Isolation, characterization and functional analysis of a bacteriophage targeting Culex pipiens pallens resistance-associated Aeromonas hydrophila.

BACKGROUND: Culex pipiens pallens is a well-known mosquito vector for several diseases. Deltamethrin, a commonly used pyrethroid insecticide, has been frequently applied to manage adult Cx. pipiens pallens. However, mosquitoes can develop resistance to these insecticides as a result of insecticide misuse and, therefore, it is crucial to identify novel methods to control insecticide resistance. The relationship between commensal bacteria and vector resistance has been recently recognized. Bacteriophages (= phages) are effective tools by which to control insect commensal bacteria, but there have as yet been no studies using phages on adult mosquitoes. In this study, we isolated an Aeromonas phage vB AhM-LH that specifically targets resistance-associated symbiotic bacteria in mosquitoes. We investigated the impact of Aeromonas phage vB AhM-LH in an abundance of Aeromonas hydrophila in the gut of Cx. pipiens pallens and its effect on the status of deltamethrin resistance. METHODS: Phages were isolated on double-layer agar plates and their biological properties analyzed. Phage morphology was observed by transmission electron microscopy (TEM) after negative staining. The phage was then introduced into the mosquito intestines via oral feeding. The inhibitory effect of Aeromonas phage vB AhM-LH on Aeromonas hydrophila in mosquito intestines was assessed through quantitative real-time PCR analysis. Deltamethrin resistance of mosquitoes was assessed using WHO bottle bioassays. RESULTS: An Aeromonas phage vB AhM-LH was isolated from sewage and identified as belonging to the Myoviridae family in the order Caudovirales using TEM. Based on biological characteristics analysis and in vitro antibacterial experiments, Aeromonas phage vB AhM-LH was observed to exhibit excellent stability and effective bactericidal activity. Sequencing revealed that the Aeromonas phage vB AhM-LH genome comprises 43,663 bp (51.6% CG content) with 81 predicted open reading frames. No integrase-related gene was detected in the vB AH-LH genome, which marked it as a potential biological antibacterial. Finally, we found that Aeromonas phage vB AhM-LH could significantly reduce deltamethrin resistance in Cx. pipiens pallens, in both the laboratory and field settings, by decreasing the abundance of Aeromonas hydrophila in their midgut. CONCLUSIONS: Our findings demonstrate that Aeromonas phage vB AhM-LH could effectively modulate commensal bacteria Aeromonas hydrophila in adult mosquitoes, thus representing a promising strategy to mitigate mosquito vector resistance.

Updates on traditional methods for combating malaria and emerging Wolbachia-based interventions.

The escalating challenge of malaria control necessitates innovative approaches that extend beyond traditional control strategies. This review explores the incorporation of traditional vector control techniques with emerging Wolbachia-based interventions. Wolbachia, a naturally occurring bacteria, offers a novel approach for combatting vector-borne diseases, including malaria, by reducing the mosquitoes' ability to transmit these diseases. The study explores the rationale for this integration, presenting various case studies and pilot projects that have exhibited significant success. Employing a multi-dimensional approach that includes community mobilization, environmental modifications, and new biological methods, the paper posits that integrated efforts could mark a turning point in the struggle against malaria. Our findings indicate that incorporating Wolbachia-based strategies into existing vector management programs not only is feasible but also heightens the efficacy of malaria control initiatives in different countries especially in Pakistan. The paper concludes that continued research and international collaboration are imperative for translating these promising methods from the laboratory to the field, thereby offering a more sustainable and effective malaria control strategy.

Mosquito Microbiomes of Rwanda: Characterizing Mosquito Host and Microbial Communities in the Land of a Thousand Hills.

Mosquitoes are a complex nuisance around the world and tropical countries bear the brunt of the burden of mosquito-borne diseases. Rwanda has had success in reducing malaria and some arboviral diseases over the last few years, but still faces challenges to elimination. By building our understanding of in situ mosquito communities in Rwanda at a disturbed, human-occupied site and at a natural, preserved site, we can build our understanding of natural mosquito microbiomes toward the goal of implementing novel microbial control methods. Here, we examined the composition of collected mosquitoes and their microbiomes at two diverse sites using Cytochrome c Oxidase I sequencing and 16S V4 high-throughput sequencing. The majority (36 of 40 species) of mosquitoes captured and characterized in this study are the first-known record of their species for Rwanda but have been characterized in other nations in East Africa. We found significant differences among mosquito genera and among species, but not between mosquito sexes or catch method. Bacteria of interest for arbovirus control, Asaia, Serratia, and Wolbachia, were found in abundance at both sites and varied greatly by species.

