Larval Molt Research Articles

Precocious Metamorphosis in the Juvenile Hormone–Deficient Mutant of the Silkworm, Bombyx mori

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several “moltinism” mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval–larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval–pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH–deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.

Alkylphenols affect lobster (Homarus americanus) larval survival, molting and metamorphosis

Alkylphenols are widely distributed in the marine environment and we have found them to be widely dispersed in New England lobster populations. In vertebrates, these compounds act as estrogenic endocrine disruptors. Here, we tested two alkylphenols, bisphenol A and 2,4-bis-(dimethylbenzyl)phenol for their effects on lobster larvae. In particular, we examined their toxicity and their effects on molting and metamorphosis. Lobsters were raised to stage 2 and were then isolated individually in ice cube trays with nylon mesh netting on the bottom giving access to flow through cooled seawater. The experimental larvae were fed with 5 or 10 ng alkylphenol per day in their diet. Controls were fed the same diet minus the test chemicals. The alkylphenols were toxic to second and third stage larvae, and fourth stage post-larvae, with 13–21% surviving to 25 days compared to 69% of controls. The compounds significantly delayed each molt by 2–3.5 days, and the surviving treated larvae were predominantly larval–juvenile intermediates at metamorphosis (62%). The differences between treatments and controls in all cases were highly significant using survival analysis based on the Weibull distribution. We conclude that alkylphenols are endocrine disruptors to lobster larvae at metamorphosis because they possess juvenile hormone activity. They also delay molting, reduce growth, and are toxic at relatively low concentrations.

PAN-1, a P-granule component important for C. elegans fertility, has dual roles in the germline and soma

In Caenorhabditis elegans, P granules are germline-specific, RNA-containing granules that segregate into the germline precursor cell during early embryogenesis. In this short report, PAN-1, which previously has been found by others in screens for genes causing larval molting defects, is identified here as a novel P-granule component and a binding partner of GLH-1 (Germline RNA Helicase-1), a constitutive, germline-specific, P-granule protein. The PAN-1 predicted protein contains multiple leucine-rich repeats (LRRs) and regions with similarities to F-box proteins. Antibodies raised against PAN-1 reveal it is present both in the soma and the germline. In the germline, PAN-1 uniquely localizes to P granules from the first larval stage onward and is unusual for a P-granule component in lacking recognizable RNA binding motifs. Homozygous pan-1(gk142) deletion worms arrest as larvae that are unable to molt and this phenotype is also seen with pan-1(RNAi) into wild type worms. pan-1(RNAi) into the somatic RNAi-defective strain rrf-1(pk1417) bypasses the larval arrest and allows an assessment of PAN-1 function in the germline. We find pan-1(RNAi) is variably effective in knocking down PAN-1 protein and results in adult progeny that display multiple germline defects. These phenocopies range from under-proliferation of the germline, as also seen with loss of GLH-1, to the induction of endomitotic replication in oocytes, both defects that result in sterility, to fertile animals with significantly reduced progeny numbers. Thus, while loss of PAN-1 in the soma inhibits molting, this report demonstrates that PAN-1 is also a P-granule component that is essential for fertility.

Evidence of extended marine planktonic larval development in far-upstream populations of the river shrimp Macrobrachium ohione (Smith, 1874) from the Mississippi River

Coastal populations of Macrobrachium ohione (Smith, 1874) have been shown previously to be amphidromous, i.e., with adults living in fresh water but with marine larval development. Larval delivery to coastal estuaries in far-upstream populations seemed unlikely because of the distances involved. Therefore, we tested the hypothesis of freshwater larval development in far-upstream populations from the Mississippi River, near Vicksburg and Greenville, Mississippi. We compared the molting success of newly hatched stage-1 (non-feeding) to stage-2 (first feeding) zoeae when exposed to fresh water and salt water (15 ppt) treatments. In addition we also tested the duration of time larvae spent in fresh water or salt water prior to major larval mortality or larval molting. In all freshwater treatments, stage-1 larvae failed to molt to stage 2; in contrast, molting success in saltwater treatments was ∼99% and after 5-6 d of exposure to salt water all surviving larvae molted to stage 2. In freshwater treatments, there was a significant decline in larval survivorship after 3-5 of exposure to fresh water. Larval survivorship declined below 50% after 5 d of “freshwater drifting.” These results suggest that far-upstream populations of M. ohione require saline environments to complete larval development. Alternate hypotheses (long-distance hatching migrations of females to and from the sea, inland brine springs producing low salinity larval nurseries, upstream population sinks) are proposed to explain the former existence of far-upstream populations.

