Cyanoprotein: Immunological properties and content changes during the development of non-diapause female bean bugs,Riptortus clavatus

Immunological properties and content changes of cyanoprotein (CP) were investigated in the bean bug, Riptortus clavatus. Anti-CPegg serum was prepared by immunizing a rabbit with CP purified from eggs (CPegg). In the Ouchterlony double diffusion test, the precipitin line between CPegg and anti-CPegg serum fused with that of non-diapause and diapause female hemolymph and anti-CPegg serum. Rocket immunoelectrophoresis (RIE) using anti-CP serum showed two types of rockets (A and B) depending on the samples. Namely, CPegg and non-diapause female adult hemolymph formed A rockets (heavy-stained with Coomassie Brilliant Blue) and early diapause female adult hemolymph formed B rockets (light-stained), but hemolymph from fifth instar nymphs formed both A and B rockets. Both rockets A and B were demonstrated by SDS-PAGE analysis of the precipitin lines to be formed from the same CP subunit (MW, 76,000). CP-1, 2 and 3 bands from native PAGE of nymphal hemolymph formed A rockets and CP-4 formed B rockets. The contents of CP-A (CP-1 to 3) and CP-B (CP-4) were separately determined by measuring the sizes of rocket A and B. CP-A and CP-B content were demonstrated to increase during the development of the last instar nymph and decrease at adult emergence by RIE analysis of non-diapause female whole body extracts. CP-B is predominant in the nymphal stage. In the early adult stage (day 2 and 3 after emergence), neither CP-A nor CP-B were detected. Only CP-A appeared again at day 4 after emergence and increased during development and vitellogenesis of non-diapause females.     

Quantitative changes and synthesis of cyanoprotein in whole body and tissues during development of the bean bug,Riptortus clavatus

Changes in biliverdin-binding cyanoprotein content in whole body and tissue extracts during development of nymphal and adult (non-diapause) bean bugs, Riptortus clavatus were analyzed by rocket immunoelectrophoresis (RIE). RIE using anti-CPegg serum can be used to determine the content of CP-A (Cp-1, 2 and 3) and CP-B (CP-4) separately. During the nymphal stage CP content of whole body changes cyclically in each instar. In the first nymphal instar, CPegg is the main CP which disappears during the first-second instar ecdysis. In nymphal bugs from the 2nd to 4th instars only CP-B (CP-4) is detected, and at the beginning of each instar the CP content is very low but increases toward the next ecdysis, after which CP decreases and disappears very rapidly. In the 5th nymphal instar, CP-B is the major CP but CP-A (CP-1, 2 and 3) is also detected. These changes in whole body CP content of 5th instar nymphs are observed in both females and males. The content of total CPs in the 5th instar nymph reaches about 1000 μg in the whole insect. During nymphal-adult ecdysis, nymphal CPs decrease and disappear at day 2 after emergence. In female adults CP-A (CP-1 only) increases rapidly after day 4 of adult emergence, while no CP is detected in male adults. In females CPs were detected only in the fat body, hemolymph and ovary. In the mid-5th-instar nymphs, CPs (CP-A and B) are mainly distributed in the hemolymph. CPs in the Hemolymph decrease during nymphal-adult ecdysis, whereas they increase in the fat body. CPs disappear from both the hemolymph and fat body by 2 days after ecdysis. Subsequently in the adult stage only CP-A increases again in the fat body and ovary. By tracer experiments using [³⁵S]-methionine, the fat body was shown to be the site of CP synthesis. CP-A and B synthetic activity was detected in nymphal females whereas, only CP-A synthesis was observed in adult females, while no CP synthesis was seen in adult males.     

