Head Capsule Slippage Research Articles

The killing speed of egt-inactivated Bombyx mori nucleopolyhedrovirus depends on the developmental stage of B. mori larvae

Several lines of evidence have shown that the deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the nucleopolyhedrovirus (NPV) genome increases the killing speed of host lepidopteran larvae. However, it has not been investigated in detail whether the effects of egt deletion depend on the larval stages of the host insect. In this study, we performed bioassays using 10 continuous larval stages of the 4th- or 5th-instar Bombyx mori larvae and B. mori NPV egt mutants. The fast-killing phenotype was observed in the egt mutants only when the infection process progressed through larval–larval transition. All day-2 4th-instar larvae infected with the egt mutants entered the molting stage and died much earlier than wild-type-infected larvae. Bodies of egt mutant-infected larvae were filled with excessive fluid immediately after head capsule slippage, owing presumably to the degeneration of Malpighian tubules. Fourth- or 5th-instar larvae infected with the egt mutants at early stages of each instar died similarly to those infected with the wild-type virus. Under infection in the middle stages of the 5th-instar, the survival time of egt mutant-infected larvae was significantly longer than that of the wild-type virus-infected larvae. These results clearly show that the effects of egt deletion on killing speed of NPV are largely dependent on the developmental stage of the host larvae infected by the virus.

Transcriptome analysis of integument differentially expressed genes in the pigment mutant (quail) during molting of silkworm, Bombyx mori.

In the silkworm Bombyx mori, pigment mutants with diverse body colors have been maintained throughout domestication for about 5000 years. The silkworm larval body color is formed through the mutual interaction of melanin, ommochromes, pteridines and uric acid. These pigments/compounds are synthesized by the cooperative action of various genes and enzymes. Previous reports showed that melanin, ommochrome and pteridine are increased in silkworm quail (q) mutants. To understand the pigment increase and alterations in pigment synthesis in q mutant, transcriptome profiles of the silkworm integument were investigated at 16 h after head capsule slippage in the fourth molt in q mutants and wild-type (Dazao). Compared to the wild-type, 1161 genes were differentially expressed in the q mutant. Of these modulated genes, 62.4% (725 genes) were upregulated and 37.6% (436 genes) were downregulated in the q mutant. The molecular function of differently expressed genes was analyzed by Blast2GO. The results showed that upregulated genes were mainly involved in protein binding, small molecule binding, transferase activity, nucleic acid binding, specific DNA-binding transcription factor activity and chromatin binding, while exclusively down-expressed genes functioned in oxidoreductase activity, cofactor binding, tetrapyrrole binding, peroxidase activity and pigment binding. We focused on genes related to melanin, pteridine and ommochrome biosynthesis; transport of uric acid; and juvenile hormone metabolism because of their importance in integument coloration during molting. This study identified differently expressed genes implicated in silkworm integument formation and pigmentation using silkworm q mutant. The results estimated the number and types of genes that drive new integument formation.

An entomopoxvirus and a granulovirus have different impacts on Adoxophyes honmai (Tortricidae, Lepidoptera) simultaneously parasitized by Ascogaster reticulatus (Braconidae: Hymenoptera)

