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Juvenile hormone control of development of selected tissues in the migratory grasshopper, Melanoplus.. Palmer, Elnora 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. Agric, University of British 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 this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1981 (c) Elnora A. Palmer, 1981 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ftpfi(  /ii ABSTRACT 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, /iii 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. /iv TABLE OF CONTENTS Page ABSTRACT ii TABLE OF CONTENTS. iv LIST OF TABLES vLIST OF FIGURES x LIST OF PLATES xii ACKNOWLEDGEMENTS v INTRODUCTION 1 A. Pest StatusB. 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 11 (c) Flight Muscle Development 1F. Mode of Action of JH on Flight Muscles 13 (a) Effect on Protein Content... 1(b) Critical Timing 14 G. JH Effects on Migration 15 H. Altering JH Levels with Precocene 16 I. Summary of Major Objectives ^ MATERIALS AND METHODS 20 A. Rearing Techniques 2B. Growth Measurements 2^ C. Protein Determinations 22 /v Page D. Chemical Treatments 23 (a) JHA Studies(b) Anti-allatotropin Studies. 25 E. Statistical Analysis 2RESULTS 26 A. Normal Development 2B. Normal Flight Muscle Protein Content 40 C. JHA Studies 46 (a) Solvent Trials 4(b) JHA Dose-response Trials 46 D. Sensitivity of Fifth Instars to R-20458 50 E. Effects of Adult Aging on JHA-treated Insects 57 F. Sensitivity of M. sanguinipes to Precocene II 64 G. JHA Effects on Precocene-treated Insects 72 (a) Precocene Effects 7(b) JHA Applied to Adultoids 7(c) JHA Applied after Precocene but Prior to the Next Molt 4 DISCUSSION 83 A. Normal Development 8B. Role of JH in 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 Flight Muscles 98 F. Grasshopper Control 102 LITERATURE CITED 104 APPENDIX v 121 /vi LIST OF TABLES Table , Page I Phase characteristics of locusts 4 II Environmental factors influencing locust phase determination 8 III Changes in various growth parameters during early adulthood in normal M. sanguinipes 27 IV Correlation between the dry weight of the flight muscles and other body measurements in normal M. sanguinipes 35 V Linear regression analyses showing relationship between fresh weight (Y) and age (X) in young adult M. sanguinipes 37 VI Analysis of variance for flight muscle dry weight and protein content during the first 9 days of adulthood 41 VII Linear regression equations showing relationship between protein content (Y) and dry weight (X) of the flight muscles during the first 9 days of adulthood 44 VIII Effects of topical application of three solvents to fifth instar nymphs 3 days after treatment 47 /vii Table Page IXa Sensitivity of different stages of M. sanguinipes to high dosages of the JHA, R-20458 48 IXb Sensitivity 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 fifth 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 fifth stadium 56 XIa Overall effects of R-20458 on adult male body measurements when applied at various times during the fifth stadium 58 Xlb Overall effects of R-20458 on adult female body measurements when applied at various times during the fifth stadium 59 XIla Mean body measurements (± S.D.) in 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.) in untreated and JHA-treated females dissected as 5- or 6-day-old adults, or 14-day-old adults 63 /viii Table Page XIII Sensitivity 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 final molt to precocene-treated adultoids 75 XVI Overall effects of 0.05 ug R-20458 applied to precocene-treated insects at various intervals prior to the next molt 78 XVII Effects of timed JHA applications to precocene-treated fourth instars. Measurements were taken 4-5 days after adult emergence 82 /ix LIST OF FIGURES Figure Page 1 Changes in total body weight of normal males and females during early adulthood. Arrow indicates approximate oviposition time 28 2 Changes in (a) tegmina and wing length, (b) tibia 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 in fat body dry weight of normal males and females during early adulthood 32 5 Changes in flight 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 (vertical 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 first 3 days of adulthood, and from 4 to 9 days after emergence 39 9 Changes in dry weight and protein content of flight muscles in normal males during early adulthood 42 10 Changes in dry weight and protein content of flight muscles in normal females during early adulthood.... 43 11 Regression lines showing the relationship between the protein content and the dry weight of flight muscles in normal males and females during early adulthood 45 12a Correlations among body measurements in male adults after R-20458 was applied during the fifth stadium 60 12b Correlations among body measurements in female adults after R-20458 was applied during the fifth stadium. 61 13a The effect of aging and JHA treatment on correlations among body measurements in 5- or 6-day-old adult males 65 /xi Figure Page 13b The effect of aging and JHA treatment on correlations among body measurements in 14-day-old adult males 66 13c The effect of aging and JHA treatment on correlations among body measurements in 5- or 6-day-old adult females 67 13d The effect of aging and JHA treatment on correlations among body measurements in 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 their precocious molt 76 14b Correlations among body measurements in female adultoids treated with precocene II as fourth instars and with R-20458 after their precocious molt 77 /xii 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 resulting from JHA application (0.375 yg) to fifth instar M. sanguinipes . For comparison, a normal untreated female is also shown 49 3b Normal, untreated male and large, green, short-winged supernumerary male resulting from JHA treatment described above. The treated insects had difficulty casting their exuvia 49 4a External morphology of 2-day-old adult females that were treated with 0.05 ug R-20458 as newly emerged fifth instar 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 fifth instar 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 fifth instar nymphs. An untreated control (d) is also indicated 54 /xiii Plate Page 6a Dorsal view of untreated male adult and precocious male adultoid resulting from precocene application (200 yg) to newly emerged fourth instars 71 6b Side view of untreated female adult and precocious female adultoid resulting from precocene application (200 yg) to newly emerged fourth instars 71 7a Fifth instar nymph and precocious adultoids resulting from precocene (300 yg) application to 1-day-old fourth instar nymphs '. 79 7b Normal-looking fifth instar 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 79 7c Two semi-adultoids which later died attempting another molt and two fifth 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 fifth 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 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.), is generally regarded as the most widespread and destructive grasshopper species in North America (Pickford and Mukerji, 1974; Hewitt, 1977; Uvarov, 1977). M. sanguinipes damages cereal grains, vegetables, forages, and even the leaves and bark of fruit trees (Metcalf and Flint, 1962; Parker and Connin, 1964). Locusts, close relatives of M. sanguinipes, are a major world pest that have caused periodic crop decimation and famine for centuries (Baron, 1972; Hill, 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 particularly 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 in the same family by their inability to transform into a gregarious, highly mobile phase during their life cycle. While locusts migrate in huge swarms over 12 vast distances and eat most of the plants in their path, grasshoppers remain relatively localized and solitary. The name "migratory grasshopper" shows the ambivalence of scientists regarding the status of M. sanguinipes. The species does not show the two extreme morphological forms common in locusts, but is capable of migrations of 10 to 50 miles per day (Willis, 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 is categorized by Rowell (1967) as containing locust species. Members of the genus exhibit the locust characteristic of green/brown polymorphism, at least in the haemolymph of individuals with high corpora allata activity (Pfeiffer, 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 it may be a rarely-occurring phase of M. sanguinipes. M. spretus is very similar to M. sanguinipes but has longer, broader wings and a darker coloring (Heifer, 1953). Perhaps M. sanguinipes is a locust whose extreme gregarious phase is not triggered by present North American conditions. /3 C. Phase Polymorphism in Locusts Locust phase theory, first expounded by Uvarov (1921, cited by Ordish, 1976) explains the natural polymorphism of many acridid populations and forms the foundation of modern locust control. Uvarov observed two extreme physiological states or phases, a solitary and a gregarious phase. Although not common to all locust species, the two phases exhibit a variety of characteristics (Table I). Locusts in the solitaria phase display grasshopper-like behavior and morphology including a more arched pronotum, shorter wings, and larger femur/head capsule ratio than gregaria adults. Solitaria are often light-colored or even green, feed and develop as isolated individuals and make only short flights. Rates of feeding, and development in solitaria are slower than in gregaria, and solitaria locusts sometimes have an extra nymphal instar. Egg-laying is