Insect Biochemistry and Molecular Biology

Els resultats d'aquesta Tesi posen de manifest el paper del cervell en el control del desenvolupament larvari de Sesamia nonagrioides. Així, S. nonagrioides és el primer lepidòpter on s' ha trobat que es pot desenvolupar de larva a adult sense cervell. Les ...

Anopheline mosquitoes are vectors of malaria parasites. Their saliva contains anti-hemostatic and immune-modulator molecules that favor blood feeding and parasite transmission. In this study, we describe the inhibition of the alternative pathway of the complement system (AP) by Anopheles aquasalis salivary gland extracts (SGE). According to our results, the inhibitor present in SGE acts on the initial step of the AP blocking deposition of C3b on the activation surfaces. Properdin, which is a positive regulatory molecule of the AP, binds to SGE. When SGE was treated with an excess of properdin, it was unable to inhibit the AP. Through SDS-PAGE analysis, A. aquasalis presented a salivary protein with the same molecular weight as recombinant complement inhibitors belonging to the SG7 family described in the saliva of other anopheline species. At least some SG7 proteins bind to properdin and are AP inhibitors. Searching for SG7 proteins in the A. aquasalis genome, we retrieved a salivar...

Small diameter (Ͻ20 µm) neurons from the sixth abdominal ganglion of the American cockroach, Periplaneta americana, were enzymatically isolated and responses to exogenously applied γ-aminobutyric acid (GABA) were recorded using the whole-cell patch clamp technique. With a minimal intracellular medium, responses to repeated applications of GABA decreased to zero within a few minutes. The rate of rundown of GABA responses was decreased by the intracellular inclusion of the phosphatase inhibitors microcystin and okadaic acid, suggesting that phosphorylation is necessary for the maintenance of cockroach GABA receptor function. ATP (5 mM) prevented GABA response rundown. ADP (5 mM) also slowed GABA response rundown, but responses stabilized at a level about half that seen with ATP. In the presence of protein kinase A inhibitory peptide (PKI), ATP was only as efficacious as ADP in slowing rundown. PKI had no effect on the ability of ADP to slow rundown, suggesting that the β-phosphate of ADP is not involved in PKA-dependent phosphorylation of the GABA receptor. These results suggest that in cockroach neurons, GABA receptor function is maintained intracellularly by adenine nucleotides, not only by phosphorylation, but also possibly by an interaction with a nucleotide recognition site unrelated to PKA-dependent phosphorylation.

In this study, we provided the demonstration of the presence of a single CHS gene in the Rhodnius prolixus (a blood-sucking insect) genome that is expressed in adults (integument and ovary) and in the integument of nymphs during development. This CHS gene appears to be essential for epidermal integrity and egg formation in R. prolixus. Because injection of CHS dsRNA was effective in reducing CHS transcript levels, phenotypic alterations in the normal course of ecdysis occurred. In addition, two phenotypes with severe cuticle deformations were observed, which were associated with loss of mobility and lifetime. The CHS dsRNA treatment in adult females affected oogenesis, reducing the size of the ovary and presenting a greater number of atresic oocytes and a smaller number of chorionated oocytes compared with the control. The overall effect was reduced oviposition. The injection of CHS dsRNA modified the natural course of egg development, producing deformed eggs that were dark in color and unable to hatch, distinct from the viable eggs laid by control females. The ovaries, which were examined under fluorescence microscopy using a probe for chitin detection, showed a reduced deposition on pre-vitellogenic and vitellogenic oocytes compared with control. Taken together, these data suggest that the CHS gene is fundamentally important for ecdysis, oogenesis and egg hatching in R. prolixus and also demonstrated that the CHS gene is a good target for controlling Chagas disease vectors. Published by Elsevier Ltd.

More than half of the proteome from mandibular glands in caterpillars is represented by chemosensory proteins. Based on sequence similarity, these proteins are putative transporters of ligands to gustatory receptors in sensory organs of insects. We sought to determine whether these proteins are inducible by comparing, both qualitatively and quantitatively, the salivary (mandibular and labial) proteomes from caterpillars (Vanessa cardui) reared on different plants and artificial diet containing either bacteria or bacterial cell-walls. We included a treatment where the caterpillars were switched from feeding on artificial diet to plant material at some point in their development. Additionally, we evaluated the degree of overlap between the proteomes in the hemolymph-filled coelom and salivary glands of caterpillars reared on plant material. We found that the quality and quantity of the identified proteins differed clearly between hemolymph-filled coelome, labial and mandibular glands. Our results indicated that even after molting and two-day feeding on a new diet, protein production is affected by the previous food source used by the caterpillar. Candidate proteins involved in chemosensory perception by insects were detected: three chemosensory (CSPs) and two odorant-binding proteins (OBPs). Using the relative amounts of these proteins across tissues and treatments as criteria for their classification, we detected hemolymph-and mandibular gland-specific CSPs and observed that their levels were affected by caterpillar diet. Moreover, we could compare the protein and transcript levels across tissues and treatment for at least one CSP and one OBP. Therefore, we have identified specific isoforms for testing the role of CSPs and OBPs in plant and pathogen recognition. We detected catalase, immune-related protein and serine proteases and their inhibitors in high relative levels in the mandibular glands in comparison to the labial glands. These findings suggest that the mandibular glands of caterpillars may play an important role protecting the caterpillar from oxidative stress, pathogens and aiding in digestion. Contamination with hemolymph proteins during dissection of salivary glands from caterpillars may occur but it is not substantial since the proteomes from hemolymph, mandibular and labial glands were easily discriminated from each other by principal component analysis of proteomic data.

