Nonshivering thermogenesis and cold resistance during seasonal acclimatization in the Djungarian hamster

Summary The absence of juvenile hormone (JH) at the time of head capsule slippage during the molt to the fifth (final) instar of the tobacco hornworm was found to cause ommochrome (primarily dihydroxanthommatin) synthesis in the epidermis during the first two days after ecdysis. Then synthesis decreased until its transient reappearance during the wandering stage. Either JH-I (ED₅₀=8x10^–4 g) or methoprene (ED₅₀=1.4x10^–2 g) applied at this critical time during the molt prevented the first synthesis. A comparison of developmental profiles of tryptophan and its metabolites, kynurenine and 3-hydroxykynurenine, in normal and allatectomized wild type larvae showed that JH at this critical time prevented both the conversion of kynurenine to 3-hydroxykynurenine and 3-hydroxykynurenine to ommochromes. A similar study in normal and methoprene-treatedblack mutant larvae showed that only the latter conversion was inhibited by JH. The accumulation of 3-hydroxykynurenine in the epidermis of the JH-treatedblack mutant is thought to be due to the altered tryptophan metabolism in these mutants in previous instars due to lower JH levels. Neither starvation of theblack mutant nor injection of 3-hydroxykynurenine significantly affected ommochrome synthesis by the epidermis. Preliminary studies of the enzymes involved showed that JH at the critical period suppressed the later activity and/or production of kynurenine 3-hydroxylase in the wild type larva, but had little effect on the particulate ommochrome synthetase activity of the epidermis.Abbreviations CA corpora allata - JH juvenile hormone - PTTH prothoracicotropic hormone     

Granular phenoloxidase involved in cuticular melanization in the tobacco hornworm: regulation of its synthesis in the epidermis by juvenile hormone

The granular phenoloxidase (PO) that is responsible for cuticular melanization in Manduca sexta larva was purified and an antibody was prepared. This granular PO was found to consist of four isozymes of 90 kDa with isoelectric points ranging from 5.7 to 5.85. The enzyme was immunologically and electrophoretically distinct from the cuticular wound PO, a second cuticular PO common to all larval cuticle, and the hemolymph PO. Both [14C]mannose and [14C]sialic acid were incorporated into the granular PO, showing that this granular PO was a glycoprotein whose sugar moiety was a complex oligosaccharide. When no juvenile hormone (JH) was present at the head capsule slippage (HCS) stage, the epidermis began synthesizing PO 6 hr later. This epidermal synthesis was maximal 12 hr after HCS at which time the PO appeared in the cuticle, and then synthesis declined. When synthesis ceased about 23 hr after HCS, no further incorporation into the cuticle was observed. As melanization proceeded, immunologically detectable cuticular PO decreased. Application of 0.1 microgram JH I at the time of HCS inhibited synthesis of PO by the epidermis and thus prevented melanization. JH application after PO synthesis had begun (8 hr after HCS) prevented its subsequent synthesis, causing partial melanization. Thus, the absence of JH is necessary during the period of epidermal synthesis of the granular PO to allow complete melanization.     

Isolation and amino terminal sequence of melanization and reddish coloration hormone (MRCH) from the silkworm,Bombyx mori

Cuticular melanization associated with the gregarious phase of the common armyworm larvae, Leucania separata, is caused by a neurohormone, melanization and reddish coloration hormone (MRCH). Two molecular species of MRCH were isolated from 211,000 heads of adult Bombyx mori with conventional column chromatography and reversed-phase high performance liquid chromatography. As little as 6 ng of purified MRCH elicited marked melanization in the cuticle of an L. separata larva. Automated Edman degradation confirmed 16 residues of N-terminal amino acid sequences of the purified MRCHs; these showed homology with each other.     

