Cuticular Genes Research Articles

Host-plant induced changes in microbial community structure and midgut gene expression in an invasive polyphage (Anoplophora glabripennis)

Polyphagous insect herbivores possess diverse mechanisms to overcome challenges of feeding in multiple plant species including, but not limited to, transcriptional plasticity and associations with obligate or facultative symbionts. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-feeder capable of developing on over 100 tree species and, like other polyphages, its genome contains amplifications of digestive and detoxification genes. This insect also possesses a diverse gut microbial community, which has the metabolic potential to augment digestive physiology. While the genomic repertoires of A. glabripennis and its microbial community have been studied previously, comparatively less is known about how the gut transcriptome and community change in response to feeding in different hosts. In this study, we show that feeding in two suitable hosts (Acer spp. and Populus nigra) altered the expression levels of multicopy genes linked to digestion and detoxification. However, feeding in a host with documented resistance (Populus tomentosa) induced changes in the transcriptome and community beyond what was observed in insects reared in P. nigra, including the downregulation of numerous β-glucosidases, odorant binding proteins, and juvenile hormone binding proteins, the upregulation of several cuticular genes, and the loss of one major bacterial family from the gut community.

Cuticle genes CpCPR63 and CpCPR47 may confer resistance to deltamethrin in Culex pipiens pallens.

Cuticular proteins (CPs) are implicated in insecticide resistance in mosquito populations. Here, we investigated the role of cuticular genes in regulation of insecticide resistance in Culex pipiens pallens. We identified two CpCPRs (CpCPR63 and CpCPR47) that exhibited higher transcript levels in pyrethroid-resistant strains than in susceptible strains. Mosquito mortality was increased after knockdown of CpCPR genes by dsRNA injection. The RNA interference experiment suggested an interaction between CpCPR63 and CpCPR47, as silencing of one gene resulted in decreased expression of the other. These findings revealed that CpCPRs may regulate pyrethroid resistance and could be used as a potential genetic marker to monitor pyrethroid resistance in mosquitoes.

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori.

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster

The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions known as corneal nipples. Although the morphology and function of the "moth-eye" structure, are relatively well studied, the mechanism of protrusion formation from cell-secreted substances is unknown. In Drosophila melanogaster, a compound eye consists of approximately 800 facets, the surface of which is formed by the corneal lens with nanoscale protrusions. In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila.

Chronic MeHg exposure modifies the histone H3K4me3 epigenetic landscape in Caenorhabditis elegans

Methylmercury (MeHg) is a persistent environmental pollutant that occurs in the food chain, at occupational sites, and via medical procedures. Exposure in humans and animal models results in renal, neuro, and reproductive toxicities. In this study, we demonstrate that chronic exposure to MeHg (10μM) causes epigenetic landscape modifications of histone H3K4 trimethylation (H3K4me3) marks in Caenorhabditis elegans using chromatin immuno-precipitation sequencing (ChIP-seq). The modifications correspond to the locations of 1467 genes with enhanced and 508 genes with reduced signals. Among enhanced genes are those encoding glutathione-S-transferases, lipocalin-related protein and a cuticular collagen. ChIP-seq enhancement of these genes was confirmed with increased mRNA expression levels revealed by qRT-PCR. Furthermore, we observed enhancement of H3K4me3 marks in these genes in animals exposed to MeHg in utero and assayed at L4 stage. In utero exposure enhanced marks without alterations in mRNA expression except for the lpr-5 gene. Finally, knockdown of lipocalin-related protein gene lpr-5, which is involved in intercellular signaling, and cuticular collagen gene dpy-7, structural component of the cuticle, by RNA interference (RNAi) resulted in increased lethality of animals after MeHg exposure. Our results provide new data on the epigenetic landscape changes elicited by MeHg exposure, as well as describe a unique model for studying in utero effects of heavy metals. Together, these findings may help to understand the toxicological effects of MeHg at the molecular level.

