Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection.

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

Transcription factor B-H2 regulates CYP9J34 expression conveying deltamethrin resistance in Culex pipiens pallens.

BACKGROUND: Mosquitoes are vectors of various diseases, posing significant health threats worldwide. Chemical pesticides, particularly pyrethroids like deltamethrin, are commonly used for mosquito control, but the emergence of resistant mosquito populations has become a concern. In the deltamethrin-resistant (DR) strain of Culex pipiens pallens, the highly expressed cytochrome P450 9 J34 (CYP9J34) gene is believed to play a role in resistance, yet the underlying mechanism remains unclear. RESULTS: Quantitative polymerase chain reaction with reverse transcription (qRT-PCR) analysis revealed that the expression of CYP9J34 was 14.6-fold higher in DR strains than in deltamethrin-susceptible (DS) strains. The recombinant production of CYP9J34 protein of Cx. pipiens pallens showed that the protein could directly metabolize deltamethrin, yielding the major metabolite 4'-OH deltamethrin. Through dual luciferase reporter assays and RNA interference, the transcription factor homeobox protein B-H2-like (B-H2) was identified to modulate the expression of the CYP9J34 gene, contributing to mosquito resistance to deltamethrin. CONCLUSIONS: Our findings demonstrate that the CYP9J34 protein could directly degrade deltamethrin, and the transcription factor B-H2 could regulate CYP9J34 expression, influencing the resistance of mosquitoes to deltamethrin. © 2023 Society of Chemical Industry.

Culex quinquefasciatus membrane-bound alkaline phosphatase is a putative receptor for Lysinibacillus sphaericus Tpp49Aa1 toxin.

The binary toxin Cry48Aa1/Tpp49Aa1 produced by Lysinibacillus sphaericus exhibits potent toxicity against Culicidae larvae. Both Cry48Aa1 and Tpp49Aa1 toxins are crucial for binding to the toxin receptor in Culex quinquefasciatus larvae, albeit with different binding sites. Previous studies have identified Glu71, a membrane-bound α-glucosidase, as a putative binding protein for the Cry48Aa1 toxin, involved in the Cry48Aa1/Tpp49Aa1 toxicity. In this study, we employed pulldown assays to identify a group of Tpp49Aa1-binding proteins from C. quinquefasciatus solubilized midgut brush-border membrane proteins (BBMFs). RNA interference assays revealed that the silencing of an alkaline phosphatase gene (referred to as ALP1263) in C. quinquefasciatus resulted in a significant reduction in larval mortality upon exposure to Cry48Aa1/Tpp49Aa1 toxin in vivo. Furthermore, the ALP1263 protein exhibited specific and high-affinity binding to the Tpp49Aa1 toxin, with a dissociation constant (Kd) of approximately 57.3 nM. The dot blot analysis demonstrated that Tpp49Aa1 C-terminal region was essential for its interaction with the ALP1263 protein. In summary, our findings establish ALP1263 as a functional receptor for Tpp49Aa1 and emphasize its role in the toxicity of Cry48Aa1/Tpp49Aa1.

A Culex quinquefasciatus strain resistant to the binary toxin from Lysinibacillus sphaericus displays altered enzyme activities and energy reserves.

BACKGROUND: The resistance of a Culex quinquefasciatus strain to the binary (Bin) larvicidal toxin from Lysinibacillus sphaericus is due to the lack of expression of the toxin's receptors, the membrane-bound Cqm1 α-glucosidases. A previous transcriptomic profile of the resistant larvae showed differentially expressed genes coding Cqm1, lipases, proteases and other genes involved in lipid and carbohydrate metabolism. This study aimed to investigate the metabolic features of Bin-resistant individuals by comparing the activity of some enzymes, energy reserves, fertility and fecundity to a susceptible strain. METHODS: The activity of specific enzymes was recorded in midgut samples from resistant and susceptible larvae. The amount of lipids and reducing sugars was determined for larvae and adults from both strains. Additionally, the fecundity and fertility parameters of these strains under control and stress conditions were examined. RESULTS: Enzyme assays showed that the esterase activities in the midgut of resistant larvae were significantly lower than susceptible ones using acetyl-, butyryl- and heptanoyl-methylumbelliferyl esthers as substrates. The α-glucosidase activity was also reduced in resistant larvae using sucrose and a synthetic substrate. No difference in protease activities as trypsins, chymotrypsins and aminopeptidases was detected between resistant and susceptible larvae. In larval and adult stages, the resistant strain showed an altered profile of energy reserves characterized by significantly reduced levels of lipids and a greater amount of reducing sugars. The fertility and fecundity of females were similar for both strains, indicating that those changes in energy reserves did not affect these reproductive parameters. CONCLUSIONS: Our dataset showed that Bin-resistant insects display differential metabolic features co-selected with the phenotype of resistance that can potentially have effects on mosquito fitness, in particular, due to the reduced lipid accumulation.

