effect growth retarded s l i g h t l y PLATE 6a: Dorsal view of untreated ( l e f t ) male adult and precocious male adultoid (right) r e s u l t i n g from precocene application (200 yg) to newly emerged fourth instars. PLATE 6b: Side view of untreated ( l e f t ) female adult and precocious female adultoid (right) r e s u l t i n g from precocene application (200 yg) to newly emerged fourth in s t a r s . Ill G. JHA Effects on Precocene-Treated Insects (a) Precocene Effects When precocene II (300 pg/insect) was applied to early- fourth i n s t a r nymphs, approximately 80% of the insects molted precociously into permanent adultoids (pseudo-adults), skipping the f i f t h i n star (Plates 6a and b). These adultoids had fresh weights about half those of normal insects (Table XIV). Gonads, fat body, and f l i g h t muscles were also about half normal size. The precocene-treated adultoids were not only smaller •v than normal, but t h e i r body proportions were also d i f f e r e n t . T i b i a length and head width were about three-quarters that of control insects, while tegmina. and wing length were less than h a l f the size of normal appendages. (b) JHA Applied to Adultoids Table XV shows the effect of a single application of JHA (0.05 pg) to precocious adultoids 6-10 days after the f i n a l molt. S i g n i f i c a n t reductions (P = 0.05) i n fat body dry weight were produced i n both males and females. In females, ovarian dry weight and t o t a l fresh body weight were s i g n i f i c a n t l y increased. No s i g n i f i c a n t difference was seen i n the size of the fixed s c l e r i t e s following JHA treatment, except for an apparently s i g n i f i c a n t increase i n t i b i a length. Figs. 14a and b show the correlations among body measurements i n precocene- and precocene-JHA-treated insects. TABLE XIV: Mean body measurements (± S.D.) of 6- to 10-day-old normal and precocene-treated adults. Precocene (300 yg) was applied to newly emerged fourth instars Male Females Body Precocene- Precocene- parameter Untreated treated Untreated treated N 6 3 6 2* Total body fresh weight (mg) 341.7±29.1 160.312.7 478.4±64.5 162.318.2 T i b i a length (mm) 10.2±0 . 4 8.1±0.1 11.510.5 8.010.0 Tegmina length (mm) 19.2±1.3 6.5±1.0 20.311.0 5.010.4 Wing length (mm) 19.2±1.3 6.5±1.0 20.311.0 5.010.4 Head width (mm) 3.9±0.1 3.1+0.2 4.210.2 3.310.1 Gonad dry weight (mg) 10.3+1.4 5.1±0.8 37.5112.8 2.110.2 Fat body dry weight (mg) 9.112.8 6.4±0.5 17.013.9 15.912.4 Fl i g h t muscle dry weight (mg) 13.2+2.3 6.1±0.4 15.013.3 7.010.4 * An anomalous female which molted precociously after precocene treatment, and produced mature eggs, was not included in the analysis. --4 TABLE XV: Effect of 0.0S ug JHA applied a f t e r the f i n a l molt to precocene-treated adultoids Body parameter Males Females Precocene only Precocene + JHA Precocene only Precocene + JHA Means ± S.D. Means ± S.D. D.F. F value Significance Means ± S.D. Means 1 S.D. D.F. F value Significance Total body fresh weight (mg) Ti b i a length (mm) Tegmina length (mm) Wing length (mm) Head length (mm) Gonad dry weight (mg) Fat body dry weight (mg) Flight muscle dry weight (mg) 160.312.7 110.0 5+1.0 5+1.0 1+0.0 1+0.8 .4+0.5 .1±0.4 143.0+12.6 8.0+0.0 6.2+0.8 6.110.8 3.2+0.0 4.7+1.0 3.5+1.8 5.211.5 1,5 1,S 1,5 1,5 1,5 1,5 1,5 1,5 5.45 1.50 0.24 0.24 0.50 0.25 6.99 1.12 .0789 NS .2879 NS .6476 NS .6476 NS .5185 NS .6453 NS .0574 .3482 NS 162.3+8.2 8.0+0.0 5.010.4 5.010.4 3.310.1 2.110.2 15.912.4 7.010.4 190.114.1 8.410.1 6.910.9 6.910.9 3.310.1 12.913.6 8.612.7 5.710.9 1.4 1,4 1,4 1,4 1,4 1.4 1,4 1,4 27.79 18.15 7.14 7.14 0.36 33.41 9.45 3.93 .0133 .0237 .0755 NS .0755 NS .5908 NS .0103 .0544 .1416 NS /75 JHA application increased the correlation amongst body parameters in males but not i n females. Comparison of Figs. 6 and 14a and b indicates that there was far less c o r r e l a t i o n between body measurements i n insects which had been treated with precocene than i n normal insects. JHA Applied After Precocene but Prior to the Next Molt Table XVI summarizes the effects of precocene II and subsequent JHA applications applied to fourth instars. Six of the 8 precocene-treated insects molted precociously into adultoids (Plate 7a) while 2 grasshoppers developed into normal adults. JHA applications at different times during the fourth instar stadium resulted i n a graded series of morphological effects. When R-20458 was applied to 4-day-old precocene-treated insects, a l l survivors molted into f i f t h instar insects (Plate 7b) and eventually into normal-looking adults. However, when the JHA was applied 1 day l a t e r i n the stadium only 2 of 7 insects eventually molted into normal-looking adults. The remaining insects molted into adult-nymphal intermediates (semi-adultoids) which subsequently died attempting a further molt (Plate 7C). When R-20458 was applied during the si x t h day of the stadium, a mixed response resulted (Plate 7d). Two insects died attempting the imaginal molt, whereas 2 molted precociously into adultoids. The remaining 4 insects developed into normal adults. Fresh Weight Tibia Length Tegmina Length Head Width ( i ) Precocene /76' Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight FIGURE 14a: Correlations among body measurements i n male adultoids treated with precocene II as fourth instars and with R-20458 after t h e i r precocious molt. ( i ) Precocene Fresh Weight Tibia Length Tegmina Length Head Width Wing Length ^Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight ( i i ) Precocene + JHA FIGURE 14b: Correlations among body measurements i n female adultoids treated with precocene II as fourth instars and with R-20458 after t h e i r precocious molt. TABLE XVI: Overall effects of insects at various 0.05 yg R-20458 intervals p r i o r applied to precoc to the next molt ene-treated Treatment # Insects Mortality # Adults # Adultoids Summary Precocene only 8 0 ? 1 eT 1 5 1 true adultoids and normal reproducing adults Precocene + JHA on day 4 8 1 ? 4 <r 3 0 0 a l l normal-looking adults Precocene + JHA on day 5 7 5 ? 1 °* 1 0 0 semi-adultoids (died molting), or non- reproducing adults Precocene + JHA on day 6 8 2 (molting) ? 