Cloning and blood‐meal dependent induction pattern of apolipophorin‐III from Anopheles sinensis

Apolipophorin‐III is known to play a role in transporting lipids in insects, and much attention has been paid to lepidopteran insects' apolipophorin. Thus, we were interested in examining the effects of blood‐meal on the expression pattern of apolipophorin‐III in mosquitoes. This led us to clone and partially characterize the full‐length cDNA of apoLp‐III (AnsiApoLp‐III) from Anopheles sinensis. Analysis of AnsiApoLp‐III cDNA shows that the 728‐bp sequence has a 582‐bp protein‐coding region with 94 bp of putative 5′ untranslated region and 152 bp of 3′ untranslated region. The deduced amino acid sequence begins with a methionine codon at position 95 and extends to position 674, encompassing a polypeptide of 193 amino acids. AnsiApoLp‐III has the highest identity (63%) to Culex quinquefasciatus apoLp‐III. Temporal expression pattern analysis shows that although AnsiApoLp‐III was expressed at all developmental stages, it was highly detected at egg and adult stages in the female mosquitoes. In addition, we found out that AnsiApoLp‐III was induced in An. sinensis adult females after uptaking a blood‐meal. Spatial expression patterns of AnsiApoLp‐III shows that AnsiApoLp‐III mRNA was strongly induced at day 1 and gradually decreased from day 1 to day 4 in the ovaries. Most interestingly, AnsiApoLp‐III mRNA in the Malpighian tubule was strongly induced at day 1, decreased during days 1–3, and then became elevated again at day 4. These data suggest that blood‐meal influences AnsiApoLp‐III mRNA induction in ovaries and Malpighian tubules. It remains to further elucidate the biological roles of AnsiApoLp‐III in these organs.     

Expression profiles of two thaumatin‐like protein (TmTLP) genes in responses to various micro‐organisms from Tenebrio molitor

It is well known that anti‐fungal thaumatin‐like proteins (TLPs) play important roles in plants. Here, we investigated the expression analysis of thaumatin‐like protein genes TmTLPs in response to various pathogens in Tenebrio molitor. Developmental expression patterns of TmTLPs show that TmTLPs are highly expressed in the early pupal and adult stages. Furthermore, tissue‐specific expression patterns of TmTLPs indicate that TmTLP1 is highly expressed in the integument and gut of last instar larvae and the integument and Malpighian tubules of 5‐day old adults. In contrast, TmTLP2 is highly expressed in the gut of both last instar larvae and 5‐day old adults. We hypothesize that the expression of TmTLP genes in developmental stages may be related to molting and body remodeling stresses. In addition, the induction patterns of TmTLP genes indicate that TmTLPs were slightly induced by Escherichia coli, Staphylococcus aureus, Candida albicans and Listeria monocytogenes. Furthermore, TmTLP1 and TmTLP2 were strongly induced in response to E. coli at 9 h post‐injection and L. monocytogenes at 3 h post‐injection. Our results suggest that TmTLPs may possess antimicrobial functions in T. molitor.     

Susceptibility of Megalurothrips sjostedti developmental stages to Metarhizium anisopliae and the effects of infection on feeding, adult fecundity, egg fertility and longevity

The susceptibility of immature stages of the legume flower thrips, Megalurothrips sjostedti, to the entomopathogenic fungus Metarhizium anisopliae, was investigated under laboratory conditions. The adult stage was found to be more susceptible to infection than the larval and pupal stages. Mortality at all stages was dose-dependent, with the highest concentration of 1×10⁸ conidia ml^–1 producing the highest mortality (26, 46 and 100% for larvae, pupae and adults, respectively) at 8 days post-inoculation. At the same concentration, daily pollen consumption was significantly reduced at 2 days after treatment in infected adults but more slowly in infected larvae. Fecundity, egg fertility and longevity in adults surviving infection as larvae were significantly reduced compared to the control.     

Armigeres subalbatus prophenoloxidase III: Cloning,characterization and potential role in morphogenesis

It has long been suggested that phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning and melanotic encapsulation of pathogens. Here we report that a mosquito PO, designated Armigeres subalbatus prophenoloxidase III (As-pro-PO III), is likely involved in the morphogenesis in mosquito. Expression profile analysis found that As-pro-PO III mRNA is persistently expressed in adult mosquitoes and is not significantly affected by blood feeding, microfilariae inoculation, or Escherichia coli inoculation, but expression levels of As-pro-PO III fluctuated in larval and pupal stages. Knockdown of As-pro-PO III expression in pupae using double-stranded RNA resulted in high pupal mortality and deformed adults that subsequently died following emergence. Promoter activity analyses by electrophoretic mobility-shift assays and transfection assays suggest that the As-pro-PO III gene is positively regulated by a putative Zeste motif, a developmental regulatory element. These results suggest that As-pro-PO III is associated with morphogenesis of mosquitoes.     