Comparative analysis of midgut bacterial communities in Chikungunya virus-infected and non-infected Aedes aegypti Thai laboratory strain mosquitoes.

Chikungunya virus (CHIKV) poses a significant global health threat, re-emerging as a mosquito-transmitted pathogen that caused high fever, rash, and severe arthralgia. In Thailand, a notable CHIKV outbreak in 2019-2020 affected approximately 20,000 cases across 60 provinces, underscoring the need for effective mosquito control protocols. Previous studies have highlighted the role of midgut bacteria in the interaction between mosquito vectors and pathogen infections, demonstrating their ability to protect the insect from invading pathogens. However, research on the midgut bacteria of Aedes (Ae.) aegypti, the primary vector for CHIKV in Thailand remains limited. This study aims to characterize the bacterial communities in laboratory strains of Ae. aegypti, both infected and non-infected with CHIKV. Female mosquitoes from a laboratory strain of Ae. aegypti were exposed to a CHIKV-infected blood meal through membrane feeding, while the control group received a non-infected blood meal. At 7 days post-infection (dpi), mosquito midguts were dissected for 16S rRNA gene sequencing to identify midgut bacteria, and CHIKV presence was confirmed by E1-nested RT-PCR using mosquito carcasses. The study aimed to compare the bacterial communities between CHIKV-infected and non-infected groups. The analysis included 12 midgut bacterial samples, divided into three groups: CHIKV-infected (exposed and infected), non-infected (exposed but not infected), and non-exposed (negative control). Alpha diversity indices and Bray-Curtis dissimilarity matrix revealed significant differences in bacterial profiles among the three groups. The infected group exhibited an increased abundance of bacteria genus Gluconobacter, while Asaia was prevalent in both non-infected and negative control groups. Chryseobacterium was prominent in the negative control group. These findings highlight potential alterations in the distribution and abundance of gut microbiomes in response to CHIKV infection status. This study provides valuable insights into the dynamic relationship between midgut bacteria and CHIKV, underscoring the potential for alterations in bacterial composition depending on infection status. Understanding the relationships between mosquitoes and their microbiota holds promise for developing new methods and tools to enhance existing strategies for disease prevention and control. This research advances our understanding of the circulating bacterial composition, opening possibilities for new approaches in combating mosquito-borne diseases.

Wolbachia infection-responsive immune genes suppress Plasmodium falciparum infection in Anopheles stephensi.

Wolbachia, a maternally transmitted symbiotic bacterium of insects, can suppress a variety of human pathogens in mosquitoes, including malaria-causing Plasmodium in the Anopheles vector. However, the mechanistic basis of Wolbachia-mediated Plasmodium suppression in mosquitoes is not well understood. In this study, we compared the midgut and carcass transcriptomes of stably infected Anopheles stephensi with Wolbachia wAlbB to uninfected mosquitoes in order to discover Wolbachia infection-responsive immune genes that may play a role in Wolbachia-mediated anti-Plasmodium activity. We show that wAlbB infection upregulates 10 putative immune genes and downregulates 14 in midguts, while it upregulates 31 putative immune genes and downregulates 15 in carcasses at 24 h after blood-fed feeding, the time at which the Plasmodium ookinetes are traversing the midgut tissue. Only a few of these regulated immune genes were also significantly differentially expressed between Wolbachia-infected and non-infected midguts and carcasses of sugar-fed mosquitoes. Silencing of the Wolbachia infection-responsive immune genes TEP 4, TEP 15, lysozyme C2, CLIPB2, CLIPB4, PGRP-LD and two novel genes (a peritrophin-44-like gene and a macro domain-encoding gene) resulted in a significantly greater permissiveness to P. falciparum infection. These results indicate that Wolbachia infection modulates mosquito immunity and other processes that are likely to decrease Anopheles permissiveness to Plasmodium infection.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

Wolbachia-carrying Aedes mosquitoes for preventing dengue infection.