Ixodid ticks on ruminants, with on-host initiated moulting (apolysis) of Ixodes, Haemaphysalis and Dermacentor larvae

To screen the host-dependent abundance of hard tick (Acari: Ixodidae) developmental stages on ruminants in South Hungary, red and roe deer, as well as goats and sheep were examined in a season, when larvae and nymphs are active. Altogether 2271 ticks were collected. In the relevant period the prevalence of tick-infestation was significantly higher among goats, than among sheep kept in the very same area, most likely in association with the browsing habit of the former. Roe deer and goats were found to be important hosts for Ixodes ricinus and Haemaphysalis concinna larvae, in contrast to the view that this stage does not usually feed on medium-sized mammals. Interestingly, one third of I. ricinus larvae and one larva of H. concinna and of Dermacentor reticulatus collected from goats in the same herd in August have started the moulting process (showed apolysis) on their host, despite being three-host ticks. This is the first survey involving four species of domestic and wild ruminants in Europe to compare the host-preference of ixodid ticks in the same region.

LIN-42/PERIOD Controls Cyclical and Developmental Progression of C. elegans Molts

Biological timing mechanisms that integrate cyclical and successive processes are not well understood. C. elegans molting cycles involve rhythmic cellular and animal behaviors linked to the periodic reconstruction of cuticles. Molts are coordinated with successive transitions in the temporal fates of epidermal blast cells, which are programmed by genes in the heterochronic regulatory network. It was known that juveniles molt at regular 8-10 hr intervals, but the anticipated pacemaker had not been characterized. We find that inactivation of the heterochronic gene lin-42a, which is related to the core circadian clock gene PERIOD (PER), results in arrhythmic molts and continuously abnormal epidermal stem cell dynamics. The oscillatory expression of lin-42a in the epidermis peaks during the molts. Further, forced expression of lin-42a leads to anachronistic larval molts and lethargy in adults. Our results suggest that rising and falling levels of LIN-42A allow the start and completion, respectively, of larval molts. We propose that LIN-42A and affiliated factors regulate molting cycles in much the same way that PER-based oscillators drive rhythmic behaviors and metabolic processes in mature mammals. Further, the combination of reiterative and stage-specific functions of LIN-42 may coordinate periodic molts with successive development of the epidermis.

Drosophila InsP3R mutants and their effects on cellular and systemic physiology

AbstractA single gene encoding two identified splice variants of the InsP3R is found in the genome of the fruit fly Drosophila melanogaster. Mutants that perturb InsP3R function differentially have helped identify systemic processes which require the InsP3R. These include larval feeding, larval molting, fluid transport in malpighian tubules, flight circuit development, and olfactory adaptation. Genetic tools that provide spatiotemporal control on overexpression or knockdown of InsP3R in the whole organism have aided in delineating cellular domains that influence these processes. Genetic interaction studies with InsP3R mutants have helped identify several intracellular signaling components that influence InsP3R function. Measurement of their Ca2+dynamics have further revealed a complex interplay between InsP3R‐mediated Ca2+ release, the endoplasmic reticular Ca2+ pump, and components of store‐operated Ca2+ entry (STIM and Orai). Together these players modulate intracellular Ca2+ signals and homeostasis. Changes in cellular Ca2+ signals brought about by altering levels of the identified intracellular Ca2+ signaling components can be correlated with the extent of changes observed in systemic phenotypes. Furthermore, molecular studies of the Drosophila InsP3R have shown that key functional properties of the InsP3R are evolutionarily conserved. Thus, insights gained from Drosophila are likely to be relevant for our understanding of human disorders caused by aberrant InsP3R‐mediated Ca2+ signaling. WIREs Membr Transp Signal 2012, 1:70–77. doi: 10.1002/wmts.7For further resources related to this article, please visit the WIREs website.