New native Bacillus thuringiensis strains induce high insecticidal action against Culex pipiens pallens larvae and adults.

Mosquitoes of many species are key disease vectors, killing millions of people each year. Bacillus thuringiensis-based insecticide formulations are largely recognized as among the most effective, ecologically safe, and long-lasting methods of managing insect pests. New B. thuringiensis strains with high mosquito control effectiveness were isolated, identified, genetically defined, and physiologically characterized. Eight B. thuringiensis strains were identified and shown to carry endotoxin-producing genes. Using a scanning electron microscope, results revealed typical crystal forms of various shapes in B. thuringiensis strains. Fourteen cry and cyt genes were found in the strains examined. Although the genome of the B. thuringiensis A4 strain had twelve cry and cyt genes, not all of them were expressed, and only a few protein profiles were observed. The larvicidal activity of the eight B. thuringiensis strains was found to be positive (LC50: 1.4-28.5 g/ml and LC95: 15.3-130.3 g/ml). Bioassays in a laboratory environment demonstrated that preparations containing B. thuringiensis spores and crystals were particularly active to mosquito larvae and adults. These new findings show that the novel preparation containing B. thuringiensis A4 spores and crystals mixture might be used to control larval and adult mosquitoes in a sustainable and ecologically friendly manner.

A deeper insight into the sialome of male and female Culex quinquefasciatus mosquitoes.

INTRODUCTION: During evolution, blood-feeding arthropods developed a complex salivary mixture that can interfere with host haemostatic and immune response, favoring blood acquisition and pathogen transmission. Therefore, a survey of the salivary gland contents can lead to the identification of molecules with potent pharmacological activity in addition to increase our understanding of the molecular mechanisms underlying the hematophagic behaviour of arthropods. The southern house mosquito, Culex quinquefasciatus, is a vector of several pathogenic agents, including viruses and filarial parasites that can affect humans and wild animals. RESULTS: Previously, a Sanger-based transcriptome of the salivary glands (sialome) of adult C. quinquefasciatus females was published based on the sequencing of 503 clones organized into 281 clusters. Here, we revisited the southern mosquito sialome using an Illumina-based RNA-sequencing approach of both male and female salivary glands. Our analysis resulted in the identification of 7,539 coding DNA sequences (CDS) that were functionally annotated into 25 classes, in addition to 159 long non-coding RNA (LncRNA). Additionally, comparison of male and female libraries allowed the identification of female-enriched transcripts that are potentially related to blood acquisition and/or pathogen transmission. CONCLUSION: Together, these findings represent an extended reference for the identification and characterization of the proteins containing relevant pharmacological activity in the salivary glands of C. quinquefasciatus mosquitoes.

Diversity oriented synthesis and SAR studies of new quinazolinones and related compounds as insecticidal agents against Culex pipiens L. Larvae and associated predator.

