Aedes aegypti post-emergence transcriptome: Unveiling the molecular basis for the hematophagic and gonotrophic capacitation.

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.

Current and potential biotechnological applications of odorant-binding proteins.

Odorant-binding proteins (OBPs) are small soluble proteins whose biological function is believed to be facilitating olfaction by assisting the transport of volatile chemicals in both vertebrate and insect sensory organs, where they are secreted. Their capability to interact with a broad range of hydrophobic compounds combined with interesting features such as being small, stable, and easy to produce and modify, makes them suitable targets for applied research in various industrial segments, including textile, cosmetic, pesticide, and pharmaceutical, as well as for military, environmental, health, and security field applications. In addition to reviewing already established biotechnological applications of OBPs, this paper also discusses their potential use in prospecting of new technologies. The development of new products for insect population management is currently the most prevailing use for OBPs, followed by biosensor technology, an area that has recently seen a significant increase in studies evaluating their incorporation into sensing devices. Finally, less typical approaches include applications in anchorage systems and analytical tools. KEY POINTS: • Odorant-binding proteins (OBPs) present desired characteristics for applied research. • OBPs are mainly used for developing new products for insect population control. • Incorporation of OBPs into chemosensory devices is a growing area of study. • Less conventional uses for OBPs include anchorage systems and analytical purposes. Graphical Abstract.

Reverse chemical ecology-based approach leading to the accidental discovery of repellents for Rhodnius prolixus, a vector of Chagas diseases refractory to DEET.

Rhodnius prolixus is one of the most important vectors of Chagas disease in Central and South America for which repellents and attractants are sorely needed. Repellents like DEET, picaridin, and IR3535 are widely used as the first line of defense against mosquitoes and other vectors, but they are ineffective against R. prolixus. Our initial goal was to identify in R. prolixus genome odorant receptors sensitive to putative sex pheromones. We compared gene expression of 21 ORs in the R. prolixus genome, identified 4 ORs enriched in male (compared with female) antennae. Attempts to de-orphanize these ORs using the Xenopus oocyte recording system showed that none of them responded to putative sex pheromone constituents. One of the them, RproOR80, was sensitive to 4 compounds in our panel of 109 odorants, namely, 2-heptanone, γ-octalactone, acetophenone, and 4-methylcychohexanol. Interestingly, these compounds, particularly 4-methylcyclohexanol, showed strong repellency activity as indicated not only by a significant decrease in residence time close to a host, but also by a remarkable reduction in blood intake. 4-Methylcyclohexanol-elicited repellency activity was abolished in RNAi-treated insects. In summary, our search for pheromone receptors led to the discovery of repellents for R. prolixus.

Functional Characterization of Odorant Binding Protein 27 (RproOBP27) From Rhodnius prolixus Antennae.

Olfactory proteins mediate a wide range of essential behaviors for insect survival. Odorant binding proteins (OBPs) are small soluble olfactory proteins involved in the transport of odor molecules (=odorants) through the sensillum lymph to odorant receptors, which are housed on the dendritic membrane of olfactory sensory neurons also known as olfactory receptor neurons. Thus, a better understanding of the role(s) of OBPs from Rhodnius prolixus, one of the main vectors of Chagas disease, may ultimately lead to new strategies for vector management. Here we aimed at functionally characterize OBPs from R. prolixus. Genes of interest were selected using conventional bioinformatics approaches and subsequent quantification by qPCR. We screened and estimated expression in different tissues of 17 OBPs from R. prolixus adults. These analyses showed that 11 OBPs were expressed in all tissues, whereas six OBP genes were specific to antennae. Two OBP genes, RproOBP6 and RproOBP13, were expressed in both male and female antennae thus suggesting that they might be involved in the recognition of semiochemicals mediating behaviors common to both sexes, such host finding (for a blood meal). Transcripts for RproOBP17 and RproOBP21 were enriched in female antennae and possibly involved in the detection of oviposition attractants or other semiochemicals mediating female-specific behaviors. By contrast, RproOBP26 and RproOBP27 might be involved in the reception of sex pheromones given that their transcripts were highly expressed in male antennae. To test this hypothesis, we silenced RproOBP27 using RNAi and examined the sexual behavior of the phenotype. Indeed, adult males treated with dsOBP27 spent significantly less time close to females as compared to controls. Additionally, docking analysis suggested that RproOBP27 binds to putative sex pheromones. We therefore concluded that RproOBP27 might be a pheromone-binding protein.

Proteomic analysis of the kissing bug Rhodnius prolixus antenna.

