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In arthropods, hemolymph carries immune cells and solubilizes and transports nutrients, hormones, and other molecules that are involved in diverse physiological processes including immunity, metabolism, and reproduction. However, despite such physiological importance, little is known about its composition. We applied mass spectrometry-based label-free quantification approaches to study the proteome of hemolymph perfused from sugar-fed female and male Aedes aegypti mosquitoes. A total of 1403 proteins were identified, out of which 447 of them were predicted to be extracellular. In both sexes, almost half of these extracellular proteins were predicted to be involved in defense/immune response, and their relative abundances (based on their intensity-based absolute quantification, iBAQ) were 37.9 and 33.2%, respectively. Interestingly, among them, 102 serine proteases/serine protease-homologues were identified, with almost half of them containing CLIP regulatory domains. Moreover, proteins belonging to families classically described as chemoreceptors, such as odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), were also highly abundant in the hemolymph of both sexes. Our data provide a comprehensive catalogue of A. aegypti hemolymph basal protein content, revealing numerous unexplored targets for future research on mosquito physiology and disease transmission. It also provides a reference for future studies on the effect of blood meal and infection on hemolymph composition.

The D7 proteins are highly expressed in the saliva of hematophagous Nematocera and bind biogenic amines and eicosanoid compounds produced by the host during blood feeding. These proteins are encoded by gene clusters expressing forms having one or two odorant-binding protein-like domains. Here we examine functional diversity within the D7 group in the genus Anopheles and make structural comparisons with D7 proteins from culicine mosquitoes in order to understand aspects of D7 functional evolution. Two domain long form (D7L) and one domain short form (D7S) proteins from anopheline and culicine mosquitoes were characterized to determine their ligand selectivity and binding pocket structures. We previously showed that a D7L protein from Anopheles stephensi, of the subgenus Cellia, could bind eicosanoids at a site in its N-terminal domain but could not bind biogenic amines in its C-terminal domain as does a D7L1 ortholog from the culicine species Aedes aegypti, raising the question of whether anopheline D7L proteins had lost their ability to bind biogenic amines. Here we find that D7L from anopheline species belonging to two other subgenera, Nyssorhynchus and Anopheles, can bind biogenic amines and have a structure much like the Ae. aegypti ortholog. The unusual D7L, D7L3, can also bind serotonin in the Cellia species An. gambiae. We also show through structural comparisons with culicine forms that the biogenic amine binding function of single domain D7S proteins in the genus Anopheles may have evolved through gene conversion of structurally similar proteins, which did not have biogenic amine binding capability. Collectively, the data indicate that D7L proteins had a biogenic amine and eicosanoid binding function in the common ancestor of anopheline and culicine mosquitoes, and that the D7S proteins may have acquired a biogenic amine binding function in anophelines through a gene conversion process., (Published by Elsevier Ltd.)

Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission., Competing Interests: The authors have declared that no competing interests exist.

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

Background: Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS) that modulate redox metabolism., Methodology/principal Findings: We demonstrate that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses to Plasmodium infection. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection., Conclusion: We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to Plasmodium infection.

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) ~ 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.

The mosquito D7 salivary proteins are encoded by a multigene family related to the arthropod odorant-binding protein (OBP) superfamily. Forms having either one or two OBP domains are found in mosquito saliva. Four single-domain and one two-domain D7 proteins from Anopheles gambiae and Aedes aegypti (AeD7), respectively, were shown to bind biogenic amines with high affinity and with a stoichiometry of one ligand per protein molecule. Sequence comparisons indicated that only the C-terminal domain of AeD7 is homologous to the single-domain proteins from A. gambiae, suggesting that the N-terminal domain may bind a different class of ligands. Here, we describe the 3D structure of AeD7 and examine the ligand-binding characteristics of the N- and C-terminal domains. Isothermal titration calorimetry and ligand complex crystal structures show that the N-terminal domain binds cysteinyl leukotrienes (cysLTs) with high affinities (50-60 nM) whereas the C-terminal domain binds biogenic amines. The lipid chain of the cysLT binds in a hydrophobic pocket of the N-terminal domain, whereas binding of norepinephrine leads to an ordering of the C-terminal portion of the C-terminal domain into an alpha-helix that, along with rotations of Arg-176 and Glu-268 side chains, acts to bury the bound ligand.

To gain insight into the molecular repertoire of the adult female salivary glands of the tiger mosquito Aedes albopictus, we performed transcriptome and proteome analysis. cDNA clones were sequenced and assembled in clusters of related sequences and the corresponding genes assigned to one of three categories: housekeeping (H; 31%), secreted (S; 34%), or unknown (U; 35%) function. Among the putative secreted factors are proteins known to be widely distributed in the saliva of blood-sucking Diptera, such as D7 and antigen 5 family members, as well as proteins that are mosquito- or culicine-specific, i.e., the 30-kDa allergen or the 62-kDa and 34-kDa families, respectively. Expression of 15 of these salivary proteins was confirmed by Edman degradation. Tissue and sex specificity of selected transcripts were evaluated by RT-PCR and identified at least 32 genes whose expression is restricted or enriched in the female salivary glands of Ae. albopictus, whereas 17 additional genes were expressed in female glands and adult males but not in other tissues of adult females. For approximately one third of the genes analyzed, involvement in blood-feeding, sugar digestion, immune response, or other more generic physiological roles can be postulated; however, no functions can be suggested for the remaining sequences, which therefore likely represent either novel functions or novel molecules recruited during the evolution of hematophagy. Supplemental spreadsheets with hyperlinks to all sequences used in this manuscript are hyperlinked throughout the text and can be found at http://www.ncbi.nlm.nih.gov/projects/omes/#salivarytranscriptomes.