Abstract Development of the endoparasitoid Ascogaster reticulatus is adversely affected when its host, Adoxophyes honmai, is simultaneously infected with either an entomopoxvirus (AdhoEPV) or a granulovirus (AdhoGV). The two viruses, however, have different effects on the survival of A. reticulatus: AdhoEPV infection prevents parasitoid emergence, whereas parasitoid larvae emerge from AdhoGV-infected hosts but die before pupation. Prevention of emergence of the parasitoid from AdhoEPV-infected hosts seems to be due to earlier death of the host during head capsule slippage in the 4th instar, which did not occur in AdhoGV-infected larvae. Previous studies showed that healthy A. honmai larvae have peaks of juvenile hormone esterase (JHE) and 20-hydroxyecdysone (20E) during the 5th instar before pupation. AdhoEPV-infected (non-parasitized) larvae do not pupate, because no peak of 20E appears in AdhoEPV-infected larvae. AdhoGV-infected larvae also do not pupate, in this case because ecdysteroid UDP-glucosyltransferase (EGT) is expressed by AdhoGV and inactivates 20E. In this study, we measured JHE and 20E levels in parasitized A. honmai larvae simultaneously infected with either AdhoEPV or AdhoGV to explain their different metamorphosis. Parasitized larvae infected with either virus alone had no JHE peak during the 4th instar. In AdhoGV-infected and parasitized larvae, however, a large peak of ecdysteroid was observed during the 4th instar, which may be abolished by EGT, whereas AdhoEPV-infected and parasitized larvae showed a small ecdysteroid peak which allows larval–larval molting. These endocrinological results are consistent with the morphology of parasitized and infected larvae, and consequently explain the fate of parasitoid larvae in virus-infected hosts.

Commencement of pupal commitment in late penultimate instar and its hormonal control in wing imaginal discs of the silkworm, Bombyx mori

Pupal commitment of the wing imaginal disc of the silkworm, Bombyx mori, is completed shortly after the final (fifth) larval ecdysis. Pupal commitment was induced by in vitro culture with 20-hydroxyecdysone (20E). Shortly after the head capsule slippage (HCS) that occurs approximately 24 h before the final larval ecdysis, the discs become competent to respond to 20E, indicating that the process of pupal commitment begins in the late penultimate (fourth) instar. The simultaneous presence of methoprene (JHA) with 20E suppressed the pupal commitment at 4 ng/ml for the discs at 12 h after HCS and at 240 ng/ml for the discs at the ecdysis. Thus, the discs rapidly lose their sensitivity to JH at the end of the fourth instar. Day 0 fourth wing discs were not pupally committed by 20E when freshly dissected discs were exposed to 20E. By contrast, exposure to 20E after a pre-culture in a hormone free medium induced the pupal commitment. In those discs, the effective JHA concentration to suppress the 20E effects was 0.1 ng/ml. The present data suggest that pupal commitment proceeds through two stages from a reversible state that begins at around HCS to an irreversible state early in the fifth instar. The loss of sensitivity to JH is the primary impetus to begin the process and 20E is the factor that drives the discs to enter the reversible state.

Ecdysteroid control of cell cycle and cellular commitment in insect wing imaginal discs

The wing imaginal disc shows two different developmental responses to the steroid hormone 20-hydroxyecdysone (20E) in conjunction with cell cycle, and the responses depend on the developmental stage and the concentration to which it is exposed. The time of head capsule slippage (HCS) in late fourth instar, which signals the onset of molting process into the fifth instar, is the boundary for the appearance of 20E-dependency. Before HCS, 20E was not effective in maintaining the cell cycle, while, after HCS, 20E drove the cell cycle (20E-dependent cell cycle). In the 20E-dependent cell cycle, S phase was induced by 20E above a threshold concentration, whereas M phase was induced in a range of concentrations, above which it was suppressed. The 20E-dependent cell cycle first appeared concomitantly with the entrance of the discs into the reversible stage of pupal commitment. The composite data suggest that the control mechanism of cell cycle is altered in the pupally committed cells so that a high ecdysteroid titer to induce a stationary molt in young instars does not affect the cell division of uncommitted imaginal disc cells while that to induce pupal ecdysis suppresses it in the committed discs, which occurs before the onset of pupal differentiation.

Ecdysone agonists: mechanism and importance in controlling insect pests of agriculture and forestry.

