Midgut Transcripts Research Articles

A multi-omics approach for understanding blood digestion dynamics in Ixodes scapularis and identification of anti-tick vaccine targets

Ixodes scapularis, the black-legged tick, is a major arthropod vector that transmits the causative agents of Lyme disease and several other pathogens of human significance. The tick midgut is the main tissue involved in blood acquisition and digestion and the first organ to have contact with pathogens ingested through the blood meal. Gene expression in the midgut before, during, and after a blood meal may vary in response to the physiological changes due to blood feeding. A systems biology approach based on RNA and protein sequencing was used to gain insight into the changes in tick midgut transcripts and proteins during blood ingestion (unfed and partially fed) and digestion (1-, 2-, 7-, and 14 days post detachment from the host) by the Ixodes scapularis female ticks. A total of 2,726 differentially expressed transcripts, and 449 proteins were identified across the time points. Genes involved in detoxification of xenobiotics, proteases, protease inhibitors, metabolism, and immunity were differentially expressed in response to blood feeding. Similarly, proteins corresponding to the same groups were also differentially expressed. Nine genes from major gene categories were chosen as potential vaccine candidates, and, using RNA interference, the effect of these gene knockdowns on tick biology was investigated. Knockdown of these genes had variable negative impacts on tick physiology, such as the inability to engorge fully and to produce eggs and increased mortality. These and additional gene targets provide opportunities to explore novel tick control strategies.

Blood-feeding adaptations and virome assessment of the poultry red mite Dermanyssus gallinae guided by RNA-seq

Dermanyssus gallinae is a blood-feeding mite that parasitises wild birds and farmed poultry. Its remarkably swift processing of blood, together with the capacity to blood-feed during most developmental stages, makes this mite a highly debilitating pest. To identify specific adaptations to digestion of a haemoglobin-rich diet, we constructed and compared transcriptomes from starved and blood-fed stages of the parasite and identified midgut-enriched transcripts. We noted that midgut transcripts encoding cysteine proteases were upregulated with a blood meal. Mapping the full proteolytic apparatus, we noted a reduction in the suite of cysteine proteases, missing homologues for Cathepsin B and C. We have further identified and phylogenetically analysed three distinct transcripts encoding vitellogenins that facilitate the reproductive capacity of the mites. We also fully mapped transcripts for haem biosynthesis and the ferritin-based system of iron storage and inter-tissue trafficking. Additionally, we identified transcripts encoding proteins implicated in immune signalling (Toll and IMD pathways) and activity (defensins and thioester-containing proteins), RNAi, and ion channelling (with targets for commercial acaricides such as Fluralaner, Fipronil, and Ivermectin). Viral sequences were filtered from the Illumina reads and we described, in part, the RNA-virome of D. gallinae with identification of a novel virus, Red mite quaranjavirus 1.

Transcriptional Analysis of Cotton Bollworm Strains with Different Genetic Mechanisms of Resistance and Their Response to Bacillus thuringiensis Cry1Ac Toxin.

Transgenic crops producing Bacillus thuringiensis (Bt) insecticidal proteins are grown widely for pest control, but the evolution of resistance in target pests could reduce their efficacy. Mutations in genes encoding cadherin, ABC transporter or tetraspanin were linked with resistance to Cry1Ac in several lepidopteran insects, including the cotton bollworm (Helicoverpa armigera), a worldwide agricultural pest. However, the detailed molecular mechanisms by which these mutations confer insect resistance to Cry1Ac remain largely unknown. In this study, we analyzed the midgut transcriptomes of a susceptible SCD strain and three SCD-derived Cry1Ac-resistant strains of H. armigera (SCD-r1, with a naturally occurring deletion mutation of cadherin; SCD-KI, with a knock-in T92C point mutation in tetraspanin; and C2/3-KO, with both ABCC2 and ABCC3 knocked out). Evaluation of midgut transcript profiles of the four strains without Cry1Ac exposure identified many constitutively differentially expressed genes (DEGs) in the resistant SCD-r1 (n = 1355), SCD-KI (n = 1254) and C2/3-KO (n = 2055) strains. Analysis of DEGs in the midguts of each strain after Cry1Ac exposure revealed similar patterns of response to Cry1Ac in the SCD and SCD-r1 strains, but unique responses in the SCD-KI and C2/3-KO strains. Expression of midgut epithelium healing and defense-related genes was strongly induced by Cry1Ac intoxication in the SCD and SCD-r1 strains, while immune-related pattern recognition receptor and effector genes were highly expressed in the SCD-KI strain after Cry1Ac exposure. This study advances our knowledge of the transcriptomic basis for insect resistance to Bt toxins and provides a valuable resource for further molecular characterization of insect response to Cry1Ac toxin in H. armigera and other pest species.

