Insulin-like peptides and ovary ecdysteroidogenic hormone differentially stimulate physiological processes regulating egg formation in the mosquito Aedes aegypti.

Mosquitoes including Aedes aegypti are human disease vectors because females must blood feed to produce and lay eggs. Blood feeding triggers insulin-insulin growth factor signaling (IIS) which regulates several physiological processes required for egg development. A. aegypti encodes 8 insulin-like peptides (ILPs) and one insulin-like receptor (IR) plus ovary ecdysteroidogenic hormone (OEH) that also activates IIS through the OEH receptor (OEHR). In this study, we assessed the expression of A. aegypti ILPs and OEH during a gonadotrophic cycle and produced each that were functionally characterized to further understand their roles in regulating egg formation. All A. aegypti ILPs and OEH were expressed during a gonadotrophic cycle. Five ILPs (1, 3, 4, 7, 8) and OEH were specifically expressed in the head, while antibodies to ILP3 and OEH indicated each was released after blood feeding from ventricular axons that terminate on the anterior midgut. A subset of ILP family members and OEH stimulated nutrient storage in previtellogenic females before blood feeding, whereas most IIS-dependent processes after blood feeding were activated by one or more of the brain-specific ILPs and/or OEH. ILPs and OEH with different biological activities also exhibited differences in IIS as measured by phosphorylation of the IR, phosphoinositide 3-kinase/Akt kinase (AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK). Altogether, our results provide the first results that compare the functional activities of all ILP family members and OEH produced by an insect.

The mosquito Aedes aegypti requires a gut microbiota for normal fecundity, longevity and vector competence.

Mosquitoes shift from detritus-feeding larvae to blood-feeding adults that can vector pathogens to humans and other vertebrates. The sugar and blood meals adults consume are rich in carbohydrates and protein but are deficient in other nutrients including B vitamins. Facultatively hematophagous insects like mosquitoes have been hypothesized to avoid B vitamin deficiencies by carryover of resources from the larval stage. However, prior experimental studies have also used adults with a gut microbiota that could provision B vitamins. Here, we used Aedes aegypti, which is the primary vector of dengue virus (DENV), to ask if carryover effects enable normal function in adults with no microbiota. We show that adults with no gut microbiota produce fewer eggs, live longer with lower metabolic rates, and exhibit reduced DENV vector competence but are rescued by provisioning B vitamins or recolonizing the gut with B vitamin autotrophs. We conclude carryover effects do not enable normal function.

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development.

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

Anti-melanization mechanism of the white spot syndrome viral protein, WSSV453, via interaction with shrimp proPO-activating enzyme, PmproPPAE2.

Inhibition of the host melanization reaction, activated by the prophenoloxidase activating (proPO) system, is one of the crucial evasion strategies of pathogens. Recently, the shrimp pathogen, white spot syndrome virus (WSSV), was found to inhibit melanization in the shrimp Penaeus monodon. The viral protein WSSV453 was previously shown to interact with PO-activating enzyme 2 (PmPPAE2) and reported to be involved in suppressing the shrimp melanization response after WSSV infection. Here, we characterized how WSSV453 inhibits melanization. WSSV453 is a non-structural viral protein, which was first detected in shrimp haemocytes at 6 hours post-infection (hpi) by WSSV and in shrimp plasma at 24 hpi. We produced recombinant proteins for three components of the P. monodon proPO system: PmproPPAE2, PmproPO1 and PmproPO2. Functional assays showed that active PmPPAE2 processed PmproPO1 and 2 to produce functional PO. Incubation of WSSV453 with PmproPPAE2 dose-dependently reduced PmPPAE2 activity toward PmPO1 or PmPO2. In contrast, WSSV453 had no effect on activated PmPPAE2. The addition of active PmPPAE2 to WSSV-infected shrimp plasma at day 2 post-infection also rescued PO activity. Taken together, these results indicate that the anti-melanization activity of WSSV is due to WSSV453, which interacts with PmproPPAE2 and interferes with its activation to active PmPPAE2.

Calcium influx enhances neuropeptide activation of ecdysteroid hormone production by mosquito ovaries.

