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

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

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

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

Wolbachia-carrying Aedes mosquitoes for preventing dengue infection.

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

The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types.

Mosquitoes are prolific vectors of human pathogens, therefore a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.

Detection and quantification of natural Wolbachia in Aedes aegypti in Metropolitan Manila, Philippines using locally designed primers.

Background: The Philippines bears health and economic burden caused by high dengue cases annually. Presently, the Philippines still lack an effective and sustainable vector management. The use of Wolbachia, a maternally transmitted bacterium, that mitigate arbovirus transmission has been recommended. Cytoplasmic incompatibility and viral blocking, two characteristics that make Wolbachia suitable for vector control, depend on infection prevalence and density. There are no current Wolbachia release programs in the Philippines, and studies regarding the safety of this intervention. Here, we screened for Wolbachia in Aedes aegypti collected from Metropolitan Manila, Philippines. We designed location-specific primers for qPCR to test whether this improved Wolbachia detection in Ae. aegypti. We explored if host sex and Wolbachia strain could be potential factors affecting Wolbachia density. Methods: Ae. aegypti mosquitoes (n=429) were screened for natural Wolbachia by taqman qPCR using location-specific Wolbachia surface protein primers (wspAAML) and known 16S rRNA primers. Samples positive for wspAAML (n=267) were processed for Sanger sequencing. We constructed a phylogenetic tree using IQ-TREE 2 to further characterize Wolbachia present in the Philippine Ae. aegypti. We then compared Wolbachia densities between Wolbachia groups and host sex. Statistical analyses were done using GraphPad Prism 9.0. Results: Wolbachia prevalence for 16S rRNA (40%) and wspAAML (62%) markers were high. Wolbachia relative densities for 16S rRNA ranged from -3.84 to 2.71 and wspAAML from -4.02 to 1.81. Densities were higher in male than female mosquitoes. Wolbachia strains detected in Ae. aegypti clustered into supergroup B. Some 54% (123/226) of these sequences clustered under a group referred to here as "wAegML," that belongs to the supergroup B, which had a significantly lower density than wAegB/wAlbB, and wAlbA strains. Conclusion: Location-specific primers improved detection of natural Wolbachia in Ae. aegypti and allowed for relative quantification. Wolbachia density is relatively low, and differed between host sexes and Wolbachia strains. An economical way of confirming sporadic or transient Wolbachia in Ae. aegypti is necessary while considering host sex and bacterial strain.

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

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

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

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

Experimental demonstration of the transmission of Spiroplasma between different arthropod taxa.

Spiroplasma (Mycoplasmatales: Spiroplasmataceae) is one of the most widely distributed symbionts of arthropods. Spiroplasma species can infect their hosts via vertical or horizontal transmission. However, the mode of transmission of Spiroplasma between different arthropod taxa has not been elucidated. In this study, we investigated the potential for the transmission of Spiroplasma to non-native arthropod species, using 2 Spiroplasma spp. isolated from ticks, namely Spiroplasma ixodetis and Spiroplasma mirum, and 3 species of mosquito laboratory colonies, namely Aedes albopictus, Aedes aegypti, and Culex pipiens pallens (Diptera: Culicidae). After feeding the adult mosquitoes with Spiroplasma-containing artificial meals, they were kept at 25 °C for 10 days. Homogenates prepared from Spiroplasma-fed mosquitoes were used to re-isolate Spiroplasma using the in vitro culture method. Nine weeks after culture initiation, the presence of Spiroplasma was tested using the polymerase chain reaction (PCR). The results revealed that only S. ixodetis was detected from all 3 species of mosquitoes and re-isolated from 2 of them. The differences in the infection ability of different Spirolasma species could be attributed to several factors, including environmental effects. Nevertheless, this is the first experimental demonstration of Spiroplasma transmission among different arthropod taxa. Further studies are needed to elucidate the evolutionary mechanism that supports the survival of Spiroplasma in nature.