delayed in solitaria, but the females are larger and their fecundity is higher than in gregaria locusts (Kennedy, 1961). Phase gregaria exhibits long-distance migrations. These flights begin under suitable weather conditions when the morphology and behavior of the new locust generation have begun to change from the solitary to the gregarious form. Dark-colored gregarious nymphs begin marching and are joined by similar individuals until a large hopper band has formed. After the nymphs molt into long-winged adults, they mill around for several days, taking short practice flights until they are all ready for flight (Chapman, 1969). Swarms form and the TABLE I: Phase characteristics of locusts Genus or Phase character Effect species References behavior color solitaria limited flight; no grouping or marching individuals avoid each other gregaria mass flight; march in groups individuals stay together solitaria green, yellow, or light beige gregaria tan to dark brown or black may have pinkish background color or yellowing general Loousta Schistocerca Loaustana grasshopper spp. Kennedy (1961) Gillett (1978) Cassier (1966) Kennedy (1961) Gillett (1978) Rowel1 (1967) morphology 1. femur/head capsule 2. wing length; length of elytron/hind femur ratio 3. pronotum compound eyes and. time of flight larger in solitaria; smaller in gregaria solitaria shorter wings slightly lower E/F ratio arched in solitaria gregaria longer wings slightly higher E/F ratio solitaria eyes have light-colored spots or bands fly at night gregaria eyes are dark and a solid color day flight Schistocerca Locusta Zonocerus variegatus Schistocerca Locusta Schistocerca Locus tana Locusta Gillett (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 is a smaller difference between the sexes. a) gregaria begin oviposit ion later than solitaria b) gregaria begin oviposition earlier than solitaria lower in 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 fly 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 until the females begin to form eggs and leave the swarm in search of oviposition sites, when the band disperses. D. Environmental Factors Influencing Locust Phase Determination Locusts benefit substantially by their ability to change state in response to their fluctuating environment. In wet weather, when food is plentiful, 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 in their 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 ideally suited to large-scale dispersal. Within a few generations locusts adapt to their new conditions. Locust phases are determined by both genetics and environment. Although selective breeding can bring about distinct solitaria and gregaria lines within four generations, the same variation in phase character can be induced within one generation by environmental stimuli such as rearing density. The altered state is then maternally inherited (cf. Kennedy, 1961). p Enviromental conditions triggering phase change are listed in Table II. Not surprisingly, form gregaria develops when the insects are densely populated. High temperatures, low relative humidity, and long days also favor phase gregaria, while solitaria is encouraged by the opposite conditions. Even the presence of faeces from certain other locust stages has been found to influence phase determination in caged nymphs (Gillett and Phillips, 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 in diet can alter the hormonal balance within insects and may increase or decrease the likelihood 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 trigger hormonal responses that initiate phase change. The solitaria phase is thought to be a TABLE II: Environmental factors influencing locust phase determination Phase character Effect Genus or species References crowding status of parents humidity-temperature photoperiod food plants extreme solitarius 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) Gillett (1978) Albrecht et al. (1978) Gillett (1978) Albrecht et al. (1978) Doane (1973) Staal (1961) McCaffery $ Page (1978) Uvarov (1966) Brett (1947) (continued). TABLE II: (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) Gillett § and Phillips (1977) /io permanently neotenized form arising from high juvenile hormone (JH) titres during the last nymphal stage (Kennedy, 1961; Doane, 1973; Cassier and Delorme-Joulie, 1976). When locust nymphs receive corpora allata (CA) implants or are treated with juvenile hormone analogs (JHA), solitaria-like adults are produced (Joly, 1960; Staal, 1961; Doane, 1973). Conversely, solitary Loousta females with one CA removed produce progeny which show gregarious characteristics (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 characteristic of the Acridoidea, and can be induced with high JH levels even in species where green individuals in the wild are rarely found (Pfeiffer, 1945). (a) Reproduction In many acridid species including M. sanguinipes, the CA have been shown to stimulate ovarian development (Gillott and Elliott, 1976; Elliot and Gillott, 1976, 1977, 1978, 1979). Since JH accelerates reproductive growth low JH levels in early adult migrants could explain the slow sexual maturation in the females of some locust species. Similarly, JH is also involved in the reproductive functions of male locusts. In S. gregaria, allatectomies performed on young males completely abolished male /II sexual behavior (Loher, 1961; Pener, 1967). Evidence for CA control of the development and functioning of the male accessory glands has been demonstrated in M. sanguinipes (Gillott and Friedel, 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 tropical pest grasshopper, Zonocerus variegatus, crowding or low JH levels during the last nymphal instar produced long-winged adults capable of long flights (McCaffery and Page, 1978). Allatectomy performed on 3-day-old fifth instars of Z. variegatus produced only long-winged adults (McCaffery and Page, 1978). In contrast, JHA applied to final instar 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 in insect flight 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 inhibits flight muscle development in nymphs /12 and encourages flight muscle degeneration in adults. In H. cecrovia, formation of many adult tissues occurs during the first 2 days of pupal development, when JH levels are low. However, when CA are implanted during early pupation, flight muscle formation is 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 flight muscle degeneration after the initial active adult flights give way to reproductive activity. However, in some of these insects flight loss can be prevented by removal of the CA. In the house cricket, Aoheta domestical, allatectomies performed during the final nymphal instar prolonged flight muscle persistance in the adults (Chudakova and Bocharova-Messner, 1968b) and resulted in a permanent retention of flight ability (Chudakova and Gutmann, 1978). Conversely, JHA application caused rapid degeneration of the flight muscles in both nymphal and adult Aoheta (Chudakova and Bocharova-Messner, 1968a, 1968b). Adult fire ants, Solenopsis inviota, failed to cast their wings or undergo normal flight muscle histolysis after allatectomy (Barker, 1979) but subsequent JHA application to the adults resulted in rapid flight muscle degeneration. Implantation of CA into adult female, Dysdercus intermedins (Edwards, 1970), also elicited flight muscle histolysis. /13 In various bark beetle species, JHA induce degeneration of the adult flight muscles (Borden and Slater, 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 flight muscles of some insects mirror their increasing flight capability during early adulthood (Poels and Bennakkers, 1969; Bursell, 1973; Panar and Nair, 1975; Baker, 1976). Protein, the major "component of muscle (Panar and Nair, 1975), is also affected by JH levels. In Poels and Beenakkers* experiments (1969), protein accumulation in adult locust flight muscles was greatly reduced, but not totally blocked, by implanting active CA during the early fifth instar. The likelihood of uneven release from the CA throughout adult development makes it impossible to determine from their experiment whether or not there is a critical stage during which JH influences flight muscle development. Perhaps protein synthesis in the flight muscles would not be affected if JH were applied at the late fifth instar stage. However, since the CA caused the greatest inhibition of flight muscle protein during the differentiation stage (2 to 5-day-old adults), JH applications to newly-emerged adults should be effective in reducing flight muscle protein content unless the tissues are already committed at this time. The /14 sensitivity of the flight muscles of fifth instar nymphs to CA implantation led Poels and Beenakkers (1969) to suggest that in normal Locusta, flight muscle development only begins when the JH titre is very low. The authors presume that JH titres in Loausta are negligible throughout the fifth nymphal stadium. Capillary gas chromatography with electron capture (Blight and Wenham, 1976a,b; Huibregetse-Minderhoud et al., 1980) and Gallevia bioassays (Johnson and Hill, 1973b) on Schistoeerca and Loausta confirmed that the high JH levels in the early fourth instar stadium decreased at the end of the stadium and were almost undetectable during much of the fifth instar stadium and early adulthood. (b) Critical Timing The sensitivity of various acridid tissues to JH-induced effects appears to vary according to the development stage. In Locusta and Schistoeerca, exogenous JHA applied in small doses at the beginning of the last larval instar cause changes in morphology whereas the same doses applied later in the same instar