Regulation of the C. elegans molt by pqn-47

C. elegans molts at the end of each of its four larval stages but this cycle ceases at the reproductive adult stage. We have identified a regulator of molting, pqn-47. Null mutations in pqn-47 cause a developmental arrest at the first larval molt, showing that this gene activity is required to transit the molt. Mutants with weak alleles of pqn-47 complete the larval molts but fail to exit the molting cycle at the adult stage. These phenotypes suggest that pqn-47 executes key aspects of the molting program including the cessation of molting cycles. The pqn-47 gene encodes a protein that is highly conserved in animal phylogeny but probably misannotated in genome sequences due to much less significant homology to a yeast transcription factor. A PQN-47::GFP fusion gene is expressed in many neurons, vulval precursor cells, the distal tip cell (DTC), intestine, and the lateral hypodermal seam cells but not in the main body hypodermal syncytium (hyp7) that underlies, synthesizes, and releases most of the collagenous cuticle . A functional PQN-47::GFP fusion protein localizes to the cytoplasm rather than the nucleus at all developmental stages, including the periods preceding and during ecdysis when genetic analysis suggests that pqn-47 functions. The cytoplasmic localization of PQN-47::GFP partially overlaps with the endoplasmic reticulum, suggesting that PQN-47 is involved in the extensive secretion of cuticle components or hormones that occurs during molts. The mammalian and insect homologues of pqn-47 may serve similar roles in regulated secretion.

Drosophila Met and Gce are partially redundant in transducing juvenile hormone action

The Drosophila Methoprene-tolerant (Met) and Germ cell-expressed (Gce) bHLH-PAS transcription factors are products of two paralogous genes. Both proteins potentially mediate the effect of juvenile hormone (JH) as candidate JH receptors. Here we report that Met and Gce are partially redundant in transducing JH action. Both Met and g ce null single mutants are fully viable, but the Met gce double mutant, Met 27 gce 2.5k , dies during the larval–pupal transition. Precocious and enhanced caspase-dependent programmed cell death (PCD) appears in fat body cells of Met 27 gce 2.5k during the early larval stages. Expression of Kr-h1, a JH response gene that inhibits 20-hydroxyecdysone (20E)-induced broad ( br) expression, is abolished in Met 27 gce 2.5k during larval molts. Consequently, expression of br occurs precociously in Met 27 gce 2.5k , which m ay cause precocious caspase-dependent PCD during the early larval stages. Defective phenotypes and gene expression changes in Met 27 gce 2.5k double mutants are similar to those found in JH-deficient animals. Importantly, exogenous application of JH agonists rescued the JH-deficient animals but not the Met 27 gce 2.5k mutants. Our data suggest a model in which Drosophila Met and Gce redundantly transduce JH action to prevent 20E-induced caspase-dependent PCD during larval molts by induction of Kr-h1 expression and inhibition of br expression.

An unusual barrier to gene flow: perpetually immature larvae from inter-population crosses in the flour beetle, Tribolium castaneum

We genetically characterize an unusual hybrid incompatibility phenotype manifest in F(1) offspring of crosses between two populations of Tribolium castaneum. Hybrid larvae cease development at the third larval instar, persisting as 'perpetually immature larvae' thereafter. Although unable to produce viable adult hybrid offspring with one another, each population produces abundant, fertile hybrids with other populations, indicating a recent origin of the incompatibility and facilitating genetic studies. We mapped the paternal component of the hybrid phenotype to a single region, which exhibits two characteristics common to hybrid incompatibility: marker transmission ratio distortion within crosses and elevated genetic divergence between populations. The incompatible variation and an elevation in between-population genetic divergence is associated with a region containing the T.castaneum ecdysone receptor homologue, a major regulatory switch, controlling larval moults, pupation and metamorphosis. This contributes to understanding the genetics of speciation in the Coleoptera, one of the most speciose of all arthropod taxa.

A novel alternative splicing site of class A chitin synthase from the insect Ostrinia furnacalis – Gene organization, expression pattern and physiological significance

Insect chitin synthase A ( CHSA) catalyzes chitin biosynthesis in tissues that develop from ectoderm. Since only one gene copy encodes CHSA, we hypothesized that CHSA is very likely to exist as isoforms through alternative splicing, and the functions of these isoforms may be tissue-specific. Besides the known alternative splicing exons in the mid-ORF region, we report here the alternative exons ( OfCHSA-2a and OfCHSA-2b) of OfCHSA, the chitin synthase A from the lepidopteran pest Ostrinia furnacalis. Sequence analysis of the 5′ upstream region of the transcription start site indicated that presences of two independent promoters for controlling the expression of OfCHSA-2a/ b. Both OfCHSA-2a and OfCHSA-2b transcripts were preferentially expressed in the epidermis. During growth and development of O. furnacalis, OfCHSA-2a was mainly expressed during larval–larval molting and larval–pupal transformation, as well as in newly-laid eggs, while OfCHSA-2b was expressed only during the larval–larval molting. Gene silencing of OfCHSA-2a caused incomplete molting, while silencing of OfCHSA-2b exclusively influenced the head cuticle formation of the 3rd instar larval. Since O. furnacalis is phylogenetically close to the model insect Bombyx mori, the same undiscovered alternative splicing exon was also identified in BmCHSA by gDNA sequence alignment. This work may lead to greater understanding of the mechanism by which a single copy of the CHSA gene could fulfill various functions with tissue specificity.

