Insulin receptor regulates food intake through sulfakinin signaling in the red flour beetle,Tribolium castaneum

Insects obtain energy and nutrients via feeding to support growth and development. The insulin signaling pathway is involved in the regulation of feeding; however, the underlying mechanisms are not fully understood. Here, we show that insulin signaling regulates food intake via crosstalk with neuropeptide sulfakinin in the red flour beetle, Tribolium castaneum. Silencing of the insulin receptor (InR) decreased the food intake in the penultimate and final instar stages, leading to a decrease of weight gain and mortality during larval-pupal metamorphosis. Interestingly, the knockdown of InR co-occurred with an increased expression of sulfakinin (sk), a gene encoding neuropeptide SK functioning as a satiety signal. In parallel, double silencing of sk and InR eliminated the inhibitory effect on food intake as induced by silencing of InR and the larvae died as prepupae. In conclusion, this study shows, for the first time, that the insulin/InR signaling regulates food intake through the sulfakinin signaling pathway in the larval stages of this important model and pest insect, indicating a novel target for pest control.     

Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti

Background

Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear.

Methodology/Principal Findings

Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production.

Conclusions/Significance

Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species.     

Silencing downstream of receptor kinase gene (drk) impairs larval-pupal ecdysis in Leptinotarsa decemlineata (Say)

In insects, an insulin-like peptide (ILP) triggers the formation of the insulin receptor (InR)/the insulin receptor substrate Chico complex. The complex then recruits downstream of receptor kinase (Drk) and phosphatidylinositol-3-kinase (PI3K) to initiate two signaling branches, i.e., Drk-mitogen-activated protein kinase (MAPK) and Pi3K-protein kinase B subdivisions. Previous findings reveal that RNA interference (RNAi) of LdILP2 or Ldchico, rather than Ldpi3k92E, impairs larval-pupal and pupal-adult molting in the Colorado potato beetle Leptinotarsa decemlineata. It is accordingly hypothesized that the Drk-MAPK branch regulates larval metamorphosis. In the present paper, we first found that silencing LdILP2, Ldchico or Ldpi3k92E did not decrease the expression level of Lddrk, indicating other receptor tyrosine kinases’ signaling except insulin pathway is not affected in the RNAi larvae. Moreover, two InRs and Torso were highly expressed in the final larval instars. Furthermore, RNAi of either Lddrk or Ldchico, or both of them equally affected larval-pupal and pupal-adult molts, and similarly repressed the expression of representative MAPK (Ldras and Ldraf), ecdysteroidogenesis (Ldphm and Ldsad), and 20E signaling (LdEcR, LdUSP, LdHR3 and LdE75) genes. 20E feeding by Lddrk RNAi larvae completely restored the reduced mRNA levels of LdEcR, LdHR3 and LdE75, but did not rescued the decreased Lddrk and LdUSP levels and the lowered pupation and emergence rates. Therefore, our findings suggest that the Drk-MAPK branch is involved in metamorphosis regulation in L. decemlineata.     

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

Background

Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis.

Results

Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage.

Conclusion

Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.     

N-β-Alanyldopamine levels and synthesis in integument and other tissues of Manduca sexta (L.) during the larval-pupal transformation

N-β-Alanyldopamine (NBAD) and other diphenols in tissues of the fifth larval instar of the tobacco hornworm, Manduca sexta (L.), were analyzed by HPLC with electrochemical detection. NBAD accumulated in the integument during the intermolt feeding period, with maximal levels in the wandering stage (6 mmol/g). It then declined to a low level during apolysis and endocuticle digestion, while hemolymph NBAD increased during the same interval to a peak concentration (3 mM) shortly before pupal ecdysis. Trachea and foregut contained lesser amounts of NBAD (0.5 mmol/g), perhaps associated with cuticle, whereas fat body, muscle, midgut and hindgut had 0.1 mmol/g or less. Dopamine (DA), N-acetyldopamine and 3,4-dihydroxyphenylalanine (DOPA) were at least 10-fold less abundant than NBAD in the integument. NBAD synthase, which catalyzes the formation of NBAD from DA and β-alanine, was assayed in both integument and fat body. Highest activity was detected in the integument, where two peaks were observed, one at day 3 near the end of larval feeding and the other at day 9 as pupal cuticle tanning was initiated. Fat body enzyme was substantially less and was detected only in the pharate pupa. Maximal NBAD synthesis by integument cultured in vitro was dependent upon DA supplementation of at least 1.4 mM. 20-Hydroxyecdysone did not alter NBAD synthesis in vitro in either the integument or the fat body, even though injection of this hormone into isolated larval abdomens induced synthesis and/or transport of integumental NBAD back into the hemolymph. The rate-limiting steps in the NBAD biosynthetic pathway appear to be the production of DOPA and DA, because β-alanine occurs in the hemolymph at relatively high levels throughout larval-pupal development.     

