INVESTIGATION OF NEW TARGETS FOR REPRODUCTIVE CONTROL OF MOSQUITO VECTORS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0213026
• Project No.: NYC-139437
• Project Start Date: Oct 1, 2007
• Project End Date: Sep 30, 2010

Project Director
Harrington, L. C.

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
ENTOMOLOGY

Non Technical Summary
There is a critical need to develop new targets for intervention and control of mosquito-borne diseases. Yet, little is known about mosquito reproductive biology and molecules regulating its reproductive physiology and behavior are not well understood. Our research will provide tools to build upon a new approach for controlling mosquito vectors through manipulation of their reproductive biology. In addition, our results will also provide novel information on major mosquito vectors in the genus Aedes that are vectors of diseases that pose a potential threat to citizens of New York State. Our proposed research will provide the groundwork leading to novel and highly applicable approaches for mosquito control that could have significant impacts of human health and well being in future years. This study is consistent with CSREES goal of promoting a healthy population and FFF priority of advancing healthy lifestyles. We hope completion of the work outlined in this proposal will provide the foundation for a larger scale proposal to NIH or NSF.

Animal Health Component

(N/A)

Research Effort Categories

Basic

50%

Applied

(N/A)

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
721	6099	1080	50%
721	6099	1130	50%

Knowledge Area
721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
6099 - People and communities, general/other;

Field Of Science
1080 - Genetics; 1130 - Entomology and acarology;

Keywords

mosquito

west nile virus

dengue fever

control

novel targets

Goals / Objectives
We propose to identify key reproductive genes in mosquitoes, by using our expertise and gene sets developed with the model-insect Drosophila. We will then select candidate reproductive molecules for use in the development of mosquito control strategies.

Project Methods
Our approach will involve four steps: 1) Identify key reproductive proteins (Accessory gland proteins=ACPs) produced by male Ae. aegypti. Acps made in insect male accessory glands (MAG) have been shown to be transferred to females during mating and to affect female subsequent reproductive capacity and behavior. This is particularly well known in Drosophila (a Dipteran, like mosquitoes), but has never been conclusively tested in mosquitoes. We will use Drosophila Acp gene sequences, and proteomics techniques that have been successful with Drosophila to identify putative Acps in Ae. aegypti; 2) verify Ae. aegypti Acp enriched MAG-predominant gene expression, using RT-PCR and, where possible, protein-based methods; 3) Identify the Ae. albopictus orthologs of the Ae. aegypti Acps; 4) Identify candidate Acps for transgenic and/or antibody study, and initiate experiments to produce a GFP-tagged Acp for use as a marker for mosquito mating for field studies. 1. Identify Ae. aegypti Acps. A. Identification of orthologs. We will search the Ae. aegypti genome for orthologs of D. melanogaster Acp genes using BLAST searches. Orthologs identified in this manner will be BLASTed against Ae. albopictus and Anopheles gambiae. B. Mass Spectrometry (MS). Male AG proteins will be separated on 1-D gels and subjected to nanoLC-MS/MS. Peptide sequences will be compared to the Ae. aegypti predicted protein database and Genbank to identify Acp genes. 2. Verify Acps AG-enriched expression. Acp genes identified by BLAST searches and MS will be checked for AG-enriched expression using RT-PCR of RNA samples from male AGs and somatic tissue, and whole females. 3. Conservation in other mosquito species. Using BLAST analysis, we will determine which Acps identified from Ae. aegypti are present in Ae. albopictus and An. gambiae. RT-PCR with species-specific or degenerate primers will be performed to test AG-enriched expression for these genes. 4A. Initiate analyses of candidate Acps for transgenic and/or antibody study. We will generate antibodies to five Acps of interest and use them for Western blotting to test for expression, transfer, and target tissues of the Acps in mated females. We will use RNA interference to generate male mosquitoes deficient in given Acps. Bioassays will be conducted to assess female behavior and reproductive capacity after mating with knockdown males. B. GFP marker for studies of mating. A GFP marker for assessing mating frequency and female choice in the large field cage setting will allow reliable assessment of the potential of sterile or modified males. We will identify an AG-specific promoter and use it to drive a seminal protein tagged with GFP (or, RFP if better for visualization). Females who have mated will receive this protein and thus fluoresce.

