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Background Arthropoda, the most numerous and diverse metazoan phylum, has species in many habitats where they encounter various microorganisms and, as a result, mechanisms for pathogen recognition and elimination have evolved. The Toll pathway, involved in the innate immune system, was first described as part of the developmental pathway for dorsal-ventral differentiation in Drosophila. Its later discovery in vertebrates suggested that this system was extremely conserved. However, there is variation in presence/absence, copy number and sequence divergence in various genes along the pathway. As most studies have only focused on Diptera, for a comprehensive and accurate homology-based approach it is important to understand gene function in a number of different species and, in a group as diverse as insects, the use of species belonging to different taxonomic groups is essential. Results We evaluated the diversity of Toll pathway gene families in 39 Arthropod genomes, encompassing 13 different Insect Orders. Through computational methods, we shed some light into the evolution and functional annotation of protein families involved in the Toll pathway innate immune response. Our data indicates that: 1) intracellular proteins of the Toll pathway show mostly species-specific expansions; 2) the different Toll subfamilies seem to have distinct evolutionary backgrounds; 3) patterns of gene expansion observed in the Toll phylogenetic tree indicate that homology based methods of functional inference might not be accurate for some subfamilies; 4) Spatzle subfamilies are highly divergent and also pose a problem for homology based inference; 5) Spatzle subfamilies should not be analyzed together in the same phylogenetic framework; 6) network analyses seem to be a good first step in inferring functional groups in these cases. We specifically show that understanding Drosophila’s Toll functions might not indicate the same function in other species. Conclusions Our results show the importance of using species representing the different orders to better understand insect gene content, origin and evolution. More specifically, in intracellular Toll pathway gene families the presence of orthologues has important implications for homology based functional inference. Also, the different evolutionary backgrounds of Toll gene subfamilies should be taken into consideration when functional studies are performed, especially for TOLL9, TOLL, TOLL2_7, and the new TOLL10 clade. The presence of Diptera specific clades or the ones lacking Diptera species show the importance of overcoming the Diptera bias when performing functional characterization of Toll pathways.

Flight dispersal represents a key aspect of the evolutionary and ecological success of insects, allowing escape from predators, mating, and colonization of new niches. The huge energy demand posed by flight activity is essentially met by oxidative phosphorylation (OXPHOS) in flight muscle mitochondria. In insects, mitochondrial ATP supply and oxidant production are regulated by several factors, including the energy demand exerted by changes in adenylate balance. Indeed, adenylate directly regulates OXPHOS by targeting both chemiosmotic ATP production and the activities of specific mitochondrial enzymes. In several organisms, cytochrome c oxidase (COX) is regulated at transcriptional, post-translational, and allosteric levels, impacting mitochondrial energy metabolism, and redox balance. This review will present the concepts on how COX function contributes to flying insect biology, focusing on the existing examples in the literature where its structure and activity are regulated not only by physiological and environmental factors but also how changes in its activity impacts insect biology. We also performed in silico sequence analyses and determined the structure models of three COX subunits (IV, VIa, and VIc) from different insect species to compare with mammalian orthologs. We observed that the sequences and structure models of COXIV, COXVIa, and COXVIc were quite similar to their mammalian counterparts. Remarkably, specific substitutions to phosphomimetic amino acids at critical phosphorylation sites emerge as hallmarks on insect COX sequences, suggesting a new regulatory mechanism of COX activity. Therefore, by providing a physiological and bioenergetic framework of COX regulation in such metabolically extreme models, we hope to expand the knowledge of this critical enzyme complex and the potential consequences for insect dispersal.