Anopheles species are vectors for malaria. To date, insecticide application has been the primary method for controlling mosquito disease vectors. Chemical interventions to control vectors may occasionally prove ineffective, due to the development of insecticide resistance. Target-site insensitivity is one of the primary mechanisms that contribute to resistance. This study aims to determine the G119S (mutation of glycine to serine) and L1014S (mutation of leucine to phenylalanine) mutation rates of Anopheles superpictus Grassi, 1899 (Culicidae: Anophelinae) and Anopheles sacharovi Favre, 1903 (Culicidae: Anophelinae) populations and their seasonal variations in the Aegean Region. For both A. superpictus and A. sacharovi, the G119S mutation was observed at a low frequency during all three periods. The mean L1014S frequency for A. sacharovi populations in the spring 2018, fall 2018, and spring 2019 periods was 0.063, 0.156, and 0.196, respectively. For A. superpictus populations, the frequencies were 0.025, 0.013, and 0.024, respectively. Pyrethroids, the most widely utilized insecticide in recent years, which are presumed to be effective, will ultimately exhibit reduced efficacy in some of these populations.

The purpose of this research is to provide empirical evidence aboutcompany characeristics such as  firm size, profitability, leverage, and  liquidity, and then corporate governance mechanismsuch as board size, and audit committe  that affect corporate risk disclosure in interim financial reports.The statistic method that used to test the hypotheses is multiple regressionanalysis. The population of this research are listed mining firms on IDX in2016-2017. Thirty-nine mining  firms listed on IDX in 2016-2017  were chosen assample. To explain the effects between the variables, agency and signallingtheories were used. And then, corporate risk disclosure was measured withcontent analysis method. The results of this research show that only firm size and board sizehave significant positive effect on corporate risk disclosure. In addition, theresults do not support that audit committe and firmcharacteristics attributes (profitability, leverage, and liquidity) have s...

To understand the evolution of premating signals in moths, it is important to know the genetic basis of these signals. We conducted Quantitative Trait Locus (QTL) analysis by hybridizing two noctuid moth species, Heliothis virescens (Hv) and Heliothis subflexa (Hs), and backcrossing the F 1 females to males of both parental species. One of these backcrosses (F 1 Â Hs) was a biological replicate of our previous study and served to test the robustness of our previous findings. The backcross to Hv was designed to reveal QTL with recessive inheritance of the Hv character state. This study confirms previously discovered QTL, but also reports new QTL. Most importantly, we found relatively large QTL affecting Z9-16:Ald, the critical sex pheromone component of Hs. For Z9-14:Ald, the critical sex pheromone component of Hv, as well as for the minor pheromone compound 14:Ald, we found QTL in which the change in pheromone ratio was opposite-to-expected. Linking QTL to the biosynthetic pathways of the pheromone compounds of Hv and Hs implicates several candidate genes in the divergence of these premating signals, the most important of which are acetyl transferase, one or more desaturase(s), and a fatty acyl reductase or alcohol oxidase.

Juvenile hormone (JH) is a major hormonal regulator in insects. In Aedes aegypti females, JH signals the completion of the ecdysis to the adult stage and initiates reproductive processes. Although the regulation of JH synthesis and titer in Ae. aegypti females has been extensively studied, relatively little is known about changes of JH synthesis and titers in male mosquitoes, as well as on the roles of JH controlling male reproductive biology. A better understanding of male mosquito reproductive biology, including an improved knowledge of the hormonal control of reproduction, could increase the likelihood of success of male-targeting vector control programs. Using a high performance liquid chromatography coupled to electrospray tandem mass spectrometry method, we measured JH biosynthesis and hemolymph levels in male mosquitoes during pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers. Synthesis of JH III was very low i...

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again "competent" to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP 3 Roperated mobilization of endoplasmic reticulum Ca 2+ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.

Juvenile hormone (JH) regulates reproductive maturation in insects; therefore interruption of JH biosynthesis has been considered as a strategy for the development of target-specific insecticides. The corpora allata (CA) from mosquitoes is highly specialized to supply variable levels of JH, which are linked to ovarian developmental stages and influenced by nutritional signals. However, very little is known about how changes in JH synthesis relate to reproductive physiology and how JH synthesis regulation is translated into changes in the CA machinery. With the advent of new methods that facilitate the analysis of transcripts, enzymes and metabolites in the minuscule CA, we were able to provide comprehensive descriptions of the mevalonic (MVA) and JH synthesis pathways by integrating information on changes in the basic components of those pathways. Our results revealed remarkable dynamic changes in JH synthesis and exposed part of a complex mechanism that regulates CA activity. Principal component (PC) analyses validated that both pathways (MVAP and JH-branch) are transcriptionally co-regulated as a single unit, and catalytic activities for the enzymes of the MVAP and JH-branch also changed in a coordinate fashion. Metabolite studies showed that global fluctuations in the intermediate pool sizes in the MVAP and JH-branch were often inversely related. PC analyses suggest that in female mosquitoes, there are at least 4 developmental switches that alter JH synthesis by modulating the flux at distinctive points in both pathways.