BACKGROUND: Dengue is a global health problem of high significance, with 3.9 billion people at risk of infection. The geographic expansion of dengue virus (DENV) infection has resulted in increased frequency and severity of the disease, and the number of deaths has increased in recent years. Wolbachia,an intracellular bacterial endosymbiont, has been under investigation for several years as a novel dengue-control strategy. Some dengue vectors (Aedes mosquitoes) can be transinfected with specific strains of Wolbachia, which decreases their fitness (ability to survive and mate) and their ability to reproduce, inhibiting the replication of dengue. Both laboratory and field studies have demonstrated the potential effect of Wolbachia deployments on reducing dengue transmission, and modelling studies have suggested that this may be a self-sustaining strategy for dengue prevention, although long-term effects are yet to be elucidated. OBJECTIVES: To assess the efficacy of Wolbachia-carrying Aedes speciesdeployments (specifically wMel-, wMelPop-, and wAlbB- strains of Wolbachia) for preventing dengue virus infection. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, four other databases, and two trial registries up to 24 January 2024. SELECTION CRITERIA: Randomized controlled trials (RCTs), including cluster-randomized controlled trials (cRCTs), conducted in dengue endemic or epidemic-prone settings were eligible. We sought studies that investigated the impact of Wolbachia-carrying Aedes deployments on epidemiological or entomological dengue-related outcomes, utilizing either the population replacement or population suppression strategy. DATA COLLECTION AND ANALYSIS: Two review authors independently selected eligible studies, extracted data, and assessed the risk of bias using the Cochrane RoB 2 tool. We used odds ratios (OR) with the corresponding 95% confidence intervals (CI) as the effect measure for dichotomous outcomes. For count/rate outcomes, we planned to use the rate ratio with 95% CI as the effect measure. We used adjusted measures of effect for cRCTs. We assessed the certainty of evidence using GRADE. MAIN RESULTS: One completed cRCT met our inclusion criteria, and we identified two further ongoing cRCTs. The included trial was conducted in an urban setting in Yogyakarta, Indonesia. It utilized a nested test-negative study design, whereby all participants aged three to 45 years who presented at healthcare centres with a fever were enrolled in the study provided they had resided in the study area for the previous 10 nights. The trial showed that wMel-Wolbachia infected Ae aegypti deployments probably reduce the odds of contracting virologically confirmed dengue by 77% (OR 0.23, 95% CI 0.15 to 0.35; 1 trial, 6306 participants; moderate-certainty evidence). The cluster-level prevalence of wMel Wolbachia-carrying mosquitoes remained high over two years in the intervention arm of the trial, reported as 95.8% (interquartile range 91.5 to 97.8) across 27 months in clusters receiving wMel-Wolbachia Ae aegypti deployments, but there were no reliable comparative data for this outcome. Other primary outcomes were the incidence of virologically confirmed dengue, the prevalence of dengue ribonucleic acid in the mosquito population, and mosquito density, but there were no data for these outcomes. Additionally, there were no data on adverse events. AUTHORS' CONCLUSIONS: The included trial demonstrates the potential significant impact of wMel-Wolbachia-carrying Ae aegypti mosquitoes on preventing dengue infection in an endemic setting, and supports evidence reported in non-randomized and uncontrolled studies. Further trials across a greater diversity of settings are required to confirm whether these findings apply to other locations and country settings, and greater reporting of acceptability and cost are important.

Efficacy of Native Bacillus thuringiensis against Mosquito Vector.

