METABOLOMIC ANALYSIS OF INSECTICIDE RESISTANCE IN INVASIVE AEDES AEGYPTI IN CALIFORNIA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017520
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Entomology and Nematology

Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) ofplant and animal procudtion, protection and health. Increasing the efficiency of vector control of mosquito borne disease is of paramount importance to animal and human health in California and globally. This research will lead to improved understanding of the mechanisms of insecticide resistance in mosquitoes and facilitate the development of rapid detection protocols for use by mosquito control operators to make decisions related to choice of insecticide formulation and type of application that will achieve maximum desired control. The biochemical and molecular approaches proposed here will provide novel insights into the mechanisms evolved by mosquitoes to survive the pesticides used to control disease vector populations. The work described in this proposal will provide proof of concept data for detection of resistance to a commonly used family of pesticides (pyrethroids) by analyzing the biochemistry of these insects. This plan aims to identify chemical markers of resistance as well as the molecular mechanisms by which these mosquitoes are able to survive exposure to pesticides. Successful implementation of these research methods will provide a pipeline by which resistance mechanisms to other pesticide types or in different mosquito populations can be characterized. Analysis of this data will be an important contribution to overcoming a significant hurdle in the implementation of effective, environmentally sound and efficient mosquito control strategies.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
721	3110	1130	30%
721	5220	1000	40%
721	3110	1040	30%

Knowledge Area
721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3110 - Insects; 5220 - Pesticides;

Field Of Science
1040 - Molecular biology; 1130 - Entomology and acarology; 1000 - Biochemistry and biophysics;

Keywords

mosquito

metabolomics

transcriptomics

next generation sequencing

metabolites

biochemistry

gene expression

Goals / Objectives
Vector-borne diseases are currently among the leading causes of global morbidity and mortality in humans and animals of agricultural importance. These diseases account for more than 17% of all infectious disease and cause more than 700,000 deaths each year (WHO Vector Borne Diseases Fact Sheet 2017) with 80% of the world's population at risk. The added variables of global climate change, increasing human populations, emerging vector borne diseases (such as Zika and Chikungunya) and resurgence of established diseases (such a Yellow Fever and Dengue) complicate an already difficult situation. The recent invasion of California by two of the most notorious vector species Aedes albopictus and Aedes aegypti (in 2011 and 2013 respectively) makes what was previously a distant threat a local one [1, 2].A primary tool in preventing vector borne disease includes the use of pesticides aimed at reducing insect vector populations around human populated areas. However, a significant and ongoing threat to this strategy is the evolution of insecticide resistance in mosquitoes and other disease vector organisms. Both Aedes albopictus and Aedes aegypti are disease vector mosquitoes that show varying and widespread levels of pesticide resistance on a global scale [3, 4]. In particular, resistance to the pyrethroid class of insecticides, which are commonly used against these species, is abundant. The genetic mechanisms of resistance are often associated with genes encoding the voltage gated sodium channel which is the target of these insecticides and gene duplications and over expression of detoxification enzyme genes such as P450-monooxygenases. However, detection of these gene variants does not necessarily correlate with observed resistance phenotypes as determined by CDC bottle test (Personal communication Anthony Cornel).The ability to quickly and accurately detect insecticide resistance in field collected mosquitoes would be an important tool to mosquito control programs in California that are evaluating the most effective control strategy for local mosquito populations. Testing for resistance conferring gene alleles is time consuming, requires lab facilities and personnel with the associated technical and analytical skills. An ideal test would be one that could be applied directly in the field or at a mosquito control facility with minimal training using low cost reagents and equipment. The development of such an assay would require the identification of a detectable physiological biomarker directly associated with insecticide resistance.The relatively recent development of metabolomics technologies has facilitated the rapid and comprehensive analysis of the relative abundance of hundreds to thousands of biological compounds within a single sample. The goals of this project are to leverage this technology to take a biochemical approach to identifying mechanisms of insecticide resistance in mosquitoes. This will be accomplished via the following aims.Identification of differential metabolite abundances between pyrethroid susceptible and resistant mosquitoes by untargeted metabolomic analysis of isogenic lines.Identification of pyrethroid resistance mechanisms by tracking the metabolic fate of topically applied stable isotope labeled pesticides in susceptible and resistant mosquitoes.Characterize and correlate differential gene expression profiles between isogenic lines of pyrethroid susceptible and resistant mosquitoes to correlate gene expression levels with resistance specific metabolic functions.