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

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

The accumulation of modular serine protease mediated by a novel circRNA sponging miRNA increases Aedes aegypti immunity to fungus.

BACKGROUND: Mosquitoes transmit many infectious diseases that affect human health. The fungus Beauveria bassiana is a biological pesticide that is pathogenic to mosquitoes but harmless to the environment. RESULTS: We found a microRNA (miRNA) that can modulate the antifungal immunity of Aedes aegypti by inhibiting its cognate serine protease. Fungal infection can induce the expression of modular serine protease (ModSP), and ModSP knockdown mosquitoes were more sensitive to B. bassiana infection. The novel miRNA-novel-53 is linked to antifungal immune response and was greatly diminished in infected mosquitoes. The miRNA-novel-53 could bind to the coding sequences of ModSP and impede its expression. Double fluorescence in situ hybridization (FISH) showed that this inhibition occurred in the cytoplasm. The amount of miRNA-novel-53 increased after miRNA agomir injection. This resulted in a significant decrease in ModSP transcript and a significant increase in mortality after fungal infection. An opposite effect was produced after antagomir injection. The miRNA-novel-53 was also knocked out using CRISPR-Cas9, which increased mosquito resistance to the fungus B. bassiana. Moreover, mosquito novel-circ-930 can affect ModSP mRNA by interacting with miRNA-novel-53 during transfection with siRNA or overexpression plasmid. CONCLUSIONS: Novel-circ-930 affects the expression level of ModSP by a novel-circ-930/miRNA-novel-53/ModSP mechanism to modulate antifungal immunity, revealing new information on innate immunity in insects.

Entomopathogenic fungi for the control of larvae and adults of Aedes aegypti (Diptera: Culicidae) vector of dengue, chikungunya and Zika viruses in Mexico.

OBJECTIVE: To assess larvicide and adulticide activity of different native strains of fungi on Aedes aegypti. MATERIALS AND METHODS: Third instar larvae were exposed for 72 h at a concentration of 1x108 conidia/ml of 15 fungi; only fungi that significantly affected the larvae were evaluated against the adult phase at a concentration of 2x1010 conidia/ml. Mortality readings were performed at 24, 48, and 72 h for larvae, and every day to 30 days for adults. RESULTS: Trichoderma longibrachiatum, Aspergillus aculeatus, and Metarhizium anisopliae had the best larvicidal activity at 24 h of exposure (p<0.05), causing mortalities of 100, 72, and 62%, respectively. Adult mosquitoes were more affected by Gliocladium virens (45% mortality), M. anisopliae (30% mortality), and T. longibrachiatum (23.33% mortality). CONCLUSION: The larval stage of Ae. aegypti was more susceptible than the adult phase to the pathogenic action of native fungi, with T. longibrachiatum being with the highest virulence.

Introducing an environmental microbiome to axenic Aedes aegypti mosquitoes documents bacterial responses to a blood meal.

IMPORTANCE: The blood meal of the female mosquito serves as a nutrition source to support egg development, so is an important aspect of its biology. Yet, the roles the microbiome may play in blood digestion are poorly characterized. We employed axenic mosquitoes to investigate how the microbiome differs between mosquitoes reared in the insectary versus mosquitoes that acquire their microbiome from the environment. Environmental microbiomes were more diverse and showed larger temporal shifts over the course of blood digestion. Importantly, only bacteria from the environmental microbiome performed hemolysis in culture, pointing to functional differences between bacterial populations. These data highlight that taxonomic differences between the microbiomes of insectary-reared and wild mosquitoes are potentially also related to their functional ecology. Thus, axenic mosquitoes colonized with environmental bacteria offer a way to investigate the role of bacteria from the wild in mosquito processes such as blood digestion, under controlled laboratory conditions.

Utility of surveillance data for planning for dengue elimination in Yogyakarta, Indonesia: a scenario-tree modelling approach.