Juvenile hormone (JH) levels must be modulated to permit the normal progress of development and reproductive maturation in mosquitoes. JH is part of a transduction system that assesses nutritional information and controls reproduction in mosquitoes. Adult female Aedes aegypti show nutritionally-dependent dynamic changes in corpora allata (CA) JH biosynthetic activities. A coordinated expression of most JH biosynthetic enzymes has been described in female pupae and adult mosquitoes; increases or decreases in transcript levels for all the enzymes were concurrent with increases or decreases in JH synthesis; suggesting that transcriptional changes are at least partially responsible for the dynamic changes of JH biosynthesis. The goal of the present study is to identify signaling network components responsible for the nutritionaldependent changes of JH synthesis in the CA of mosquitoes. The insulin/TOR signaling network plays a central role in the transduction of nutritional signals that regulate cell growth and metabolism in insects. These pathways have also been suggested as a link between nutritional signals and JH synthesis regulation in the CA of cockroaches and flies. We used a combination of in vitro studies and in vivo genetic knockdown experiments to explore nutritional signaling pathways in the CA. Our results suggest that the insulin/TOR pathway plays a role in the transduction of the nutritional information that regulates JH synthesis in mosquitoes. Transcriptional regulation of the genes encoding JH biosynthetic enzymes is at least partially responsible for these nutritionally modulated changes of JH biosynthesis.

Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterwards IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg 2+ or Mn 2+ but not Zn 2+ for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis . This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect.

Helicoverpa armigerautilizes (Z)-11-hexadecenal (Z11-16:Ald) as its major sex pheromone component. Three pheromone binding proteins (PBPs) and two general odorant binding proteins (GOBPs) are abundantly expressed in male antennae ofH. armigera. However, their precise roles in the olfactory detection of Z11-16:Ald remain enigmatic. To answer this question, we first synthesized the antibody against HarmOR13, a pheromone receptor (PR) primarily responding to Z11-16:Ald and mapped the local associations between PBPs / GOBPs and HarmOR13. Immunostaining showed that HarmPBPs and HarmGOBPs were localized in the supporting cells of sensilla trichodea and sensilla basiconica respectively. In particular, HarmPBP1 and HarmPBP2 were colocalized in the cells surrounding the olfactory receptor neurons (ORNs) expressing HarmOR13. Next, using two noninterfering binary expression tools, we heterologously expressed HarmPBP1, HarmPBP2 and HarmOR13 inDrosophilaT1 sensilla to validate the functional int...

Insect gustatory systems play important roles in food selection and feeding behaviors. In spite of the enormous progress in understanding gustation in Drosophila, for other insects one of the key elements in gustatory signaling, the gustatory receptor (GR), is still elusive. In this study, we report that fructose elicits behavioral and physiological responses in Helicoverpa armigera (Harm) to fructose and identify the gustatory receptor for this sugar. Using the proboscis extension reflex (PER) assays we found that females respond to fructose following stimulation of the distal part of the antenna, where we have identified contact chemosensilla tuned to fructose in tip recording experiments. We isolated three full-length cDNAs encoding candidate HarmGRs based on comparison with orthologous GR sequences in Heliothis virescens and functionally characterized the responses of HarmGR4 to 15 chemicals when this receptor was expressed in Xenopus oocytes with two-electrode voltage-clamp rec...

A new neuropeptide hormone was isolated from the corpora cardiaca of the periodical cicadas, Magicicada species. Primary structure of the peptide as determined by a combination of automated Edman degradation after enzymatic deblocking with pyroglutamate aminopeptidase and mass spectrometery is: pGlu-VaI-Asn-Phe-Ser-Pro-Ser-Trp-Giy-Asn-NHz. Synthetic peptide assayed in the green stink bug Nezara viridula caused a 112% increase in hemolymph lipids at a dose of 0.625 pmol, and a 67% increase in hemolymph carbohydrates at a dose of 2.5 pmol. Based on these results we designate this peptide, a first from order Homoptera, as Magicicada species-adipokinetic hormone (Mcsp-AKH). Adipokinetic hormone Neuropeptide Corpora cardiaca Lipid mobilization Periodical cicada Magicicada species Nezara viridula

Olfaction is crucial for short distance host location and pheromone detection by insects. Complexes of olfactory receptors (ORs) are composed of odor-specific ORs and OR co-receptors (Orco). Orcos are widely co-expressed with odor-specific ORs and are conserved across insect taxa. A number of Orco orthologs have been studied to date, although none has been identified in cereal aphids. In this study, an Orco gene ortholog was cloned from the grain aphid, Sitobion avenae, and named "SaveOrco"; RNA interference (RNAi) reduced the expression of SaveOrco to 34.11% in aphids, resulting in weaker EAG (electroantennogram) responses to plant volatiles (Z-3-hexene-1-ol; methyl salicylate, MeSA) and aphid alarm pheromone (E-β-farnesene, EBF). Aphid wing differentiation induced by EBF was investigated in both RNAi treated and untreated aphids. EBF induced production of winged aphids in both pre-natal and post-natal periods in untreated aphids, but no such induction was observed in the...

