Showing papers in "Insect Molecular Biology in 2012"
TL;DR: It is shown that feeding DWV to larvae causes wing deformity in adult bees in the absence of varroa mites and decreases survival rates of adult bees relative to bees not fed DWV, which suggests that dsRNA is non‐toxic to larvae.
Abstract: Deformed wing virus (DWV) is a serious pathogen of the honey bee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In the present study we reduced DWV infection by feeding both first instar larvae and adult A. mellifera with a double-stranded (ds) RNA construct, DWV-dsRNA, which is specific to DWV in DWV-inoculated bees, by mixing it with their food. We showed that feeding DWV to larvae causes wing deformity in adult bees in the absence of varroa mites and decreases survival rates of adult bees relative to bees not fed DWV. Feeding larvae with DWV-dsRNA in advance of inoculation with virus reduced the DWV viral level and reduced wing deformity relative to larvae fed DWV or DWV with green fluorescent protein-dsRNA (probably a result of RNA silencing), but did not affect survival to the adult stage. Feeding DWV-dsRNA did not affect larval survival rates, which suggests that dsRNA is non-toxic to larvae. Feeding adult workers with DWV-dsRNA in advance of inoculation with virus increased their longevity and reduced DWV concentration relative to controls.
120 citations
TL;DR: There is a clear relationship between CSP gene transcription and host searching behaviour and genome annotation and phylogenetic analyses comparing G.’morsitans morsitans CSPs with those of other Diptera showed rapid evolution after speciation of mosquitoes.
Abstract: Chemosensory proteins (CSPs) are a class of soluble proteins present in high concentrations in the sensilla of insect antennae It has been proposed that they play an important role in insect olfaction by mediating interactions between odorants and odorant receptors Here we report, for the first time, the presence of five CSP genes in the tsetse fly Glossina morsitans morsitans, a major vector transmitting nagana in livestock Real-time quantitative reverse transcription PCR showed that three of the CSPs are expressed in antennae One of them, GmmCSP2, is transcribed at a very high level and could be involved in olfaction We also determined expression in the antennae of both males and females at different life stages and with different blood feeding regimes The transcription of GmmCSP2 was lower in male antennae than in females, with a sharp increase in 10-week-old flies, 48 h after a bloodmeal Thus there is a clear relationship between CSP gene transcription and host searching behaviour Genome annotation and phylogenetic analyses comparing G morsitans morsitans CSPs with those of other Diptera showed rapid evolution after speciation of mosquitoes
91 citations
TL;DR: New information is provided on the miRNA abundance of honey bees, and the understanding of miRNA function in the regulation of honey bee development is enhanced, according to next‐generation high‐throughput sequencing technology.
Abstract: imb_1135 297..303 MicroRNAs (miRNAs) are endogenous small noncoding RNAs regulating gene expression in animals and plants. To find some differentially expressed miRNAs that may be associated with age-dependent behavioural changes in honey bees (Apis mellifera), we applied next-generation high-throughput sequencing technology to detect small RNAs in nurses and foragers. Our results showed that both nurses and foragers had a complicated small RNA population, and the length of small RNAs varied, 22 nucleotides being the predominant length. Combining deep sequencing and bioinformatic analysis, we discovered that nine known miRNAs were significantly different between nurses and foragers (P < 0.01; absolute value of fold-change 1). Some of their target genes were related to neural function. Moreover, 67 novel miRNAs were identified in nurses and foragers. Ame-miR-31a and ame-miR-13b were further validated using quantitative reverse-transcription PCR assays. The present study provides new information on the miRNA abundance of honey bees, and enhances our understanding of miRNA function in the regulation of honey bee development.
77 citations
TL;DR: The lack of uniformity in the genes that responded to artificial selection as well as differences in the direction of their responses challenges the assumption that one or a few genes control permethrin metabolic resistance.
Abstract: Changes in gene expression before, during and after five generations of permethrin laboratory selection were monitored in six strains of Aedes aegypti: five F2–F3 collections from the Yucatan Peninsula of Mexico and one F2 from Iquitos, Peru. Three biological replicate lines were generated for each strain. The response to selection was measured as changes in the lethal and knockdown permethrin concentrations (LC50, KC50) and in the frequency of the Ile1,016 substitution in the voltage-gated sodium channel (para) gene. Changes in expression of 290 metabolic detoxification genes were measured using the ‘Aedes Detox’ microarray. Selection simultaneously increased the LC50, KC50 and Ile1,016 frequency. There was an inverse relationship between Ile1,016 frequency and the numbers of differentially transcribed genes. The Iquitos strain lacked the Ile1,016 allele and 51 genes were differentially transcribed after selection as compared with 10–18 genes in the Mexican strains. Very few of the same genes were differentially transcribed among field strains but 10 cytochrome P450 genes were upregulated in more than one strain. Laboratory adaptation to permethrin in Ae. aegypti is genetically complex and largely conditioned by geographic origin and pre-existing target site insensitivity in the para gene. The lack of uniformity in the genes that responded to artificial selection as well as differences in the direction of their responses challenges the assumption that one or a few genes control permethrin metabolic resistance. Attempts to identify one or a few metabolic genes that are predictably associated with permethrin adaptation may be futile.