A critical step in mosquito reproduction is the ingestion of a blood meal from a vertebrate host. In mosquitoes like Aedes aegypti, blood feeding stimulates the release of ovary ecdysteroidogenic hormone (OEH) and insulin-like peptide 3 (ILP3). This induces the ovaries to produce ecdysteroid hormone (ECD), which then drives egg maturation. In many immature insects, prothoracicotropic hormone (PTTH) stimulates the prothoracic glands to produce ECD that directs molting and metamorphosis. The receptors for OEH, ILP3 and PTTH are different receptor tyrosine kinases with OEH and ILP3 signaling converging downstream in the insulin pathway and PTTH activating the mitogen-activated protein kinase pathway. Calcium (Ca(2+)) flux and cAMP have also been implicated in PTTH signaling, but the role of Ca(2+) in OEH, ILP3, and cAMP signaling in ovaries is unknown. Here, we assessed whether Ca(2+) flux affects OEH, ILP3, and cAMP activity in A. aegypti ovaries and also asked whether PTTH stimulated ovaries to produce ECD. Results indicated that Ca(2+) flux enhanced but was not essential for OEH or ILP3 activity, whereas cAMP signaling was dependent on Ca(2+) flux. Recombinant PTTH from Bombyx mori fully activated ECD production by B. mori PTGs, but exhibited no activity toward A. aegypti ovaries. Recombinant PTTH from A. aegypti also failed to stimulate either B. mori PTGs or A. aegypti ovaries to produce ECD. We discuss the implications of these results in the context of mosquito reproduction and ECD biosynthesis by insects generally.

Ovary ecdysteroidogenic hormone functions independently of the insulin receptor in the yellow fever mosquito, Aedes aegypti.

Most mosquito species must feed on the blood of a vertebrate host to produce eggs. In the yellow fever mosquito, Aedes aegypti, blood feeding triggers medial neurosecretory cells in the brain to release insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH). Theses hormones thereafter directly induce the ovaries to produce ecdysteroid hormone (ECD), which activates the synthesis of yolk proteins in the fat body for uptake by oocytes. ILP3 stimulates ECD production by binding to the mosquito insulin receptor (MIR). In contrast, little is known about the mode of action of OEH, which is a member of a neuropeptide family called neuroparsin. Here we report that OEH is the only neuroparsin family member present in the Ae. aegypti genome and that other mosquitoes also encode only one neuroparsin gene. Immunoblotting experiments suggested that the full-length form of the peptide, which we call long OEH (lOEH), is processed into short OEH (sOEH). The importance of processing, however, remained unclear because a recombinant form of lOEH (rlOEH) and synthetic sOEH exhibited very similar biological activity. A series of experiments indicated that neither rlOEH nor sOEH bound to ILP3 or the MIR. Signaling studies further showed that ILP3 activated the MIR but rlOEH did not, yet both neuropeptides activated Akt, which is a marker for insulin pathway signaling. Our results also indicated that activation of TOR signaling in the ovaries required co-stimulation by amino acids and either ILP3 or rlOEH. Overall, we conclude that OEH activates the insulin signaling pathway independently of the MIR, and that insulin and TOR signaling in the ovaries is coupled.

Ovary ecdysteroidogenic hormone activates egg maturation in the mosquito Georgecraigius atropalpus after adult eclosion or a blood meal.

The rockpool mosquito, Georgecraigius atropalpus, is a facultatively autogenous species that produces its first egg clutch without a blood meal shortly after emergence. Several days after depositing this clutch, females must take a blood meal to produce a second egg clutch. Decapitation of females shortly after emergence or blood ingestion prevents egg maturation. Here, we report that a single injected dose of the neuropeptide ovary ecdysteroidogenic hormone (OEH) fully restored egg maturation in decapitated females in both circumstances. This neuropeptide and two insulin-like peptides (ILPs) are potent gonadotropins in the related yellow fever mosquito, Aedes aegypti. ILP3 was marginally restorative in decapitated G. atropalpus, and ILP4 had no effect. Egg maturation in non- and blood-fed G. atropalpus was dependent on the enzymatic mobilization of amino acids from stored protein or the blood meal for yolk protein (vitellogenin, VG) synthesis and uptake by oocytes. We further show that OEH stimulates serine protease activity in the fat body of newly eclosed females or in the midgut of blood-fed ones, and ecdysteroid hormone production by the ovaries of both females. In contrast, only 20-hydroxyecdysone stimulated VG synthesis in the fat body of non- and blood-fed females. Using RNA interference to knock down expression of the insulin receptor, we found that OEH still fully restored autogenous egg maturation. In summary, our results identify OEH as a primary regulator of egg maturation in both autogenous and blood-fed G. atropalpus females and suggest the shift from blood meal-dependent to blood meal-independent release of OEH is a key factor in the evolution of autogeny in this species.

Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses.

BACKGROUND: Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized. METHODOLOGY/PRINCIPAL FINDINGS: Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or -170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (K(D) = 0.4 nM) and to the active site of FXa (K(D) = 3.07 nM). We propose the name "Simukunin" for this novel protein. CONCLUSIONS: We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.

Characterization and kinetic analysis of protein tyrosine phosphatase-H2 from Microplitis demolitor bracovirus.