BACKGROUND: Larval source management is an effective measure to control mosquito-borne diseases. Bacillus thuringiensis produces specific insecticidal crystal proteins toxic to mosquito larvae. In many parts of the South East Asian region, Bacillus thuringiensis is used for larval source management. In Nepal, larvicidal Bacillus thuringiensis is not available. The study aims to isolate larvicidal Bacillus thuringiensis from soil samples of Nepal to control mosquitoes. METHODS: Native Bacillus thuringiensis was obtained from soil samples by the acetate selection method. It was identified by observing crystal protein with Coomassie Brilliant Blue stain in a light microscope. The mosquito larvae were collected from different breeding habitats. A preliminary bioassay was performed by inoculating three loopful of 48 hours culture of spherical crystal protein producing Bacillus thuringiensis in a plastic cup containing 25 larvae and 100 ml of sterile distilled water. The cup was incubated at room temperature for 24 hours to observe the mortality of larvae. Further selective bioassay was performed with the isolate which showed 100% mortality, as described above in four replicates along with the negative and positive control. RESULTS: Out of 1385 Bacillus thuringiensis obtained from 454 soil samples, 766 (55.30%) were spherical crystal protein producers, among them, a single strain (14P2A) showed 100% mortality against mosquito larvae. The lethal concentration doses required to kill 50% and 90% of the larval population were 32.35 and 46.77 Parts per million respectively. CONCLUSIONS: The native Bacillus thuringiensis produces the crystal protein effective in killing mosquito larvae. The native Bacillus thuringiensis should be included as a tool to control mosquito-borne diseases in Nepal.

Wolbachia invasion dynamics of a random mosquito population model with imperfect maternal transmission and incomplete CI.

In this work, we formulate a random Wolbachia invasion model incorporating the effects of imperfect maternal transmission and incomplete cytoplasmic incompatibility (CI). Under constant environments, we obtain the following results: Firstly, the complete invasion equilibrium of Wolbachia does not exist, and thus the population replacement is not achievable in the case of imperfect maternal transmission; Secondly, imperfect maternal transmission or incomplete CI may obliterate bistability and backward bifurcation, which leads to the failure of Wolbachia invasion, no matter how many infected mosquitoes would be released; Thirdly, the threshold number of the infected mosquitoes to be released would increase with the decrease of the maternal transmission rate or the intensity of CI effect. In random environments, we investigate in detail the Wolbachia invasion dynamics of the random mosquito population model and establish the initial release threshold of infected mosquitoes for successful invasion of Wolbachia into the wild mosquito population. In particular, the existence and stability of invariant probability measures for the establishment and extinction of Wolbachia are determined.

Revealing the microbiome diversity and biocontrol potential of field Aedes ssp.: Implications for disease vector management.

The mosquito Aedes spp. holds important relevance for human and animal health, as it serves as a vector for transmitting multiple diseases, including dengue and Zika virus. The microbiome's impact on its host's health and fitness is well known. However, most studies on mosquito microbiomes have been conducted in laboratory settings. We explored the mixed microbial communities within Aedes spp., utilizing the 16S rRNA gene for diversity analysis and shotgun metagenomics for functional genomics. Our samples, which included Ae. aegypti and Ae. albopictus, spanned various developmental stages-eggs, larvae, and adults-gathered from five semiurban areas in Mexico. Our findings revealed a substantial diversity of 8,346 operational taxonomic units (OTUs), representing 967 bacterial genera and 126,366 annotated proteins. The host developmental stage was identified as the primary factor associated with variations in the microbiome composition. Subsequently, we searched for genes and species involved in mosquito biocontrol. Wolbachia accounted for 9.6% of the 16S gene sequences. We observed a high diversity (203 OTUs) of Wolbachia strains commonly associated with mosquitoes, such as wAlb, with a noticeable increase in abundance during the adult stages. Notably, we detected the presence of the cifA and cifB genes, which are associated with Wolbachia's cytoplasmic incompatibility, a biocontrol mechanism. Additionally, we identified 221 OTUs related to Bacillus, including strains linked to B. thuringiensis. Furthermore, we discovered multiple genes encoding insecticidal toxins, such as Cry, Mcf, Vip, and Vpp. Overall, our study contributes to the understanding of mosquito microbiome biodiversity and metabolic capabilities, which are essential for developing effective biocontrol strategies against this disease vector.

Successful introgression of wMel Wolbachia into Aedes aegypti populations in Fiji, Vanuatu and Kiribati.