The ongoing study reports the synthesis, spectroscopic analyses and larvicidal efficacy of novel series of quinazolinone derivatives and related compounds. The structures of the products were confirmed relied on their analytical and spectral data (IR, 1H NMR, and 13C NMR). The spectral documentation promoted the successful isolation of the desirable compounds. The insecticidal activities of the synthesized compounds were assessed against laboratory and field strains of Culex pipiens larvae and a predator from the same ecological niche, Cybister tripunctatus. The results revealed that most of the tested compounds showed high potencies against lab strain of C. pipiens larvae with low resistance ratios in filed strain. In particular, compounds 15, 6 and 16 showed low LC50 values, 0.094, 0.106, 0.129 (µg/mL), respectively against lab strain of C. pipiens larvae. The present study also explored the toxicity of tested compounds against field strain of non-target C. tripunctatus. Most of tested compounds were safer than temephos, especially 15 and 6 with SI/PSF values 96.746 and 83.167, respectively. Structure-activity relationship (SAR) was discussed the effect of substituents insertion on the derivatives activities. Quinazolinone derivatives and related compounds are promising compounds in the mosquito control programs and further studies are recommended to develop more effective derivatives and reveal their mode of action.

Synthesis, larvicidal efficiency and molecular docking studies of novel annulated pyrano[2,3-c]pyrazoles against Culex pipiens L. and Musca domestica L. larvae.

A number of novel annulated pyrazolopyranopyrimidines were prepared via reaction of iminoether of the corresponding 6-amino-5-cyano-pyrano[2,3-c]pyrazole derivative 1 with different nitrogen nucleophiles. The structure of the synthesized compounds was deduced based on IR, MS, 1H NMR and 13C NMR spectroscopic data. The larvicidal potency of the synthesized compounds against the lab and field strains of Culex pipiens and Musca domestica larvae was evaluated and the structure-activity relationship (SAR) was discussed. The assay revealed that the tested pyranopyrazole derivatives exhibited good larvicidal bio-efficacy whereas iminoether 4 exhibited the highest efficiency, for lab more than field strains of both species. Also, M. domestica larvae were more sensitive to tested compounds than C. pipiens. The field strain showed low resistance ratios to all compounds with only about 2 folds. The inhibitory effects of synthesized molecules on nAChRs were evaluated by molecular docking. Moreover, the cytotoxicity of the newly synthesized compounds against normal human fibroblasts (WI-38) was investigated. The cytotoxic assay showed that derivatives 4 and 5 were not harmful to normal fibroblasts.

The Crystal Structure of Bacillus thuringiensis Tpp80Aa1 and Its Interaction with Galactose-Containing Glycolipids.

Tpp80Aa1 from Bacillus thuringiensis is a Toxin_10 family protein (Tpp) with reported action against Culex mosquitoes. Here, we demonstrate an expanded target range, showing Tpp80Aa1 is also active against the larvae of Anopheles gambiae and Aedes aegypti mosquitoes. We report the first crystal structure of Tpp80Aa1 at a resolution of 1.8 Å, which shows Tpp80Aa1 consists of two domains: an N-terminal ß-trefoil domain resembling a ricin B lectin and a C-terminal putative pore-forming domain sharing structural similarity with the aerolysin family. Similar to other Tpp family members, we observe Tpp80Aa1 binds to the mosquito midgut, specifically the posterior midgut and the gastric caecum. We also identify that Tpp80Aa1 can interact with galactose-containing glycolipids and galactose, and this interaction is critical for exerting full insecticidal action against mosquito target cell lines.

Cry4Aa and Cry4Ba Mosquito-Active Toxins Utilize Different Domains in Binding to a Particular Culex ALP Isoform: A Functional Toxin Receptor Implicating Differential Actions on Target Larvae.

The three-domain Cry4Aa toxin produced from Bacillus thuringiensis subsp. israelensis was previously shown to be much more toxic to Culex mosquito larvae than its closely related toxin-Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from Culex quinquefasciatus larvae, Cq-mALP1263and Cq-mALP1264, predicted to be GPI-linked was cloned and functionally expressed in Spodoptera frugiperda (Sf9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that both Cq-mALP isoforms share significant sequence similarity to Aedes aegypti-mALP-a Cry4Ba toxin receptor. In cytotoxicity assays, Sf9 cells expressing Cq-mALP1264, but not Cq-mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each Cq-mALP expressed on the cell membrane surface. Molecular docking of the Cq-mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to Cq-mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain-DII, including Thr512, Tyr513 and Lys514 in the ß10-ß11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain-DIII to interact with such a Culex counterpart receptor. Ala-substitutions of selected ß10-ß11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against C. quinquefasciatus larvae. Further substitution of Lys514 with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔGbind) in Cry4Aa-Cq-mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔGbind value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position. Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same Culex membrane-bound ALP isoform-Cq-mALP1264 for mediating differential toxicity against Culex target larvae.