2 c 2 1 1 died molting, or true adultoids, or reproducing adults CO /79 PLATE 7a: F i f t h instar nymph (right) and three precocious adultoids ( l e f t ) r e s u l t i n g from precocene (300 ug) application to 1-day-old fourth instar nymphs. PLATE 7b: Normal-looking nymphs which received a single precocene application (300 yg) as 1-day-old fourth instars followed by 0.05 yg R-20458 on day 4 of the same stadium. /80 PLATE 7c: Two semi-adultoids (far left and far right) which later died attempting another molt, and two f i f t h instar nymphs (center) which became non-reproducing adults. The insects were treated as mentioned previously, except that the JHA was applied on day 5 of the fourth stadium. PLATE 7d: Two true adultoids (left) and two f i f t h instar nymphs which later became reproductive adults. The insects were treated as above, except that the JHA was applied on day 6 of the fourth stadium. /81 The morphological effects of precocene II and subsequent JHA applications applied to fourth instars are shown i n Table XVII. When R-20458 was applied to 4-day-old precocene-treated insects, the surviving adults of both sexes had smaller than normal t i b i a length and head width, and shortened t i b i a and wings. Mean wing length was also shorter than tegmina length. Total fresh body weight, f l i g h t muscle dry weight, and gonad dry weight were reduced i n both sexes, but p a r t i c u l a r l y i n the females. When the JHA was applied one day l a t e r , the insects which molted into adults exhibited a pronounced reduction i n fresh body weight and gonad dry weight i n both sexes. Head width and tegmina and wing length were s l i g h t l y reduced, but the tegmina and wings were of equal length. In the females, the dry weight of the f l i g h t muscles, but not of the fat body, was lower than i n normal insects. In the male, however, both the fat body and f l i g h t muscles were severely reduced. Survivors of R-20458 application to 6-day-old fourth instars previously treated with precocene produced 2 precocious adultoids s i m i l a r to those described previously (Table XIV). However, the f l i g h t muscle dry weight of males was even less than that seen i n insects treated with precocene alone. The 4 insects which became r e l a t i v e l y normal adults nevertheless exhibited shortened wing and tegmina lengths and s l i g h t l y reduced f l i g h t muscle dry weight, p a r t i c u l a r l y i n the males. Wings were shorter than tegmina. TABLE XVII: Effects of timed JHA applications to precocene-treated fourth i n s t a r s . Measurements were taken 4-5 days after adult emergence Males Females Time of JHA application Time of JHA application Precocene Day 6 Precocene Day 6 Body parameter only Day 4 Day 5 precocious normal only Day 4 Day 5 precocious normal N 1 3 1 1 2 5 3 1 1 2 Total body fresh weight (mg) 193.6 270.0+22.7 133.9 143.9 312.0+13.2 178.8123.8 321.0+22.3 261.2 185.2 399.5139.; T i b i a length (mm) 8.5 9.6+0.2 10.5 7.8 10.310.6 8.110.3 10.310.9 10.5 9.9 11.110.4 Tegmina length (mm) 5.9 • 16.4+2.3 17.7 6.3 18.4+0.1 6.610.6 17.011.2 17.2 6.2 19.210.7 Wing length (mm) 5.9 15.2±2.7 17.7 6.3 17.411.3 6.610.6 14.610.9 17.2 6.2 18.510.7 Head width (mm) 3.4 3.6±0.2 3.6 2.9 3.710.1 3.310.2 3.810.2 3.7 3.2 4.110.1 Gonad dry weight (mg) 6.3 7.1*1.1 3.0 4.3 9.3+1.3 2.010.5 5.2+5.6 2.2 1.8 40.813.0 Fat body dry weight (mg) 9.5 7.613.7 0.5 4.0 7.810.8 14.312.1 15.016.4 15.2 14.2 12.411.4 Flight muscle dry 7.1 7.5±1.9 2.7 3.7 8.3+1.3 7.2+0.7 8.510.9 9.5 5.7 11.710.8 weight (mg) 00 /83 DISCUSSION A. Normal Development As i n other a c r i d i d species ( P f e i f f e r , 1945; H i l l et al., 1968) newly emerged females of M. sanguinipes undergo two d i s t i n c t phases of development ( G i l l o t t and E l l i o t t , 1976). The i n i t i a l phase of somatic development, which was reported to involve growth of the c u t i c l e , f l i g h t muscles and alimentary t r a c t , i s completed within 3 days of emergence. After reaching a basic weight, mated females undergo successive periods of reproductive growth that take 2 to 4 days to complete. In the current study, a biphasic growth pattern was demonstrated i n both sexes. At a l l adult stages investigated, the t o t a l body weight of males was consistently less than that of females. However, during the somatic growth phase, the overall growth rates, as judged by the regression slopes, were comparable i n the two sexes, e.g. D m a i e = 23.8, D £ e m a i e = 26.3. After t h i s period, growth rates diff e r e d markedly. In females, the t o t a l body weight increased s i g n i f i c a n t l y (b = 15.8) u n t i l day 8 when oviposition occurred. As reported previously ( G i l l o t t and E l l i o t t , 1976; E l l i o t t and G i l l o t t , 1978), t h i s increase i s largely due to ovarian growth and protein accumulation by the developing oocytes. In contrast, the t o t a l body weight of males did not increase s i g n i f i c a n t l y 4 to 9 days after emergence (b = 4.6). The absence of weight increases i n the male comparable to those i n the female reflects.- several fundamental differences /84 i n the reproductive physiology of the sexes. F i r s t l y , the dry weight of the testes-accessory gland complex contributes l i t t l e to the t o t a l body weight of the male. The complex, which weighed 6 mg on day 3, reached a maximum of 10 mg on day 5 when the weight s t a b i l i z e d . This increase i s largely attributable to the growth of the accessory glands. Apart from demonstrating that males become reproductively mature e a r l i e r than females, the stable weight of the testes-accessory gland complex appears to resul t from the promiscuous behavior observed i n male M. sanguinipes (Friedel and G i l l o t t , 1977). After day 5, reproductively-active males may copulate several times daily^and, on each occassion, transfer about seven spermatophores into the female (Pickform and G i l l o t t , 1972). Proteins, which are a major constituent of the spermatophore, are synthesized i n the fat body and sequestered by the accessory glands p r i o r to mating ( G i l l o t t and F r i e d e l , 1976). Like v i t e l l o g e n i c proteins ( E l l i o t t and G i l l o t t , 1979), these accessory gland proteins are eventually deposited as yolk i n the developing oocytes (Friedel and G i l l o t t , 1977). Therefore, i n females, proteins produced by both sexes accumulate i n the ovary throughout the entire reproductive period u n t i l p r i o r to oviposition. In contrast, multiple copulations by the male re s u l t i n the d a i l y discharge of proteinaceous products by the accessory glands. Thus, cumulative weight changes i n the gonads and t o t a l body weight observed in females are not evident i n males. Changes i n the dry weight of the fat body i n females