Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
FL Medical Entomology Lab
Non Technical Summary
Mosquitoes transmit viruses and parasites that can cause serious disease in humans. To reduce the impact of these diseases, we need to develop new tools to control mosquito populations and to block infection with key pathogens. An important step in this process is to improve our understanding of mosquito biology and interactions between mosquitoes and pathogens. A key player in these interactions is bacteria, as they inhabit mosquitoes in large numbers and can affect the mosquito immune response. This project seeks to understand how bacteria that naturally associate with mosquitoes affect mosquito biology and their likelihood of transmitting pathogens like dengue virus.In order to achieve this goal, we will make use of standard microbiology techniques to collect bacteria from different mosquito populations in nature and build a library of these bacteria to use in controlled inoculation of laboratory mosquitoes. We will use standard entomology techniques to assess the impact of these bacterial inoculations on mosquito biology, including traits such as lifespan, reproductive biology, and physical size. We will used tissue culture to grow virus and perform experimental oral infection to assess the impact of bacterial inoculation on the ability of mosquitoes to transmit viruses. Finally, we will use bacterial sequencing and multivariate statistical analysis to assess how key factors linked to the environment and mosquito biology affect which bacteria they become associated with.We will inform target audiences of our findings through publication of scientific papers, though talks and posters presented at scientific conferences, and through communication of key findings over social media, and web bulletins distributed over the institutional web portal or the lab website. Any essential findings will be communicated to stakeholders via public outreach.Through this project we expect to generate improved understandings of the types of bacteria found in mosquitoes, how mosquito-bacteria relationships are affected by changes in the environment and by changes to mosquito immune and metabolic processes. We expect to generate a library of mosquito-associated bacteria and use that library to improve our understanding of the roles that specific bacteria play in mosquito biology and during infection with important arboviruses. Through the advancements in knowledge generated by this project we anticipate identifying mosquito-associated bacteria that are: (1) widely found in mosquitoes, which stand a greater chance of affecting mosquito biology, (2) capable of killing mosquitoes, which could be used to develop new insecticides, and (3) capable of making mosquitoes less likely to transmit key viruses, which could be used to develop new therapies to combat viruses, like dengue.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Goals:This project will improve understanding of the biological roles that bacteria play in mosquitoes. It will do so through the meeting four objectives:Goal 1:Identify bacteria associated with different mosquito populations. For this objective, we will collect populations of different mosquito species, isolate the various bacteria that are found within them, use sequencing to identify specific isolated bacteria and to compare microbiome composition across populations and/or species.Goal 2:Examine extrinsic factors that moderate the composition of the mosquito microbiome.For this objective, we will assess the role of key extrinsic variables play in determining the composition of the mosquito microbiome. We will vary environmental conditions linked to the larval aquatic habitat, specifically the physicochemical parameters of the water, and also the composition of the mosquito diet. We will then compare the composition of the microbiome of mosquitoes reared under these different treatments.Goal 3:Examine intrinsic factors that moderate the composition of the mosquito microbiome.For this objective, we will assess the role that key intrinsic factors linked to mosquito biology play in determining the composition of the mosquito microbiome. We will seek to modify the host immune response or key host metabolic processes linked to bacterial replication, and then compare the composition of the microbiome of mosquitoes reared under these different treatments.Goal 4:Assess the roles different bacteria play in mosquito biology and vector competence. For this objective, we will use controlled inoculation of mosquitoes with key bacteria that were identified in the first three objectives to assess how the presence, absence of relative abundance of these bacteria impact mosquito life history, vector competence and behavior.