Molting is the result of the expression of a cascade of genes that is sequentially both up and down-regulated by the molting hormone, 20-hydroxyecdysone (20E), which is secreted as a pulse during each instar. Benzoyl hydrazine analogs of 20E act like the native molting hormone at the molecular level by binding with the ecdysone receptor complex and transactivating a succession of molt initiating transcription factors that, in turn, induce the expression of a group of molt-related genes. As a result of the expression of these up-regulated genes, the larva undergoes apolysis and head capsule slippage and takes on the appearance of the pharate larva. However, unlike 20E, which is cleared at this juncture, allowing the down-regulated genes to be expressed, these synthetic analogs bind strongly to the receptors and remain in place and repress all the down-regulatory genes such as the ones necessary for cuticle elaboration, sclerotization, and ecdysis resulting in a developmental arrest in this state. As a result, the treated larva goes into a precocious incomplete molt that is lethal. Two of the analogs, tebufenozide and methoxyfenozide, are lepidopteran specific and have good control potential for open feeding larvae that ingest this material while a third one, halofenozide, acts on coleopteran larvae. Since they specifically act through an insect receptor complex, they have little or no effect on non-target species, making them environmentally attractive pest control agents. Some insects, however, show resistance to these analogs and this could be, inter alia, due to an ATP Binding Cassette Transporter like system that selectively pumps out the analogs.

Biochemical and biological mode of action of ecdysone agonists on the spruce budworm

The mode of action of RH-5992 (tebufenozide), a non-steroidal ecdysone agonist, on the eastern spruce budworm, Choristoneura fumiferana, was investigated. This diacyl hydrazine compound, upon ingestion, initiates a precocious incomplete molt that is lethal in most lepidopteran larvae including the spruce budworm. This was found to be induced when the larvae ingested the compound early in the stadium prior to the appearance of the ecdysone peak in the hemolymph. The larvae stopped feeding within 8h post feeding (PF) and remained quiescent just as they do in preparation for a normal molt. Head capsule slippage started at 12h PF, became pronounced by 24h, and by 48h an untanned new head capsule was visible behind the old one. The lack of tanning of the new cuticle was due to the failure of dopadecarboxylase gene expression. Although the old cuticle was loose around the entire body, indicating that apolysis had occurred, there was no evidence of ecdysis of the old cuticle, suggesting that eclosion hormone was probably not released. The transcription factor, Choristoneura hormone receptor 3 (CHR3), which is normally expressed at the onset of the hemolymph ecdysone peak, was expressed in the epidermis 1h PF of RH-5992 confirming that this analogue acts through the ecdysone receptor system. This unique mode of action at the molecular level of this ecdysone agonist and its effectiveness as an environmentally benign control agent for the spruce budworm are described.

Synergistic Effects of Verbutin with Dibenzoylhydrazine-Type Ecdysteroid Agonists on Larvae of Aedes aegypti (Diptera: Culicidae)

There were no significant differences between the activities of RH-5849, tebufenozide, and halofenozide on 4th instars of Aedes aegypti (L.). Methoxyfenozide was almost 10 times more active (LC50 = 0.22 mg kg-1) than the other ecdysteroid agonists. Halofenozide, methoxyfenozide, tebufenozide, and RH-5849 caused premature wing disk evagination and also caused the larval thorax to bloat on the dorsoventral sides resulting in a larval-pupal intermediate thorax. Methoxyfenozide induced these effects in the lowest concentration. Further molting disturbances (e.g., hanging larval exuvium, head capsule slippage failure) and a more intense sclerotization and melanization of the thorax also were noted. Although verbutin did not show significant effects with any of the tested ecdysteroid agonists under actual test conditions, some improvements could be found in the use of RH-5849 and tebufenozide.

EFFECT OF RH-5992, A NONSTEROIDAL ECDYSONE AGONIST, ON THE SPRUCE BUDWORM,CHORISTONEURA FUMIFERANA(LEPIDOPTERA: TORTRICIDAE): LABORATORY, GREENHOUSE, AND GROUND SPRAY TRIALS