Leishmania infection induces a limited differential gene expression in the sand fly midgut

BackgroundSand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages.ResultsThe combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1–4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics.ConclusionOverall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

Assessment of midgut enteroendocrine peptide complement in the honey bee, Apis mellifera

Peptides modulate physiological/behavioral control systems in all animals. In arthropods, midgut epithelial endocrine cells are one of the largest sources of these signaling agents. At present, little is known about the identity of the peptides that form arthropod midgut enteroendocrine peptidomes. While many techniques can be used for peptide structural identification, in silico transcriptome mining is one that has been used extensively for arthropod neuropeptidome prediction; this strategy has yet to be used for large-scale arthropod enteroendocrine peptide discovery. Here, a tissue-specific transcriptome was used to assess putative enteroendocrine peptide complement in the honey bee, Apis mellifera, midgut. Searches for transcripts encoding members of 42 peptide families were conducted, with evidence of expression for 15 groups found in the assembly: adipokinetic hormone, allatostatin A, allatostatin C, bursicon, CCHamide, CNMamide, diuretic hormone 31, diuretic hormone 44, insulin-like peptide, myosuppressin, neuropeptide F, pigment dispersing hormone, pyrokinin, short neuropeptide F, and tachykinin-related peptide. The proteins deduced from the midgut transcripts are identical in sequence, or nearly so, to those of Apis pre/preprohormones deposited previously into NCBI, providing increased confidence in the accuracy of the reported data. Seventy-five peptides were predicted from the deduced precursor proteins, 26 being members of known peptide families. Comparisons to previously published mass spectrometric data support the existence of many of the predicted Apis peptides. This study is the first prediction of an arthropod midgut peptidome using transcriptomics, and provides a powerful new resource for investigating enteroendocrine peptide signaling within/from the Apis midgut, a species of significant ecological/economic importance.

An insight into the sialome, mialome and virome of the horn fly, Haematobia irritans

BackgroundThe horn fly (Haematobia irritans) is an obligate blood feeder that causes considerable economic losses in livestock industries worldwide. The control of this cattle pest is mainly based on insecticides; unfortunately, in many regions, horn flies have developed resistance. Vaccines or biological control have been proposed as alternative control methods, but the available information about the biology or physiology of this parasite is rather scarce.ResultsWe present a comprehensive description of the salivary and midgut transcriptomes of the horn fly (Haematobia irritans), using deep sequencing achieved by the Illumina protocol, as well as exploring the virome of this fly. Comparison of the two transcriptomes allow for identification of uniquely salivary or uniquely midgut transcripts, as identified by statistically differential transcript expression at a level of 16 x or more. In addition, we provide genomic highlights and phylogenetic insights of Haematobia irritans Nora virus and present evidence of a novel densovirus, both associated to midgut libraries of H. irritans.ConclusionsWe provide a catalog of protein sequences associated with the salivary glands and midgut of the horn fly that will be useful for vaccine design. Additionally, we discover two midgut-associated viruses that infect these flies in nature. Future studies should address the prevalence, biological effects and life cycles of these viruses, which could eventually lead to translational work oriented to the control of this economically important cattle pest.