result in phase change effects (Nemec, 1970). In terms of the effects of JH on wing and flight muscle development, several authors (Staal, 1975; McCaffery and Page, 1978; Chapman et al., 1978) believe the last nymphal instar to be the critical period. Doane (1973), however, states that in 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 failed to affect locust flight 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 flight muscle dry weight and protein content. The JH sensitivity 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 tropical grasshopper, Z. variegatus, wing length is highly correlated with migratory capacity (McCaffery and Page, 1978), and short-winged insects have poorly developed flight muscles (Chapman et al., 1978). JHA applications to final nymphal instar grasshoppers resulted in progressively shorter wings as JH dosage increased (McCaffery and Page, 1978). Chapman et al. (1978) state that in 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 critical final nymphal instar stage inhibit flight muscle as well as wing development in solitaria locusts. Unfavorable JH levels for maximum flight muscle /16 development could be a major cause of non-migration in many insects. If migrating insects possessed low JH levels in early adulthood, flight muscle development would be favored and reproductive development delayed. These characteristics are indeed seen in gregaria locust females. There may be a different response to JH of flight muscles and gonads in the two sexes during development, but this possibility 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 anti-allatotropic effect (Bowers et al., 1976). Actions of the compound on various insects include a reduction of CA activity, CA degeneration, precocious metamorphosis, and sterility (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 is capable of directly inactivating the CA (Pratt and Bowers, 1977; Muller et al., 1979). CA of Oncopeltus, excised and incubated in vitro with precocene, lost the ability to induce supernumerary molting which normally results in many CA-implanted insects (Muller et al., 1979). This work has been supported by further in vitro experiments on Schistocerca. Precocene caused CA parenchyma cell degeneration in second, third, and fourth instar Schistocerca nymphs (Unnithan et al., 1980). Bowers /17 and Aldrich (1980) confirmed that the brain and neurosecretion are not involved in precocene inactivation 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 this process may form the basis of precocene resistance in non-target tissues. Precocene sensitivity 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 in insects in which precocene is rapidly degraded, the compound is less bioactive, and that this may be a basis for its selectivity. Precocene applications are only effective 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 later applications after a blood meal slowed ovarian maturation and produced abnormal oviposition. Unnithan and Nair (1979) applied precocene II to fourth and fifth instar Oncopeltus fasciatus and found antiallatotropic effects in the earlier instars but not during the final instar. 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 is critical in Locusta as well (Kruse Pedersen, 1978). /18 Although the degeneration of precocene-treated CA is permanent, some precocene effects seem to be short-lived. Sahota and Farris (1980) temporarily arrested the normal degeneration of flight muscles in log-colonizing female spruce bark bettles (Dendroctonus rufipennis) by applications of precocene to adults. The normal increase in flight muscle DNA/protein ratio 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 transcription 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 in Oncopeltus (Masner et al., 1979). I. Summary of Major Objectives The main purpose of this study was to investigate the role of JH on the development of selected tissues in M. sanguinipes from the following aspects: 1. What is the normal pattern of growth during early adulthood in male and female M. sanguinipes? What is the normal change in flight muscle protein content during this period? 2. How is the normal developmental pattern changed by topical JHA treatments? Is timing of JHA application critical 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 in this study were of the non-diapause strain of M. sanguinipes(Pickford and Randell, 1969). Stock colonies were reared under crowded conditions (ca. 30 to .100 grasshoppers) in 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 alfalfa meal mixture (100 g alfalfa meal:100 g bran:10 g brewer's yeast:12 ml corn oil) supplemented by daily additions of fresh lettuce (Gillott and Dogra, 1972). B. Growth Measurements The initial experiments were aimed, at establishing the normal growth patterns of adult males and females. Within 3 to 4 hrs after the imaginal molt, insects in the stock colony were transferred into glass jar cages (Plate 2). At various intervals after ecdysis, the grasshoppers were decapitated and weighed on a Mettler microbalance. The wing length, tibia length, and head width were measured with vernier calipers. Dissections were performed under a stereo-microscope. Using the methods described by Hill et at. (1968), the fat body, gonads, and flight muscles were removed, dried to constant weight at 35°C, and weighed on a microbalance. Flight muscles were later 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 daily changes in total body weight, the fresh weight was measured in the same insects during the first 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 in glass jars (Plate 2), and reared under conditions identical to those in the stock colony. Each day after the imaginal molt, the insects were anaesthetized with. C02 and the fresh weight determined. The data were subjected to regression analysis to determine the overall relationship between age and fresh weight in both sexes. As distinct phases of somatic and reproductive development were evident in 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 flight 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 solubilized in 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 tartrate, and 0.05% copper sulfate, but lacking NaOH), 4.0 ml of phenol reagent (Fisher 723 Scientific 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 in a Spectronic 20. By knowing the total dry weight of the flight muscles and the amount used in each protein determination, the total protein content of the flight muscle was calculated, D. Chemical Treatments The role of JH in somatic and reproductive development was investigated using a juvenile hormone analogue (JHA) and an anti-allatotropin. Newly ecdysed fourth instars, fifth instars, and adults were removed from the stock colony. They were isolated in glass jars in 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 individually with dots of Testor's PLA enamel paint (Testor Corp.). (a) JHA Studies At selected intervals, 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 biologically active in M. sanguinipes (Elliott and Gillott, 1978), the compound was diluted appropriately. To determine the best solvent for the JHA trials, 1.0 ul samples /24 of acetone, olive oil, or an acetone/olive oil mixture (1:1) were applied using a micropipette to the abdomenal terga of six insects. The olive oil and acetone-oil mixture inhibited molting whereas acetone alone was non-inhibitory. To determine the lethal and morphological effects of R-20458, the JHA was serially diluted in acetone. The JHA doses examined ranged from 0.375-0.0375 yg. One yl samples were applied topically with a micropipette to fourth and fifth instar nymphs. Treated insects were kept in glass jar cages until 4 to 5 days after adult emergence, when mortality and external appearance were noted. The sensitivity of different stages of fifth instar nymphs to R-20458 was investigated by treating 1- to 6-day-old fifth 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 if there were any significant differences between various body measurements with different 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 fifth instar nymphs. The treated insects were dissected 5 and 14 days after adult emergence and their body measurements compared. /25 (b) Anti-allatotropin Studies The insect anti-allatotropin, precocene II (6,7-dimethoxy-2,2-dimethyl chromene; Aldrich Chem. Co.) was stored under nitrogen at 6°C. Stock solutions were prepared by dilution in dimethyl sulfoxide (Pound and Oliver, 1979; Verma, 1981). Since 1 yl of the solvent exhibited no lethal or other deleterious properties (see Appendix 8), solvent-treated controls were not used in all the precocene experiments. Instead, the normal growth patterns established in the preceeding studies were used. In order to determine a biologically-active but non-lethal dosage of precocene, fourth instar, fifth instar, 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 instar nymphs were treated with 300 yg precocene II. 