INVESTIGATING THE BIOLOGY AND INSECTICIDE RESISTANCE OF CALIFORNIA MOSQUITO DISEASE VECTORS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017221
• 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) of plant and animal production, protection, and health. Increasing the efficiency of mosquito disease vector control is of paramount importance to animal and human health. This research will lead to improved interpretations and guidelines that will be used by mosquito control operators to make decisions related to choice of insecticide formulation and type of application that will achieve maximum desired control. Novel genetic approaches using various algorithms based on sequence data will be tested for use on mosquitoes to determine their population sizes, dispersal and mating behavior. These genetic approaches will provide critical data needed to design the implementation of environmentally friendly 21st century control strategies including Sterile insect technique, auto-dissemination of mosquito specific insect growth regulators and GMM approaches.

Animal Health Component

45%

Research Effort Categories

Basic

40%

Applied

45%

Developmental

15%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3110	1130	20%
722	3110	1130	80%

Knowledge Area
311 - Animal Diseases; 722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
3110 - Insects;

Field Of Science
1130 - Entomology and acarology;

Keywords

mosquito

insecticide resistance

population size estimates

population structure

male and female dispersal

next generation sequencing

Goals / Objectives
1. Improve our understanding of the data derived from mosquito insecticide resistance bio-assays and other assays to better predict expected outcomes of field applications of insecticides. The overall purpose is to combine knock down and mortality rates and knowledge from what is known about the resistance mechanisms in the targeted mosquitoes to better advise the mosquito abatement personnel what insecticides to use to achieve maximum control in urban and agricultural environs.2. Examine the population structure and local and long distance dispersal of Aedes aegypti in California. Multiple novel control mechanisms such as auto-dissemination of insecticides, trap-kill systems, biopesticides, SIT and genetic modification strategies are receiving more acceptance as next generation control strategies. However, all of these strategies require thorough knowledge of population abundances, mating behaviors and flight patterns of the males and females. These biological issues all in turn guide the decisions that need to be made about trap placement densities and locations, and mosquito release locations and numbers to achieve success.3. Determine degrees of polyandry and satyrization of Aedes aegypti and Aedes albopictus in California. Sterile insect and other kinds of genetic modification release control strategies require knowledge of male mating propensities and sperm usage by females. Degrees of multiple mating within Aedes aegypti and between Ae. aegypti and Aedes albopictus in areas where they sympatric and non-sympatric will be determined based on comparative microsatellite profiles of the sperm (within the spermathecae of the female) and from the females offspring.

Project Methods
Collection sites for mosquitoes for both objectives: Major mosquito disease vectors comprising species such as Aedes aegypti, Aedes albopictus, Aedes notoscriptus, Culex pipiens s.l. and Culex tarsalis will be collected from at least three locations each in Northern, Central and Southern California.Objective 1. Insecticide (pyrethroids and OPs) resistance/susceptibility profiles of F1 offspring from field collected mosquitoes will be determined in bottle bio-assays and Ultra Low Volume (ULV) sentinel cage trials. ULV trials will be performed in residential and agricultural environments where the mosquitoes commonly occur. Truck-mounted and aerial ULV trials will be evaluated with formulations of compounds registered for that environment and the bottles coated with technical grade material of the active ingredient of the corresponding formulation. The bottle bio-assays will be performed on the same F1 batch of mosquitoes used for the corresponding ULV trial. Kdr genotype and metabolite (of known resistance mediating proteins and enzymes- P450s and co-factors, esterases and GSTs) profiles will be determined and measured on the same F1 generations as insecticide resistance assays to determine correlations between the resistance mechanisms and knock down and mortalities in the bottle bio-assays and ULV trials. In ULV applications performed in natural environments, pre- and post-trapping will be conducted to ascertain effectiveness of applications on local wild mosquito populations.R program will be used for statistical analysis to determine strength of correlations between, bottle bio-assay, ULV sentinel trials, kdr genotypes and metabolic resistance mechanism data.Objective 2. We will collect Aedes aegypti throughout California where they have invaded have become overwintering established populations. Genome wide sequencing will be done at UC Davis using Hi-seq 400 instrumentation. Sequences will be mapped to the AaefL4 reference genome using BWA-MEM aligner. Freebayes will be used for variant calling. FST and nucleotide diversity will be computed using VCFtools. We will use likelihood-based inferences on isolation-by distance models for estimating dispersal implemented in Raddle. We will also apply the Bayesian MCMC based SpaceMix model to identify isolated pockets of Ae. aegypti populations suitable for future field trials aimed at eradicating this mosquito. We will also use the MSMC model on whole genome sequence data to calculate effective population sizes.Dispersal will also be determined using a direct Mark Release Recapture methodology and these results will be compared to the genetic sequencing approach. If the sequencing approach gives similar dispersal values then future estimates of dispersal can be done via genetics and not by resorting to more ethically controversial MRR studies.Objective 3. At least 60 gravid females of Ae. aegypti will be collected from Central (Fresno Clovis cities) and also from southern California (Los Angeles). Iso-female lines will be reared from each female. The parent mother's spermathecae will be dissected and the sperm content within will be extracted and prepared for DNA based analysis. The profiles of at least 9 microsatellites will be compared between sperm profile genomes of the sperm in the spermathecae of the parent mother and her offspring. Sperm microsatellite profiles will determine if she mated more than once and microstellite profiles of 30 of her offspring will reveal if her progeny was derived from sperm from more than one male. Ae. aegypti and Ae. albopictus genetic markers will be designed to establish if Ae. albopictus cross mate with Ae. aegypti and vice versa to determine satyrization effects where the populations of these two species are sympatric.Cage trials with combinations of numbers of male and female Ae. aegypti of known genetic marker profiles, will also be done to determine if California Ae. aegypti mate multiple times. If they do, based on the microsatellite profiles of the offspring sperm precedence will be determined.