Insecticidal efficacy of nanomaterials used to control mosquito, Culex quinquefasciatus Say, 1823 with special reference to their hepatotoxicity in rats.

The present study aimed to develop a novel methodology for controlling the mosquito larvae using different nanoparticles, with special reference to their effect on rats (a non-target mammalian model). The mosquito species of Culex quinquefasciatus was reared in the laboratory. Chitosan, silver nanoparticles and their combination as well as lavender (Lavandula officinalis) nanoemulsion with different concentrations were tested as biological insecticides against the mosquito larvae. Mammalian toxicity of the used nanoparticles were evaluated using 27 adult male rats, experimental rats were divided into 9 equal groups (n=3). The nanoparticles were added to the drinking water for 30 days. At the end of the study, blood and tissue samples were collected to assess the levels of the serum alanine aminotransferase and aspartate aminotransferase, different genes expression as interleukin 6 (IL-6) and IL-1ß activity. Histopathological and immunohistochemical studies using two markers (TNF-α and BAX expression) were also applied. The LC50 and LC90 were recorded for each tested nanoparticles, and also the changes of the treated mosquito larvae cuticle were assessed using the scanning electron microscopy. Green nanoemulsion (Lavandula officinalis) was more effective than metal (silver) or even biodegradable (chitosan) nanoparticles in controlling of Culex quiquefasciatus mosquito larvae, and also it proved its safety by evaluation of the mammalian hepatotoxicity of the tested nanoparticles.

Culex quinquefasciatus alpha-glucosidase serves as a putative receptor of the Cry48Aa toxin from Lysinibacillus sphaericus.

The Cry48Aa/Cry49Aa toxin of Lysinibacillus sphaericus shows specific toxicity towards larvae of Culex spp. Individual Cry48Aa and Cry49Aa subunits interact with distinct target sites in the larval midgut and overcome the resistance of Culex to the Bin toxin. However, the toxin-binding proteins have not yet been identified. The present study aimed to identify Cry48Aa-binding proteins in Culex quinquefasciatus. Pulldown assays using C. quinquefasciatus midgut brush-border membrane fractions (BBMFs) identified a class of proteins, including aminopeptidases (APNs), protease m1 zinc metalloproteases, alkaline phosphatases (ALPs), and maltases, that could be potentially involved in the mode of action of this toxin. RNA interference analysis showed that silenced larvae treated with dsRNA of the alpha-glucosidase (named Glu71) gene were more tolerant of the Cry48Aa/Cry49Aa toxin, which induced less than 20% mortality. The amino acid sequence of Glu71 exhibited 42% identity with Cqm1/Cpm1, which acted as a Bin toxin receptor. Toxin binding assays showed that Cry48Aa had a high specific binding capacity for the Glu71 protein, whereas Cry49Aa exhibited no specific binding. Overall, our results showed that Glu71 is a Cry48-binding protein involved in Cry48Aa/Cry49Aa toxicity.

Screening of olfactory genes related to blood-feeding behaviors in Culex pipiens quinquefasciatus and Culex pipiens molestus by transcriptome analysis.