Pacific Island countries have experienced periodic dengue, chikungunya and Zika outbreaks for decades. The prevention and control of these mosquito-borne diseases rely heavily on control of Aedes aegypti mosquitoes, which in most settings are the primary vector. Introgression of the intracellular bacterium Wolbachia pipientis (wMel strain) into Ae. aegypti populations reduces their vector competence and consequently lowers dengue incidence in the human population. Here we describe successful area-wide deployments of wMel-infected Ae. aegypti in Suva, Lautoka, Nadi (Fiji), Port Vila (Vanuatu) and South Tarawa (Kiribati). With community support, weekly releases of wMel-infected Ae. aegypti mosquitoes for between 2 to 5 months resulted in wMel introgression in nearly all locations. Long term monitoring confirmed a high, self-sustaining prevalence of wMel infecting mosquitoes in almost all deployment areas. Measurement of public health outcomes were disrupted by the Covid19 pandemic but are expected to emerge in the coming years.

Efficacy of Wolbachia-mediated sterility to reduce the incidence of dengue: a synthetic control study in Singapore.

BACKGROUND: Due to the absence of available therapeutics and good vaccines, vector control solutions are needed to mitigate the spread of dengue. Matings between male Aedes aegypti mosquitoes infected with the wAlbB strain of Wolbachia and wildtype females yield non-viable eggs. We evaluated the efficacy of releasing wAlbB-infected A aegypti male mosquitoes to suppress dengue incidence. METHODS: In this synthetic control study, we conducted large-scale field trials in Singapore involving release of wAlbB-infected A aegypti male mosquitoes for dengue control via vector population suppression, from epidemiological week (EW) 27, 2018, to EW 26, 2022. We selected two large towns (Yishun and Tampines) to adopt an expanding release strategy and two smaller towns (Bukit Batok and Choa Chu Kang) to adopt a targeted-release approach. Releases were conducted two times a week in high-rise public housing estates. All intervention and control locations practised the same baseline dengue control protocol. The main outcome was weekly dengue incidence rate caused by any dengue virus serotype. We used incidence data collected by the Singapore Ministry of Health to assess the efficacy of the interventions. To compare interventions, we used the synthetic control method to generate appropriate counterfactuals for the intervention towns using a weighted combination of 30 control towns between EW 1, 2014 and EW 26, 2022. FINDINGS: Our study comprised an at-risk population of 607 872 individuals living in intervention sites and 3 894 544 individuals living in control sites. Interventions demonstrated up to 77·28% (121/156, 95% CI 75·81-78·58) intervention efficacy despite incomplete coverage across all towns until EW 26, 2022. Intervention efficacies increased as release coverage increased across all intervention sites. Releases led to 2242 (95% CI 2092-2391) fewer cases per 100 000 people in intervention sites during the study period. Secondary analysis showed that these intervention effects were replicated across all age groups and both sexes for intervention sites. INTERPRETATION: Our results demonstrated the potential of Wolbachia-mediated incompatible insect technique for strengthening dengue control in tropical cities, where dengue burden is the greatest. FUNDING: Singapore Ministry of Finance, Ministry of Sustainability, and the National Environment Agency, and the Singapore National Robotics Program.

Response of the mosquito immune system and symbiotic bacteria to pathogen infection.

Mosquitoes are the deadliest animal in the word, transmitting a variety of insect-borne infectious diseases, such as malaria, dengue fever, yellow fever, and Zika, causing more deaths than any other vector-borne pathogen. Moreover, in the absence of effective drugs and vaccines to prevent and treat insect-borne diseases, mosquito control is particularly important as the primary measure. In recent decades, due to the gradual increase in mosquito resistance, increasing attention has fallen on the mechanisms and effects associated with pathogen infection. This review provides an overview of mosquito innate immune mechanisms in terms of physical and physiological barriers, pattern recognition receptors, signalling pathways, and cellular and humoral immunity, as well as the antipathogenic effects of mosquito symbiotic bacteria. This review contributes to an in-depth understanding of the interaction process between mosquitoes and pathogens and provides a theoretical basis for biological defence strategies against mosquito-borne infectious diseases.

Aedes aegypti microbiome composition covaries with the density of Wolbachia infection.