Project Methods
Mosquitoes utilized for the proposed assays will be generated from single gravid Aedes aegypti females (isogenic female lines) collected from Fresno, Clovis or Los Angeles. Representative offspring from these lines will be tested for pyrethroid resistance by CDC bottle assay. An isogenic line derived from the pyrethroid susceptible Rockefeller strain of Aedes aegypti will be generated as a control. Lines demonstrating a resistance phenotype relative to the susceptible Rockefeller will be inbred to ensure genetic homogeneity and reduce phenotypic heterogeneity. Respective susceptible and resistant strains will be reared and maintained in captivity in the Tupper Hall arthropod containment level 2 insectary in the UC Davis School of Veterinary Medicine using established rearing protocols.Aim 1: To identify baseline differences in metabolic function between pyrethroid susceptible and resistant Aedes aegypti, mosquitos at different physiological states will be collected and submitted for metabolomic analysis. Female mosquitoes will be collected at 0-24 hours post-eclosion (teneral), 96-120 hours post eclosion (pre-vitellogenic), 2-4 hrs post blood meal, 24 hours post blood meal (vitellogenic), 72 hours post blood meal (gravid). Mosquitoes will be anesthetized on ice and snap frozen in liquid nitrogen. Pools of 5 mosquitoes will be collected for each condition with at least 6 replicates per condition from the representative susceptible and resistant colonies. The samples will be submitted to the NIH: West Coast Metabolomics Center at UC Davis for untargeted metabolomic analysis. The untargeted analyses consist of 3 panels of metabolites including primary metabolites (carbohydrates and sugar phosphates, amino acids, hydroxyl acids, free fatty acids, purines, pyrimidines, aromatics, exposome-derived chemicals), complex lipids (ceramides, sphingomyelins, cholesteryl esters, oxysterols, lyso- and phospholipids, mono-, di- and triacylglycerols, galactosyl- and glucuronyllipids) and biogenic amines (acylcarnitines, TMAO, cholines, betaines, SAM, SAH, nucleotides and nucleosides, methylated and acetylated amines, di- and oligopeptides) for a total of ~800 metabolites. Differential abundance of individual metabolites will be determined by Welshes two sample t-test followed by a False Discovery Rate Test (FDR) to determine confidence levels (q-values) for individual P-value scores. Metabolites with a P-value of < 0.05 and a Q-value of <0.05 will be considered to have differential abundances between samples. Metabolites with differential abundances will be mapped to the KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway library [5] to identify resistance associated pathways.Aim 2: Susceptible and resistant mosquitoes at 5 days post eclosion will be given topical treatments of stable isotope (13C) labeled pyrethroids (Sigma/Aldrich) and parallel groups will be treated with unlabeled pesticide suspended in acetone. Control mosquitoes will be treated with an equivalent volume of acetone alone. At 15 minutes post treatment, mosquitoes will be anesthetized on ice, collected, flash frozen in liquid nitrogen and stored at -80°C. The samples will be submitted to the NIH: West Coast Metabolomics Center at UC Davis for targeted quantitation of 13C labeled pesticides and associated 13C labeled secondary compounds derived metabolic processes within the mosquito. The methodologies for sample preparation, isolation and quantitation of these compounds will be determined in consultation with the metabolomics center to optimize the identification of relevant metabolites. Mosquitoes treated with unlabeled pesticides will be used in a subtractive manner to eliminate peaks associated with unlabeled compounds and emphasize labeledcompounds resulting from a shift in mass. This analysis will determine differences in metabolic flux and the ultimate fate of applied pyrethroids in the context of susceptible and resistant phenotypes.Aim 3: To determine differential gene expression profiles between susceptible and resistant mosquitoes we will isolate total RNA from pyrethroid susceptible and resistant mosquitoes and perform high throughput RNA-seq analysis. Mosquitoes will be collected at 0-24 hours post-eclosion (teneral), 96-120 hours post eclosion (pre-vitellogenic), 2-4 hrs post blood meal, 24 hours post blood meal (vitellogenic), 72 hours post blood meal (gravid). Each time point will include 3 replicates of 5 mosquitoes for susceptible and resistant mosquitoes. Mosquitoes will be anesthetized on ice and homogenized in Trizol reagent (Invitrogen). Total RNA will be isolated via the Direct-zol kit (Zymo) according to the kit protocol. RNA quantity will be determined by nanodrop