The polydnavirus Microplitis demolitor bracovirus (MdBV) encodes 13 genes that share homology with classical protein tyrosine phosphatases (PTPs). Prior sequence analysis suggested that five members of the MdBV PTP gene family (ptp-H2, -H3, -H5, -N1 and -N2) encode PTPs, seven family members encode pseudophosphatases, and one family member is a pseudogene. Prior experimental studies further implicated PTP-H2 in disabling the function of host hemocytes following infection by MdBV. Here we report expression of PTP-H2 and selected mutants in Escherichia coli cells as non-fusion or thioredoxin-fusion proteins. Following purification by nickel affinity chromatography, the full-length and mutant proteins ran as single bands of predicted size on SDS-PAGE gels under reducing conditions. The non-fusion form of PTP-H2 exhibited classical Michaelis-Menten kinetics using the phosphopeptide END(pY)INASL and difluoro-4-methylumbiliferyl phosphate (DiFMUP) as substrates. As expected, the non-fusion mutant PTP-H2(C236S) had no enzymatic activity, while the thioredoxin-fusion form of PTP-H2 had low levels of activity. PTP-H2 exhibited optimal activity at pH 4.0 and 26 degrees C in sodium acetate buffer, and its activity was diminished by increasing buffer ionic strength. Activity was also greatly reduced by the presence of copper, heparin, and the classical PTP inhibitor vanadate. Using an anti-PTP-H2 antibody, immunoblotting and immunocytochemical studies only detected PTP-H2 in hemocytes from MdBV-infected Pseudoplusia includens. Overall, our results indicate that PTP-H2 is a functional tyrosine phosphatase that is specifically expressed in MdBV-infected hemocytes.

Proteomic analysis of parasitized Plutella xylostella larvae plasma.

Insects use their innate immunity to defend themselves against foreign invaders, such as microorganisms, nematodes and parasites. Cotesia plutellae, an endoparasitoid wasp that parasitizes the diamondback moth Plutella xylostella, uses several strategies to attack the host immune system, such as injection of viruses, venom, and serosal membrane-derived cells denoted teratocytes. However, the proteome profiles related to these immune deficiency systems have yet to be clearly defined. In this study, we investigate differences in protein expression patterns in parasitized P. xylostella larvae, with a view to identifying parasitism-specific factors. Using 2D polyacrylamide gel electrophoresis, proteins in the host plasma were assessed every 48 h after parasitism by C. plutellae. A large number of protein spots (350 in total) were detected, and approximately 50 spots were differentially expressed in the parasitized P. xylostella larvae every 48 h. In total, 26 potential candidates, including P. xylostella Serpin 2 (pxSerpin 2), translationally controlled tumor protein, signal transduction histidine kinase, apolipophorin-III, and fatty-acid binding protein were identified through quadrupole time-of-flight tandem mass spectrometry and sequence homology analysis. These proteins were classified into the following functional groups: immunity, signaling, lipid metabolism, energy metabolism, amino acid/nucleotide metabolism, and others. The pxSerpin 2 gene was cloned, and its expression profile investigated during the course of parasitism. Real-time PCR analysis of pxSerpin 2 revealed a poor correlation between the mRNA level and protein abundance. Our results clearly suggest that parasitism-specific proteins participate in suppression of the host immune response.

Analysis of the immune-inducible genes of Plutella xylostella using expressed sequence tags and cDNA microarray.

In the present study, the complex gene expression responses of Plutella xylostella to microbial challenges and injury were surveyed using a newly constructed expressed sequence tag (EST) clone collection and cDNA microarray analysis. A total of 1132 P. xylostella ESTs were cloned, annotated and categorized by their putative functions; these included proteases, protease inhibitors, recognition molecules and anti-microbial peptides. GeneOntology revealed that 4% of the P. xylostella ESTs corresponded to immunity-related genes potentially involved in innate immunity. We then used microarray analysis to identify 44 genes that were differentially expressed with at least a two-fold expression difference in P. xylostella before and after pathogen challenge. Together, our EST categorization and microarray profiling analyses allowed us to identify 70 genes that should be considered candidate immune response genes, providing important new insights into the molecular events that occur during the innate immune response in P. xylostella.

Characterization of a novel repetitive secretory protein specifically expressed in the modified salivary gland of Hydropsyche sp. (Trichoptera; Hydropsychidae).

Here, we report the cloning and characterization of a common salivary gland-specific gene, Nf-1, from late instar Hydropsyche sp. larvae, and show that the corresponding gene product is translated in the gland and secreted to the gland lumen. The deduced Nf-1 protein is primarily composed of five repetitive sequence units of 63-65 amino acids, and contains a putative signal sequence composed of 19 amino acids. Secreted Nf-1 (approximately 37 kDa) was localized to the gland lumen by Western blotting of gland and lumen fractions. Together, the structure, expression pattern and protein localization of Nf-1 indicate that this protein is likely to be a major component of the silk shields and nets produced by the aquatic insect, Trichoptera.