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    Optimal release programs for dengue prevention using Aedes aegypti mosquitoes transinfected with wMel or wMelPop Wolbachia strains. Cardona-Salgado Daiver,Campo-Duarte Doris Elena,Sepulveda-Salcedo Lilian Sofia,Vasilieva Olga,Svinin Mikhail Mathematical biosciences and engineering : MBE In this paper, we propose a dengue transmission model of SIR(S)-SI type that accounts for two sex-structured mosquito populations: the wild mosquitoes (males and females that are Wolbachia-free), and those deliberately infected with either wMel or wMelPop strain of Wolbachia. This epidemiological model has four possible outcomes: with or without Wolbachia and with or without dengue. To reach the desired outcome, with Wolbachia and without dengue, we employ the dynamic optimization approach and then design optimal programs for releasing Wolbachia-carrying male and female mosquitoes. Our discussion is focused on advantages and drawbacks of two Wolbachia strains, wMelPop and wMel, that are recommended for dengue prevention and control. On the one hand, the wMel strain guarantees a faster population replacement, ensures durable Wolbachia persistence in the wild mosquito population, and requiters fewer releases. On the other hand, the wMelPop strain displays better results for averting dengue infections in the human population. 10.3934/mbe.2021149
    RNA-Seq analysis of blood meal induced gene-expression changes in Aedes aegypti ovaries. Nag Dilip K,Dieme Constentin,Lapierre Pascal,Lasek-Nesselquist Erica,Kramer Laura D BMC genomics BACKGROUND:Transmission of pathogens by vector mosquitoes is intrinsically linked with mosquito's reproductive strategy because anautogenous mosquitoes require vertebrate blood to develop a batch of eggs. Each cycle of egg maturation is tightly linked with the intake of a fresh blood meal for most species. Mosquitoes that acquire pathogens during the first blood feeding can transmit the pathogens to susceptible hosts during subsequent blood feeding and also vertically to the next generation via infected eggs. Large-scale gene-expression changes occur following each blood meal in various tissues, including ovaries. Here we analyzed mosquito ovary transcriptome following a blood meal at three different time points to investigate blood-meal induced changes in gene expression in mosquito ovaries. RESULTS:We collected ovaries from Aedes aegypti that received a sugar meal or a blood meal on days 3, 10 and 20 post blood meal for transcriptome analysis. Over 4000 genes responded differentially following ingestion of a blood meal on day 3, and 660 and 780 genes on days 10 and 20, respectively. Proteins encoded by differentially expressed genes (DEGs) on day 3 include odorant binding proteins (OBPs), defense-specific proteins, and cytochrome P450 detoxification enzymes. In addition, we identified 580 long non-coding RNAs that are differentially expressed at three time points. Gene ontology analysis indicated that genes involved in peptidase activity, oxidoreductase activity, extracellular space, and hydrolase activity, among others were enriched on day 3. Although most of the DEGs returned to the nonsignificant level compared to the sugar-fed mosquito ovaries following oviposition on days 10 and 20, there remained differences in the gene expression pattern in sugar-fed and blood-fed mosquitoes. CONCLUSIONS:Enrichment of OBPs following blood meal ingestion suggests that these genes may have other functions besides being part of the olfactory system. The enrichment of immune-specific genes and cytochrome P450 genes indicates that ovaries become well prepared to protect their germ line from any pathogens that may accompany the blood meal or from environmental contamination during oviposition, and to deal with the detrimental effects of toxic metabolites. 10.1186/s12864-021-07551-z
    A Thioester-Containing Protein Controls Dengue Virus Infection in Through Modulating Immune Response. Weng Shih-Che,Li Hsing-Han,Li Jian-Chiuan,Liu Wei-Liang,Chen Chun-Hong,Shiao Shin-Hong Frontiers in immunology Complement-like proteins in arthropods defend against invading pathogens in the early phases of infection. Thioester-containing proteins (TEPs), which exhibit high similarity to mammalian complement C3, are thought to play a key role in the innate immunity of arthropods. We identified and characterized anti-dengue virus (DENV) host factors, in particular complement-like proteins, in the mosquito . Our results indicate that TEP1 limits DENV infection in We showed that transcription is highly induced in mosquitoes following DENV infection. Silencing resulted in the up-regulation of viral RNA and proteins. In addition, the production of infectious virus particles increased in the absence of . We generated a transgenic mosquito line with a TEP1 loss-of-function phenotype under a blood meal-inducible promoter. We showed that viral protein and titers increased in transgenic mosquitoes after an infectious blood meal. Interestingly, expression of transcription factor Rel2 and certain anti-microbial peptides (AMPs) were inhibited in transgenic mosquitoes. Overall, our results suggest that TEP1 regulates the immune response and consequently controls the replication of dengue virus in mosquitoes. This finding provides new insight into the molecular mechanisms of mosquito host factors in the regulation of DENV replication. 10.3389/fimmu.2021.670122
    The microbiome and mosquito vectorial capacity: rich potential for discovery and translation. Cansado-Utrilla Cintia,Zhao Serena Y,McCall Philip J,Coon Kerri L,Hughes Grant L Microbiome Microbiome research has gained considerable interest due to the emerging evidence of its impact on human and animal health. As in other animals, the gut-associated microbiota of mosquitoes affect host fitness and other phenotypes. It is now well established that microbes can alter pathogen transmission in mosquitoes, either positively or negatively, and avenues are being explored to exploit microbes for vector control. However, less attention has been paid to how microbiota affect phenotypes that impact vectorial capacity. Several mosquito and pathogen components, such as vector density, biting rate, survival, vector competence, and the pathogen extrinsic incubation period all influence pathogen transmission. Recent studies also indicate that mosquito gut-associated microbes can impact each of these components, and therefore ultimately modulate vectorial capacity. Promisingly, this expands the options available to exploit microbes for vector control by also targeting parameters that affect vectorial capacity. However, there are still many knowledge gaps regarding  mosquito-microbe interactions  that need to be addressed in order to exploit them efficiently. Here, we review current evidence of impacts of the microbiome on aspects of vectorial capacity, and we highlight likely opportunities for novel vector control strategies and areas where further studies are required. Video abstract. 10.1186/s40168-021-01073-2
    Trained Immunity in : Antibacterial Immunity Is Enhanced by Priming via Sugar Meal Supplemented With a Single Gut Symbiotic Bacterial Strain. Kulkarni Aditi,Pandey Ashmita,Trainor Patrick,Carlisle Samantha,Chhilar Jainder S,Yu Wanqin,Moon Alex,Xu Jiannong Frontiers in microbiology Mosquitoes have evolved an effective innate immune system. The mosquito gut accommodates various microbes, which play a crucial role in shaping the mosquito immune system during evolution. The resident bacteria in the gut microbiota play an essential role in priming basal immunity. In this study, we show that antibacterial immunity in can be enhanced by priming via a sugar meal supplemented with bacteria. S1 and sp. Ag1 are gut bacteria in mosquitoes. The intrathoracic injection of the two bacteria can result in an acute hemocoelic infection in the naïve mosquitoes with mortality of ∼40% at 24 h post-infection. However, the or primed mosquitoes showed a better 24 h survival upon the bacterial challenge. The priming confers the protection with a certain degree of specificity, the primed mosquitoes had a better survival upon the but not challenge, and the primed mosquitoes had a better survival upon the but not challenge. To understand the priming-mediated immune enhancement, the transcriptomes were characterized in the mosquitoes of priming as well as priming plus challenges. The RNA-seq was conducted to profile 10 transcriptomes including three samples of priming conditions (native microbiota, priming, and priming), six samples of priming plus challenges with the two bacteria, and one sample of injury control. The three priming regimes resulted in distinctive transcriptomic profiles with about 60% of genes affected by both bacteria. Upon challenges, different primed mosquitoes displayed different transcriptomic patterns in response to different bacteria. When a primed cohort was challenged with a heterogenous bacterium, more responsive genes were observed than when challenged with a homogenous bacterium. As expected, many canonical immune genes were responsive to the priming and challenge, but much more non-immune genes with various functions were also responsive in the contexts, which implies that the prior priming triggers a delicately coordinated systemic regulation that results in an enhanced immunity against the subsequent challenge. Besides the participation of typical immune pathways, the transcriptome data suggest the involvement of lysosome and metabolism in the context. Overall, this study demonstrated a trained immunity via priming with bacteria in diet. 10.3389/fmicb.2021.649213
    Metagenomic sequencing reveals viral abundance and diversity in mosquitoes from the Shaanxi-Gansu-Ningxia region, China. He Xiaozhou,Yin Qikai,Zhou Liwei,Meng Lei,Hu Weijun,Li Fan,Li Yang,Han Kun,Zhang Shaobai,Fu Shihong,Zhang Xiaoshu,Wang Ji,Xu Songtao,Zhang Yi,He Ying,Dong Maoxing,Shen Xinxin,Zhang Zheng,Nie Kai,Liang Guodong,Ma Xuejun,Wang Huanyu PLoS neglected tropical diseases BACKGROUND:Mosquitoes host and transmit numerous arthropod-borne viruses (arboviruses) that cause disease in both humans and animals. Effective surveillance of virome profiles in mosquitoes is vital to the prevention and control of mosquito-borne diseases in northwestern China, where epidemics occur frequently. METHODS:Mosquitoes were collected in the Shaanxi-Gansu-Ningxia region (Shaanxi Province, Gansu Province, and Ningxia Hui Autonomous Region) of China from June to August 2019. Morphological methods were used for taxonomic identification of mosquito species. High-throughput sequencing and metagenomic analysis were used to characterize mosquito viromes. RESULTS:A total of 22,959 mosquitoes were collected, including Culex pipiens (45.7%), Culex tritaeniorhynchus (40.6%), Anopheles sinensis (8.4%), Aedes (5.2%), and Armigeres subalbatus (0.1%). In total, 3,014,183 (0.95% of clean reads) viral sequences were identified and assigned to 116 viral species (including pathogens such as Japanese encephalitis virus and Getah virus) in 31 viral families, including Flaviviridae, Togaviridae, Phasmaviridae, Phenuiviridae, and some unclassified viruses. Mosquitoes collected in July (86 species in 26 families) showed greater viral diversity than those from June and August. Culex pipiens (69 species in 25 families) and Culex tritaeniorhynchus (73 species in 24 families) carried more viral species than Anopheles sinensis (50 species in 19 families) or Aedes (38 species in 20 families) mosquitoes. CONCLUSION:Viral diversity and abundance were affected by mosquito species and collection time. The present study elucidates the virome compositions of various mosquito species in northwestern China, improving the understanding of virus transmission dynamics for comparison with those of disease outbreaks. 10.1371/journal.pntd.0009381
    Effects of Ascogregarina culicis (Eugregarinorida: Lecudinidae) on Mosquito Size and Dengue Virus Infection in Aedes aegypti (Diptera: Culicidae). Thongsripong Panpim,Wesson Dawn M Journal of medical entomology Dengue virus infection, transmitted via mosquito bites, poses a substantial risk to global public health. Studies suggest that the mosquito's microbial community can profoundly influence vector-borne pathogen transmissions, including dengue virus. Ascogregarina culicis (Ross) of the phylum Apicomplexa is among the most common parasites of Aedes aegypti (Linnaeus), the principal vector of dengue. Despite a high prevalence worldwide, including in the areas where dengue is endemic, the impact of A. culicis on Ae. aegypti vector competence for dengue virus is unknown. This study aimed to investigate the effects of A. culicis infection on mosquito size and fitness, as measured by wing length, and the susceptibility to dengue virus infection in Ae. aegypti. Our results showed that there was no statistically significant difference in wing lengths between Ae. aegypti infected and not infected with A. culicis. Furthermore, A. culicis infection did not significantly affect dengue virus infection or disseminated infection rate. However, there was a significant association between shorter wings and higher dengue virus infection rate, whereby a 0.1-mm increase in wing length decreased the odds of the mosquito being infected by 32%. Thus, based on our result, A. culicis infection does not influence the body size and dengue virus infection in Ae. aegypti. This study helps to shed light on a common but neglected eukaryotic mosquito parasite. 10.1093/jme/tjaa280
    The Aedes albopictus (Diptera: Culicidae) microbiome varies spatially and with Ascogregarine infection. Seabourn Priscilla,Spafford Helen,Yoneishi Nicole,Medeiros Matthew PLoS neglected tropical diseases The mosquito microbiome alters the physiological traits of medically important mosquitoes, which can scale to impact how mosquito populations sustain disease transmission. The mosquito microbiome varies significantly within individual mosquitoes and among populations, however the ecological and environmental factors that contribute to this variation are poorly understood. To further understand the factors that influence variation and diversity of the mosquito microbiome, we conducted a survey of the bacterial microbiome in the medically important mosquito, Aedes albopictus, on the high Pacific island of Maui, Hawai'i. We detected three bacterial Phyla and twelve bacterial families: Proteobacteria, Acitinobacteria, and Firmicutes; and Anaplasmataceae, Acetobacteraceae, Enterobacteriaceae, Burkholderiaceae, Xanthobacteraceae, Pseudomonadaceae, Streptomycetaceae, Staphylococcaceae, Xanthomonadaceae, Beijerinckiaceae, Rhizobiaceae, and Sphingomonadaceae. The Ae. albopictus bacterial microbiota varied among geographic locations, but temperature and rainfall were uncorrelated with this spatial variation. Infection status with an ampicomplexan pathosymbiont Ascogregarina taiwanensis was significantly associated with the composition of the Ae. albopictus bacteriome. The bacteriomes of mosquitoes with an A. taiwanensis infection were more likely to include several bacterial symbionts, including the most abundant lineage of Wolbachia sp. Other symbionts like Asaia sp. and several Enterobacteriaceae lineages were less prevalent in A. taiwanensis-infected mosquitoes. This highlights the possibility that inter- and intra-domain interactions may structure the Ae. albopictus microbiome. 10.1371/journal.pntd.0008615
    Antibody-Dependent Enhancement: A Challenge for Developing a Safe Dengue Vaccine. Shukla Rahul,Ramasamy Viswanathan,Shanmugam Rajgokul K,Ahuja Richa,Khanna Navin Frontiers in cellular and infection microbiology In 2019, the United States Food and Drug Administration accorded restricted approval to Sanofi Pasteur's Dengvaxia, a live attenuated vaccine (LAV) for dengue fever, a mosquito-borne viral disease, caused by four antigenically distinct dengue virus serotypes (DENV 1-4). The reason for this limited approval is the concern that this vaccine sensitized some of the dengue-naïve recipients to severe dengue fever. Recent knowledge about the nature of the immune response elicited by DENV viruses suggests that all LAVs have inherent capacity to predominantly elicit antibodies (Abs) against the pre-membrane (prM) and fusion loop epitope (FLE) of DENV. These antibodies are generally cross-reactive among DENV serotypes carrying a higher risk of promoting Antibody-Dependent Enhancement (ADE). ADE is a phenomenon in which suboptimal neutralizing or non-neutralizing cross-reactive antibodies bind to virus and facilitate Fcγ receptor mediated enhanced entry into host cells, followed by its replication, and thus increasing the cellular viral load. On the other hand, antibody responses directed against the host-cell receptor binding domain of DENV envelope domain-III (EDIII), exhibit a higher degree of type-specificity with lower potential of ADE. The challenges associated with whole DENV-based vaccine strategies necessitate re-focusing our attention toward the designed dengue vaccine candidates, capable of inducing predominantly type-specific immune responses. If the designed vaccines elicited predominantly EDIII-directed serotype specific antibodies in the absence of prM and FLE antibodies, this could avoid the ADE phenomenon largely associated with the prM and FLE antibodies. The generation of type-specific antibodies to each of the four DENV serotypes by the designed vaccines could avoid the immune evasion mechanisms of DENVs. For the enhanced vaccine safety, all dengue vaccine candidates should be assessed for the extent of type-specific (minimal ADE) vs. cross-reactive (ADE promoting) neutralizing antibodies. The type-specific EDIII antibodies may be more directly related to protection from disease in the absence of ADE promoted by the cross-reactive antibodies. 10.3389/fcimb.2020.572681
    Cryoprotectant toxicity and hypothermic sensitivity among Anopheles larvae. Nesbitt Jenny E,Swei Anisa,Hunt Catherine,Dotson Ellen M,Toner Mehmet,Sandlin Rebecca D Cryobiology Laboratory rearing of mosquitoes is commonly practiced by researchers studying mosquito-borne infectious diseases and vector control methods. In the absence of cryopreservation methods to stabilize unique or genetically modified strains, mosquito lines must be continuously maintained, a laborious process that risks selection effects, contamination, and genetic drift. Towards the development of a cryopreservation protocol, several commonly used cryoprotectants were systematically characterized here both individually and as cocktails. Among first instar, feeding-stage An. gambiae and An. stephensi larvae, cryoprotectant toxicity followed the order of dimethyl sulfoxide > ethylene glycol > methanol. The resulting LD values were used as the basis for the development of cryoprotectant cocktail solutions, where formulation optimization was streamlined using Taguchi methods of experimental design. Sensitivity to hypothermia was further evaluated to determine the feasibility of cryoprotectant loading at reduced temperatures and slow cooling approaches to cryopreservation. The information described here contributes to the knowledge base necessary to inform the development of a cryopreservation protocol for Anopheles larvae. 10.1016/j.cryobiol.2020.12.007