Cyanoprotein: Developmental stage,sex and diapause-dependent expression,and synthesis regulation by juvenile hormone in the bean bug,Riptortus clavatus

Cyanoprotein (CP) synthesis was studied in nymphal and nondiapause adult, diapause adult, and juvenile hormone (JH) treated adult bean bugs, Riptortus clavatus. Hemolymph collected from bugs injected with [³⁵S]-methionine was analyzed by native polyacrylamide gel electrophoresis (PAGE) and fluorography, and CP synthesis was also determined quantitatively by rocket immunoelectrophoresis of hemolymph and counting. In the nymphal stages synthesis of CP-1, 2, 3, and 4 (with CP-4 synthesis predominant) reached a maximum at mid-instar and was not detected at each ecdysis, so that the synthetic activity of CPs changed cyclically in each instar. During the nymphal stages CP synthesis showed the same pattern and level in both females and males, and both diapause and nondiapause oriented bugs. In the adult stage, however, CP synthesis differed in the two sexes and nondiapause or diapause conditions. In nondiapause males CP synthesis was not detected in the adult, but in nondiapause females CP (only CP-1) was synthesized through the reproductive stages. CP-1 accumulated in the egg yolk together with vitellogenin. In diapause adults (both females and males) CP-1 to 4 were synthesized at a very low rate for over 2 months and accumulated in the hemolymph. JH or JH analog (JHA) methoprene and also long day condition, which terminate diapause, switched the main CP synthesis CP-4 to CP-1 in females. CP synthesis in diapause males stopped after JH(A) treatment. The activities of CP synthesis, thus, changed in developmental stages, sexes, and diapause. This is an excellent system for study of specific gene expression and switching controlled by insect hormones and sex. © 1992 Wiley-Liss, Inc.     

Influence of methoprene and temperature on diapause termination in adult females of the over-wintering solitary bee, Osmia rufa L

Females of Osmia rufa, as most species in this genus, enter an obligatory diapause, overwintering as an imago inside a cocoon until the ensuing spring when after emergence – mating, egg development and oviposition occur. Diapause in this species is initiated in November, undergoes 2 months of a pre-wintering period that is terminated at the end of January, after 1 month of maintenance. In this study, factors that affect the termination of adult diapause in the female of this species were investigated. The experimental material consisted of bees that were brought from nests kept in natural conditions 1 month prior to natural termination of diapause. Three different experimental treatments were planned to evaluate the potential effect of methoprene and temperature on diapause termination. During the 5 day experimental period the first group of females was kept at 4 °C, the second group at 15 °C and the last group of females was kept at 20 °C. All groups of females were treated with methoprene topically at a dose of 200 μg. After methoprene application a significant increase (p < 0.001) in the size of terminal oocytes was recorded in the three experimental groups. However, no changes in the size of terminal oocytes between acetone treated and untreated control groups were observed. The number of oocytes progressively increased following topical application of methoprene compared to non-treated or acetone treated females. In successive applications of 200 μg methoprene gradual changes in ovary and fat body protein concentration were observed. As compared to controls, protein content in ovaries isolated from methoprene-treated females increased, whereas it decreased in fat body. The least differences in oocyte size and protein concentration in ovary and fat body between control groups and with methoprene application occurred at 4 °C. Differences increased and were higher in females kept at 20 °C and increased rapidly after methoprene application. Exposure to increasing temperature regimes accelerated the juvenile hormone (JH) induced termination of diapause. Taken together, our results indicate that temperature may play an important role in termination of diapause in O. rufa, but its role is secondary to that played by JH.     

Effects of nutrition and methoprene treatment upon reproductive diapause in Caloptilia fraxinella (Lepidoptera: Gracillariidae)