Gene expression changes in the tyrosine metabolic pathway regulate caste-specific cuticular pigmentation of termites

In social insects, all castes have characteristic phenotypes suitable for their own tasks and to engage in social behavior. The acquisition of caste-specific phenotypes was a key event in the course of social insect evolution. However, understanding of the genetic basis and the developmental mechanisms that produce these phenotypes is still very limited. In particular, termites normally possess more than two castes with specific phenotypes (i.e. workers, soldiers, and reproductives), but proximate developmental mechanisms are far from being fully understood. In this study, we focused on the pigmentation of the cuticle as a model trait for caste-specific phenotypes, during the molts of each caste; workers, soldiers, presoldiers (intermediate stage of soldiers), and alates (primary reproductives) in Zootermopsis nevadensis. Expression patterns of cuticular tanning genes (members of the tyrosine metabolic pathway) were different among each molt, and high expression levels of several “key genes” were observed during each caste differentiation. For the differentiation of castes with well-tanned cuticles (i.e. soldiers and alates), all focal genes except DDC in the former were highly expressed. On the other hand, high expression levels of yellow and aaNAT were observed during worker and presoldier molts, respectively, but most other genes in the pathway were expressed at low levels. RNA interference (RNAi) of these key genes affected caste-specific cuticular pigmentation, leading to soldiers with yellowish-white heads and pigmented mandibular tips, presoldiers with partly pigmented head cuticles, and alates with the yellow head capsules. These results suggest that the pigmentation of caste-specific cuticles is achieved by the regulation of gene expression in the tyrosine metabolic pathway.

De novo transcriptome assembly of the grapevine phylloxera allows identification of genes differentially expressed between leaf- and root-feeding forms.

BackgroundGrapevine phylloxera, an insect related to true aphids, is a major historic pest of viticulture only controlled through the selection of resistant rootstocks or through quarantine regulations where grapevine is cultivated own-rooted. Transcriptomic data could help understand the bases of its original life-traits, including a striking case of polyphenism, with forms feeding on roots and forms feeding in leaf-galls. Comparisons with true aphids (for which complete genomes have been sequenced) should also allow to link differences in life-traits of the two groups with changes in gene repertoires or shifts in patterns of expression.ResultsWe sequenced transcriptomes of the grapevine phylloxera (Illumina technology), choosing three life-stages (adults on roots or on leaf galls, and eggs) to cover a large catalogue of transcripts, and performed a de novo assembly. This resulted in 105,697 contigs, which were annotated: most contigs had a best blastx hit to the pea aphid (phylogenetically closest complete genome), while very few bacterial hits were recorded (except for Probionibacterium acnes). Coding sequences were predicted from this data set (17,372 sequences), revealing an extremely high AT-bias (at the third codon position). Differential expression (DE) analysis among root-feeding and gall-feeding showed that i) the root-feeding form displayed a much larger number of differentially expressed transcripts ii) root-feeding biased genes were enriched in some categories, for example cuticular proteins and genes associated with cell-cell signaling iii) leaf-galling-biased genes were enriched in genes associated with the nucleus and DNA-replication, suggesting a metabolism more oriented towards fast and active multiplication. We also identified a gene family with a very high expression level (copies totaling nearly 10 % of the reads) in the grapevine phylloxera (both in root and leaf galling forms), but usually expressed at very low levels in true aphids (except in sexual oviparous females). These transcripts thus appear to be associated with oviparity.ConclusionsOur study illustrated major intraspecific changes in transcriptome profiles, related with different life-styles (and the feeding on roots versus in leaf-galls). At a different scale, we could also illustrate one major shift in expression levels associated with changes in life-traits that occurred along evolution and that respectively characterize (strictly oviparous) grapevine phylloxera and (mostly viviparous) true aphids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2530-8) contains supplementary material, which is available to authorized users.