    E93 confers steroid hormone responsiveness of digestive enzymes to promote blood meal digestion in the midgut of the mosquito Aedes aegypti. He Ya-Zhou,Ding Yike,Wang Xueli,Zou Zhen,Raikhel Alexander S Insect biochemistry and molecular biology Anautogenous female mosquitoes obtain the nutrients needed for egg development from vertebrate blood, and consequently they transmit numerous pathogens of devastating human diseases. Digestion of blood proteins into amino acids that are used for energy production, egg maturation and replenishment of maternal reserves is an essential part of the female mosquito reproductive cycle. However, the regulatory mechanisms underlying this process remain largely unknown. Here, we report that the transcription factor E93 is a critical factor promoting blood meal digestion in adult females of the major arboviral vector Aedes aegypti in response to the steroid hormone 20-hydroxyecdysone (20E). E93 was upregulated in the female mosquito midgut after a blood meal, and RNA interference (RNAi)-mediated knockdown of E93 inhibited midgut blood digestion. E93 RNAi depletion repressed late trypsin (LT), serine protease I (SPI), SPVI and SPVII, and activated early trypsin (ET) expression in the female mosquito midgut after a blood meal. Injection of 20E activated E93, LT, SPI, SPVI and SPVII, and repressed ET expression, whereas RNAi knockdown of the ecdysone receptor (EcR) repressed E93, LT, SPI, SPVI and SPVII, and activated ET expression in the midgut. Furthermore, E93 depletion resulted in a complete loss of 20E responsiveness of LT, SPVI and SPVII. Our findings reveal important mechanisms regulating blood meal digestion in disease-transmitting mosquitoes. 10.1016/j.ibmb.2021.103580
    Towards a method for cryopreservation of mosquito vectors of human pathogens. Gallichotte Emily N,Dobos Karen M,Ebel Gregory D,Hagedorn Mary,Rasgon Jason L,Richardson Jason H,Stedman Timothy T,Barfield Jennifer P Cryobiology Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes. 10.1016/j.cryobiol.2021.02.001
    Metagenomic shotgun sequencing reveals host species as an important driver of virome composition in mosquitoes. Thongsripong Panpim,Chandler James Angus,Kittayapong Pattamaporn,Wilcox Bruce A,Kapan Durrell D,Bennett Shannon N Scientific reports High-throughput nucleic acid sequencing has greatly accelerated the discovery of viruses in the environment. Mosquitoes, because of their public health importance, are among those organisms whose viromes are being intensively characterized. Despite the deluge of sequence information, our understanding of the major drivers influencing the ecology of mosquito viromes remains limited. Using methods to increase the relative proportion of microbial RNA coupled with RNA-seq we characterize RNA viruses and other symbionts of three mosquito species collected along a rural to urban habitat gradient in Thailand. The full factorial study design allows us to explicitly investigate the relative importance of host species and habitat in structuring viral communities. We found that the pattern of virus presence was defined primarily by host species rather than by geographic locations or habitats. Our result suggests that insect-associated viruses display relatively narrow host ranges but are capable of spreading through a mosquito population at the geographical scale of our study. We also detected various single-celled and multicellular microorganisms such as bacteria, alveolates, fungi, and nematodes. Our study emphasizes the importance of including ecological information in viromic studies in order to gain further insights into viral ecology in systems where host specificity is driving both viral ecology and evolution. 10.1038/s41598-021-87122-0
    Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes. Jové Veronica,Venkataraman Krithika,Gabel Thomas M,Duvall Laura B Journal of visualized experiments : JoVE Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females. Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito. These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions. 10.3791/61835
    Measuring and reducing biofilm in mosquito rearing containers. Hunt Catherine M,Collins C Matilda,Benedict Mark Q Parasites & vectors BACKGROUND:Mosquito rearing containers contain organic-rich water that nourishes numerous bacteria, some of which are capable of forming biofilms. Biofilm is broadly an extracellular polymeric matrix (EPS) in which living bacteria occur, and the accumulation of biofilm is possible during routine stock-keeping as most of these containers are re-used. Whether biofilm has an effect on the mosquito rearing is not a question that has been investigated, nor have measures to reduce biofilm in this context been systematically studied. METHODS:We measured biofilm accumulation in standard rearing containers by staining with crystal violet and determining the OD using a spectrophotometer. We also treated rearing containers with 0.1% sodium hypochlorite to determine its effectiveness in reducing biofilm abundance. Lastly, we performed an analysis of the relationship between the occurrence of biofilm and the likelihood of microbial blooms that were associated with larval death during trials of larval diets. RESULTS:We observed that soaking rearing containers overnight in 0.1% sodium hypochlorite greatly reduced biofilm, but we observed no relationship between the use of containers that had not been treated with bleach and subsequent microbial blooms. CONCLUSIONS:Larva rearing leaves detectable biofilm. While we were unable to correlate microbial blooms with the presence of biofilm, as a precaution, we recommend that plastic containers that are re-used be treated with 0.1% sodium hypochlorite occasionally. 10.1186/s13071-020-04315-8
    Mosquito Trilogy: Microbiota, Immunity and Pathogens, and Their Implications for the Control of Disease Transmission. Gabrieli Paolo,Caccia Silvia,Varotto-Boccazzi Ilaria,Arnoldi Irene,Barbieri Giulia,Comandatore Francesco,Epis Sara Frontiers in microbiology In mosquitoes, the interaction between the gut microbiota, the immune system, and the pathogens that these insects transmit to humans and animals is regarded as a key component toward the development of control strategies, aimed at reducing the burden of severe diseases, such as malaria and dengue fever. Indeed, different microorganisms from the mosquito microbiota have been investigated for their ability to affect important traits of the biology of the host insect, related with its survival, development and reproduction. Furthermore, some microorganisms have been shown to modulate the immune response of mosquito females, significantly shaping their vector competence. Here, we will review current knowledge in this field, focusing on i) the complex interaction between the intestinal microbiota and mosquito females defenses, both in the gut and at humoral level; ii) how knowledge on these issues contributes to the development of novel and targeted strategies for the control of mosquito-borne diseases such as the use of paratransgenesis or taking advantage of the relationship between and mosquito hosts. We conclude by providing a brief overview of available knowledge on microbiota-immune system interplay in major insect vectors. 10.3389/fmicb.2021.630438
    Making gene drive biodegradable. Zapletal Josef,Najmitabrizi Neda,Erraguntla Madhav,Lawley Mark A,Myles Kevin M,Adelman Zach N Philosophical transactions of the Royal Society of London. Series B, Biological sciences Gene drive systems have long been sought to modify mosquito populations and thus combat malaria and dengue. Powerful gene drive systems have been developed in laboratory experiments, but may never be used in practice unless they can be shown to be acceptable through rigorous field-based testing. Such testing is complicated by the anticipated difficulty in removing gene drive transgenes from nature. Here, we consider the inclusion of self-elimination mechanisms into the design of homing-based gene drive transgenes. This approach not only caused the excision of the gene drive transgene, but also generates a transgene-free allele resistant to further action by the gene drive. Strikingly, our models suggest that this mechanism, acting at a modest rate (10%) as part of a single-component system, would be sufficient to cause the rapid reversion of even the most robust homing-based gene drive transgenes, without the need for further remediation. Modelling also suggests that unlike gene drive transgenes themselves, self-eliminating transgene approaches are expected to tolerate substantial rates of failure. Thus, self-elimination technology may permit rigorous field-based testing of gene drives by establishing strict time limits on the existence of gene drive transgenes in nature, rendering them essentially biodegradable. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'. 10.1098/rstb.2019.0804
    Laboratory transmission potential of British mosquitoes for equine arboviruses. Chapman Gail E,Sherlock Ken,Hesson Jenny C,Blagrove Marcus S C,Lycett Gareth J,Archer Debra,Solomon Tom,Baylis Matthew Parasites & vectors BACKGROUND:There has been no evidence of transmission of mosquito-borne arboviruses of equine or human health concern to date in the UK. However, in recent years there have been a number of outbreaks of viral diseases spread by vectors in Europe. These events, in conjunction with increasing rates of globalisation and climate change, have led to concern over the future risk of mosquito-borne viral disease outbreaks in northern Europe and have highlighted the importance of being prepared for potential disease outbreaks. Here we assess several UK mosquito species for their potential to transmit arboviruses important for both equine and human health, as measured by the presence of viral RNA in saliva at different time points after taking an infective blood meal. RESULTS:The following wild-caught British mosquitoes were evaluated for their potential as vectors of zoonotic equine arboviruses: Ochlerotatus detritus for Venezuelan equine encephalitis virus (VEEV) and Ross River virus (RRV), and Culiseta annulata and Culex pipiens for Japanese encephalitis virus (JEV). Production of RNA in saliva was demonstrated at varying efficiencies for all mosquito-virus pairs. Ochlerotatus detritus was more permissive for production of RRV RNA in saliva than VEEV RNA. For RRV, 27.3% of mosquitoes expectorated viral RNA at 7 days post-infection when incubated at 21 °C and 50% at 24 °C. Strikingly, 72% of Cx. pipiens produced JEV RNA in saliva after 21 days at 18 °C. For some mosquito-virus pairs, infection and salivary RNA titres reduced over time, suggesting unstable infection dynamics. CONCLUSIONS:This study adds to the number of Palaearctic mosquito species that demonstrate expectoration of viral RNA, for arboviruses of importance to human and equine health. This work adds to evidence that native mosquito species should be investigated further for their potential to vector zoonotic mosquito-borne arboviral disease of equines in northern Europe. The evidence that Cx. pipiens is potentially an efficient laboratory vector of JEV at temperatures as low as 18 °C warrants further investigation, as this mosquito is abundant in cooler regions of Europe and is considered an important vector for West Nile Virus, which has a comparable transmission ecology. 10.1186/s13071-020-04285-x
    Living with predators at the larval stage has differential long-lasting effects on adult life history and physiological traits in two anopheline mosquito species. Roux Olivier,Renault David,Mouline Karine,Diabaté Abdoulaye,Simard Frederic Journal of insect physiology 10.1016/j.jinsphys.2021.104234
    Natural Wolbachia infection in field-collected Anopheles and other mosquito species from Malaysia. Wong Meng Li,Liew Jonathan Wee Kent,Wong Wai Kit,Pramasivan Sandthya,Mohamed Hassan Norzihan,Wan Sulaiman Wan Yusoff,Jeyaprakasam Nantha Kumar,Leong Cherng Shii,Low Van Lun,Vythilingam Indra Parasites & vectors BACKGROUND:The endosymbiont bacterium Wolbachia is maternally inherited and naturally infects some filarial nematodes and a diverse range of arthropods, including mosquito vectors responsible for disease transmission in humans. Previously, it has been found infecting most mosquito species but absent in Anopheles and Aedes aegypti. However, recently these two mosquito species were found to be naturally infected with Wolbachia. We report here the extent of Wolbachia infections in field-collected mosquitoes from Malaysia based on PCR amplification of the Wolbachia wsp and 16S rRNA genes. METHODS:The prevalence of Wolbachia in Culicinae mosquitoes was assessed via PCR with wsp primers. For some of the mosquitoes, in which the wsp primers failed to amplify a product, Wolbachia screening was performed using nested PCR targeting the 16S rRNA gene. Wolbachia sequences were aligned using Geneious 9.1.6 software, analyzed with BLAST, and the most similar sequences were downloaded. Phylogenetic analyses were carried out with MEGA 7.0 software. Graphs were drawn with GraphPad Prism 8.0 software. RESULTS:A total of 217 adult mosquitoes representing 26 mosquito species were screened. Of these, infections with Wolbachia were detected in 4 and 15 mosquito species using wsp and 16S rRNA primers, respectively. To our knowledge, this is the first time Wolbachia was detected using 16S rRNA gene amplification, in some Anopheles species (some infected with Plasmodium), Culex sinensis, Culex vishnui, Culex pseudovishnui, Mansonia bonneae and Mansonia annulifera. Phylogenetic analysis based on wsp revealed Wolbachia from most of the mosquitoes belonged to Wolbachia Supergroup B. Based on 16S rRNA phylogenetic analysis, the Wolbachia strain from Anopheles mosquitoes were more closely related to Wolbachia infecting Anopheles from Africa than from Myanmar. CONCLUSIONS:Wolbachia was found infecting Anopheles and other important disease vectors such as Mansonia. Since Wolbachia can affect its host by reducing the life span and provide resistance to pathogen infection, several studies have suggested it as a potential innovative tool for vector/vector-borne disease control. Therefore, it is important to carry out further studies on natural Wolbachia infection in vector mosquitoes' populations as well as their long-term effects in new hosts and pathogen suppression. 10.1186/s13071-020-04277-x
    Hatching pattern and coexistence of Aedes aegypti and Aedes albopictus (Culicidae) in a subtropical city, Argentina, after three decades of coexistence. Faraone Janinna,Fischer Sylvia,Aponte Carla Agustina,Etchepare Eduardo,Stechina Ornela Sofia,Stein Marina Acta tropica Aedes aegypti and Aedes albopictus are mosquito vectors of numerous arboviruses of sanitary importance. Presently in Argentina, neither Ae. aegypti nor Ae. albopictus, have displaced the other species in the places where they coexist, since the introduction of the latter in 1998. In this study, we evaluated whether these species coexist at different scales (ovitrap, microhabitat and habitat) in the city of Eldorado, Misiones province, northeast Argentina. We also analyzed the seasonal variation and climate variables related to the delay in egg hatching of both species. Mosquitoes were collected weekly, from June 2017 to May 2018, using ovitraps placed in urban areas. We conclude that Ae. aegypti and Ae. albopictus coexist in the study area, at the ovitrap, microhabitat and habitat scales. Furthermore, no pronounced pattern of delayed hatching has been observed for either species; however, eggs of Ae. albopictus laid during colder weeks and less rainfall needed a greater number of immersions to hatch, while for Ae. aegypti those laid during weeks with low rainfall and high temperatures showed the longest delay in hatching response. 10.1016/j.actatropica.2021.105885
    Monitoring Mosquito-Borne Arbovirus in Various Insect Regions in China in 2018. Fang Yuan,Zhang Wei,Xue Jing-Bo,Zhang Yi Frontiers in cellular and infection microbiology Background:Increases in global travel and trade are changing arbovirus distributions worldwide. Arboviruses can be introduced by travelers, migratory birds, or vectors transported international trade. Arbovirus surveillance in field-collected mosquitoes may provide early evidence for mosquito-borne disease transmission. Methods:During the seasons of high mosquito activity of 2018, 29,285 mosquitoes were sampled from seven sentinel sites in various insect regions. The mosquitoes were analyzed by RT-PCR for alphaviruses, flaviviruses, and orthobunyaviruses. Results:We detected three strains of Japanese encephalitis virus (JEV), five strains of Getah virus (GETV), and 45 strains of insect-specific flaviviruses including Aedes flavivirus (AeFV, 1), Chaoyang virus (CHAOV, 1), Culex flavivirus (CxFV, 17), Hanko virus (HANKV, 2), QuangBinh virus (QBV, 22), and Yunnan Culex flavivirus (YNCxFV, 2). Whole genomes of one strain each of GETV, CxFV, CHAOV, and AeFV were successfully amplified. Phylogenetic analysis revealed that the new JEV strains detected in the Shanghai and Hubei Provinces belong to the GI-b strain and are phylogenetically close to the NX1889 strain (MT134112) isolated from a patient during a JE outbreak in Ningxia in 2018. GETVs were found in Inner Mongolia, Hubei, and Hainan and belonged to Group III. They were closely related to strains isolated from swine. HANKV was recorded for the first time in China and other ISFVs were newly detected at several sentinel sites. The bias-corrected maximum likelihood estimation value for JEV in Jinshan, Shanghai was 4.52/1,000 (range 0.80-14.64). Hence, there is a potential risk of a JEV epidemic in that region. Conclusion:GI-b is the dominant circulating JEV genotype in nature and poses a health risk to animals and humans. The potential threat of widespread GETV distribution as a zoonosis is gradually increasing. The present study also disclosed the dispersion and host range of ISFVs. These findings highlight the importance of tracing the movements of the vectors and hosts of mosquito-borne pathogens in order to prevent and control arbovirus outbreaks in China. 10.3389/fcimb.2021.640993
    Diversity of Culturable Bacteria Isolated From the Feces of Wild Anopheles darlingi (Diptera: Culicidae) Mosquitoes From the Brazilian Amazon. Arruda Andrelisse,Ferreira Gabriel E M,Santos Júnior Antônio,Matos Najla B,Carvalho Tatiane S,Ozaki Luiz S,Stabeli Rodrigo G,Silva Alexandre A E Journal of medical entomology Microorganisms living in the midgut of Anopheles mosquitoes have been studied to fight vector-borne diseases, such as malaria. Studies on the microbiota of the Neotropical Anopheles darlingi, the most important Brazilian vector for malaria, have been reported for the same purpose. Our aims were to isolate and identify culturable bacteria from An. darlingi mosquito guts through their feces and to estimate the species richness and the frequency distribution of the sampled bacteria. Sixty wild females of An. darlingi mosquitoes were captured at two rural locations, near Porto Velho, Rondônia, Brazil. Bacteria were isolated from mosquito feces, which were collected using cages which permit the collection of feces on LB nutrient agar plates. Sixty bacterial colonies were isolated and stored in glycerol at -80°C. Bacteria were identified by sequencing their 16S rRNA gene obtained using PCR and Sanger sequencing. To aid in species identification, MALDI-TOF, VITEK2, and BBL Crystal were used as complementary protocols. The sequences obtained from the 60 bacterial isolates were compared to sequences deposited in GenBank (NCBI) using BLAST. Homology greater than 97% between the query and the subject was used as the criteria for assigning the identity of each isolate. Fourteen species from eight different genera were identified among the 60 isolates. The most frequent species were Serratia liquefaciens (20%) and Serratia marcescens (15%). Due to their established apathogenicity and according to previous studies, we suggest Serratia and Pantoea species as suitable for paratransgenesis development to fight malaria in Brazilian Amazon. 10.1093/jme/tjab028
    Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host-microbiota interactions. Romoli Ottavia,Schönbeck Johan Claes,Hapfelmeier Siegfried,Gendrin Mathilde Nature communications The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development. 10.1038/s41467-021-21195-3
    Use of Microbiota to Fight Mosquito-Borne Disease. Huang Wei,Wang Sibao,Jacobs-Lorena Marcelo Frontiers in genetics Mosquito-borne diseases cause more than 700 million people infected and one million people die (Caraballo and King, 2014). With the limitations of progress toward elimination imposed by insecticide- and drug-resistance, combined with the lack of vaccines, innovative strategies to fight mosquito-borne disease are urgently needed. In recent years, the use of mosquito microbiota has shown great potential for cutting down transmission of mosquito-borne pathogens. Here we review what is known about the mosquito microbiota and how this knowledge is being used to develop new ways to control mosquito-borne disease. We also discuss the challenges for the eventual release of genetically modified (GM) symbionts in the field. 10.3389/fgene.2020.00196
    Multiple Factors Determine the Structure of Bacterial Communities Associated With Under Artificial Rearing Conditions. Chen Shi,Zhang Dongjing,Augustinos Antonios,Doudoumis Vangelis,Bel Mokhtar Naima,Maiga Hamidou,Tsiamis George,Bourtzis Kostas Frontiers in microbiology Insect symbionts are major manipulators of host's behavior. Their effect on parameters such as fecundity, male mating competitiveness, and biological quality in general, can have a major influence on the effectiveness of the sterile insect technique (SIT). SIT is currently being developed and applied against human disease vectors, including , as an environment-friendly method of population suppression, therefore there is a renewed interest on both the characterization of gut microbiota and their exploitation in artificial rearing. In the present study, bacterial communities of eggs, larvae, and adults (both males and females) of artificially reared , were characterized using both culture-dependent and culture-independent approaches. Mosquito-associated bacteria corresponding to thirteen and five bacteria genera were isolated from the larval food and the sugar solution (adult food), respectively. The symbiont community of the females was affected by the provision of a blood meal. and were either introduced or enhanced with the blood meal, whereas were relatively stable during the adult stage of females. Maintenance of these taxa in female guts is probably related with blood digestion. Gut-associated microbiota of males and females were different, starting early after emergence and continuing in older stages. Our results indicate that eggs contained bacteria from more than fifteen genera including , , and , which were also main components of gut microbiota of female adults before and after blood feeding, indicating potential transmission among generations. Our results provided a thorough study of the egg- and gut-associated bacteria of artificially reared , which can be important for further studies using probiotic bacteria to improve the effectiveness of mosquito artificial rearing and SIT applications. 10.3389/fmicb.2020.00605
    The Midgut Microbiota of Colombian Populations with Different Levels of Resistance to the Insecticide Lambda-cyhalothrin. Arévalo-Cortés Andrea,Mejia-Jaramillo Ana M,Granada Yurany,Coatsworth Heather,Lowenberger Carl,Triana-Chavez Omar Insects Insecticide resistance in populations is a problem that hinders vector control and dengue prevention programs. In this study, we determined the susceptibility of populations from six Colombian regions to the pyrethroid lambda-cyhalothrin and evaluated the presence of the V1016I mutation in the sodium channel gene, which has been broadly involved in the resistance to this insecticide. The diversity of the gut microbiota of these mosquito populations was also analyzed. Only mosquitoes from Bello were susceptible to lambda-cyhalothrin and presented a lower allelic frequency of the V1016I mutation. Remarkably, there was not an important change in allelic frequencies among populations with different resistance ratios, indicating that other factors or mechanisms contributed to the resistant phenotype. Treatment of mosquitoes with antibiotics led us to hypothesize that the intestinal microbiota could contribute to the resistance to lambda-cyhalothrin. Beta diversity analysis showed significant differences in the species of bacteria present between susceptible and resistant populations. We identified 14 OTUs of bacteria that were unique in resistant mosquitoes. We propose that mutations are important in the development of resistance to lambda-cyhalothrin at low insecticide concentrations but insect symbionts could play an essential role in the metabolization of pyrethroid insecticides at higher concentrations, contributing to the resistant phenotype in . 10.3390/insects11090584
    Microbiome Interaction Networks and Community Structure From Laboratory-Reared and Field-Collected , , and Mosquito Vectors. Hegde Shivanand,Khanipov Kamil,Albayrak Levent,Golovko George,Pimenova Maria,Saldaña Miguel A,Rojas Mark M,Hornett Emily A,Motl Greg C,Fredregill Chris L,Dennett James A,Debboun Mustapha,Fofanov Yuriy,Hughes Grant L Frontiers in microbiology Microbial interactions are an underappreciated force in shaping insect microbiome communities. Although pairwise patterns of symbiont interactions have been identified, we have a poor understanding regarding the scale and the nature of co-occurrence and co-exclusion interactions within the microbiome. To characterize these patterns in mosquitoes, we sequenced the bacterial microbiome of , , and caught in the field or reared in the laboratory and used these data to generate interaction networks. For collections, we used traps that attracted host-seeking or ovipositing female mosquitoes to determine how physiological state affects the microbiome under field conditions. Interestingly, we saw few differences in species richness or microbiome community structure in mosquitoes caught in either trap. Co-occurrence and co-exclusion analysis identified 116 pairwise interactions substantially increasing the list of bacterial interactions observed in mosquitoes. Networks generated from the microbiome of often included highly interconnected hub bacteria. There were several instances where co-occurring bacteria co-excluded a third taxa, suggesting the existence of tripartite relationships. Several associations were observed in multiple species or in field and laboratory-reared mosquitoes indicating these associations are robust and not influenced by environmental or host factors. To demonstrate that microbial interactions can influence colonization of the host, we administered symbionts to larvae that either possessed or lacked their resident microbiota. We found that the presence of resident microbiota can inhibit colonization of particular bacterial taxa. Our results highlight that microbial interactions in mosquitoes are complex and influence microbiome composition. 10.3389/fmicb.2018.02160
    Blood meal-derived heme decreases ROS levels in the midgut of Aedes aegypti and allows proliferation of intestinal microbiota. Oliveira Jose Henrique M,Gonçalves Renata L S,Lara Flavio A,Dias Felipe A,Gandara Ana Caroline P,Menna-Barreto Rubem F S,Edwards Meredith C,Laurindo Francisco R M,Silva-Neto Mário A C,Sorgine Marcos H F,Oliveira Pedro L PLoS pathogens The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme. 10.1371/journal.ppat.1001320
    Persistent Infection by AlbB Has No Effect on Composition of the Gut Microbiota in Adult Female . Chen Shicheng,Zhao Jiangchao,Joshi Deepak,Xi Zhiyong,Norman Beth,Walker Edward D Frontiers in microbiology The bacteria in the midgut of adult females from laboratory colonies were studied by sequencing the V4 region of 16S rRNA genes, with respect to three experimental factors: stable or cured infection; sugar or blood diet; and age. and dominated the community [>90% of operational taxonomic units (OTUs)]; most taxa were in the classes , , and , and were assigned to (46.9%), (6.4%) and (6.0%), or unclassified (37.2%). Bacterial communities were similar between -cured and -infected mosquito lines, indicating that the gut microbiota were not dysregulated in the presence of . The proportion of was higher in mosquitoes fed a blood meal compared to those provided a sugar meal. Collectively, the bacterial community had a similar structure in older -infected mosquitoes 8 days after the blood meal, as in younger -infected mosquitoes before a blood meal, except that older mosquitoes had a higher proportion of and lower proportion of . Consistent presence of certain predominant bacteria (, , , and ) suggests they would be useful for paratransgenesis to control malaria infection, particularly when coupled to a -based intervention strategy. 10.3389/fmicb.2016.01485
    Mutual exclusion of Asaia and Wolbachia in the reproductive organs of mosquito vectors. Rossi Paolo,Ricci Irene,Cappelli Alessia,Damiani Claudia,Ulissi Ulisse,Mancini Maria Vittoria,Valzano Matteo,Capone Aida,Epis Sara,Crotti Elena,Chouaia Bessem,Scuppa Patrizia,Joshi Deepak,Xi Zhiyong,Mandrioli Mauro,Sacchi Luciano,O'Neill Scott L,Favia Guido Parasites & vectors BACKGROUND:Wolbachia is a group of intracellular maternally inherited bacteria infecting a high number of arthropod species. Their presence in different mosquito species has been largely described, but Aedes aegypti, the main vector of Dengue virus, has never been found naturally infected by Wolbachia. Similarly, malaria vectors and other anophelines are normally negative to Wolbachia, with the exception of an African population where these bacteria have recently been detected. Asaia is an acetic acid bacterium stably associated with several mosquito species, found as a dominant microorganism of the mosquito microbiota. Asaia has been described in gut, salivary glands and in reproductive organs of adult mosquitoes in Ae. aegypti and in anophelines. It has recently been shown that Asaia may impede vertical transmission of Wolbachia in Anopheles mosquitoes. Here we present an experimental study, aimed at determining whether there is a negative interference between Asaia and Wolbachia, for the gonad niche in mosquitoes. METHODS:Different methods (PCR and qPCR, monoclonal antibody staining and FISH) have been used to address the question of the co-localization and the relative presence/abundance of the two symbionts. PCR and qPCR were performed to qualitatively and quantitatively verify the distribution of Asaia and Wolbachia in different mosquito species/organs. Monoclonal antibody staining and FISH were performed to localize the symbionts in different mosquito species. RESULTS:Here we provide evidence that, in Anopheles and in other mosquitoes, there is a reciprocal negative interference between Asaia and Wolbachia symbionts, in terms of the colonization of the gonads. In particular, we have shown that in some mosquito species the presence of one of the symbionts prevented the establishment of the second, while in other systems the symbionts were co-localized, although at reduced densities. CONCLUSIONS:A mutual exclusion or a competition between Asaia and Wolbachia may contribute to explain the inability of Wolbachia to colonize the female reproductive organs of anophelines, inhibiting its vertical transmission and explaining the absence of Wolbachia infection in Ae. aegypti and in the majority of natural populations of Anopheles mosquitoes. 10.1186/s13071-015-0888-0
    Diverse laboratory colonies of Aedes aegypti harbor the same adult midgut bacterial microbiome. Dickson Laura B,Ghozlane Amine,Volant Stevenn,Bouchier Christiane,Ma Laurence,Vega-Rúa Anubis,Dusfour Isabelle,Jiolle Davy,Paupy Christophe,Mayanja Martin N,Kohl Alain,Lutwama Julius J,Duong Veasna,Lambrechts Louis Parasites & vectors BACKGROUND:Host-associated microbes, collectively known as the microbiota, play an important role in the biology of multicellular organisms. In mosquito vectors of human pathogens, the gut bacterial microbiota influences vectorial capacity and has become the subject of intense study. In laboratory studies of vector biology, genetic effects are often inferred from differences between geographically and genetically diverse colonies of mosquitoes that are reared in the same insectary. It is unclear, however, to what extent genetic effects can be confounded by uncontrolled differences in the microbiota composition among mosquito colonies. To address this question, we used 16S metagenomics to compare the midgut bacterial microbiome of six laboratory colonies of Aedes aegypti recently derived from wild populations representing the geographical range and genetic diversity of the species. RESULTS:We found that the diversity, abundance, and community structure of the midgut bacterial microbiome was remarkably similar among the six different colonies of Ae. aegypti, regardless of their geographical origin. We also confirmed the relatively low complexity of bacterial communities inhabiting the mosquito midgut. CONCLUSIONS:Our finding that geographically diverse colonies of Ae. aegypti reared in the same insectary harbor a similar gut bacterial microbiome supports the conclusion that the gut microbiota of adult mosquitoes is environmentally determined regardless of the host genotype. Thus, uncontrolled differences in microbiota composition are unlikely to represent a significant confounding factor in genetic studies of vector biology. 10.1186/s13071-018-2780-1
    Microbial interactions in the mosquito gut determine Serratia colonization and blood-feeding propensity. Kozlova Elena V,Hegde Shivanand,Roundy Christopher M,Golovko George,Saldaña Miguel A,Hart Charles E,Anderson Enyia R,Hornett Emily A,Khanipov Kamil,Popov Vsevolod L,Pimenova Maria,Zhou Yiyang,Fovanov Yuriy,Weaver Scott C,Routh Andrew L,Heinz Eva,Hughes Grant L The ISME journal How microbe-microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that, Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however, one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behavior; Serratia exposure to Ae. aegypti disrupted their feeding behavior, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host-microbe interactions and provides evidence that microbial interactions influence mosquito behavior. 10.1038/s41396-020-00763-3
    Flow cytometry analysis of the microbiota associated with the midguts of vector mosquitoes. Habtewold Tibebu,Duchateau Luc,Christophides George K Parasites & vectors BACKGROUND:The scientific interest to understand the function and structure of the microbiota associated with the midgut of mosquito disease vectors is increasing. The advancement of such a knowledge has encountered challenges and limitations associated with conventional culture-based and PCR techniques. METHODS:Flow cytometry (FCM) combined with various cell marking dyes have been successfully applied in the field of ecological microbiology to circumvent the above shortcomings. Here, we describe FCM technique coupled with live/dead differential staining dyes SYBR Green I (SGI) and Propidium Iodide (PI) to quantify and study other essential characteristics of the mosquito gut microbiota. RESULTS:A clear discrimination between cells and debris, as well as between live and dead cells was achieved when the midgut homogenate was subjected to staining with 5 × 103 dilution of the SGI and 30 μM concentration of the PI. Reproducibly, FCM event collections produced discrete populations including non-fluorescent cells, SYBR positive cells, PI fluorescing cells and cells that fluoresce both in SYBR and PI, all these cell populations representing, respectively, background noise, live bacterial, dead cells and inactive cells with partial permeability to PI. The FCM produced a strong linear relationship between cell counts and their corresponding dilution factors (R (2) = 0.987), and the technique has a better precision compared to qRT-PCR. The FCM count of the microbiota reached a peak load at 18 h post-feeding and started declining at 24 h. The present FCM technique also successfully applied to quantify bacterial cells in fixed midgut samples that were homogenized in 4 % PFA. CONCLUSION:The FCM technique described here offers enormous potential and possibilities of integration with advanced molecular biochemical techniques for the study of the microbiota community in disease vector mosquitoes. 10.1186/s13071-016-1438-0
    Mosquitoes host communities of bacteria that are essential for development but vary greatly between local habitats. Coon Kerri L,Brown Mark R,Strand Michael R Molecular ecology Mosquitoes are insects of interest because several species vector disease-causing pathogens to humans and other vertebrates. We previously reported that mosquitoes from long-term laboratory cultures require living bacteria in their gut to develop, but development does not depend on particular species of bacteria. Here, we focused on three distinct but interrelated areas of study to better understand the role of bacteria in mosquito development by studying field and laboratory populations of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from the southeastern United States. Sequence analysis of bacterial 16S rRNA gene amplicons showed that bacterial community composition differed substantially in larvae from different collection sites, whereas larvae from the same site shared similarities. Although previously unknown to be infected by Wolbachia, results also indicated that Ae. aegypti from one field site hosted a dual infection. Regardless of collection site or factors like Wolbachia infection, however, each mosquito species required living bacteria in their digestive tract to develop. Results also identified several concerns in using antibiotics to eliminate the bacterial community in larvae in order to study its developmental consequences. Altogether, our results indicate that several mosquito species require living bacteria for development. We also hypothesize these species do not rely on particular bacteria because larvae do not reliably encounter the same bacteria in the aquatic habitats they develop in. 10.1111/mec.13877
    Reduced diversity of gut microbiota in two Aedes mosquitoes species in areas of recent invasion. Rosso Fausta,Tagliapietra Valentina,Albanese Davide,Pindo Massimo,Baldacchino Frédéric,Arnoldi Daniele,Donati Claudio,Rizzoli Annapaola Scientific reports Aedes mosquitoes are considered highly successful global invasive species and vectors of several pathogens of relevance for public health. Their midgut's microbiota can play an important role in affecting not only their vectorial competence but also their fitness, physiology, food digestion, metabolism, immunity and adaptation to new environmental conditions. Using high-throughput sequencing we compared the microbiota of Aedes albopictus collected in Italy with those reported in populations from France and Vietnam. We also analysed Aedes koreicus gut microbiota for the first time. We found remarkable individual difference along with common bacterial taxa in both species. Ae. albopictus collected in Italy had a lower richness and a different composition of microbiota in respect to specimens collected in France and Vietnam. It also showed a core microbiota formed mainly of bacteria of the genus Pseudomonas. Overall, the two Aedes species (Ae. albopictus and Ae. koreicus) collected in Italy, showed a large core microbiota with 75.98% of the identified Operational Taxonomic Units. Furthermore, Ae. albopictus had 2.5% prevalence of Wolbachia and 0.07% of Asaia spp, while Ae. koreicus had 14.42% of Asaia spp. and no Wolbachia. This study provides new informations on the spatial variation of the midgut bacterial communities in mosquitoes of medical relevance within areas of recent invasion and provide the basis for further studies aimed at assessing the effects of such variation on vectorial capacity for a range of pathogens. 10.1038/s41598-018-34640-z
    Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections. Kalappa Devaiah Monnanda,Subramani Pradeep Annamalai,Basavanna Sowmya Kanchanahalli,Ghosh Susanta Kumar,Sundaramurthy Varadharajan,Uragayala Sreehari,Tiwari Satyanarayan,Anvikar Anupkumar R,Valecha Neena Malaria journal BACKGROUND:The native gut microbiota of Anopheles mosquitoes is known to play a key role in the physiological function of its host. Interestingly, this microbiota can also influence the development of Plasmodium in its host mosquitoes. In recent years, much interest has been shown in the employment of gut symbionts derived from vectors in the control of vector-borne disease transmission. In this study, the midgut microbial diversity has been characterized among laboratory-reared adult Anopheles stephensi mosquitoes, from the colony created by rearing progeny of wild-caught mosquitoes (obtained from three different locations in southern India) for multiple generations, using 16S ribosomal RNA (rRNA) gene sequencing approach. Further, the influence of native midgut microbiota of mosquitoes on the development of rodent malaria parasite Plasmodium berghei in its host has been studied. METHODS:The microbial diversity associated with the midgut of An. stephensi mosquitoes was studied by sequencing V3 region of 16S ribosomal RNA (rRNA) gene. The influence of native midgut microbiota of An. stephensi mosquitoes on the susceptibility of the mosquitoes to rodent malaria parasite P. berghei was studied by comparing the intensity and prevalence of P. berghei infection among the antibiotic treated and untreated cohorts of mosquitoes. RESULTS:The analysis of bacterial diversity from the midguts of An. stephensi showed Proteobacteria as the most dominant population among the three laboratory-reared strains of An. stephensi studied. Major genera identified among these mosquito strains were Acinetobacter, Pseudomonas, Prevotella, Corynebacterium, Veillonella, and Bacillus. The mosquito infectivity studies carried out to determine the implication of total midgut microbiota on P. berghei infection showed that mosquitoes whose native microbiota cleared with antibiotics had increased susceptibility to P. berghei infection compared to the antibiotic untreated mosquitoes with its natural native microbiota. CONCLUSIONS:The use of microbial symbiont to reduce the competence of vectors involved in disease transmission has gained much importance in recent years as an emerging alternative approach towards disease control. In this context, the present study was aimed to identify the midgut microbiota composition of An. stephensi, and its effect on the development of P. berghei. Interestingly, the analysis of midgut microbiota from An. stephensi revealed the presence of genus Veillonella in Anopheles species for the first time. Importantly, the study also revealed the negative influence of total midgut microbiota on the development of P. berghei in three laboratory strains of An. stephensi, emphasizing the importance of understanding the gut microbiota in malaria vectors, and its relationship with parasite development in designing strategies to control malaria transmission. 10.1186/s12936-018-2535-7
    Changes in Microbiota Across Developmental Stages of , an Invasive Mosquito Vector in Europe: Indications for Microbiota-Based Control Strategies. Alfano Niccolò,Tagliapietra Valentina,Rosso Fausta,Manica Mattia,Arnoldi Daniele,Pindo Massimo,Rizzoli Annapaola Frontiers in microbiology Since it has been understood that gut microbiota of vector mosquitoes can influence their vector competence, efforts have been undertaken to develop new control strategies based on host microbiota manipulation, and aimed at suppressing the vector population or replacing it with a less competent one. For the proper design of such control strategies it is necessary to know the microbiota composition of the target vector species, how it is acquired, and how it changes throughout the host's life cycle. In this study, 16S rRNA amplicon high-throughput sequencing was used to characterize the changes in microbiota from the aquatic environment (larval breeding sites) to the different developmental stages of field-collected in Italy, an emerging invasive mosquito species in Europe and a potential vector of several pathogens. The bacterial communities of the aquatic breeding sites, larvae, pupae and adults showed distinctive structures to one another. Indeed, 84% of community members were unique to a given sample type. Nevertheless, almost 40% of the sequences generated were assigned to bacteria detected in all sample types, suggesting the importance of bacteria transstadially transmitted from water to the adult stage in constituting mosquito microbiota. Among these, genus largely constituted water microbiota, family was the most abundant in larvae and pupae, and genus dominated adult communities. In addition, constituted a core microbiota across all sample types. Our results suggest that the microbiota of mosquitoes is composed by a community which derives from the aquatic bacteria of the larval breeding sites, is then filtered by the larval gut, where only certain members are able to persist, rearranged by metamorphosis and finally modified by the change in diet at the adult stage. Understanding how the microbiota of changes through the mosquito life cycle represents a first step in selecting bacterial candidates for use in microbiota-based intervention measures for this species. The properties which exhibits in this species, such as dominance, high prevalence and transstadial transmission, prevent the use of but make an ideal candidate for paratransgenesis. 10.3389/fmicb.2019.02832
    [Taxonomic composition of metagenomic community in the larval gut of mosquito Anopheles sinensis (Diptera: Culicidae)]. Nan Chun-Yan,Ma Ya-Jun,Xu Jian-Nong,Liang Jian Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases OBJECTIVE:To investigate the bacteria diversity in larval gut of field-collected Anopheles sinensis. METHODS:The 16S rDNA V4 region of An. sinensis larvae collected from paddy on Jiading District of Shanghai (L1/L2) and small seeping water on Wenchang City of Hainan (AS) was sequenced by high-throughput pyrosequencing. Using Qiime and Mothur softwares, the number of sequences and operational taxonomic units (OTUs) for each sample was sorted and calculated, the species abundance and distribution, Alpha diversity index and difference times of species abundance among samples were analyzed. RESULTS:The number of sequences and OTUs for each sample were 253 724/3 930 (L1), 225 203/4 312 (L2) and 73 990/2 380 (AS). The rarefaction curves showed that adequate sampling was achieved. The number of OTUs was close to actual situation. The value of richness index was 5 942.61/6 534.88 (L1), 6 328.17/7 235.89 (L2) and 4228.66/5 651.20 (AS); diversity index was 4.63/0.03 (L1), 5.10/0.02 (L2) and 0.14/3.94 (AS). The dominant species of An. sinensis larvae gut microbiota all belonged to the phylum Proteobacteria, with a percentage of 87% (AS) and 90% (L). In addition, the dominant phyla among them were Firmicutes, Bacteroidetes and Actinobacteria. The comparison of bacterial abundance between L and AS showed that there were 18 phyla with significant difference, except the Proteobacteria and Deinococcus-Thermus; only 9 phyla were different significantly between L1 and L2. CONCLUSION:Evenness and richness of bacteria flora in the An. sinensis larvae gut collected from paddy and small seeping waters were obtained.
    Mosquito C-type lectins maintain gut microbiome homeostasis. Pang Xiaojing,Xiao Xiaoping,Liu Yang,Zhang Rudian,Liu Jianying,Liu Qiyong,Wang Penghua,Cheng Gong Nature microbiology The long-term evolutionary interaction between the host immune system and symbiotic bacteria determines their cooperative rather than antagonistic relationship. It is known that commensal bacteria have evolved a number of mechanisms to manipulate the mammalian host immune system and maintain homeostasis. However, the strategies employed by the microbiome to overcome host immune responses in invertebrates still remain to be understood. Here, we report that the gut microbiome in mosquitoes utilizes C-type lectins (mosGCTLs) to evade the bactericidal capacity of antimicrobial peptides (AMPs). Aedes aegypti mosGCTLs facilitate colonization by multiple bacterial strains. Furthermore, maintenance of the gut microbial flora relies on the expression of mosGCTLs in A. aegypti. Silencing the orthologues of mosGCTL in another major mosquito vector (Culex pipiens pallens) also impairs the survival of gut commensal bacteria. The gut microbiome stimulates the expression of mosGCTLs, which coat the bacterial surface and counteract AMP activity. Our study describes a mechanism by which the insect symbiotic microbiome offsets gut immunity to achieve homeostasis. 10.1038/nmicrobiol.2016.23
    Shifts in the microbiota associated with male mosquitoes (Aedes aegypti) exposed to an obligate gut fungal symbiont (Zancudomyces culisetae). Frankel-Bricker Jonas Scientific reports Research characterizing arthropod-associated microbiota has revealed that microbial dynamics can have an important impact on host phenotypic traits. The influence of fungi on these interactions are emerging as targets for research, especially in organisms associated with global human health. A recent study demonstrated colonization of a widespread gut fungus (Zancudomyces culisetae) in a larval mosquito (Aedes aegypti) digestive tract affected microbiomes in larvae and newly emerged adult females (Frankel-Bricker et al. Appl Environ Microbiol, 2020. https://doi.org/10.1128/AEM.02334-19) but did not investigate these processes in males. The objective of the study presented here was to assess fungal influences on adult male mosquito microbiomes to enable a more complete assessment of fungal-bacterial-host interactions in the A. aegypti-Z. culisetae system. Sequencing of 16S rRNA gene amplicons from microbiomes harbored in adult males directly after emerging from pupae revealed larval fungal exposure significantly decreased overall microbial community diversity, altered microbiome composition and structure, and decreased within-group microbiome variation across individuals. Further, bacteria in the family Burkholderiaceae were present in high abundance in fungal-exposed males, likely contributing to the disparate microbiota between treatment groups. Comparisons between male and the female microbiomes analyzed in Frankel-Bricker et al. (2020), showed distinct shifts in bacterial communities incurred by larval exposure to fungi, potentially revealing sex-specific fungal-bacterial-host dynamics in A. aegypti. These findings highlight the complex role a gut fungus can play in influencing the microbial communities harbored in an important insect and emphasize the significance of accounting for an organism's sex when studying fungal-bacterial-host dynamics. 10.1038/s41598-020-69828-9
    Factors shaping the gut bacterial community assembly in two main Colombian malaria vectors. Bascuñán Priscila,Niño-Garcia Juan Pablo,Galeano-Castañeda Yadira,Serre David,Correa Margarita M Microbiome BACKGROUND:The understanding of the roles of gut bacteria in the fitness and vectorial capacity of mosquitoes that transmit malaria, is improving; however, the factors shaping the composition and structure of such bacterial communities remain elusive. In this study, a high-throughput 16S rRNA gene sequencing was conducted to understand the effect of developmental stage, feeding status, species, and geography on the composition of the gut bacterial microbiota of two main Colombian malaria vectors, Anopheles nuneztovari and Anopheles darlingi. RESULTS:The results revealed that mosquito developmental stage, followed by geographical location, are more important determinants of the gut bacterial composition than mosquito species or adult feeding status. Further, they showed that mosquito gut is a major filter for environmental bacteria colonization. CONCLUSIONS:The sampling design and analytical approach of this study allowed to untangle the influence of factors that are simultaneously shaping the microbiota composition of two Latin-American malaria vectors, essential aspect for the design of vector biocontrol strategies. 10.1186/s40168-018-0528-y
    Molecular characterization of midgut microbiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India. Yadav Kamlesh K,Bora Ajitabh,Datta Sibnarayan,Chandel Kshitij,Gogoi Hemant K,Prasad G B K S,Veer Vijay Parasites & vectors BACKGROUND:Microbiota inhabiting midguts of mosquitoes play a key role in the host - parasite interaction and enhance vectorial capacity of viral diseases like dengue and chikungunya fevers. Mosquito midgut is considered to be an important site for host-pathogen interaction and pathogen survival is thought to be an outcome of this interaction. In the present study we examined the bacterial community in the midgut of Aedes mosquitoes in Arunanchal Pradesh, India, a subtropical zone where dengue fever is reported to be emerging. METHOD:Larvae and pupa of Aedes mosquitoes were collected from a biodiversity hotspot, Bhalukpong, Arunachal Pradesh, India. 16S rRNA gene sequences were used for identification of isolated bacterial population from each species of mosquitoes. We used various diversity indices to assess the diversity and richness of the bacterial isolates in both mosquito species. RESULT:On the basis of 16S rRNA gene sequence analysis a total of 24 bacterial species from 13 genera were identified belonging to 10 families of four major phyla. Phylum Proteobacteria was dominant followed by Firmicutes, Bacteroidetes and Actinobacteria. The midgut bacteria belonging to the phylum Proteobacteria and Firmicutes were isolated from both Ae. albopictus and Ae. aegypti, whereas, bacteria belonging to phylum Bacteroidetes and Actinobacteria were isolated only from Ae. albopictus and Ae. aegypti respectively. Enterobacter cloacae was the dominant bacterial species in both Ae. albopictus (33.65%) and Ae. aegypti (56.45%). Bacillus aryabhattai (22.78%) was the second most common bacterial species in Ae. albopictus whereas, in Ae. aegypti the second most common bacterial species was Stenotrophomonas maltophilia (7.44%). CONCLUSION:The family Enterobacteriaceae of phylum Proteobacteria was dominant in both species of Aedes mosquitoes. To the best of our knowledge, this is the first attempt to study midgut microbiota from a biodiversity hotspot in Northeastern India. Some bacterial genera Enterobacter and Acinetobacter isolated in this study are known to play important roles in parasite-vector interaction. Information on midgut microflora may lead towards the development of novel, safe, and effective strategies to manipulate the vectorial capacity of mosquitoes. 10.1186/s13071-015-1252-0
    Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictus. Bennett Kelly L,Gómez-Martínez Carmelo,Chin Yamileth,Saltonstall Kristin,McMillan W Owen,Rovira Jose R,Loaiza Jose R Scientific reports Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that influence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We find both species share a limited, yet highly variable core microbiota, reflecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We find significant differences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we find little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama. 10.1038/s41598-019-48414-8
    Trans-stadial fate of the gut bacterial microbiota in Anopheles albimanus. Galeano-Castañeda Yadira,Bascuñán Priscila,Serre David,Correa Margarita M Acta tropica Gut microbiota communities in mosquitoes are influenced among others, by developmental stage. There is evidence that the aquatic environment where larvae feed influences the mosquito gut bacterial community composition with only a subgroup of these bacteria been transmitted trans-stadially to adults. This study evaluated the gut bacterial composition of Anopheles albimanus larvae, emerged and circulating mosquitoes, as well as water from the larval habitat, to elucidate transitions in these bacterial communities and determine the final composition in circulating mosquitoes. A 16S rRNA Illumina sequencing allowed to determine that Proteobacteria was the most abundant phylum in larvae (72.4%), emerged mosquitoes (75%), circulating adults (45.4%) and water from the larval habitat (79.1%). A core microbiome analysis evidenced that Enterobacter, Bacillus and Staphylococcus genera were the core bacterial microbiota (OTUs detected in >90%) in the four groups evaluated. PCoA cluster based on Jaccard and Bray Curtis distances showed two main bacterial clusters, one comprising the emerged and circulating adults, and the other the larvae. The results indicated that the gut microbiota of An. albimanus larvae is composed of bacteria acquired from the larval habitat; then, a rearrangement of the bacterial communities occurs in the trans-stadial passage. However, the higher bacterial richness detected in circulating adults suggests bacterial acquisition from the terrestrial environment where the mosquito feeds. Finally, the trans-stadially passage of some bacteria makes of interest their evaluation as candidates for paratransgenic control. 10.1016/j.actatropica.2019.105204
    Role of gut inhabitants on vectorial capacity of mosquitoes. Jayakrishnan Lekshmi,Sudhikumar Ambalaparambil Vasu,Aneesh Embalil Mathachan Journal of vector borne diseases Mosquito-borne diseases are spreading at an alarming rate. Globally millions of deaths occur due to the diseases transmitted by mosquitoes, next to AIDS and tuberculosis. Several methods have been used to control these vectors and the diseases caused by them. Earlier studies have shown the potential role of mosquito gut inhabitants on disease transmission. Their findings can be used as an innovative approach for devising strategies to modify the survival of mosquitoes by reducing their lifespan, reproduction and disease transmission abilities. In this study, microbiome of the three genera of mosquitoes, namely Aedes, Anopheles, and Culex along with their vectorial capacity have been reviewed for assessing their role in mosquito control and transmission. Relevant articles were accessed using different databases, including LILACS, Embase, Science Direct and PubMed from inception to June 2017. The search keywords included "Aedes", "Anopheles", "Culex", "gut inhabitants", "vectors", and "mosquito". The titles, abstract, and keywords of the retrieved articles were screened, and eligible research articles were sorted. The review indicates that paratransgenesis may be considered as a versatile and effective strategy to eradicate the spurt of mosquito transmitting diseases. Enterobacter species is the most common type of gram-negative bacteria associated with the gut of all the three genera of mosquitoes. It was found to have a beneficial effect on humans as it helps in destroying dreadful disease-transmitting vectors. These symbiotic qualities of the microbes need to be thoroughly investigated further to reveal their antipathogenic effect on the vector. 10.4103/0972-9062.242567
    Effects of Organic Amendments on Microbiota Associated with the Mosquito Vector of the Saint Louis Encephalitis and West Nile Viruses. Duguma Dagne,Hall Michael W,Smartt Chelsea T,Neufeld Josh D mSphere Pollution from nutrients in aquatic habitats has been linked to increases in disease vectors, including mosquitoes and other pestiferous insects. One possibility is that changes in mosquito microbiomes are impacted by nutrient enrichments and that these changes affect various traits, including larval development, susceptibility to larval control agents, and susceptibility of the adult mosquitoes to pathogens. We tested this hypothesis using field mesocosms supplemented with low- and high-organic-nutrient regimens and then sampled microbial communities associated with the naturally colonizing mosquito vector. By high-throughput sequencing of 16S rRNA gene sequences, we found no significant differences in overall microbial communities associated with sampled mosquitoes, despite detecting discernible differences in environmental variables, including pH, dissolved oxygen, and nutrient amendments. Nevertheless, indicator species analysis revealed that members of the were significantly associated with mosquitoes that originated from high-nutrient enrichments. In contrast, members of the were associated with mosquitoes from the low-nutrient enrichment. High bacterial variability associated with the life stages of the was largely unaffected by levels of nutrient enrichments that impacted larval microbial resources, including bacteria, ciliates, and flagellates in the larval environments. Mosquito microbiota provide important physiological and ecological attributes to mosquitoes, including an impact on their susceptibility to pathogens, fitness, and sensitivity to mosquito control agents. mosquito populations transmit various pathogens, including the Saint Louis and West Nile viruses, and proliferate in nutrient-rich environments, such as in wastewater treatment wetlands. Our study examined whether increases in nutrients within larval mosquito developmental habitats impact microbial communities associated with mosquitoes. We characterized the effects of organic enrichments on microbiomes associated with mosquitoes and identified potential bacterial microbiota that will be further investigated for whether they alter mosquito life history traits and for their potential role in the development of microbial-based control strategies. 10.1128/mSphere.00387-16
    Composition of Anopheles coluzzii and Anopheles gambiae microbiota from larval to adult stages. Gimonneau Geoffrey,Tchioffo Majoline T,Abate Luc,Boissière Anne,Awono-Ambéné Parfait H,Nsango Sandrine E,Christen Richard,Morlais Isabelle Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases During their immature life stages, malaria mosquitoes are exposed to a wide array of microbes and contaminants from the aquatic habitats. Although prior studies have suggested that environmental exposure shapes the microbial community structure in the adult mosquito, most reports have focused on laboratory-based experiments and on a single mosquito epithelium, the gut. In this study, we investigated the influence of the breeding site on the development of the Anopheles coluzzii and Anopheles gambiae microbiota in natural conditions. We characterized bacterial communities from aquatic habitats, at surface microlayer and subsurface water levels, to freshly emerge adult mosquitoes using multiplexed 16S rRNA gene pyrosequencing and we separately analyzed the microbiota associated with the different epithelia of adult individual, midguts, ovaries and salivary glands. We found that the distribution of bacterial communities in the aquatic habitats differed according to the depth of water collections. Inter-individual variation of bacterial composition was large in larvae guts but adult mosquitoes from a same breeding site shared quite similar microbiota. Although some differences in bacterial abundances were highlighted between the different epithelia of freshly emerged An. coluzzii and An. gambiae, an intriguing feature from our study is the particular similarity of the overall bacterial communities. Our results call for further investigations on the bacterial population dynamics in the different tissues to determine the distinctive characteristics of each microbiota during the mosquito lifespan and to identify specific interactions between certain key phyla or species and the insect life history traits. 10.1016/j.meegid.2014.09.029
    Deep sequencing reveals extensive variation in the gut microbiota of wild mosquitoes from Kenya. Osei-Poku J,Mbogo C M,Palmer W J,Jiggins F M Molecular ecology The mosquito midgut is a hostile environment that vector-borne parasites must survive to be transmitted. Commensal bacteria in the midgut can reduce the ability of mosquitoes to transmit disease, either by having direct anti-parasite effects or by stimulating basal immune responses of the insect host. As different bacteria have different effects on parasite development, the composition of the bacterial community in the mosquito gut is likely to affect the probability of disease transmission. We investigated the diversity of mosquito gut bacteria in the field using 454 pyrosequencing of 16S rRNA to build up a comprehensive picture of the diversity of gut bacteria in eight mosquito species in this population. We found that mosquito gut typically has a very simple gut microbiota that is dominated by a single bacterial taxon. Although different mosquito species share remarkably similar gut bacteria, individuals in a population are extremely variable and can have little overlap in the bacterial taxa present in their guts. This may be an important factor in causing differences in disease transmission rates within mosquito populations. 10.1111/j.1365-294X.2012.05759.x
    Who is eating fructose within the Aedes albopictus gut microbiota? Guégan Morgane,Tran Van Van,Martin Edwige,Minard Guillaume,Tran Florence-Hélène,Fel Benjamin,Hay Anne-Emmanuelle,Simon Laurent,Barakat Mohamed,Potier Patrick,Haichar Feth El Zahar,Valiente Moro Claire Environmental microbiology The Asian tiger mosquito Aedes albopictus is a major public health concern because of its invasive success and its ability to transmit pathogens. Given the low availability of treatments against mosquito-borne diseases, vector control remains the most suitable strategy. The methods used thus far are becoming less effective, but recent strategies have emerged from the study of mosquito-associated microorganisms. Although the role of the microbiota in insect biology does not require further proof, much remains to be deciphered in mosquitoes, especially the contribution of the microbiota to host nutrient metabolism. Mosquitoes feed on plant nectar, composed of mostly fructose. We used stable isotope probing to identify bacteria and fungi assimilating fructose within the gut of Ae. albopictus. Mosquitoes were fed a C-labelled fructose solution for 24 h. Differences in the active microbial community according to the sex of mosquitoes were highlighted. The bacterium Lelliottia and the fungi Cladosporium and Aspergillus dominated the active microbiota in males, whereas the bacterium Ampullimonas and the yeast Cyberlindnera were the most active in females. This study is the first to investigate trophic interactions between Ae. albopictus and its microbiota, thus underscoring the importance of the microbial component in nectar feeding in mosquitoes. 10.1111/1462-2920.14915
    Pyrosequencing 16S rRNA genes of bacteria associated with wild tiger mosquito Aedes albopictus: a pilot study. Minard Guillaume,Tran Florence-Hélène,Dubost Audrey,Tran-Van Van,Mavingui Patrick,Moro Claire Valiente Frontiers in cellular and infection microbiology The Asian tiger mosquito Aedes (Stegomya) albopictus is an invasive species that has spread across the world in the last two decades, showing a great capacity to adapt to contrasting climates and environments. While demonstrated in many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that needs to be investigated. Also some bacterial species have already been identified in Ae. albopictus using classical methods, but a more accurate survey of mosquito-associated bacterial diversity is needed to decipher the potential biological functions of bacterial symbionts in mediating or constraining insect adaptation. We surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar by pyrosequencing 16S rRNA gene amplicons. Different aspects of amplicon preparation and sequencing depth were tested to optimize the breadth of bacterial diversity identified. The results revealed that all mosquitoes collected from different sites have a bacterial microbiota dominated by a single taxon, Wolbachia pipientis, which accounted for about 99% of all 92,615 sequences obtained. As Ae. albopictus is known to harbor two Wolbachia strains (wAlbA and wAlbB), a quantitative PCR was used to estimate the relative densities, (i.e., the bacteria-to-host gene ratios) of each strains in individual mosquitoes. Relative densities were between 6.25 × 10(0.01) and 5.47 × 10(0.1) for wAlbA and between 2.03 × 10(0.1) and 1.4 × 10(1) for wAlbB. Apart from Wolbachia, a total of 31 bacterial taxa were identified at the genus level using different method variations. Diversity index values were low and probably underestimated the true diversity due to the high abundance of Wolbachia sequences vastly outnumbering sequences from other taxa. Further studies should implement alternative strategies to specifically discard from analysis any sequences from Wolbachia, the dominant endosymbiotic bacterium in Ae. albopictus from this area. 10.3389/fcimb.2014.00059
    Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, Aedes albopictus from Tezpur, India. Yadav Kamlesh K,Datta Sibnarayan,Naglot Ashok,Bora Ajitabh,Hmuaka Vanlal,Bhagyawant Sameer,Gogoi Hemanta K,Veer Vijay,Raju P Srinivas PloS one Aedes aegypti and Ae. albopictus are among the most important vectors of arboviral diseases, worldwide. Recent studies indicate that diverse midgut microbiota of mosquitoes significantly affect development, digestion, metabolism, and immunity of their hosts. Midgut microbiota has also been suggested to modulate the competency of mosquitoes to transmit arboviruses, malaria parasites etc. Interestingly, the midgut microbial flora is dynamic and the diversity changes with the development of vectors, in addition to other factors such as species, sex, life-stage, feeding behavior and geographical origin. The aim of the present study was to investigate the midgut bacterial diversity among larva, adult male, sugar fed female and blood fed female Ae. albopictus collected from Tezpur, Northeastern India. Based on colony morphological characteristics, we selected 113 cultivable bacterial isolates for 16S rRNA gene sequence based molecular identification. Of the 113 isolates, we could identify 35 bacterial species belonging to 18 distinct genera under four major phyla, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Phyla Proteobacteria and Firmicutes accounted for majority (80%) of the species, while phylum Actinobacteria constituted 17% of the species. Bacteroidetes was the least represented phylum, characterized by a single species- Chryseobacterium rhizoplanae, isolated from blood fed individuals. Dissection of midgut microbiota diversity at different developmental stages of Ae. albopictus will be helpful in better understanding mosquito-borne diseases, and for designing effective strategies to manage mosquito-borne diseases. 10.1371/journal.pone.0167409
    Curious entanglements: interactions between mosquitoes, their microbiota, and arboviruses. Caragata Eric P,Tikhe Chinmay V,Dimopoulos George Current opinion in virology Mosquitoes naturally harbor a diverse community of microorganisms that play a crucial role in their biology. Mosquito-microbiota interactions are abundant and complex. They can dramatically alter the mosquito immune response, and impede or enhance a mosquito's ability to transmit medically important arboviral pathogens. Yet critically, given the massive public health impact of arboviral disease, few such interactions have been well characterized. In this review, we describe the current state of knowledge of the role of microorganisms in mosquito biology, how microbial-induced changes to mosquito immunity moderate infection with arboviruses, cases of mosquito-microbial-virus interactions with a defined mechanism, and the molecular interactions that underlie the endosymbiotic bacterium Wolbachia's ability to block virus infection in mosquitoes. 10.1016/j.coviro.2019.05.005
    Mosquito gut antiparasitic and antiviral immunity. Saraiva Raúl G,Kang Seokyoung,Simões Maria L,Angleró-Rodríguez Yesseinia I,Dimopoulos George Developmental and comparative immunology Mosquitoes are responsible for the transmission of diseases with a serious impact on global human health, such as malaria and dengue. All mosquito-transmitted pathogens complete part of their life cycle in the insect gut, where they are exposed to mosquito-encoded barriers and active factors that can limit their development. Here we present the current understanding of mosquito gut immunity against malaria parasites, filarial worms, and viruses such as dengue, Chikungunya, and West Nile. The most recently proposed immune mediators involved in intestinal defenses are discussed, as well as the synergies identified between the recognition of gut microbiota and the mounting of the immune response. 10.1016/j.dci.2016.01.015
    Shared larval rearing environment, sex, female size and genetic diversity shape Ae. albopictus bacterial microbiota. Minard Guillaume,Tran Florence-Hélène,Tran Van Van,Fournier Corentin,Potier Patrick,Roiz David,Mavingui Patrick,Valiente Moro Claire PloS one The Asian tiger mosquito Aedes albopictus became of public health concern as it can replicate and transmit viral and filarial pathogens with a strong invasive success over the world. Various strategies have been proposed to reduce mosquito population's vectorial capacity. Among them, symbiotic control of mosquito borne disease offers promising perspectives. Such method is likely to be affected by the dynamics of mosquito-associated symbiotic communities, which might in turn be affected by host genotype and environment. Our previous study suggested a correlation between mosquitoes' origin, genetic diversity and midgut bacterial diversity. To distinguish the impact of those factors, we have been studying the midgut bacterial microbiota of two Ae. albopictus populations from tropical (La Réunion) and temperate (Montpellier) origins under controlled laboratory conditions. the two populations experienced random mating or genetic bottleneck. Microbiota composition did not highlight any variation of the α and β-diversities in bacterial communities related to host's populations. However, sizes of the mosquitoes were negatively correlated with the bacterial α-diversity of females. Variations in mosquito sex were associated with a shift in the composition of bacterial microbiota. The females' mosquitoes also exhibited changes in the microbiota composition according to their size and after experiencing a reduction of their genetic diversity. These results provide a framework to investigate the impact of population dynamics on the symbiotic communities associated with the tiger mosquito. 10.1371/journal.pone.0194521
    Effects of environment, dietary regime and ageing on the dengue vector microbiota: evidence of a core microbiota throughout Aedes aegypti lifespan. David Mariana Rocha,Santos Lilha Maria Barbosa Dos,Vicente Ana Carolina Paulo,Maciel-de-Freitas Rafael Memorias do Instituto Oswaldo Cruz Mosquito midgut microbiota is a key component of vector competence, as gut bacteria can disturb pathogen development. In this study, we addressed the microbiota composition of Aedes aegypti during its lifespan, under field conditions. We also investigated the possible effects of environment, dietary regime and ageing on the gut community composition. We employed culture independent and dependent approaches to characterise vector microbiota. There was evidence of a lifelong stable core microbiota after mosquitoes were released into an urban settlement, where they presumably fed on a range of vertebrate hosts and carbohydrate sources. This core was formed mainly of bacteria belonging to the genera Pseudomonas, Acinetobacter, Aeromonas and Stenotrophomonas and to the families Oxalobacteraceae, Enterobacteriaceae and Comamonadaceae. We showed that both dietary regime and age were associated with the abundance of some bacterial groups in the Ae. aegypti microbiota. The majority of the bacterial groups we identified have been detected in the midgut of Ae. aegypti from laboratory and wild populations, indicating a possible core microbiota associated with this mosquito species. Our findings suggest that Ae. aegypti harbours a stable bacterial community during its adult life, similar to mosquito populations from distinct geographic areas, which may be further explored for arbovirus biocontrol strategies. 10.1590/0074-02760160238
    A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes. Liu Xinyi,Wu Si,Li Wenqian,Zhang Meihong,Wu Yan,Zhou Ning,Wu Pa Journal of visualized experiments : JoVE The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism. This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species. 10.3791/61341
    Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus. Valiente Moro Claire,Tran Florence Hélène,Raharimalala Fara Nantenaina,Ravelonandro Pierre,Mavingui Patrick BMC microbiology BACKGROUND:The microbiota has been shown to play an important role in the biology of insects. In recent decades, significant efforts have been made to better understand the diversity of symbiotic bacteria associated with mosquitoes and assess their influence on pathogen transmission. Here, we report the bacterial composition found in field-caught Aedes albopictus populations by using culture-dependent methods. RESULTS:A total of 104 mosquito imagos (56 males and 48 females) were caught from four contrasting biotopes of Madagascar and their bacterial contents were screened by plating whole body homogenates on three different culture media. From 281 bacterial colony types obtained, amplified ribosomal DNA restriction analysis (ARDRA) showed they had 40 distinct ribotypes. Sequencing and BLAST analysis of the 16S rDNA genes responsible for each representative profile made it possible to identify 27 genera distributed in three major phyla. In female mosquitoes, bacterial isolates were mostly Proteobacteria (51.3%) followed by Firmicutes (30.3%) and Actinobacteria (18.4%). Conversely, Actinobacteria was the most abundant phylum in male mosquitoes (48%) followed by Proteobacteria (30.6%) and Firmicutes (20.4%). The relative abundance and composition of isolates also varied between sampling sites, ranging from 3 distinct families in Ankazobe to 8 in Tsimbazaza Park, and Toamasina and Ambohidratrimo. Pantoea was the most common genus in both females and males from all sampling sites, except for Ambohidratrimo. No differences in genome size were found between Pantoea isolates from mosquitoes and reference strains in pulse field gel electrophoresis. However, according to the numbers and sizes of plasmids, mosquito isolates clustered into three different groups with other strains isolated from insects but distinct from isolates from the environment. CONCLUSIONS:The recent upsurge in research into the functional role of the insect microbiota prompts the interest to better explore the role some bacteria detected here may have in the mosquito biology. Future studies of culturable bacteria might decipher whether they have a biological role in the invasiveness of Ae. albopictus. As a possible candidate for paratransgenesis, the predominant genus Pantoea will be characterized to better understand its genetic contents and any possible influence it may have on vector competence of Ae. albopictus. 10.1186/1471-2180-13-70
    Characterization of Bacterial Communities in Breeding Waters of Anopheles darlingi in Manaus in the Amazon Basin Malaria-Endemic Area. Nilsson Louise K J,de Oliveira Marta Rodrigues,Marinotti Osvaldo,Rocha Elerson Matos,Håkansson Sebastian,Tadei Wanderli P,de Souza Antonia Queiroz Lima,Terenius Olle Microbial ecology The microbiota in mosquito breeding waters can affect ovipositing mosquitoes, have effects on larval development, and can modify adult mosquito-gut bacterial composition. This, in turn, can affect transmission of human pathogens such as malaria parasites. Here, we explore the microbiota of four breeding sites for Anopheles darlingi, the most important malaria vector in Latin America. The sites are located in Manaus in the Amazon basin in Brazil, an area of active malaria transmission. Using 16S rRNA gene sequencing by MiSeq, we found that all sites were dominated by Proteobacteria and Firmicutes and that 94% of the total number of reads belonged to 36 operational taxonomic units (OTUs) identified in all sites. Of these, the most common OTUs belonged to Escherichia/Shigella, Staphylococcus, and Pseudomonas. Of the remaining 6% of the reads, the OTUs found to differentiate between the four sites belonged to the orders Burkholderiales, Actinomycetales, and Clostridiales. We conclude that An. darlingi can develop in breeding waters with different surface-water bacteria, but that the common microbiota found in all breeding sites might indicate or contribute to a suitable habitat for this important malaria vector. 10.1007/s00248-019-01369-9
    Culex pipiens and Culex restuans mosquitoes harbor distinct microbiota dominated by few bacterial taxa. Muturi Ephantus J,Kim Chang-Hyun,Bara Jeffrey,Bach Elizabeth M,Siddappaji Madhura H Parasites & vectors BACKGROUND:Mosquitoes host diverse microbial communities that influence many aspects of their biology including reproduction, digestion, and ability to transmit pathogens. Unraveling the composition, structure, and function of these microbiota can provide new opportunities for exploiting microbial function for mosquito-borne disease control. METHODS:MiSeq® sequencing of 16S rRNA gene amplicons was used to characterize the microbiota of adult females of Culex pipiens L. and Cx. restuans Theobald collected from nine study sites in central Illinois. RESULTS:Out of 195 bacterial OTUs that were identified, 86 were shared between the two mosquito species while 16 and 93 OTUs were unique to Cx. pipiens and Cx. restuans, respectively. The composition and structure of microbial communities differed significantly between the two mosquito species with Cx. restuans hosting a more diverse bacterial community compared to Cx. pipiens. Wolbachia (OTU836919) was the dominant bacterial species in Cx. pipiens accounting for 91% of total microbiota while Sphingomonas (OTU817982) was the dominant bacterial species in Cx. restuans accounting for 31% of total microbiota. Only 3 and 6 OTUs occurred in over 60% of individuals in Cx. pipiens and Cx. restuans, respectively. There was little effect of study site on bacterial community structure of either mosquito species. CONCLUSION:These results suggest that the two mosquito species support distinct microbial communities that are sparsely distributed between individuals. These findings will allow investigations of the role of identified microbiota on the spatial and temporal heterogeneity in WNV transmission and their potential application in disease control. 10.1186/s13071-016-1299-6
    The tripartite interactions between the mosquito, its microbiota and Plasmodium. Romoli Ottavia,Gendrin Mathilde Parasites & vectors The microbiota of Anopheles mosquitoes interferes with mosquito infection by Plasmodium and influences mosquito fitness, therefore affecting vectorial capacity. This natural barrier to malaria transmission has been regarded with growing interest in the last 20 years, as it may be a source of new transmission-blocking strategies. The last decade has seen tremendous progress in the functional characterisation of the tripartite interactions between the mosquito, its microbiota and Plasmodium parasites. In this review, we provide insights into the effects of the mosquito microbiota on Plasmodium infection and on mosquito physiology, and on how these aspects together influence vectorial capacity. We also discuss three current challenges in the field, namely the need for a more relevant microbiota composition in experimental mosquitoes involved in vector biology studies, for a better characterisation of the non-bacterial microbiota, and for further functional studies of the microbiota present outside the gut. 10.1186/s13071-018-2784-x
    Temporal Variations of Microbiota Associated with the Immature Stages of Two Florida Culex Mosquito Vectors. Duguma Dagne,Hall Michael W,Smartt Chelsea T,Neufeld Josh D Microbial ecology Microbiota associated with mosquito vector populations impact several traits of mosquitoes, including survival, reproduction, control, and immunity against pathogens. The influence of seasonal variations and mosquito species on mosquito gut microbiota is poorly understood. We sought to determine whether the mosquito microbiota associated with immature stages of two congeners (Culex coronator and Culex nigripalpus) differ temporally and between the two species. Using high throughput 16S rRNA gene sequence analysis, we characterized bacterial and archaeal communities found in the immature stages of the two Culex mosquito species sampled over three seasons to compare the diversity of bacteria between the two species. Beta diversity analyses of the larval microbiota sequences revealed that the two Culex species differed significantly, both temporally within each species and between the two species. Bacteria in Cx. coronator larvae were dominated by Alphaproteobacteria, mainly associated with Roseoccocus and unidentified species of Rhizobiales, and two unidentified species of Cyanobacteria. In contrast, Cx. nigripalpus was dominated by Thorsellia anophelis (Gammaproteobacteria), Clostridium, an unidentified species of Ruminococcacae (Clostridiales), and additional unidentified species associated with Erysipelotrichaceae (Erysipelotrichales), Bacteroidales, and Mollicutes. Results of our study revealed both seasonal and interspecies differences in bacterial community composition associated with the immature stages of Cx. coronator and Cx. nigripalpus vector populations in Florida. These results have important implications for our understanding of the underlying factors of variations in disease transmission among seasons, susceptibility to various pesticides, and other biotic factors, including the role of the microbiota on the spread of invasive species. In addition, our results suggest close associations of certain bacteria species with each of the two Culex species that will be further targeted for their potential in the development of microbial-based control strategies. 10.1007/s00248-017-0988-9
    Insect pathogenic fungus interacts with the gut microbiota to accelerate mosquito mortality. Wei Ge,Lai Yiling,Wang Guandong,Chen Huan,Li Fang,Wang Sibao Proceedings of the National Academy of Sciences of the United States of America The insect gut microbiota plays crucial roles in modulating the interactions between the host and intestinal pathogens. Unlike viruses, bacteria, and parasites, which need to be ingested to cause disease, entomopathogenic fungi infect insects through the cuticle and proliferate in the hemolymph. However, interactions between the gut microbiota and entomopathogenic fungi are unknown. Here we show that the pathogenic fungus interacts with the gut microbiota to accelerate mosquito death. After topical fungal infection, mosquitoes with gut microbiota die significantly faster than mosquitoes without microbiota. Furthermore, fungal infection causes dysbiosis of mosquito gut microbiota with a significant increase in gut bacterial load and a significant decrease in bacterial diversity. In particular, the opportunistic pathogenic bacterium overgrows in the midgut and translocates to the hemocoel, which promotes fungal killing of mosquitoes. We further reveal that fungal infection down-regulates antimicrobial peptide and dual oxidase expression in the midgut. Duox down-regulation in the midgut is mediated by secretion of the toxin oosporein from Our findings reveal the important contribution of the gut microbiota in -killing activity, providing new insights into the mechanisms of fungal pathogenesis in insects. 10.1073/pnas.1703546114
    Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota. Tandina Fatalmoudou,Almeras Lionel,Koné Abdoulaye K,Doumbo Ogobara K,Raoult Didier,Parola Philippe Parasites & vectors BACKGROUND:Mosquitoes transmit a wide range of human parasitic and viral diseases. In recent years, new techniques such as MALDI-TOF MS have been developed to identify mosquitoes at the species level, which is key for entomological surveys. Additionally, there is increasing interest in the mosquito microbiota and its role in vector capacity. METHODS:The culturomics approach previously used in our laboratory to study human gut microbiota was applied to evaluate the midgut bacterial diversity of Anopheles gambiae (wild and laboratory strains), Aedes albopictus (wild and laboratory strains) and Culex quinquefasciatus (wild strains) in order to determine the influence of the environmental status on the midgut microbiota of the mosquitoes. RESULTS:Mosquitoes collected in the field were accurately identified by MALDI-TOF MS analysis of their legs. Adult mosquito midgut microbiota was composed of four phyla, including Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The majority of the bacteria detected in the microbiota of mosquitoes were gram-negative and belong to the phylum Proteobacteria. MALDI-TOF MS identified for the first time a new bacterial species from An. gambiae midgut microbiota. CONCLUSION:In this study, the culturomics approach was found to be a reliable technique for exploring the diversity of the mosquito microbiota. MALDI-TOF MS was confirmed as a promising technique to identify mosquitoes collected in the field. Culturomics allowed the isolation of a new bacterial species not previously associated with mosquito vectors. The environment plays a role in the bacterial diversity of the microbiota, which could enable the development of new control strategies for mosquito-borne disease. 10.1186/s13071-016-1776-y
    Composition and functional roles of the gut microbiota in mosquitoes. Strand Michael R Current opinion in insect science An estimated 3500 species of mosquitoes (family Culicidae) exist worldwide of which several are known vectors of pathogens that cause disease in humans and other vertebrates. Mosquitoes also host communities of microbes in their digestive tract that form a gut microbiota. Recent studies provide important insights on how mosquitoes acquire a gut microbiota and the community of microbes that are present. Results also indicate that the gut microbiota affects several aspects of mosquito biology. Altogether, these effects impact mosquito fitness with potential consequences for disease prevalence. 10.1016/j.cois.2018.05.008
    Naturally Occurring Microbiota in Dengue Vector Mosquito Breeding Habitats and Their Use as Diet Organisms by Developing Larvae in the Kandy District, Sri Lanka. Ranasinghe H A K,Amarasinghe L D BioMed research international Naturally occurring microbiota in mosquito larval habitats are among biotic factors which affect the population dynamics of developing larvae. Many microbiota species serve as food items for vector mosquito larvae, and food limitations within habitats adversely affect larval survival, developmental rate, adult fitness, and thereby vector competence. Therefore, identification of microbiota as associates with larvae reveals their relationship between each other as parasites, pathogens, epibionts, or diet organisms. Analysis of associated microbiota species in the dengue vector larval breeding habitats ( = 40) and the mosquito larval gut content were conducted in Kandy District in Sri Lanka. Study revealed that a total of 22 microbiota species belong to nine phyla (Amoebozoa, Bacillariophyta, Ciliophora, Chlorophyta, Sarcodina, Cyanobacteria/Cyanophyta, Euglenozoa, Ochrophyta/Heterokontophyta, and Rotifera) were encountered from different mosquito breeding habitats while 26 microbiota species that belonged to ten phyla were recorded from mosquito breeding habitats with one additional phylum Arthropoda. Considering breeding habitats, only in low roof gutters existed as constant species. Considering breeding habitats, in plastic containers, in coconut shells, in concrete slabs, and sp. in tree holes existed as constant species. The rest of the microbiota existed as common or accidental/rare species in a variety of habitat types. The Shannon-Weiner diversity (21.01 and 19.36) and gamma diversity (eight and eight) of the microbiota associated with and larvae, respectively, in ponds were found to be higher than other types of breeding habitats recorded during the study. Twelve microbiota species were recorded from larval gut analysis as food organisms of both species of mosquito larvae. However, the distribution of gut microbiota species differed between and (Chi - square = 21.294, = 0.002). Identification of microbiota as food items of vector mosquito larvae led to a focus on larval food limitation by introducing food competitors, which could be a potential additional tool for integrated vector control approaches within the country. 10.1155/2020/5830604
    Mosquito microbiota cluster by host sampling location. Muturi Ephantus J,Lagos-Kutz Doris,Dunlap Christopher,Ramirez Jose L,Rooney Alejandro P,Hartman Glen L,Fields Christopher J,Rendon Gloria,Kim Chang-Hyun Parasites & vectors BACKGROUND:Microbial communities that inhabit the mosquito body play an import role in host biology and may have potential for mosquito control. However, the forces that shape these microbial communities are poorly understood. METHODS:To gain a better understanding of how host location influences the composition and diversity of mosquito microbiota, we performed a survey of microbial communities in mosquito samples collected from six USA states using HiSeq sequencing of the 16S rRNA gene. RESULTS:A total of 284 bacterial operational taxonomic units (OTUs) belonging to 14 phyla were detected in nine mosquito species, with Proteobacteria, Firmicutes and Actinobacteria accounting for 95% of total sequences. OTU richness varied markedly within and between mosquito species. The microbial composition and diversity was heavily influenced by the site of mosquito collection, suggesting that host location plays an important role in shaping the mosquito microbiota. CONCLUSIONS:Variation in microbial composition and diversity between mosquitoes from different locations may have important implications on vector competence and transmission dynamics of mosquito-borne pathogens. Future studies should investigate the environmental factors responsible for these variations and the role of key bacteria characterized in this study on mosquito biology and their potential application in symbiotic control of mosquito-borne diseases. 10.1186/s13071-018-3036-9
    Influences of a Prolific Gut Fungus () on Larval and Adult Mosquito (Aedes aegypti)-Associated Microbiota. Frankel-Bricker Jonas,Buerki Sven,Feris Kevin P,White Merlin M Applied and environmental microbiology Adult mosquitoes inherit a bacterial community from larvae via transstadial transmission, an understudied process that may influence host-microbe interactions. Microbes contribute to important host life history traits, and analyzing transmitted microbial communities, the interrelationship between larval and adult-associated microbiota, and factors influencing host-microbe relationships provides targets for research. During its larval stage, the yellow fever mosquito () hosts the trichomycete gut fungus , and fungal colonization coincides with environmental perturbations in the digestive tract microecosystem. Natural populations are differentially exposed to fungi, thereby potentially harboring distinct microbiota and experiencing disparate host-microbe interactions. This study's objectives were to characterize larval and initial adult microbiomes, investigate variation in diversity and distribution of microbial communities across individuals, and assess whether larval fungal colonization impacted microbiomes at these developmental stages. Laboratory-based fungal infestation assays, sequencing of 16S rRNA gene amplicons, and bacterial load quantification protocols revealed that initial adult microbiomes varied in diversity and distribution. Larval fungal colonization had downstream effects on initial adult microbiomes, significantly reducing microbial community variation, shifting relative abundances of certain bacterial families, and influencing transstadial transmission outcomes of particular genera. Further, abundances of several families consistently decreased in adults relative to levels in larvae, possibly reflecting impacts of host development on specific bacterial taxa. These findings demonstrated that a prolific gut fungus impacted mosquito-associated microbiota at two developmental stages in an insect connected with global human health. Mosquitoes are widespread vectors of numerous human pathogens and harbor microbiota known to affect host phenotypic traits. However, little research has directly investigated how bacterial communities associated with larvae and adults are connected. We characterized whole-body bacterial communities in mosquito larvae preceding pupation and in newly emerged adults, and investigated whether a significant biotic factor, fungal colonization of the larval hindgut, impacted these microbiomes. Results showed that fungal colonization reduced microbial community variation across individuals and differentially impacted the outcomes of transstadial transmission for certain bacterial genera, revealing downstream effects of the fungus on initial adult microbiomes. The importance of our research is in providing a thorough comparative analysis of whole-body microbiota harbored in larvae and adults of the yellow fever mosquito () and in demonstrating the important role a widespread gut fungus played in a host-associated microbiome. 10.1128/AEM.02334-19
    Comparative Analysis of Gut Microbiota of Culex restuans (Diptera: Culicidae) Females From Different Parents. Muturi Ephantus J,Ramirez Jose L,Rooney Alejandro P,Kim Chang-Hyun Journal of medical entomology The potential for gut microbiota to impede or enhance pathogen transmission is well-documented but the factors that shape this microbiota in mosquito vectors are poorly understood. We characterized and compared the gut microbiota of adult females of Culex restuans (Theobald; Diptera: Culicidae) from different parents. Cx. restuans larvae from nine field-collected egg rafts were reared on a common diet and gut microbiota of newly emerged adult females characterized by MiSeq sequencing of the V4 hypervariable region of the 16S rRNA gene. Bacterial diversity and evenness in individuals from one egg raft were significantly lower compared to those of three of the other eight egg rafts. The gut microbiota of adult females reared from seven of the nine egg rafts clustered together suggesting that individuals from most egg rafts had similar profiles of gut microbiota. These findings suggest that the microbiota of adult females from the same parents do not differ appreciably from the microbiota of adult females from different parents. However, additional studies using mosquitoes separated by geographic distances greater than those studied here and estimating the genetic distances between populations from different egg rafts are needed to provide further insights into the influence of host genetics on gut microbiota. Also worthwhile are studies evaluating how individuals from different egg rafts and harboring different gut microbiota compare in relation to vector competence for different pathogens. 10.1093/jme/tjx199
    Microbiota variations in disease vector mosquito of West Nile virus and Saint Louis Encephalitis from different geographic origins. Duguma Dagne,Hall Michael W,Smartt Chelsea T,Debboun Mustapha,Neufeld Josh D PeerJ Although mosquito microbiota are known to influence reproduction, nutrition, disease transmission, and pesticide resistance, the relationship between host-associated microbial community composition and geographical location is poorly understood. To begin addressing this knowledge gap, we characterized microbiota associated with adult females of mosquito vectors of Saint Louis Encephalitis and West Nile viruses sampled from three locations in Florida (Vero Beach, Palmetto Inland, and Palmetto Coast). High-throughput sequencing of PCR-amplified 16S rRNA genes demonstrated significant differences among microbial communities of mosquitoes sampled from the three locations. Mosquitoes from Vero Beach (east coast Florida) were dominated by uncultivated sp. (), whereas microbiota associated with mosquitoes collected from two mosquito populations at Palmetto (west coast Florida) sites were dominated by uncultured (), (), (), uncultured , Candidatus Megaira (; ), and (). The variation in taxonomic profiles of gut microbial communities, especially with respect to dominating taxa, is a potentially critical factor in understanding disease transmission and mosquito susceptibility to insecticides among different mosquito populations. 10.7717/peerj.6168
    Mosquitoes rely on their gut microbiota for development. Coon Kerri L,Vogel Kevin J,Brown Mark R,Strand Michael R Molecular ecology Field studies indicate adult mosquitoes (Culicidae) host low diversity communities of bacteria that vary greatly among individuals and species. In contrast, it remains unclear how adult mosquitoes acquire their microbiome, what influences community structure, and whether the microbiome is important for survival. Here, we used pyrosequencing of 16S rRNA to characterize the bacterial communities of three mosquito species reared under identical conditions. Two of these species, Aedes aegypti and Anopheles gambiae, are anautogenous and must blood-feed to produce eggs, while one, Georgecraigius atropalpus, is autogenous and produces eggs without blood feeding. Each mosquito species contained a low diversity community comprised primarily of aerobic bacteria acquired from the aquatic habitat in which larvae developed. Our results suggested that the communities in Ae. aegypti and An. gambiae larvae share more similarities with one another than with G. atropalpus. Studies with Ae. aegypti also strongly suggested that adults transstadially acquired several members of the larval bacterial community, but only four genera of bacteria present in blood fed females were detected on eggs. Functional assays showed that axenic larvae of each species failed to develop beyond the first instar. Experiments with Ae. aegypti indicated several members of the microbial community and Escherichia coli successfully colonized axenic larvae and rescued development. Overall, our results provide new insights about the acquisition and structure of bacterial communities in mosquitoes. They also indicate that three mosquito species spanning the breadth of the Culicidae depend on their gut microbiome for development. 10.1111/mec.12771
    Effect of life stage and pesticide exposure on the gut microbiota of Aedes albopictus and Culex pipiens L. Juma Elijah O,Allan Brian F,Kim Chang-Hyun,Stone Christopher,Dunlap Christopher,Muturi Ephantus J Scientific reports Pesticides commonly contaminate the aquatic environments inhabited by mosquito juveniles. However, their role in shaping the mosquito microbiota is not well understood. We hypothesized that environmentally relevant concentrations of atrazine, permethrin and malathion will mediate a shift in the mosquito gut bacterial community structure due to their toxic effect on the aquatic bacterial communities, and reduce mosquito gut bacterial diversity by enriching pesticide-degrading bacterial communities over susceptible taxa. Illumina MiSeq sequencing of the V3-V4 hypervariable regions of the 16 S rRNA gene was used to characterize the microbial communities of larval and adult stages of the two mosquito species and the water samples from microcosms treated with each of the pesticides, separately. Bacterial community composition differed by sample type (larval stage vs. adult stage) and water sampling date (day 3 vs. day 7), but not by pesticide treatment. In larval stages, bacterial OTU richness was highest in samples exposed to malathion, intermediate in permethrin, and lowest in controls. Bacterial richness was significantly higher in larval stages compared to adult stages for all treatments. This study provides a primer for future studies evaluating mosquito microbial responses to exposures to chemical pesticides and the possible implications for mosquito ecology. 10.1038/s41598-020-66452-5
    Mosquito vector-associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod-borne diseases. Thongsripong Panpim,Chandler James Angus,Green Amy B,Kittayapong Pattamaporn,Wilcox Bruce A,Kapan Durrell D,Bennett Shannon N Ecology and evolution Vector-borne diseases are a major health burden, yet factors affecting their spread are only partially understood. For example, microbial symbionts can impact mosquito reproduction, survival, and vectorial capacity, and hence affect disease transmission. Nonetheless, current knowledge of mosquito-associated microbial communities is limited. To characterize the bacterial and eukaryotic microbial communities of multiple vector species collected from different habitat types in disease endemic areas, we employed next-generation 454 pyrosequencing of and amplicon libraries, also known as metabarcoding. We investigated pooled whole adult mosquitoes of three medically important vectors, , , and collected from different habitats across central Thailand where we previously characterized mosquito diversity. Our results indicate that diversity within the mosquito microbiota is low, with the majority of microbes assigned to one or a few taxa. Two of the most common eukaryotic and bacterial genera recovered ( and respectively) are known mosquito endosymbionts with potentially parasitic and long evolutionary relationships with their hosts. Patterns of microbial composition and diversity appeared to differ by both vector species and habitat for a given species, although high variability between samples suggests a strong stochastic element to microbiota assembly. In general, our findings suggest that multiple factors, such as habitat condition and mosquito species identity, may influence overall microbial community composition, and thus provide a basis for further investigations into the interactions between vectors, their microbial communities, and human-impacted landscapes that may ultimately affect vector-borne disease risk. 10.1002/ece3.3676
    Predaceous mosquitoes require a living gut microbiota to develop. Coon Kerri L,Valzania Luca,Brown Mark R,Strand Michael R Proceedings. Biological sciences Most species of mosquitoes are detritivores that feed on decaying plant and animal materials in their aquatic environment. Studies of several detritivorous mosquito species indicate that they host relatively low diversity communities of microbes that are acquired from the environment while feeding. Our recent results also indicate that detritivorous species normally require a living gut microbiota to grow beyond the first instar. Less well known is that some mosquitoes, including those belonging to the genus , are predators that feed on other species of mosquitoes and nektonic prey. In this study, we asked whether predaceous larvae still require living microbes in their gut in order to develop. Using the detritivorous mosquito as prey, we found that larvae harbour bacterial communities that are highly similar to that of their prey. Functional assays showed that first instars provided axenic (i.e. bacteria-free) prey failed to develop, while two bacterial species present in gnotobiotic (i.e. colonized by one or more known bacterial species) prey successfully colonized the gut and rescued development. Axenic larvae also displayed defects in growth consistent with previously identified roles for microbe-mediated gut hypoxia in nutrient acquisition and assimilation in Collectively, these results support a conserved role for gut microbes in regulating the development of mosquitoes with different feeding strategies. 10.1098/rspb.2019.2705
    Diversity and Species Composition of Microbiota Associated with Mosquito Breeding Habitats: A Study from Kurunegala District in Sri Lanka. Amarasinghe L D,Ranasinghe H A K BioMed research international The pool of microbiota associated with mosquito breeding habitats varies with the habitat type and its characteristic features. The pool of microbiota in a given mosquito breeding habitat can include free living, symbiotic, noncompetitive, parasitic, predatory, and toxin producing species. However, in Sri Lanka the studies on the microbiota associated with mosquito breeding habitats are scarce. The present study was conducted to identify microbiota species/taxa associated with a variety of mosquito breeding habitats in selected areas of the Kurunegala district in Sri Lanka to determine the relationship, if any, the microbiota has with mosquito larvae breeding. A total of 44 microbiota species/taxa belonging to 10 phyla, namely, Bacillariophyta, Charophyta, Chlorophyta, Cyanobacteria/Cyanophyta, Ochrophyta/Heterokontophyta, Amoebozoa, Euglenozoa, Ciliophora, Arthropoda, and Rotifera were identified. (Ciliophora) showed a constant occurrence frequency in rice field habitats occupied mainly by while the rest of the species had an accidental or rare frequency of occurrence. Nineteen species/taxa were identified as common species. Trophont stages of and spp. were found attached to the cuticle of mosquito larvae but only caused a lethal effect. The autotrophic protist, , spp., and spp. served as the diet items to mosquito larvae. The majority of the microbiota identified had no observable effect on mosquito larvae breeding. 10.1155/2019/5897317
    The transcriptome of the mosquito Aedes fluviatilis (Diptera: Culicidae), and transcriptional changes associated with its native Wolbachia infection. Caragata E P,Pais F S,Baton L A,Silva J B L,Sorgine M H F,Moreira L A BMC genomics BACKGROUND:Wolbachia is a bacterial endosymbiont that naturally infects a wide range of insect species, and causes drastic changes to host biology. Stable infections of Wolbachia in mosquitoes can inhibit infection with medically important pathogens such as dengue virus and malaria-causing Plasmodium parasites. However, some native Wolbachia strains can enhance infection with certain pathogens, as is the case for the mosquito Aedes fluviatilis, where infection with Plasmodium gallinaceum is enhanced by the native wFlu Wolbachia strain. To better understand the biological interactions between mosquitoes and native Wolbachia infections, and to investigate the process of pathogen enhancement, we used RNA-Seq to generate the transcriptome of Ae. fluviatilis with and without Wolbachia infection. RESULTS:In total, we generated 22,280,160 Illumina paired-end reads from Wolbachia-infected and uninfected mosquitoes, and used these to make a de novo transcriptome assembly, resulting in 58,013 contigs with a median sequence length of 443 bp and an N50 of 2454 bp. Contigs were annotated through local alignments using BlastX, and associated with both gene ontology and KEGG orthology terms. Through baySeq, we identified 159 contigs that were significantly upregulated due to Wolbachia infection, and 98 that were downregulated. Critically, we saw no changes to Toll or IMD immune gene transcription, but did see evidence that wFlu infection altered the expression of several bacterial recognition genes, and immune-related genes that could influence Plasmodium infection. wFlu infection also had a widespread effect on a number of host physiological processes including protein, carbohydrate and lipid metabolism, and oxidative stress. We then compared our data set with transcriptomic data for other Wolbachia infections in Aedes aegypti, and identified a core set of 15 gene groups associated with Wolbachia infection in mosquitoes. CONCLUSIONS:While the scale of transcriptional changes associated with wFlu infection might be small, the scope is rather large, which confirms that native Wolbachia infections maintain intricate molecular relationships with their mosquito hosts even after lengthy periods of co-evolution. We have also identified several potential means through which wFlu infection might influence Plasmodium infection in Ae. fluviatilis, and these genes should form the basis of future investigation into the enhancement of Plasmodium by Wolbachia. 10.1186/s12864-016-3441-4
    Both living bacteria and eukaryotes in the mosquito gut promote growth of larvae. Valzania Luca,Martinson Vincent G,Harrison Ruby E,Boyd Bret M,Coon Kerri L,Brown Mark R,Strand Michael R PLoS neglected tropical diseases We recently reported that larval stage Aedes aegypti and several other species of mosquitoes grow when living bacteria are present in the gut but do not grow when living bacteria are absent. We further reported that living bacteria induce a hypoxia signal in the gut, which activates hypoxia-induced transcription factors and other processes larvae require for growth. In this study we assessed whether other types of organisms induce mosquito larvae to grow and asked if the density of non-living microbes or diet larvae are fed obviate the requirement for living organisms prior results indicated are required for growth. Using culture conditions identical to our own prior studies, we determined that inoculation density of living Escherichia coli positively affected growth rates of Ae. aegypti larvae, whereas non-living E. coli had no effect on growth across the same range of inoculation densities. A living yeast, alga, and insect cell line induced axenic Ae. aegypti first instars to grow, and stimulated similar levels of midgut hypoxia, HIF-α stabilization, and neutral lipid accumulation in the fat body as E. coli. However, the same organisms had no effect on larval growth if heat-killed. In addition, no axenic larvae molted when fed two other diets, when fed diets supplemented with heat-killed microbes or lysed and heat-killed microbes. Experiments conducted with An. gambiae yielded similar findings. Taken together, our results indicate that organisms from different prokaryotic and eukaryotic groups induce mosquito larvae to grow, whereas no conditions were identified that stimulated larvae to grow in the absence of living organisms. 10.1371/journal.pntd.0006638
    Extensive genetic diversity of Rickettsiales bacteria in multiple mosquito species. Guo Wen-Ping,Tian Jun-Hua,Lin Xian-Dan,Ni Xue-Bing,Chen Xiao-Ping,Liao Yong,Yang Si-Yuan,Dumler J Stephen,Holmes Edward C,Zhang Yong-Zhen Scientific reports Rickettsiales are important zoonotic pathogens, causing severe disease in humans globally. Although mosquitoes are an important vector for diverse pathogens, with the exception of members of the genus Wolbachia little is known about their role in the transmission of Rickettsiales. Herein, Rickettsiales were identified by PCR in five species of mosquitoes (Anopheles sinensis, Armigeres subalbatus, Aedes albopictus, Culex quinquefasciatus and Cu. tritaeniorhynchus) collected from three Chinese provinces during 2014-2015. Subsequent phylogenetic analyses of the rrs, groEL and gltA genes revealed the presence of Anaplasma, Ehrlichia, Candidatus Neoehrlichia, and Rickettsia bacteria in mosquitoes, comprising nine documented and five tentative species bacteria, as well as three symbionts/endosybionts. In addition, bacteria were identified in mosquito eggs, larvae, and pupae sampled from aquatic environments. Hence, these data suggest that Rickettsiales circulate widely in mosquitoes in nature. Also of note was that Ehrlichia and Rickettsia bacteria were detected in each life stage of laboratory cultured mosquitoes, suggesting that Rickettsiales may be maintained in mosquitoes through both transstadial and transovarial transmission. In sum, these data indicate that mosquitoes may have played an important role in the transmission and evolution of Rickettsiales in nature. 10.1038/srep38770
    Characterization of the Aedes albopictus (Diptera: Culicidae) holobiome: bacterial composition across land use type and mosquito sex in Malaysia. Lee J M,Yek S H,Wilson R F,Rahman S Acta tropica Understanding the diversity and dynamics of the microbiota within the mosquito holobiome is of great importance to apprehend how the microbiota modulates various complex processes and interactions. This study examined the bacterial composition of Aedes albopictus across land use type and mosquito sex in the state of Selangor, Malaysia using 16S rRNA sequencing. The bacterial community structure in mosquitoes was found to be influenced by land use type and mosquito sex, with the environment and mosquito diet respectively identified to be the most likely sources of microbes. We found that approximately 70% of the microbiota samples were dominated by Wolbachia and removing Wolbachia from analyses revealed the relatively even composition of the remaining bacterial microbiota. Furthermore, microbial interaction network analysis highlighted the prevalence of co-exclusionary patterns in all networks regardless of land use and mosquito sex, with Wolbachia exhibiting co-exclusionary interactions with other residential bacteria such as Xanthomonas, Xenophilus and Zymobacter. 10.1016/j.actatropica.2020.105683
    Generation of axenic Aedes aegypti demonstrate live bacteria are not required for mosquito development. Correa Maria A,Matusovsky Brian,Brackney Doug E,Steven Blaire Nature communications The mosquito gut microbiome plays an important role in mosquito development and fitness, providing a promising avenue for novel mosquito control strategies. Here we present a method for rearing axenic (bacteria free) Aedes aegypti mosquitoes, consisting of feeding sterilized larvae on agar plugs containing a high concentration of liver and yeast extract. This approach allows for the complete development to adulthood while maintaining sterility; however, axenic mosquito's exhibit delayed development time and stunted growth in comparison to their bacterially colonized cohorts. These data challenge the notion that live microorganisms are required for mosquito development, and suggest that the microbiota's main role is nutritional. Furthermore, we colonize axenic mosquitoes with simplified microbial communities ranging from a single bacterial species to a three-member community, demonstrating the ability to control the composition of the microbiota. This axenic system will allow the systematic manipulation of the mosquito microbiome for a deeper understanding of microbiota-host interactions. 10.1038/s41467-018-07014-2