Effects of nonsteroidal ecdysone agonist RH-5992 and chitin biosynthesis inhibitor lufenuron on Spodoptera littoralis (Boisduval, 1833)

Abstract Comparative studies of the effects of two compounds, tebufenozide (an ecdysone agonist) and lufenuron (an insect growth regulator inhibiting chitin synthesis), were conducted on Spodoptera littoralis (Boisduval, 1833). The compounds, orally administered, caused larval mortality proportional to the concentrations in the food source. Tebufenozide initiated precocious molting, and lufenuron, and inhibited chitin synthesis. In both cases, larvae were unable to complete the molting process and died in the old larval cuticle. Larvae contaminated by sublethal doses completed their development to adulthood. Lufenuron is more active than is tebufenozide. LD-50 for lufenuron is 0.0001ppm and for tebufenozide 0.001ppm. Topical application of the test compounds to eggs caused dose- and agedependent inhibition of embryonic development. Application of tebufenozide in the second half of embryogenesis caused precocious molting of eclosed larvae of the 1st instar. Some morphological changes in the process of larval-pupal transformation were also observed. Tested compounds also reduced reproduction in adult individuals that had been treated by the tested compounds in the larval stage.

Large-scale RNAi screen of G protein-coupled receptors involved in larval growth, molting and metamorphosis in the red flour beetle

BackgroundThe G protein-coupled receptors (GPCRs) belong to the largest superfamily of integral cell membrane proteins and play crucial roles in physiological processes including behavior, development and reproduction. Because of their broad and diverse roles in cellular signaling, GPCRs are the therapeutic targets for many prescription drugs. However, there is no commercial pesticide targeting insect GPCRs. In this study, we employed functional genomics methods and used the red flour beetle, Tribolium castaneum, as a model system to study the physiological roles of GPCRs during the larval growth, molting and metamorphosis.ResultsA total of 111 non-sensory GPCRs were identified in the T. castaneum genome. Thirty-nine of them were not reported previously. Large-scale RNA interference (RNAi) screen was used to study the function of all these GPCRs during immature stages. Double-stranded RNA (dsRNA)-mediated knockdown in the expression of genes coding for eight GPCRs caused severe developmental arrest and ecdysis failure (with more than 90% mortality after dsRNA injection). These GPCRs include dopamine-2 like receptor (TC007490/D2R) and latrophilin receptor (TC001872/Cirl). The majority of larvae injected with TC007490/D2R dsRNA died during larval stage prior to entering pupal stage, suggesting that this GPCR is essential for larval growth and development.ConclusionsThe results from our study revealed the physiological roles of some GPCRs in T. castaneum. These findings could help in development of novel pesticides targeting these GPCRs.

Evaluation of time toxicity, residual effect, and growth-inhibiting property of Carapa guianensis and Copaifera sp. in Aedes aegypti

Oils of Carapa guianensis and Copaifera spp. are well-known in the Amazonian region as natural insect repellents, and studies have reported their efficiency as larvicide against some mosquito species. However, toxicity persistence and effect on mosquito development have not yet been evaluated. Thus, the objective of this study was to evaluate the initial time of larvicidal activity, residual effect, and the effect of very low concentrations of these oils on Aedes aegypti. Different concentrations of the oils were used to evaluate the initial time of larval mortality and residual effect, as well as, the development of larvae, pupae, and adults. Results demonstrated that the lethal effect started mainly between the first 2 and 3 h of larvae exposure to oils, when using concentrations which ranged from 500 mg/L of C. guianensis and 90 mg/L of Copaifera sp. The toxic effect remained with total efficiency (100% mortality) until the sixth day for Copaifera sp. and 12th day for C. guianensis. When using sublethal dosages (ranging from 140 mg/L of C. guianensis to 26 mg/L of Copaifera sp.) mortality was observed after the larval molt. Also, imperfection of pupae and adult development and unsuccessful emergence of adults were observed. A product of botanical origin that could break the development of immature stage of mosquitoes and inhibit the emergence of adults should be essential in vector control. Thus, our results provide new information for a better understanding in using C. guianensis and Copaifera sp. oils with a potential to be used as a natural insecticide.