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 prior to the molt and 6 to 10 days after the molt. E. Statistical Analyses Analysis of variance, correlation matrices, and linear 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 total body weight of males and females increased significantly during the first 9 days of adulthood (Table III). The daily increments are shown in Fig. 1. In both sexes, the fresh weight increased markedly during the first 3 days. However, after this, growth patterns were distinctly different. In males, the total body weight increased marginally to reach a maximum on day 5 when mating normally begins. Concurrent with this, the fresh weight declined and remained relatively stable for the remainder of the assessment period. In contrast, the total body weight of females increased significantly until day 8 when oviposition occurred. In both sexes, no signficant change in tegmina and wing length, tibia length, or head width occurred after emergence (Table III, Fig. 2). However, significant changes in the dry weight of various internal organs were evident (Table III). In newly-emerged females, the ovary was poorly developed and weighed less than 2 mg (Fig. 3). The dry weight remained relatively constant until day 3, after which a pronounced increase occurred until day 8. At this point, the ovary contained mature eggs in the oviducts and weighed nearly 46 mg. The sharp decrease in 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 III: Analysis of daily Changes in various growth parameters during early adulthood in normal M. sanguinipes Males Females Growth parameter D.F. F value Significance D.F. F value Significance Total body fresh weight (mg) 8,44 5.49 .0034 7,39 5.92 .0001 Tibia 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 Flight muscle dry weight (mg) 8,44 6.88 .0000 7,40 4.89 .0005 NS = not significant (P = 0.05) * wing length comparable /28 100 H i i i i i t I.I i i 1234567 G9 AGE (days) FIGURE 1: Changes in total 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 in (a) tegmina and wing length, (b) tibia length, and (c) head width in 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 in 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 stabilized at approximately 10 mg. Highly significant (P = 0.01) changes in both fat body and flight muscle dry weight took place in males and females during early adulthood (Table III). Although the fat body was larger in females than in males, the pattern of daily changes in the dry weight of the tissue was similar in 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. Similarly, flight muscle dry weight was greater in females than in males (Fig. 5). However, in both sexes, the dry weight of the flight muscles followed a pattern similar to that of the fat body viz. rising sharply after adult emergence, peaking on days 5-6, then declining. All body measurements were highly correlated with each other in normal M. sanguinipes (Fig. 6). The exception was the gonads, which were not correlated with tibia length or head width in either sex, nor with tegmina and wing length in females. Flight muscle dry weight was highly correlated with the other body measurements in normal M. sanguinipes (Table IV). Although the former fluctuated after imaginal ecdysis, it was still 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 in 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 flight muscle dry weight of normal males (A) and females (o) during early adulthood. Fresh Weight Tibia Length Tegmina Length /34 Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Head Width Gonad Dry Weight a) Males FIGURE 6: Correlations among body measurements in normal adult M. sanguinipes. In this and remaining figures, a double line joining two points denotes a significant correlation at the 1% level; a single line denotes significance at the 5% level. When the growth of two body parameters was not correlated, they were not joined with a line. There were on negative correlations. TABLE IV: Correlation between the dry weight of the flight muscles and other body measurements in normal M. sanguinipes Growth parameter correlated with flight muscle dry weight D.F. Males R Significance D.F. Females R Significance Total body fresh weight (mg) 54 .86 1% 54 .86 1% Tibia 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 this study. Analysis of variance revealed a significant variation in fresh body weight of both sexes during early adulthood (Table V). The increments in fresh weight were highly dependent upon age, yielding significant regressions in males and females for the 9-day period (Table V; Fig. 7). Analysis of the residuals confirmed the normal distribution of residual sizes, and the random distribution of the residuals with respect to age. Regression equations of Yfemale = 275.7 + 21.IX and vma^e = 242.4 + 9.8X show a faster growth rate for females than for males. Examination of changes in the fresh weight and dry weight of the internal organs in normal adults indicated that there were two growth periods occurring in grasshoppers during the first 9 days of adulthood (Figs. 1-5). During the first 3 days, limited reproductive growth occurred in both sexes so the increase in fresh weight was due primarily to growth of somatic tissues including the fat body, flight muscles and cuticle. However, after this the converse was true so that the increase in total 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 first 3 days, the slopes of the regression lines for males and females were similar, being 28.8 and 26.3, respectively (Fig. 8). Dependency of fresh body weight on age was significant in both sexes at this time (Table V). However, after day 3, the regression equations TABLE V: Linear regression analyses showing relationship between fresh weight (Y) and age (X) in 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 in normal males and females during early adulthood. Mean fresh weights of males (A) and females (o) with standard errors (vertical 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 in normal males and females during the first 3 days of adulthood, and from 4 to 9 days after emergence. /4 0 for both sexes were quite different. In males, there was no significant change in fresh body weight with age (Table V); the slope of the regression line being only 4.6 (Fig. 8). However, in females, fresh body weight increased significantly with age (Table V) and yielded a regression slope of 15.6. B. Normal Flight Muscle Protein Content With the exception of Trial 1, flight muscle dry weight and protein content usually varied significantly (P = 0.05) with age in normal males and females (Table VI). In males, changes in the protein content and dry weight of the flight muscles paralleled each other (Fig. 9). In both trials, these two parameters increased rapidly immediately after ecdysis until days 5-7, when they decreased. Similar results were also observed in females (Fig. 10). Although the protein content and dry weight of the flight muscles were highly correlated, it is 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 similar for the two sexes in both trials. In trial 1, the regression slopes for males and females were 0.54 and 0.46, respectively, whereas in trial 2 they were 0.62 and 0.58. Analysis of the residuals confirmed the normal distribution of residuals with respect to age. TABLE VI: Analysis of variance for flight muscle dry weight and protein content during the first 9 days of adulthood Males Females Tissue parameter Trial D.F. F value Significance Trial 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 significant (P = 0.05) /4'2 Trial 1 18 M H 10 c3 UJ I i i 2 3 4 5 6 AGE (days) 8 9 Trial 2 J T t 5 $ I $ 2 J !_ 1 2 3 4 5 6 7 AGE (days) 8 9 FIGURE 9: Changes in dry weight (A) and protein content (A) of flight muscles in normal males during early adulthood. /43 20 15 HH 10 Trial 1 _1_ 2 3 4 5 6 AGE (days) 5 I Trial 2 I P 1 JL 8 a 3 4 5 6 AGE (days) 8 9 FIGURE 10: Changes in dry weight (®) and protein content (o) of flight muscles in normal females during early adulthood. For significance of daily changes in- dry weight see Appendix 3. TABLE VII: Linear regression equations showing relationship between protein content (Y) and dry weight (X) of the flight muscles during the first 9 days of adulthood Sex Trial 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 Trial 1 r8 -r Z o " 6 w 5 E~ O U J 1 2 1 234567 89 10 11 Y_ , = 0.88 + 0.58 X female Y . = 0.13 + 0.62 X male Trial 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 flight muscles in normal males ( ) and females ( ) during early adulthood. /46 C. JHA Studies (a) Solvent Trials The sensitivity of fifth instar nymphs to topical application of 1 ul of three solvents is shown in Table VIII. Three days after application, both olive oil and a 1:1 mixture of olive oil/acetone resulted in delayed nymphal development and at least 50% mortality. In all instances, mortality occurred during the imaginal molt as the insects appeared unable to cast their exuvium. Because acetone exhibited no deleterious effects, it was chosen as the appropriate solvent in all subsequent JHA studies. (b) JHA Dose-Response Trials The sensitivity of different stages of M. sanguinipes to high dosages of R-20458 is shown in Table IXa. At higher doses (0.375 and 0.75 ug), the JHA killed most of the grasshoppers. Mortality was higher when R-20458 was applied to young and old fifth 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 in the middle of the fifth instar 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 topical application of three solvents to fifth instar nymphs 3 days after treatment No. insects No. surviving No. surviving No. Solvent treated nymphs adults died olive oil 6 1 2 3 oil/acetone (1:1) 6 1 1 4 acetone 6 0 6 0 TABLE IXa: Sensitivity 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 00 /49 PLATE 3a: Abnormally large female in supernumerary stadium (right) resulting from JHA application (0.375 yg) to fifth instar M. sanguinipes. For comparison, a normal untreated female (left) is also shown. PLATE 3b: Normal, untreated male (left) and large, green, short-winged supernumerary male (right) resulting from JHA treatment described above. The treated insects had difficulty casting their 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 negligible mortality and a high frequency of easily observable JHA