77 citations
TL;DR: RNA interference‐mediated gene knockdown resulted in a loss of CO2 sensitivity in both male and female mosquitoes, suggesting that these two proteins, like the Drosophila orthologues, function as a heterodimer.
Abstract: Carbon dioxide (CO(2)) is an important long-range chemosensory cue used by blood-feeding female mosquitoes to find their hosts. The CO(2) receptor in Drosophila melanogaster was previously determined to be a heterodimer comprised of two gustatory receptor (Gr) proteins, DmGr21a and DmGr63a. In the mosquito Aedes aegypti, two putative orthologous genes, AaGr1 and AaGr3, were identified in the genome database, along with an apparent paralogue of AaGr1, AaGr2. In this study, RNA interference (RNAi)-mediated gene knockdown of either AaGr1 or AaGr3 resulted in a loss of CO(2) sensitivity in both male and female mosquitoes, suggesting that these two proteins, like the Drosophila orthologues, function as a heterodimer. RNAi-mediated knockdown of AaGr2 expression had no impact on CO(2) reception. All three Gr genes were expressed in the maxillary palps of both Ae. aegypti and the West Nile virus vector mosquito, Culex pipiens quinquefasciatus. Interestingly, expression of the two CO(2) receptor genes was not equivalent in the two sexes and the implications of differential sex expression of the CO(2) receptor in different species are discussed. The functional identification of the CO(2) receptor in a mosquito could prove invaluable in the strategic design of compounds that disrupt the mosquito's ability to find hosts.
75 citations
TL;DR: It is shown that in Nasonia vitripennis all Dnmt1 messenger RNAs andDnmt3 mRNA are maternally provided to the embryo and, of these, DnMT1a is essential during early embryogenesis, suggesting evolutionary conservation of the function of DNmt1 during embryogenesis.
Abstract: Although the role of DNA methylation in insect development is still poorly understood, the number and role of DNA methyltransferases in insects vary strongly between species. DNA methylation appears to be widely present among the social hymenoptera and functional studies in Apis have suggested a crucial role for de novo methylation in a wide variety of developmental processes. The sequencing of three parasitoid Nasonia genomes revealed the presence of three Dnmt1 (Dnmt1a, Dnmt1b and Dnmt1c) genes and one Dnmt2 and Dnmt3 gene, suggesting a role of DNA methylation in Nasonia development. In the present study we show that in Nasonia vitripennis all Dnmt1 messenger RNAs (mRNAs) and Dnmt3 mRNA are maternally provided to the embryo and, of these, Dnmt1a is essential during early embryogenesis. Lowering of maternal Dnmt1a mRNA results in embryonic lethality during the onset of gastrulation. This dependence on maternal Dnmt1a during embryogenesis in an organismal group outside the vertebrates, suggests evolutionary conservation of the function of Dnmt1 during embryogenesis.
70 citations
TL;DR: The identification of 37 putative P450s expressed in the antennae of the pest moth Spodoptera littoralis and the characterization of a redox partner, cytochrome P450 reductase (CPR) are reported, suggesting that the antennaes of insects are a key site for P450‐mediated metabolism of a large range of exogenous and endogenous molecules.
Abstract: Cytochrome P450 enzymes (P450s) are involved in many physiological functions in insects, such as the metabolism of signal molecules, adaptation to host plants and insecticide resistance. Several P450s have been reported in the olfactory organs of insects, the antennae, and have been proposed to play a role in odorant processing and/or xenobiotic metabolism. Despite recent transcriptomic analyses in several species, the diversity of antennal P450s in insects has not yet been investigated. Here, we report the identification of 37 putative P450s expressed in the antennae of the pest moth Spodoptera littoralis, as well as the characterization of a redox partner, cytochrome P450 reductase (CPR). Phylogenetic analysis revealed that S. littoralis P450s belong to four clades defined by their conservation with vertebrate P450s and their cellular localization. Interestingly, the CYP3 and CYP4 clans, which have been described to be mainly involved in the metabolism of plant compounds and xenobiotics, were largely predominant. More surprisingly, two P450s related to ecdysteroid metabolism were also identified. Expression patterns in adult and larval tissues were studied. Eight P450s appeared to be specific to the chemosensory organs, ie the antennae and proboscis, suggesting a specific role in odorant and tastant processing. Moreover, exposure of males to a plant odorant down-regulated the transcript level of CPR, revealing for the first time the regulation of this gene by odorants within insect antennae. This work suggests that the antennae of insects are a key site for P450-mediated metabolism of a large range of exogenous and endogenous molecules.