Abstract Female Caloptilia fraxinella exhibit a prolonged reproductive diapause immediately post adult emergence in mid‐summer until the next spring when mating, egg development and oviposition on fresh Fraxinus spp. leaflets occur. Factors that effect the termination of reproductive diapause are investigated in this species. Caloptilia fraxinella diapausing adults held in overwintering conditions (2 °C, LD 0 : 24 h) for 24 weeks terminate diapause after placement for 2 weeks in simulated summer conditions (24 °C, LD 16 : 8 h) only if they are provided with 10% sugar water. Exogenous application of the Juvenile Hormone (JH) analogue methoprene to moths in both early‐ (summer) and mid‐ (autumn) reproductive diapause demonstrates that JH affects diapause termination but a carbohydrate nutrition source also mediates mating and vitellogenesis. Mating between moth pairs early in diapause occurs only after treatment with methoprene and provision with sugar water. However, there is no impact of mating on the propensity of females to produce vitellogenic oöcytes. Moths collected in the autumn in mid‐diapause respond in a dose‐dependent fashion to methoprene treatment and the response is greater than that of moths early in diapause collected in the summer. Treatment with methoprene and access to sugar water results in vitellogenic oöcytes in 18.75% of females from mid‐diapause moth pairs treated with 0.01 μg methoprene per insect and in all females from pairs treated at the two highest doses of methoprene (0.1 and 1 μg per insect). Mating occurs only between moths in mid‐diapause treated with the two highest doses of methoprene and these doses induce 91% and 100% mating, respectively. Both control and methoprene‐treated males in mid‐diapause held under summer conditions mate successfully and pass a spermatophore to their methoprene‐treated female partner. These data demonstrate that female C. fraxinella undergo a prolonged reproductive diapause in which termination is dependent on JH and further mediated by a carbohydrate nutrition source. The production of vitellogenic oöcytes is independent of mating. These data also provide evidence that response of moths in diapause to exogenous applications of methoprene differs throughout the diapause period and between male and female C. fraxinella.     

Relationship between the fatty acids of fat body triacylglycerol and lipophorin diacylglycerol of non-diapause and diapause larvae of the southwestern corn borer,Diatraea grandiosella

The fatty acids of the triacylglycerol reserves in the fat body and of the diacylglycerol of lipophorin in the hemolymph of non-diapause and diapause larvae of D. grandiosella were compared. For both non-diapause and diapause larvae palmitate, palmitoleate, oleate, and linoleate were the predominant fatty acids present in fatty body triacylglycerol, and palmitate, oleate, and linoleate were the predominant fatty acids present in lipophorin diacylglycerol. However, differences were detected in the relative amounts of oleate and linoleate present in lipophorin diacylglycerol of non-diapause and diapause larvae. The relative amount of linoleate in lipophorin diacylglycerol declined during diapause, whereas that of oleate remained relatively high during diapause. The fatty acid profile of lipophorin diacylglycerol from non-diapause larvae treated with a juvenile hormone analog to induce a diapause-like state more closely matched that of diapause larvae than that of non-diapause larvae. The differences detected in the fatty acid composition of lipophorin diacylglycerol in non-diapause and diapause larvae appear to be due mainly to the different physiological states rather than to the different rearing temperatures employed. The results are discussed in relation to the essential role fatty acids, especially oleate, play in the survival of diapause larvae.     

Vitellogenin mRNA in locust fat body: coordinate induction of two genes by a juvenile hormone analog

Levels of vitellogenin (Vg) mRNA in Locusta migratoria fat body were determined as indicators of gene expression induced by the juvenile hormone analog methoprene. After injection of methoprene into juvenile hormone-deprived locusts, excised fat bodies were cultured with [3H]leucine for immunochemical assay of Vg synthesis, and RNA was assayed for Vg mRNA content by hybridization with probes from the previously cloned locust Vg genes A and B. In general, the rise in Vg mRNA paralleled the rise in Vg synthesis. During the primary response to methoprene (in female locusts in which the corpora allata had been destroyed immediately after emergence), Vg mRNA was first detected after 18-24 hr and accumulated rapidly between 36 and 48 hr. The secondary response (in locusts allatectomized during vitellogenesis and kept until Vg disappeared) was accelerated, as Vg mRNA was detectable at 12 hr and titers rose steeply after 18 hr. When Vg synthesis was prematurely induced by injection of methoprene into fifth-stage female larvae, the kinetics of mRNA accumulation were similar to those of primary stimulation in the adult. After allatectomy of vitellogenic females, fat body Vg mRNA decayed with a half-life of about 24 hr, roughly paralleling the decline in Vg synthesis. Assays with the two Vg probes showed coordinate accumulation of gene A and gene B messages under all conditions tested: during primary and secondary stimulation in adult females and in the low-level response obtained by treating male larvae with methoprene.     