BACKGROUND: Wolbachia is a widespread bacterial endosymbiont that can inhibit vector competency when stably transinfected into the mosquito, Aedes aegypti, a primary vector of the dengue virus (DENV) and other arboviruses. Although a complete mechanistic understanding of pathogen blocking is lacking, it is likely to involve host immunity induction and resource competition between Wolbachia and DENV, both of which may be impacted by microbiome composition. The potential impact of Wolbachia transinfection on host fitness is also of importance given the widespread release of mosquitos infected with the Drosophila melanogaster strain of Wolbachia (wMel) in wild populations. Here, population-level genomic data from Ae. aegypti was surveyed to establish the relationship between the density of wMel infection and the composition of the host microbiome. RESULTS: Analysis of genomic data from 172 Ae. aegypti females across six populations resulted in an expanded and quantitatively refined, species-level characterization of the bacterial, archaeal, and fungal microbiome. This included 844 species of bacteria across 23 phyla, of which 54 species were found to be ubiquitous microbiome members across these populations. The density of wMel infection was highly variable between individuals and negatively correlated with microbiome diversity. Network analyses revealed wMel as a hub comprised solely of negative interactions with other bacterial species. This contrasted with the large and highly interconnected network of other microbiome species that may represent members of the midgut microbiome community in this population. CONCLUSION: Our bioinformatic survey provided a species-level characterization of Ae. aegypti microbiome composition and variation. wMel load varied substantially across populations and individuals and, importantly, wMel was a major hub of a negative interactions across the microbiome. These interactions may be an inherent consequence of heightened pathogen blocking in densely infected individuals or, alternatively, may result from antagonistic Wolbachia-incompatible bacteria that could impede the efficacy of wMel as a biological control agent in future applications. The relationship between wMel infection variation and the microbiome warrants further investigation in the context of developing wMel as a multivalent control agent against other arboviruses. Video Abstract.

Adhesion and virulence properties of native Metarhizium fungal strains from Burkina Faso for the control of malaria vectors.

BACKGROUND: Local strains of the entomopathogenic fungus Metarhizium pingshaense in Burkina Faso have demonstrated remarkable virulence against malaria vectors, positioning them as promising candidates for inclusion in the future arsenal of malaria control strategies. However, the underlying mechanisms responsible for this virulence remain unknown. To comprehend the fungal infection process, it is crucial to investigate the attachment mechanisms of fungal spores to the mosquito cuticle and explore the relationship between virulence and attachment kinetics. This study aims to assess the adhesion and virulence properties of native Metarhizium fungal strains from Burkina Faso for controlling malaria vectors. METHODS: Fungal strains were isolated from 201 insects and 1399 rhizosphere samples, and four strains of Metarhizium fungi were selected. Fungal suspensions were used to infect 3-day-old female Anopheles coluzzii mosquitoes at three different concentrations (106, 107, 108 conidia/ml). The survival of the mosquitoes was measured over 14 days, and fungal growth was quantified after 1 and 24 h to assess adhesion of the fungal strains onto the mosquito cuticle. RESULTS: All four fungi strains increased mosquito mortality compared to control (Chi-square test, χ2 = 286.55, df = 4, P < 0.001). Adhesion of the fungal strains was observed on the mosquito cuticle after 24 h at high concentrations (1 × 108 conidia/ml), with one strain, having the highest virulent, showing adhesion after just 1 h. CONCLUSION: The native strains of Metarhizium spp. fungi found in Burkina Faso have the potential to be effective biocontrol agents against malaria vectors, with some strains showing high levels of both virulence and adhesion to the mosquito cuticle.

Wolbachia dominance influences the Culex quinquefasciatus microbiota.

Microorganisms present in mosquitoes and their interactions are key factors affecting insect development. Among them, Wolbachia is closely associated with the host and affects several fitness parameters. In this study, the bacterial and fungal microbiota from two laboratory Culex quinquefasciatus isolines (wild type and tetracycline-cured) were characterized by metagenome amplicon sequencing of the ITS2 and 16S rRNA genes at different developmental stages and feeding conditions. We identified 572 bacterial and 61 fungal OTUs. Both isolines presented variable bacterial communities and different trends in the distribution of diversity among the groups. The lowest bacterial richness was detected in sugar-fed adults of the cured isoline, whereas fungal richness was highly reduced in blood-fed mosquitoes. Beta diversity analysis indicated that isolines are an important factor in the differentiation of mosquito bacterial communities. Considering composition, Penicillium was the dominant fungal genus, whereas Wolbachia dominance was inversely related to that of Enterobacteria (mainly Thorsellia and Serratia). This study provides a more complete overview of the mosquito microbiome, emphasizing specific highly abundant components that should be considered in microorganism manipulation approaches to control vector-borne diseases.