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B . subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B . subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The cDNA corresponding to an inhibitor of apoptosis (IAP) from the Egyptian armyworm, Spodoptera littoralis, was cloned by RT-PCR. Sequence analysis showed that the IAP of S. littoralis (SlIAP) contains two baculoviral IAP repeat (BIR) motifs, followed by a RING finger, an organization which is very similar to that of other lepidopteran IAPs. SlIAP mRNA was detected in ovary, testis, salivary gland, fat body, epidermis, brain and midgut of S. littoralis. During the last larval instar, prepupal and pupal stages, brain mRNA levels remained approximately constant, whereas those of midgut showed a large peak centred in the prepupal stage. Midgut morphology changed during metamorphosis from a semi-transparent, cylindrical structure in last instar larvae to a brownish globular mass in pupae. TUNEL assays, LysoTracker staining and caspase-3 immunohistochemistry, indicated that programmed cell death in midgut starts actively at the onset of pupation process, coinciding with the dramatic decrease of SlIAP mRNA levels observed at the same time.

Metabolic studies on insect allatostatins have suggested that the dipeptide Leu-Tyr may be a target for endopeptidases. In order to increase resistance to degradation, methyleneamino ⌿[CH 2 NH] and ketomethylene ⌿[COCH 2 ] peptide bond surrogates have been introduced at the position Leu 3 -Tyr 4 of the allatostatin Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide (BLAST-2), and Leu 3 -Phe 4 of [Phe 4 ]BLAST-2, respectively. Assays of inhibition of juvenile hormone (JH) synthesis in vitro by corpora allata from the cockroach Blattella germanica showed that both analogues were similarly active to the respective model peptides. The methyleneamino analogue was further tested in vivo as an inhibitor of JH synthesis, and in vivo and in vitro as an inhibitor of vitellogenin production by the fat body of B. germanica. The analogue was less active than BLAST-2 when tested in vitro, but more active than it when tested in vivo.

Allatectomy showed that Juvenile Hormone (JH) induced the accumulation of proteins in the accessory reproductive glands (ARC) of male Blattella germanica. During mating, the formation of the spermatophore and its transfer to the female was observed, and a selective depletion of most ARG proteins occurred in parallel, as shown by electrophoretic studies carried out at selected moments of the process. The synthetic activity of corpora allata incubated in vitro, measured at the same times, indicated that JH production is inhibited during the formation and transfer of the spermatophore. It is suggested that this inhibition may be necessary to start a new cycle of ARG maturation. @ 1996 LVliey-Liss, Inc.

Studies have shown that the East African mosquito strain, Culex pipiens fatigans, is attracted to a synthetic oviposition attractant pheromone in the laboratory. Chemical analysis has shown that the main volatile component of the apical droplets is ( -)-(5R,6R) erythro-6-acetoxy-5-hexadecanolide. In contrast to the lack of response in the Sri Lankan strain of the same mosquito, the East African strain shows remarkable similarity in attraction response to the West African, American and Japanese strains of C. p. fatigans.

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Instant and refrigerated acid soaking are commonly used in cocoon production to prevent or break diapause, and provide developable silkworm eggs for sericulture, while their mechanisms have not been fully understood. This study aims to investigate the mechanisms by which hydrochloric acid (HCl) or dimethyl sulfoxide (DMSO) promotes embryonic development in silkworm Bombyx mori, focusing on the chloride ion (Cl-) related gene expression profiles. Our results revealed that the HCl treatment of up to 6 min enhanced hatchability in freshly picked and cold-stored eggs, whereas a slight decrease in hatchability was observed in those treated with DMSO for 40 min. The genes encoding chloride ion channel proteins were cloned and transcriptionally analyzed by quantitative real-time PCR in B. mori 932 strain. Bioinformatics analysis showed that BmCLIC, BmGluCl, and Bmγ-Cl mainly clustered with Lepidopteran insect homologs, while there existed a relatively low conservation from B. mori to mammals. We further explored the mRNA levels of BmCLIC, BmGluCl, and Bmγ-Cl in different egg processing conditions and embryonic developmental stages following HCl or DMSO treatments. Our results revealed that the mRNA expression profiles of BmCLIC, BmGluCl, and Bmγ-Cl increased in 20 h post-egg oviposition and refrigerated eggs after acid soaking. Moreover, the relative expression level of BmCLIC and BmGluCl gene was the highest on the first day, and maximum relative expression level of Bmγ-Cl gene presented on the second day in diapause eggs after treatment, and then it showed diversity. The expression profiles of the candidate genes seem like the same trend of changes during the development of eggs treated with instant acid soaking or DMSO treatment. In general, in the production practice of soaking silkworm eggs with HCl or DMSO, Cl-channel is likely to play an important subsidiary role to promot the relieving of diapause. 1 | Introduction Diapause is a unique phase of insect growth and development that delays insect embryonic or pupae development to cope with changing climatic conditions and environments. It can occur in the egg, larval, pupal, or adult stages of insects undergoing complete metamorphosiswith each stage having distinct regulatory mechanisms. As is well known, hormones are important intrinsic factors in regulating the growth and development of Bombyx mori, which mainly include diapause hormones (DH, Isobe, Hasegawa, and Goto 1975; Imai et al. 1991), juvenile hormones (JH, Riddiford and

The olfactory system of insects is important for behavioral activities as it recognizes internal and external volatile stimuli in the environment. Insect odorant degrading enzymes (ODEs), including antennal-specific carboxylesterases (CXEs), are known to degrade redundant odorant molecules or to hydrolyze important olfactory sex pheromone components and plant volatiles. Compared to many well-studied Type-I sex pheromone-producing lepidopteran species, the molecular mechanisms of the olfactory system of Type-II sex pheromone-producing Hyphantria cunea (Drury) remain poorly understood. In the current study, we first identified a total of ten CXE genes based on our previous H. unea antennal transcriptomic data. We constructed a phylogenetic tree to evaluate the relationship of HcunCXEs with other insects’ CXEs, and used quantitative PCR to investigate the gene expression of H. cunea CXEs (HcunCXEs). Our results indicate that HcunCXEs are highly expressed in antennae, legs and wings, su...