A shortening effect of KK-42 on the moult cycle of juvenile Macrobrachium nipponense (De Haan, 1849) (Decapoda, Palaemonidae)

The imidazole derivative KK-42 is frequently used as an insect growth regulator. However, the effect of KK-42 on prawn growth remains largely unknown. To explore the effect of KK-42 on the moult cycle of juvenileMacrobrachium nipponense(De Haan, 1849), 1.2-2.0 cm long prawns were collected from aquaculture grounds, and acclimated at 26 ± 1°C in running-water tanks for one week before the experiments. The prawns were soaked with KK-42 solution at a concentration of 1.95 × 10−4 mol/l (treatment group) or without KK-42 solution (control group) for 1 min. Thereafter, a part of the prawns was used to measure body growth rate and moult cycle, and the rest, being at intermoult stage (C) and premoult stage (D), were used to quantitatively analyse the mRNA level of the cuticular chitinase1 gene (Mnchi-1) and chitinase activity. The results showed that the average body weight of the treatment group was significantly higher than that of the controls. Meanwhile, the body growth rate increased more than 16% in the first two weeks after KK-42 treatment. The duration of the moulting period of the juvenile prawns was shortened by KK-42 treatment in the first two cycles among four consecutively-determined moult cycles, from 8.70 ± 1.07 and 9.81 ± 0.43 days to 6.93 ± 0.97 and 8.11 ± 1.20 days, respectively. The cuticularMnchi-1mRNA level as well as the chitinase activity at stage C showed an over 10- and 2-fold increase, respectively, after KK-42 treatment. However, at stage D, only amodestrise was observed at 12 h (). The results suggest that KK-42 can significantly up-regulate the cuticularMnchi-1gene expression and chitinase activity of the prawns at stage C, which may be one of the potential mechanisms of producing the KK-42-shortened moult cycle for juvenileM. nipponense.

Parallel molecular routes to cold adaptation in eight genera of New Zealand stick insects.

The acquisition of physiological strategies to tolerate novel thermal conditions allows organisms to exploit new environments. As a result, thermal tolerance is a key determinant of the global distribution of biodiversity, yet the constraints on its evolution are not well understood. Here we investigate parallel evolution of cold tolerance in New Zealand stick insects, an endemic radiation containing three montane-occurring species. Using a phylogeny constructed from 274 orthologous genes, we show that stick insects have independently colonized montane environments at least twice. We compare supercooling point and survival of internal ice formation among ten species from eight genera, and identify both freeze tolerance and freeze avoidance in separate montane lineages. Freeze tolerance is also verified in both lowland and montane populations of a single, geographically widespread, species. Transcriptome sequencing following cold shock identifies a set of structural cuticular genes that are both differentially regulated and under positive sequence selection in each species. However, while cuticular proteins in general are associated with cold shock across the phylogeny, the specific genes at play differ among species. Thus, while processes related to cuticular structure are consistently associated with adaptation for cold, this may not be the consequence of shared ancestral genetic constraints.

Analysis of cuticular wax constituents and genes that contribute to the formation of 'glossy Newhall', a spontaneous bud mutant from the wild-type 'Newhall' navel orange.

Navel orange (Citrus sinensis [L.] Osbeck) fruit surfaces contain substantial quantities of cuticular waxes, which have important eco-physiological roles, such as water retention and pathogen defense. The wax constituents of ripe navel orange have been studied in various reports, while the wax changes occurring during fruit development and the molecular mechanism underlying their biosynthesis/export have not been investigated. Recently, we reported a spontaneous bud mutant from the wild-type (WT) 'Newhall' Navel orange. This mutant displayed unusual glossy fruit peels and was named 'glossy Newhall' (MT). In this study, we compared the developmental profiles of the epicuticular and intracuticular waxes on the WT and MT fruit surfaces. The formation of epicuticular wax crystals on the navel orange surface was shown to be dependent on the accumulation of high amounts of aliphatic wax components with trace amounts of terpenoids. In sharp contrast, the underlying intracuticular wax layers have relatively low concentrations of aliphatic wax components but high concentrations of cyclic wax compounds, especially terpenoids at the late fruit developmental stages. Our work also showed that many genes that are involved in wax biosynthesis and export pathways were down-regulated in MT fruit peels, leading to a decrease in aliphatic wax component amounts and the loss of epicuticular wax crystals, ultimately causing the glossy phenotype of MT fruits.