Project Methods
Objective 1: We will collect mosquito populations from different areas, particularly in Florida due to its proximity to our laboratory, using adult and egg collections. Adult collections will use traps that target female mosquitoes seeking a blood meal or a site to lay their eggs. We will target mosquito populations from different regions in order to be able to isolate more unique bacteria. These mosquitoes will be returned to our laboratory in cold storage (dry ice or cold packs) to preserve the bacteria that naturally infect them. We will isolate these bacteria by dissecting out mosquito midguts as this tissue is where most mosquito-associated bacteria dwell. These midguts will be plated on bacterial growth media and morphologically distinct bacterial colonies isolated. We will identify these bacteria to the genus or the species level by sequencing the DNA of their 16S rRNA gene, which is commonly used for bacterial barcoding, using sanger sequencing, and by comparing those sequences to databases of bacterial genomes. Distinct bacterial isolates will then be stored as a reference library of mosquito-associated bacteria.Objective 2: We will examine the role of different extrinsic factors that might affect the composition of the mosquito microbiome. Specifically, we are interested in identifying environmental variables that determine which bacteria associate with mosquitoes. We expect that these variables will include important physicochemical properties of the larval aquatic environment such as conductivity, salinity, pH, levels of micronutrients. These are all easily quantified and levels in the larval environment can be easily manipulated, which is how we will assess the impact of the variables on the mosquito microbiome. For instance, we will rear larvae in water with different concentrations of essential micronutrients or in water with conductivity altered changing salt content. We will also look at the impact of diet, varying the composition of larval mosquito diet, which is typically nutrient-rich and protein-based, and adult mosquito diet, which is typically carbohydrate-based. We will look at the presence or absence of key nutrients, concentrations of key nutrients, or ratios of different nutrients.The choice of specific variables will depend on literature review of studies highlighting links between particular variables, the microbiome or mosquito immunity. We will conduct small-scale pilot assays that look at the impact of these variables on mosquito survival, as this will help to determine whether assessment of any particular variable is feasible. We will evaluate the impact of the different rearing and/or dietary regimes by observing changes to the composition of the mosquito microbiome. We will predominantly look at the adult microbiome, as that is the most critical to mosquito-arbovirus interactions that facilitate arbovirus infection and transmission. We will compare the impact of key variables across different mosquito species, particularly vectors including Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. Microbiome sequencing will be performed using the industry standard 2x300bp 16S rRNA Illumina MiSeq approach. Data produced during sequencing will be analyzed using the R package vegan() which is used with microbial ecology data and offers easy interpretation of microbiome differences between treatment groups.Objective 3: We will examine the role of key factors linked to mosquito biology in determining the composition of the mosquito microbiome. We will focus on intrinsic factors associated with host immunity, which can serve to suppress microbial proliferation and impact arboviral infection. We will also focus on host metabolism, as mosquito metabolic products might be used as food by the bacteria that live in the mosquito gut, while the bacteria themselves might serve as a nutritional resource for the mosquito host leading to changes in abundance of certain metabolites.These factors will predominantly be targeted through RNAi-based gene silencing through double-stranded RNA injection in mosquito adults, which will reduce transcription levels of target genes. We will use this approach to reduce immune activity by targeting immune effector genes or increase immune activity by targeting immune suppressing genes. For metabolism, we will seek to disrupt essential biosynthetic pathways by targeting certain enzymes, which will alter levels of key metabolites. The microbiome composition for mosquitoes subject to these treatments will be compared through 16S rRNA sequencing, as described for Objective 2.Objective 4: Here, we will utilize key bacteria identified in the first three objectives as having (1) differential abundance between mosquito populations, mosquito species, or ecological regions; (2) responsiveness to specific extrinsic factors, or (3) responsiveness to specific intrinsic factors. Bacteria that meet at least one of these criteria will be used for controlled inoculation of mosquitoes. This will transpire through one of two methodologies. First, gnotobiotic larval rearing, where mosquito eggs are hatched under sterile conditions, and larvae are exposed to specific bacteria. Second, adult inoculation via feeding, where newly eclosed adults are fed sucrose or water dosed with specific bacteria. Both of these methodologies are expected to produce mosquitoes with a microbiome entirely consisting of, or at least dominated by the bacteria of interest. The efficacy of inoculation will be evaluated via custom qPCR assays that measure the proportion of total bacterial reads represented by the bacteria of interest for individual mosquitoes. We will use mosquitoes inoculated in this manner to perform a series of experiments designed to gauge the impact of infection with those bacteria on mosquito life history and vector competence.Life history studies are critical determinants of mosquito fitness and vectorial capacity. We will assess the following parameters: survival time post-inoculation, fecundity and fertility, blood meal intake, and post-blood meal bacteria proliferation, or others if need arises. Survival time will be monitored through daily counts of dead mosquitoes. Fecundity and fertility by counting numbers of eggs laid and hatched for individual mosquitoes. Blood meal intake by measuring mosquito mass before and after taking a blood meal. Bacterial proliferation will be measured through 16S-based qPCR. The effects of different bacteria on these traits will be evaluated using basic statistical analyses in Graphpad prism and/or R, including t-tests, ANOVAs and general linear models of regression. These results will help us understand how association with different bacteria affect mosquito biology and fitness.We will assay vector competence against prominent arboviruses, with the choice depending on the mosquito species we are working with in any particular experiment. This list could include DENV, CHIKV, WNV and/or ZIKV, but also others, where appropriate. Vector competence will be assessed through experimental oral infection, with viral load quantified in mosquito tissues including whole bodies, midguts, heads/thoraces, and/or saliva. Virus quantification will be performed via one of two methods, either plaque forming assay, or absolute viral quantification via Taqman-based RT-qPCR. Either of these techniques is an appropriate choice for quantifying arbovirus levels in mosquitoes, and they can both be easily adapted for use with different arboviruses. Data for these experiments will be analyzed using statistical analyses in Graphpad prism and/or R, including t-tests, ANOVAs, general linear models of regression and multivariate regression. These data will help us to identify bacteria that interact with arboviruses and alter host vector competence. These bacteria could be used to develop new mosquito control tools or act as diagnostic markers for susceptibility to arboviral infection.