Regulation of vitellogenesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)

Studies were undertaken to investigate vitellogenesis and its regulation in female adults of the fall armyworm, Spodoptera frugiperda. A single female-specific protein, likely to be the S. frugiperda vitellogenin (Vg), appeared approximately 5 h after adult eclosion in the hemolymph of virgin females. The concentration of the protein increased with age as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed. A protein with the same relative molecular mass was also present in egg extracts, but absent from hemolymph samples from male moths. The relative molecular mass of the designated S. frugiperda Vg was determined as 164.5+/-2.5 kDa. Vitellogenic oocytes became visible 36-48 h after emergence and egg deposition began on day 3 of adult life. Vg could not be detected in the hemolymph of females decapitated directly after eclosion. When decapitated virgin females were injected with the JH-mimic methoprene (MP), the level of Vg was comparable to that in non-decapitated moths, indicating that vitellogenesis in S. frugiperda depends on juvenile hormone (JH). However, the number of vitellogenic oocytes was somewhat lower than in non-decapitated virgin females. Injection of 20-hydroxyecdysone (20E) promoted Vg production to a similar extent in decapitated female moths, but in contrast to methoprene injection, treatment with 20E never resulted in the production of vitellogenic oocytes. In vitro cultivated ovaries of adult females dissected directly after eclosion produced lower amounts of ecdysteroids than those isolated on day 1 after emergence. Our results suggest a crucial role for 20E in the induction of vitellogenesis in the noctuid S. frugiperda, while JH seems to be essential for the continued uptake of Vg by developing oocytes and may trigger 20E biosynthesis in the ovary.     

Methoprene-induced matings of young Queensland fruit fly males are effective at inducing sexual inhibition in females

Pre-release dietary treatment with methoprene, a juvenile hormone analogue, decreases the age at which male Queensland fruit flies mature and hence may decrease the post-release delay until released sterile flies participate in sterile insect technique (SIT) programmes. However, if matings of young methoprene-treated males are not effective at inducing sexual inhibition in their mates, then this treatment may not enhance SIT. The present study investigates efficacy of matings of methoprene-treated males at inducing sexual inhibition in their mates. Methoprene incorporated into a diet of sugar and yeast hydrolysate (w/w 3:1) for 48 hr after emergence resulted in significantly increased male mating propensity when flies were <10 days of age, but not when older, and longer copulations. Copula latency did not vary with methoprene treatment but did decrease with age. The matings of young methoprene-treated males were effective at inducing sexual inhibition in their mates, matching the efficacy of untreated mature males. Regardless of treatment, females had reduced tendency to remate if their first mate was 15 days of age than if their first mate was younger (6, 8 days) or older (20, 25, 30 days). Females mated by methoprene-treated males that did remate tended to remate later in the day than females mated by untreated males. Also, second copula durations of females first mated by a 6- to 10-day-old male were shorter if the male was methoprene treated. These patterns in remating females may indicate greater efficacy of the initial mating of methoprene-treated males. Overall, we find that the additional matings of young methoprene-treated male Queensland fruit flies are effective at inducing sexual inhibition in their mates. This finding supports the incorporation of methoprene into pre-release diet for SIT.     