spectrophotometry. Isolated RNA will be submitted to the UC Davis Genome Center Expression Analysis Core. Sample RNAs will be used to generate multiplexed single stand paired end polyA specific RNA-seq libraries which will be sequenced on an Illumina HiSeq 4000 sequencer. Sequencing data will be aligned to the Aedes aegypti transcriptome using the Star sequence alignment software package [6]. Differential gene expression will be determined using the count data output from STAR using the EdgeR differential expression analysis package in R [7]. Genes identified as differentially expressed will be analyzed for functional enrichment using the topGO package in R [8]. Differentially expressed enzyme functions will be compared with the metabolomics data generated in Aims 1 + 2 to determine metabolic overlap between insecticide resistance associated metabolites and resistance associated gene expression. Transcriptomic and metabolomic data will be analyzed using the KEGG mapper software suite [9] to correlate genes and metabolites with differential abundances with metabolic pathways associated with pyrethroid resistance.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The goal of this project is to study the physiological, genetic and metabolic changes associated with resistance to the pyrethroid class of insecticides in invasive Aedes aeypti mosquitoes. These mosquitoes have undergone a rapid spread throughout most of the state of California. This in part has been facilitated by the resistance of these populations to pyrethroid type insecticides utilized to manage adult mosquito populations during periods when risk of vector borne disease is predicted to be high. This species thrives in human associated habitats and are aggressive day feeding mosquitoes that prefer human hosts and pose a significant biting nuisance to affected human populations. In addition, this mosquito species can carry and transmit the Zika, dengue, yellow fever, and chikungunya viruses to humans. While local transmission of these diseases has not yet been detected, the establishment of this species throughout California and inability to control its spread means the potential for a mosquito borne viral epidemic is a possibility. This rapidly evolving situation is of public health importance to the entire population of California and to those stakeholders tasked with ensuring the health and safety of Californian's. These include the California Department of Public Health (CDPH), the various disease vector abatement districts throughout the state and research groups in the field of vector biology/control. Changes/Problems:A criticism of our initial proposal was the use of a control insecticide susceptible strain that is derived from the lab and has a distinct genetic background from that of the wild resistant mosquitoes. We proposed to address this by using a hybridization strategy where a hybrid strain will be generated by mating resistant Californian Aedes aegypti with the susceptible Rockerfeller lab strain over three generations. This strategy should produce hybrid offspring displaying a wide array of phenotypes while maintaining a common genetic background. This will eliminate potential confounding genetic and biochemical differences resulting from adaptations to lab rearing and extensive inbreeding in the control strain. The hybrids will be classified by analysis of their resistance phenotype followed by correlational analysis of genetic and biochemical features linked to the phenotype. The work described in the NIH proposal aims at identifying biochemical, molecular and morphological changes associated with the resistance phenotype. This work expands upon our initially proposed work by providing an in-depth analysis of identified biochemical and genetic features and their role in pyrethroid resistance. We aim to visualize the location within the mosquito where these genes are being expressed by in situ staining of RNAs in whole mounted tissues. It also proposes to explore the potential role of modification of the mosquitoes exoskeleton to prevent the uptake of insecticides. We plan to achieve this using scanning and transmission electron microscopy of the mosquito's exoskeleton to measure its thickness. We also proposed to perform internal structural analysis of the mosquito using microCT scanning to generate a virtual 3-dimensional visualization of the external and internal features of the mosquito to identify structural changes that may facilitate resistance mechanisms such as increased internal surface area and changes in organ morphology/structure. The proposal also aims to investigate the role that environmental adaptation by invasive Californian Aedes aegypti is playing in the resistance