Progress 10/01/07 to 09/30/10

Outputs
OUTPUTS: Our goal for this project was to identify key reproductive genes in mosquitoes, by using our expertise and gene sets developed with the model-insect Drosophila. Among these activities, we selected candidate reproductive molecules for use in the development of mosquito control strategies. Another major activity was the training of two postdoctoral researchers and one graduate student on the new techniques required to study seminal fluid proteins in mosquitoes. In addition, we established a collaboration with Dr. Jose Ribiero at the NIH to identify male-derived small peptides. A major product of this project was the development of stable-isotope (15N) labeling to distinguish between male- and female-derived proteins in the reproductive tracts of freshly-mated females. This method was first described for use in Drosophila by Findlay et al. (2008), but has never been applied to mosquitoes. We developed a method for labeling all female-derived proteins with 15N by feeding larvae 15N -labeled yeast. In addition, we developed a method to label bacteria used as a nutrient substrate for larvae, by using inoculums from previous cohorts of mosquitoes reared on 15N-labeled yeast. After adult labeled females became reproductively mature, they were mated to unlabeled males. Female reproductive organs were dissected immediately after mating. Protein samples were separated on a gel and identified using mass spectrometry. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera. Two of these candidate proteins are currently under further investigation for functional analysis using transient knockdown in mosquitoes. A major manuscript reporting this work is in press in the PLos Neglected Tropical Diseases. We also disseminated our results to on this project with 3 presentations at the 2010 American Society for Tropical Medicine and Hygiene meetings. PARTICIPANTS: Two postdoctoral researchers and one graduate student received training on this project. In particular, they learned new techniques required to study seminal fluid proteins in mosquitoes. In addition, we established a collaboration with Dr. Jose Ribiero at the National Institutes of Health to identify male-derived small peptides. TARGET AUDIENCES: Out target audiences for this project included other scientists working to develop novel approaches for mosquito control. Mosquito abatement professionals and citizens of New York State. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our research has resulted in a change in knowledge about the role of male mosquitoes in female biology and fitness. Our study is the first to address the reproductive molecules regulating mosquito reproductive physiology and behavior. We have now identified a large number of proteins that are transferred from males to females and are studying the detailed function of two candidate proteins from this pool. Our research has provided new tools to build upon a new approach for controlling mosquito vectors through manipulation of their reproductive biology. In addition, our results have provided novel information on major mosquito vectors in the genus Aedes that is applicable to vectors of diseases such as the Asian Tiger mosquito (Aedes albopictus) that pose a potential threat to citizens of New York State.