Reception of odorants is essential in insects' life since the chemical signals in the environment (=semiochemicals) convey information about availability of hosts for a blood meal, mates for reproduction, sites for oviposition and other relevant information for fitness in the environment. Once they reach the antennae, these semiochemicals bind to odorant-binding proteins and are transported through the sensillar lymph until reach the odorant receptors. Such perireceptor events, particularly the interactions with transport proteins, are the liaison between the external environment and the entire neuroethological system and, therefore, a potential target to disrupt insect chemical communication. In this study, a proteomic profile of female and male antennae of Rhodnius prolixus, a vector of Chagas disease, was obtained in an attempt to unravel the entire repertoire of olfactory proteins involved in perireceptor events. Using shotgun proteomics and two-dimensional gel electrophoresis approaches followed by nano liquid chromatography coupled with tandem LTQ Velos Orbitrap mass spectrometry, we have identified 581 unique proteins. Putative olfactory proteins, including 17 odorant binding proteins, 6 chemosensory proteins, 2 odorant receptors, 3 transient receptor channels and 1 gustatory receptor were identified. Proteins involved in general cellular functions such as generation of precursor metabolites, energy generation and catabolism were expressed at high levels. Additionally, proteins that take part in signal transduction, ion binding, and stress response, kinase and oxidoreductase activity were frequent in antennae from both sexes. This proteome strategy unraveled for the first time the complex nature of perireceptor and other olfactory events that occur in R. prolixus antennae, including evidence for phosphorylation of odorant-binding and chemosensory proteins. These findings not only increase our understanding of the olfactory process in triatomine species, but also identify potential molecular targets to be explored for population control of such insect vectors.

Comparative proteome analysis reveals that blood and sugar meals induce differential protein expression in Aedes aegypti female heads.

Aedes aegypti females ingest sugar or blood to obtain the nutrients needed to maintain cellular homeostasis. During human blood ingestion, female mosquitoes may transmit different viruses such as dengue, yellow fever and, more recently, zika and chikungunya. Here, we report changes in protein expression in the heads of adult female Ae. aegypti mosquitoes in response to the ingestion of blood or sugar. Proteins extracted from the heads of Ae. aegypti fed exclusively on blood (BF) or sugar (SF) were trypsin hydrolyzed (off-gel) and analyzed by the reverse-phase nano-liquid chromatography coupled with hybrid mass spectrometry. A total of 1139 proteins were identified in female heads, representing 7.4% of the predicted proteins in Ae. aegypti genome (total = 15 419 active genes). Gene ontology annotation and categories showed that, in this insect, the head was rich in proteins involved in the metabolic process, proton transport, organelle, macromolecular complex, structural molecule activity, antioxidant activity, and catalytic activity. Our report is the first indicating that many of the annotated genes are translated into functional proteins in heads of adult female Ae. aegypti. Interestingly, we identified 8.7 times more exclusively expressed proteins involved in signal transduction, replication-transcription-translation (5.5 x), and transport (2.9 x) activity in BF than in SF groups. This paper discusses the protein profile of Ae. aegypti female heads and its implications for blood ingestion and carbohydrate intake.

Silencing the odorant receptor co-receptor RproOrco affects the physiology and behavior of the Chagas disease vector Rhodnius prolixus.

Olfaction is one of the main sensory modalities that allow insects to interpret their environment. Several proteins, including odorant-binding proteins (OBPs) and odorant receptors (ORs), are involved in this process. Odorant receptors are ion channels formed by a binding unit OR and an odorant receptor co-receptor (Orco). The main goal of this study was to characterize the Orco gene of Rhodnius prolixus (RproOrco) and to infer its biological functions using gene silencing. The full-length RproOrco gene sequence was downloaded from VectorBase. This gene has 7 introns and is located in the genome SuperContig GL563069: 1,017,713-1,023,165. RproOrco encodes a protein of 473 amino acids, with predicted 7 transmembrane domains, and is highly expressed in the antennae during all R. prolixus developmental stages. The RNAi technique effectively silenced RproOrco, reducing the gene's expression by approximately 73%. Interestingly, the effect of gene silencing persisted for more than 100 days, indicating a prolonged effect of dsRNA that was maintained even after molting. The phenotypic effects of silencing involved the following: (1) loss of the ability to find a vertebrate host in a timely manner, (2) decreased ingested blood volume, (3) delayed and decreased molt rate, (4) increased mortality rate, and (5) decreased egg laying. Our data strongly suggest that dsOrco disrupts R. prolixus host-finding behavior, which is further reflected in the blood ingestion, molting, mortality, and egg laying data. This study clearly demonstrates that Orco is an excellent target for controlling triatomine populations. Thus, the data presented here open new possibilities for the control of vector-borne diseases.