effects. To further minimize lethal effects, 0.05 yg R-20458 was applied in subsequent studies. D. Sensitivity of Fifth Instars to R-20458 Single applications of 0.05 yg R-20458 to fifth instar insects of slightly varying ages resulted in dramatically different external and internal effects. JHA application to newly emerged fifth instar males and females produced mixed effects, ranging from insects which molted into normal-looking adults to those which became adultoids with pronounced nymphal characteristics. Juvenile characteristics exhibited by the latter included short, wings, reddish heads and pale cuticles marked with black (Plate 4a). A distinct, pale stripe extended the length of the pronotum. In normal adults, the pronotal stripe extends only halfway up the pronotum then blends into the beige-brown cuticle. When the same dose of JHA was applied to 4-day-old fifth instar nymphs, the insects molted into normal-looking adults with long wings, beige-brown cuticle, and an indistinct pronotal stripe TABLE IXb: Sensitivity of different stages of M. sanguinipes to low dosages of the JHA, R-20458 No. No. No. JHA 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 all slightly 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 fifth instar nymphs.' An untreated adult is shown on the far right. PLATE 4b: Two-day-old adult males showing the effect of a single application of 0.05 yg R-20458 to (left to right) 4-, 5-, and 6-day-old fifth instar nymphs. An untreated grasshopper of comparable age is shown on the far right. /53 (Plate 4a). Similar JH treatments applied to fifth instars from late day 4 to early day 5 usually resulted in adults with green-brown to bright green cuticles and slightly shortened to very short tegmina and wings (Plates 4b, 5). Maximum green color was usually seen around day 5 of adulthood. The distinct pale pronotal stripe observed in grasshoppers which received JHA at fifth instar emergence was also present in the short-winged, green insects. Although the tegmina and wings are of equal length in normal M. sanguinipes, treated insects often had shorter wings than tegmina. Furthermore, in 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 fifth-instars produced little effect, although a faint green coloration and slight wing shortening occurred in some of the adults. Tables Xa and b compare the body measurements of normal 5-day-old adults (average of two previous trials) and those that had been treated with 0.05 yg JHA during the fifth instar. In males, JHA application resulted in significant (P = 0.05) reductions in total fresh body weight, tegmina length, wing length, and flight muscle dry weight. Except in trial 2, the tegmina-wing length and flight muscle dry weight were significantly reduced in JHA-treated females. Both sexes experienced significant changes in gonad dry weight after JHA application in trial 1 but not in trial 2. Tibia lengths, head widths, and fat body dry weights were not significantly different 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 fifth instar nymphs. An untreated control (d) is 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 fifth stadium* Body parameter Untreated JHA Trial 1 JHA Trial 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 Tibia 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 fifth stadium in Trial 1 whereas in Trial 2, the JHA was applied on days 1, 2, 4, 4 1/2, 4 3/4 or 5 of the fifth 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 fifth stadium* Body parameter Untreated JHA Trial 1 JHA Trial 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 Tibia 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 fifth stadium. In general, the timing of JHA application only had a significant effect upon the fat body, tegmina, and wings of 5-day-old adults. In some cases significant changes in ovarian dry weight, tibia length, and flight 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 in JHA-treated than in normal grasshoppers, and varied with sex and with trial (Figs. 12a and b). E. Effects of Adult Aging on JHA-Treated Insects Tables XIla and b show the various body measurements in normal insects and those treated with R-20425 as newly emerged fifth instars. The measurements were taken 5 or 6 and 14 days after adult emergence. Treated insects had shorter than normal tegmina and wings in both age groups and female gonad dry weight was variably altered. Five-to 6-day-old males and females exhibited reduced flight muscle dry weight after fifth instar JHA treatment. However, no reduction in flight muscle dry weight was seen in 14-day-old adults after the same treatment. The main effect of aging on the body parameters was reduced fat body dry weight viz. in both sexes, the weight in 14-day-old insects was about half 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 fifth stadium Body parameter Trial 1 Trial 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 Tibia 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 Flight 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 fifth stadium Body parameter Trial 1 Trial 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 Tibia 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) Trial 1 Fresh Weight Tibia Length Tegmina Length Head Width (ii) Trial 2 Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight FIGURE 12a: Correlations among body measurements in male adults after R-20458 was applied during the fifth stadium. Negative correlations in this and succeeding figure are denoted by dotted lines. /61 Fresh Weight Tibia Length Tegmina Length Head Width Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight (i) Trial 1 Fresh Weight Tibia Length Tegmina Length Head Width (ii) Trial 2 Wing Length Flight Muscle Dry Weight Fat Body Dry Weight Gonad Dry Weight FIGURE 12b: Correlations among body measurements in female adults after R-20458 was applied during the fifth stadium. TABLE Xlla: Mean body measurements (1S.D.) in 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 Tibia 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 Xllb: Mean body measurements (±S.D.) in 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 in both sexes (Fig. 13a-d). In 13-day-old, JHA-treated males and females, the only significant 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, fifth, and adult instars. Precocious metamorphosis was produced in 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 XIII). Two of the survivors of these high precocene doses exhibited JHA-like effects, namely short wings and juvenile coloration. Precocene II did not result in precocious metamorphosis when applied to fifth instars, 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 this finding. (i) Untreated insects FIGURE 13a: The e'ffect of aging and JHA treatment on correlations among body measurements in 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 (ii) 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 in 14-day-old on correlations adult males. FIGURE 13c: The effect of aging and JHA treatment on correlations among body measurements in 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 in 14-day-old adult females. TABLE XIII: Sensitivity 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 " 31 0 9 0" 27  " 6 0" 7 0" 33 1 undeveloped ovary 4  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 slightly PLATE 6a: Dorsal view of untreated (left) male adult and precocious male adultoid (right) resulting from precocene application (200 yg) to newly emerged fourth instars. PLATE 6b: Side view of untreated (left) female adult and precocious female adultoid (right) resulting from precocene application (200 yg) to newly emerged fourth instars. Ill G. JHA Effects on Precocene-Treated Insects (a) Precocene Effects When precocene II (300 pg/insect) was applied to early-fourth instar nymphs, approximately 80% of the insects molted precociously into permanent adultoids (pseudo-adults), skipping the fifth instar (Plates 6a and b). These adultoids had fresh weights about half those of normal insects (Table XIV). Gonads, fat body, and flight muscles were also about half normal size. The precocene-treated adultoids were not only smaller •v than normal, but their body proportions were also different. Tibia length and head width were about three-quarters that of control insects, while tegmina. and wing length were less than half 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 final molt. Significant reductions (P = 0.05) in fat body dry weight were produced in both males and females. In females, ovarian dry weight and total fresh body weight were significantly increased. No significant difference was seen in the size of the fixed sclerites following JHA treatment, except for an apparently significant increase in tibia length. Figs. 14a and b show the correlations among body measurements in 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 Tibia 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 Flight 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 after the final molt to precocene-treated adultoids Body parameter Males FemalePrecocene 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) Tibia 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 in females. Comparison of Figs. 6 and 14a and b indicates that there was far less correlation between body measurements in insects which had been treated with precocene than in 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 in a graded series of morphological effects. When R-20458 was applied to 4-day-old precocene-treated insects, all survivors molted into fifth instar insects (Plate 7b) and eventually into normal-looking adults. However, when the JHA was applied 1 day