The adult females of Aedes aegypti mosquitoes are facultative hematophagous insects but they are unable to feed on blood right after pupae emergence. The maturation process that takes place during the first post-emergence days, hereafter named hematophagic and gonotrophic capacitation, comprises a set of molecular and physiological changes that prepare the females for the first gonotrophic cycle. Notwithstanding, the molecular bases underlying mosquito hematophagic and gonotrophic capacitation remain obscure. Here, we investigated the molecular and biochemical changes in adult Ae. aegypti along the first four days post-emergence, prior to a blood meal. We performed a RNA-Seq analysis of the head and body, comparing male and female gene expression time courses. A total of 811 and 203 genes were differentially expressed, respectively in the body and head, and both body parts showed early, mid, and late female-specific expression profiles. Female-specific up-regulation of genes involved in muscle development and the oxidative phosphorylation pathway were remarkable features observed in the head. Functional assessment of mitochondrial oxygen consumption in heads showed a gradual increase in respiratory capacity and ATP-linked respiration as a consequence of induced mitochondrial biogenesis and content over time. This pattern strongly suggests that boosting oxidative phosphorylation in heads is a required step towards blood sucking habit. Several salivary gland genes, proteases, and genes involved in DNA replication and repair, ribosome biogenesis, and juvenile hormone signaling were up-regulated specifically in the female body, which may reflect the gonotrophic capacitation. This comprehensive description of molecular and biochemical mechanisms of the hematophagic and gonotrophic capacitation in mosquitoes unravels potentially new targets for vector control., Author summary Aedes aegypti is a vector of several human diseases such as Dengue, Zika, Chikungunya, and Yellow Fever. Only the adult females can feed on blood, important for the production of their offspring, and therefore transmitting these pathogens for the human host. Interestingly, right after their emergence as adults, females show a preference of feeding on nectar, and only after three days, they are fully capacitated to perform a blood meal. Understanding the molecular mechanisms that orchestrate this physiological and biochemical switch is the main topic of the present study. The transcriptome analysis of males and females during the first hours post-emergence reveals that the gene expression in females is tightly regulated, showing three time-dependent profiles of expression. There is an increase in genes related to the oxidative phosphorylation pathway in the female head. Assessment of mitochondrial function revealed a gradual increase in respiration linked to ATP synthesis and mitochondrial content. Also, there is an increase in genes related to the muscular function, suggesting a putative role in muscle development and contraction, required, for example, for pumping of blood. The female body showed an increase in salivary gland related genes and serine proteases, probably related to the ingestion and digestion of the blood. Also, the increase in DNA replication, ribosome biogenesis, and juvenile hormone signaling pathways together with changes in cell cycle control suggest the gonotrophic capacitation for the massive synthesis of yolk proteins precursors during vitellogenesis. All these genes were enhanced even before the first blood meal, which reveals insightful information about the biology of anautogenous mosquitoes. Our data showed the molecular machinery that stands the females hematophagy and gonotrophy, leading to potential new targets for vector control.

Triatominae is a subfamily of the order Hemiptera whose species are able to feed in the vertebrate blood (i.e., hematophagy). This feeding behavior presents a great physiological challenge to insects, especially in Hemipteran species with a digestion performed by lysosomal-like cathepsins instead of the more common trypsin-like enzymes. With the aim of having a deeper understanding of protease involvement in the evolutionary adaptation for hematophagy in Hemipterans, we screened peptidases in the Rhodnius prolixus genome and characterized them using common blast (NCBI) and conserved domain analyses (HMMER/blast manager software, FAT, plus PFAM database). We compared the results with available sequences from other hemipteran species and with 18 arthropod genomes present in the MEROPS database. Rhodnius prolixus contains at least 433 protease coding genes, belonging to 71 protease families. Seven peptidase families in R. prolixus presented higher gene numbers when compared to other arthropod genomes. Further analysis indicated that a gene expansion of the protease family A1 (Eukaryotic aspartyl protease, PF00026) might have played a major role in the adaptation to hematophagy since most of these peptidase genes seem to be recently acquired, are expressed in the gut and present putative secretory pathway signal peptides. Besides that, most R. prolixus A1 peptidases showed high frequencies of basic residues at the protein surface, a typical structural signature of Cathepsin D-like proteins. Other peptidase families expanded in R. prolixus (i.e., C2 and M17) also presented significant differences between hematophagous (higher number of peptidases) and non-hematophagous species. This study also provides evidence for gene acquisition from microorganisms in some peptidase families in R. prolixus: (1) family M74 (murein endopeptidase), (2) family S29 (Hepatitis C virus NS3 protease), and (3) family S24 (repressor LexA). This study revealed new targets for studying the adaptation of these insects for digestion of blood meals and their competence as vectors of Chagas disease.