AbstractIngestion of 0.1 μg of RH-5992, tebufenozide, by early 6th instar larvae of the spruce budworm,Choristoneura fumiferana(Clem.), prior to the appearance of the ecdysone peak in the hemolymph, resulted in the induction of a precocious incomplete moult that was lethal. The larvae stopped feeding within 8 h post ingestion and remained quiescent just as they do in preparation for a normal moult. Head capsule slippage started at 12 h post ingestion, became pronounced by 24 h, and by 48 h an untanned new head capsule was visible behind the old one. The lack of tanning of the new cuticle was due to the failure of dopadecarboxylase gene expression. Although the old cuticle was loose around the entire body, indicating that apolysis had occurred, there was no evidence of ecdysis of the old cuticle, suggesting that eclosion hormone was probably not released. Earlier instars required a lower dose than the later ones to elicit an "all or none" type of moulting response. The most effective routes of entry were by intrahemocoelic injection, followed by ingestion. Topical application was effective only when nonaqueous carriers such as acetone or dimethyl sulfoxide were used. The larvae were unable to discriminate between treated and untreated diet over a 48-h period. The transcription factor,Choristoneurahormone receptor 3, which is normally expressed at the onset of the hemolymph ecdysone peak, was expressed in the epidermis 1 h post ingestion of RH-5992, reached a peak level by 3 h, and became undetectable by 24 h, confirming that this analogue acts through the ecdysone receptor system. Greenhouse tests using potted white spruce trees sprayed with RH-5992 and colonized with 4th-instar spruce budworm indicated that field dosages of 35, 70, 140, and 280 g/ha would all be effective. Ground spray trials conducted in a spruce budworm infested white spruce stand in Zee Casault, Gaspé, Quebec, using a backpack sprayer showed that ≥ 70 g/ha of RH-5992 reduced the insect population by 100% with very little defoliation and was better than Chlorfluazuron®(an analogue of the chitin synthesis inhibitor, diflubenzuron or Dimilin®) treatment, which was used as a positive control. The unique mode of action of this ecdysone agonist and its effectiveness as an environmentally benign control agent for the spruce budworm are discussed.

Ultrastructural Effects of a Non-Steroidal Ecdysone Agonist, RH-5992, on the Sixth Instar Larva of the Spruce Budworm, Choristoneura fumiferana

Force feeding of RH-5992 (Tebufenozide), a non-steroidal ecdysone agonist to newly moulted sixth instar larvae of the spruce budworm, Choristoneura fumiferana, (Lepidoptera: Tortricidae) initiates a precocious, incomplete moult. Within 6 h post treatment (pt) the larva stops feeding and remains quiescent. Around 12 h pt, the head capsule slips partially revealing an untanned new head capsule that appears wrinkled and poorly formed. By 24 hr pt, the head capsule slippage is pronounced and there is a mid-dorsal split of the old cuticle in the thoracic region but there is no ecdysis. The larva remains moribund in this state and ultimately dies of starvation and desiccation. The temporal sequence of the external and internal changes of the integument were studied using both scanning and transmission electron microscopy. Within 3 h pt, there is hypertrophy of the Golgi complex indicating synthetic activity and soon after, large, putative ecdysial droplets are seen. Within 24 h, a new cuticle that lacks the endocuticular lamellae is formed. The formation of the various cuticular components, the degradation of the old cuticle and changes in the organelles of the epidermal cells of the mesothoracic tergite are described. The difference between the natural moult and the one induced by RH-5992 are explained on the basis of molecular events that take place during the moulting cycle. The persistence of this ecdysone agonist in the tissues permits the expression of all the genes that are up-regulated by the presence of the natural hormone but those that are turned on in the absence of the hormone are not expressed.

625 Establishment of stage-specific pure oligodendrocyte cultures and oligodendrocyte lineage

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 μg juvenile hormone I at HCS. Infusion of 1 μg/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 μg/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1–3 μg/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED50=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED50 = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.

Establishment of stage-specific pure oligodendrocyte cultures and oligodendrocyte lineage

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 μg juvenile hormone I at HCS. Infusion of 1 μg/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 μg/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1–3 μg/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.