later in 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 sixth 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 in male adultoids treated with precocene II as fourth instars and with R-20458 after their 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 (ii) Precocene + JHA FIGURE 14b: Correlations among body measurements in female adultoids treated with precocene II as fourth instars and with R-20458 after their precocious molt. TABLE XVI: Overall effects of insects at various 0.05 yg R-20458 intervals prior 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 all 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: Fifth instar nymph (right) and three precocious adultoids (left) resulting 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 fifth 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 fifth 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 in Table XVII. When R-20458 was applied to 4-day-old precocene-treated insects, the surviving adults of both sexes had smaller than normal tibia length and head width, and shortened tibia and wings. Mean wing length was also shorter than tegmina length. Total fresh body weight, flight muscle dry weight, and gonad dry weight were reduced in both sexes, but particularly in the females. When the JHA was applied one day later, the insects which molted into adults exhibited a pronounced reduction in fresh body weight and gonad dry weight in both sexes. Head width and tegmina and wing length were slightly reduced, but the tegmina and wings were of equal length. In the females, the dry weight of the flight muscles, but not of the fat body, was lower than in normal insects. In the male, however, both the fat body and flight muscles were severely reduced. Survivors of R-20458 application to 6-day-old fourth instars previously treated with precocene produced 2 precocious adultoids similar to those described previously (Table XIV). However, the flight muscle dry weight of males was even less than that seen in insects treated with precocene alone. The 4 insects which became relatively normal adults nevertheless exhibited shortened wing and tegmina lengths and slightly reduced flight muscle dry weight, particularly in the males. Wings were shorter than tegmina. TABLE XVII: Effects of timed JHA applications to precocene-treated fourth instars. 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.; Tibia 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 in other acridid species (Pfeiffer, 1945; Hill et al., 1968) newly emerged females of M. sanguinipes undergo two distinct phases of development (Gillott and Elliott, 1976). The initial phase of somatic development, which was reported to involve growth of the cuticle, flight muscles and alimentary tract, is 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 in both sexes. At all adult stages investigated, the total 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 in the two sexes, e.g. Dmaie = 23.8, D£emaie = 26.3. After this period, growth rates differed markedly. In females, the total body weight increased significantly (b = 15.8) until day 8 when oviposition occurred. As reported previously (Gillott and Elliott, 1976; Elliott and Gillott, 1978), this increase is largely due to ovarian growth and protein accumulation by the developing oocytes. In contrast, the total body weight of males did not increase significantly 4 to 9 days after emergence (b = 4.6). The absence of weight increases in the male comparable to those in the female reflects.- several fundamental differences /84 in the reproductive physiology of the sexes. Firstly, the dry weight of the testes-accessory gland complex contributes little to the total 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 stabilized. This increase is largely attributable to the growth of the accessory glands. Apart from demonstrating that males become reproductively mature earlier than females, the stable weight of the testes-accessory gland complex appears to result from the promiscuous behavior observed in male M. sanguinipes (Friedel and Gillott, 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 Gillott, 1972). Proteins, which are a major constituent of the spermatophore, are synthesized in the fat body and sequestered by the accessory glands prior to mating (Gillott and Friedel, 1976). Like vitellogenic proteins (Elliott and Gillott, 1979), these accessory gland proteins are eventually deposited as yolk in the developing oocytes (Friedel and Gillott, 1977). Therefore, in females, proteins produced by both sexes accumulate in the ovary throughout the entire reproductive period until prior to oviposition. In contrast, multiple copulations by the male result in the daily discharge of proteinaceous products by the accessory glands. Thus, cumulative weight changes in the gonads and total body weight observed in females are not evident in males. Changes in the dry weight of the fat body in females concurred with those recorded for female M. sanguinipes (Gillott and /85 Elliott, 1976) and S. gregaria (Hill et al., 1968). The dry weight was small from adult emergence until day 3, when a rapid increase began. The dry weight peaked at day 5, then decreased until oviposition occurred. The decrease in the dry weight of the fat body is accompanied by a rapid increase in ovarian dry weight. Elliott and Gillott (1976, 1978) have shown that in female M. sanguinipes the CA controls yolk protein synthesis in 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 in the dry weight of the fat body also closely paralleled those of the testes-accessory gland complex during the first 5 days of adulthood. For the next 4 days, however, the dry weight of the complex remained fairly constant while that of the fat body decreased. These results are consistent with the findings of Friedel and Gillott (1976a,b) who showed in male M. sanguinipes that the fat body aids in 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 in body weight during early adulthood are associated with rapid flight muscle development. However, the above studies did not extend into the reproductive period. In adult female S. gregaria such studies by Hill et al. (1968) have shown a rapid, steady increase in flight muscle dry weight until /86 soon after yolk deposition begins. After this, the weight remained relatively steady. In M. sanguinipes, similar changes in the dry weight of the flight muscles in males and females were evident. Flight muscle size increased rapidly during somatic growth, then fluctuated after somatic maturity. Contrary to previous studies on M. sanguinipes (Gillott and Elliott, 1976), the present investigation has shown that flight muscle development is not complete by day 3 but that the dry weight increases until day 5 when it declines. Flight muscle dry weight and protein content were interdependent and followed a nearly-parallel, fluctuating pattern. Similar findings have been reported in Loausta (Poels and Beenakkers, 1969; van Marrewijk et al., 1980). The slopes of the regression lines showing the relationship between flight muscle protein content and dry weight in both sexes also indicate that there was more protein per unit weight in small flight muscles than in the large ones. Therefore, some of the increase in flight muscle dry weight around day 5 appears to be due to the accumulation of non-proteinaceous materials. Protein synthesis in the flight muscles of Loausta remains high even after completion of adult development (van Marrewijk et al., 1980). The latter authors have proposed that in Locusta the high level of protein synthesis after maturity is due to high flight muscle metabolism and turnover of tissue materials. In both sexes of M. sanguinipes, peaks in the dry weight of the flight muscle roughly corresponded to those of the fat body. /87 The dry weight of these tissues in both sexes decreased as yolk deposition proceeded in the females. A similar trend is found in several other insects which exhibit high teneral flight activity, followed by flight muscle autolysis (Chapman, 1969). At this 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* flight muscles do not completely degenerate, they merely decrease in size prior to the first oviposition. It would be interesting to see if materials from the flight muscles are incorporated into the fat body to be used in spermatophore or ovarian development. Analysis of protein in the flight muscles using methods similar to those of Gillott and Friedel (1976), would provide a firmer indication of the potential role of the flight muscles in reproduction. In the present studies, the significant correlation among the dry weights of the gonads, fat body, and flight muscle in normal grasshoppers of each sex emphasizes the coordinated growth patterns of these organs within individual insects. In addition, the similarity in growth patterns of the internal organs such as fat body and flight muscles of males and females may indicate some type of synchronization between the two sexes. The maturity of male locusts and grasshoppers is 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 via spermatophores (Friedel and Gillott, 1977). If the fat body and flight muscles of both males .and females were contributing proteins to the oocytes, similar patterns in the development of these internal organs in the two sexes would be expected. There is even recent evidence that JH can be passed from the male to the female during mating in some insects (#. cecvopia; Shirk et al., 1980). If a comparable situation exists in M. sanguinipes, it would account for the synchrony of development between the two sexes. Although the influence of the CA on flight muscle development has not been established