E-β-Farnesene, the alarm pheromone of many aphid species, and E-β-caryophyllene are considered as two sesquiterpenes attractive for aphid predators, among which Syrphidae species. Both compounds were formulated in alginate gel beads as slow-release devices in a biological control approach against aphids. Semiochemical diffusion from beads was studied in the laboratory according to abiotic parameters. Efficiency of formulations as hoverfly attractant was demonstrated in field experiments from June to August 2009. The diffusion of semiochemicals from alginate bead formulations was principally limited by high values (>85%) of relative humidity in the air. Temperature also impacts the release of volatile compounds. In field experiments, these two abiotic factors were supposed to highly condition the trapping of hoverflies. These field trappings demonstrated the efficiency of both semiochemical formulations compared to control (formulation without semiochemical) to catch females of Syrphidae during three months. This research proved the efficiency of semiochemical alginate formulations as potential biological control tool to attract aphid predators.

The Phasmatodea (stick insects) have multiple, endogenous, highly expressed copies of glycoside hydrolase family 9 (GH9) genes. The purpose for retaining so many was unknown. We cloned and expressed the enzymes in transfected insect cell lines, and tested the individual proteins against different plant cell wall component poly-and oligosaccharides. Nearly all isolated enzymes were active against carboxymethylcellulose, however most could also degrade glucomannan, and some also either xylan or xyloglucan. The latter two enzyme groups were each monophyletic, suggesting the evolution of these novel substrate specificities in an early ancestor of the order. These are respectively the first, true, cellulase-xylanases in the GH9 family and the first xyloglucanases of any kind, let alone bifunctional cellulase-xyloglucanases, to be discovered in Metazoa. Phasmatodea gut extracts could degrade multiple plant cell wall components fully into sugar monomers, suggesting that enzymatic breakdown of plant cell walls by the entire Phasmatodea digestome may contribute to the Phasmatodea nutritional budget. The duplication and neofunctionalization of GH9s in the ancestral Phasmatodea may have enabled them to specialize as folivores and diverge from their omnivorous ancestors. The structural changes enabling these unprecedented activities in the cellulases require further study.

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22%. The cDNAs encompassed 11 trypsinlike sequences classifiable into three families and 3 chymotrypsin-like sequences belonging to a single family. Using a combination of 5´ and 3´ RACE the full length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine genes existed in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse Northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors SKTI and BTCI, expression of one of the trypsin-like sequences was up-regulated while expression of the two chymotrypsin-like sequences was down-regulated. However, these effects were compensative and the overall expression of the four serine proteinase genes decreased little.

Hylobius abietis (Linnaeus), or large pine weevil (Coleoptera, Curculionidae), is a pest of European coniferous forests. In order to gain understanding of the functional physiology of this species, we have assembled a de novo transcriptome of H. abietis, from sequence data obtained by Next Generation Sequencing. In particular, we have identified genes encoding neuropeptides, peptide hormones and their putative G-protein coupled receptors (GPCRs) to gain insights into neuropeptide-modulated processes. The transcriptome was assembled de novo from pooled paired-end, sequence reads obtained from RNA from whole adults, gut and central nervous system tissue samples. Data analysis was performed on the transcripts obtained from the assembly including, annotation, gene ontology and functional assignment as well as transcriptome completeness assessment and KEGG pathway analysis. Pipelines were created using Bioinformatics tools and techniques for prediction and identification of neuropeptides and neuropeptide receptors. Peptidomic analysis was also carried out using a combination of MALDI-TOF as well as Q-Exactive Orbitrap mass spectrometry to confirm the identified neuropeptide. 41 putative neuropeptide families were identified in H. abietis, including Adipokinetic hormone (AKH), CAPA and DH31. Neuropeptide F, which has not been yet identified in the model beetle T. castaneum, was identified. Additionally, 24 putative neuropeptide and 9 leucine-rich repeat containing G protein coupled receptor-encoding transcripts were determined using both alignment as well as non-alignment methods. This information, submitted to the NCBI sequence read archive repository (SRA accession: SRP133355), can now be used to inform understanding of neuropeptide-modulated physiology and behaviour in H. abietis; and to develop specific neuropeptide-based tools for H. abietis control.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for ins...