Induction of apoptosis by protein kinase inhibitors in the cells of Drosophila neuronal cell line following treatment with 20-hydroxyecdysone

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 μg juvenile hormone I at HCS. Infusion of 1 μg/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 μg/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1–3 μg/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.

Ecdysteroid receptor binding activity and ecdysteroid agonist activity at the level of gene expression are correlated with the activity of dibenzoyl hydrazines in larvae of Bombyx mori

Ecdysteroid activities of two dibenzoyl hydrazines (RH 5849 and RH 5992) were examined using the Kc cell line. Gene expression activity was determined in the transient assay utilizing the ecdysteroid responsive Drosophila melanogaster hsp27 promoter gene. Ecdysteroid receptor binding (EC 50 = 3.0 × 10 −7 m), induction of ecdysteroid responsive gene expression (EC 50 = 3.0 × 10 −6 m) and induction of cell morphological change (EC 50 = 3.0 × 10 −7 m) were 13-fold, 7-fold and 13-fold higher in RH 5992 than in RH 5849, respectively. The addition of 10 ppm RH 5849 to the diet induced head capsule slippage in Bombyx mori larvae and most of the larvae died. RH 5992 caused the same response at 1 ppm. The higher insecticidal activity of RH 5992 compared to RH 5849 is likely caused by its higher ecdysteroid agonist activity at the molecular level.

Hormonally-regulated differential expression of the two duplicated insecticyanin genes, ins-a and ins-b, during development of the tobacco hornworm, Manduca sexta.

The effects of juvenile hormone (JH) and 20-hydroxyecdysone (20E) on the developmental expression of the two insecticyanin genes, ins-a and ins-b, were investigated with two gene-specific probes. Removal of the corpora allata (-CA, source of JH) clearly delayed and down-regulated the epidermal expression of these genes but enhanced their expression in the fat body during the early development of the fifth instar. Application of JH I to the -CA larvae at the time of head capsule slippage completely restored the normal epidermal expression pattern of the two genes in the early fifth instar, then INS-a mRNA declined prematurely whereas INS-b mRNA remained similar to that in the intact larvae. By contrast, in the fat body of -CA larvae, the exogenous JH had little effect on the levels of INS-a mRNA, but enhanced expression of INS-b mRNA relative to intact larvae. Culture of epidermis from day 1 fifth instar larvae with 40 ng/ml 20E for up to 24 h accelerated the loss of INS-a mRNA without affecting the levels of INS-b mRNA. Both mRNAs declined in isolated larval abdomens over a 24 h period, and this decline was slowed by 1 μg methoprene (a JH analog). Together these results indicate that JH controls the levels of the two mRNAs in both the epidermis and fat body, with additional factors involved in regulating these genes in the fat body during the molt and in the epidermis during the growth phase.

Simultaneous effects of potassium, rutin and temperature on performance of Manduca sexta caterpillars

AbstractThe simultaneous effects on an insect herbivore (third instar tobacco hornworm Manduca sexta (L.): Sphingidae) of temperature (daytime temperatures of 20 °C, 25 °C and 30 °C), a mineral that may play a role in plant defense (potassium) and a common allelochemical (rutin) were examined in a factorial experiment. To manipulate potassium levels, a modified diet with limited plant material was used as the base and KCl and rutin added. Temperature affected efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), time to head‐capsule slippage, stadium duration, relative consumption rate (RCR) and relative growth rate (RGR) but not food consumed, biomass gained and approximate digestibility (AD). Potassium concentration influenced all of the variables except AD, time to head‐capsule slippage (HCS), duration of the stadium and percent of stadium time to HCS. Rutin impacted negatively on all of the variables except food consumed. Compared to larvae on the non‐rutin diets, fewer larvae fed rutin survived through molt initiation to ecdysis and fewer successfully completed ecdysis. Temperature and rutin had interactive effects for AD, ECD, RCR, RGR, time to HCS, and percent of stadium required to reach HCS. Rutin and potassium had interactive effects for biomass gained, RCR, ECI, time to HCS, duration of stadium, and percent of stadium required to reach HCS. Comparison of larval responses on an average potassium concentration (3.1%) versus high concentration (6.1%) showed that at the low daytime temperature increasing potassium concentration depressed biomass gained, but at the warmer temperatures potassium concentration had little effect unless rutin was present. In addition, potassium concentration had little impact on ECI unless rutin was present. These results indicate that significant interactive effects occur among temperature, potassium and rutin, and thus suggest that such interactive effects on larval performance may be common under field conditions, which are characterized by varying temperature and different concentrations of minerals and allelochemicals in hostplants.