    Presence of Aedes and Anopheles mosquito larvae is correlated to bacteria found in domestic water-storage containers. Nilsson Louise K J,Sharma Anil,Bhatnagar Raj K,Bertilsson Stefan,Terenius Olle FEMS microbiology ecology Water-storage containers are common in households where access to water is scarce and often act as breeding sites for vector mosquitoes. Bacteria in these containers may be important for attracting or repelling ovipositing mosquitoes. We hypothesized that bacterial community composition in water-storage containers would represent either inhibitory or suitable environmental conditions for mosquito larvae. To investigate this, we characterized the bacterial community composition in water-storage containers and correlated these communities to Aedes and Anopheles larval densities. Water samples were collected over two years from 13 containers in an Indian village and analyzed by high throughput 16S rRNA gene amplicon sequencing. Comparisons of bacterial community composition between water with and without mosquito larvae showed that Xanthomonadaceae, Comamonadaceae and Burkholderiaceae were more common (P < 0.05) in absence of larvae, while Lachnospiraceae, Synechococcaceae, Alcaligenaceae and Cryomorphaceae were more common (P < 0.05) in presence of larvae. Indicator analysis identified operational taxonomic units designated as CL500-29 marine group (Acidimicrobiaceae) and FukuN101 (Microbacteriaceae) for absence and presence of larvae, respectively. These results contribute to the understanding of which bacteria, directly or indirectly, can be linked to absence or presence of mosquitoes around households and set the basis for potential measures to be taken against these vector mosquitoes. 10.1093/femsec/fiy058
    Metagenome and Culture-Based Methods Reveal Candidate Bacterial Mutualists in the Southern House Mosquito (Diptera: Culicidae). Telang Aparna,Skinner Jessica,Nemitz Robert Z,McClure Alexander M Journal of medical entomology Mosquitoes are intensely studied as vectors of disease-causing pathogens, but we know relatively less about microbes that naturally reside in mosquitoes. Profiling resident bacteria in mosquitoes can help identify bacterial groups that can be exploited as a strategy of controlling mosquito populations. High-throughput 16S rRNA gene sequencing and traditional culture-based methods were used to identify bacterial assemblages in Culex quinquefasciatus Say (Diptera: Culicidae) in a tissue- and stage-specific design. In parallel, wild host Cx. quinquefasciatus was compared with our domestic strain. 16S rRNA genes survey finds that Cx. quinquefasciatus has taxonomically restricted bacterial communities, with 90% of its bacterial microbiota composed of eight distinctive bacterial groups: Nocardioidaceae (Actinomycetales), Microbacteriaceae (Actinomycetales), Flavobacteriaceae, Rhizobiales, Acetobacteraceae, Rickettsiaceae, Comamondaceae (Burkholderiales), and Enterobacteriaceae. Taking into account both metagenome- and culture-based methods, we suggest three bacterial groups, Acetobacteraceae, Flavobacteriaceae, and Enterobacteriaceae, as candidates for mutualists in Cx. quinquefasciatus. Members of these three bacterial families have been studied as mutualists, or even as symbionts, in other insect groups, so it is quite possible they play similar roles in mosquitoes. 10.1093/jme/tjy056
    Establishment of a medium-scale mosquito facility: tests on mass production cages for Aedes albopictus (Diptera: Culicidae). Zhang Dongjing,Li Yongjun,Sun Qiang,Zheng Xiaoying,Gilles Jeremie R L,Yamada Hanano,Wu Zhongdao,Xi Zhiyong,Wu Yu Parasites & vectors BACKGROUND:Mass egg production is an important component of Aedes albopictus mosquito control programs, such as the sterile insect technique and incompatible insect technique, which requires the releases of large number of sterile males. Developing standard operating procedures and optimized cages for adult maintenance of Ae. albopictus can improve the mass rearing efficiency. METHODS:Three different sex ratios of females to males with a total number of 4,000 mosquitoes were tested by evaluating the insemination rate, egg production (total number of eggs per cage), female fecundity and egg hatch rate in small cage (30 × 30 × 30 cm). Blood meals with adenosine triphosphate (ATP, 0.05 g/ml), cage structures (Big cage A: 90 × 30 × 30 cm; Big cage B: 90 × 30 × 50 cm or 90 × 50 × 30 cm) and rearing densities (12,000, 16,000 and 20,000 mosquitoes, corresponding to 0.9 cm/mosquito, 0.675 cm/mosquito and 0.54 cm/mosquito, respectively) were also tested and evaluated on the basis of egg production, female fecundity and egg hatch rate. An adult rearing unit holding 15 of Big cage A with optimal egg production was designed to produce 10 million eggs per rearing cycle in a 1.8 m space. RESULTS:Female to male ratios at 3:1 in small cages resulted in higher egg production but did not affect insemination rate, female fecundity and egg hatch rate. A concentration of 0.05 g/ml of ATP added to blood meals improved the blood-feeding frequency and thus increased the overall egg production per cage. Cage structures affected the egg production per cage, but not egg hatch rate. A medium rearing density at 0.675 cm/mosquito (16,000 mosquitoes) resulted in higher egg production compared to both low and high densities. An adult rearing unit for Ae. albopictus on the basis of Big cage A has been developed with the capacity of producing 10 million eggs within 15 days. CONCLUSIONS:Our results have indicated that the adult rearing methods and adult maintenance unit are recommended for Ae. albopictus mass rearing in support of the establishment of a medium-sized mosquito factory. 10.1186/s13071-018-2750-7
    A Gut Commensal Bacterium Promotes Mosquito Permissiveness to Arboviruses. Wu Pa,Sun Peng,Nie Kaixiao,Zhu Yibin,Shi Mingyu,Xiao Changguang,Liu Han,Liu Qiyong,Zhao Tongyan,Chen Xiaoguang,Zhou Hongning,Wang Penghua,Cheng Gong Cell host & microbe Mosquitoes are hematophagous vectors that can acquire human viruses in their intestinal tract. Here, we define a mosquito gut commensal bacterium that promotes permissiveness to arboviruses. Antibiotic depletion of gut bacteria impaired arboviral infection of a lab-adapted Aedes aegypti mosquito strain. Reconstitution of individual cultivable gut bacteria in antibiotic-treated mosquitoes identified Serratia marcescens as a commensal bacterium critical for efficient arboviral acquisition. S. marcescens facilitates arboviral infection through a secreted protein named SmEnhancin, which digests membrane-bound mucins on the mosquito gut epithelia, thereby enhancing viral dissemination. Field Aedes mosquitoes positive for S. marcescens were more permissive to dengue virus infection than those free of S. marcescens. Oral introduction of S. marcescens into field mosquitoes that lack this bacterium rendered these mosquitoes highly susceptible to arboviruses. This study defines a commensal-driven mechanism that contributes to vector competence, and extends our understanding of multipartite interactions among hosts, the gut microbiome, and viruses. 10.1016/j.chom.2018.11.004
    [Mosquito microbiota and its influence on disease vectorial transmission]. Heu Katy,Gendrin Mathilde Biologie aujourd'hui Mosquitoes (Diptera: Culicidae) are found worldwide. Around 100 among 3500 mosquito species are known to be vectors of parasites and viruses, responsible for infectious diseases including malaria and dengue. Mosquitoes host diverse microbial communities that influence disease transmission, either by direct interference or via affecting host immunity and physiology. These microbial communities are present within diverse tissues, including the digestive tract, and vary depending on the sex of the mosquito, its developmental stage, and ecological factors. This review summarizes the current knowledge about the mosquito microbiota, defined as a community of commensal, symbiotic or pathogenic microbes harboured by a host. We first describe the current knowledge on the diversity of the microbiota, that includes bacteria, fungi, parasites and viruses and on its modes of acquisition throughout the mosquito life cycle. We then focus on microbial interactions within the mosquito gut, which notably affect vector competence, and on host-microbe interactions affecting mosquito fitness. Finally, we discuss current or potential methods based on the use of microbes or microbial products to interfere with pathogen transmission or to reduce mosquito lifespan and reproduction. 10.1051/jbio/2019003
    Mosquito Microbiota and Implications for Disease Control. Gao Han,Cui Chunlai,Wang Lili,Jacobs-Lorena Marcelo,Wang Sibao Trends in parasitology Mosquito-transmitted diseases account for about 500 000 deaths every year. Blocking these pathogens in the mosquito vector before they are transmitted to humans is an effective strategy to prevent mosquito-borne diseases. Like most higher organisms, mosquitoes harbor a highly diverse and dynamic microbial flora that can be explored for prevention of pathogen transmission. Here we review the structure and function of the mosquito microbiota, including bacteria, fungi, and viruses, and discuss the potential of using components of the microbiota to thwart pathogen transmission. 10.1016/j.pt.2019.12.001
    Comparative analysis of gut microbiota of mosquito communities in central Illinois. Muturi Ephantus J,Ramirez Jose L,Rooney Alejandro P,Kim Chang-Hyun PLoS neglected tropical diseases BACKGROUND:The composition and structure of microbial communities that inhabit the mosquito midguts are poorly understood despite their well-documented potential to impede pathogen transmission. METHODOLOGY/PRINCIPAL FINDINGS:We used MiSeq sequencing of the 16S rRNA gene to characterize the bacterial communities of field-collected populations of 12 mosquito species. After quality filtering and rarefaction, the remaining sequences were assigned to 181 operational taxonomic units (OTUs). Approximately 58% of these OTUs occurred in at least two mosquito species but only three OTUs: Gluconobacter (OTU 1), Propionibacterium (OTU 9), and Staphylococcus (OTU 31) occurred in all 12 mosquito species. Individuals of different mosquito species shared similar gut microbiota and it was common for individuals of the same species from the same study site and collection date to harbor different gut microbiota. On average, the microbiota of Aedes albopictus was the least diverse and significantly less even compared to Anopheles crucians, An. quadrimaculatus, Ae. triseriatus, Ae. vexans, Ae. japonicus, Culex restuans, and Culiseta inornata. The microbial community of Cx. pipiens and Ae. albopictus differed significantly from all other mosquitoes species and was primarily driven by the dominance of Wolbachia. CONCLUSION AND SIGNIFICANCE:These findings expand the range of mosquito species whose gut microbiota has been characterized and sets the foundation for further studies to determine the influence of these microbiota on vector susceptibility to pathogens. 10.1371/journal.pntd.0005377
    The mosquito holobiont: fresh insight into mosquito-microbiota interactions. Guégan Morgane,Zouache Karima,Démichel Colin,Minard Guillaume,Tran Van Van,Potier Patrick,Mavingui Patrick,Valiente Moro Claire Microbiome The holobiont concept was first developed for coral ecosystems but has been extended to multiple organisms, including plants and other animals. Studies on insect-associated microbial communities have produced strong evidence that symbiotic bacteria play a major role in host biology. However, the understanding of these symbiotic relationships has mainly been limited to phytophagous insects, while the role of host-associated microbiota in haematophagous insect vectors remains largely unexplored. Mosquitoes are a major global public health concern, with a concomitant increase in people at risk of infection. The global emergence and re-emergence of mosquito-borne diseases has led many researchers to study both the mosquito host and its associated microbiota. Although most of these studies have been descriptive, they have led to a broad description of the bacterial communities hosted by mosquito populations. This review describes key advances and progress in the field of the mosquito microbiota research while also encompassing other microbes and the environmental factors driving their composition and diversity. The discussion includes recent findings on the microbiota functional roles and underlines their interactions with the host biology and pathogen transmission. Insight into the ecology of multipartite interactions, we consider that conferring the term holobiont to the mosquito and its microbiota is useful to get a comprehensive understanding of the vector pathosystem functioning so as to be able to develop innovative and efficient novel vector control strategies. 10.1186/s40168-018-0435-2
    Gut bacteria differentially affect egg production in the anautogenous mosquito Aedes aegypti and facultatively autogenous mosquito Aedes atropalpus (Diptera: Culicidae). Coon Kerri L,Brown Mark R,Strand Michael R Parasites & vectors BACKGROUND:Aedes aegypti and A. atropalpus are related mosquitoes that differ reproductively. Aedes aegypti must blood-feed to produce eggs (anautogenous) while A. atropalpus always produces a first clutch of eggs without blood-feeding (facultatively autogenous). We recently characterized the gut microbiota of A. aegypti and A. atropalpus that were reared identically in the laboratory. Here, we assessed the effects of specific members of the gut microbiota in A. aegypti and A. atropalpus on female fitness including egg production. METHODS:Gnotobiotic A. aegypti and A. atropalpus larvae were colonized by specific members of the gut microbiota. Survival, development time, size and egg production for each treatment was then compared to axenic and conventionally reared larvae. RESULTS:Most species of bacteria we tested supported normal development and egg production by A. aegypti but only one betaproteobacterium, a Comamonas, supported development and egg production by A. atropalpus to equivalent levels as conventionally reared females. Aedes atropalpus females colonized by Comamonas contained similar stores of glycogen and protein as conventionally reared females, whereas females colonized by Aquitalea did not. Small differences in bacterial loads were detected between gnotobiotic and conventionally reared A. aegypti and A. atropalpus, but this variation did not correlate with the beneficial effects of Comamonas in A. atropalpus. CONCLUSIONS:Specific members of the gut microbiota more strongly affected survival, size and egg production by A. atropalpus than A. aegypti. 10.1186/s13071-016-1660-9
    Bacteria-mediated modification of insecticide toxicity in the yellow fever mosquito, Aedes aegypti. Scates Sara S,O'Neal Scott T,Anderson Troy D Pesticide biochemistry and physiology The incidence of mosquito-borne disease poses a significant threat to human and animal health throughout the world, with effective chemical control interventions limited by widespread insecticide resistance. Recent evidence suggests that gut bacteria of mosquitoes, known to be essential in nutritional homeostasis and pathogen defense, may also play a significant role in facilitating insecticide resistance. This study investigated the extent to which bacteria contribute to the general esterase and cytochrome P450 monooxygenase (P450)-mediated detoxification of the insecticides propoxur and naled, as well as the insecticidal activity of these chemistries to the yellow fever mosquito, Aedes aegypti. Experiments conducted using insecticide synergists that reduce general esterase and P450 activity demonstrate a role for both groups of enzymes in the metabolic detoxification of propoxur and naled. Furthermore, reduction of bacteria in mosquito larvae using broad-spectrum antibiotics was found to decrease the metabolic detoxification of propoxur and naled, suggesting that the bacteria themselves may be contributing to the in vivo metabolic detoxification of these insecticides. This was supported by in vitro assays using culturable gut bacteria isolated from mosquito larvae which demonstrated that the bacteria were capable of reducing insecticide toxicity. More work is needed, however, to fully elucidate the contribution of bacteria in Ae. aegypti larvae to the metabolic detoxification of insecticides. 10.1016/j.pestbp.2019.07.016
    Identification of symbiotic bacteria in the midgut of the medically important mosquito, Culiseta longiareolata (Diptera: Culicidae). Ghahvechi Khaligh Fereshteh,Vahedi Mozaffar,Chavshin Ali Reza BMC research notes OBJECTIVE:The potential use of symbiotic bacteria for the control of mosquito-borne diseases has attracted the attention of scientists over the past few years. Culiseta longiareolata is among the medically important mosquitoes that transmit a wide range of vector-borne diseases worldwide. However, no extensive studies have been done on the identification of its symbiotic bacteria. Given the role of this species in the transmission of some important diseases and its widespread presence in different parts of the world, including northwestern parts and the West Azerbaijan Province in Iran, a knowledge about the symbiotic bacteria of this species may provide a valuable tool for the biological control of this mosquito. Accordingly, the present study was conducted to isolate and identify the cultivable isolates bacterial symbionts of Culiseta longiareolata using 16S rRNA fragment analysis. RESULTS:The midguts of 42 specimens of Cs. longiareolata were dissected, and the bacteria were cultured on agar plates. After the purification of the bacterial colonies, 16srRNA region amplification and gene sequence analysis were performed, and the sequences were confirmed by biochemical methods. In the present study, 21 isolates belonging to the genera Acinetobacter, Aerococcus, Aeromonas, Bacillus, Carnobacterium, Klebsiella, Morganella, Pseudomonas, Shewanella and Staphylococcus were identified. 10.1186/s13104-020-05220-0
    A Diverse Microbial Community Supports Larval Development and Survivorship of the Asian Tiger Mosquito (Diptera: Culicidae). Travanty Nicholas V,Apperson Charles S,Ponnusamy Loganathan Journal of medical entomology Laboratory microcosm experiments were conducted to evaluate effects of bacteria isolated from senescent white oak leaves on the growth and survivorship of larval Aedes albopictus (Skuse). Larvae hatched from surface-sterilized eggs were reared in microcosms containing individual bacterial isolates, combined isolates (Porphyrobacter sp., Enterobacter asburiae, Acidiphilium rubrum, Pseudomonas syringae, and Azorhizobium caulinodans), a positive control containing a microbial community from an infusion of white oak leaves, and a negative control consisting of sterile culture media. Experiments were conducted for 21 d after which microcosms were deconstructed, larval survivorship was calculated, and bacteria contained in pupae, and adults that developed were quantified to determine rates of transstadial transmission. Positive control microcosms containing diverse microbial communities had an average (±SE) pupation rate of 89.3 (±5.8)% and average larval survivorship of 96.0 (± 2.3)%. Pupation in microcosms with bacterial isolates only occurred twice among all experimental replications; average larval survivorship ranged from 19 to 56%, depending on treatment. Larval growth was not found to be dependent on bacterial isolate density or isolate species, and larval survivorship was dependent on bacterial isolate density, not on isolate species. Potential mechanisms for failed development of larvae in microcosms with bacterial isolates are discussed. Bacterial isolates alone did not support larval development. High larval survivorship in positive control microcosms suggests that a diverse microbial community is required to complete larval development. Additional studies are needed to evaluate larval growth and survivorship on nonbacterial microbes, such as fungi and protozoa. 10.1093/jme/tjz003