InR gene expression and octopamine metabolism in Drosophila melanogaster females

The influence of suppression of the expression of the Drosophila insulin-like receptor gene (InR) in corpus allatum (the gland-synthesizing juvenile hormone) on octopamine (OA) and juvenile hormone metabolism and on the development of the stress-reaction in Drosophila melanogaster females was studied. It was demonstrated that the suppression of InR gene expression in corpus allatum induces in D. melanogaster females an increase in the activity of the enzyme that limits the rate of octopamine synthesis (tyrosine decarboxylase), as well as in the level of juvenile hormone degradation and the intensity of the response of octopamine and juvenile hormone metabolism systems to heat stress. It was mentioned that a decrease in InR gene expression in corpus allatum does not influence the activity of OA-dependent N-acetyltransferase (the enzyme that degrades octopamine). It was established that the influence of suppression of the InR gene expression in corpus allatum on octopamine metabolism is mediated by juvenile hormone, since the treatment of flies by exogenous juvenile hormone restores the activity of tyrosine decarboxylase in flies with decreased InR expression in corpus allatum up to the normal level.     

Changes in fat body urate synthesizing capacity during the larval-pupal transformation of the tobacco hornworm, Manduca sexta

Large quantities of uric acid or urates are deposited in the fat body of tobacco hornworms, Manduca sexta, between the larval and pupal stages in development. The cause of this increased deposition was investigated by measuring fat body urate synthesizing capacity (USC) during the larval-pupal transformation (LPT). An 85% loss in USC occurs between the late-feeding larval and newly-ecdysed pupal stages. Urate synthesizing capacity, per se, is not responsible for the increase in fat body urate deposition, as evidenced by comparable rates of urate deposition in insects whose USCs differ by a factor of three. Rather, the increased deposition is caused by an increase in substrate availability. The loss in USC is programmed in two steps. The first programmed loss occurs by the end of the feeding fifth larval instar, since hornworms ligated at the pink stripe (PS) stage and measured at the time of the larval-pupal ecdysis (LPE) exhibit an increased retention of USC relative to controls. The second programmed loss in USC occurs between PS + one and PS + two day stages in development. A single administration of 20-hydroxyecdysone to hornworms ligated at the PS stage causes a restoration of this loss in USC by PS + two days, which is further sustained until the LPE. Unexpectedly, when measured immediately after the LPE, the second programmed loss in USC can be delayed until PS + 3 days if non-ligated hornworms are daily administered 20-hydroxyecdysone. The possibility is raised that 20-hydroxyecdysone does not act alone in causing the loss in fat body USC.     

Comparison of physiological functions of antagonistic insulin-like peptides,INS-23 and INS-18, in Caenorhabditis elegans

In Caenorhabditis elgans, insulin-like peptides have significant roles in modulating larval diapause and adult lifespan via the insulin/IGF-1 signaling (IIS) pathway. Although 40 insulin-like peptides (ILPs) have been identified, it remains unknown how ILPs act as either agonists or antagonists for their sole receptor, DAF-2. Here we found 1) INS-23 functions as an antagonistic ILP to promote larval diapause through the IIS pathway like a DAF-2 antagonist, INS-18, 2) INS-23 and INS-18 have similar biochemical functions. In addition, our molecular modeling suggests that INS-23 and INS-18 have characteristic insertions in the B-domain, which are crucial for the recognition of the insulin receptor, when compared with DAF-2 agonists. These characteristic insertions in the B-domain of INS-23 and INS-18 would modulate their intermolecular interactions with the DAF-2 receptor, which may lead these molecules to act as antagonistic ligands. Our study provides new insight into the function and structure of ILPs.     

Genome-wide Investigation of Intron Length Polymorphisms and Their Potential as Molecular Markers in Rice (Oryza sativa L.)

Intron length polymorphisms (ILPs) have been used as geneticmarkers in some studies. However, a systematic investigationand large-scale exploitation of ILP markers has not been reported.In this study, we performed a genome-wide search of ILPs betweentwo subspecies (indica and japonica) in rice using the draftgenomic sequences of cultivars 93-11 (indica) and Nipponbare(japonica) and 32 127 full-length cDNA sequences of Nipponbareobtained from public databases. We identified 13 308 putativeILPs. Based on these putative ILPs, we developed 5811 candidateILP markers via electronic-PCR with primers designed in flankingexons. We further conducted experiment to verify the candidateILP markers. Out of 215 candidate ILP markers tested on 93-11,Nipponbare and their hybrid, we successfully exploited 173 codominantILP markers. Further analyses on 10 rice accessions showed thatthese ILP markers were widely applicable and most (71.1%) exhibitedsubspecies specificity. This feature suggests that ILPs wouldbe useful for the studies of genome evolution and inter-subspeciesheterosis and for cross-subspecies marker-assisted selectionin rice. In addition, by testing 51 pairs of the ILP primerson five Gramineae plants and three dicot plants, we found anotherdesirable characteristic of rice ILP markers that they havehigh transferability to other plants.