Vitellogenins and vitellins in the bean bug,Riptortus clavatus (hemiptera: alydidae): Purification,immunological identification,and induction by juvenile hormone

Vitellogenins (Vgs) and vitellins (Vns) were purified from reproductive female adult hemolymph (HL) and newly laid eggs of the bean bug, Riptortus clavatus. They were separated into two (Vg-1 and 2) and three (Vn-1′, Vn-1, and Vn-2) sub-components, respectively, by ion exchange chromatography. Fused rocket immunoelectrophoresis using anti-(Vn-1) serum showed that they are distinguished clearly into two immunologically distinct groups, i.e., Vg-1/Vn-1/Vn-1′ and Vg-2/Vn-2. SDS-PAGE analysis showed Vn-1 (Vn-1′) and Vn-2 have the same and/or smaller subunit component polypeptides as Vg-1 and Vg-2, respectively. Vn-1′ has some lower molecular weight peptides than Vn-1. Quantitative rocket immunoelectrophoretic analysis showed that Vg-1 and Vn-1 (plus V-1′) were detected first on day 3 in the HL and day 4 in the ovary, respectively, after adult emergence, and increased by day 10 in non-diapause female adults. Vg-1 synthesis is induced in diapause female adults in 1 day after the treatment with juvenile hormone (JH) and JH analog (JHA) or in a few days after transfer from short day to long day conditions. © 1996 Wiley-Liss, Inc.     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

JH III production, titers and degradation in relation to reproduction in male and female Anthonomus grandis

Juvenile hormone (JH) is necessary for the production of vitellogenin (Vg) in the boll weevil, Anthonomus grandis. Occurrence of Vg in this species is typically restricted to reproductively competent females, and is not detected in untreated males. However, the JH analog, methoprene stimulates Vg production in intact males and in the isolated abdomens of both male and female boll weevils (where in each case no Vg is detected without treatment), suggesting that males are competent to produce Vg but are normally not stimulated to do so. Preliminary work indicating that male boll weevil corpora allata (CA) produced little or no JH in vitro suggested that failure of males to produce Vg might be due to very low JH levels compared to females. This study re-examines the question of JH in male boll weevils by determining in vitro production of JH III by male CA during the first 10 days after adult emergence, determining hemolymph JH esterase activity during this same time period and hemolymph JH III titers in adults of both sexes. We also re-examine the ability of isolated male abdomens to produce Vg in response to hormonal stimulation, analyzing the effect of a wide range of methoprene and JH III dosages. Results indicate that male A. grandis have circulating JH titers and JH production similar to females. JH esterase activity is slightly but significantly higher in males than females. Vg production by isolated abdomens of both sexes after stimulation with methoprene or JH III was confirmed. Dose response studies indicated that high levels of methoprene were less effective than intermediate doses in stimulating Vg synthesis in both sexes. We conclude that the sexually dimorphic effect of JH on Vg synthesis is not due to differences in JH production or differences in JH titer between the sexes.     

Prothoracicotropic Hormone Acts as a Neuroendocrine Switch between Pupal Diapause and Adult Development

Diapause is a programmed developmental arrest that has evolved in a wide variety of organisms and allows them survive unfavorable seasons. This developmental state is particularly common in insects. Based on circumstantial evidence, pupal diapause has been hypothesized to result from a cessation of prothoracicotropic hormone (PTTH) secretion from the brain. Here, we provide direct evidence for this classical hypothesis by determining both the PTTH titer in the hemolymph and the PTTH content in the brain of diapause pupae in the cabbage army moth Mamestra brassicae. For this purpose, we cloned the PTTH gene, produced PTTH-specific antibodies, and developed a highly sensitive immunoassay for PTTH. While the hemolymph PTTH titer in non-diapause pupae was maintained at high levels after pupation, the titer in diapause pupae dropped to an undetectable level. In contrast, the PTTH content of the post-pupation brain was higher in diapause animals than in non-diapause animals. These results clearly demonstrate that diapause pupae have sufficient PTTH in their brain, but they do not release it into the hemolymph. Injecting PTTH into diapause pupae immediately after pupation induced adult development, showing that a lack of PTTH is a necessary and sufficient condition for inducing pupal diapause. Most interestingly, in diapause-destined larvae, lower hemolymph titers of PTTH and reduced PTTH gene expression were observed for 4 and 2 days, respectively, prior to pupation. This discovery demonstrates that the diapause program is already manifested in the PTTH neurons as early as the mid final instar stage.     