phenotype. The hot and dry environments found in the Central Valley of California and the significant temperature shifts observed across the seasons are very different from the native habitat of Aedes aegypti which is tropical rather than temperate. Adaptations allowing these mosquitoes to live in the Californian climate would likely be associated with thermal and dehydration stressors. We believe that these adaptations may also be tied to pyrethroid resistance as cuticular modification has been associated with desiccation tolerance and higher temperatures may facilitate higher metabolic rates. This will be tested by rearing susceptible and resistant strains under internal (climate controlled Lab) and external (California Central Valley) conditions for 10 generations to determine if adaptation to differential rearing conditions impacts the pyrethroid resistance phenotype. What opportunities for training and professional development has the project provided?This project is the primary research focus of my two graduate students (Taylor Kelly and Lindsey Mack). The work associated with this project has provided a valuable opportunity to learn how to maintain, handle and design experiments for an important disease vector. This research is in part dependent on relationships with vector abatement groups, Anton Cornell at the Kearny Agricultural Station and the California Department of Public Health. These relationships have provided Taylor and Lindsey with valuable connections with personnel actively working on solutions to mitigate vector borne disease. The project has also provided Taylor and Lindsey with opportunities to learn new techniques in physiological, molecular biology, biochemistry, bioinformatic analyses. It has also provided opportunities for Undergraduate training in these techniques and mentorship opportunities for Taylor and Lindsey. They have been responsible for supervision and mentorship of three undergraduate students (Aamina Zahid, Tess Van Schoor and Victoria Shen). They are all authors on the resulting paper which is currently under review. How have the results been disseminated to communities of interest?The results derived from the project have been communicated to communities of interest via teleconferences with Vicki Kramer and her team at the California Department of Public Health as well as members of the Shasta, Sacramento/Yolo, Greater LA and Delta Vector Districts. The results of the population genetics analysis were presented at the Mosquito and Vector Control Association of California (MVCAC) meeting in January of 2020. In addition, the results of the metabolomics analysis were presented at the annual meeting of the Pacific Southwest Center of Excellence in Vectorborne Diseases. This analysis was also presented as a virtual poster by Taylor at the Entomological Society of America National Meeting in 2020. Her poster received 1st prize in the competition for posters within the category of Insect Physiology. What do you plan to do during the next reporting period to accomplish the goals?A five-year NIH R01 proposal on this work was submitted and reviewed by NIH. The proposal was not funded, however valuable feedback was received and a revised proposal that addresses reviewer critiques was resubmitted in November and will be reviewed again in February of 2021. We have generated high throughput gene expression datasets from resistant Clovis mosquitoes before and after exposure to pyrethroids (relative to an unexposed control group). We will use this dataset to identify genes that demonstrate altered expression profiles in response to insecticide exposure. In particular we are interested in identifying genes coding for enzymes associated with insecticide detoxification as well as genes associated with metabolic pathways required to support the function of detoxification enzymes. The next steps planned are to perform an in depth metabolomic analysis of mosquitoes from the Clovis strain that demonstrate significant variability in their susceptibility pyrethroid exposure within the population. This will be a correlational analysis in which Clovis strain mosquitoes will be categorized by knockdown time after exposure by bottle bioassay and will then be analyzed using untargeted metabolomics to identify differences between mosquitoes with the same genetic background demonstrating different levels of resistance. The results of this analysis will identify changes in biochemical compound abundances and metabolic pathways correlating with the phenotypic intensity of pyrethroid resistance. This will provide an array of candidate compounds with the potential to function as biomarkers in development of a biochemical resistance monitoring assay.

