Characterization of essential eggshell proteins from Aedes aegypti mosquitoes

Background: Up to 40% of the world population live in areas where mosquitoes capable of transmitting the dengue virus, including Aedes aegypti, coexist with humans. Understanding how mosquito egg development and oviposition are regulated at the molecular level may provide new insights into novel mosquito control strategies. Previously, we identified a protein named eggshell organizing factor 1 (EOF1) that when knocked down with RNA interference (RNAi) resulted in non-melanized and fragile eggs that did not contain viable embryos. Results: In this current study, we performed a comprehensive RNAi screen of putative A. aegypti eggshell proteins to identify additional proteins that interact with intracellular EOF1. We identified several proteins essential for eggshell formation in A. aegypti and characterized their phenotypes through a combination of molecular and biochemical approaches. We found that Nasrat, Closca, and Polehole structural proteins, together with the Nudel serine protease, are indispensable for eggshell melanization and egg viability. While all four proteins are predominantly expressed in ovaries of adult females, Nudel messenger RNA (mRNA) expression is highly upregulated in response to blood feeding. Furthermore, we identified four additional secreted eggshell enzymes that regulated mosquito eggshell formation and melanization. These enzymes included three dopachrome-converting enzymes (DCEs) and one cysteine protease. All eight of these eggshell proteins were essential for proper eggshell formation. Interestingly, their eggshell surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1, suggesting that additional mechanisms may influence how EOF1 regulates eggshell formation and melanization. Conclusions: While our studies did not identify a definitive regulator of EOF1, we did identify eight additional proteins involved in mosquito eggshell formation that may be leveraged for future control strategies

In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti

<p>Abstract</p> <p>Background</p> <p>The major Dengue virus vector <it>Aedes aegypti </it>requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of <it>Ae. aegypti </it>midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.</p> <p>Results</p> <p>We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (k_cat/K_M) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.</p> <p>Conclusions</p> <p>These data show that <it>in vitro </it>activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.</p

Alpha-COPI Coatomer Protein Is Required for Rough Endoplasmic Reticulum Whorl Formation in Mosquito Midgut Epithelial Cells

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta'), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases

Comparative analysis of the vitellogenin genes of the Culicidae

A comparative sequence analysis of mosquito vitellogenin genes was done to gain a better understanding of the evolution of vitellogenin genes in mosquitoes. Genomic clones of vitellogenin genes were isolated from Aedes aegypti , Ae. atropalpus, Culex quinquefasciatus , Toxorhynchites amboinensis, and Anopheles albimanus. Vitellogenin genes were also cloned using degenerate PCR primers from 34 species of mosquitoes representing genera (Aedes, Anopheles, Armigeres, Coquillettidia, Culex, Culiseta, Deinocerites, Psorophora, Mansonia, Mimomyia, Toxorhynchites, and Wyeomyia). Analysis of mosquito vitellogenin gene sequences suggests that a majority of nonsynonymous amino acid substitutions were due to conserved and moderately conserved changes. The vitellogenin genes of the three anautogenous mosquito species had very high synonymous codon usage biases. On the other hand, the vitellogenin genes of autogenous mosquitoes exhibited low synonymous codon usage bias. An unusual pattern of synonymous codon usage was observed in the first 15 amino acid residues encoding the signal peptide in the mosquito vitellogenin genes, where a significantly high number of rarely used synonymous codons have accumulated. A phylogenetic footprinting analysis detected several evolutionarily conserved sequence elements in the 5' regulatory regions of some of the mosquito vitellogenin genes. Mosquito phylogenetic trees reconstructed from maximum parsimony, maximum likelihood, and distance methods based on vitellogenin gene sequences are highly supported and robust, and vitellogenin gene sequences can be utilized to infer the phylogenetic history of mosquitoes, and perhaps other animals

<i>Aedes aegypti</i> mosquito eggshell LC-MS/MS proteomics data

Two independent biological replicates from both RNAi-Fluc and RNAi-EOF1 were used in the Aedes aegypti mosquito eggshell proteomic analysis. Ovary tissues were dissected from RNAi-mosquitoes 4days PBM. Mature primary follicles were removed from dissected ovaries and thoroughly washed in 1X PBS to completely remove shed follicular epithelial cells, secondary follicles, the germarium, and the ovarian tissues including common and lateral oviducts.The enriched eggshells were homogenized in 6.0 M guanidine hydrochloride and incubated at 37oC overnight. Eggshell protein samples were separated about 1.0 cm into resolving gel on SDS-12% PAGE gels. LC-MS/MS analysis was performed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an EASY-Spray nanoESI source at the University of Arizona Analytical and Biological Mass Spectrometry Facility.For inquiries regarding the contents of this dataset, please contact the Corresponding Author listed in the README.txt file. Administrative inquiries (e.g., removal requests, trouble downloading, etc.) can be directed to [email protected]</p

Urea synthesis and excretion in Aedes aegypti mosquitoes are regulated by a unique cross-talk mechanism.

Aedes aegypti mosquitoes do not have a typical functional urea cycle for ammonia disposal such as the one present in most terrestrial vertebrates. However, they can synthesize urea by two different pathways, argininolysis and uricolysis. We investigated how formation of urea by these two pathways is regulated in females of A. aegypti. The expression of arginase (AR) and urate oxidase (UO), either separately or simultaneously (ARUO) was silenced by RNAi. The amounts of several nitrogen compounds were quantified in excreta using mass spectrometry. Injection of mosquitoes with either dsRNA-AR or dsRNA-UO significantly decreased the expressions of AR or UO in the fat body (FB) and Malpighian tubules (MT). Surprisingly, the expression level of AR was increased when UO was silenced and vice versa, suggesting a cross-talk regulation between pathways. In agreement with these data, the amount of urea measured 48 h after blood feeding remained unchanged in those mosquitoes injected with dsRNA-AR or dsRNA-UO. However, allantoin significantly increased in the excreta of dsRNA-AR-injected females. The knockdown of ARUO mainly led to a decrease in urea and allantoin excretion, and an increase in arginine excretion. In addition, dsRNA-AR-injected mosquitoes treated with a specific nitric oxide synthase inhibitor showed an increase of UO expression in FB and MT and a significant increase in the excretion of nitrogen compounds. Interestingly, both a temporary delay in the digestion of a blood meal and a significant reduction in the expression of several genes involved in ammonia metabolism were observed in dsRNA-AR, UO or ARUO-injected females. These results reveal that urea synthesis and excretion in A. aegypti are tightly regulated by a unique cross-talk signaling mechanism. This process allows blood-fed mosquitoes to regulate the synthesis and/or excretion of nitrogen waste products, and avoid toxic effects that could result from a lethal concentration of ammonia in their tissues

Effect of arginase (AR), urate oxidase (UO) and ARUO knockdown on nitrogen waste excretion.

<p><i>A. aegypti</i> females were injected with dsRNA-firefly luciferase (dsRNA-FL), dsRNA-AR, dsRNA-UO or dsRNA-ARUO and then fed with a blood meal. A–C. Urea, allantoin and arginine concentrations measured in the female excreta by mass spectrometry at 48 h after blood feeding. Data are presented as the mean ± SEM of five independent samples. *<i>p</i><0.05, ***<i>p</i><0.001 (when compared to dsRNA-FL by ANOVA).</p