INTRODUCTION: Field trials and modelling studies suggest that elimination of dengue transmission may be possible through widespread release of Aedes aegypti mosquitoes infected with the insect bacterium Wolbachia pipientis (wMel strain), in conjunction with routine dengue control activities. This study aimed to develop a modelling framework to guide planning for the potential elimination of locally acquired dengue in Yogyakarta, a city of almost 400 000 people in Java, Indonesia. METHODS: A scenario-tree modelling approach was used to estimate the sensitivity of the dengue surveillance system (including routine hospital-based reporting and primary-care-based enhanced surveillance), and time required to demonstrate elimination of locally acquired dengue in Yogyakarta city, assuming the detected incidence of dengue decreases to zero in the future. Age and gender were included as risk factors for dengue, and detection nodes included the probability of seeking care, probability of sample collection and testing, diagnostic test sensitivity and probability of case notification. Parameter distributions were derived from health system data or estimated by expert opinion. Alternative simulations were defined based on changes to key parameter values, separately and in combination. RESULTS: For the default simulation, median surveillance system sensitivity was 0.131 (95% PI 0.111 to 0.152) per month. Median confidence in dengue elimination reached 80% after a minimum of 13 months of zero detected dengue cases and 90% confidence after 25 months, across different scenarios. The alternative simulations investigated produced relatively small changes in median system sensitivity and time to elimination. CONCLUSION: This study suggests that with a combination of hospital-based surveillance and enhanced clinic-based surveillance for dengue, an acceptable level of confidence (80% probability) in the elimination of locally acquired dengue can be reached within 2 years. Increasing the surveillance system sensitivity could shorten the time to first ascertainment of elimination of dengue and increase the level of confidence in elimination.

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

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

Hierarchical shift of the Aedes albopictus microbiota caused by antimicrobiota vaccine increases fecundity and egg-hatching rate in female mosquitoes.

Recent studies show that mosquito-microbiota interactions affects vector competence and fitness. We investigated if host antibodies modifying microbiota impact mosquito physiology. We focused on three prevalent bacteria (Acinetobacter, Pantoea, and Chryseobacterium), originally isolated from the Asian tiger mosquito Aedes albopictus. Our goal was to assess the impact of host antibodies on mosquito microbiota and life traits. Female mosquitoes were fed with blood from rabbits immunized with each bacterium or a mock vaccine. We compared various factors, including feeding behavior, survival rates, and reproductive success of the mosquitoes. Interestingly, mosquitoes fed with blood from a Chryseobacterium-immunized rabbit showed a significant increase in fecundity and egg-hatching rate. This outcome correlated with a decrease in the abundance of Chryseobacterium within the mosquito microbiota. While no significant changes were observed in the alpha and beta diversity indexes between the groups, our network analyses revealed an important finding. The antimicrobiota vaccines had a considerable impact on the bacterial community assembly. They reduced network robustness, and altered the hierarchical organization of nodes in the networks. Our findings provide the basis for the rational design of antimicrobiota vaccines to reduce mosquito fitness and potentially induce infection-refractory states in the microbiota to block pathogen transmission.

Variable microbiomes between mosquito lines are maintained across different environments.

The composition of the microbiome is shaped by both environment and host in most organisms, but in the mosquito Aedes aegypti the role of the host in shaping the microbiome is poorly understood. Previously, we had shown that four lines of Ae. aegypti harbored different microbiomes when reared in the same insectary under identical conditions. To determine whether these lines differed from each other across time and in different environments, we characterized the microbiome of the same four lines of Ae. aegypti reared in the original insectary and at another institution. While it was clear that the environment influenced the microbiomes of these lines, we did still observe distinct differences in the microbiome between lines within each insectary. Clear differences were observed in alpha diversity, beta diversity, and abundance of specific bacterial taxa. To determine if the line specific differences in the microbiome were maintained across environments, pair-wise differential abundances of taxa was compared between insectaries. Lines were most similar to other lines from the same insectary than to the same line reared in a different insectary. Additionally, relatively few differentially abundant taxa identified between pairs of lines were shared across insectaries, indicating that line specific properties of the microbiome are not conserved across environments, or that there were distinct microbiota within each insectary. Overall, these results demonstrate that mosquito lines under the same environmental conditions have different microbiomes across microbially- diverse environments and host by microbe interactions affecting microbiome composition and abundance is dependent on environmentally available bacteria.