Impacts
What was accomplished under these goals? We have established partnerships with multiple mosquito abatement groups as well as the California Department of Public Health to obtain representative mosquito samples from throughout California. We have also developed an informative, high throughput genetic analysis that allows us to discriminate the population origins of Californian Aedes aegypti as well as monitor their movement throughout the state. The baseline physiological parameters of pyrethroid resistant mosquitoes have been established and significant differences in life history parameters have been noted (including lifespan, developmental times and reproductive capacity) which have allowed us to focus our efforts on identifying the physiological costs of resistance and the mechanisms that might be underlying them. These findings are allowing us to develop target hypotheses for testing to define potentially exploitable weaknesses associated with the resistance phenotype. We have performed initial analyses defining the biochemical differences between resistant and susceptible strains of Aedes aegypti. The results of these analyses have identified significant differences between these strains and highlight changes in biochemical pathway function as well as identification of individual biochemical features with the potential to act as diagnostic markers for the presence and intensity of the pyrethroid resistance phenotype. We have also performed high throughput gene expression analyses on the resistant Clovis strain mosquitoes to identify genetic responses to exposure to pyrethroids over time. The sequencing for this analysis is complete and bioinformatic analysis and identification of pyrethroid responsive gene expression is in progress.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: L. K. Mack, et al., Association Between Pyrethrum Knockdown Time and Sodium Channel Genotypes in California Aedes Aegypti (2020) (https://www.researchsquare.com/article/rs-84448/v1).

