Juvenile hormone-mediated termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

Larvae of the bamboo borer, Omphisa fuscidentalis are in diapause for more than nine months (Singtripop, T., Wanichaneewa, S., Tsuzuki, S., Sakurai, S. 1999. Larval growth and diapause in a tropical moth, Omphisa fuscidentalis Hampson. Zool. Sci. 16, 725-733). To examine the endocrine mechanisms underlying this larval diapause, we assayed the responsiveness of the diapausing larvae to 20-hydroxyecdysone (20E) and a juvenile hormone analogue (JHA: S-methoprene). 20E injection caused the larvae to halt movement, followed by deposition of a pupal cuticle. Topical application of JHA induced pupation in a dose-dependent manner. JHA also induced pupation of the larvae whose brains were removed before JHA application. In those larvae, the prothoracic glands became active and competent to respond to brain extracts within seven days after JHA treatment, and the hemolymph ecdysteroid concentration began to increase 12 days after JHA application. These results indicate that JHA stimulates the prothoracic glands of diapausing Omphisa larvae, terminating larval diapause, in contrast with previous findings that JH inhibits the brain-prothoracic gland axis and thus maintains the larval diapause. Current results therefore suggest a novel regulatory mechanism for larval diapause in this species.     

Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.     

Stimulatory effect of organic solvents on initiating development in diapausing pupae of the flesh fly, Sarcophaga crassipalpis, and the tobacco hornworm, Manduca sexta

ABSTRACT. Alkanes, diethyl ether, and various other organic solvents proved to be potent stimulants of development in diapausing pupae of Sarcophaga crassipalpis Macquart and of Manduca sexta (Johansson). Topical application of 2μl or vapour exposure for 1–2h was sufficient stimulation for the flies, but the solvent had to be injected to elicit the response in the hornworms. In flies, oxygen consumption increased nearly 100-fold within 15 min of hexane application, and thereafter persisted at non-diapause levels. Sensitivity of fly pupae to hexane remained high throughout diapause but acetone sensitivity dropped sharply after the second day in diapause. Acetone applied the day before the onset of diapause averted diapause in flies, and topical application to hornworm larvae 3 days before pupation likewise prevented pupal diapause. Debrained pupae failed to respond to solvent treatment, and we conclude that it is the brain, rather than the prothoracic gland, that responds directly to stimulation.     

Diapause hormone directly stimulates the prothoracic glands of diapause larvae under juvenile hormone regulation in the bamboo borer,Omphisa fuscidentalis Hampson

Larval diapause in many lepidopteran insects is induced and maintained by high juvenile hormone (JH). In the case of the bamboo borer, Omphisa fuscidentalis, the effect of JH is the opposite: The application of juvenile hormone analog (JHA: S‐methoprene) terminates larval diapause, unlike in other insect species. Here, we analyzed the expression of JH‐receptor Met, DH‐PBAN, and Kr‐h1 in the subesophageal ganglion (SG) from October to April using semi‐quantitative polymerase chain reaction (PCR). The results show that OfMet and OfDH‐PBAN messenger RNA in the SG are mainly expressed during the larval diapause stage, while OfKr‐h1 increases during the pupal stage. Using tissue culture techniques and an enzyme‐linked immunosorbent assay (ELISA), diapause hormone (DH) was found to induce ecdysteroidogenesis in the culture medium of the prothoracic gland (PG) after incubation for 30 min with 25 ng and 50 ng of DH. Thus, DH is a novel stimulator for the PG. We identified a DHR homolog in the bamboo borer and confirmed that it is expressed in the PG. In addition, for in vitro experiments, DH increased the expression levels of OfDHR, OfEcR‐A, and ecdysone‐inducible genes in the PG. These results demonstrate that DH can function as a prothoracicotropic factor, and this function of DH might be through of DHR expressed on PG cells. Consequently, DH is one of the key factors in larval diapause break which is triggered by JH in the bamboo borer, O. fuscidentalis.     