concurred with those recorded for female M. sanguinipes ( G i l l o t t and /85 E l l i o t t , 1976) and S. gregaria ( H i l l et al., 1968). The dry weight was small from adult emergence u n t i l day 3, when a rapid increase began. The dry weight peaked at day 5, then decreased u n t i l oviposition occurred. The decrease i n the dry weight of the fat body i s accompanied by a rapid increase i n ovarian dry weight. E l l i o t t and G i l l o t t (1976, 1978) have shown that i n female M. sanguinipes the CA controls yolk protein synthesis i n the fat body and subsequent protein uptake by the ovary. Therefore, the CA are responsible for the coordinated.development of these two tissues during early adulthood. In male M. sanguinipes, changes i n the dry weight of the fat body also closely p a r a l l e l e d those of the testes-accessory gland complex during the f i r s t 5 days of adulthood. For the next 4 days, however, the dry weight of the complex remained f a i r l y constant while that of the fat body decreased. These results are consistent with the findings of Friedel and G i l l o t t (1976a,b) who showed i n male M. sanguinipes that the fat body aids i n spermatophore production by synthesizing protein for the male accessory glands. In female Locusta (Poels and Bennakkers, 1969) and Schistocerca (Panar and Nair, 1975), rapid increases i n body weight during early adulthood are associated with rapid f l i g h t muscle development. However, the above studies did not extend into the reproductive period. In adult female S. gregaria such studies by H i l l et al. (1968) have shown a rapid, steady increase i n f l i g h t muscle dry weight u n t i l /86 soon after yolk deposition begins. After t h i s , the weight remained r e l a t i v e l y steady. In M. sanguinipes, sim i l a r changes i n the dry weight of the f l i g h t muscles i n males and females were evident. F l i g h t muscle size increased ra p i d l y during somatic growth, then fluctuated after somatic maturity. Contrary to previous studies on M. sanguinipes ( G i l l o t t and E l l i o t t , 1976), the present investigation has shown that f l i g h t muscle development i s not complete by day 3 but that the dry weight increases u n t i l day 5 when i t declines. F l i g h t muscle dry weight and protein content were interdependent and followed a n e a r l y - p a r a l l e l , fluctuating pattern. Similar findings have been reported i n Loausta (Poels and Beenakkers, 1969; van Marrewijk et al., 1980). The slopes of the regression lines showing the relationship between f l i g h t muscle protein content and dry weight i n both sexes also indicate that there was more protein per unit weight i n small f l i g h t muscles than i n the large ones. Therefore, some of the increase i n f l i g h t muscle dry weight around day 5 appears to be due to the accumulation of non-proteinaceous materials. Protein synthesis i n the f l i g h t muscles of Loausta remains high even after completion of adult development (van Marrewijk et al., 1980). The l a t t e r authors have proposed that i n Locusta the high l e v e l of protein synthesis after maturity i s due to high f l i g h t muscle metabolism and turnover of tissue materials. In both sexes of M. sanguinipes, peaks i n the dry weight of the f l i g h t muscle roughly corresponded to those of the fat body. /87 The dry weight of these tissues i n both sexes decreased as yolk deposition proceeded i n the females. A sim i l a r trend i s found i n several other insects which exhibit high teneral f l i g h t a c t i v i t y , followed by f l i g h t muscle autolysis (Chapman, 1969). At t h i s time, fat body size increases and adult reproductive development begins. "In general it is believed that the degenerating muscles provide essential reserves for egg development. .." (Chapman, 1969). However, M. sanguinipes* f l i g h t muscles do not completely degenerate, they merely decrease i n size p r i o r to the f i r s t oviposition. I t would be interesting to see i f materials from the f l i g h t muscles are incorporated into the fat body to be used i n spermatophore or ovarian development. Analysis of protein i n the f l i g h t muscles using methods sim i l a r to those of G i l l o t t and Friedel (1976), would provide a firmer indication of the potential role of the f l i g h t muscles i n reproduction. In the present studies, the s i g n i f i c a n t correlation among the dry weights of the gonads, fat body, and f l i g h t muscle i n normal grasshoppers of each sex emphasizes the coordinated growth patterns of these organs within individual insects. In addition, the s i m i l a r i t y i n growth patterns of the internal organs such as fat body and f l i g h t muscles of males and females may indicate some type of synchronization between the two sexes. The maturity of male locusts and grasshoppers i s known to influence the developmental rate of females (Riegert,1965), possibly by means of pheromones (Doane, 1973). Interestingly, males may also contribute to the developing oocytes by transferring protein /88 to the females v i a spermatophores (Friedel and G i l l o t t , 1977). I f the fat body and f l i g h t muscles of both males .and females were contributing proteins to the oocytes, s i m i l a r patterns i n the development of these internal organs i n the two sexes would be expected. There i s even recent evidence that JH can be passed from the male to the female during mating i n some insects (#. cecvopia; Shirk et al., 1980). I f a comparable s i t u a t i o n exists i n M. sanguinipes, i t would account for the synchrony of development between the two sexes. Although the influence of the CA on f l i g h t muscle development has not been established i n M. sanguinipes, JH i s known to affect the development of the fat body and gonads. Therefore, i t may be that coordinated changes i n JH levels within a grasshopper or locust population would result i n synchronzied development. B. Role of JH in Development The CA are known to play a c r i t i c a l role i n insect development (Doane, 1973). The c l a s s i c experiments which Wigglesworth performed on Rhodnius prolixus during the 1930's demonstrated that when the CA of young nymphs were s u r g i c a l l y inactivated (decapitation, e x t i r p a t i o n ) , the insects molted prematurely into adults. In contrast, when the CA i n f i n a l i n s t a r nymphs were a r t i f i c i a l l y activated (CA implantation), the nymphs underwent an extra nymphal molt before