Genetic manipulation is a powerful technique for addressing research questions in arthropods of medical importance. Current approaches rely upon delivering DNA or endonucleases to preblastoderm embryos via embryonic microinjection. However, embryonic microinjection is technically challenging, is limited to a small number of arthropod taxa, and is inefficient even in optimized species. As such, there is a critical need to develop methods for arthropod genetic manipulation that are simple, accessible for many researchers and generally compatible for a large variety of arthropod species. During oogenesis, insects transfer yolk protein precursors to developing oocytes by receptor-mediated endocytosis (RME). When Drosophila melanogaster yolk protein 1 (DmYP1) is injected into pre-vitellogenic adult female Anopheles mosquitoes, it is transduced into the germline by RME. We show that DmYP1 can be used to transduce cargo such as protein or DNA with 100% efficiency to the developing Anopheles germline. We term this technique "Receptor-Mediated Ovary Transduction of Cargo, or "ReMOT Control", which we hypothesize can be used to transduce cargo into the Anopheles germline for stable and heritable editing of the mosquito chromosomal genetic sequence. This hypothesis will be investigated by two specific Aims.
The first aim i s to use ReMOT Control to specifically delete genes in the germline of Anopheles stephensi. DmYP1 will be fused to transcription activator-like effector nucleases (TALENs) targeting GFP. After injection into GFP-transgenic mosquitoes, deletion lines will be identified by loss of fluorescence and sequencing.
The second aim i s to use ReMOT Control to create transgenic Anopheles stephensi by transduction of transposable elements into the mosquito germline. DmYP1 will be used to transduce GFP-containing transposons into transgenic transposase-expressing mosquitoes or co-injected with DmYP1-transposase fusion enzymes. Transgenic lines will be identified by GFP gain-of- function;insertion sites will be identified by PCR, sequencing and southern blot. Once optimized, ReMOT Control will dramatically change the landscape of molecular entomology research, allowing easy, flexible genetic manipulation of a wide variety of vector arthropods and non-model species.

Public Health Relevance

Human malaria, transmitted by Anopheles mosquitoes, is the most important vector- borne disease in the world. Genetic manipulation of Anopheles mosquitoes is a critical part of research in this important vector genus, but techniques for genetic manipulation are very difficult and inefficient. In this research we will develop a novel, easy and efficient technique, called ReMOT Control, for genetic manipulation of Anopheles mosquitoes.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Vector Biology Study Section (VB)
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Costero, Adriana
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Pennsylvania State University
Schools of Earth Sciences/Natur
University Park
United States
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Hughes, Grant L; Dodson, Brittany L; Johnson, Rebecca M et al. (2014) Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes. Proc Natl Acad Sci U S A 111:12498-503
Provost-Javier, K N; Rasgon, J L (2014) 20-hydroxyecdysone mediates non-canonical regulation of mosquito vitellogenins through alternative splicing. Insect Mol Biol 23:407-16
Ren, Xiaoxia; Hughes, Grant L; Niu, Guodong et al. (2014) Anopheles gambiae densovirus (AgDNV) has negligible effects on adult survival and transcriptome of its mosquito host. PeerJ 2:e584
Suzuki, Yasutsugu; Niu, Guodong; Hughes, Grant L et al. (2014) A viral over-expression system for the major malaria mosquito Anopheles gambiae. Sci Rep 4:5127
Dodson, Brittany L; Hughes, Grant L; Paul, Oluwatobi et al. (2014) Wolbachia enhances West Nile virus (WNV) infection in the mosquito Culex tarsalis. PLoS Negl Trop Dis 8:e2965
Hughes, G L; Samuels, S K; Shaikh, K et al. (2014) Discrimination of the Plasmodium mexicanum vectors Lutzomyia stewarti and Lutzomyia vexator by a PCR-RFLP assay and Wolbachia infection. J Vector Ecol 39:224-7