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.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

Disturbance of Adult Eclosion by Fenoxycarb,a Juvenile Hormone Mimic,in the Beet Armyworm,Spodoptera exigua

Effect of exogenous juvenile hormones (JHs) on pupal development was assayed in the beet armyworm, Spodoptera exigua. Fenoxycarb, a potent JH mimic, was applied topically to different ages of the pupae, and showed significant inhibition of normal adult eclosion even at 0.1 μg dose when it was applied at the early pupal stage (day 0). As the pupal development underwent, the susceptibility of the pupae fenoxycarb decreased. RH5992, a potent ecdysteroid mimic, did not, however, any similar inhibitory effect on the pupae. Natural JH types (JH I, JH II, and JH III) were applied on day 0 pupae to compare their inhibitory effects on adult eclosion. Both JH I and JH II significantly inhibited adult eclosion at 1.0 μg dose, but JH III did not even at 10.0 μg dose. It was noted that fenoxycarb-treated pupae showed little rectum development. Fenoxycarb did not, however, show any negative effect on the development of compound eye and wing imaginal discs, and on the pupal hemolymph protein pattern. These results suggest that there should be a commitment period requiring an absence of JH for a normal adult metamorphosis during early pupal development and that the endogenous type of JH in S. exigua is JH I or JH II or both JHs like other lepidopteran species.     

METHOPRENE INFLUENCES REPRODUCTION AND FLIGHT CAPACITY IN ADULTS OF THE RICE LEAF ROLLER,Cnaphalocrocis Medinalis (GUENỂE) (LEPIDOPTERA: PYRALIDAE)

Juvenile hormone (JH) influences many aspects of insect biology, including oogenesis‐flight syndrome tradeoffs between migration and reproduction. Drawing on studies of many migratory insects, we posed the hypothesis that JH influences migratory capacity and oogenesis in the rice leaf roller, Cnaphalocrocis medinalis. We treated adults moths (days 1, 2 and 3 postemergence) with the JH analog (JHA), methoprene, and then recorded the influences of JHA treatments on reproduction. JHA treatment on day 1 postemergence, but not on the other days, shortened the preoviposition period, although JHA did not influence total fecundity, oviposition period, or longevity. We infer day 1 postemergence is the JH‐sensitive stage to influence reproduction. Therefore, we treated moths on day 1 postemergence with JHA and recorded flight capacity, flight muscle mass, and triacylglycerol (TAG) accumulation. JHA treatments did not influence flight speed, but led to reductions in flight durations and flight distances. At day 3 posttreatment (PT), JHA‐treated females flew shorter times and less distance than the controls; JHA‐treated males, however, only flew shorter times than the controls. JHA treatments led to reductions in flight muscle mass in females at days 2–3 PT and reductions in TAG content in females at day 3 PT, but, these parameters were not influenced by JHA in males. These findings strongly support our hypothesis, from which we infer that JH is a major driver in C. medinalis oogenesis‐flight syndrome tradeoffs. Our data also reveal a JH‐sensitive stage in adulthood during which JH influences the oocyte‐flight syndrome in C. medinalis.     

Macromolecular synthesis in wing discs of Spodoptera mauritia Boisd: effects of a juvenile hormone analogue

Last instar larvae of S. mauritia treated topically on day 0, day 1, day 2 and day 3 with a daily (dose of 25 microg juvenile hormone analogue (JHA) moulted into supernumerary larvae. The imaginal discs of the supernumerary larvae especially those of mouthparts and thoracic appendages showed pupal characteristics. However the wing discs, which showed only partial differentiation, were uneverted and highly tanned. In an effort to provide an explanation to this anomaly the RNA, DNA and protein profile in the wing discs of supernumerary larvae were studied. Quantitative analysis of DNA, RNA and protein showed a considerable increase in the amount of DNA and protein and a decline in RNA level. SDS-PAGE analysis of wing disc proteins of JHA treated larvae showed a reduction in the expression of many major proteins that were predominant in the wing discs of control larvae. The results suggest that JHA induced inactivation of genes involved in the synthesis of proteins needed for evagination process may be responsible for the formation of uneverted, partially differentiated pupal wing discs in supernumerary larvae.     