In vivo and in vitro effects of 20-hydroxyecdysone and methoprene on diapause maintenance and reproductive development in Musca autumnalis

Abstract. Face flies overwinter as adults in reproductive diapause. Administration of 20-hyroxyecdysone and/or methoprene induced reproductive development in diapausing flies which were maintained in a diapause-inducing environment. Hormone effects were additive and female flies were more sensitive than males. Release of vitellogenin from cultured fat body was stimulated by 20-hydroxyecdysone or methoprene. Transfer of flies from diapause to diapause-breaking environments induced some to break diapause, but this decreased with the time flies had been in a diapause-inducing environment. In contrast, topical application of methoprene to diapausing flies induced reproductive development irrespective of their ages even when they were kept in the diapause-inducing environment for 80 day degrees above a 12°C base temperature (14.5 days). Therefore diapause induction must depend on hormone levels less than some threshold level. The putative threshold varied according to diapause propensities of different genetic lines. Lines showing high frequencies of diapause required greater amounts of methoprene for reproductive development in diapause conditions than did lines showing low frequencies of diapause.     

Neuroendocrine control of diapause hormone secretion in the silkworm,Bombyx mori

To clarify the control mechanism of diapause hormone (DH) secretion in the silkworm Bombyx mori a series of anatomical and pharmacological experiments were carried out. The arrangement of 'diapause' and 'non-diapause' eggs in the ovarioles of the moths was determined by the coloration method to estimate the accumulation of 3-hydroxykynurenine in the eggs. The females destined to lay non-diapause eggs (non-diapause producers) had diapause eggs in their ovaries if their subesophageal ganglions (Sg) had been surgically removed at 2days after larval-pupal ecdysis or later. In contrast when the surgical extirpation extended to the brain and the corpora cardiaca (CC)-corpora allata (CA) complex in addition to the Sg, the non-diapause producers had no diapause eggs. When the Sg was removed from the females destined to lay diapause eggs (diapause-producers), diapause eggs appeared in response to the treatment at 2days after larval-pupal ecdysis, but the appearance of diapause eggs was delayed by 2days when the brain-CC-CA complex was included among the organs removed. These observations suggested that DH is produced in Sg and transferred to the CC-CA complex, and that the secretion of DH from the complex is suppressed in non-diapause producers. The pattern of diapause and non-diapause eggs induced by the transection of the subesophageal connective in diapause and non-diapause producers suggested a regenerative and secretory capacity of the neurosecretory cells after the operation. The appearance of diapause eggs in non-diapause producers with transected protocerebrum of the brain confirmed that there was an inhibitory center in the protocerebrum. Changes in parts of the ovarioles containing diapause and non-diapause eggs with time of injection of gamma-aminobutyric acid (GABA) and picrotoxin suggested that a GABAergic inhibitory mechanism in DH secretion may be active in non-diapause producers but inactive in diapause producers throughout the pupal stage.     

Juvenile hormone and the induction of larval polymorphism and the diapause of the southwestern corn borer, Diatraea grandiosella

The southwestern corn borer, Diatraea grandiosella, enters diapause as an immaculate mature larva which is a polymorphic variant of the spotted non-diapause larva. Because dormant immaculate larvae could be obtained by treating last stage non-diapause larvae with a juvenile hormone mimic (JHM), experiments were conducted to determine whether these hormonally induced immaculate larvae (HIL) were physiologically comparable to the normal environmentally induced immaculate diapause larvae (EIL). Comparative data obtained about pupation rates, response to JHM, metabolic reserves, oxygen consumption, and the state of spermatogenesis of HIL and EIL led to the conclusion that the HIL were physiologically similar to the EIL. The results demonstrate that the developmental programme of non-diapause larvae could be switched and ‘diapause’ induced solely by the topical application of JHM. We believe that the data further support our hypothesis that the larval diapause of D. grandiosella is initiated and maintained by the juvenile hormone.     

