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.     

Juvenile Hormone and Insulin Regulate Trehalose Homeostasis in the Red Flour Beetle,Tribolium castaneum

Insulin/IGF-1 signaling (IIS) has been well studied for its role in the control of life span extension and resistance to a variety of stresses. The Drosophila melanogaster insulin-like receptor (InR) mutant showed extended life span due to reduced juvenile hormone (JH) levels. However, little is known about the mechanism of cross talk between IIS and JH in regulation of life span extension and resistance to starvation. In the current study, we investigated the role of IIS and JH signaling in regulation of resistance to starvation. Reduction in JH biosynthesis, JH action, or insulin-like peptide 2 (ILP2) syntheses by RNA interference (RNAi)-aided knockdown in the expression of genes coding for juvenile hormone acid methyltransferase (JHAMT), methoprene-tolerant (Met), or ILP2 respectively decreased lipid and carbohydrate metabolism and extended the survival of starved beetles. Interestingly, the extension of life span could be restored by injection of bovine insulin into JHAMT RNAi beetles but not by application of JH III to ILP2 RNAi beetles. These data suggest that JH controls starvation resistance by regulating synthesis of ILP2. More importantly, JH regulates trehalose homeostasis, including trehalose transport and metabolism, and controls utilization of stored nutrients in starved adults.     

EVIDENCE THAT ECDYSIS IN THE LARVAL COCKROACH,Periplaneta americana L. IS TRIGGERED BY AN INCREASE IN THE CONCENTRATION OF HEMOLYMPH SUGAR

Ecdysis in insects can be defined as shedding of the cuticle at the end of a larval stadium. This event can only occur after the peak titer of ecdysteroid in the hemolymph has returned to a low level. In the cockroach Periplaneta americana, ecdysis is strongly correlated with a rise in the concentration of trehalose and glucose in the hemolymph, leading to the idea that a causal relationship may exist between both events. The objective in this study was to determine if an increase in hemolymph sugar level would shorten the time to ecdysis in cockroach larvae with experimentally delayed ecdysis. The last larval stadium of P. americana averages 33.5 days but this increases significantly if the larva is injected with a small volume of saline. Injection of 10 μl of saline on day 20 and on four successive days lengthened the stadium by as much as 2 weeks. If, however, trehalose or glucose is incorporated into the saline, approximately 40% of the treated larvae undergo ecdysis at the same time as uninjected larvae. Injection of Peram‐AKH, the hypertrehalosemic hormone, also decreases the time for ecdysis to occur. This suggests that peak levels of ecdysteroid trigger the release of Peram‐AKH, which then leads to activation of trehalose synthesis. The results support the hypothesis that elevated hemolymph sugar is a contributing factor in the removal of ecdysteroid from the hemolymph.     

In vitro study of lipid metabolism in the mealworm larval fat body

Lipid metabolism in Tenebrio larval fat body has been studied in vitro. Lipid release required the presence of diluted hemolymph in the incubation medium. This time-dependent release of lipid was strongly stimulated in a dose-dependent manner by Tenebrio corpora cardiaca (CC) extracts or synthetic adipokinetic hormone (AKH I). Furthermore, some glycerol was released when larval fat body was incubated without hemolymph, and this phenomenon was also dose dependent for added CC extracts. Lipid synthesis was estimated in vitro by following the incorporation of radioactivity from [6-¹⁴C] glucose into fatty acids. Lipogenesis occurred in the absence of added carbohydrates in the medium, but it was stimulated by the addition of glucose, and especially trehalose (10 mg ml^?1). Intestinal insulin-like peptide (ILP) also stimulated in vitro lipogenesis in a dose-dependent fashion. We conclude that lipolytic and lipogenetic activities of larval mealworm fat body in vitro are effectively under hormonal control.     