Malpighian tubules of adult female yellow fever mosquitoes Aedes aegypti express three inward rectifier K + (Kir) channel subunits: AeKir1, AeKir2B and AeKir3. Here we 1) elucidate the cellular and membrane localization of these three channels in the Malpighian tubules, and 2) characterize the effects of small molecule inhibitors of AeKir1 and AeKir2B channels (VU compounds) on the transepithelial secretion of fluid and electrolytes and the electrophysiology of isolated Malpighian tubules. Using subunit-specific antibodies, we found that AeKir1 and AeKir2B localize exclusively to the basolateral membranes of stellate cells and principal cells, respectively; AeKir3 localizes within intracellular compartments of both principal and stellate cells. In isolated tubules bathed in a Ringer solution containing 34 mM K + , the peritubular application of VU590 (10 µM), a selective inhibitor of AeKir1, inhibited transepithelial fluid secretion 120 min later. The inhibition brings rates of transepithelial KCl and fluid secretion to 54% of the control without a change in transepithelial NaCl secretion. VU590 had no effect on the basolateral membrane voltage (V bl ) of principal cells, but it significantly reduced the cell input conductance (g in ) to values 63% of the control within ~90 min. In contrast, the peritubular application of VU625 (10 µM), an inhibitor of both AeKir1 and AeKir2B, started to inhibit transepithelial fluid secretion as early as 60 min later. At 120 min after treatment, VU625 was more efficacious than VU590, inhibiting transepithelial KCl and fluid secretion to ~35% of the control without a change in transepithelial NaCl secretion. Moreover, VU625 caused the V bl and g in of principal cells to respectively drop to values 62% and 56% of the control values within only ~30 min. Comparing the effects of VU590 with those of VU625 allowed us to estimate that AeKir1 and AeKir2B respectively contribute to 46% and 20% of the transepithelial K + secretion when the tubules are bathed in a Ringer solution containing 34 mM K + . Thus, we uncover an important role of AeKir1 and stellate cells in transepithelial K + transport under conditions of peritubular K + challenge. The physiological role of AeKir3 in intracellular membranes of both stellate and principal cells remains to be determined.

Epicuticular lipids in many terrestrial arthropods consist of vast numbers of polar and non-polar aliphatic compounds, which are mainly responsible for the water balance in these animals but can also affect conidia germination of entomopathogenic fungi. In this work the qualitative and quantitative profiles of cuticular fatty acids from three insect species differing in their susceptibility to fungal infection were studied. In an innovative approach, laser light scattering detection was coupled with HPLC in order to identify the non-chromophoric chemicals usually present in cuticular extracts. The acids identified contained from 5 to 20 carbon atoms in the alkyl chain and included unsaturated entities such as C 16:1 , C 18:1 , C 18:2 , C 18:3 and C 20:1 . There was a marked dominance of acids containing 16-18 carbon atoms. The relative contents of fatty acids in the extracted waxes varied from trace amounts to 44%. Cuticular fatty acids profile of Calliphora vicina (species resistant to fungal infection) significantly differs from profiles of Dendrolimus pini and Galleria mellonella (both species highly susceptible to fungal infection). The major difference is the presence of C 14:0 , C 16:1 and C 20:0 in the cuticle of C. vicina. These three fatty acids are absent in the cuticle of D. pini while G. mellonella cuticle contains their traces. The concentrations of four fatty acids dominating in the G. mellonella larval cuticle (C 16:0 , C 18:0 , C 18:1 and C 18:2 ) were found to fluctuate during the final larval instar and correlate with fluctuations in the susceptibility of larvae to fungal infection. The possible role of cuticular fatty acids in preventing fungal infection is discussed.

Dipeptidase activity in Rhynchosciara americana (Diptera: Sciaridae) is found mainly in midgut caeca cells. The caecal dipeptidase activity is partly soluble and partly membrane bound. Differential centrifugation of midgut caeca homogenates, followed by assays of enzyme markers and dipeptidase, suggest that soluble dipeptidase is cytosoHe or weakly associated with the cell glyeoealyx. Membranebound dipeptidase is Hkely a microvillar enzyme. Soluble dipeptidase activity is resolved by gel filtration and ion exchange chromatography into two enzymes (M, 63,000 and 73,000), which hydrolyze both Gly-Leu and Pro-Gly, although with different efficiency. The two enzymes also differ in their stability in the presence of EDTA and degree of inhibition by phenanthroline and aminoacyl hydroxamates. Dipeptidase inhibition by phenanthroline is reversed by dialysis. Membrane-bound dipeptidase activity was solubilized by Triton X-100 and papain. Density-gradient ultracentrifugation, gel filtration, and ion-exchange chromatography suggest that there is only one detergent (Mr 86,000) form of this enzyme, which is active on Gly-Leu and Pro-Gly. No activity upon Gly-Pro was found in R. americana midguts, whereas the weak activity observed upon carnosine is independent of the enzymes hydrolyzing Gly-Leu and Pro-Gly. Thus, R. americana midguts seem to have major soluble and membrane-bound dipeptide hydrolases (EC 3.4.13.11), which in contrast to the mammalian enzyme, are very active upon Pro-Gly. R. americana also has a minor carnosinase (EC 3.4.13.3).

Brush borders (microvilli) were isolated from Spodoptera frugiperda posterior midgut cells by using the cation differential precipitation technique. These microvilli preparations still contained some cytoskeleton elements visible by electron microscope. These elements are removed by Tris treatment followed by dilution and ultracentrifugation. Tris treatment solubilizes protein with M r values similar to some cytoskeleton proteins, and causes an increase in the specific activity of the marker enzyme (aminopeptidase) and in the lipid-protein ratio of the purified microvillar membrane. Microvilli preparations from anterior midgut cells seem to be free from cytoskeleton. This is supported by electron microscopy and by the absence, after Tris treatment, of changes in the specific activity of the marker enzyme and in the lipid-protein ratio. The cytoskeleton of these microvilli should assemble and disassemble to permit the passage of vesicles observed migrating through them. As a result, it is likely that the cytoskeleton is not rigid and is lost during microvilli preparation by differential precipitation. In comparison with other insects, S. frugiperda purified microvillar membranes have low cholesterol, carbohydrate and lipid-protein ratios. Because S. frugiperda midgut microvillar membranes almost lack intrinsic digestive enzymes, their low lipid-protein ratios might result from a large amount of carrier or other proteins.