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

Summary WhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both -MDH and -fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 g/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.Abbreviations -MDH L-3-(3,4 dihydroxyphenyl)-2-hydrazine-methylpropionic acid - -FM-dopa R-S--fluoromethyl-dopa - DCC dopa decarboxylase - 20-HE 20-hydroxyecdysone - JH juvenile hormone - HCS head capsule slippage     

Hormonal control of larval coloration in the armyworm, Leucania separata

Leucania separata larvae show various degrees of darkening depending on the population density. A ligature applied behind the thorax of crowded or yellow solitary larvae caused black or reddish brown pigmentation in the anterior part after the larval ecdysis. Extirpation of the brain, the corpus cardiacumcorpus allatum complexes, or the suboesophageal ganglion reduced the degree of melanization in the crowded larvae, lack of the suboesophageal ganglion having a particularly striking effect. Transplantation of 3 complexes of brain-corpora cardiaca-corpora allata-suboesophageal ganglion induced intense black pigmentation in the isolated abdomens of crowded larvae and reddish brown pigmentation with some melanization in the isolated abdomens of yellow solitary larvae, though the melanization in the latter was weaker than in the former. Implantation of these organs or of the suboesophageal ganglia into yellow solitary larvae caused black and reddish brown pigmentation after a larval ecdysis. In the pieces of integument implanted into the body cavity of crowded larvae, melanization occurred after ecdysis, whereas it did not occur in most of the fragments implanted in yellow solitary larvae. Transplantation of corpora allata and other organs from solitary larvae or injection of juvenile hormone into crowded larvae did not inhibit melanization.     

Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae

The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.     

Pheromonotropic activity in the gypsy moth Lymantria dispar: evidence for a neuropeptide

The brain-suboesophageal ganglion complex of the gypsy moth, Lymantria dispar, contains pheromonotropic activity detectable using a Helicoverpa zea in vivo bioassay for pheromone-biosynthesis-activating neuropeptide. Pheromonotropic activity was detected as early as the third larval instar and was present throughout development and through day 6 post-eclosion. Activity in the adult is presumably associated with pheromone production, while it is speculated that larval activity may be related to melanization. Adult pheromonotropic activity is associated with a peptide of approximately 3.500 kDa. It is heat labile and only partially stable when incubated at 35°C or exposed to freeze-thawing. Isolation of L. dispar pheromonotropic factor should facilitate the elucidation of the mechanism of pheromone production in this insect pest.Abbreviations ED ₅₀ dose at which one-half maximal response is observal - eq equivalent - MRCH melanization and reddish colorization hormone - MW molecular weight - PBAN pheromone biosynthesis activating neuropeptide - SOG suboesophageal ganglion - TFA trifluoroacetic acid - Z11-16: Ald (Z)-11-hexadecenal     

Induction of cuticular melanization in Spodoptera littoralis larvae by PBAN/MRCH: Development of a quantitative bioassay and structure function analysis

PBAN (also termed melanization and reddish coloration hormone, MRCH) is a cerebral factor known to regulate sex pheromone biosynthesis and cuticular melanization in moths. In the present study we developed a quantitative method (based on computerized image analysis of cuticles) to determine the effect of Helicoverpa zea PBAN (Hez-PBAN) on cuticular melanization and to study the structure-activity relationship of the neuropeptide in Spodoptera littoralis larvae. The results indicate that Hez-PBAN stimulates cuticular melanization in an interspecific manner, and that the minimal dose evoking formation of melanins is between 3–10 pmol/larva. Higher doses of Hez-PBAN did not stimulate melanization any further. Examination of the structure-activity relationship of Hez-PBAN revealed that the first eight N-terminal amino acids are not essential for the melanotropic activity and that the activity resides in the C-terminal region. Within this region the C-terminal amide was found to play a very important role. © 1996 Wiley-Liss, Inc.     

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 g 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.     

Relationship between photoperiod and secretion of the diapause hormone during larval stages of the silkworm, Bombyx mori L., reared on an artificial diet

The suboesophageal ganglion of the silkworm, Bombyx mori synthesizes sufficient diapause hormone to produce diapause eggs, regardless of the photoperiodic conditions experienced during the larval stages. When larvae destined to produce non-diapause eggs are implanted with the brain-suboesophageal ganglion complex from larvae which have been reared under short-day conditions, the resulting adults lay diapause eggs. The larvae receiving the complex from larvae reared under long-day conditions gave rise to adults which did not produce any diapause eggs. The brains from pupae which have been reared under long-day conditions show an activity inhibiting the secretion of diapause hormone by the suboesophageal ganglion. The mechanism through which the brain controls the secretion of diapause hormone from the suboesophageal ganglion can be modified by photoperiodic conditions during the larval stages.     