Identification of a hypertrehalosemic factor in Spodoptera exigua

Trehalose is a major blood sugar in insects with a range of physiological functions, including an energy source and a cryoprotectant. Hemolymph trehalose concentrations are tightly regulated according to physiological conditions. An insulin‐like peptide, SeILP1, downregulates hemolymph trehalose concentrations in Spodoptera exigua. Here, we identified a factor that upregulates hemolymph trehalose concentration in S. exigua. Hemolymph trehalose concentrations were significantly increased after immune challenge or under starvation in a time‐dependent manner. To determine endocrine factors responsible for the upregulation, stress‐associated mediators, such as octopamine, serotonin, or eicosanoids were injected, but they did not upregulate hemolymph trehalose. On the other hand, injection with Schistocerca gregaria adipokinetic hormone (AKH) significantly increased hemolymph trehalose concentration in S. exigua. During upregulation of hemolymph trehalose by AKH injection, trehalose degradation appeared to be inhibited because expression of trehalase and SeILP1 were significantly suppressed while that of trehalose phosphate synthase was not significantly changed. Interrogation of a Spodoptera genome database identified an S. exigua AKH‐like gene and its expression was confirmed. During starvation, its expression concentrations were increased, although RNA interference specific to the AKH‐like hypertrehalosemic factor (SeHTF) gene significantly prevented the upregulation of hemolymph trehalose concentrations during starvation. A synthetic peptide of SeHTF was prepared and injected into S. exigua larvae. At nanomolar concentration, the synthetic SeHTF peptide effectively upregulated hemolymph trehalose concentrations. Here we report a novel hypertrehalosemic factor in S. exigua (SeHTF).     

Oviposition preference and development of Maruca vitrata (Fabricius) (Lepidoptera: Crambidae) on different radiofrequency fields

Maruca vitrata is considered as one of the major impediments in boosting up the production of tropical legumes. Since chemical pesticides have an adverse effect on human health and environment, there is an urgent need for developing a non‐chemical based method to replace chemical insecticides for controlling of pests. We investigated the oviposition, development and adult performances of M. vitrata at five radiofrequency levels of 0 (control without frequency), 5, 10, 20, and 30 kHz on artificial diet. In addition, the effects were also studied in three successive generations (Parent, F1‐ and F2‐ generations) of M. vitrata. The results showed that radiofrequency application had significant effects on the life‐variables of M. vitrata. Radiofrequency exposures on M. vitrata not only impacted on oviposition, developmental time, adult longevity, and adult weight but negative effects were also transmitted to the successive generations. Oviposition was found to reduce as generation passed after exposed to radiofrequencies. Total developmental times from egg to adult emergence were longer with shorter longevity in case of radiofrequency treatment than untreated. Decreasing the radiofrequency level further increased time for development of adults as generation passed. The shortest adult longevity occurred at 5 kHz treatment. The outcomes of this study are discussed in terms of targeting susceptibilities to radiofrequency as an alternative to chemical treatments.     

Trehalose extends longevity in the nematode Caenorhabditis elegans

Trehalose is a disaccharide of glucose found in diverse organisms and is suggested to act as a stress protectant against heat, cold, desiccation, anoxia, and oxidation. Here, we demonstrate that treatment of Caenorhabditis elegans with trehalose starting from the young‐adult stage extended the mean life span by over 30% without any side effects. Surprisingly, trehalose treatment starting even from the old‐adult stage shortly thereafter retarded the age‐associated decline in survivorship and extended the remaining life span by 60%. Demographic analyses of age‐specific mortality rates revealed that trehalose extended the life span by lowering age‐independent vulnerability. Moreover, trehalose increased the reproductive span and retarded the age‐associated decrease in pharyngeal‐pumping rate and the accumulation of lipofuscin autofluorescence. Trehalose also enhanced thermotolerance and reduced polyglutamine aggregation. These results suggest that trehalose suppressed aging by counteracting internal or external stresses that disrupt protein homeostasis. On the other hand, the life span‐extending effect of trehalose was abolished in long‐lived insulin/IGF‐1‐like receptor (daf‐2) mutants. RNA interference‐mediated inactivation of the trehalose‐biosynthesis genes trehalose‐6‐phosphate synthase‐1 (tps‐1) and tps‐2, which are known to be up‐regulated in daf‐2 mutants, decreased the daf‐2 life span. These findings indicate that a reduction in insulin/IGF‐1‐like signaling extends life span, at least in part, through the aging‐suppressor function of trehalose. Trehalose may be a lead compound for potential nutraceutical intervention of the aging process.     