Temporal Gene Expression Profiles of Pre Blood-Fed Adult Females Immediately Following Eclosion in the Southern House Mosquito Culex Quinquefasciatus.

Prior to acquisition of the first host blood meal, the anautogenous mosquito Culex quinquefasciatus requires a period of time in order to prepare for the blood feeding and, later, vitellogenesis. In the current study, we conducted whole transcriptome analyses of adult female Culex mosquitoes to identify genes that may be necessary for both taking of the blood meal, and processing of the blood meal in adult female mosquitoes Cx. quinquefasciatus. We examined temporal expression of genes for the periods of post eclosion and prior to the female freely taking a blood meal. We further evaluated the temporal expression of certain genes for the periods after the taking of a blood meal to identify genes that may be necessary for both the taking of the blood meal, and the processing of the blood meal. We found that adult females required a minimum of 48 h post-eclosion before they freely took their first blood meal. We hypothesized that gene expression signatures were altered in the mosquitoes before blood feeding in preparation for the acquisition of the blood meal through changes in multiple gene expression. To identify the genes involved in the acquisition of blood feeding, we quantified the gene expression levels of adult female Cx. quinquefasciatus using RNA Seq throughout a pre-blooding period from 2 to 72 h post eclosion at 12 h intervals. A total of 325 genes were determined to be differentially-expressed throughout the pre-blooding period, with the majority of differentially-expressed genes occurring between the 2 h and 12 h post-eclosion time points. Among the up-regulated genes were salivary proteins, cytochrome P450s, odorant-binding proteins, and proteases, while the majority of the down-regulated genes were hypothetical or cuticular genes. In addition, Trypsin was found to be up-regulated immediately following blood feeding, while trypsin and chymotrypsin were up-regulated at 48h and 60h post blood-feeding, respectively, suggesting that these proteases are likely involved in the digestion of the blood meal. Overall, this study reviewed multiple genes that might be involved in the adult female competency for blood meal acquisition in mosquitoes.

Use of an Activated Beta-Catenin to Identify Wnt Pathway Target Genes inCaenorhabditis elegans, Including a Subset of Collagen Genes Expressed in Late Larval Development

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin−dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Expression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfish Procambarus clarkii during premolt.

The rigid crustacean exoskeleton, the cuticle, is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression in the hypodermis underlying the cuticle of the carapace at seven time points along ~14 premolt days. Next generation sequencing was used to construct a multi-tissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysis revealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure.

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

BackgroundNutritional symbioses play a central role in insects’ adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology.ResultsWe used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch over to the late embryonic stages in pea aphid development.ConclusionsOur data show that, in the development of A. pisum, a specific host gene set regulates the biosynthetic pathways of amino acids, demonstrating how the regulation of gene expression enables an insect to control the production of metabolites crucial for its own development and symbiotic metabolism.

Functional genomic and phenotypic responses to desiccation in natural populations of a desert drosophilid.