Background Mosquitoes feed on plant-derived fluids such as nectar and sap and are exposed to bioactive molecules found in this dietary source. However, the role of such molecules on mosquito vectorial capacity is unknown. Weather has been recognized as a major determinant of the spread of dengue, and plants under abiotic stress increase their production of polyphenols. Results Here, we show that including polyphenols in mosquito meals promoted the activation of AMP-dependent protein kinase (AMPK). AMPK positively regulated midgut autophagy leading to a decrease in bacterial proliferation and an increase in vector lifespan. Suppression of AMPK activity resulted in a 6-fold increase in midgut microbiota. Similarly, inhibition of polyphenol-induced autophagy induced an 8-fold increase in bacterial proliferation. Mosquitoes maintained on the polyphenol diet were readily infected by dengue virus. Conclusion The present findings uncover a new direct route by which exacerbation of autophagy through activation of the AMPK pathway leads to a more efficient control of mosquito midgut microbiota and increases the average mosquito lifespan. Our results suggest for the first time that the polyphenol content and availability of the surrounding vegetation may increase the population of mosquitoes prone to infection with arboviruses., Author Summary The dramatic climate changes currently occurring on our planet may likely influence the biology and the distribution of mosquitoes. Aedes aegypti is a major vector of arboviruses. However, females feed on plants for a few days before feeding on blood for the first time. Plants are sessile and cannot move to search for better environmental conditions. In times of extreme temperatures, drought, or UV radiation plants produce polyphenols that allow plants to survive under such extreme conditions. Polyphenol ingestion by mosquitoes enhances the activity of an enzyme named AMP-activated protein kinase (AMPK). Mosquitoes emerging from pupae leave the aquatic environment and are readily colonized by many bacterial strains. Polyphenol-fed mosquitoes display a huge activation of AMPK which enhances autophagy in the midgut cells that increases the capture and killing of midgut bacteria, thus enhancing vector lifespan. Therefore, early meals on polyphenol sources allow for the regulation of vector microbiota. These data may provide the basis of a novel strategy that promotes bacterial proliferation and a shortening of vector lifespan.

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immunedeficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods., La lista completa de autores que integran el documento puede consultarse en el archivo, Este documento tiene una corrección (ver documento relacionado)., Centro Regional de Estudios Genómicos, Instituto de Investigaciones Bioquímicas de La Plata

BackgroundProtein Tyrosine Phosphatases (PTPs) are enzymes that catalyze phosphotyrosine dephosphorylation and modulate cell differentiation, growth and metabolism. In mammals, PTPs play a key role in the modulation of canonical pathways involved in metabolism and immunity. PTP1B is the prototype member of classical PTPs and a major target for treating human diseases, such as cancer, obesity and diabetes. These signaling enzymes are, hence, targets of a wide array of inhibitors. Anautogenous mosquitoes rely on blood meals to lay eggs and are vectors of the most prevalent human diseases. Identifying the mosquito ortholog of PTP1B and determining its involvement in egg production is, therefore, important in the search for a novel and crucial target for vector control.Methodology/principal findingsWe conducted an analysis to identify the ortholog of mammalian PTP1B in the Aedes aegypti genome. We identified eight genes coding for classical PTPs. In silico structural and functional analyses of proteins coded by such genes revealed that four of these code for catalytically active enzymes. Among the four genes coding for active PTPs, AAEL001919 exhibits the greatest degree of homology with the mammalian PTP1B. Next, we evaluated the role of this enzyme in egg formation. Blood feeding largely affects AAEL001919 expression, especially in the fat body and ovaries. These tissues are critically involved in the synthesis and storage of vitellogenin, the major yolk protein. Including the classical PTP inhibitor sodium orthovanadate or the PTP substrate DiFMUP in the blood meal decreased vitellogenin synthesis and egg production. Similarly, silencing AAEL001919 using RNA interference (RNAi) assays resulted in 30% suppression of egg production.Conclusions/significanceThe data reported herein implicate, for the first time, a gene that codes for a classical PTP in mosquito egg formation. These findings raise the possibility that this class of enzymes may be used as novel targets to block egg formation in mosquitoes.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet., Journal Article, Research Support, N.I.H. Intramural, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

BackgroundRhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.Methodology/principal findingsHere we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities.Conclusions/significanceTaken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB) proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1). The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich) C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.

Germline mutations that inactivate BRCA1 are responsible for breast and ovarian cancer susceptibility. One possible outcome of genetic testing for BRCA1 is the finding of a genetic variant of uncertain significance for which there is no information regarding its cancer association. This outcome leads to problems in risk assessment, counseling and preventive care. The purpose of the present study was to functionally evaluate seven unclassified variants of BRCA1 including a genomic deletion that leads to the in-frame loss of exons 16/17 (Delta exons 16/17) in the mRNA, an insertion that leads to a frameshift and an extended carboxy-terminus (5673insC), and five missense variants (K1487R, S1613C, M1652I, Q1826H and V1833M). We analyzed the variants using a functional assay based on the transcription activation property of BRCA1 combined with supervised learning computational models. Functional analysis indicated that variants S1613C, Q1826H, and M1652I are likely to be neutral, whereas variants V1833M, Delta exons 16/17, and 5673insC are likely to represent deleterious variants. In agreement with the functional analysis, the results of the computational analysis also indicated that the latter three variants are likely to be deleterious. Taken together, a combined approach of functional and bioinformatics analysis, plus structural modeling, can be utilized to obtain valuable information pertaining to the effect of a rare variant on the structure and function of BRCA1. Such information can, in turn, aid in the classification of BRCA1 variants for which there is a lack of genetic information needed to provide reliable risk assessment.