Tsetse fly (Glossina pallidipes) midgut responses to Trypanosoma brucei challenge

BackgroundTsetse flies (Glossina spp.) are the prominent vector of African trypanosome parasites (Trypanosoma spp.) in sub-Saharan Africa, and Glossina pallidipes is the most widely distributed species in Kenya. This species displays strong resistance to infection by parasites, which are typically eliminated in the midgut shortly after acquisition from the mammalian host. Although extensive molecular information on immunity for the related species Glossina morsitans morsitans exists, similar information is scarce for G. pallidipes.MethodsTo determine temporal transcriptional responses of G. pallidipes to Trypanosoma brucei brucei challenge, we conducted Illumina based RNA-seq on midgut organ and carcass from teneral females G. pallidipes at 24 and 48 h post-challenge (hpc) with T. b. brucei relative to their respective controls that received normal blood meals (without the parasite). We used a suite of bioinformatics tools to determine differentially expressed and enriched transcripts between and among tissues, and to identify expanded transcripts in G. pallidipes relative to their orthologs G. m. morsitans.ResultsMidgut transcripts induced at 24 hpc encoded proteins were associated with lipid remodelling, proteolysis, collagen metabolism, apoptosis, and cell growth. Midgut transcripts induced at 48 hpc encoded proteins linked to embryonic growth and development, serine endopeptidases and proteosomal degradation of the target protein, mRNA translation and neuronal development. Temporal expression of immune responsive transcripts at 48 relative to 24 hpc was pronounced, indicative of a gradual induction of host immune responses the following challenge. We also searched for G. m. morsitans orthologous groups that may have experienced expansions in the G. pallidipes genome. We identified ten expanded groups in G. pallidipes with putative immunity-related functions, which may play a role in the higher refractoriness exhibited by this species.ConclusionsThere appears to be a lack of strong immune responses elicited by gut epithelia of teneral adults. This in combination with a compromised peritrophic matrix at this stage during the initial phase of T. b. brucei challenge may facilitate the increased parasite infection establishment noted in teneral flies relative to older adults. Although teneral flies are more susceptible than older adults, the majority of tenerals are still able to eliminate parasite infections. Hence, robust responses elicited at a later time point, such as 72 hpc, may clear parasite infections from the majority of flies. The expanded G. m. morsitans orthologous groups in G. pallidipes may also be functionally associated with the enhanced refractoriness to trypanosome infections reported in G. pallidipes relative to G. m. morsitans.

De novo assembly and analysis of midgut transcriptome of Haemaphysalis flava and identification of genes involved in blood digestion, feeding and defending from pathogens

Bioactive components in the midgut of ticks play a key role in tick blood digestion, feeding and pathogen transmission. The study of protein and gene targets in midgut provides opportunities to explore novel tick control strategies. Only a few nucleotide sequences are available in public databases for Haemaphysalis flava, an important disease vector for humans and animals. Knowledge of the process of blood digestion by the ticks and protein expression in the digestive tract is limited. Here, we utilize high-throughput sequencing to characterize the midgut transcriptome of fully engorged (FE, average length of 10mm) and partially engorged (PE, average length of 5mm) female H. flava. 6.8GB and 8.3GB of high-quality sequence data were obtained using Illumina sequencing technology. 54,357,490 and 66,116,050 reads were finally assembled into 76,556 unigenes with mean length of 704bp. The transcripts involved in blood meal digestion were classified into eight large categories, including peptidase inhibitor, peptidase (serine-, metallo-, cysteine-, aspartic-peptidase), phospholipase, carbohydrate digestion/hydrolases, lipid binding, immunity-related proteins, iron/heme metabolism and secreted proteins. A total of 5508 differentially expressed genes (DEGs) were identified between FE and PE. To confirm the DEG results, ten genes involved in blood digestion, feeding and defending from pathogens, were validated using qPCR. Our results not only contribute to better understanding of the changes in midgut transcript expression during different blood feeding stages, but also provide a valuable resource for identifying targets for future tick control studies.