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The goal of this project is to study the physiological, genetic and metabolic changes associated with resistance to the pyrethroid class of insecticides in invasive Aedes aegypti which were recently discovered in Southern and Central California. As such, there are a number of stakeholders associated with this project. Our primary targets for distribution of the resulting experimental outcomes are: The California Department of Public Health (CDPH), California Vector Abatement districts, scientists in the field of vector biology/control and the public at large. Changes/Problems:We have made some changes to our experimental strategy in regards to the comparisons we are making between susceptible and resistant mosquito strains. The lab susceptible strain of Aedes aegypti (Rockerfeller) has been in culture for decades and is highly inbred. This raises the concern of whether metabolic differences we are detecting are due to the resistance phenotype or to adaptations to colonization. To address this we will generate a hybrid strain by crossing Rockerfeller mosquitoes with resistant mosquitoes from Clovis California. The hybrid F1s will be tested by bottle bioassay to observe how the resistance phenotype manifests in offspring from this cross to determine the level of penetrance and variance of the phenotype. The F1 generation will be crossed amongst themselves to generate the F2 generation. Individual female and male F2s will be paired for mating and then blood fed individually. A subset of eggs from each individual pairing will be reared out to adults and tested via bottle bioassay to identify the pairings with offspring showing the highest and lowest resistance phenotypes. Females with offspring demonstrating the extremes of the phenotype will be maintained for egg production. Egg papers will be collected from these females over multiple reproductive cycles. These eggs will be reared and utilized for metabolomic and gene expression analysis according to the protocols described in the initial submission. It was initially proposed perform these analyses over multiple physiological time points in the resistant and susceptible strains. However, due to the number of samples required to be performed with the addition of the hybrid strains these analyses will be limited to mosquitoes at five days post eclosion. This time point has been demonstrated to be informative based upon the initial metabolomics data we have received. In the initial proposal, stable isotope tracking of pyrethroids was put forth for Aim 2. However, after consultation with the metabolomics core we concluded that this may pose a technical challenge as the resulting metabolites of these compounds may be difficult to identify due to lack of appropriate characterized mass spectra to compare against. It is possible to identify these unknown metabolites individually, however this is a slow and expensive process and the investment may not be worth the outcome. As an alternative, it is proposed to perform a Genome Wide Association Study (GWAS) to identify genomic loci correlated with the insecticide resistance phenotype. This will be performed using the Rock and Clovis strains as susceptible and resistant strains respectively as references and then using the susceptible and resistant hybrid F2 strains to identify genetic loci that correlate with the resistance phenotype in the hybrids. High throughput short read and long read sequencing of genomic DNA from these mosquitoes will be used to generate data on genetic loci/features that associate with the resistance phenotype in both the Clovis strain and the resistant hybrid strain. This work will be performed in collaboration with the UC Davis Genomics Facility. This research is being proposed in an NIH R01 proposal in February. What opportunities for training and professional development has the project provided?This project has been the focus of the thesis work of my two Ph.D. students Erin "Taylor" Kelly and Lindsey Mack. They have developed the proposed research into the metabolic changes and reproductive impacts correlated with the pyrethroid resistance phenotype into experimental plans for their thesis projects. In addition, Taylor and Lindsey have been mentoring two undergraduate students, Victoria Shen and Aamina Zahid who have been assisting with the research for this project and who will be authors on resulting papers. Taylor and Lindsey have also had the opportunity to establish relationships with members of the local mosquito abatement district and present their work at the Entomological Society of America. Both Taylor and Lindsey are excellent scientists and have grown over the past year. They have learned new software and analytical techniques to manipulate and analyze the large and complex datasets associated with these experiments. They have both formulated their projects into research proposals which have been put into fellowship proposals to the National Science Foundation, Department of Defense and next year the National Institutes of Health. How have the results been disseminated to communities of interest?Since the initiation of this project we have touched base with representatives from the California Department of Public Health (CDPH) and the heads of four mosquito abatement districts (listed below) to share our current findings. The Attardo and Cornel labs held a group Skype call with Vicki Kramer and five other members of her team at CDPH to share our results from the project and to touch base with them regarding ways in which we could collaborate to utilize samples collected previously to expand the scope of this project. In addition we (myself and/or my two graduate students) have met with: Jennifer Henke (Lab Manager) and Jeremy Wittie (General Manager) of the Coachella Valley Mosquito and Vector Control District. Angela Caranci (Vector Ecologist) and Bill van Dyke (Public Information Officer) at Northwest Mosquito and Vector Control in Corona California. Gary Goodman (District Manager), Marcia Reed (Laboratory Director) and Samer Elkashef (Assistant Manager) at the Sacramento-Yolo Mosquito and Vector Control District. Susanne Kluh (Scientific-Technical Services Director) at the Greater Los Angeles County Vector Control District. The spread of Aedes aegypti through California has made it such that collaboration with these groups has allowed us to expand the scope of our studies on insecticide resistance and develop location specific mosquito colonies with which to determine the resistance phenotypes of populations from different geographic foci. We plan on maintaining these relationships into the future for related and new projects. Both of my graduate students attended the Entomological Society of America National Meeting this year and presented posters on their work to date and proposed work on this project. They will both be attending the Mosquito and Vector Control Association of California (MVCAC) annual meeting this coming January as well as the annual meeting for the Pacific Southwest Center of Excellence in Vector-Borne Diseases to present their work. The results we have developed based on our analyses of pyrethroid resistance associated target gene mutations and population genetics are being developed into two manuscripts for submission to the Journal of Medical Entomology and PLoS Neglected Tropical Diseases respectively. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we plan to publish the results from the bioassay and genetic testing analyses. We have also been in touch with mosquito abatement districts throughout the state and are obtaining additional field caught Aedes aegypti from different locations for analysis via our SNP assay. We will attempt to obtain eggs so that phenotypic data can be obtained via bioassay prior to genetic testing. This will aid in understanding the movement of these mosquitoes throughout California as well as the genetic interactions or lack thereof between populations. The data resulting from the metabolomics analyses will be analyzed to identify biochemical compounds showing differential abundances between the susceptible and wild strains then determine the potential biological significance of those observations by associating them with related biochemical pathways/functions/enzymatic reactions. We will then test these individual compounds to determine if they are informative as to the insecticide resistance state of our other collected Californian strains as well as F2 offspring of hybrid lines derived from crosses of the susceptible and resistant strains. Comparative gene expression analysis of these two strains of mosquitoes will be performed using mosquitoes from the same time point as the metabolomic samples. The resulting data will be used to identify differentially expressed genes. Differential gene expression data will be annotated and analyzed to identify enrichments in function as well as to identify correlations with the metabolic data in terms of differential biochemical abundances and differentially expressed enzyme coding genes associated with metabolism of those compounds.