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

Outputs
Target Audience:Public health personnel from Guam, Yap, Palau, Marshall Islands and Kosrae. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A document describing all aspects of arboviral vector surveillance, control and emergency response plan is being prepared for control of Dengue virus for all unincorporated territories of the US in Micronesia and neighboring Islands. How have the results been disseminated to communities of interest?Ongoing monthly Zoom meetings with personnel from Guam, Yap, Palau, Marshall Islands and Kosrae to develop the vector control plan document for these Islands. What do you plan to do during the next reporting period to accomplish the goals? Perform ULV insecticide trials in Clovis with new formulations of malathion - pending uplifting of Covid-19 restrictions. Perform RNA seq analysis of Aedes aegypti exposed to permethrin to determine potential novel resistance mechanisms. Conduct field trials to determine efficacy of a small device that attracts urban populations ovipositing Aedes aegypti to replace the currently deployed large more cumbersome trap. Determine efficacy of different concentrations of a novel synthetic oviposition compound that showed promise in antennal electrograms on Aedes aegypti. Conduct Vector control training workshop in Guam and Yap if travel is allowed.

Impacts
What was accomplished under these goals? Analysis of bottle bio-assay and urban ULV applications revealed that 1 hour knock down time points of mosquitoes exposed to deltamethrin, and permethrin correlated best with mortality in sentinel ULV cage trial exposures placed within 300 foot swaths from ULV application point in urban environs. Aedes aegypti from Los Angeles show some degree of correlation with knock down in bottle bio-assays but those from Fresno County do not. This is likely due to differences in the genetic background and pyrethroid resistance mechanisms between the two populations. Analysis of Mark-Release Recapture results performed on Aedes aegypti in Clovis was completed and manuscript is in preparation. Cage mating experiments between Aedes aegypti and Aedes albopictus were done. Genome analyses of sperm and offspring is ongoing.

Publications

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

Outputs
Target Audience:Insecticide resistance assay workshops were organized and attended by personnel from Mosquito Abatement Districts in 1) March 20th Sante Fe Springs, attended by five Southern California MADs, 2) May 1st and June 19th and August 21st Parlier, California, attended by 7 central California MADs, 3) April 3rd Phoenix, Arizona, attended by 3 MADs (lecture series), 4) April 4th Phoenix, Arizona, attended by 6 MADs (hands on training), 5) May 22nd Elk Grove, California, attended by 5 MADs (lecture and hands on training), June 13th- 14th Las Vegas, Nevada, attended by 1 MAD (lectures and hands on training), and August 15th Kona, Hawaii, attended by 2 MADs (lectures and hands on training). Evaluation of Field application of aerial insecticide applications were also done in collaboration with Consolidated and Sacramento Yolo MADs and an autodissemination In2 Care trap evaluation was done in collaboration with Hawaii (Big Island) public health personnel. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided 6 insecticide resistance training workshops for MAD personnel that were performed in California, Nevada, Arizona and Hawaii. How have the results been disseminated to communities of interest?1) Results were presented at Annual California Mosquito and Vector Control Association meeting in Sacramento (February 3-6th 2019) . Two presentations were given: a) Title: Synopsis of 2018 bottle bioassay and ULV sentinel cage trial data MAD annual conference (attended by 600 personnel from all MADs in California). b) Title: Insecticide resistance testing with a meaning "operationally." (Attended by 300 personnel from multiple California MADs.) 2) Presentation at Annual USA Society of Vector Ecology meeting in Puerto Rico (22-26th September 2019). Title: The link between bottle-bio-assay and truck mounted ULV sentinel cage trial data. (Approximately 250 attended.) 3) Presentation at annual Arizona Vector Conference, Maricopa, Arizona. Title: Insecticide Resistance & Bottle Bioassay 101: Why should Vector Control Agencies Care? (Approximately 100 attendees.) 4) Northern San Joaquin Continuing education seminar series, Stanislaus County Dept. of Agriculture, October 22 2019, Title: Aedes aegypti resistance in California. (Approximately 300 attended.) What do you plan to do during the next reporting period to accomplish the goals?1) Continue organizing and running insecticide resistance bio-assay training workshops. 2) Conduct ULV sentinel cage trial evaluations. 3) Examine methods of pyrethroid resistance in Aedes aegypti. 4) Still under development: Evaluate pyreproxyfen as a method of control in cemetery's on Big Island, Hawaii.

Impacts
What was accomplished under these goals? 1) Concomitant bottle bio-assay and Ultra-low volume aerial applications were performed to assemble data to ascertain relationship and predictability of resistance/susceptibilities of mosquitoes (Aedes aegypti, Culex quinquefasciatus and Culex tarsalis) between bottle bio-assay profiles and sentinel ULV field cage trials in urban and rice cultivated fields. 2) Conducted experiments to prove that kdr genotype alone does not correlate with knock down phenotype in bottle bio-assays in California Aedes aegypti populations. This proves that other pyrethroid resistance mechanisms must be responsible for conferring pyrethroid resistance in this mosquito. 3) An In2 Care trap auto-dissemination method for control of Aedes aegypti and Aedes albopictus in a village in Hawaii was performed. Preliminary data analysis indicates this method did not effectively control these mosquitoes in a field trial situation.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Brelsfoard, C. L, Mains, J. W., Mulligan, S., Cornel, A. J., Holeman, J., Kluh, S., Leal, A., Hribar, L., Morales,, H., Posey., and Dobson, S. L. Aedes aegypti males as vehicle for insecticide delivery. Insects, 10(8): E230.