metamorphosis into the adult stage. These studies prompted Wigglesworth to hypothesize that elevated JH t i t r e s i n the haemolymph of younger nymphal stages /89 suppresses adult d i f f e r e n t i a t i o n and favours the retention of juvenile c h a r a c t e r i s t i c s . Conversely, a precipitous decline i n JH haemolymph t i t r e s during the f i n a l i nstar results i n metamorphosis and the expression of adult c h a r a c t e r i s t i c s . Confirmation of t h i s hypothesis i n Loausta males and females has been provided by more recent studies which have i n d i r e c t l y (Galleria bioassay) or d i r e c t l y (radio-imrnuno assay, electron-capture gas chromatography) measured JH haemolymph levels during nymphal development (Johnson and H i l l , 1973a,b, 1975; Baehr et al., 1979; Huibregtse-Minderhoud et al., 1980). C o l l e c t i v e l y , these studies have shown that high JH levels are present i n the haemolymph throughout most of the fourth (penultimate) stadium whereas JH i s absent throughout most of the f i n a l stadium. However, Baehr et al. (1979) reported a temporary surge i n JH levels at the beginning of the f i f t h i n s tar. In a c r i d i d species, JHA and precocene have been employed as chemical probes to a r t i f i c i a l l y manipulate JH levels during nymphal and adult development. In M.- sanguinipes, the effects of these compounds on development depend upon the stage to which they are applied. As reported i n other species (Kelly and Fuchs, 1978; Kruse Pedersen, 1978; Unnithan and Nair, 1979), t o p i c a l application of 200-300 yg precocene II to recently molted fourth instar nymphs of M. sanguinipes resulted i n a high proportion of the insects molting precociously into diminutive adults. Precocene II tends to have a variable success rate, and 50% effectiveness i s common (Muller et al., /90 1979; Masner et al., 1980; Unnithan et al., 1980). The precocene apparently inactivates the CA (Unnithan and Nair, 1979), and without s u f f i c i e n t endogenous JH, the insects are unable to maintain juvenile c h a r a c t e r i s t i c s for a f i f t h stadium. However, when even higher precocene doses were applied to f i f t h i n s t a r M. sanguinipes, molting and metamorphosis were not disrupted. Comparable findings i n Oncopeltus led Unnithan and Nair (1979) to theorize that prococene only affects insects during those developmental stages i n which the CA are active. I f t h i s theory i s tenable i n M. sanguinipes, the results of the present study substantiate the premise that JH i s absent during part or most of the f i f t h i n star stadium. Two of 6 insects treated with 500 yg precocene as fourth instars and 1 of 7 insects receiving the same dose as mid - f i f t h instars retained juvenile characteristics as adults. The effects resembled those of R-20458 and included shortened wings, uneven tegmina and wings, and juvenile coloration. Characteristics of JH excess preceeding CA degeneration have also been observed when comparable stages i n Locusta were treated with precocene (Fridman-Cohen and Pener, 1980; M i a l l and Mordue, 1980). These results led M i a l l and Mordue (1980) to suggest that "the effect is probably the result of synthesis or release of JH during the breakdown of the glands". C. Reversing Precocious Metamorphosis with JHA Precocious metamorphosis induced by precocene i n Locusta (Kruse Pedersen, 1978) and Oncopeltus (Masner et al., 1979) has been reversed /91 by applying JHA during the same stadium. However, i n both species, observations were only carried out into the f i f t h i n star so that the effect on adult morphology was not determined. In the present experiments, a gradation of effects was observed when 0.05 yg R-20458 was applied to fourth instars 4 to 6 days after they were treated with precocene I I . When JHA was applied 4 days after the precocene treatment, the insects molted into f i f t h instars and eventually into normal-looking adults. Apparently the dosage of R-20458 applied at t h i s time was high enough to maintain the juvenile characteristics into the f i f t h and f i n a l nymphal stadium but also low enough so as not to interfere with metamorphosis. The same JHA treatment applied one day l a t e r resulted i n a high number of semi-precocious adults which l a t e r died attempting another molt. R-20458 applied at t h i s time seemed to be too late to program a f i f t h nymphal stadium. Instead, the epidermal c e l l s received c o n f l i c t i n g instructions. I n i t i a l l y , the absence of JH caused by precocene appeared to res u l t i n the epidermal c e l l s being committed to produce an adult c u t i c l e but l a t e r being programmed by the JHA to produce nymphal c u t i c a l and undergo an additional molt. Results of the present study agree with those of Kruse Pedersen (1978), who noted that timing of JHA application i n such experiments was c r i t i c a l i n Locusta, and that the optimum time for JHA-reversal of precocious metamorphosis was between the t h i r d and fourth days of the fourth stadium. In addition, the current results indicate that during normal development, JH levels must be low on the t h i r d and /92 fourth days of the f i f t h i n s t a r stadium. However, when JHA was applied 6 days after the precocene treatment, a mixed effect was observed. The two insects which died attempting a f i n a l molt were probably s t i l l young enough to be affected as i n the previous study. However, two other insects became precocious adultoids, so that the JHA was applied too late to counteract the effects of precocene. It i s d i f f i c u l t to explain why the other four insects molted to normal-looking adults, unless JH application immediately before the molt can also i n i t i a t e nymphal commitment, which seems u n l i k e l y . D. JHA Studies on Molting and Metamorphosis JHA-induced supernumerary molting has been observed i n Zonoaerus (McCaffery and Page, 1978) and i n Locusta (Vogel et al., 1978). CA implants produced s i m i l a r effects i n Locusta and Schistoeerca (Nemec, 1970; Van den Hondel-Franken et al., 1980). None of these papers dealt with the precise JH application time necessary to produce an extra nymphal molt. Theoretically, i f the CA are inactive throughout part of the f i f t h i n star stadium, then timing of JHA applications should be important i n determining the re s u l t i n g effects. This i s p a r t i c u l a r l y true since J H - s e n s i t i v i t y of different