Enantiomerization and Enantioselective Bioaccumulation of Metalaxyl in Tenebrio molitor Larvae

The enantiomerization and enantioselective bioaccumulation of metalaxyl by a single dose of exposure to Tenebrio molitor larvae under laboratory condition were studied by high‐performance liquid chromatography tandem mass spectroscopy (HPLC‐MS/MS) based on a ChiralcelOD‐3R [cellulosetris‐tris‐(3, 5‐dichlorophenyl‐carbamate)] column. Exposure of enantiopure R‐metalaxyl and S‐metalaxyl in Tenebrio molitor larvae exhibited significant enantiomerization, with formation of the R enantiomers from the S enantiomers, and vice versa, which might be attributed to the chiral pesticide catalyzed by a certain enzyme in Tenebrio molitor larvae. Enantiomerization was not observed in wheat bran during the period of 21 d. In addition, bioaccumulation of rac‐metalaxyl in Tenebrio molitor larvae was enantioselective with a preferential accumulation of S‐metalaxyl. These results showed that enantioselectivity was caused not only by actual degradation and metabolism but also by enantiomerization, which was an important process in the environmental fate and behavior of metalaxyl enantiomers. Chirality 26:88–94, 2014. © 2013 Wiley Periodicals, Inc.     

Developmental and myotropic effects of the Led-NPF-I peptide in tenebrionid beetles

Effects of Led-NPF-I peptide hormone (Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide) and its analogues were tested on the contractile activity of the oviduct of two beetle species Tenebrio molitor and Zophobas atratus. The influence of Led-NPF-I on the developmental processes of T. molitor was also examined. The unmodified Led-NPF-I molecule exerts inhibitory effects on contractions of the oviduct in both beetles; however, the efficiency of inhibition was stronger in T. molitor females. Structure–activity studies showed that replacement of Arg residue in positions 2, 7 and 9 in the amino acid sequence by His, D-Arg or Lys led to changes in myoinhibitory properties of almost all of the analogues in both species. In the majority of the analogues, the effect was weaker when compared to the native molecule. In the developmental bioassays, injections of Led-NPF-I peptide delayed the moulting process of T. molitor larvae and accelerated pupal moult. It also increased the growth in pre-starved and non-starved larvae. Our results indicate that short neuropeptides F are involved in the regulation of growth, developmental processes and some reproductive functions in beetles.     

MOLECULAR CHARACTERIZATION OF AN APOLIPOPHORIN‐III GENE FROM THE CHINESE OAK SILKWORM,Antheraea pernyi (LEPIDOPTERA: SATURNIIDAE)

Apolipophorin‐III (ApoLp‐III) acts in lipid transport, lipoprotein metabolism, and innate immunity in insects. In this study, an ApoLp‐III gene of Antheraea pernyi pupae (Ap‐ApoLp‐III) was isolated and characterized. The full‐length cDNA of Ap‐ApoLp‐III is 687 bp, including a 5′‐untranslated region (UTR) of 40 bp, 3′‐UTR of 86 bp and an open reading frame of 561 bp encoding a polypeptide of 186 amino acids that contains an Apolipophorin‐III precursor domain (PF07464). The deduced Ap‐apoLp‐III protein sequence has 68, 59, and 23% identity with its orthologs of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap‐apoLp‐III was close to that of Bombycoidea. qPCR analysis revealed that Ap‐ApoLp‐III expressed during the four developmental stages and in integument, fat body, and ovaries. After six types of microorganism infections, expression levels of the Ap‐ApoLp‐III gene were upregulated significantly at different time points compared with control. RNA interference (RNAi) of Ap‐ApoLp‐III showed that the expression of Ap‐ApoLp‐III was significantly downregulated using qPCR after injection of E. coli. We infer that the Ap‐ApoLp‐III gene acts in the innate immunity of A. pernyi.     

CHANGES IN THE HEMOLYMPH PROTEIN PROFILES IN Galleria mellonella INFECTED WITH Bacillus thuringiensis INVOLVE APOLIPOPHORIN III. THE EFFECT OF HEAT SHOCK

This report concerns the effect of heat shock on host–pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis. We show enhanced activity against Gram‐positive bacteria in the hemolymph of larvae pre‐exposed to heat shock before infection with B. thuringiensis. Heat shock influenced the protein pattern in the hemolymph of infected larvae: more peptides with a molecular weight below 10 kDa were detected in comparison with nonshocked animals. Additionally, we noticed that the amount of apolipophorin III (apoLp‐III) in the hemolymph decreased transiently following infection, which was considerably higher in larvae pre‐exposed to heat shock. On the other hand, its expression in the fat body showed a consequent infection‐induced decline, observed equally in shocked and nonshocked animals. This suggests that the amount of apoLp‐III in the hemolymph of G. mellonella larvae is regulated at multiple levels. We also report that this protein is more resistant to degradation in the hemolymph of larvae pre‐exposed to heat shock in comparison to nonshocked larvae. Two‐dimensional analysis revealed the presence of three isoforms of apoLp‐III, all susceptible to proteolytic degradation. However, one of them was the most abundant, both in the protease‐treated and untreated hemolymph. Taking into consideration that, in general, apoLp‐III has a stimulative effect on different immune‐related hemolymph proteins and peptides, the reported findings bring us closer to understanding the effect of heat shock on the resistance of G. mellonella to infection.     

