@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Land and Food Systems, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Palmer, Elnora"@en ; dcterms:issued "2010-03-26T03:25:00Z"@en, "1981"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "Changes in total body weight and in 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 in the two sexes during the somatic growth phase but differed markedly during the reproductive phase. Reasons for these differences are discussed in light of the behavior and physiology of the sexes. Head width, tibia length, tegmina length, and dry weights of gonads, fat body, and flight muscles were highly correlated in normal adults. This indicated that growth patterns were highly coordinated within individual insects. Fluctuations in the dry weights of the fat body and flight muscles during reproductive development indicated that these tissues were a source of protein for the developing ovaries or accessory glands. The similarity in the pattern of changes in males and females indicated that development may be synchronized between the two sexes. Factors contributing to this apparent synchrony are discussed. Depending upon the time of application, both the anti-allatotropin, precocene II and the juvenile hormone analog (JHA), R-20458 have been shown to drastically alter the development of various tissues in 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, somatic growth, coloration, wing length, and development of the flight muscles and fat body during the fifth instar. Precocene applied to fourth instars caused precocious metamorphosis and the production of diminutive adults. However, nearly normal development was produced in pxecocene-treated insects when R-20458 was applied 4 days later. Later JHA treatments resulted in the production of nymphal-adult intermediates. Intermediates were also produced when JHA alone was applied to fifth instars. However, specific effects depended upon precise application time. Supernumerary molting occurred only in insects treated with JH during the middle of the fifth instar stadium. Therefore, absence of JH at this precise time seems to be necessary to permit the imaginal molt. Green-colored and short-winged adults characteristic of locust solitarious phase, were produced when JHA was applied at certain times within the fifth instar. JHA application to fifth instars resulted in a significant reduction in wing length and in dry weight of the flight muscles. Flight muscles were sensitive to JHA throughout the fifth stadium whereas wing length was only significantly affected by JHA during certain periods of the stadium. Therefore, the flight muscles must be developing separately from the wings. JHA applications to precocious adultoids were too late to change the commitment of flight muscles in either sex, or of the male gonads. However, the fat body of males and females, and the female gonads were still susceptible to JHA at this time, and the precocene-induced sterility of females was reversed."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/22574?expand=metadata"@en ; skos:note "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 -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 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