Background Due to an abundance of repetitive DNA, the annotation of heterochromatic regions of the genome such as the Y chromosome is problematic. The Y chromosome is involved in key biological functions such as male-fertility and sex-determination and hence, accurate identification of its sequences is vital. The hemipteran insect Rhodnius prolixus is an important vector of Chagas disease, a trypanosomiasis affecting 6–7 million people worldwide. Here we report the identification of the first Y-linked genes of this species. Results The R. prolixus genome was recently sequenced using separate libraries for each sex and the sequences assembled only with male reads are candidates for Y linkage. We found 766 such candidates and PCR tests with the ten largest ones, confirmed Y-linkage for all of them, suggesting that "separate libraries" is a reliable method for the identification of Y-linked sequences. BLAST analyses of the 766 candidate scaffolds revealed that 568 scaffolds contained genes or part of putative genes. We tested Y-linkage for 36 candidates and found that nine of them are Y-linked (the PCR results for the other 25 genes were inconclusive or revealed autosomal/X-linkage). Hence, we describe in this study, for the first time, Y-linked genes in the R. prolixus genome: two zinc finger proteins (Znf-Y1 and Znf-Y2), one metalloproteinase (Met-Y), one aconitase/iron regulatory protein (Aco-Y) and five genes devoid of matches in any database (Rpr-Y1 to Rpr-Y5). Expression profile studies revealed that eight genes are expressed mainly in adult testis (some of which presented a weak expression in the initial developmental stages), while Aco-Y has a gut-restricted expression. Conclusions In this study we showed that the approach used for the R. prolixus genome project (separate sequencing of male and female DNA) is key to easy and fast identification of sex-specific (e.g. Y chromosome sequences). The nine new R. prolixus Y-linked genes reported here provide unique markers for population and phylogenetic analysis and further functional studies of these genes may answer some questions about sex determination, male fertility and Y chromosome evolution in this important species. Fundo Devido à abundância de DNA repetitivo, a anotação de regiões heterocromáticas do genoma, como o cromossomo Y, é problemática. O cromossomo Y está envolvido em funções biológicas importantes, como fertilidade masculina e determinação do sexo e, portanto, a identificação precisa de suas sequências é vital. O inseto hemíptero Rhodnius prolixus é um importante vetor da doença de Chagas, uma tripanossomíase que afeta 6 a 7 milhões de pessoas em todo o mundo. Aqui relatamos a identificação dos primeiros genes ligados ao Y desta espécie. Resultados O genoma de R. prolixus foi recentemente sequenciado usando bibliotecas separadas para cada sexo e as sequências montadas apenas com reads masculinos são candidatas à ligação Y. Encontramos 766 desses candidatos e testes de PCR com os dez maiores, confirmaram a ligação Y para todos eles, sugerindo que "bibliotecas separadas" é um método confiável para a identificação de sequências ligadas ao Y. Análises BLAST dos 766 scaffolds candidatos revelaram que 568 scaffolds continham genes ou parte de genes putativos. Testamos a ligação Y para 36 candidatos e descobrimos que nove deles estão ligados ao Y (os resultados da PCR para os outros 25 genes foram inconclusivos ou revelaram ligação autossômica/X). Assim, descrevemos neste estudo, pela primeira vez, genes ligados ao Y no genoma de R. prolixus: duas proteínas dedo de zinco (Znf-Y1 e Znf-Y2), uma metaloproteinase (Met-Y), uma aconitase/ferro proteína reguladora (Aco-Y) e cinco genes desprovidos de correspondências em qualquer banco de dados (Rpr-Y1 a Rpr-Y5). Estudos de perfil de expressão revelaram que oito genes são expressos principalmente em testículos adultos (alguns dos quais apresentaram uma expressão fraca nos estágios iniciais de desenvolvimento), enquanto o Aco-Y tem uma expressão restrita ao intestino. Conclusões Neste estudo, mostramos que a abordagem usada para o projeto genoma de R. prolixus (sequenciamento separado de DNA masculino e feminino) é a chave para a identificação fácil e rápida de sexo específico (por exemplo, sequências do cromossomo Y). Os nove novos genes ligados ao Y de R. prolixus relatados aqui fornecem marcadores únicos para análise populacional e filogenética e estudos funcionais adicionais desses genes podem responder a algumas perguntas sobre determinação do sexo, fertilidade masculina e evolução do cromossomo Y nesta importante espécie.

Tsetse flies are responsible for the transmission of Trypanossoma sp. to vertebrate animals in Africa causing huge health issues and economic loss. The availability of the genome sequence of Glossina morsitans enabled the discovery of several genes related to medically important phenotypes and novel physiological features. However, a genome-wide scan for coding regions that underwent positive selection is still missing, which is surprising given the evolution of traits associated with the hematophagy in this lineage. In this study, we employed an experimental design that controlled for the rate of false positives and we performed a scan of 3,318 G. morsitans genes. We found 145 genes with significant historical signal of positive selection. These genes were categorized into 18 functional classes after careful manual annotation. Based on their attributed functions, we identified candidate genes related with feeding habits and embryonic development. When our results were contrasted with gene expression data, we confirmed that most genes that underwent adaptive molecular evolution were frequently expressed in organs associated with key physiological evolutionary innovations in the G. morsitans lineage, namely, the salivary gland, the midgut, fat body tissue, and in the spermatophore.