Factors influencing juvenile hormone esterase activity in the wax moth, Galleria mellonella

The effects of juvenile hormone, antiallatotropins, selected surgical procedures and starvation on the juvenile hormone esterase levels in Galleria larvae and pupae were investigated. JH reduced JH esterase activity in larvae but induced the enzyme in 1-day-old pupae. In vitro studies confirmed that the peak of synthesis and/or release of JH esterase from the fat body of last instar larvae occurred 4 days after ecdysis. These studies also showed that fat body from JH-treated larvae released much less enzyme than controls. Antiallatotropins, precocene 2 and ZR 2646 also reduced JH esterase levels in larvae, but ZR 2646 induced JH esterase in pupae. In starved larvae, JH esterase did not increase during the first five days. A minimum of 36 hr of feeding was necessary for the larval esterase activity to increase on schedule on day 4 of the last larval stadium. When day-l larvae were ligated behind the head or the prothorax, they had lower JH esterase levels and yet showed a slight increase in the enzyme when the larvae reached the age of 4 days. The significance of these results is discussed in relation to the possible control of esterase activity during metamorphosis.     

The juvenile hormone titer of Trichoplusia ni and its potential role in embryogenesis of the polyembryonic wasp CopidosomaFloridanum

The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum, was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of (RS)-methoprene or JH II had a juvenilizing effect on parasitized T. ni, and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.     

Juvenile hormone esterases of Lepidoptera

Summary The juvenile hormone esterase (JHE) titer was measured during the last larval instar of 11 species of Lepidoptera (Pieris rapae, Junonia coenia, Danaus plexippus, Hemileuca nevadensis, Pectinophora gossypiella, Spodoptera exigua, Orgyia vetusta, Ephestia elutella, Galleria mellonella, Manduca sexta andEstigmene acrea). All species had a peak of JHE at or near the time of wandering. The peak activity at this time ranged from 0.8 to 388 nmoles JH III cleaved/min·ml. All species exceptJ. coenia had a second peak of JHE during the late prepupal stage. The height of the second peak ranged from 0.4 to 98.4 nmoles/min·ml. However, there was no apparent correlation between size of the first and second JHE activity peaks for the lepidopteran species examined. There was an apparent relationship between the height of the first and second JHE peaks and reports on titer of JH just prior to these peaks. These data support, with some qualifications, the extension of developmental information obtained on several well studied species to a variety of Lepidoptera.Abbreviations JH juvenile hormone - JHE juvenile hormone esierase - PTTH prothoracotropic hormone - R _o -10-3108 1-(4-ethylphenoxy)-6,7-epoxy-3-ethyl-7-methylnonane     

Temporal patterns of protein synthesis inManduca epidermis during the change to pupal commitment in vitro: their modulation by 20-hydroxyecdysone and juvenile hormone

Summary The epidermis of final instar tobacco hornworm larvae,Manduca sexta, becomes committed to pupal differentiation in response to ecdysteroid in the absence of juvenile hormone (JH). Many changes in protein synthetic patterns have been noted during this time (Kiely and Riddiford 1985). To determine which of these changes are caused by ecdysteroid and which are important for the change of commitment, we have incubated larvally-committed epidermis for 24 h with 1 g/ml 20-hydroxyecdysone (20HE) and 3 g/ml epoxygeranylsesamole (EGS) (a JH mimic), with 3 g/ml EGS alone, or in hormone-free medium. Synthesis of larval-specific proteins such as insecticyanin and larval cuticular proteins was reduced to trace amounts or was undetectable after culture with 20HE for 24 h. The larval cuticular proteins that are greatly increasedin vivo on day 3 were not synthesized after exposure to 20HEin vitro. Ecdysteroid increased the synthesis of many of the proteins first seenin vivo on day 3 or during the wandering stage. The synthesis of about half of these latter proteins was inhibited by JH, indicating that they were likely part of the change of commitment. Other proteins that appear at this stagein vivo showed increased synthesis also in hormone-free medium and therefore were independent of the change of commitment.     