Transcriptional analysis of susceptible and resistant European corn borer strains and their response to Cry1F protoxin

BackgroundDespite a number of recent reports of insect resistance to transgenic crops expressing insecticidal toxins from Bacillus thuringiensis (Bt), little is known about the mechanism of resistance to these toxins. The purpose of this study is to identify genes associated with the mechanism of Cry1F toxin resistance in European corn borer (Ostrinia nubilalis Hübner). For this, we compared the global transcriptomic response of laboratory selected resistant and susceptible O. nubilalis strain to Cry1F toxin. We further identified constitutive transcriptional differences between the two strains.ResultsAn O. nubilalis midgut transcriptome of 36,125 transcripts was assembled de novo from 106 million Illumina HiSeq and Roche 454 reads and used as a reference for estimation of differential gene expression analysis. Evaluation of gene expression profiles of midgut tissues from the Cry1F susceptible and resistant strains after toxin exposure identified a suite of genes that responded to the toxin in the susceptible strain (n = 1,654), but almost 20-fold fewer in the resistant strain (n = 84). A total of 5,455 midgut transcripts showed significant constitutive expression differences between Cry1F susceptible and resistant strains. Transcripts coding for previously identified Cry toxin receptors, cadherin and alkaline phosphatase and proteases were also differentially expressed in the midgut of the susceptible and resistant strains.ConclusionsOur current study provides a valuable resource for further molecular characterization of Bt resistance and insect response to Cry1F toxin in O. nubilalis and other pest species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1751-6) contains supplementary material, which is available to authorized users.

Unravelling the Evolution of the Allatostatin-Type A, KISS and Galanin Peptide-Receptor Gene Families in Bilaterians: Insights from Anopheles Mosquitoes.

Allatostatin type A receptors (AST-ARs) are a group of G-protein coupled receptors activated by members of the FGL-amide (AST-A) peptide family that inhibit food intake and development in arthropods. Despite their physiological importance the evolution of the AST-A system is poorly described and relatively few receptors have been isolated and functionally characterised in insects. The present study provides a comprehensive analysis of the origin and comparative evolution of the AST-A system. To determine how evolution and feeding modified the function of AST-AR the duplicate receptors in Anopheles mosquitoes, were characterised. Phylogeny and gene synteny suggested that invertebrate AST-A receptors and peptide genes shared a common evolutionary origin with KISS/GAL receptors and ligands. AST-ARs and KISSR emerged from a common gene ancestor after the divergence of GALRs in the bilaterian genome. In arthropods, the AST-A system evolved through lineage-specific events and the maintenance of two receptors in the flies and mosquitoes (Diptera) was the result of a gene duplication event. Speciation of Anopheles mosquitoes affected receptor gene organisation and characterisation of AST-AR duplicates (GPRALS1 and 2) revealed that in common with other insects, the mosquito receptors were activated by insect AST-A peptides and the iCa2+-signalling pathway was stimulated. GPRALS1 and 2 were expressed mainly in mosquito midgut and ovaries and transcript abundance of both receptors was modified by feeding. A blood meal strongly up-regulated expression of both GPRALS in the midgut (p < 0.05) compared to glucose fed females. Based on the results we hypothesise that the AST-A system in insects shared a common origin with the vertebrate KISS system and may also share a common function as an integrator of metabolism and reproduction. Highlights: AST-A and KISS/GAL receptors and ligands shared common ancestry prior to the protostome-deuterostome divergence. Phylogeny and gene synteny revealed that AST-AR and KISSR emerged after GALR gene divergence. AST-AR genes were present in the hemichordates but were lost from the chordates. In protostomes, AST-ARs persisted and evolved through lineage-specific events and duplicated in the arthropod radiation. Diptera acquired and maintained functionally divergent duplicate AST-AR genes.

Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.)