The potential of trehalose to replace insect hemolymph in artificial media for Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Insect additives have been shown to improve the value of artificial media for Trichogramma species, but at the same time maintain dependence on parallel cultures of host insects. In the present study, Trichogramma dendrolimi Matsumura was reared in vitro from egg to adult on artificial media with different contents of pupal hemolymph of Chinese oak silkmoth Antheraea pernyi (Guérin‐Méneville) and with supplements of distilled water, or of trehalose dissolved in water or in Grace's insect medium. The results indicated insect hemolymph was the key component of artificial medium. Developmental parameters, including rates of parasitism, pupation, adult emergence and normal adults, and numbers of produced adults, were increased on media supplemented with trehalose even when the proportion of pupal hemolymph was reduced. Two artificial media, the first containing 30% hemolymph and 10% trehalose in water with 98.9% parasitism rate, 77.7.0% pupation rate, 77.2% emergence rate, 80.0% normal adult rate and 333 produced adults, and the second containing 25% hemolymph and 15% trehalose in Grace's insect medium with 97.8% parasitism rate, 91.0% pupation rate, 85.2% emergence rate, 76.1% normal adult rate and 757 produced adults, were believed to hold potential to mass produce T. dendrolimi. The use of trehalose to partially replace pupal hemolymph in artificial medium of this and other Trichogramma species may contribute to a significant reduction in their production cost and may as such help to evade problems related to short supplies of lepidopteran eggs, which currently constitute the main factitious host for the mass rearing of the parasitoids.     

Effects of food stress and density in different life stages on reproduction in a butterfly

Availability of adequate nutrition and (rearing) density are among the most important factors affecting growth, development and reproduction in animals. In holometabolous insects diets and energetic needs change between life stages, with storing of larval resources, adult feeding and reproduction being linked strategies. Nevertheless, studies investigating nutritional (and density) effects across metamorphic boundaries are largely lacking. We aim at disentangling the functional basis of reproductive patterns by independently manipulating larval and adult (1) density and (2) access to food, respectively, in the tropical butterfly, Bicyclus anynana. (1) A high larval rearing density had, contrary to common wisdom, very little impact on body size, but reduced larval development time through increased growth rates. The latter is thought to be an adaptation to high densities, driven by the risk of larval food resources becoming exhausted before reaching metamorphosis. Larval density and male company during oviposition (i.e. adult density) had no detectable effects on female reproduction. (2) Larval food stress prolonged larval development time and reduced larval growth rate, body size, fecundity and reproductive investment. Detrimental effects on female reproduction were mediated through a reduction in body size. Additional negative effects of adult food stress on fecundity were largely confined to females being fed as larvae ad libitum, while those being previously starved showed reduced performance regardless of adult income. Effects on egg size were inconsistent and, overall, marginal. Our results show that restricted food access in different developmental stages may set different limits to reproduction, either posed by shortage of larval‐derived storage reserves (i.e. nitrogenous compounds) or adult income (i.e. carbohydrates). Thus, one should be cautious when stating that one or the other type of nutrients is ultimately limiting to reproduction. Rather, our findings highlight the importance of resource congruence and of considering both, larval‐ and adult‐derived resources for reproduction.     

Honey bee PTEN--description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes

Background

Phosphatase and TENsin (PTEN) homolog is a negative regulator that takes part in IIS (insulin/insulin-like signaling) and Egfr (epidermal growth factor receptor) activation in Drosophila melanogaster. IIS and Egfr signaling events are also involved in the developmental process of queen and worker differentiation in honey bees (Apis mellifera). Here, we characterized the bee PTEN gene homologue for the first time and begin to explore its potential function during bee development and adult life.

Results

Honey bee PTEN is alternatively spliced, resulting in three splice variants. Next, we show that the expression of PTEN can be down-regulated by RNA interference (RNAi) in the larval stage, when female caste fate is determined. Relative to controls, we observed that RNAi efficacy is dependent on the amount of PTEN dsRNA that is delivered to larvae. For larvae fed queen or worker diets containing a high amount of PTEN dsRNA, PTEN knockdown was significant at a whole-body level but lethal. A lower dosage did not result in a significant gene down-regulation. Finally, we compared same-aged adult workers with different behavior: nursing vs. foraging. We show that between nurses and foragers, PTEN isoforms were differentially expressed within brain, ovary and fat body tissues. All isoforms were expressed at higher levels in the brain and ovaries of the foragers. In fat body, isoform B was expressed at higher level in the nurse bees.

Conclusion

Our results suggest that PTEN plays a central role during growth and development in queen- and worker-destined honey bees. In adult workers, moreover, tissue-specific patterns of PTEN isoform expression are correlated with differences in complex division of labor between same-aged individuals. Therefore, we propose that knowledge on the roles of IIS and Egfr activity in developmental and behavioral control may increase through studies of how PTEN functions can impact bee social phenotypes.     

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.     

Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae.