Effect of Microsporidia MB infection on the development and fitness of Anopheles arabiensis under different diet regimes.

BACKGROUND: Microsporidia MB (MB) is a naturally occurring symbiont of Anopheles and has recently been identified as having a potential to inhibit the transmission of Plasmodium in mosquitoes. MB intensity is high in mosquito gonads, with no fitness consequences for the mosquito, and is linked to horizontal (sexual) and vertical (transovarial) transmission from one mosquito to another. Maximising MB intensity and transmission is important for maintaining heavily infected mosquito colonies for experiments and ultimately for mosquito releases. We have investigated how diet affects the MB-Anopheles arabiensis symbiosis phenotypes, such as larval development and mortality, adult size and survival, as well as MB intensity in both larvae and adults. METHODS: F1 larvae of G0 females confirmed to be An. arabiensis and infected with MB were either combined (group lines [GLs]) or reared separately (isofemale lines [IMLs]) depending on the specific experiment. Four diet regimes (all mg/larva/day) were tested on F1 GLs: Tetramin 0.07, Tetramin 0.3, Gocat 0.3 and Cerelac 0.3. GLs reared on Tetramin 0.3 mg/larva/day were then fed either a 1% or 6% glucose diet to determine adult survival. Larvae of IMLs were fed Tetramin 0.07 mg and Tetramin 0.3 mg for larval experiments. The mosquitoes in the adult experiments with IMLs were reared on 1% or 6% glucose. RESULTS: Amongst the four larval diet regimes tested on An. arabiensis development in the presence of MB, the fastest larval development highest adult emergence, largest body size of mosquitoes, highest prevalence and highest density of MB occurred in those fed Tetramin 0.3 mg/larva/day. Although adult MB-positive mosquitoes fed on 6% glucose survived longer than MB-negative mosquitoes, there was no such effect for those fed on the 1% glucose diet. Development time, wing length and adult survival were not significantly different between MB-infected and uninfected An. arabiensis fed on the Tetramin 0.07 mg/larva/day diet, demonstrating that the MB-conferred fitness advantage was diet-dependent. CONCLUSIONS: Microsporidia MB does not adversely impact the development and fitness of An. arabiensis, even under limited dietary conditions. The diet regime of Tetramin 0.3 mg/larva/day + 6% glucose for adults is the superior diet for the mass rearing of MB-infected An. arabiensis mosquitoes. These results are important for rearing MB-infected An. arabiensis in the laboratory for experiments and the mass rearing required for field releases.

Molecular identification and GC-MS analysis of a newly isolated novel bacterium (Lysinibacillus sp. VCRC B655) for mosquito control.

BACKGROUND: Mosquitoes are widespread globally and have contributed to transmitting pathogens to humans and the burden of vector-borne diseases. They are effectively controlled at their larval stages by biocontrol agents. Unravelling natural sources for microbial agents can lead us to novel potential candidates for managing mosquito-borne diseases. In the present study, an attempt was made to isolate a novel bacterium from the field-collected agricultural soil for larvicidal activity and promising bacterial metabolites for human healthcare. METHODS AND RESULTS: Field-collected soil samples from the Union territory of Puducherry, India, have been used as the source of bacteria. Isolate VCRC B655 belonging to the genus Lysinibacillus was identified by 16S rRNA gene sequencing and exhibited promising larvicidal activity against different mosquito species, including Culex (Cx.) quinquefasciatus, Anopheles (An.) stephensi, and Aedes (Ae.) aegypti. The lethal concentration (LC) of Lysinibacillus sp. VCRCB655 was observed to be high for Cx. quiquefasciatus: LC50 at 0.047 mg/l, LC90 at 0.086 mg/l, followed by An. stephensi and Ae. aegypti (LC50: 0.6952 mg/l and 0.795 mg/l) respectively. Additionally, metabolic profiling of the culture supernatant was carried out through Gas chromatography and Mass spectrophotometry (GC/MS) and identified 15 major secondary metabolites of different metabolic classes. Diketopiperazine (DKPs), notably pyro lo [1, 2-a] pyrazine1, 4-dione, are the abundant compounds reported for antioxidant activity, and an insecticide compound benzeneacetic acid was also identified. CONCLUSIONS: A new bacterial isolate, Lysinibacillus sp. VCRC B655 has been identified with significant larvicidal activity against mosquito larvae with no observed in non-target organisms. GC-MS analysis revealed diverse bioactive compounds with substantial biological applications. In conclusion, Lysinibacillus sp. VCRC B655 showed promise as an alternative biocontrol agent for mosquito vector control, with additional biological applications further enhancing its significance.

Basophil-Derived IL-4 and IL-13 Protect Intestinal Barrier Integrity and Control Bacterial Translocation during Malaria.

Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1ß (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.

Microbiome profile of South Korean vector mosquitoes.

This research offers a comprehensive exploration of the microbial communities associated with vector mosquitoes from South Korea. Aedes albopictus, Anopheles sinensis, and Culex molestus are vectors of pathogens, and understanding the intricacies of their microbiome profile is paramount for unraveling their roles in disease transmission dynamics. In this study, we characterized the microbiome of the midguts of adult female vector mosquitoes collected from different locations in South Korea. After DNA extraction from dissected mosquito midguts, we used the Illumina MiSeq next-generation sequencing to obtain sequences spanning the V4 hypervariable region of the bacteria 16S rRNA. Morphological and molecular characterization using 506-bp mitochondrial 16S rRNA was used to identify the mosquito species before amplicon sequencing. Across the three vector mosquitoes surveyed, 21 bacteria genera belonging to 20 families and 5 phyla were discovered. Proteobacteria and Bacteriodota were the major phyla of bacteria associated with the three mosquito species. There were significant differences in the gut microbiome genera composition between the species and little variation in the gut microbiome between individuals of the same mosquito species. Wolbachia is the most dominant genus in Aedes while Aeromonas, Acinetobacter, and unassigned taxa are the most common in An. sinensis. In addition to that, Chromobacterium, Chryseobacterium, and Aeromonas are dominant in Cx. molestus. This study sheds light on the complex interactions between mosquitoes and their microbiome, revealing potential implications for vector competence, disease transmission, and vector control strategies.

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.

Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response.

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

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.

Delftia tsuruhatensis TC1 symbiont suppresses malaria transmission by anopheline mosquitoes.

Malaria control demands the development of a wide range of complementary strategies. We describe the properties of a naturally occurring, non-genetically modified symbiotic bacterium, Delftia tsuruhatensis TC1, which was isolated from mosquitoes incapable of sustaining the development of Plasmodium falciparum parasites. D. tsuruhatensis TC1 inhibits early stages of Plasmodium development and subsequent transmission by the Anopheles mosquito through secretion of a small-molecule inhibitor. We have identified this inhibitor to be the hydrophobic molecule harmane. We also found that, on mosquito contact, harmane penetrates the cuticle, inhibiting Plasmodium development. D. tsuruhatensis TC1 stably populates the mosquito gut, does not impose a fitness cost on the mosquito, and inhibits Plasmodium development for the mosquito's life. Contained field studies in Burkina Faso and modeling showed that D. tsuruhatensis TC1 has the potential to complement mosquito-targeted malaria transmission control.

Characterizations of Larval Gut Bacteria of Anopheles subpictus Grassi (1899) and their Role in Mosquito Development in Hooghly, West Bengal, India.

Malaria is a serious vector borne disease transmitted by different species of Anopheles mosquitoes. The present study was aimed to isolate and characterize the bacterial flora from the gut of larvae of An. subpictus Grassi (1899) prevalent in Hooghly and explore their roles in host survival and development. Mosquito larvae and adults were collected from field and were maintained in laboratory. Bacterial load in the larval mid-gut was determined, and predominant strains were isolated and characterized by polyphasic approach. Role of these bacteria in larval survival and development were assayed. Bacterial load in the gut of larvae was found to vary in field-collected and lab-reared mosquitoes in different seasons. Morphological, bio-chemical, and molecular analyses explored four common bacterial isolates, namely Bacillus subtilis, Bacillus pumilus, Bacillus cereus, and Proteus vulgaris in the larval gut throughout the year. Larval survival rate was greatly reduced (0.06) and time of pupation was prolonged (17.8 ± 0.57) [days] in the absence of their gut bacteria. Total tissue protein (7.78 ± 0.56) [µg/mg], lipid (2.25 ± 0.19) [µg/mg] & carbohydrate (16.5 ± 0.79) [µg/mg] contents of larvae, and body weight & wing length of adult male (0.17 ± 0.02 & 1.74 ± 0.43) [mm] & female (0.19 ± 0.02 & 1.99 ± 0.46) [mm] mosquitoes were also found to be greatly reduced in the absence of gut bacteria. Developmental characteristics were restored with the introduction of culture suspension of all four resident gut bacterial isolates. Present study indicates that the mosquitoes largely depend on their gut bacteria for their survival and development. So, manipulation or control of this gut bacterial communities might inhibit survival and development of vector mosquitoes.

Detection of Wolbachia in Mosquitoes (Diptera: Culicidae) in the State of Maranhão, Brazil.

Recently, the endobacteria Wolbachia has emerged as a biological tool for the control of arboviruses. Thus, we investigated the rate of natural infection by Wolbachia in Culicidae species from Maranhão, Brazil. For this, we amplified the Wolbachia surface protein gene (wsp) from mosquitoes collected in six localities of Maranhão, and positive samples were subjected to new analysis using group-specific primers. In total, 448 specimens comprising 6 genera and 18 species of mosquitoes were analyzed. Wolbachia DNA was PCR-detected in 7 species, three of which are new records: Aedes scapularis (Rondani, 1848), Coquillettidia juxtamansonia (Chagas, 1907) and Cq. venezuelensis (Theobald, 1912), in addition to Ae. albopictus (Skuse, 1894) and Culex quinquefasciatus Say, 1823, which are commonly described as permissive to maintain this bacterium in natural environments, and two species of the subgenera Anopheles (Nyssorhynchus) Blanchard, 1902 and Culex (Melanoconion) Theobald, 1903 which could not be identified at species level. The infection rate of all species ranged from 0 to 80%, and the average value was 16.5%. This study increases the knowledge about the prevalence of Wolbachia in the culicid fauna and may help in selecting strains for biological control purposes.

High Prevalence of Rickettsia bellii in Mosquitoes From Eastern China.

Mosquitoes are the most important vectors carrying significant numbers of human pathogens. Recent studies implicated that mosquitoes play an important role in circulation and transmission of multiple Rickettsia species. In this study, Rickettsia bellii was identified in four mosquito species (Culex pipiens, C. tritaeniorhynchus, Aedes albopictus, and Anopheles sinensis) collected from three Eastern China provinces during 2019-2020. Rickettsia bellii was detected in 37.50 and 26.32% of the C. pipiens pools from Beijing and Jiangsu province, respectively. In C. tritaeniorhynchus and An. sinensis from Shandong, the infection rate is 20.00 and 6.25%, respectively. Additionally, three Ae. albopictus pools (3/42, 7.14%) from Beijing were also detected positive for R. bellii. Genetic and phylogenetic analysis on 16S, gltA, and groEL genes indicates that sequences from all these strains are highly homologous and closely related to other R. bellii strains. This is the first report that Ae. albopictus and C. tritaeniorhynchus harbor R. bellii. The wide host range and high infection rate in certain areas may dramatically increase the exposure of R. bellii to human and other vertebrates. The role of mosquitoes in transmission of rickettsiosis and its potential risk to public health should be further considered.

Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission.

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

Wolbachia cifB induces cytoplasmic incompatibility in the malaria mosquito vector.

Wolbachia, a maternally inherited intracellular bacterial species, can manipulate host insect reproduction by cytoplasmic incompatibility (CI), which results in embryo lethality in crosses between infected males and uninfected females. CI is encoded by two prophage genes, cifA and cifB. Wolbachia, coupled with the sterile insect technique, has been used in field trials to control populations of the dengue vector Aedes albopictus, but CI-inducing strains are not known to infect the malaria vector Anopheles gambiae. Here we show that cifA and cifB can induce conditional sterility in the malaria vector An. gambiae. We used transgenic expression of these Wolbachia-derived genes in the An. gambiae germline to show that cifB is sufficient to cause embryonic lethality and that cifB-induced sterility is rescued by cifA expression in females. When we co-expressed cifA and cifB in male mosquitoes, the CI phenotype was attenuated. In female mosquitoes, cifB impaired fertility, which was overcome by co-expression of cifA. Our findings pave the way towards using CI to control malaria mosquito vectors.

Increased insulin signaling in the Anopheles stephensi fat body regulates metabolism and enhances the host response to both bacterial challenge and Plasmodium falciparum infection.

In vertebrates and invertebrates, the insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) cascade is highly conserved and plays a vital role in many different physiological processes. Among the many tissues that respond to IIS in mosquitoes, the fat body has a central role in metabolism, lifespan, reproduction, and innate immunity. We previously demonstrated that fat body specific expression of active Akt, a key IIS signaling molecule, in adult Anopheles stephensi and Aedes aegypti activated the IIS cascade and extended lifespan. Additionally, we found that transgenic females produced more vitellogenin (Vg) protein than non-transgenic mosquitoes, although this did not translate into increased fecundity. These results prompted us to further examine how IIS impacts immunity, metabolism, growth and development of these transgenic mosquitoes. We observed significant changes in glycogen, trehalose, triglycerides, glucose, and protein in young (3-5 d) transgenic mosquitoes relative to non-transgenic sibling controls, while only triglycerides were significantly changed in older (18 d) transgenic mosquitoes. More importantly, we demonstrated that enhanced fat body IIS decreased both the prevalence and intensity of Plasmodium falciparum infection in transgenic An. stephensi. Additionally, challenging transgenic An. stephensi with Gram-positive and Gram-negative bacteria altered the expression of several antimicrobial peptides (AMPs) and two anti-Plasmodium genes, nitric oxide synthase (NOS) and thioester complement-like protein (TEP1), relative to non-transgenic controls. Increased IIS in the fat body of adult female An. stephensi had little to no impact on body size, growth or development of progeny from transgenic mosquitoes relative to non-transgenic controls. This study both confirms and expands our understanding of the critical roles insulin signaling plays in regulating the diverse functions of the mosquito fat body.

Overabundance of Asaia and Serratia Bacteria Is Associated with Deltamethrin Insecticide Susceptibility in Anopheles coluzzii from Agboville, Côte d'Ivoire.

Insecticide resistance among mosquito species is now a pervasive phenomenon that threatens to jeopardize global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterized and compared the microbiota of Anopheles coluzzii in relation to their deltamethrin resistance and exposure profiles. Comparisons between 2- and 3-day-old deltamethrin-resistant and -susceptible mosquitoes demonstrated significant differences in microbiota diversity. Ochrobactrum, Lysinibacillus, and Stenotrophomonas genera, each of which comprised insecticide-degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals, with Asaia and Serratia dominating microbial profiles. There was no significant difference in deltamethrin-exposed and -unexposed 5- to 6-day-old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. Serratia and Asaia were also dominant in 5- to 6-day-old mosquitoes, which had reduced microbial diversity compared to 2- to 3-day-old mosquitoes. Our findings revealed significant alterations of Anopheles coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of Serratia and Asaia was consistent with 16S rRNA sequencing, suggesting that population-level field screening of bacterial microbiota may be feasibly integrated into wider resistance monitoring, if reliable and reproducible markers associated with phenotype can be identified. IMPORTANCE Control of insecticide-resistant vector populations remains a significant challenge to global malaria control and while substantial progress has been made elucidating key target site mutations, overexpressed detoxification enzymes and alternate gene families, the contribution of the mosquito microbiota to phenotypic insecticide resistance has been largely overlooked. We focused on determining the effects of deltamethrin resistance intensity on Anopheles coluzzii microbiota and identifying any microbial taxa associated with phenotype. We demonstrated a significant reduction in microbial diversity between deltamethrin-resistant and -susceptible mosquitoes. Insecticide degrading bacterial species belonging to Ochrobactrum, Lysinibacillus, and Stenotrophomonas genera were significantly enriched in resistant mosquitoes, while Asaia and Serratia dominated microbial profiles of susceptible individuals. Our results revealed significant alterations of Anopheles coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for surveillance and opportunities for designing innovative control techniques to prevent the further evolution and spread of insecticide resistance.

Molecular detection and maternal transmission of a bacterial symbiont Asaia species in field-caught Anopheles mosquitoes from Cameroon.

BACKGROUND: Malaria control relies mainlyon insecticide-based tools. However, the effectiveness of these tools is threatened by widespread insecticide resistance in malaria vectors, highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated the prevalence of Asaia and its maternal transmission in the natural population of Anopheles mosquitoes in Cameroon. METHODS: Indoor-resting adult mosquitoes belonging to four species (An. coluzzii, An. arabiensis, An. funestus and An. gambiae) were collected from eight localities across Cameroon from July 2016 to February 2020. PCR was performed on the Asaia-specific 16S ribosomal RNA gene, and samples positive by PCR for Asaia were confirmed by Sanger sequencing and phylogenetic analysis. The vertical transmission of Asaia was investigated by screening F1 mosquitoes belonging to F0 Asaia-positive females. RESULTS: A total of 895 mosquitoes were screened. We found 43% (384) Asaia infection prevalence in four mosquito species. Phylogenetic analysis revealed that Asaia from Cameroon clustered together with the strains of Asaia isolated from other parts of the world. In addition, seven nucleotide sequence variants were found with low genetic diversity (π = 0.00241) and nucleotide sequence variant diversity (Hd = 0.481). Asaia was vertically transmitted with high frequency (range from 42.5 to 100%). CONCLUSIONS: This study provides field-based evidence of the presence of Asaia in Anopheles mosquitoes in Cameroon for exploitation as a symbiont in the control of malaria in sub-Saharan Africa.

Comprehensive characterization of internal and cuticle surface microbiota of laboratory-reared F1 Anopheles albimanus originating from different sites.

BACKGROUND: Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings. METHODS: Using high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2-5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed. RESULTS: Results showed location-associated heterogeneity in both F1 larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F1 adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche. CONCLUSIONS: These results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F1 progeny under normal laboratory conditions, but this may not last beyond the F1 larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F1 An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings.

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens and Cx. tritaeniorhynchus in China.

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.

Pathogenic fungi infection attributes of malarial vectors Anopheles maculipennis and Anopheles superpictus in central Iran.

BACKGROUND: Due to the effect of synthetic and commercial insecticides on non-target organisms and the resistance of mosquitoes, non-chemical and environmentally friendly methods have become prevalent in recent years. The present study was to isolate entomopathogenic fungi with toxic effects on mosquitoes in natural larval habitats. METHODS: Larvae of mosquitoes were collected from Central, Qamsar, Niasar, and Barzok Districts in Kashan County, Central Iran by standard dipping method, from April to late December 2019. Dead larvae, live larvae showing signs of infection, and larvae and pupae with a white coating of fungal mycelium on the outer surface of their bodies were isolated from the rest of the larvae and sterilized with 10% sodium hypochlorite for 2 min, then washed twice with distilled water and transferred to potato-dextrose-agar (PDA) and water-agar (WA) media and incubated at 25 ± 2 °C for 3-4 days. Larvae and fungi were identified morphologically based on identification keys. RESULTS: A total of 9789 larvae were collected from urban and rural areas in Kashan County. Thirteen species were identified which were recognized to belong to three genera, including Anopheles (7.89%), Culiseta (17.42%) and Culex (74.69%). A total of 105 larvae, including Anopheles superpictus sensu lato (s.l), Anopheles maculipennis s.l., Culex deserticola, Culex perexiguus, and Culiseta longiareolata were found to be infected by Nattrassia mangiferae, Aspergillus niger, Aspergillus fumigatus, Trichoderma spp., and Penicillium spp. Of these, Penicillium spp. was the most abundant fungus isolated and identified from the larval habitats, while An. superpictus s.l. was the most infected mosquito species. CONCLUSIONS: Based on the observations and results obtained of the study, isolated fungi had the potential efficacy for pathogenicity on mosquito larvae. It is suggested that their effects on mosquito larvae should be investigated in the laboratory. The most important point, however, is the proper way of exploiting these biocontrol agents to maximize their effect on reducing the population of vector mosquito larvae without any negative effect on non-target organisms.

Microsporidia MB is found predominantly associated with Anopheles gambiae s.s and Anopheles coluzzii in Ghana.

A vertically transmitted microsporidian, Microsporidia MB, with the ability to disrupt Plasmodium development was reported in Anopheles arabiensis from Kenya, East Africa. To demonstrate its range of incidence, archived DNA samples from 7575 Anopheles mosquitoes collected from Ghana were screened. MB prevalence was observed at 1.8%. An. gambiae s.s constituted 87% of positive mosquitoes while the remaining were from An. coluzzii. Both sibling species had similar positivity rates (24% and 19%; p = 0.42) despite the significantly higher number of An. gambiae s.s analysed (An. gambiae s.s = 487; An. coluzzii = 94; p = 0.0005). The microsporidian was also more prevalent in emerged adults from field-collected larvae than field-caught adults (p < 0.0001) suggestive of an efficient vertical transmission and/or horizontal transfer among larvae. This is the first report of Microsporidia MB in Anopheles mosquitoes in West Africa. It indicates possible widespread among malaria vector species and warrants investigations into the symbiont's diversity across sub-Saharan Africa.