Biopesticides or transgenic crops based on Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) effectively control agricultural insect pests. The sustainable use of Bt biopesticides and Bt crops is threatened, however, by the development of Cry resistance in the target pests. The diamondback moth, Plutella xylostella (L.), is the first pest that developed resistance to a Bt biopesticide in the field, and a recent study has shown that the resistance of P. xylostella to Cry1Ac is caused by a mutation in an ATP-binding cassette (ABC) transporter gene (ABCC2). In this study, we report that down-regulation of a novel ABC transporter gene from ABCG subfamily (Pxwhite) is associated with Cry1Ac resistance in P. xylostella. The full-length cDNA sequence of Pxwhite was cloned and analyzed. Spatial-temporal expression detection revealed that Pxwhite was expressed in all tissues and developmental stages, and highest expressed in Malpighian tubule tissue and in egg stage. Sequence variation analysis of Pxwhite indicated the absence of constant non-synonymous mutations between susceptible and resistant strains, whereas midgut transcript analysis showed that Pxwhite was remarkably reduced in all resistant strains and further reduced when larvae of the moderately resistant SZ-R strain were subjected to selection with Cry1Ac toxin. Furthermore, RNA interference (RNAi)-mediated suppression of Pxwhite gene expression significantly reduced larval susceptibility to Cry1Ac toxin, and genetic linkage analysis confirmed that down-regulation of Pxwhite gene is tightly linked to Cry1Ac resistance in P. xylostella. To our knowledge, this is the first report indicating that Pxwhite gene is involved in Cry1Ac resistance in P. xylostella.

In depth annotation of the Anopheles gambiae mosquito midgut transcriptome.

BackgroundGenome sequencing of Anopheles gambiae was completed more than ten years ago and has accelerated research on malaria transmission. However, annotation needs to be refined and verified experimentally, as most predicted transcripts have been identified by comparative analysis with genomes from other species. The mosquito midgut—the first organ to interact with Plasmodium parasites—mounts effective antiplasmodial responses that limit parasite survival and disease transmission. High-throughput Illumina sequencing of the midgut transcriptome was used to identify new genes and transcripts, contributing to the refinement of An. gambiae genome annotation.ResultsWe sequenced ~223 million reads from An. gambiae midgut cDNA libraries generated from susceptible (G3) and refractory (L35) mosquito strains. Mosquitoes were infected with either Plasmodium berghei or Plasmodium falciparum, and midguts were collected after the first or second Plasmodium infection. In total, 22,889 unique midgut transcript models were generated from both An. gambiae strain sequences combined, and 76% are potentially novel. Of these novel transcripts, 49.5% aligned with annotated genes and appear to be isoforms or pre-mRNAs of reference transcripts, while 50.5% mapped to regions between annotated genes and represent novel intergenic transcripts (NITs). Predicted models were validated for midgut expression using qRT-PCR and microarray analysis, and novel isoforms were confirmed by sequencing predicted intron-exon boundaries. Coding potential analysis revealed that 43% of total midgut transcripts appear to be long non-coding RNA (lncRNA), and functional annotation of NITs showed that 68% had no homology to current databases from other species. Reads were also analyzed using de novo assembly and predicted transcripts compared with genome mapping-based models. Finally, variant analysis of G3 and L35 midgut transcripts detected 160,742 variants with respect to the An. gambiae PEST genome, and 74% were new variants. Intergenic transcripts had a higher frequency of variation compared with non-intergenic transcripts.ConclusionThis in-depth Illumina sequencing and assembly of the An. gambiae midgut transcriptome doubled the number of known transcripts and tripled the number of variants known in this mosquito species. It also revealed existence of a large number of lncRNA and opens new possibilities for investigating the biological function of many newly discovered transcripts.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-636) contains supplementary material, which is available to authorized users.

Gossypol‐enhanced P450 gene pool contributes to cotton bollworm tolerance to a pyrethroid insecticide

Cotton plants accumulate phytotoxins, including gossypol and related sesquiterpene aldehydes, to resist insect herbivores and pathogens. To counteract these defensive plant secondary metabolites, cotton bollworms (Helicoverpa armigera) elevate their production of detoxification enzymes, including cytochrome P450 monooxygenases (P450s). Besides their tolerance to phytotoxin, cotton bollworms have quickly developed resistance to deltamethrin, a widely used pyrethroid insecticide in cotton field. However, the relationship between host plant secondary metabolites and bollworm insecticide resistance is poorly understood. Here, we show that exogenously expressed CYP6AE14, a gossypol-inducible P450 of cotton bollworm, has epoxidation activity towards aldrin, an organochlorine insecticide, indicating that gossypol-induced P450s participate in insecticide metabolism. Gossypol-ingested cotton bollworm larvae showed higher midgut P450 enzyme activities and exhibited enhanced tolerance to deltamethrin. The midgut transcripts of bollworm larvae administrated with different phytochemicals and deltamethrin were then compared by microarray analysis, which showed that gossypol and deltamethrin induced the most similar P450 expression profiles. Gossypol-induced P450s exhibited high divergence and at least five of them (CYP321A1, CYP9A12, CYP9A14, CYP6AE11 and CYP6B7) contributed to cotton bollworm tolerance to deltamethrin. Knocking down one of them, CYP9A14, by plant-mediated RNA interference (RNAi) rendered the larvae more sensitive to the insecticide. These data demonstrate that generalist insects can take advantage of secondary metabolites from their major host plants to elaborate defence systems against other toxic chemicals, and impairing this defence pathway by RNAi holds a potential for reducing the required dosages of agrochemicals in pest control.

RNAi‐mediated knockdown of a Spodoptera frugiperda trypsin‐like serine‐protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin

SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis.

Expression of diverse midgut serine proteinases in the sericigenous Lepidoptera Antheraea assamensis (Helfer) is influenced by choice of host plant species

Antheraea assamensis is reared on various species of the Lauraceae family from north-east India for its distinctive cocoon silk. We demonstrate differential expression of digestive trypsin and chymotrypsins in larvae feeding on a primary host, Persea bombycina Kosterm., in comparison to larvae feeding on Litsea monopetala Roxb. using in vitro proteolytic assays, zymogram analyses with proteinase inhibitors, restriction digestion of RNA-PCR amplicons and quantitative real-time PCR (RT-PCR). Eight novel members of the serine proteinase gene family were identified, including an intron-spliced trypsin (AaPb4) and seven putative chymotrypsins (AaPb2, AaPb4, AaPb12, AaLm4, AaLm6, AaLm19 and AaLm29). Midgut transcript levels of the putative trypsin were higher in larvae fed P. bombycina whereas levels of transcripts encoding putative chymotrypsins were higher in larvae reared on L. monopetala. Complex, differential expression of sequence divergent midgut serine proteinases may reflect the ability of lepidopteran larvae to feed on different species of host plants. Possible implications of host plant choice on the digestive physiology of A. assamensis are discussed.

Analysis of Rickettsia typhi-infected and uninfected cat flea (Ctenocephalides felis) midgut cDNA libraries: deciphering molecular pathways involved in host response to R. typhi infection.

Murine typhus is a flea-borne febrile illness that is caused by the obligate intracellular bacterium, Rickettsia typhi. The cat flea, Ctenocephalides felis, acquires R. typhi by imbibing a bloodmeal from a rickettsemic vertebrate host. To explore which transcripts are expressed in the midgut in response to challenge with R. typhi, cDNA libraries of R. typhi-infected and uninfected midguts of C. felis were constructed. In this study, we examined midgut transcript levels for select C. felis serine proteases, GTPases and defence response genes, all thought to be involved in the fleas response to feeding or infection. An increase in gene expression was observed for the serine protease inhibitors and vesicular trafficking proteins in response to feeding. In addition, R. typhi infection resulted in an increase in gene expression for the chymotrypsin and rab5 that we studied. Interestingly, R. typhi infection had little effect on expression of any of the defence response genes that we studied. We are unsure as to the physiological significance of these gene expression profiles and are currently investigating their potential roles as it pertains to R. typhi infection. To our knowledge, this is the first report of differential expression of flea transcripts in response to infection with R. typhi.

Cowpea bruchid midgut transcriptome response to a soybean cystatin – costs and benefits of counter‐defence

The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real-time PCR (RT-PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN-responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a Blast search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, whereas others exhibited developmental stage-specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN whereas structural, defence and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.

The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies

BackgroundIn the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi.ResultsComparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5), two peritrophin like proteins, a trypsin like protein (Lltryp1), two chymotrypsin like proteins (LuloChym1A and 2) and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3), a trypsin like protein (Lltryp2) and an astacin-like metalloprotease (LuloAstacin). Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5), a Chymotrypsin (LuloChym1A) and a carboxypeptidase (LuloCpepA1), among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1), a trypsin-like protein (Lltryp2) and an unknown protein.ConclusionThis transcriptome analysis represents the largest set of sequence data reported from a specific sand fly tissue and provides further information of the transcripts present in the sand fly Lutzomyia longipalpis. This analysis provides the detailed information of molecules present in the midgut of this sand fly and the transcripts potentially modulated by blood feeding and by the presence of the Leishmania parasite. More importantly, this analysis suggests that Leishmania infantum chagasi alters the expression profile of certain midgut transcripts in the sand fly during blood meal digestion and that this modulation may be relevant for the survival and establishment of the parasite in the gut of the fly. Moreover, this analysis suggests that these changes may be occurring during the digestion of the blood meal and not afterwards.

Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugar-fed, blood-fed and Leishmania major-infected sandflies

BackgroundIn sandflies, the blood meal is responsible for the induction of several physiologic processes that culminate in egg development and maturation. During blood feeding, infected sandflies are also able to transmit the parasite Leishmania to a suitable host. Many blood-induced molecules play significant roles during Leishmania development in the sandfly midgut, including parasite killing within the endoperitrophic space. In this work, we randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR.ResultsTranscriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3), a peritrophin (PpPer1), trypsin 4 (PpTryp4), chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major.ConclusionThis tissue-specific transcriptome analysis provides a comprehensive look at the repertoire of transcripts present in the midgut of the sandfly P. papatasi. Furthermore, the customized bioinformatic analysis allowed us to compare and identify the overall transcript abundance from sugar-fed, blood-fed and Leishmania-infected sandflies. The suggested upregulation of specific transcripts in a blood-fed cDNA library were validated by real-time PCR, suggesting that this customized bioinformatic analysis is a powerful and accurate tool useful in analysing expression profiles from different cDNA libraries. Additionally, the findings presented in this work suggest that the Leishmania parasite is modulating key enzymes or proteins in the gut of the sandfly that may be beneficial for its establishment and survival.

Transcripts of the malaria vector Anopheles gambiae that are differentially regulated in the midgut upon exposure to invasive stages of Plasmodium falciparum.

Understanding the interactions between the most deadly malaria parasite, Plasmodium falciparum, and its main vector, Anopheles gambiae, would be of great help in developing new malaria control strategies. The malaria parasite undergoes several developmental transitions in the mosquito midgut and suffers population losses to which mosquito factors presumably contribute. To identify such factors, we analysed An. gambiae midgut transcripts whose expression is regulated upon ingestion of invasive or non-invasive forms of P. falciparum using a differential display approach. Sixteen cDNA were studied in detail; 12 represent novel genes of An. gambiae including a gene encoding profilin. Four transcripts were specifically regulated by P. falciparum gametocytes (invasive forms), whereas the others were regulated by either non-invasive or both non-invasive and invasive forms of the parasite. This differential regulation of some genes may reflect the adaptation of P. falciparum to its natural vector. These genes may be involved in the development of P. falciparum in An. gambiae or in the defence reaction of the mosquito midgut towards the parasite.