BACKGROUND: Culex pipiens quinquefasciatus Say (Cx. quinquefasciatus) and Culex pipiens form molestus Forskal (Cx. molestus) in the Culex pipiens complex group show considerable differences in host seeking, blood feeding, mating behavior and in vector competence. Blood-feeding mosquito behaviors are closely related to their olfactory gene expression and olfactory gene repertoire composition. Comparing olfactory genes between these two subspecies with significantly different blood-feeding behaviors can support further research on the molecular mechanism of the Culex pipiens complex olfactory sensory system, providing a new approach for determining candidate attractant or repellent compounds. METHODS: Non-blood-feeding (NBF) and post-blood-feeding (PBF) olfactory system transcriptomes of the two subspecies were sequenced, and the biological functions of their differentially expressed genes were described by bioinformatics analysis. A quantitative polymerase chain reaction (qPCR) was applied to validate the RNA-seq data. The roles of particular olfactory receptors in Cx. quinquefasciatus blood-feeding behaviors were evaluated by RNAi. RESULTS: Five, 7, 24, and 3 Cx. quinquefasciatus-specific OBPs, Cx. molestus-specific OBPs, Cx. quinquefasciatus-specific ORs and Cx. molestus-specific ORs were identified, respectively. The majority of selected ORs were consistent with the predicted transcriptome sequencing results after qRT-PCR validation. OR5 was expressed only in Cx. quinquefasciatus, and OR65 was the only gene upregulated after blood feeding in Cx. molestus. The blood-feeding rates of the OR5 and OR78 dsRNA groups were significantly lower (4.3%±3.1% and 13.3%±11.5%) than those of the enhanced green fluorescence protein (EGFP) group (64.5%±8.7%). CONCLUSION: Most OBPs and ORs were expressed in both subspecies but showed divergence in expression level. OR5 and OR65 might be species-specific expressed genes that regulate the olfactory behaviors of Cx. quinquefasciatus and Cx. molestus, respectively. The RNA interference of OR5 and OR78 could inhibit the blood-feeding behavior of Cx. quinquefasciatus, providing new targets for screening effective repellent compounds to control mosquito-borne diseases effectively and efficiently.

Deltamethrin interacts with Culex quinquefasciatus odorant-binding protein: a novel potential resistance mechanism.

BACKGROUND: Odorant-binding proteins (OBPs) play important roles in many physiological processes of mosquitoes. Previous high-throughput sequencing studies have revealed that some OBPs of Culex quinquefasciatus might be involved in the development of resistance to insecticides. METHODS: Based on the results of sequencing analyses, the OBP28 gene was selected for evaluation in this study. Three laboratory strains of Cx. quinquefasciatus [susceptible strain (SS), deltamethrin-resistant strain 1 (HN) and deltamethrin-resistant strain 2 (RR)] were first examined by using the Centers for Disease Control and Prevention bottle bioassay, after which the expression level of the OBP28 gene in the susceptible and deltamethrin-resistant strains was determined by real-time quantitative polymerase chain reaction. The OBP28 gene in deltamethrin-resistant strain RR was silenced using RNA interference technology. The expression level of OBP28 and the resistance level were tested in the silenced strain and control strain after microinjection of double-stranded RNA for a 48-h interference period. Four field-collected strains (henceforth 'field strains') of Cx. quinquefasciatus were also examined for their resistance to deltamethrin and levels of OBP28 expression. Finally, a correlation analysis between deltamethrin resistance and gene expression was carried out for all seven strains, i.e. the four field strains and the three laboratory strains. RESULTS: In the bioassay, the mortality of SS, HN and RR was 100%, 21.33% and 1.67%, respectively. The relative expression levels of OBP28 in strains HN and RR were 6.30- and 6.86-fold higher, respectively, than that of strain SS. After silencing of the OBP28 gene, the mortality of strain RR was 72.20% and that of the control strain 26.32%. The mortality of strain RR increased significantly after interference compared to that of the control strain. There was a negative correlation between OBP28 gene expression and mortality in adult mosquitoes after exposure to deltamethrin. CONCLUSIONS: To our knowledge, this study shows for the first time a correlation between the expression of a gene coding for OBP and insecticide resistance in mosquitoes. The potential resistance mechanism that was elucidated provides a new target gene for the surveillance of resistance in mosquitoes.

Susceptibility of Culex quinquefasciatus (Say) Larvae to Methanolic Extracts of Annona reticulata.