Impacts
What was accomplished under these goals? In the year since the initiation of this project, we have been working in close collaboration with Dr. Anton Cornel and his technician Katherine Brisco to colonize and characterize invasive Aedes aegypti populations. We have successfully acquired and colonized mosquitoes from the areas of Greater LA, Kingsburg, Clovis, Sanger and Dinuba. The goal of establishing these colonies was to understand the genetic relationships between these populations across geographic space and to identify populations at the extremes of the pyrethroid resistance phenotype. The phenotype was documented for these populations by CDC bottle bioassay testing of representative mosquitoes from these populations in groups and individually. Group testing of 25 mosquitoes per bottle was utilized to determine the overall level of resistance within the population. Testing of individual mosquitoes was used to record the level of resistance for later association with genetic information from those individuals. After individual testing, mosquitoes were homogenized in DNA/RNA homogenization buffer followed by column based DNA and RNA extraction for each individual. The DNA was analyzed to characterize the presence or absence of pyrethroid resistance associated mutations and genetic features show to be informative for geographic localization. The analysis was performed on 309 individual mosquitoes and was very informative as to the frequency of resistance associated mutations in these populations and the correlation between the mutations and the resistance phenotype. The geographically informative SNPs have provided us with information allowing the discrimination of the genetic relatedness of individuals from the different populations. The results of this analysis showed a clear separation in the genetic relationships between populations from the Central Valley (Dinuba, Sanger, Clovis, Kingsburg) and the Greater Los Angeles areas. This result agrees with previous work describing two separate introductions of genetically unrelated strains of Aedes aegypti into California. In addition it showed that, mutations associated with pyrethroid resistance are almost completely fixed (99-100%) in the populations from the Central Valley while the frequency in mosquitoes from the Greater LA area is around 55-56%. Comparison of the phenotypic resistance data with the presence or absence of resistance associated mutations in the para gene provided interesting results. The presence or absence of para gene resistance mutations in the mosquitoes from Greater LA appears to have a positive correlation with the bottle bioassay determined phenotype. However, the mosquitoes derived from the Central Valley populations are all fixed for the resistance mutations yet they display a wide variance of phenotypic resistance with little correlation between genotype and phenotype. This suggests screening for pyrethroid resistance within this population using these genetic markers is likely of little informative value in terms of predicting the actual level of resistance. In addition it suggests that there are other uncharacterized resistance mechanisms at play in these mosquitoes. Based on the phenotypic analysis of these populations, the Clovis strain shows the highest level of pyrethroid resistance relative to the other populations tested. Based on this observation, mosquitoes from this location will be used as the foundation for our physiological analyses of resistance with the hope of uncovering additional resistance mechanisms and additional mutations that are informative for resistance prediction. The assay utilized to detect these genetic features is rapid and high-throughput which will allow us to expand our analysis of the presence or absence of resistance loci and the genetic relationships between populations. We are working with mosquito abatement districts to obtain Aedes aegypti from untested or newly discovered populations. In particular, Aedes aegypti were recently discovered outside of Sacramento. We are working with the Sac/Yolo Mosquito abatement district to obtain samples to determine the frequency of pyrethroid resistance associated mutations in these mosquitoes and to determine their genetic relationship relative to previously tested populations. The Clovis strain has been established in our lab and has been used to perform a comparative metabolomic analysis of biochemical differences relative to our insecticide susceptible lab strain. The mosquitoes for this analysis were reared under identical conditions during larval development. Age matched adult mosquitoes (within 24 hours of pupal eclosion) were reared for 5 days then collected and snap frozen in liquid nitrogen in 10 replicates of 10 mosquitoes each replicate. Mosquitoes were only provided water in the two days prior to sample collection to reduce the potential confounding factor of sugar feeding. The frozen mosquitoes were analyzed to measure strain specific differences in their biochemistry by gas/liquid chromatography followed by mass spectrometry. This analysis covers >1000 biochemicals and includes primary metabolites, lipids and biogenic amines. Preliminary analysis of the primary metabolites shows a clear separation between the two strains in terms of the composition of primary metabolites. This data was just received last week, so analysis is ongoing. The lipids and biogenic amine data will be coming in the next few weeks.

Publications