Molting Success of Ixodes scapularis Varies Among Individual Blood Meal Hosts and Species

The blacklegged tick (Ixodes scapularis) is an important vector of emerging human pathogens. It has three blood-feeding stages, as follows: larva, nymph, and adult. Owing to inefficient transovarial transmission, at least for the Lyme disease agent (Borrelia burgdorferi), larval ticks rarely hatch infected, but they can acquire infection during their larval blood meal. Nymphal ticks are primarily responsible for transmitting pathogens to hosts, including humans. The transition from uninfected host-seeking larva to infectious host-seeking nymph is therefore a key aspect of human risk of infection. It can be divided into a series of steps, as follows: finding a host, taking a blood meal, becoming infected, molting, and overwintering. The chance of succeeding in each of these steps may depend on the species identity of the blood meal host. We used a Bayesian method to estimate the molting success of larval I. scapularis collected from four commonly parasitized species of birds and eight commonly parasitized small and mid-sized mammals found in the forests of Dutchess County, New York. We show that molting success varies substantially among host species; white-footed mice, veeries, and gray catbirds support particularly high molting success, whereas ticks feeding on short-tailed shrews, robins, and wood thrushes were less successful. We also show that larval molting success varies substantially between individual blood meal hosts, and that this intraspecific variability is much higher in some species than in others. The causes of both inter- and intraspecific variation in molting success remain to be determined.

The apoptosis inhibitor survivin prevents insect midgut from cell death during postembryonic development

The programmed cell death (PCD) is important in maintaining the cell number homoeostasis of tissues and organs in metazoan. This process is regulated by the inhibitors of apoptosis proteins (IAPs). The function and mechanism of IAPs have been well studied in animal embryonic development and human cancers. However, the roles and hormonal regulation of IAPs in the postembryonic development are not well understood. We report that an IAP survivin (Ha-survivin) played roles in the postembryonic development of the midgut in a lepidopteran insect Helicoverpa armigera. Ha-survivin was transcribed not only in the embryo, but also in the haemocytes, fat body and midgut during larval molting or pupal adulting. The transcription of Ha-survivin was upregulated by the steroid hormone 20-hydroxyecdysone (20E). Ha-survivin was located in the embryonic cells around the periplasm of the eggs during embryonic development. It was also located in the epithelium cells of the midgut in the fifth molting larvae and later pupae. Knockdown of Ha-survivin by RNAi in the epidermal cell line caused cell apoptosis. These results indicated that Ha-survivin played roles not only in the embryonic development, but also in the postembryonic tissue development by preventing cell death.

Modification of Hormonal Balance in Larvae of the Corn Borer Sesamia nonagrioides (Lepidoptera: Noctuidae) Due to Sublethal Bacillus thuringiensis Protein Ingestion

Bacillus thuringiensis (Bt) corn, Zea mays L., is highly efficient against the corn borer Sesamia nonagrioides (Lefèbvre) (Lepidoptera: Noctuidae) when the larvae feed only on the transgenic plants. However, when they feed on Bt leaves during only part of their development, thus ingesting sublethal amounts of Bt toxins, some larvae survive. A previous study reported a prolonged development and precocious diapause induction in larvae fed on a diet with sublethal amounts of Cry1Ab protein. To determine whether these effects were accompanied by a modification of the hormonal balance, S. nonagrioides larvae were fed on sublethal amounts of Bt protein provided in Bt leaves or in the diet. The larvae that survived had higher levels of juvenile hormone (JH), whereas their level of ecdysteroids did not increase sufficiently to allow pupation, leading to a longer larval development and more larval molts. This response may be considered a defense mechanism that allows some larvae to survive toxin ingestion; it is similar the response to insecticidal toxins or viruses observed in other larvae. Changes in the hormone levels in diapausing larvae were undetectable, probably because these changes were masked by the higher level of JH in the hemolymph of diapausing larvae and because of lack of ecdysteroid titer increase, a phenomenon that is usually observed a few days before pupation in nondiapausing larvae. These results should be taken into account in the establishment of non-Bt refuges to prevent development of Bt-resistance in S. non-agrioides populations.