Wolbachia interferes with Zika virus replication by hijacking cholesterol metabolism in mosquito cells.

IMPORTANCE: Arthropod-borne viruses are emerging pathogens that are spread widely by mosquitos. Zika virus is an arbovirus that can infect humans and be transmitted from an infected mother to the fetus, potentially leading to microcephaly in infants. One promising strategy to prevent disease caused by arboviruses is to target the insect vector population. Recent field studies have shown that mosquito populations infected with Wolbachia bacteria suppress arbovirus replication and transmission. Here, we describe how intracellular bacteria redirect resources within their host cells and suppress Zika virus replication at the cellular level. Understanding the mechanism behind Wolbachia-induced interference of arbovirus replication could help advance strategies to control arbovirus pathogens in insect vectors and human populations.

Exploring dose-response relationships in Aedes aegypti survival upon bacteria and arbovirus infection.

A detailed understanding of how host fitness changes in response to variations in microbe density (an ecological measure of disease tolerance) is an important aim of infection biology. Here, we applied dose-response curves to study Aedes aegypti survival upon exposure to different microbes. We challenged female mosquitoes with Listeria monocytogenes, a model bacterial pathogen, Dengue 4 virus and Zika virus, two medically relevant arboviruses, to understand the distribution of mosquito survival following microbe exposure. By correlating microbe loads and host health, we found that a blood meal promotes disease tolerance in our systemic bacterial infection model and that mosquitoes orally infected with bacteria had an enhanced defensive capacity than insects infected through injection. We also showed that Aedes aegypti displays a higher survival profile following arbovirus infection when compared to bacterial infections. Here, we applied a framework for investigating microbe-induced mosquito mortality and details how the lifespan of Aedes aegypti varies with different inoculum sizes of bacteria and arboviruses.

Screening of natural Wolbachia infection in mosquitoes (Diptera: Culicidae) from the Cape Verde islands.

BACKGROUND: Wolbachia pipientis is an endosymbiont bacterium that induces cytoplasmic incompatibility and inhibits arboviral replication in mosquitoes. This study aimed to assess Wolbachia prevalence and genetic diversity in different mosquito species from Cape Verde. METHODS: Mosquitoes were collected on six islands of Cape Verde and identified to species using morphological keys and PCR-based assays. Wolbachia was detected by amplifying a fragment of the surface protein gene (wsp). Multilocus sequence typing (MLST) was performed with five housekeeping genes (coxA, gatB, ftsZ, hcpA, and fbpA) and the wsp hypervariable region (HVR) for strain identification. Identification of wPip groups (wPip-I to wPip-V) was performed using PCR-restriction fragment length polymorphism (RFLP) assay on the ankyrin domain gene pk1. RESULTS: Nine mosquito species were collected, including the major vectors Aedes aegypti, Anopheles arabiensis, Culex pipiens sensu stricto, and Culex quinquefasciatus. Wolbachia was only detected in Cx. pipiens s.s. (100% prevalence), Cx. quinquefasciatus (98.3%), Cx. pipiens/quinquefasciatus hybrids (100%), and Culex tigripes (100%). Based on the results of MLST and wsp hypervariable region typing, Wolbachia from the Cx. pipiens complex was assigned to sequence type 9, wPip clade, and supergroup B. PCR/RFLP analysis revealed three wPip groups in Cape Verde, namely wPip-II, wPip-III, and wPip-IV. wPip-IV was the most prevalent, while wPip-II and wPip-III were found only on Maio and Fogo islands. Wolbachia detected in Cx. tigripes belongs to supergroup B, with no attributed MLST profile, indicating a new strain of Wolbachia in this mosquito species. CONCLUSIONS: A high prevalence and diversity of Wolbachia was found in species from the Cx. pipiens complex. This diversity may be related to the mosquito's colonization history on the Cape Verde islands. To the best of our knowledge, this is the first study to detect Wolbachia in Cx. tigripes, which may provide an additional opportunity for biocontrol initiatives.