Immunohistological evidence of dopamine cells in the cephalic nervous system of the silkworm Bombyx mori. Coexistence of dopamine and α endorphin-like substance in neurosecretory cells of the suboesophageal ganglion

Summary Evidence of dopamine cells in the brain and the suboesophageal ganglion of the silkworm Bombyx mori was obtained immunohistologically in larvae and pupae. From six to eight and eight (two symmetrical groups of four) immunoreactive cells are present respectively in median and lateral protocerebral areas of the brain. In the suboesophageal ganglion, two cell clusters with dopamine immunoreactivity were observed. There was no clear difference in the nature of the immunohistochemical reaction and the number of cells between diapause- and non-diapause-egg producers, in both brains and suboesophageal ganglia. By examination of adjacent sections, it was possible to show that dopamine-immunoreactive cells in larval suboesophageal ganglia also contain an endorphin-like substance.     

The endocrine basis for developmentally stationary prepupae in larvae ofTrichoplusia ni pseudoparasitized byChelonus insularis

Summary Larvae of the cabbage looper,Trichoplusia ni, precociously initiate metamorphosis in the penultimate instar when parasitized byChelonus insularis. Some larvae developing from stung eggs precociously spin cocoons, but upon dissection contain no live or obvious parasites. Such pseudoparasitized larvae greatly slow down in development as prepupae, due to a suppressed ecdysteroid titer which in turn may be caused by a suppressed juvenile hormone titer.Abbreviations JH juvenile hormone - JHA juvenile hormone analog - JHE juvenile hormone esterase     

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.     

Juvenile hormone stimulation of ornithine decarboxylase activity during vitellogenesis in Drosophila melanogaster

Summary The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, becomes elevated in intact female Drosophila melanogaster shortly after adult eclosion. This activity reaches a peak at 24 h following eclosion, and then drops to lower levels by 48 h. This pattern is not observed in males, consistent with the hypothesis that polyamine synthesis is involved in ovarian maturation in Drosophila. Abdomens isolated within 2 h of adult eclosion do not display elevated ODC activity or ovarian maturation. However, a 250-ng dose of the juvenile hormone analog methoprene (ZR-515) applied in acetone to these abdomens, recovers ovarian maturation and causes a 5–10 fold increase in enzyme activity over controls treated with acetone alone. The same dose of the inactive precursor methyl farnesoate caused no such increase, whereas a 500-ng dose of the newly discovered natural Drosophila JHB₃ stimulated a four-fold response. The response to methoprene was dose-dependent, showing stimulatory activity at a dose as low as 10 ng. This stimulation by JHA is rapid, occurring between 1 and 3 h following hormone treatment, reminiscent of JH induction of fat body vitellogenin synthesis in Drosophila. Elevated ODC activity appeared to be localized in the adult fat body. During embryogenesis, ODC activity remained undetectable until just prior to hatching, when a large increase was detected. We postulate that JH may, either directly or indirectly, regulate polyamine biosynthesis in vivo, and that this synthesis may be required for the production of macromolecules during Drosophila vitellogenesis or embryogenesis.Abbreviations JH juvenile hormone - JHA juvenile hormone analog - ODC ornithine decarboxylase - SAMDC S-adenosyl-methionine decarboxylase - JHB ₃ juvenile hormone III bisepoxide     

Effect of juvenile hormone and pyriproxyfen treatments on the production of Spodoptera litura nuclear polyhedrosis virus

Efficient production is essential for developing baculoviruses into marketable bioinsecticides. In this study, we evaluated how juvenile hormone (JH) III and the JH analog pyriproxyfen affected the production efficiency of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) Nucleopolyhedrovirus (SpltNPV) in SpltNPV‐inoculated early sixth instars. Both the JH III and pyriproxyfen treatments significantly enhanced the body size and weight of S. litura, which resulted in a 1.5‐ to 2‐fold increase in the production of occlusion bodies (OBs). The OBs obtained from JH‐ and pyriproxyfen‐treated larvae were as pathogenic as those produced in untreated larvae. More importantly, JH‐ or pyriproxy‐fen‐treated larvae consumed less than twice the amount of food consumed by two untreated larvae. These results indicate that JH and pyriproxyfen treatments improve the production efficiency of SpltNPV in S. litura. SpltNPV infection affected the development of S. litura not only as larvae, but also during subsequent growth stages and generations.