tissues i n insects varies with development time (Luscher et al., 1971; W i l l i s , 1974). In M. sanguinipes, supernumerary molting, a symptom of JH-excess, occurred i n approximately one quarter of the insects treated with 0.0375 to 0.375 ug R-20458 i n the middle of the f i f t h stadium. However, /93 no supernumerary molts developed when R-20458 was applied to early or late f i f t h instars. Apparently JHA applications during the mi d - f i f t h i n s t a r stadium are capable of preventing the commitment of epidermal c e l l s and other tissues to adult development. This again suggests that low JH t i t r e s during the middle of the f i n a l stadium are necessary to permit an imaginal molt. However, the present results also indicate that high JH levels during the end of the f i n a l stadium are inconsequential i n terms of molting and metamorphosis. Locust size i s influenced by JH le v e l s . High endogenous JH t i t r e such as those presumably present i n solitaria locusts, favor increased sexual dimorphism with the development of large females and small males (Kennedy, 1961; Poels and Beenakkers, 1969; Beenakkers, 1973; Beenakkers and Van den Broek, 1974). This d i f f e r e n t i a l effect of JH on the two sexes was also observed i n the present experiments. JHA application to f i f t h i n star female M. sanguinipes produced no s i g n i f i c a n t overall change i n fresh body weight of the adult, whereas i n males, the fresh body weight of the adults was s i g n i f i c a n t l y reduced. Contrary to the conclusions of Senhal (1971), the current findings c l e a r l y indicate that JH can serve as a general growth hormone during nymphal development. Correlations among adult body measurements were greatly reduced after precocene or JHA treatment of nymphs. Therefore, not only the size of the insects, but also the body proportions changed and became less related. Although precocious adults generally looked /94 normal (except for the t i n y wings),, there were small changes i n r e l a t i v e body measurements. However, Bowers et al. (1976) writes that i n Oneopeltus "the morphology and coloration of the precocious adults is identical with that of normal adults". Later additions of JHA to precocious adult males increased the correlations among body measurements, as f l i g h t muscle and gonad dry weights were more closely proportional to the size of some of the fixed s c l e r i t e s . However, female precocious adults, treated with JHA, showed no improvement i n body measurement correlations. These results indicate that i n males, JH stimulates development of internal organs u n t i l they reach a basic r e l a t i v e l y stable l e v e l i n proportion to the other body parameters. However, i n females, JH stimulates ovarian dry weight independently from that of most other body parts. The metamorphic and coloration changes that occur with phase change i n many species have been attributed to JH (Table 1). Characteristics of the r e l a t i v e l y sedentary solitarious phase have been produced by CA implants or JHA applications (Joly, 1960, cit e d i n Uvarov, 1966; Staal, 1961; Doane, 1973). CA-implanted or JHA-injected grasshoppers (Rowell, 1967) and locusts (Kruse Pedersen, 1978) usually turned green after the next molt although green haemolymph could be observed i n some cases within the same instar (Rowell, 1967). Results of the present experiments agree with these findings. Reasons for t h i s time lag are probably complex, but may be due i n part to the natural cycle of endogenous JH levels within the insects. In /95 the present experiments, JHA treatment of f i n a l nymphal instar males and females produced a variety of coloration and wing length effects, depending upon when the JHA was added. Males and females treated with 0.05 ug R-20458 just after the middle of the f i f t h instar (days 4-5) became green adults, while those treated on day 6 were only f a i n t l y green or normal-colored. Insects treated with JHA on day 3 became normal-colored adults, while those treated on the f i r s t 2 days of the ins t a r retained juvenile black and beige markings as adults. Wing length was also altered when JHA applications were made at various times during the f i f t h nymphal stadium. Effects on insects treated at the beginning of the f i f t h i n star (newly emerged to day 3) ranged from l i t t l e effect to pronounced metathetaly. Grasshoppers treated on days 3-4 had a normal appearance, where those treated towards the end of the f i f t h i n star (days 4-5) possessed shortened wings. By day 6 JHA application was too late to produce much effe c t , and the insects became nearly normal adults. Apparently JH i s involved i n determining wing length i n M. sanguinipes at the beginning of the f i f t h stadium and around days 4-5, but not during the remainder of the stadium. Interestingly, these two periods correspond with the JH peaks i n f i f t h stadium Locusta (Baehr et al., 1979). I f such peaks are also present i n M. sanguinipes, the addition of JHA during periods of high endogenous JH t i t r e might be implicated i n the production of short wings. I f JH must be low i n m i d - f i f t h instars to allow the imaginal molt, and since no effect on color or wing length i s produced /96 at t h i s time, i t appears that both endogenous JH and JHA applications are needed to affect these two parameters. In M. sanguinipes, JHA applications can bring about the locust solitarious phase characteristics of shortened wings and juvenile coloration. Therefore, M. sanguinipes must have the genetic potential to undergo phase changes i n response to increased JH t i t r e s at pa r t i c u l a r nymphal stages. Results of the present JHA experiments are supported by si m i l a r effects of timed JHA applications to f i n a l nymphal instar Locusta and Schistocerca (Nemec, 1970; Joly and Meyer, 1970). Nemec (1970) categorized the JHA effects on locust species into three groups, including (1) an action on metamorphosis, (2) changes i n phase coloration, and (3) no effect. He concluded that JHA induces multiple effects and that "merely the time of application determined whether a block of metamorphosis or the phase change will occur" (Nemec, 1970). He also stated that the JHA must break down within the insects' bodies i n order for the two diff e r e n t effects to occur. The l a t t e r hypothesis f a i l s to consider the p o s s i b i l i t y that JHA, even when d i r e c t l y applied at a la t e