Failure to avoid rutin diets results in altered food utilization and reduced growth rate of Manduca sexta larvae

AbstractThe possibility of avoidance of and dietary self‐selection relative to the flavonol rutin was examined for third instar Manduca sexta for two thermal regimes and for larvae previously reared on plain or rutin diet. Temperature affected all of the performance indices examined, except efficiency of conversion of digested food. Significant interactive effects between temperature and diet occurred for relative consumption rate, relative growth rate and the food utilization efficiencies. For example, at the warm daytime temperature, relative growth rate prior to head capsule slippage was lower for caterpillars previously reared on rutin diet compared to those reared on plain diet. In contrast, at the cool daytime temperature, the relative growth rates were similar for caterpillars reared on plain diet and on 6 μmoles rutin diet. Consequently, in some treatments (5 of 12), caterpillars with a history of rutin in their diet had lower relative growth rates than those experiencing rutin for the first time. Despite rutin's impact on food utilization indices and negative effect on relative growth rate, the caterpillars did not avoid rutin nor was there evidence of regulation of the intake of rutin. The lack of metabolic feedback is discussed.

Lower temperature influences developmental rhythms of the wax moth Galleria mellonella: Putative role of ecdysteroids

1. 1. Development of the wax moth Galleria mellonella is strongly affected by transferring to a lower rearing temperature 2. 2. When the insects of different developmental stages (from 6th instar head capsule slippage larvae up to 8-day-old pupae) were transferred from optimal temperature (30°C) to 18°C their further development was markedly prolonged and desynchronized. 3. 3. One-day-old last instar larvae transferred to 18°C arrested their development completely (did not pupate) but went through all behavioural phases characteristic of 30°C-reared larvae including formation of cocoons in which they remained as dauer larvae. 4. 4. Surgical manipulations, such as head-ligation, nerve cord-severance, and implantation of brains and prothoracic glands accompanied by ecdysteroid titre measurements indicated that diapause-like arrest of larval development at 18°C might be due to the nervous inhibition of their prothoracic glands. 5. 5. The dauer larvae pupated at 18°C after implanting active prothoracic glands or applying a non-steroidal ecdysone agonist RH-5849. The putative mechanism of these effects is discussed.

A neuroendocrine factor essential for cuticular melanization in the tobacco hornworm, Manduca sexta

The decline in juvenile hormone before the head capsule slippage stage during the moult is essential for cuticular melanization in Manduca sexta larvae. Yet when larvae were allatectomized 15–16 h before head capsule slippage, then 10 h later ligated anterior to the 5th abdominal segment, only the posterior portion melanized. The ligation had no effect on dopa decarboxylase activity in the epidermis in either portion. By contrast, no premelanin granules containing phenoloxidase were deposited in the anterior portion. Injection of an extract of the ventral nerve cord prior to head capsule slippage induced the deposition of these granules followed by melanization, but purified melanization and reddish colouration hormone (MRCH) was not effective. When the integuments of larvae at the head capsule slippage stage were cultured in vitro, no deposition of premelanin granules occurred, although the epidermis synthesized the granular phenoloxidase. Addition of either a nerve cord extract and/or haemolymph was ineffective. Implantation of the integument into allatectomized larvae, however, induced melanization of the implants. Thus, both a factor from the ventral nerve cord and another unidentified humoral factor are important for the deposition of premelanin granules.