Publications

• Sirot LK, Hardstone MC, Helinksi MEH, Marinotti O, Kimura M, Deewatthanawong P, Wolfner MF and LC Harrington. 2011(In press). Towards an ejaculatome of the dengue vector mosquito: Protein identification and potential functions. PLoS Neglected Tropical Diseases.
• Seminal fluid protein identification and potential functions in the dengue vector, Ae. aegypti. 2010. Laura C. Harrington, Laura K. Sirot, Melissa C. Hardstone, Michelle E. Helinski, Mari Kimura, Prasit Deewathanawong, Mariana F. Wolfner 83(5): 225.
• Multiple mating in Ae. aegypti: sperm transfer and usage patterns. 2010. Prasit Deewatthanawong, Laura C. Harrington 83(5): 183.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Our goal for this project was to identify key reproductive genes in mosquitoes, by using our expertise and gene sets developed with the model-insect Drosophila. Among these activities, we selected candidate reproductive molecules for use in the development of mosquito control strategies. Another major activity was the training of two postdoctoral researchers and one graduate student on the new techniques required to study seminal fluid proteins in mosquitoes. A major product of this project was the development of stable-isotope (15N) labeling to distinguish between male- and female-derived proteins in the reproductive tracts of freshly-mated females. This method was first described for use in Drosophila by Findlay et al. (2008), but has never been applied to mosquitoes. We developed a method for labelling all female-derived proteins with 15N by feeding larvae 15N -labeled yeast. In addition, we developed a method to label bacteria used as a nutrient substrate for larvae, by using inoculums from previous cohorts of mosquitoes reared on 15N-labeled yeast. After adult labeled females became reproductively mature, they were mated to unlabeled males. Female reproductive organs were dissected immediately after mating. Protein samples were separated on a gel and identified using mass spectrometry. In two independent replicates, we identified ninety-seven proteins with this approach. We know that 71 of these proteins are found in the male reproductive accessory glands. These proteins fall into a range of interesting classes that are likely to have major effects on male and female mosquito biology including fibrinogen/ fibronectins, vitellogenins, lipases, proteases, venom allergens, lectins and protease inhibitors. Two of these candidate proteins are currently under further investigation for functional analysis using transient knockdown in mosquitoes. One additional product of this investigation was the establishment of a collaboration with Dr. Jose Ribiero at the NIH to identify male-derived small peptides. We disseminated our results to date on this project at a variety of conferences and workshops including a presentation by Sirot entitled "Identification of seminal fluid proteins in the mosquito, Aedes aegypti" as part of the Post-Doctoral Research Day at Cornell University. This talk received the "Best Presentation" Award. Our research also was presented at the Aedes/Dengue Workshop, North Carolina State University, Raleigh, NC in 2008 and Society for Integrative and Comparative Biology, San Antonio, TX in 2008. Two additional presentations of our results were made at the 2008 and 2009 American Society for Tropical Medicine and Hygiene meetings. In addition, one presentation was made to the reproductive physiology group at Cornell University. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our research has resulted in a change in knowledge about the role of male mosquitoes in female biology and fitness. Our study is the first to address the reproductive molecules regulating mosquito reproductive physiology and behavior. We have now identified a large number of proteins that are transferred from males to females and are studying the detailed function of two candidate proteins from this pool. Our research has provided new tools to build upon a new approach for controlling mosquito vectors through manipulation of their reproductive biology. In addition, our results have provided novel information on major mosquito vectors in the genus Aedes that is applicable to vectors of diseases such as the Asian Tiger mosquito (Aedes albopictus) that pose a potential threat to citizens of New York State.

Publications

• Helinski, M.E.H., Sirot, L.K., Wolfner,M.F. and L.C. Harrington. 2009. The effect of male mating history on female fecundity and longevity in the dengue vector Aedes aegypti. American Journal of Tropical Medicine and Hygiene. 79(6):293.
• Harrington,L.C., Connors, K.J., Cator, L.J. and M. E. Helinski. 2009. Assortative mating in the dengue vector mosquito, Aedes aegypti. American Journal of Tropical Medicine and Hygiene. 79(6):293.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: We have made significant progress over the past year. We have identified 63 new male reproductive proteins to bring our total number to 126 for Ae. aeygpti. We have also evaluated expression in male and female mosquitoes. Outputs include several publications on our work as well as new collaborations with scientists at NIH regarding bioinformatic tools. In addition to publications, Harrington and Sirot presented research related to this project at several meetings in 2008. PARTICIPANTS: Professor Mariana Wolfner and postdoctoral associate Laura Sirot collaborated with Harrington on the project. In addition, we collaborated with Sheng Zhang, Director of Proteomics and Mass Spectrometry Core Facility at Cornell, to optimize identification of seminal proteins and peptides. Several graduate students in Harrington's lab helped with the project as well as undergraduates. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have identified additional proteins that are transferred from males to females during mating. In addition we have developed a method to reverse-label male reproductive proteins using stable isotopes. Using this approach we can differentiate male from female derived proteins. We have also developed methods for microinjection that will be employed during the coming year of the project.

Publications

• Cator, LJ, Arthur, BJ, Harrington, LC and RR Hoy. 2009 In press. Mosquito hearing reaches new heights: acoustic communication by the dengue vector. Science.
• Sirot, L.K. Poulson, R.L, McKenna, M.C., Girnary, H., Wolfner, M.F. and L.C.Harrington. 2008. Identity and transfer of male reproductive gland proteins of the dengue vector mosquito, Aedes aegypti: potential tools for control of female feeding and reproduction. Insect. Biochem. Mol. Biol. Volume 38 (2):176-189.
• Ponlawat, A., and L.C.Harrington. 2008. Factors associated with male mating success of the dengue vector mosquito, Aedes aegypti. Amer. Soc. Trop. Med. Hyg. 79(3):312-8.