r stage i n the in s t a r , may be too late to produce metamorphic effects i n these insects. I t may even be that JH must be maintained at a certain l e v e l for a p a r t i c u l a r period of time for various observable effects to occur (Van den Hondel-Franken et al., 1980). Unfortunately, i t i s impossible to discern from the present experiments and those of Nemec (1970) the extent to which JHA persistence i s influencing the r e s u l t s . Differences between the results of Nemec and the present experiments may be due to species characteristics or to the JHA /97 applied. In the present investigation, there was considerable v a r i a t i o n of effects between t r i a l s . Since locust development i s extremely sensitive to environmental conditions, d i f f i c u l t y i n obtaining repeatable results i s experienced (Beenakkers and Van den Brock, 1976). Rowell (1967) succeeded i n producing green coloration i n four grasshopper species, some of which never produced green adults i n the wild. However, the CA implants rarely produced metathetalic individuals. The r e l a t i v e absence of t h i s type of effect may be due to the lack of consideration accorded the timing of the implants within the instar i n Rowell's experiments. This oversight places i n doubt the conclusions of Staal (1961), supported by Rowell (1967), "that corpus allatum hormone levels in Locusta had profound effects on green/brown polymorphism, but relatively slight effects on morphometries...it seems clear that there must be in the locusts an inhibitory mechanism responsible for green coloration and that responsible for the gregarious phase, but it cannot be therefore assumed that these effects are opposite extremes of a continuous series". In f a c t , the present experiments and those of Joly and Meyer (1970) and Nemec (1970) showed that JH causes both coloration and morphological effects depending upon application time. I t may be that timing of endogenous JH peaks r e l a t i v e to tissue development i n each individual insect, i s responsible for the wide variety of effects observed i n M. sanguinipes and other species (Riegert, 1965; Beenakkers and Van den Brock, 1974). /98 E. JH Effects on the Development of Gonads, Fat Body, and Flight Muscles High JH levels caused by CA implantations into young f i f t h i n s t a r Locusta females have been implicated as a cause of poor f l i g h t muscle development (Poels and Bennakkers, 1969; Van den Hondel-Franken et al., 1980). In the present experiments, JHA applications to f i f t h i n s tar M. sanguinipes of both sexes produced a s i g n i f i c a n t (P = 0.01) reduction i n the f l i g h t muscle dry weight. However, no s i g n i f i c a n t difference was produced i n f l i g h t muscle dry weight by varying JHA application time within the instar. Apparently the f l i g h t muscles of M. sanguinipes are JHA-sensitive throughout the f i f t h instar.stadium, even though wing length i s only affected at certain times within the stadium. Results of the present experiments do not support the theory of Chudakova et al. (1976) that "the flight apparatus develops as an integrated functioning system rather than as a result of im- aginisation of individual components". After JH treatment, the correlation between various body measurements i s usually reduced, indicating that each tissue responds i n a unique way to the hormone. Adding JHA to precocious adultoids did not s i g n i f i c a n t l y affect f l i g h t muscle dry weight. Apparently JHA added after early adulthood i s too late to affect f l i g h t muscle growth. This indicates that previous commitment of the tissues may be important i n the case of f l i g h t muscles, although not of the fat body as mentioned below. It seems that after early adulthood, JHA cannot a l t e r f l i g h t muscle synthesis even though i t i s capable of stimulating fat body and /99 ovarian development at that time. The presence of some type of programming within insect muscles which determines the timing of their' response to hormones has also been suggested by Poels and Beenakkers (1969), Unnithan and Nair (1977), and Van den Hondel-Franken et al. (1980). Bioassays of haemolymph JH levels i n gregarious adult male Locusta and Schistocerca (Johnson and H i l l , 1973b) and adult female Locusta (Johnson and H i l l , 1975) indicated that i n both sexes the hormone was present i n r e l a t i v e l y small amounts immediately a f t e r imaginal ecdysis. However, JH then became almost undetectable u n t i l just before the beginning of sexual maturation which began i n both sexes around day 10. Following sexual maturity, JH levels i n males remained uniformly high whereas i n females the hormone lev e l peaked near oocyte maturation and dropped to a low t i t r e at oviposition. Subsequent chromatographic studies confirmed the presence of JH i n 18-day-old mature adult female Locusta. In an in vitro radiochemical assay on excised CA from adult female Schistocerca, Tobe and Pratt (1975) showed that JH peaks near the onset of vitellogenesis of each successive egg batch. The studies of E l l i o t t and G i l l o t t (1976, 1978, 1979) strongly indicate that i n adult female M.. sanguinipes the CA stimulate the synthesis of yolk proteins and t h e i r uptake by the ovary. JH i s also known to enhance reproductive functions i n male adult grasshoppers of t h i s species ( G i l l o t t and F r i e d e l , 1976). It i s possible, /100 however, that high JH levels i n the late l a r v a l stages also affect gonad maturation, since i n solitaria locusts the rate of reproductive development i s usually different from that of gregaria ( c f . Kennedy, 1961). When JHA was applied to f i f t h instar M. sanguinipes of both sexes, gonad dry weight increased or decreased. Attempts to i n i t i a t e reproducible JHA-effects on gonad dry weight did not produce clear consistent trends, even though the insects within each j a r usually exhibited s i m i l a r development. Overall, gonad dry weight of males and females was s i g n i f i c a n t l y reduced i n the f i r s t , but not in the second t r i a l . These results indicate that precise timing of JHA applications to f i f t h instars r e l a t i v e to each insect's stage of development results i n a wide range of effects on the gonads of both sexes. With the intention of lowering JH t i t r e s by inactivating the CA, 300 yg precocene II was applied to 1-day-old fourth instar nymphs. In the supposed absence of JH, most