Further Study on the Esterase Patterns of Sibling Species in the Drosophila saltans Subgroup (saltans Group): Intraspecific and Interspecific Variations in the Development

Twenty of the 32 esterase bands previously detected in the adults of D. prosaltans, D. saltans and D.␣austrosaltans were found in larvae and pupae studied in this work. The results showed that, in addition to expressing the highest number of esterase bands, the adult stage of the three species exhibited the highest degree of expression (amount of synthesis) for most of the bands. Differences between larval and pupal stages were detected in the degree of expression (amount of synthesis) of the bands and in the frequency of samples expressing them. The frequencies of expression of the bands corresponding to genes in loci 1–3 were greater in pupae than in larvae while the frequencies of expression of the bands corresponding to genes in loci 4–9 were predominantly expressed in larvae or were equal in both developmental stages. Like the adults, larvae, pupae and empty pupal cases (which were also studied in this work) showed specific esterases. Taken together, the observations showed that, in the species studied, every developmental stage is characterized by specific bands and by specific frequency and degree of expression of the bands shared with other stages.     

GABAergic modulation of motor‐driven behaviors in juvenile Drosophila and evidence for a nonbehavioral role for GABA transport

We have identified specific GABAergic‐modulated behaviors in the juvenile stage of the fruit fly, Drosophila melanogaster via systemic treatment of second instar larvae with the potent GABA transport inhibitor DL‐2,4‐diaminobutyric acid (DABA). DABA significantly inhibited motor‐controlled body wall and mouth hook contractions and impaired rollover activity and contractile responses to touch stimulation. The perturbations in locomotion and rollover activity were reminiscent of corresponding DABA‐induced deficits in locomotion and the righting reflex observed in adult flies. The effects were specific to these motor‐controlled behaviors, because DABA‐treated larvae responded normally in olfaction and phototaxis assays. Recovery of these behaviors was achieved by cotreatment with the vertebrate GABA_A receptor antagonist picrotoxin. Pharmacological studies performed in vitro with plasma membrane vesicles isolated from second instar larval tissues verified the presence of high‐affinity, saturable GABA uptake mechanisms. GABA uptake was also detected in plasma membrane vesicles isolated from behaviorally quiescent stages. Competitive inhibition studies of [³H]‐GABA uptake into plasma membrane vesicles from larval and pupal tissues with either unlabeled GABA or the transport inhibitors DABA, nipecotic acid, or valproic acid, revealed differences in affinities. GABAergic‐modulation of motor behaviors is thus conserved between the larval and adult stages of Drosophila, as well as in mammals and other vertebrate species. The pharmacological studies reveal shared conservation of GABA transport mechanisms between Drosophila and mammals, and implicate the involvement of GABA and GABA transporters in regulating physiological processes distinct from neurotransmission during behaviorally quiescent stages of development. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Effect of omeprazole-evoked hypergastrinemia on ultrastructure of enterochromaffin-like cells in the stomach of portacaval-shunted rats

Immunocytochemical staining of the nervous system of larva, pupa, and adult stage of Tenebrio molitor with anti-insulin serum demonstrated insulin-like peptides in the protocerebrum, corpora allata, and suboesophageal ganglion. During pupal development, marked changes in staining intensity of the protocerebral cells were detected. The staining pattern suggests release of insulin-like peptides early on day 0 and again on day 3 of the stadium. Injections of anti-insulin at these times caused significant delays in the timing of pupal/adult ecdysis. An immunoblot of haemolymph from day-3 pupae revealed a 6.5-kDa insulin-like molecule. These results suggest that the prothoracicotropic hormone of T. molitor is an insulin-like molecule.     