in M. sanguinipes, JH is known to affect the development of the fat body and gonads. Therefore, it may be that coordinated changes in JH levels within a grasshopper or locust population would result in synchronzied development. B. Role of JH in Development The CA are known to play a critical role in insect development (Doane, 1973). The classic experiments which Wigglesworth performed on Rhodnius prolixus during the 1930's demonstrated that when the CA of young nymphs were surgically inactivated (decapitation, extirpation), the insects molted prematurely into adults. In contrast, when the CA in final instar nymphs were artificially 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 titres in the haemolymph of younger nymphal stages /89 suppresses adult differentiation and favours the retention of juvenile characteristics. Conversely, a precipitous decline in JH haemolymph titres during the final instar results in metamorphosis and the expression of adult characteristics. Confirmation of this hypothesis in Loausta males and females has been provided by more recent studies which have indirectly (Galleria bioassay) or directly (radio-imrnuno assay, electron-capture gas chromatography) measured JH haemolymph levels during nymphal development (Johnson and Hill, 1973a,b, 1975; Baehr et al., 1979; Huibregtse-Minderhoud et al., 1980). Collectively, these studies have shown that high JH levels are present in the haemolymph throughout most of the fourth (penultimate) stadium whereas JH is absent throughout most of the final stadium. However, Baehr et al. (1979) reported a temporary surge in JH levels at the beginning of the fifth instar. In acridid species, JHA and precocene have been employed as chemical probes to artificially 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 in other species (Kelly and Fuchs, 1978; Kruse Pedersen, 1978; Unnithan and Nair, 1979), topical application of 200-300 yg precocene II to recently molted fourth instar nymphs of M. sanguinipes resulted in a high proportion of the insects molting precociously into diminutive adults. Precocene II tends to have a variable success rate, and 50% effectiveness is 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 sufficient endogenous JH, the insects are unable to maintain juvenile characteristics for a fifth stadium. However, when even higher precocene doses were applied to fifth instar M. sanguinipes, molting and metamorphosis were not disrupted. Comparable findings in Oncopeltus led Unnithan and Nair (1979) to theorize that prococene only affects insects during those developmental stages in which the CA are active. If this theory is tenable in M. sanguinipes, the results of the present study substantiate the premise that JH is absent during part or most of the fifth instar stadium. Two of 6 insects treated with 500 yg precocene as fourth instars and 1 of 7 insects receiving the same dose as mid-fifth 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 in Locusta were treated with precocene (Fridman-Cohen and Pener, 1980; Miall and Mordue, 1980). These results led Miall 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 in Locusta (Kruse Pedersen, 1978) and Oncopeltus (Masner et al., 1979) has been reversed /91 by applying JHA during the same stadium. However, in both species, observations were only carried out into the fifth instar 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 II. When JHA was applied 4 days after the precocene treatment, the insects molted into fifth instars and eventually into normal-looking adults. Apparently the dosage of R-20458 applied at this time was high enough to maintain the juvenile characteristics into the fifth and final nymphal stadium but also low enough so as not to interfere with metamorphosis. The same JHA treatment applied one day later resulted in a high number of semi-precocious adults which later died attempting another molt. R-20458 applied at this time seemed to be too late to program a fifth nymphal stadium. Instead, the epidermal cells received conflicting instructions. Initially, the absence of JH caused by precocene appeared to result in the epidermal cells being committed to produce an adult cuticle but later being programmed by the JHA to produce nymphal cutical and undergo an additional molt. Results of the present study agree with those of Kruse Pedersen (1978), who noted that timing of JHA application in such experiments was critical in Locusta, and that the optimum time for JHA-reversal of precocious metamorphosis was between the third and fourth days of the fourth stadium. In addition, the current results indicate that during normal development, JH levels must be low on the third and /92 fourth days of the fifth instar stadium. However, when JHA was applied 6 days after the precocene treatment, a mixed effect was observed. The two insects which died attempting a final molt were probably still young enough to be affected as in 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 is difficult to explain why the other four insects molted to normal-looking adults, unless JH application immediately before the molt can also initiate nymphal commitment, which seems unlikely. D. JHA Studies on Molting and Metamorphosis JHA-induced supernumerary molting has been observed in Zonoaerus (McCaffery and Page, 1978) and in Locusta (Vogel et al., 1978). CA implants produced similar effects in 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, if the CA are inactive throughout part of the fifth instar stadium, then timing of JHA applications should be important in determining the resulting effects. This is particularly true since JH-sensitivity of different tissues in insects varies with development time (Luscher et al., 1971; Willis, 1974). In M. sanguinipes, supernumerary molting, a symptom of JH-excess, occurred in approximately one quarter of the insects treated with 0.0375 to 0.375 ug R-20458 in the middle of the fifth stadium. However, /93 no supernumerary molts developed when R-20458 was applied to early or late fifth instars. Apparently JHA applications during the mid-fifth instar stadium are capable of preventing the commitment of epidermal cells and other tissues to adult development. This again suggests that low JH titres during the middle of the final stadium are necessary to permit an imaginal molt. However, the present results also indicate that high JH levels during the end of the final stadium are inconsequential in terms of molting and metamorphosis. Locust size is influenced by JH levels. High endogenous JH titre such as those presumably present in 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 differential effect of JH on the two sexes was also observed in the present experiments. JHA application to fifth instar female M. sanguinipes produced no significant overall change in fresh body weight of the adult, whereas in males, the fresh body weight of the adults was significantly reduced. Contrary to the conclusions of Senhal (1971), the current findings clearly 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 tiny wings),, there were small changes in relative body measurements. However, Bowers et al. (1976) writes that in 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 flight muscle and gonad dry weights were more closely proportional to the size of some of the fixed sclerites. However, female precocious adults, treated with JHA, showed no improvement in body measurement correlations. These results indicate that in males, JH stimulates development of internal organs until they reach a basic relatively stable level in proportion to the other body parameters. However, in females, JH stimulates ovarian dry weight independently from that of most other body parts. The metamorphic and coloration changes that occur with phase change in many species have been attributed to JH (Table 1). Characteristics of the relatively sedentary solitarious phase have been produced by CA implants or JHA applications (Joly, 1960, cited in 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 in some cases within the same instar (Rowell, 1967). Results of the present experiments agree with these findings. Reasons for this time lag are probably complex, but may be due in part to the natural cycle of endogenous JH levels within the insects. In /95 the present experiments, JHA treatment of final 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 fifth instar (days 4-5) became green adults, while those treated on day 6 were only faintly green or normal-colored. Insects treated with JHA on day 3 became normal-colored adults, while those treated on the first 2 days of the instar retained juvenile black and beige markings as adults. Wing length was also altered when JHA applications were made at various times during the fifth nymphal stadium. Effects on insects treated at the beginning of the fifth instar (newly emerged to day 3) ranged from little effect to pronounced metathetaly. Grasshoppers treated on days 3-4 had a normal appearance, where those treated towards the end of the fifth instar (days 4-5) possessed shortened wings. By day 6 JHA application was too late to produce much effect, and the insects became nearly normal adults. Apparently JH is involved in determining wing length in M. sanguinipes at the beginning of the fifth stadium and around days 4-5, but not during the remainder of the stadium. Interestingly, these two periods correspond with the JH peaks in fifth stadium Locusta (Baehr et al., 1979). If such peaks are also present in M. sanguinipes, the addition of JHA during periods of high endogenous JH titre might be implicated in the production of short wings. If JH must be low in mid-fifth instars to allow the imaginal molt, and since no effect on color or wing length is produced /96 at this time, it 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 in response to increased JH titres at particular nymphal stages. Results of the present JHA experiments are supported by similar effects of timed JHA applications to final 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 in 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 in order for the two different effects to occur. The latter hypothesis fails to consider the possibility that JHA, even when directly applied at a later stage in the instar, may be too late to produce metamorphic effects in these insects. It may even be that JH must be maintained at a certain level for a particular period of time for various observable effects to occur (Van den Hondel-Franken et al., 1980). Unfortunately, it is impossible to discern from the present experiments and those of Nemec (1970) the extent to which JHA persistence is influencing the results. 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 variation of effects between trials. Since locust development is extremely sensitive to environmental conditions, difficulty in obtaining repeatable results is experienced (Beenakkers and Van den Brock, 1976). Rowell (1967) succeeded in producing green coloration in four grasshopper species, some of which never produced green adults in the wild. However, the CA implants rarely produced metathetalic individuals. The relative absence of this type of effect may be due to the lack of consideration accorded the timing of the implants within the instar in Rowell's experiments. This oversight places in 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 fact, 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. It may be that timing of endogenous JH peaks relative to tissue development in each individual insect, is responsible for the wide variety of effects observed in 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 fifth instar Locusta females have been implicated as a cause of poor flight muscle development (Poels and Bennakkers, 1969; Van den Hondel-Franken et al., 1980). In the present experiments, JHA applications to fifth instar M. sanguinipes of both sexes produced a significant (P = 0.01) reduction in the flight muscle dry weight. However, no significant difference was produced in flight muscle dry weight by varying JHA application time within the instar. Apparently the flight muscles of M. sanguinipes are JHA-sensitive throughout the fifth instar.stadium, even though wing length is 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 is usually reduced, indicating that each tissue responds in a unique way to the hormone. Adding JHA to precocious adultoids did not significantly affect flight muscle dry weight. Apparently JHA added after early adulthood is too late to affect flight muscle growth. This indicates that previous commitment of the tissues may be important in the case of flight muscles, although not of the fat body as mentioned below. It seems that after early adulthood, JHA cannot alter flight muscle synthesis even though it is 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 in gregarious adult male Locusta and Schistocerca (Johnson and Hill, 1973b) and adult female Locusta (Johnson and Hill, 1975) indicated that in both sexes the hormone was present in relatively small amounts immediately after imaginal ecdysis. However, JH then became almost undetectable until just before the beginning of sexual maturation which began in both sexes around day 10. Following sexual maturity, JH levels in males remained uniformly high whereas in females the hormone level peaked near oocyte maturation and dropped to a low titre at oviposition. Subsequent chromatographic studies confirmed the presence of JH in 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 Elliott and Gillott (1976, 1978, 1979) strongly indicate that in adult female M.. sanguinipes the CA stimulate the synthesis of yolk proteins and their uptake by the ovary. JH is also known to enhance reproductive functions in male adult grasshoppers of this species (Gillott and Friedel, 1976). It is possible, /100 however, that high JH levels in the late larval stages also affect gonad maturation, since in solitaria locusts the rate of reproductive development is usually different from that of gregaria (cf. Kennedy, 1961). When JHA was applied to fifth instar M. sanguinipes of both sexes, gonad dry weight increased or decreased. Attempts to initiate reproducible JHA-effects on gonad dry weight did not produce clear consistent trends, even though the insects within each jar usually exhibited similar development. Overall, gonad dry weight of males and females was significantly reduced in the first, but not in the second trial. These results indicate that precise timing of JHA applications to fifth instars relative to each insect's stage of development results in a wide range of effects on the gonads of both sexes. With the intention of lowering JH titres 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 sterile, possessing greatly reduced reproductive organs. The single exception was an anomalous precocious female which contained eggs. Sterility is a common result of precocene treatment (Bowers et al., 1976) in both males and females. As reported in Oncopeltus (Bowers et al., 1976; Bowers and Martinez-Pardo, 1977; Unnithan and Nair, 1979), JHA reversed the sterility of female M. sanguinipes. However, after JHA was applied to precocious adult male M. sanguinipes, no significant change in gonad dry weight was /101 observed. This indicates that in males of this species, JH must be present during nymphal or early adult development for normal gonad size to be reached. Gonad size in males does not increase if JHA is added after early adulthood. The reduction in general fat body dry weight after JHA application, seen in the present experiments, was also recorded in Locusta after CA implantation into final stadium nymphs (Poels and Beenakkers, 1969), and in allatectomized females M. sanguinipes adults after JHA treatment (Gillott and Elliott, 1976; Elliott and Gillott, 1978). In Locusta, the extra JH produced no observable effect until after the imaginal molt. The present experiments show, however, that in M. sanguinipes the fat body is also susceptible to JH during the fifth instar. Although JHA treatment of fifth instar males and females generally had no significant influence on fat body dry weight, the effects of varying application time within the fifth instar were highly significant. In determining the amount of subsequent fat body reduction, precise timing of JHA application is critical but no clear-cut period of JHA-sensitivity of the fat body was observed. As in the previous JHA trials on normal fifth instars, fat body size decreased significantly when precocious adults of both sexes were treated with JHA. Therefore, the development of the fat body of M. sanguinipes is not entirely pre-programmed because the tissue is sensitive to JHA after adulthood is reached. /102 As adult M. sanguinipes males and females age, the effects of the JHA treatment during the fifth 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 similar loss of developmental synchrony also takes place in grasshoppers. F. Grasshopper Control Depending upon the timing of the application, both precocene II and R-20458 have been shown to drastically alter the development of various tissues in 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, particularly that of the wing and flight muscles. Depending upon when JHA and precocene were applied, the development of the tissues was so drastically altered that the compounds were lethal to the grasshoppers. Therefore, theoretically, the compounds are potentially insecticidal and, in addition, act as chemosterilants or inhibit wing development. 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APPENDIX 1: Means and standard errors for growth measurements in early adult M. sanguinipes Age (days) Total body fresh weight (mg) Tibia 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 total body fresh weight in early adult M. sanguinipes (N = 4) Mean total body Age fresh weight Standard (days) (mg) error Males 1 228.4 11.6 2 239.0 8.8 3 276.1 18.4 288.S 16.7 5 296.3 9 8 288.0 15.9 325.0 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 flight muscle dry weight and protein content in normal early adult M. sanguinipes (N = 2 to 3) Males -• Females ;  Mean flight / Mean flight Mean flight Mean flight muscle dry muscle protein muscle dry muscle protein Age weight content Age weight content (days) (mg) (mg) (days) (mg) (mg) Trial la 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 Trial 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 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 significant b D4, D6 >D0, Dl, D2; D5>D1, DO; D8>D1 c. D4, D5>D2 d D4, D6>D1; D6>D2 Trial lc 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 Trial 2d 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 fifth instar HALES  Total body fresh Fat body dry Flight muscle Day of weight Tibia length Head width Tegmina width Wing length Gonad dry weight weight dry weight Trial 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 fifth instar FEMALES Trial Day of application Total body fresh weight (mg) Tibia 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 Flight 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 later treatment with JHA Body Measurements Gonad Flight Insect Tibia 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 Flight Insect Tibia 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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