<b>Background and Objective:</b> Environment pollution and resistance of many pests to the most frequently used chemical insecticides gave the rationale of altering to replace them with natural herbal extracts for pests and vector control. This study investigated the effect of methanolic extract of <i>Annona reticulata</i> on the mortality and development of larvae of <i>Culex quinquefasciatus</i>. <b>Materials and Methods:</b> Methanolic herbal extracts were analyzed using Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) to identify the phytochemical compounds in them. Ten mosquito larvae were used as replicates and exposed to each of the five concentrations of the plant extract (30, 100, 150, 200 and 250 mg mL¹) and ten larvae were exposed to double distilled water and considered as control. <b>Results:</b> Phytochemical analysis revealed the presence of phenols, steroids, quinones, tannins and saponins. Statistical analysis showed a significant strong correlation and regression between exposure to the different concentrations of the extract and mortality of <i>Culex quinquefasciatus</i> larvae where R² = 0.982, the Correlation value is 0.991099 (p<0.05). Results also showed that the extracts affect the development of larvae more than mortality. The effect of the extracts on the early larval stages was significantly high compared to the late stages of larvae. <b>Conclusion:</b> According to our knowledge results of this study has been reported for the first time in Saudi Arabia where <i>Annona reticulata</i> is neglected regionally and no study approved its efficacy as a botanical herbal extract against mosquitoes.

Microbiome reduction prevents lipid accumulation during early diapause in the northern house mosquito, Culex pipiens pipiens.

The mosquito microbiome is critical to multiple facets of their biology, including larval development and disease transmission. For mosquitoes that reside in temperate regions, periods of diapause are critical to overwintering survival, but how the microbiome impacts this state is unknown. In this study, we compared the midgut microbial communities of diapausing and non-diapausing Culex pipiens and assessed how a reduced midgut microbiome influences diapause preparation. High community variability was found within and between non-diapausing and diapausing individuals, but no specific diapause-based microbiome was noted. Emergence of adult, diapausing mosquitoes under sterile conditions generated low bacterial load (LBL) lines with nearly a 1000-fold reduction in bacteria levels. This reduction in bacterial content resulted in significantly lower survival of diapausing females after two weeks, indicating acquisition of the microbiome in adult females is critical for survival throughout diapause. LBL diapausing females had high carbohydrate levels, but did not accumulate lipid reserves, suggesting an inability to process ingested sugars necessary for diapause-associated lipid accumulation. Expression patterns of select genes associated with mosquito lipid metabolism during diapause showed no significant differences between LBL and control lines, suggesting transcriptional changes may not underlie impaired lipid accumulation. Overall, a diverse, adult-acquired microbiome is critical for diapause in C. pipiens to process sugar reserves and accumulate lipids that are necessary to survive prolonged overwintering.

Physiological characterization of chitin synthase A responsible for the biosynthesis of cuticle chitin in Culex pipiens pallens (Diptera: Culicidae).

BACKGROUND: The pathogens transmitted by mosquitoes to humans and animals cause several emerging and resurgent infectious diseases. Increasing insecticide resistance requires rational action to control the target vector population. Chitin is indispensable for insect growth and development and absent from vertebrates and higher plants. Chitin synthase A (CHSA) is a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines chitin content may contribute to the development of novel vector control strategies. RESULTS: The injection of small interfering RNA targeting CHSA (siCHSA) to knockdown CHSA transcripts in larval, pupal and adult stages of Culex pipiens pallens resulted in the appearance of different lethal phenotypes. When larval and pupal stages were injected with siCHSA, CHSA knockdown prevented larval molting, pupation and adult eclosion, and affected the production of chitin and chitin degradation, which resulted in an ecdysis defect phenotype of mosquitoes. When siCHSA was injected into mosquitoes in the adult stage, CHSA knockdown also affected the laminar organization of the mesoderm and the formation of pseudo-orthogonal patterns of the large fibers of the endoderm. CONCLUSION: We provide a systematic and comprehensive description of the effects of CHSA on morphogenesis and metamorphosis. The results show that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our results further show that CHSA is important for the structural integrity of the adult mosquito cuticle.

Whole blood and blood components from vertebrates differentially affect egg formation in three species of anautogenous mosquitoes.