We used whole-transcriptome microarrays to assess changes in gene expression and monitored mortality rates and epicuticular hydrocarbons (CHCs) in response to desiccation stress in four natural populations of Drosophila mojavensis from Baja California and mainland Mexico. Desiccation had the greatest effect on gene expression, followed by biogeographical variation at regional and population levels. Genes involved in environmental sensing and cuticular structure were up-regulated in dry conditions, while genes involved in transcription itself were down-regulated. Flies from Baja California had higher expression of reproductive and mitochondrial genes, suggesting that these populations have greater fecundity and higher metabolic rates. Host plant differences had a surprisingly minor effect on the transcriptome. In most cases, desiccation-caused mortality was greater in flies reared on fermenting cactus tissues than that on laboratory media. Water content of adult females and males was significantly different and was lower in Baja California males. Different groups of CHCs simultaneously increased and decreased in amounts due to desiccation exposure of 9 and 18 h and were population-specific and dependent on larval rearing substrates. Overall, we observed that changes in gene expression involved a coordinated response of behavioural, cuticular and metabolic genes. Together with differential expression of cuticular hydrocarbons, this study revealed some of the mechanisms that have allowed D. mojavensis to exploit its harsh desert conditions. Certainly, for D. mojavensis that uses different host plants, population-level understanding of responses to stressors associated with future climate change in desert regions must be evaluated across geographical and local ecological scales.

Proteome Response of Tribolium castaneum Larvae to Bacillus thuringiensis Toxin Producing Strains

Susceptibility of Tribolium castaneum (Tc) larvae was determined against spore-crystal mixtures of five coleopteran specific and one lepidopteran specific Bacillus thuringiensis Cry toxin producing strains and those containing the structurally unrelated Cry3Ba and Cry23Aa/Cry37Aa proteins were found toxic (LC50 values 13.53 and 6.30 µg spore-crystal mixture/µL flour disc, respectively). Using iTRAQ combined with LC-MS/MS allowed the discovery of seven novel differentially expressed proteins in early response of Tc larvae to the two active spore-crystal mixtures. Proteins showing a statistically significant change in treated larvae compared to non-intoxicated larvae fell into two major categories; up-regulated proteins were involved in host defense (odorant binding protein C12, apolipophorin-III and chemosensory protein 18) and down-regulated proteins were linked to metabolic pathways affecting larval metabolism and development (pyruvate dehydrogenase Eα subunit, cuticular protein, ribosomal protein L13a and apolipoprotein LI-II). Among increased proteins, Odorant binding protein C12 showed the highest change, 4-fold increase in both toxin treatments. The protein displayed amino acid sequence and structural homology to Tenebrio molitor 12 kDa hemolymph protein b precursor, a non-olfactory odorant binding protein. Analysis of mRNA expression and mortality assays in Odorant binding protein C12 silenced larvae were consistent with a general immune defense function of non-olfactory odorant binding proteins. Regarding down-regulated proteins, at the transcriptional level, pyruvate dehydrogenase and cuticular genes were decreased in Tc larvae exposed to the Cry3Ba producing strain compared to the Cry23Aa/Cry37Aa producing strain, which may contribute to the developmental arrest that we observed with larvae fed the Cry3Ba producing strain. Results demonstrated a distinct host transcriptional regulation depending upon the Cry toxin treatment. Knowledge on how insects respond to Bt intoxication will allow designing more effective management strategies for pest control.

Mass spectrometrical analysis of cuticular proteins from the wing of Hebemoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Although several insect cuticular genes and proteins are annotated and an arthropod cuticular database is available, mass spectrometrical data on cuticular proteins and their post-translational modifications are limited. Wings from Hebemoia glaucippe were analyzed by scanning electron microscopy or homogenized, proteins were extracted and run on 2DE. In-gel digestion was carried out by using trypsin, chymotrypsin and Asp-N and subsequently the resulting peptides and post-translational modifications were identified by ion trap tandem mass spectrometry (nano-LC-ESI-MS/MS; HCT). A complex wing skeleton and the cuticle of H. glaucippe were demonstrated. Cuticle protein 18.6, isoform A, pupal cuticle protein, cuticular protein CPR59A and two putative proteins, putative cuticular protein B2DBJ and putative cuticle protein CPG31 with two expression forms were identified. Two phosphorylation sites on the same peptide, T213 and S214, were identified on putative cuticle protein CPG31, quinone formation was observed at Y76 on cuticular protein CPR59A probably indicating the presence of post-translational modifications. The results may be relevant for the interpretation of mechanoelastic and physical properties of these proteins. Along with the extraordinary architecture the proteinaceous matrix is probably representing or allowing the unusual aerodynamic function of the butterfly wing. Moreover, the results may be important for mechanisms of insecticide and drought resistance.