68 citations
TL;DR: It is determined that DNA from Wolbachia‐infected mosquito Aedes albopictus (Aa23) cells shows a higher amount of the base 8‐oxo‐deoxyguanosine, a marker of oxidative DNA damage, than DNA from uninfected cells, and that Wolbachian density is sensitive to redox balance in these flies.
Abstract: Molecular interactions between symbiotic bacteria and their animal hosts are, as yet, poorly understood The most widespread bacterial endosymbiont, Wolbachia pipientis, occurs in high density in testes of infected Drosophila simulans and causes cytoplasmic incompatibility (CI), a form of male-derived zygotic lethality Wolbachia grow and divide within host vacuoles that generate reactive oxygen species (ROS), which in turn stimulate the up-regulation of antioxidant enzymes These enzymes appear to protect the host from ROS-mediated damage, as there is no obvious fitness cost to Drosophila carrying Wolbachia infections We have now determined that DNA from Wolbachia-infected mosquito Aedes albopictus (Aa23) cells shows a higher amount of the base 8-oxo-deoxyguanosine, a marker of oxidative DNA damage, than DNA from uninfected cells, and that Wolbachia infection in D simulans is associated with an increase in DNA strand breaks in meiotic spermatocytes Feeding exogenous antioxidants to male and female D simulans dramatically increased Wolbachia numbers with no obvious effects on host fitness These results suggest that ROS-induced DNA damage in sperm nuclei may contribute to the modification characteristic of CI expression in Wolbachia-infected males and that Wolbachia density is sensitive to redox balance in these flies
64 citations
TL;DR: Using liquid chromatography‐mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphid producing sexual morphs under short‐day conditions than in aphids producing parthenogenetic morphs in response to short photoperiods.
Abstract: Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive-mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive-mode change. Using liquid chromatography-mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short-day conditions than in aphids producing parthenogenetic morphs under long-day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real-time quantitative reverse-transcription-PCR across the reproductive-mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short-day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short-days, leading to the production of sexual morphs.
62 citations
TL;DR: The study showed that cold acclimatization clearly decreases chill coma recovery times in both Drosophila montana and D. virilis, whereas rapid cold hardening did not have a significant effect.
Abstract: The importance of high and low temperature tolerance in adaptation to changing environmental conditions has evoked new interest in modulations in gene expression and metabolism linked with stress tolerance. We investigated the effects of rapid cold hardening and cold acclimatization on the chill coma recovery times of two Drosophila virilis group species, Drosophila montana and D. virilis, with different distributions and utilized a candidate gene approach to trace changes in their gene expression during and after the cold treatments. The study showed that cold acclimatization clearly decreases chill coma recovery times in both species, whereas rapid cold hardening did not have a significant effect. Microarray analysis revealed several genes showing expression changes during different stages of cold response. Amongst the 219 genes studied, two genes showed rather consistent expression changes: hsr-omega, which was up-regulated in both study species during cold acclimatization, and Eip71CD, which was down-regulated in nearly all of the cold treatments. In addition, 29 genes showed expression changes that were more treatment- and/or species specific. Overall, different stages of cold response elicited changes mainly in genes involved in heat shock response, circadian rhythm and metabolism.
61 citations
TL;DR: Temperature stress appears to have activated immunity before the pathogen elicited a response from the host, and this early activity prevented infection under stressful conditions in this insect.
Abstract: Environmental stresses are thought to be associated with increases in disease suceptibility, attributable to evolutionary trade-offs between the energy demands required to deal with stress vs pathogens. We compared the effects of temperature stress and pathogen exposure on the immune response of a solitary bee, Megachile rotundata. Using an oligonucleotide microarray with 125 genes (375 probes), we determined that both high and low temperatures increased the expression of immune response genes in M. rotundata and reduced levels of a disease called chalkbrood. In the absence of the pathogen, trypsin-like serine and pathogen recognition proteases were most highly expressed at the lowest rearing temperature (20°C), while immune response signalling pathways and melanization were highly expressed at the warmest temperature tested (35°C). In pathogen-exposed bees, immune response genes tended to be most highly expressed at moderate temperatures, where we also saw the greatest infection levels. Temperature stress appears to have activated immunity before the pathogen elicited a response from the host, and this early activity prevented infection under stressful conditions. In this insect, the trade-off in energetic costs associated with stress and infection may be partially avoided by the use of conserved responses that reduce the effects of both.
TL;DR: The assembly of the transcriptome was carried out in a manner that enabled identification of putative single nucleotide polymorphisms (SNPs) and alternative splices, and that provided expression estimates for various life history stages and for salivary tissue.
Abstract: The blow fly Lucilia sericata (Diptera: Calliphoridae) (Meigen) is a nonmodel organism with no reference genome that is associated with numerous areas of research spanning the ecological, evolutionary, medical, veterinary and forensic sciences. To facilitate scientific discovery in this species, the transcriptome was assembled from more than six billion bases of Illumina and twenty-one million bases of 454 sequence derived from embryonic, larval, pupal, adult and larval salivary gland libraries. The assembly was carried out in a manner that enabled identification of putative single nucleotide polymorphisms (SNPs) and alternative splices, and that provided expression estimates for various life history stages and for salivary tissue. The assembled transcriptome was also used to identify transcribed transposable elements in L. sericata. The results of this study will enable blow fly biologists, dipterists and comparative genomicists to more rapidly develop and test molecular and genetic hypotheses, especially those regarding blow fly development and salivary gland biology.
TL;DR: The results indicate that Hsp70 and small Hsps (sHsps) are probably the major players in midgut metamorphosis in S.’litura.
Abstract: Heat shock proteins (Hsps) are important chaperones, which are involved in various signal pathways and regulate lots of physiological processes. Early research suggested that some Hsps are involved in insect development. However, few studies have been carried out to explore the roles of Hsps, especially in larval-pupal metamorphosis. In the present study, 49 Hsp unigenes were identified in the Spodoptera litura transcriptome and their mRNA expression profiles during midgut metamorphosis were examined using a tag-based digital gene expression system. The genes with the most different levels of expression were then cloned and their expression patterns in midguts from sixth instar larvae to pupae were analysed using real time quantitative PCR. The responses of these genes to juvenile hormone (JH) and 20-hydroxyecdysone (20E) were also studied. The results showed that the mRNA levels of 22 Hsp unigenes changed significantly during midgut metamorphosis. Amongst these 22 unigenes, hsp70, hsp20.4 and hsp20.8 were the most up-regulated members, and hsp15.9, hsp19.3 and hsp22.0 were the most down-regulated ones. Further studies showed that hsp70, hsp20.4 and hsp20.8 were remarkably up-regulated by JH. In addition, 20E slightly increased the mRNA levels of both hsp20.4 and hsp20.8. However, hsp15.9, hsp19.3 and hsp22.0 did not respond to either JH or 20E. These results indicate that Hsp70 and small Hsps (sHsps) are probably the major players in midgut metamorphosis in S. litura. The current findings provide valuable insights into the roles of the Hsp superfamily in insect metamorphosis.
TL;DR: Germline transformation of the diamondback moth is reported, using the piggyBac transposable element, by embryo microinjection to permit the development of RIDL strains for use against this pest and facilitate the wider use of this species as a model organism for basic studies.
Abstract: The diamondback moth, Plutella xylostella, is one of the most economically important agricultural pests. The larvae of this moth cause damage by feeding on the foliage of cruciferous vegetables such as cabbage, broccoli, cauliflower and rapeseed. Control generally comprises chemical treatment; however, the diamondback moth is renowned for rapid development of resistance to pesticides. Other methods, such as biological control, have not been able to provide adequate protection. Germline transformation of pest insects has become available in recent years as an enabling technology for new genetics-based control methods, such as the Release of Insects carrying a Dominant Lethal (RIDL(®) ). In the present study, we report the first transformation of the diamondback moth, using the piggyBac transposable element, by embryo microinjection. In generating transgenic strains using four different constructs, the function of three regulatory sequences in this moth was demonstrated in driving expression of fluorescent proteins. The transformation rates achieved, 0.48-0.68%, are relatively low compared with those described in other Lepidoptera, but not prohibitive, and are likely to increase with experience. We anticipate that germline transformation of the diamondback moth will permit the development of RIDL strains for use against this pest and facilitate the wider use of this species as a model organism for basic studies.
TL;DR: Few differences exist, which supports the hypothesis that head and body lice are ecotypes of the same species, and one novel microRNA was predicted in both lice species.
Abstract: Head and body lice are both blood-feeding parasites of humans although only the body louse is a potent disease vector. In spite of numerous morphological and life history differences, head and body lice have recently been hypothesized to be ecotypes of the same species. We took a comparative genomics approach to measure nucleotide diversity by comparing expressed sequence tag data sets from head and body lice. A total of 10 771 body louse and 10 770 head louse transcripts were predicted from a combined assembly of Roche 454 and Illumina sequenced cDNAs from whole body tissues collected at all life stages and during pesticide exposure and bacterial infection treatments. Illumina reads mapped to the 10 775 draft body louse gene models from the whole genome assembly predicted nine presence/absence differences, but PCR confirmation resulted in a single gene difference. Read per million base pair estimates indicated that 14 genes showed significant differential expression between head and body lice under our treatment conditions. One novel microRNA was predicted in both lice species and 99% of the 544 transcripts from Candidatus riesia indicate that they share the same endosymbiont. Overall, few differences exist, which supports the hypothesis that these two organisms are ecotypes of the same species.
TL;DR: The isolation and characterization of a heat shock protein 70 (hsp70) gene, the hsp83 gene and two genes that encode small Hsps from the Australian sheep blowfly, Lucilia cuprina, a major agricultural pest are reported.
Abstract: In this study we report the isolation and characterization of a heat shock protein 70 (hsp70) gene, the hsp83 gene and two genes that encode small Hsps (Lchsp23 and Lchsp24) from the Australian sheep blowfly, Lucilia cuprina, a major agricultural pest. Phylogenetic analyses indicate that the LcHsp23 protein is the orthologue of Drosophila melanogaster Hsp23 and LcHsp24 is the orthologue of Sarcophaga crassipalpis Hsp23. Quantitative reverse-transcriptase PCR analysis showed that the basal level of Lchsp83 RNA is relatively high at all developmental stages and only moderately induced by heat shock. In contrast, Lchsp70 transcripts are present at low levels and strongly induced by heat shock at all stages. The basal levels of expression and degrees of heat induction of the Lchsp23 and Lchsp24 transcripts were more variable across the different developmental stages. Putative heat shock factor binding sites were identified in the Lchsp24, Lchsp70 and Lchsp83 gene promoters. The isolation of these hsp gene promoters will facilitate constitutive or conditional expression of a gene of interest in transgenic Lucilia.
TL;DR: The normalized EST library provides a comprehensive genetic resource of C. formosanus and will serve a diverse range of research areas, and represents one of the repositories of enzymes useful for physiological studies and applications in sugar‐based biofuel production.
Abstract: Coptotermes formosanus is one of the most destructive wood-feeding termites. To understand the molecular mechanisms that regulate the development of the termite, a normalized C. formosanus cDNA library was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. The sequencing of this library generated 131 636 expressed sequence tags (ESTs) and 25 939 assembled unigenes. The carbohydrate-active enzymes (CAZymes) revealed in this library were analysed in the present report. A total of 509 putative CAZymes were identified. Diverse cellulolytic enzymes were uncovered from both the host termite and from symbionts harboured by the termite, which were possibly the result of the high efficiency of cellulose utilization. CAZymes associated with trehalose biosynthetic and metabolic pathways were also identified, which are potential regulators of the physiological activities of trehalose, an important insect blood sugar. Representative CAZyme coding genes in glycoside hydrolase family 1 (GH1) were quantitatively analysed. The results showed that the five GH1 β-glucosidase genes were expressed differentially among different castes and one of them was female alate-specific. Overall, the normalized EST library provides a comprehensive genetic resource of C. formosanus and will serve a diverse range of research areas. The CAZymes represent one of the repositories of enzymes useful for physiological studies and applications in sugar-based biofuel production.
TL;DR: It is concluded that biogenic amine receptors are expressed in the ovaries and are likely to be directly influential in the regulation of worker sterility in honey bees.
Abstract: In the honey bee Apis mellifera loss of the queen from a colony induces increased levels of the biogenic amine dopamine in the brain of workers, and this elevation is correlated with ovary activation. In the present study we use real-time quantitative PCR to investigate expression of five biogenic amine receptor genes. We show that biogenic amine receptors are expressed in ovarian tissue, and that their expression is strongly influenced by the presence or absence of a queen in the colony. In contrast to the brain, where all three dopamine receptors are expressed, only two dopamine receptors are expressed in the ovaries, and their expression is strongly correlated with the reproductive status of workers. We conclude that biogenic amine receptors are expressed in the ovaries and are likely to be directly influential in the regulation of worker sterility in honey bees.
TL;DR: A strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene, the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species.
Abstract: Spiromesifen is a novel insecticide and is classed as a tetronic acid derivative. It targets the insects' acetyl-coenzyme A carboxylase (ACCase) enzyme, causing a reduction in lipid biosynthesis. At the time of this publication, there are no reports of resistance to this class of insecticides in insects although resistance has been observed in several mite species. The greenhouse whitefly Trialeurodes vaporariorum (Westwood) is a serious pest of protected vegetable and ornamental crops in temperate regions of the world and spiromesifen is widely used in its control. Mortality rates of UK and European populations of T. vaporariorum to spiromesifen were calculated and up to 26-fold resistance was found. We therefore sought to examine the molecular mechanism underlying spiromesifen resistance in this important pest. Pre-treatment with piperonyl butoxide did not synergize spiromesifen, suggesting a target-site resistance mechanism. The full length ACCase gene was sequenced for a range of T. vaporariorum strains and a strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene. A TaqMan allelic discrimination assay confirmed these findings. Although this resistance is not considered sufficient to compromise the field performance of spiromesifen, this association of E645K with resistance is the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species.
TL;DR: Structural comparison and phylogenetic analysis showed that GmAtg8 belong to the Atg8 family of ubiquitin‐like proteins, and expression of autophagy seems to precede apoptosis in the midgut transformation from larva to pupa and pupa to adult during metamorphosis.
Abstract: A 345 base pair cDNA encoding autophagy 8 (Atg8) of Galleria mellonella (GmAtg8) was cloned and sequenced The deduced protein was estimated to be 118 amino acids long Structural comparison and phylogenetic analysis showed that GmAtg8 belong to the Atg8 family of ubiquitin-like proteins It is predicted to contain four β-sheets and four α-helices It also contains a highly conserved glycine residue at the C-terminal, as well as highly conserved Phe77 and Ph79 at a recognition cleavage site of Atg4 and Tyr49, and Leu50 at a site for activation of the lipidated form of Atg8 by Atg7 and Atg3 The developmental expression profile demonstrated that GmAtg8 transcript and its protein products are expressed in such organs as the midgut, ovary, Malpighian tubules, fat body and silk gland In the midgut and silk gland, GmAtg8 transcript and its protein products increased during metamorphosis and under starvation, but decreased after re-feeding Expression of autophagy seems to precede apoptosis in the midgut transformation from larva to pupa and pupa to adult during metamorphosis Some waves overlap with apoptotic waves, particularly at early stages, but others are unique in terms of site and timing
TL;DR: It is shown that the transcription factor Grainy head and the steroid hormone ecdysone drive the production of two partially overlapping sets of cuticle factors, suggesting that at least two independently triggered pathways have evolved in parallel to cooperatively ensure the stereotypic implementation of the cuticle.
Abstract: The arthropod epidermis is an epithelium that deposits the apical cuticle, which is a stratified extracellular matrix (ECM) protecting the animal against pathogens, preventing dehydration and also serving as an exoskeleton Differentiation of the cuticle conceivably implies coordinated production, secretion and localization of its components The underlying molecular mechanisms are poorly explored In this work, we show that the transcription factor Grainy head and the steroid hormone ecdysone drive the production of two partially overlapping sets of cuticle factors Nevertheless, Grainy head is needed to modulate the expression of ecdysone signalling factors; the significance of this cross-talk is yet unclear In addition, we found that ecdysone signalling negatively regulates its own impact In conclusion, our findings suggest that at least two independently triggered pathways have evolved in parallel to cooperatively ensure the stereotypic implementation of the cuticle As Grainy head is also essential for epithelial differentiation in vertebrates, we speculate that it acts to decode the ancient skin programme common to all animals Full differentiation of the skin necessitates a second, complementing taxon-specific programme that requires its own decoder, which is represented by ecdysone in arthropods, whereas the vertebrate specific one remains to be identified
TL;DR: This work reports that OfCHSA from the lepidopteran Ostrinia furnacalis contains two alternative‐splicing exons, exons 2a and 2b and exons 19a and 19b, and finds that transcripts containing exon 2a were dominant during most developmental stages, including embryonic development, larval−larval moulting, the larval+pupal transition and pupal−adult metamorphosis.
Abstract: Insect chitin synthase is an essential enzyme involved in chitin biosynthesis in insects. Chitin synthase A (CHSA) is expressed in different insect tissues during different developmental stages. CHSA contains alternative-splicing exons that allow tissue- and development-specific chitin synthesis. Here, we report that OfCHSA from the lepidopteran Ostrinia furnacalis contains two alternative-splicing exons, exons 2a and 2b and exons 19a and 19b. Although four combinations of these exons are theoretically possible, we found that transcripts containing exon 2a were dominant during most developmental stages, including embryonic development, larval-larval moulting, the larval-pupal transition and pupal-adult metamorphosis. Unexpectedly, 2b-containing transcripts were much more responsive to 20-hydroxyecdysone regulation than 2a-containing ones, suggesting that although OfCHSA isoforms encoded by 2b-containing transcripts are normally expressed at very low levels, they play unique roles. Spliced exons 2a and 2b have also been observed in Bombyx mori; therefore, this work provides new insights into the regulation of insect chitin synthase, particularly in lepidopteran insects.
TL;DR: This study constructed an unprecedented four gigabase pair transcriptome of P. citri, which was assembled into 64 149 unique transcripts, the functions of which were annotated by five public databases and identified as representatives of potential involvement in the detoxification of xenobiotics.
Abstract: The citrus red mite, Panonychus citri, is known for its ability rapidly to evolve resistance to insecticides/acaricides and to adapt to hosts that produce toxins. In this study, we constructed an unprecedented four gigabase pair transcriptome of P. citri, which was assembled into 64 149 unique transcripts, the functions of which were annotated by five public databases. A total of 116 unique transcripts were identified as representatives of potential involvement in the detoxification of xenobiotics. Genes recorded to encoding insecticide/acaricide target proteins were also obtained from the P. citri transcriptome. In order to explore novel candidate genes potentially involved in the pesticide detoxification of P. citri, we also constructed digital gene expression libraries of short-term transcriptome responses of P. citri to pesticides, which resulted in the identification of 120 unique transcripts potentially associated with insecticide/acaricide detoxification. Our study will facilitate molecular research on pesticide resistance in citrus red mites, as well as in other phytophagous mites.
TL;DR: Analysis of the developmental expression of Corazonin and the corazonin receptor revealed that transcription of both follows a bimodal distribution, with highest mRNA levels in 2nd instar larvae and during the pupa to adult transition.
Abstract: Haemolymph flow in mosquitoes is primarily driven by the contraction of a dorsal vessel that is subdivided into an abdominal heart and a thoracic aorta. The factors that regulate mosquito heart contractions are not understood, but in other insects heart physiology is partially controlled by several neurohormones. One of these is corazonin, a neuropeptide initially discovered because of its cardioacceleratory activity in the cockroach Periplaneta americana. In the present study, we describe the corazonin gene and transcript structure in the mosquito Anopheles gambiae, characterize its developmental expression, and test its role in modulating heart physiology. We show that the A. gambiae corazonin gene encodes the most common form of the corazonin peptide ([Arg7]-corazonin) and that it is alternatively spliced, with the only difference between the transcripts occurring in the 5′ untranslated region. Analysis of the developmental expression of corazonin and the corazonin receptor revealed that transcription of both follows a bimodal distribution, with highest mRNA levels in 2nd instar larvae and during the pupa to adult transition. Finally, experiments where mosquitoes were injected with various doses of corazonin and experiments where the transcription of corazonin and the corazonin receptor were reduced by RNA interference failed to detect a significant role for this neuropeptide in modulating mosquito heart physiology.
TL;DR: It is shown that virgin females injected with Drosophila melanogaster SP, in addition to inhibition of pheromone production, also exhibited a suppression of calling behaviour and a significant reduction in the gene expression levels of the PBAN‐receptor.
Abstract: Female Helicoverpa armigera sex pheromone production is under the control of pheromone biosynthesis-activating neuropeptide (PBAN). After mating, females undergo suppression of sex pheromone production and enhanced oviposition as a result of the transfer of male-derived seminal peptides. In a previous study we identified a putative H. armigera sex-peptide receptor (HeaSP-R) and demonstrated a significant up-regulation in gene expression levels of this receptor in brains and pheromone glands of mated females, thereby implicating a regulatory role for sex peptide in the reproductive behaviour of H. armigera. In the present study, we show that virgin females injected with Drosophila melanogaster SP (DrmSP), in addition to inhibition of pheromone production, also exhibited a suppression of calling behaviour and a significant reduction in the gene expression levels of the PBAN-receptor. In addition, RNA interference (RNAi) silencing of the HeaSP-R expression by 50-60% prevented DrmSP-suppression of pheromone production and calling behaviour. Moreover, mated, silenced females failed to increase their oviposition rates as is normally observed in mated females, and their behaviour did not differ from that of virgin females. However, sex pheromone production by mated, silenced females remained low, comparable to mated, normal females, thereby indicating the probable involvement of additional factors in the suppression of sex pheromone production after mating.
TL;DR: The results indicate that cells that express either AgOBP48, Ag OBP1 or AgOR1 are housed together in distinct olfactory sensilla and that an interplay of the proteins may contribute to the specific responsiveness of the sensillum to distinct odorants.
Abstract: In the malaria mosquito Anopheles gambiae (Ag), olfaction plays a crucial role in various behaviours, most strikingly in the seeking of females after a blood meal. The first step of odorant recognition in antennal sensilla involves soluble odorant binding proteins (OBPs), which transfer odorous compounds to olfactory receptors (ORs) in the dendritic membrane of sensory neurons. A particular OBP subtype of the 'Plus-C' class, called AgOBP48, is abundantly transcribed in female antennae and partially down-regulated after a blood meal, suggesting a possible role in host detection. In the present study, we have identified the AgOBP48-expressing cells, explored their antennal topography and determined their position relative to cells that express the 'classic' AgOBP1, the AgOR co-receptor (AgOrco) and the receptor AgOR1. By means of two-colour whole-mount fluorescence in situ hybridization it was found that AgOBP48 was expressed in cells, which are closely associated with AgOrco-expressing sensory neurons. Furthermore, AgOBP48 was not expressed in the same cells as AgOBP1, but subpopulations of AgOBP48- and of AgOBP1-expressing cells were found closely associated and adjacent to sensory neurons expressing AgOR1. Together, the results indicate that cells that express either AgOBP48, AgOBP1 or AgOR1 are housed together in distinct olfactory sensilla and that an interplay of the proteins may contribute to the specific responsiveness of the sensillum to distinct odorants.
TL;DR: This short review aims to provide an update on current understanding of the proteins expressed by mosquito cells that mediate DENV binding as a prerequisite to DENV entry and replication.
Abstract: Dengue is the most important mosquito transmitted viral disease of humans worldwide. Despite intensive study over several decades, many of the fine details of the dengue virus (DENV) replication cycle remain unknown, although generally more is known about the phase of the replication cycle in mammalian cells as compared to the phase in mosquito cells. This results from a combination of less research emphasis on the mosquito stage, as well as fewer tools such as specific antibodies against mosquito proteins and insect informatics databases. The binding of a virus to a host cell is a first and critical stage in the infectious process and the mechanism and identity of cellular proteins involved in this process remains largely unknown. This short review aims to provide an update on our current understanding of the proteins expressed by mosquito cells that mediate DENV binding as a prerequisite to DENV entry and replication.
TL;DR: It is reported here that haemocytes play roles in fat body degradation by expressing a cysteine proteinase cathepsin L in the lepidopteran Helicoverpa armigera and it is suggested that cathep L is related to the transformation of granulocyte to macrogranulocytes to enter the fat body, and induce haemocyte apoptosis for further tissue degradation.
Abstract: Insect haemocytes are known to participate in innate immunity via the phagocytosis of pathogens. However, the function of haemocytes in tissue remodelling is less understood. We report here that haemocytes play roles in fat body degradation by expressing a cysteine proteinase cathepsin L in the lepidopteran Helicoverpa armigera. During metamorphosis, haemocytes undergo morphological changes by increasing their cell size and transforming their granulocytes into macrogranulocytes. The population of haemocytes also changes with increased number of granulocytes and decreased plasmatocytes. The expression level of cathepsin L in haemocytes, mainly in granulocytes and plasmatocytes, increases. The steroid hormone 20-hydroxyecdysone is able to promote the transformation of granulocytes into macrogranulocytes, and up-regulate the expression level of cathepsin L. The knock-down of the cathepsin L gene by RNA interference in haemocytes in vitro results in deficient granulocytes transforming into macrogranulocytes. Haemocytes are able to enter the decomposed fat body during metamorphosis. The over-expression of the proteinase domain C1A of cathepsin L results in cell apoptosis. Haemocytes, especially macrogranulocytes, undergo apoptosis and cathepsin L is released into haemolymph and the fat body during metamorphosis for fat body decomposition and degradation. These results suggest that cathepsin L is related to the transformation of granulocytes to macrogranulocytes to enter the fat body, and induce haemocyte apoptosis for further tissue degradation.
TL;DR: Results indicate that β‐glucosidase is an endogenous, midgut‐origin termite digestive enzyme that may have applications in understanding the mechanism of lignocellulose degradation in fungus‐growing termites.
Abstract: β-glucosidase from the midgut of the fungus-growing termite Macrotermes barneyi was first cloned and characterized to gain a better understanding of cellulolytic systems in fungus-growing termites. β-glucosidase activity was proven to present primarily in the midgut of M. barneyi and two β-glucosidases were partially purified from the midgut. Based on the N-terminus sequence of one of the β-glucosidases, a full-length cDNA fragment of 1708 bp was obtained. This sequence encodes a 493 amino acid protein belonging to glycoside hydrolase family 1. Quantitative real-time PCR analysis proved that the β-glucosidase gene was primarily expressed in the midgut. β-glucosidase was expressed heterologously and biochemically characterized. Results indicate that β-glucosidase is an endogenous, midgut-origin termite digestive enzyme. It may have applications in understanding the mechanism of lignocellulose degradation in fungus-growing termites.
TL;DR: Three‐dimensional structure modelling suggested that ClAChEs and ClSChE shared structural similarities, but had some differences in the residues forming the acyl pocket and oxyanion hole, which should provide valuable insights into the evolution and functional diversification of insect cholinesterase.
Abstract: We identified and characterized the full-length cDNA sequences encoding two acetylcholinesterases (ClAChE1 and ClAChE2) and a salivary gland-specific cholinesterase-like protein (ClSChE) from the common bed bug, Cimex lectularius. All three cholinesterase genes (Clac1, Clace2 and Clsce) have conserved motifs, including a catalytic triad, a choline-binding site and an acyl pocket. Phylogenetic analysis showed that ClAChE1 belongs to the insect AChE1 clade, whereas ClAChE2 belongs to the insect AChE2 clade. ClSChE was grouped into the clade containing all AChE1s, suggesting a paralogous relationship to ClAChE1. Transcription levels of Clace1 were higher than those of Clace2 in all tissues examined, including the central nervous system (CNS). In contrast, the Clsce transcript was not detected in the CNS but specifically found in the salivary gland at much higher levels (>3000-fold) than those of Clace1 and Clace2. Western blot analysis using anti-ClAChE antibodies, in conjunction with activity staining, revealed that ClAChE1 is more active than ClAChE2, whereas ClSChE has little enzyme activity. Three-dimensional structure modelling suggested that ClAChEs and ClSChE shared structural similarities, but had some differences in the residues forming the acyl pocket and oxyanion hole. The current findings should provide valuable insights into the evolution and functional diversification of insect cholinesterase.