Diapause phenomena in non-diapausing last instar larvae,pupae, and pharate adults of the Colorado beetle

From the first day of the last (fourth) larval instar no trace of juvenile hormone (JH) can be detected in the haemolymph by Galleria bioassay. Three specific diapause proteins, which are also found in diapausing adults, appear in the haemolymph. These proteins disappear towards the end of the pupal stage. Study of the ultrastructure of the fat body revealed the formation from lysosomes of proteinaceous bodies which are also characteristic for adult diapause. The behaviour of last instar larvae and pupae resembles that of prediapausing and diapausing adults respectively. Injection of synthetic JH delays the appearance of the diapause proteins in the haemolymph and of proteinaceous bodies in the fat body for 2 to 3 days. The absence of JH seems to trigger off these diapause phenomena.     

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.     

Infradian cycles of oxygen consumption in diapausing pupae of the flesh fly, Sarcophaga crassipalpis, monitored by a scanning microrespirographic method.

Fine details of the infradian O2 consumption cycles that characterize pupal diapause in flesh flies have been monitored by a newly designed microrespirographic method coupled with an electronically regulated O2 generator. During the 4-5 days between the peaks of elevated O2 consumption, the diapausing pupae maintained a very low and fairly constant respiratory rate (13 microl O2 x g-1.h-1). During the intercalated peaks of increased respiratory metabolism, which lasted an average of 33.6 h to 24-27 degrees C, the average maximum rate of O2 consumption was 86.9 microl.g-1.h-1, a value of 6.7 times higher than the interpeak values. The respiratory peaks started abruptly in some cases while the decline was consistently gradual. During the periods between the peaks there were no discontinuous bursts of CO2 release, a feature common to diapause in many other insects. Diapause was characteristically terminated during a peak of the O2 consumption cycle. At diapause termination O2 consumption remained at the maximum values of the peak for many hours and then gradually increased to levels characteristic of nondiapause development.     

Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris

At 25 degrees C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at approximately 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of approximately 1.1 ng/ml was maintained even 30-60 days into the instar, when they were in diapause. Ecdysteroid titers remained approximately 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers.     

Control of pupal commitment in the imaginal disks of Precis coenia (Lepidoptera: Nymphalidae)

When final (5th) instar larvae of Precis coenia were treated with the juvenile hormone analog (JHA) methoprene, they underwent a supernumerary larval molt, except for certain regions of their imaginal disks, which deposited a normal pupal cuticle. Evidently those regions had already become irreversibly committed to pupal development at the time JHA was applied. By applying JHA at successively later times in the instar, the progression of pupal commitment could be studied. Pupal commitment in the proboscis, antenna, eye, leg and wing imaginal disks occurred in disk-specific patterns. In each imaginal disk there were distinct initiation sites where pupal commitment began during the first few hours of the final larval instar, and from which commitment spread across the remainder of the disk over a 2- to 3-day period. The initiation sites were not always located in homologous regions of the various disks. As a rule, pupal commitment also spread from imaginal disk tissue to surrounding epidermal tissue. The regions of pupal commitment in all disks except those of the wings, coincided with the regions of growth of the disk. Only portions of the disk that had undergone cell division and growth underwent pupal commitment. Shortening the growth period did not prevent pupal commitment in the wing imaginal disk, indicating that, in this disk at least, a normal number of cell divisions was not crucial in reprogramming of disk cells for pupal cuticle synthesis. The apparent growth spurt of imaginal disks that occurs during the last part of the final larval instar is merely the final stage of normal and constant exponential growth. Juvenile hormone (JH) and ecdysteroids appeared to play little role in the regulation of normal imaginal disk growth. Instead, growth of the disks may be under intrinsic control. Interestingly, even though endogenous fluctuation in JH titers do not affect imaginal disk growth, exogenous JHA proved able to inhibit both pupal commitment, cell movement, and growth of the disks during the last larval instar. This function of JH could be important under certain adverse conditions, such as when metamorphosis is delayed in favor of a supernumerary larval molt.