Cloning and expression of the nuclear transcription factor SlβFTZ-F1 cDNA from Spodoptera litura

Insect molting and metamorphosis are regulated by 20-hydroxyecdysone, which activates the ecdysone receptors, followed by expression of a series of transcription factors. Beta Fushi Tarazu-Factor 1 (βFTZ-F1) is one of these ecdysis-related early-expressed transcription factors. A βFTZ-F1 complementary DNA (cDNA) was cloned from Spodoptera litura. The full-length cDNA was 2 194 bp in length and the open reading frame of SlβFTZ-F1 cDNA consisted of 1 659 bp and encoded a 552-amino acid protein with a molecular mass of 61.4 kDa. Homology analysis revealed that SlβFTZ-F1 protein shared high identities to its homologues from other species. The A/B domains and the D domains were diverse among the insect βFTZ-F1 proteins, but DNA binding domains (C domain) and the ligand binding domains (E domain) were highly conserved. Northern blot analysis detected a transcript of 2.2 kb in the epidermis of fifth to sixth molting larvae. Reverse transcription polymerase chain reaction revealed that SlβFTZ-F1 gene started to increasingly and periodically express before each molt, followed by a decrease during the intermolt stages.

Molecular characterization of an ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia nonagrioides

We obtained a full-length cDNA encoding a carboxylesterase in Sesamia nonagrioides. The complete cDNA sequence is comprised of 1838bp with an open reading frame encoding 576 amino acid residues with predicted molecular mass of 64.24kDa. The deduced amino acid sequence showed high identity to JHE-Related of Trichoplusia ni (65% amino acid identity) and 49–46% amino acid identity to JHEs of other lepidopterans and contained all five functional motifs of insect JHEs. The gene has been termed as SnJHE-Related (SnJHER) to denote its similarity to other insect JHE genes and the occurrence of an unusual cysteine residue immediately adjacent to the catalytic serine, instead of the conventional alanine residue. Phylogenetic analyses localised SnJHER together with TnJHER in a branch of the lepidopteran's JHEs group, with other carboxylesterases (COEs) occuring in separated groups. The JH analog methoprene did not affect the expression of SnJHER in contrast to other insect JHEs. Additionally, ecdysteroid analogs induced SnJHER gene expression. The SnJHER mRNA levels were higher in long-day non-diapausing larvae than in short-day diapausing ones. In the fifth instar of non-diapausing and ninth instar of diapausing larvae, the SnJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) non-diapausing larval instar, SnJHER mRNA levels peaked in the intermolt period but were lower than during the fifth instar.

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

Insulin-regulated growth of the prothoracic glands appears to play a critical role in timing the last larval molt, and hence metamorphosis. The present study examined insulin signaling in relation to the growth and secretory activity of prothoracic glands in the tobacco hornworm, Manduca sexta. As larvae feed during the first half of the final larval stage, the prothoracic glands grow and ecdysone secretory capacity increases. During this period of growth, we verified the presence of insulin receptor transcript in the prothoracic glands and demonstrated that the glands were responsive to insulin, as evidenced by the in vitro phosphorylation of signaling proteins in the insulin pathway such as Akt/protein kinase B and FOXO. It was predicted that starvation would reduce ecdysone secretion with concomitant changes in insulin signaling. To test this prediction, larvae were starved and changes were quantified in two nutritionally sensitive transcripts, insulin receptor and the translation inhibitor 4EBP. In glands from starved larvae, growth and ecdysone secretory capacity were reduced, and insulin receptor and 4EBP transcripts were increased. The latter changes would be expected to accompany starvation in conjunction with enhanced insulin sensitivity and reduced protein synthesis. Increased transcription of insulin receptor and 4EBP strongly suggest that nutritional deprivation reduces the secretion of endogenous insulin-like hormones. When injected with insulin, 4EBP levels in the prothoracic glands of starved larvae decreased. Thus, insulin appeared to correct starvation-induced deficits in glandular protein synthesis. However, insulin injection did not enhance ecdysone secretion. Thus, although the prothoracic glands are insulin-responsive and insulin-like hormones may promote glandular growth as larvae feed, the effects of nutritional depletion on steroidogenesis in Manduca cannot be explained solely by reduced insulin.