Spodoptera frugiperda development and midgut/3-glucosidase activity are not affected by salicin and amygdalin present in their diets up to a concentration of at least 0.5%, suggesting that glycoside tolerance results from aglycone detoxification. Both salicin and amygdalin affect Diatraea saccharalis development, whereas only 0.5 % amygdalin causes a decrease in midgut /3-glucosidase activity. There are three electrophoretically-resolved/3-glucosidases in D. saccharalis midguts. /3-Glucosidases 1 and 3 are thought to hydrolyze amygdalin to glucose and prunasin, and/3-glucosidase 2 to hydrolyze prunasin to glucose and the cyanogenic mandelonitrile. A short (48-h) exposure to 0.5% amygdalin causes a decrease of/3-glucosidase 1 and 3, whereas a long exposure (whole larval stage) depresses/3-glucosidase 2 activity. This suggests that glycoside tolerance may result from reduction in the midgut aryl/3-glucosidase activity without affecting the glycosyl/3-glucosidase activity, which hydrolyzes cellobiose and other/3glycans.

Biochemical studies of mammalian synaptosomal nerve fractions indicate the existence of multiple transporter proteins important for the termination of synaptic transmission by each of several monoamines. In insects, however, data on monoamine uptake has been limited to the study of whole tissue preparations, making it unclear whether neuronal (as opposed to glial) uptake is a significant mechanism in the insect. The present experiments elucidate the difficulties that have limited the use of insect synaptosomai preparations for characterizing amine reuptake. Key procedural improvements, including the utilization of carrier protein for tracer separation and the use of receptor antagonists to decrease non-specific membrane binding are described. With these and other modifications, reproducible sodium-dependent and cocaine-inhibitable dopamine and octopamine uptake are described in synaptosomal-containing preparations from insect brain and ganglia. These studies therefore support the existence of specific Na+-dependent uptake mechanisms in insect neurons. Transporter Dopamine Octopamine Reuptake Monamine Synaptosome

EFSA workshop «Risk assessment considerations for RNAi-based GM plants» 4-5 June 2014 Brussels, Belgium Wheeler et al. 2001. The Phylogeny of the Extant Hexapod Orders Evolution of the Insects. Grimaldi & Engel Drosophilids: 0.3% Towards the end of Drosophila dictatorship Drosophilids represent 0.3% of total described insect species Papers on Drosophilids represent 71% of total papers published on insect functional genomics (PubMed, 1980-2007) Drosophilids Papers on Drosophilids Brussels City Hall Tower, 96 m Papers on Drosophila: 68 m Drosophila: 20 cm

Background & objectives: Increase in prevalence and intensity of insecticide-resistance in vectors of vector-borne diseases is a major threat to sustainable disease control; and, for their effective management, studies on resistance mechanisms are important to come out with suitable strategies. Esterases are major class of detoxification enzymes in mosquitoes, which confers protection against insecticides in causing resistance. This study was aimed at biochemical characterization of esterases responsible for malathion resistance in Anopheles stephensi mosquitoes, along with its validation through biochemical techniques and native-PAGE assays. Methods: Laboratory maintained susceptible and resistant An. stephensi mosquitoes were used for assessing the activity and effect of α-and β-esterases on malathion. Bioassay, synergist bioassay, biochemical assay and native-PAGE were employed to characterize the role of esterases in conferring malathion-resistance. Results: Notably significant (p < 0.0001) enhancement in α-and β-esterases activity was observed with 2-fold increase in resistant An. stephensi GOA compared to susceptible An. stephensi BB. native-PAGE depicted two major bands 'a' (R f = 0.80) and 'b' (R f = 0.72) in susceptible An. stephensi BB , while one intense band 'b' (R f = 0.72) was visible in resistant An. stephensi GOA . Inhibition assay revealed complete inhibition of α-and β-esterases activity in presence of 1 mM malathion in susceptible strain compared to observed partial inhibition in resistant strain on native-PAGE. This study provides a better understanding on the role of esterase enzyme (carboxylesterase) in conferring malathion-resistance in An. stephensi mosquitoes, as evident from the native-PAGE assay results. The study results could be used in characterizing the resistance mechanisms in vectors and for suggesting alternative chemical insecticide based resistance management strategies for effective vector-borne disease control.

Epoxyoctadecamonoenoic acids (EpOMEs) are epoxide derivatives of linoleic acid (9,12-octadecadienoic acid: LA). They are metabolized into dihydroxyoctadecamonoenoic acids (DiHOMEs) in mammals. Unlike in mammals where they act as adipokines or lipokines, EpOMEs act as immunosuppressants in insects. However, the functional link between EpOMEs and pro-immune mediators such as PGE2is not known. In addition, the physiological significance of DiHOMEs is not clear in insects. This study analyzed the physiological role of these C18 oxylipins using a lepidopteran insect pest,Spodoptera exigua. Immune challenge ofS. exiguarapidly upregulated the expression of the phospholipase A2gene to trigger C20 oxylipin biosynthesis, followed by the upregulation of genes encoding EpOME synthase (SE51385) and a soluble epoxide hydrolase (Se-sEH). The sequential gene expression resulted in the upregulations of the corresponding gene products such as PGE2, EpOMEs, and DiHOMEs. Interestingly, only PGE2injecti...