Rapid cold hardening in the predatory mite Euseius (Amblyseius) finlandicus (Acari: Phytoseiidae)

A rapid cold hardening response was studied in diapause and non-diapause females of the predatory mite Euseius finlandicus. When laboratory reared diapause and non-diapause females were transferred and maintained from the rearing temperature of 20 degrees C for 2 h to -11.5 degrees C and -10 degrees C, 10 to 20% survived respectively. However, conditioning of diapause females for 4 h at a range of temperatures from 0 to 10 degrees C before their exposure for 2 h to -11.5 degrees C, increased survival to approximately 90%. Similarly, conditioning of non-diapause females for 4 h at 5 degrees C before their exposure for 2 h to -10 degrees C increased survival to 90%. A similar rapid cold hardening response in both diapause and non-diapause females was also induced through gradual cooling of the mites, at a rate of approximately 0.4 degrees C per min. The rapid increase in cold tolerance after prior conditioning of the mites to low temperatures, was rapidly lost when they returned to a higher temperature of 20 degrees C. Rapid cold hardening extended the survival time of diapause and non-diapause females at sub-zero temperatures. The cost of rapid cold hardening in reproductive potential after diapause termination was negligible. In non-diapause females, however, the increase in cold tolerance gained through gradual cooling could not prevent cold shock injuries, as both fecundity and survival were reduced.     

Sensitivities to juvenile hormone and ecdysteroid in the diapause larvae of Omphisa fuscidentalis based on the hemolymph trehalose dynamics index

The final instar larva of the bamboo borer, Omphisa fuscidentalis, is in diapause for 9 months from September to the following June. Trehalose and ecdysteroid concentrations in hemolymph were measured through the larval diapause period and in the pupal stage. The ecdysteroid concentration remained low until November, followed by a gradual increase to about 30 ng/ml in May. The trehalose concentration remained at levels ranging between 40-50 mM until May, and decreased to an almost undetectable level after pupation. Since a juvenile hormone analogue (JHA), methoprene, is capable of terminating diapause by stimulating larval prothoracic glands, we examined its effects on ecdysteroid and trehalose concentrations in larvae in December and February. The hemolymph ecdysteroid increased more quickly in February than in December, indicating that the sensitivity of the prothoracic glands to JHA increased towards the end of diapause termination. Similarly, hemolymph trehalose in February decreased within a few days after JHA application, while in December the decrease occurred in the third week. Exogenous 20-hydroxyecdysone (20E) caused a decrease in trehalose concentration in a dose-dependent manner. The effective dose of 20E, however, did not change from January until April, implying that the sensitivity of tissue(s) to 20E may not change until the end of diapause. Taken together, our results suggest that the sensitivities of tissues to JH and 20E do not increase simultaneously with the progress of diapause development and that termination of larval diapause is not associated simply with the restoration of hormone deficiencies.     

A juvenile hormone analogue,methoprene as a circadian and developmental modulator in Diatraea grandiosella (Pyralidae)

A juvenile hormone analogue (methoprene) affected the circadian system controlling the adult eclosion rhythm in Diatraea grandiosella. The effect of methroprene was compared with those of photoperiod, sex and temperature. The phase difference (ψ&#x0303;rl) between the rhythm and the light-dark cycle and its variance increased as the photoperiod increased. Males emerged earlier than females. Higher temperatures induced earlier eclosion and reduced the variance. A parallel phase advance was observed in papae treated topically with methoprene. At the higher dose tested, methoprene induced earlier eclosion. The effect of methoprene was both dose and age dependent, with earlier application more effective. A dosage of 3.0 μg was lethal, however, if applied in the first 2 days of pupal stage. Synthetic juvenile hormone I had less effect than methoprene. Methoprene accelerated adult differentiation and emergence in both sexes.