Threshold dynamics of a stochastic mathematical model for Wolbachia infections.

A stochastic mathematical model is proposed to study how environmental heterogeneity and the augmentation of mosquitoes with Wolbachia bacteria affect the outcomes of dengue disease. The existence and uniqueness of the positive solutions of the system are studied. Then the V-geometrically ergodicity and stochastic ultimate boundedness are investigated. Further, threshold conditions for successful population replacement are derived and the existence of a unique ergodic steady-state distribution of the system is explored. The results show that the ratio of infected to uninfected mosquitoes has a great influence on population replacement. Moreover, environmental noise plays a significant role in control of dengue fever.

Microplastic ingestion perturbs the microbiome of Aedes albopictus (Diptera: Culicidae) and Aedes aegypti.

Microplastics (MPs) are common environmental pollutants; however, little is known about their effects after ingestion by insects. Here we fed Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) mosquito larvae 1 µm polystyrene MPs and examined the impacts of ingestion on adult emergence rates, gut damage, and fungal and bacterial microbiota. Results show that MPs accumulate in the larval guts, resulting in gut damage. However, little impact on adult emergence rates was observed. MPs are also found in adult guts postemergence from the pupal stage, and adults expel MPs in their frass after obtaining sugar meals. Moreover, MPs effects on insect microbiomes need to be better defined. To address this knowledge gap, we investigated the relationship between MP ingestion and the microbial communities in Ae. albopictus and Ae. aegypti. The microbiota composition was altered by the ingestion of increasing concentrations of MPs. Amplicon sequence variants (ASVs) that contributed to differences in the bacterial and fungal microbiota composition between MP treatments were from the genera Elizabethkingia and Aspergillus, respectively. Furthermore, a decrease in the alpha diversity of the fungal and bacterial microbiota was observed in treatments where larvae ingested MPs. These results highlight the potential for the bacterial and fungal constituents in the mosquito microbiome to respond differently to the ingestion of MPs. Based on our findings and the effects of MP ingestion on the mosquito host micro- and mycobiome, MP pollution could impact the vector competence of important mosquito-transmitted viruses and parasites that cause human and animal diseases.

Aedes albopictus microbiome derives from environmental sources and partitions across distinct host tissues.

The mosquito microbiome consists of a consortium of interacting microorganisms that reside on and within culicid hosts. Mosquitoes acquire most of their microbial diversity from the environment over their life cycle. Once present within the mosquito host, the microbes colonize distinct tissues, and these symbiotic relationships are maintained by immune-related mechanisms, environmental filtering, and trait selection. The processes that govern how environmental microbes assemble across the tissues within mosquitoes remain poorly resolved. We use ecological network analyses to examine how environmental bacteria assemble to form bacteriomes among Aedes albopictus host tissues. Mosquitoes, water, soil, and plant nectar were collected from 20 sites in the Manoa Valley, Oahu. DNA was extracted and associated bacteriomes were inventoried using Earth Microbiome Project protocols. We find that the bacteriomes of A. albopictus tissues were compositional taxonomic subsets of environmental bacteriomes and suggest that the environmental microbiome serves as a source pool that supports mosquito microbiome diversity. Within the mosquito, the microbiomes of the crop, midgut, Malpighian tubules, and ovaries differed in composition. This microbial diversity partitioned among host tissues formed two specialized modules: one in the crop and midgut, and another in the Malpighian tubules and ovaries. The specialized modules may form based on microbe niche preferences and/or selection of mosquito tissues for specific microbes that aid unique biological functions of the tissue types. A strong niche-driven assembly of tissue-specific microbiotas from the environmental species pool suggests that each tissue has specialized associations with microbes, which derive from host-mediated microbe selection.