Outbreaks of the black army cutworm, Actebia fennica, are associated with recently burned sites, where larvae feed on early successional plants. In the present paper we show that smoke volatiles stimulate juvenile hormone biosynthesis in virgin females, resulting in a more rapid rate of oocyte maturation and a significant advance in the age of first calling (the release of the sex pheromone) compared to control females. The ecological implications of this physiological effect are discussed.

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltagegated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V 50,act ) from À17.3 mV (wild-type) to À13.1 and À13.5 mV respectively, whereas L1014S caused no shift in V 50,act but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (!1 nM), permethrin (!30 nM) or DDT (!1 mM) resulted in hyperpolarizing shifts in V 50,act . Deltamethrin, permethrin and DDT also produced "tail currents" with EC 50 s of 0.043, 0.40 and 65 mM and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V 50,act . We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.

Voltage-gated sodium channels (VGSCs) are a major target site for the action of pyrethroid insecticides and resistance to pyrethroids has been ascribed to mutations in the VGSC gene. VGSCs in insects are encoded by only one gene and their structural and functional diversity results from posttranscriptional modification, particularly, alternative splicing. Using whole cell patch clamping of neurons from pyrethroid susceptible (wild-type) and resistant strains (s-kdr) of housefly, Musca domestica, we have shown that the V50 for activation and steady state inactivation of sodium currents (INa+) is significantly depolarised in s-kdr neurons compared with wild-type and that 10nM deltamethrin significantly hyperpolarised both of these parameters in the neurons from susceptible but not s-kdr houseflies. Similarly, tail currents were more sensitive to deltamethrin in wild-type neurons (EC15 14.5 nM) than s-kdr (EC15 133 nM). We also found that in both strains INa+ are of two types: a strongly inactivating (to 6.8% of peak) current, and a more persistent (to 17.1 % of peak) current. Analysis of tail currents showed that the persistent current in both strains (wild-type EC15 5.84 nM) was more sensitive to deltamethrin than was the inactivating type (wild-type EC15 35.1 nM). It has been shown previously, that the presence of exon l in the Drosophila melanogaster VGSC gives rise to a more persistent INa+ than does the alternative splice variant containing exon k and we used PCR with housefly head cDNA to confirm the presence of the housefly orthologues of splice variants k and l. Their effect on deltamethrin sensitivity was determined by examining INa+ in Xenopus oocytes expressing either the k or l variants of the Drosophila para VGSC. Analysis of tail currents, in the presence of various concentrations of deltamethrin, showed that the l splice variant was significantly more sensitive (EC50 42nM) than the k splice variant (EC50 866nM). We conclude that in addition to the presence of point mutations, target site resistance to pyrethroids may involve the differential expression of splice variants.

Most temperate multivoltine insects enter diapause, a hormonally controlled developmental suspension, in response to seasonal photoperiodic and/or thermal cues. Some insect species exhibit maternal regulation of diapause in which developmental trajectories of the offspring are determined by mothers in response to environmental cues that the mother received. Although maternally regulated diapause is common among insects, the maternal endocrinological mechanisms are largely veiled. To approach this issue, we used the parasitic jewel wasp Nasonia vitripennis, which produces non-diapause-destined offspring under long days and diapause-destined offspring under short days or low temperatures. Comparative transcriptomics of these wasps revealed possible involvement of the juvenile hormone (JH) biosynthetic cascade in maternal diapause regulation. JH acid methyltransferase was typically downregulated in short-day wasps, and this was reflected by a reduction in haemolymph JH concentrations. RNAi targeted at jhamt reduced haemolymph JH concentration and induced wasps to produce diapause-destined offspring even under long days. In addition, topical application of JH suppressed the production of diapause-destined offspring under short days or low temperatures. These results indicate that diapause in N. vitripennis is determined by maternal jhamt expression and haemolymph JH concentration in response to day length. We, therefore, report a novel role for JH in insect seasonality.

Cry proteins from Bacillus thuringiensis (Bt) have been used to control insect pests either as formulated sprays or as in Bt-crops. However, field-evolved resistance to Bt proteins is threatening the long-term use of Bt products. The SeABCC2 locus has been genetically linked to resistance to a Bt bioinsecticide (Xentari TM ) in Spodoptera exigua (a mutation producing a truncated form of the transporter lacking an ATP binding domain was found in the resistant insects). Here, we investigated the role of SeABCC2 in the mode of action of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and two Cry1A-1Ca hybrids by expressing the receptor in Sf21 and HEK293T cell lines. Cell toxicity assays showed that Sf21 cells expressing SeABCC2 become susceptible to Cry1A proteins. HEK293T cells expressing the transporter were found susceptible to Cry1A proteins but not to Cry1Ca. The results with the Cry1A-1Ca hybrids suggest that domain II from Cry1Ab/c is crucial for the toxicity to Sf21 cells, whereas domain III from Cry1Aa/b is crucial for the toxicity to HEK293T cells. Binding assays showed that the Cry1Ac binding is of high affinity and specific to cells expressing the SeABCC2 transporter. Heterologous competition experiments support a model in which domain II of Cry1Ab/c has a common binding site in the SeABCC2 protein, whereas domain III of Cry1Aa/b binds to a different binding site in the SeABCC2 protein.