BACKGROUND: Most female mosquitoes are anautogenous and must blood feed on a vertebrate host to produce eggs. Prior studies show that the number of eggs females lay per clutch correlates with the volume of blood ingested and that protein is the most important macronutrient for egg formation. In contrast, how whole blood, blood fractions and specific blood proteins from different vertebrates affect egg formation is less clear. Since egg formation is best understood in Aedes aegypti, we examined how blood and blood components from different vertebrates affect this species and two others: the malaria vector Anopheles gambiae and arbovirus vector Culex quinquefasciatus. METHODS: Adult female mosquitoes were fed blood, blood fractions and purified major blood proteins from different vertebrate hosts. Markers of reproductive response including ovary ecdysteroidogenesis, yolk deposition into oocytes and number of mature eggs produced were measured. RESULTS: Ae. aegypti, An. gambiae and C. quinquefasciatus responded differently to meals of whole blood, plasma or blood cells from human, rat, chicken and turkey hosts. We observed more similarities between the anthropophiles Ae. aegypti and An. gambiae than the ornithophile C. quinquefasciatus. Focusing on Ae. aegypti, the major plasma-derived proteins (serum albumin, fibrinogen and globulins) differentially stimulated egg formation as a function of vertebrate host source. The major blood cell protein, hemoglobin, stimulated yolk deposition when from pigs but not humans, cows or sheep. Serum albumins from different vertebrates also variably affected egg formation. Bovine serum albumin (BSA) stimulated ovary ecdysteroidogenesis, but more weakly induced digestive enzyme activities than whole blood. In contrast, BSA-derived peptides and free amino acids had no stimulatory effects on ecdysteroidogenesis or yolk deposition into oocytes. CONCLUSIONS: Whole blood, blood fractions and specific blood proteins supported egg formation in three species of anautogenous mosquitoes but specific responses varied with the vertebrate source of the blood components tested.

Determining the effects of nutrition on the reproductive physiology of male mosquitoes.

Nutrition affects multiple aspects of insect physiology such as body size and fecundity, but we lack a detailed understanding of how nutrition influences the reproductive physiology of male insects such as mosquitoes. Given that female mosquitoes are vectors of many deadly diseases and can quickly proliferate, understanding how male nutrition impacts female fecundity could be of critical importance. To uncover the relationship between nutrition in adult male mosquitoes and its impacts on reproductive physiology, we reared larvae of the Northern house mosquito, Culex pipiens, on a standard lab diet and divided adult males among three different dietary treatments: low (3%), moderate (10%), and high (20%) sucrose. We found that although overall body size did not differ among treatments, one-week-old males raised on the 3% sucrose diet had significantly smaller male accessory glands (MAGs) compared to males that consumed the 10% and the 20% sucrose diets. Diet affected whole-body lipid content but did not affect whole-body protein content. Using nuclear magnetic resonance (NMR) spectroscopy, we found that diet altered the metabolic composition of the MAGs, including changes in lactic acid, formic acid, and glucose. We also observed changes in protein and lipid abundance and composition in MAGs. Females who mated with males on the 3% diet were found to produce significantly fewer larvae than females who had mated with males on the 10% diet. Taken together, our results demonstrate that the diet of adult male mosquitoes clearly affects male reproductive physiology and female fecundity.

Do energy reserves and cold hardiness limit winter survival of Culex pipiens?

The risks of depletion of energy reserves and encountering lethally low temperatures are considered as two important mortality factors that may limit winter survival of mosquito, Culex pipiens f. pipiens populations. Here we show that the autumn females carry lipid reserves, which are safely sufficient for at least two overwintering periods, provided the females diapausing at temperatures typical for underground spaces (0 °C - 8 °C) would continuously rest at a standard metabolic rate (SMR). The overwintering females, however, switch from SMR to much higher metabolic rate during flight, either seeking for optimal microhabitat within the shelter or in response to disturbances by air current or predator attack. These behaviors result in fast oxidation of lipid reserves and, therefore, the autumn load of energy reserves may actually limit winter survival under specific circumstances. Next, we show that the level of females' cold hardiness is physiologically set relatively weak for overwintering in open field, above-ground habitats, but is ecologically entirely sufficient for overwintering in most underground spaces. The characteristics of suitable overwintering shelters are: no or limited risk of contact with ice crystals, no or limited air movements, winter temperatures relatively stable between +2 and + 6 °C, winter minimum does not drop below -4 °C for longer than one week, or below -8 °C for longer than 1 day.