Disturbance and investment: developmental responses of tropical lotic midges to repeated tube destruction in the juvenile stages

Abstract. 1. Investment in silken tubes may give tubicolous chironomid larvae more to lose than non‐tube building taxa when physical disturbances or the actions of other organisms induce dispersal. In this study, two chironomid species from flood‐prone Australian tropical streams were subjected to repeated experimental destruction of their larval and pupal tubes in laboratory experiments, with life‐history parameter responses recorded. 2. When subjected to the tube destruction treatments, Echinocladius martini (Orthocladiinae), whose larvae construct branched loose silken tubes in fast flow, showed no reduction in survivorship to adult, whereas Polypedilum australotropicus (Chironominae), which builds compact tightly woven tubes incorporating detrital material within pool leaf packs, showed reduced survivorship of about 25%. 3. For both species, tube destruction reduced pupal duration, regardless of whether destruction occurred in the larval stage only, or in both the larval and pupal stages. This may be a response to the risks of exposure outside tubes in nature, particularly for pupae, which are incapable of spinning silk. 4. Tube destruction caused little or no reduction in adult size, longevity, or female fecundity (oocyte number) for either species, but oocyte development and size was retarded in E. martini females, reflecting the energetic and nutritive costs of the tube destruction treatments. 5. Contrasts in the responses of the two species reflect differences in tube‐building behaviour and in situ habitat use. These results demonstrate that tube loss can affect the fitness of individual chironomids, though both species proved generally resilient, which may reflect their long‐term exposure to variable tropical lotic conditions.     

Effects of the mixed biocide Bacillus thuringiensis–abamectin on the development of the parasitoid Microplitis mediator and its host Helicoverpa armigera

The effects of the mixed biocide Bacillus thuringiensis Berliner with abamectin (BtA) on the development of the parasitoid Microplitis mediator (Haliday) (Hymenoptera: Braconidae) and its cotton bollworm host, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), were evaluated in the laboratory. Weight gain in larvae of H. armigera was initially delayed, but larval developmental period increased and pupal weight increased when they were fed on a diet containing BtA. Due to increased longevity of the host larvae, the susceptible period to parasitization of H. armigera by M. mediator increased when the host larvae were reared on diets containing BtA at concentrations of 0.5, 1, 2, and 4 μg g^?1. The longevity of female and male parasitoids significantly decreased when newly emerged wasps were fed a honey solution containing 200 μg ml^?1 BtA in comparison with those fed only a honey solution. Mean longevity was significantly prolonged when parasitoids were fed a honey solution and BtA–honey solution in comparison with those fed BtA–distilled water, distilled water, or nothing. There were no significant differences compared with the control in any biological characteristics for the offspring of female parasitoids fed the honey solutions containing BtA at concentrations of 50, 100, and 200 μg ml^?1; characteristics measured include the egg‐larval period, pupal weight, male and female pupal periods, adult fresh weight, and adult longevity. When female parasitoids parasitized host larvae that had been fed the diet containing BtA, their male and female pupal periods were significantly prolonged compared with the control (without BtA).     

Juvenile hormone esterase activity during the last larval and pupal stages of Tenebrio molitor

In vitro analysis of juvenile hormone esterase activity of haemolymph of T. molitor was performed during the end of post-embryonic development. Weak activity was found in penultimate stage larvae as in the major part (except the last day) of last-larval instar, while very high activity was monitored in the early pupae (female or male).This pupal peak was the only one detected during development in the insect, coinciding with the pupal juvenile hormone sensitive period. The first juvenile hormone sensitive period, during the lastlarval instar, does not seem to be protected by any juvenile hormone esterase activity in contrast to other species. These results suggest a central control for the drop in juvenile hormone level ceasing synthesis by the corpora allata after integration of external stimuli. This hypothesis could explain the natural occurrence of prothetelic larvae, the absence of pupal adult intermediates and the variable number of instars in Tenebrio.     

The fate of specific motoneurons and sensory neurons of the pregenital abdominal segments inTenebrio molitor (Insecta : Coleoptera) during metamorphosis

The anatomy and innervation of the lateral external muscle and sensory cells located in the ventral region of pregenital abdominal segments were examined at the larval and adult stages ofTenebrio molitor (Coleoptera). All seven muscles located in this region degenerate during the pupal stage, whilst only the lateral external median (lem) appears in the adult. Backfillings of the motor nerve innervating this muscle reveal that, at both larval and adult stages, it is innervated by ten neurons. Intracellular records from the muscle fibres show that two neurons are inhibitory, and at least five are excitatory. There are also two unpaired neurons. A variety of sensory organs are located in the ventral region of the larvae, whilst only campaniform sensilla are found in the adult. At both stages, the innervation pattern of the sensory nerve branches is very similar. Also, the central projections of the sensory cells occupy similar neuropilar areas. Finally, prolonged intracellular records from the lem muscle revealed that, at the larval stage, it participates only in segmental or intersegmental reflexes, whilst in the adult it has a primary expiratory role in ventilation. The results show that extensive changes occur in the number of muscles located in the ventral region of the pregenital abdominal segments, as well as in the arrangement and number of sensory neurons, in the structure of the exoskeleton, and even in the central nervous system. In contrast, only minor changes are observed in the sensory and motor nerve branches, in the sensory projections, and in the number and the location of the motoneurons innervating the lateral external median muscle. Correspondence to: G. Theophilidis