of the precocious adult insects were apparently s t e r i l e , possessing greatly reduced reproductive organs. The single exception was an anomalous precocious female which contained eggs. S t e r i l i t y i s a common result of precocene treatment (Bowers et al., 1976) i n both males and females. As reported i n Oncopeltus (Bowers et al., 1976; Bowers and Martinez-Pardo, 1977; Unnithan and Nair, 1979), JHA reversed the s t e r i l i t y of female M. sanguinipes. However, after JHA was applied to precocious adult male M. sanguinipes, no s i g n i f i c a n t change i n gonad dry weight was /101 observed. This indicates that i n males of th i s species, JH must be present during nymphal or early adult development for normal gonad size to be reached. Gonad size i n males does not increase i f JHA i s added af t e r early adulthood. The reduction i n general fat body dry weight after JHA application, seen i n the present experiments, was also recorded i n Locusta after CA implantation into f i n a l stadium nymphs (Poels and Beenakkers, 1969), and i n allatectomized females M. sanguinipes adults after JHA treatment ( G i l l o t t and E l l i o t t , 1976; E l l i o t t and G i l l o t t , 1978). In Locusta, the extra JH produced no observable effect u n t i l after the imaginal molt. The present experiments show, however, that i n M. sanguinipes the fat body i s also susceptible to JH during the f i f t h i n s t a r . Although JHA treatment of f i f t h i n s t a r males and females generally had no s i g n i f i c a n t influence on fat body dry weight, the effects of varying application time within the f i f t h i n s tar were highly s i g n i f i c a n t . In determining the amount of subsequent fat body reduction, precise timing of JHA application i s c r i t i c a l but no clear-cut period of JHA-sensitivity of the fat body was observed. As i n the previous JHA t r i a l s on normal f i f t h i n s t a r s , fat body size decreased s i g n i f i c a n t l y when precocious adults of both sexes were treated with JHA. Therefore, the development of the fat body of M. sanguinipes i s not e n t i r e l y pre-programmed because the tissue i s sensitive to JHA after adulthood i s reached. /102 As adult M. sanguinipes males and females age, the effects of the JHA treatment during the f i f t h stadium appear to decrease. Correlation among body measurements also decreased within each group of insects as they aged, regardless of treatment. Synchronization of development appears to be most important during early adulthood. Such a system would favor locust survival since large numbers of insects must develop simultaneously for swarming to take place. After reproductive development occurs, swarms disperse, and coordinated maturation rates are no longer necessary. Apparently, a sim i l a r loss of developmental synchrony also takes place i n grasshoppers. F. Grasshopper Control Depending upon the timing of the application, both precocene II and R-20458 have been shown to d r a s t i c a l l y a l t e r the development of various tissues i n M. sanguinipes. The present studies have substantiated previous reports that JH regulates fat body metabolism and the development of the gonads. In addition, JH has been shown to regulate somatic growth, p a r t i c u l a r l y that of the wing and f l i g h t muscles. Depending upon when JHA and precocene were applied, the development of the tissues was so d r a s t i c a l l y altered that the compounds were l e t h a l to the grasshoppers. Therefore, t h e o r e t i c a l l y , the compounds are p o t e n t i a l l y i n s e c t i c i d a l and, i n addition, act as chemosterilants or i n h i b i t wing development. However, the wide variety of effects produced by changes i n c r i t i c a l timing of both precocene and JHA appliations makes these compounds u n l i k e l y as agents /103 of grasshopper control, at least at the present time. Grasshopper emergence i s asynchronous and the insects' habits are s o l i t a r y . Timing of sprays r e l a t i v e to the insects' development would be c r i t i c a l . In addition, grasshoppers have time to cause s i g n i f i c a n t economic damage before they reach the f i n a l nymphal stadium i n which they are most susceptible to the l e t h a l effects of JHA. /104 LITERATURE CITED Albrecht, F. 0., Michel, R. and Casanova, D. (1978) The temperature and photoperiodic control of f l i g h t a c t i v i t y i n crowded desert locusts, Schistoeerca gregaria (Forsk.). 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APPENDIX 1: Means and standard errors for growth measurements i n early adult M. sanguinipes Age (days) Total body fresh weight (mg) Ti b i a length (mm) Tegmina and wing length (mm) Head width (mm) Gonad dry weight (mg) Fat body dry weight (mg) Flight muscle dry weight (mg) Males Females 230.8±23.7 10.6+0.4 19.0+0.7 - 4.010.1 4.210.5 5.110.5 6.010.4 286.1±23.3 10.410.2 19.010.5 3.910.1 4.810.5 5.810.9 7.210.7 337.5±16.3 10.210.2 19.210.4 3.910.1 6.010.5 8.311.6 9.410.7 340.4±12.3 10.210.2 19.010.4 3.910.1 8.810.5 9.910.6 11.711.0 387.4±27.3 10.810.3 19.310.6 4.010.1 10.311.1 11.711.6 14.111.5 349.5+13.2 10.210.1 19.310.3 3.910.0 10.610.5 10.811.0 14.1+1.0 349.4±14.5 10.310.3 19.310.8 3.9+0.1 10.610.6 8.811.6 13.211.3 340.2±14.0 10.110.2 18.910.8 3.910.1 9.710.4 8.111.3 12.711.0 327.6+6.5 10.010.1 19.2+0.3 3.8+0.0 10.210.5 8.5+0.5 12.810.6 219.9±8.9 11.110.1 19.210.6 3.910.1 1.6+0.1 5.910.3 6.110.5 328.2111.2 11.310.2 19.710.4 4.310.1 1.7+0.2 9.110.7 8.310.7 383.4126.9 11.210.3 20.1+0.6 4.210.1 2.410.3 13.5+2.1 11.0+1.3 434.1+17.6 11.7+0.2 20.710.2 4.310.1 7.511.5 24.312.1 15.310.7 475.8134.8 11.410.2 20.010.7 4.210.1 26.611.6 24.813.5 17.5+2.0 471.1117.7 11.110.2 19.9+0.4 4.210.1 35.011.6 17.811.3 15.011.2 509.1124.9 11.810.3 20.610.3 4.310.1 40.216.9 18.511.4 16.312.1 504.8+32.9 11.9+0.2 20.6+0.6 4.3+0.1 45.6+6.3 16.0+2.0 14.811.4 428.7118.4 11.410.2 20.010.2 4.110.1 29.112.4 16.0+1.8 14.0+0.8 /122 APPENDIX 2: Means and standard errors for normal t o t a l body fresh weight in early adult M. sanguinipes (N = 4) Mean t o t a l body Age fresh weight Standard (days) (mg) error Males 1 228.4 11.6 2 239.0 8.8 3 276.1 18.6 4 288.S 16.7 5 296.3 16.9 8 288.0 15.7 9 325.0 18.7 Females 1 263.6 0.8 2 292.1 1.7 3 316.2 12.3 4 249.7 2.0 5 381.9 11.2 8 421.6 28.8 9 432.0 13.5 APPENDIX 3: Means and standard errors for f l i g h t muscle dry weight and protein content in normal early adult M. sanguinipes (N = 2 to 3) Males -• Females ; Mean f l i g h t / Mean f l i g h t Mean f l i g h t Mean f l i g h t muscle dry muscle protein muscle dry muscle protein Age weight content Age weight content (days) (mg) (mg) (days) (mg) (mg) T r i a l l a 2 8.6±0.6 3.910.2 3 10.211.2 7.511.1 4 11.410.6 6.410.4 5 11.411.8 7.211.1 6 13.811.2 7.711.4 7 10.310.5 6.710.5 8 11.8+0.1 5.510.8 9 12.010.6 7.410.9 T r i a l 2° 1 6.010.4 3.710.4 2 5.910.3 3.610.0 3 8.510.5 5.510.5 4 12.012.2 7.711.5 5 16.910.6 10.410.6 6 14.411.7 9.511.0 7 16.010.8 10.6+0.3 8 13.311.6 7.5+0.8 9 13.510.9 8.510.5 a not si g n i f i c a n t b D4, D6 >D0, Dl, D2; D5>D1, DO; D8>D1 c. D4, D5>D2 d D4, D6>D1; D6>D2 T r i a l l c 2 7.911.4 3.910.2 3 12.611.7 6.310.6 4 14.811.1 8.511.4 5 16.212.8 8.211.2 6 14.310.5 8.410.6 7 12.310.6 6.610.7 8 13.912.2 8.610.4 9 12.310.1 6.610.9 T r i a l 2 d 1 6.110.5 3.510.4 2 8.710.8 7.110.7 3 9.411.8 5.810.8 4 15.811.0 10.411.3 5 18.713.1 12.111.6 6 15.712.3 9.711.5 7 20.312.3 11.612.0 8 15.811.9 10.410.5 9 15.610.8 9.010.6 APPENDIX 4: Mean (± S.D.) body parameters of S day-old adult males treated with 0.0S ug R-20458 at various intervals during the f i f t h i n s t a r HALES Total body fresh Fat body dry Fl i g h t muscle Day of weight T i b i a length Head width Tegmina width Wing length Gonad dry weight weight dry weight T r i a l application (mg) (mm) (mm) (mm) (mm) (mg) (mg) (mg) 1 1 351.3122.4 10.810.3 4.010.2 9.811.2 10.011.8 8.111.6 13.712.5 10.711.4 3 304.1143.9 11.010.3 3.910.2 13.712.1 11.111.5 6.511.6 6.510.9 6.911.1 4 294.3129.2 10.010.5 3.610.3 17.110.7 17.110.7 8.110.7 8.312.7 10.112.3 5 361.2130.5 11.110.5 4.010.1 16.411.3 11.811.0 8.411.3 8.013.2 9.710.6 6 307.3124 . 8 10 . 710.1 3.810.1 18.910.4 18.610 . 7 8 . 410 . 3 6 . 2 1 2.3 11.611 .0 Grand mean 323.6138.5 10.710.5 3.910.2 15.213.5 13.713.7 7.911.3 8.513.5 9.812.0 2 1 318.6122.7 10.610.0 3.910.1 10.411.6 10.411.6 11.711.8 5.711.5 12.010.7 2 323.2151.4 10.610.8 3.810.2 10.512.2 10.512.2 8.611.7 8.712.1 8.014.3 4 340.0130.4 10.610.4 3.410.2 15.610.6 14.310.6 8.911.7 10.412.6 9.011.4 4 1/2 304.4119.5 10.310.5 3.710.2 18.210.1 18.210.1 7.811.1 10.610.9 9.611.8 4 3/4 341.5127.9 11.110.2 3.910.2 18.310.8 15.911.8 8.610.2 10.712.4 9.411.9 5 345.1145.0 10.810.6 3.810.1 19.011.4 19.011.4 9.310.1 11.911.6 11.612.9 Grand mean 327.8131.7 10.710.5 3.810.2 15.113.9 14.513.6 9.211.7 9.512.6 9.812.5 APPENDIX 5: Mean (± S.D.) body parameters of S day-old adult females treated with 0.05 ug R-20458 at various intervals during the f i f t h instar FEMALES T r i a l Day of application Total body fresh weight (mg) T i b i a length (mm) Head width (mm) Tegmina width (mm) 1 3 4 5 6 Grand mean 491.5*29.2 396.9*90.9 478.8*19.5 383.7+80.3 408.U22.4 431.8±66.8 12.6±0.6 11.9±0.4 11.5*0.3 11.4+0.0 11.2+0.3 11.7+0.6 4.4+0.2 4.2+0.3 4.2+0.1 4.1+0.2 4.0+0.1 4.2+0.2 12.1+2.3 14.9+3.9 20.0+0.5 14.6+2.4 18.6t0.5 16.0±3.6 King length (mm) 12.111.9 12.311.7 20.0+0.5 12.611.6 18.211.1 15.113.7 Gonad dry weight (mg) 2.510.4 4.111.5 29.813.0 6.712.4 24.312.1 13.5111.9 Fat body dry weight (mg) 28.4+4.4 14.613.7 23.4+3.9 19.917.0 15.011.8 20.216.6 Fl i g h t muscle dry weight (mg) 11.111.8 10.2+3.5 15.511.9 11.012.6 12.110'. 9 12.012.7 1/2 3/4 Grand mean 420.1156.7 452.5169.4 467.6148.9 418.8142.2 483.8+8.1 448.6147.0 11.110.6 11.510.5 12.010.3 11.810.2 12.010.2 11.710.4 4.210.1 4.2+0.2 4.210.1 4.210.1 4.210.1 4.210.1 13.012.8 16.210.8 19.510.4 18.510.8 20.3+0.1 17.512.9 11.911.6 14.410.7 19.210.4 16.510.8 19.710.6 16.413.1 51.8122.2 27.514.4 35.1113.7 Zi.'hi.z 26.6110.5 33.9114.7 7.112.0 23.514.4 20.512.5 1<?.7U.Z 23.613.2 18.816.7 2.911.2 12.0+1.0 12.0+1.0 12.110.9 14.212.2 12.011.8 Cn APPENDIX 6: Raw data: Precocene-treated adultoids ± a l a t e r treatment with JHA Body Measurements Gonad F l i g h t Insect T i b i a Tegmina Head dry Fat body muscle Wing fresh weight length length width weight dry weight dry weight length Treatment (mg) (mm) (mm) (mm) (mg) (mg) (mg) (mm) Control 212.8 8.2 6.3 3.4 24 .7 5.5 6.3 6.3 (Precocene only) 168.1 8.0 4.7 3.3 2 .2 17.6 7.3 4.7 Adultoids 156.5 8.0 5.2 3.2 1, .9 14.2 6.7 5.2 JHA-treated 188.8 8.3 7.3 3.4 21, .6 6.4 4.7 7.3 Adultoids 186.9 8.3 7.5 3.3 14, .8 7.9 6.3 ' 7.5 194.7 8.5 5.8 3.2 16, .2 11.6 6.0 5.8 Control 161.7 8.1 7.5 3.1 5. .7 7.0 5.7 7.5 (Precocene only) 157.2 8.2 5.5 3.2 4. .2 6.0 6.3 5.5 Adultoids 162.0 8.0 6.6 3.1 5. ,4 6.2 6.4 6.6 JHA-treated 129.7 7.9 5.7 3.1 3. ,7 1.4 3.4 5.7 Adultoids 154.7 8.1 7.1 3.2 5. ,7. 4.4 6.1 7.8 144.5 8.0 5.7 3.2 4. 8 4.7 6.0 5.7 APPENDIX 7: Raw data: reversing precocious matamorphosis by timed applications of JHA to fourth instar precocene-treated M. aanguinipee Body Measurements Gonad Fl i g h t Insect T i b i a Tegmina Wing Head dry Fat body muscle fresh weight length length length width weight dry weight dry weight Treatment (mg) (mm) (mm) (mg) (mm) (mg) (mg) (mg) Resulting type A. MALES Control 193.6 8.5 5.9 5.9 3.4 6.3 9.5 7, .1 (Precocene only) Adultoid Control for more control insects, see Table 21 JHA applied 295.9 9.7 18.6 17.8 3.7 7.7 11.7 9. .6 day 4 260.6 253.6 9.7 9.3 14.0 16.7 12.4 15.5 3.6 3.4 7.8 5.8 4.4 6.8 6. 6, .5 .3 Normal-looking adults JHA applied 133.9 10.5 17.7 17.7 3.6 3.0 0.5 2 .7 day 5 JHA applied 302.6 9.9 18.4 16.5 3.8 8.3 7.2 9, .2 Reproductive day 6 321.3 10.7 18.3 18.3 3.6 10.2 8.3 7. .4 adults 143.9 7.8 6.3 6.3 2.9 4.3 4.0 3, .7 Adultoid B. FEMALES Control 161.8 7.9 6.5 6.5 3.2 1.8 14.9 6. .8 (Precocene 185.0 8.2 6.0 6.0 3.3 2.7 14.1 7. .6 only) 217.9 8.6 7.5 7.5 3.6 - 1.6 16.2 8. .0 Adultoids 164.9 8.1 7.0 7.0 3.2 1.6 10.7 7. .3 164.2 7.7 6.2 6.2 3.2 2.2 15.4 6. ,2 JHA added 297.1 9.8 16.3 13.8 3.6 1.8 14.0 8. ,9 day 4 324.6 341.2 9.7 11.3 16.2 18.4 14.5 15.5 3.8 4.0 11.7 2.2 9.2 21.8 7. 9. ,4 ,1 Normal-looking adults JHA added 261.2 10.5 17.2 17.2 3.7 2.2 15.2 9. ,5 day 5 JHA added 371.8 11.4 19.7 19.0 4.1 42.9 13.4 11 . 1 Reproduct ive day 6 427.2 10.9 18.7 18.0 4.0 38.7 11.4 12. .2 adults 185.2 9.9 6.2 6.2 3.2 1.8 14.2 5. .7 Adultoid APPENDIX 8: The effect of DMSO (1 yl) application to fourth instar nymphs 6-day-old adult body measurements Tegmina Gonad Flight Fresh body Tibia and Head dry Fat body muscle weight length wing length width weight dry weight dry weight Treatment Sex (mg) (mm) (mm) (mm) (mg) (mg) (mg) Untreated M 343.3 10.7 20.4 3.8 9.3 9.1 12.0 M 391.3 10.9 20.5 3.8 11.7 12.4 12.1 F 386.1 11.1 19.7 3.9 15.3 17.7 11.2 DMSO M 394.0 11.2 20.2 4.0 10.8 13.8 12.5 (1 yl) F 399.5 11.3 21.6 4.1 5.9 16.8 14.9 F 328.5 10.8 19.6 3.8 6.1 14.2 9.3 F 476.2 11.0 20.1 4.3 7.6 23.3 13.2

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