A De Novo Expression Profiling of Anopheles funestus, Malaria Vector in Africa, Using 454 Pyrosequencing

Background Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.ResultsUsing a pool of life stages (pupae, larvae, adults: females and males) for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp). De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs) and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.ConclusionThis study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

Developmental and evolutionary basis for drought tolerance of the Anopheles gambiae embryo

During the evolution of the Diptera there is a dramatic modification of the embryonic ectoderm, whereby mosquitoes contain separate amnion and serosa lineages while higher flies such as Drosophila melanogaster contain a single amnioserosa. Whole-genome transcriptome assays were performed with isolated serosa from Anopheles gambiae embryos. These assays identified a large number of genes implicated in the production of the larval cuticle. In D. melanogaster, these genes are activated just once during embryogenesis, during late stages where they are used for the production of the larval cuticle. Evidence is presented that the serosal cells secrete a dedicated serosal cuticle, which protects A. gambiae embryos from desiccation. Detailed temporal microarray assays of mosquito gene expression profiles revealed that the cuticular genes display biphasic expression during A. gambiae embryogenesis, first in the serosa of early embryos and then again during late stages as seen in D. melanogaster. We discuss how evolutionary modifications in the well-defined dorsal–ventral patterning network led to the wholesale deployment of the cuticle biosynthesis pathway in early embryos of A. gambiae.

Expression of the cytochrome P450s, CYP6P3 and CYP6M2 are significantly elevated in multiple pyrethroid resistant populations of Anopheles gambiae s.s. from Southern Benin and Nigeria.

BackgroundInsecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. This is particularly true in Benin where pyrethroid resistance has been linked to the failure of insecticide treated bed nets. The role of mutations in the insecticide target sites in conferring resistance has been clearly established. In this study, the contribution of other potential resistance mechanisms was investigated in Anopheles gambiae s.s. from a number of localities in Southern Benin and Nigeria. The mosquitoes were sampled from a variety of breeding sites in a preliminary attempt to investigate the role of contamination of mosquito breeding sites in selecting for resistance in adult mosquitoes.ResultsAll mosquitoes sampled belonged to the M form of An. gambiae s.s. There were high levels of permethrin resistance in an agricultural area (Akron) and an urban area (Gbedjromede), low levels of resistance in mosquito samples from an oil contaminated site (Ojoo) and complete susceptibility in the rural Orogun location. The target site mutation kdrW was detected at high levels in two of the populations (Akron f = 0.86 and Gbedjromede f = 0.84) but was not detected in Ojoo or Orogun. Microarray analysis using the Anopheles gambiae detox chip identified two P450s, CYP6P3 and CYP6M2 up regulated in all three populations, the former was expressed at particularly high levels in the Akron (12.4-fold) and Ojoo (7.4-fold) populations compared to the susceptible population. Additional detoxification and redox genes were also over expressed in one or more populations including two cuticular pre-cursor genes which were elevated in two of the three resistant populations.ConclusionMultiple resistance mechanisms incurred in the different breeding sites contribute to resistance to permethrin in Benin. The cytochrome P450 genes, CYP6P3 and CYP6M2 are upregulated in all three resistant populations analysed. Several additional potential resistance mechanisms were also identified that warrant further investigation. Metabolic genes were over expressed irrespective of the presence of kdr, the latter resistance mechanism being absent in one resistant population. The discovery that mosquitoes collected from different types of breeding sites display differing profiles of metabolic genes at the adult stage may reflect the influence of a range of xenobiotics on selecting for resistance in mosquitoes.