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-Saint Denis, Adriana
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Pennsylvania State University
Schools of Earth Sciences/Natur
University Park
United States
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Chaverra-Rodriguez, Duverney; Macias, Vanessa M; Hughes, Grant L et al. (2018) Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing. Nat Commun 9:3008
Tsujimoto, Hitoshi; Sakamoto, Joyce M; Rasgon, Jason L (2017) Functional characterization of Aquaporin-like genes in the human bed bug Cimex lectularius. Sci Rep 7:3214
Barik, Tapan K; Suzuki, Yasutsugu; Rasgon, Jason L (2016) Factors influencing infection and transmission of Anopheles gambiae densovirus (AgDNV) in mosquitoes. PeerJ 4:e2691
Raygoza Garay, Juan Antonio; Hughes, Grant L; Koundal, Vikas et al. (2016) Genome Sequence of Elizabethkingia anophelis Strain EaAs1, Isolated from the Asian Malaria Mosquito Anopheles stephensi. Genome Announc 4:
Henning, Tyler C; Orr, John M; Smith, Joshua D et al. (2016) Discovery of filarial nematode DNA in Amblyomma americanum in Northern Virginia. Ticks Tick Borne Dis 7:315-8
Liu, Kun; Tsujimoto, Hitoshi; Huang, Yuzheng et al. (2016) Aquaglyceroporin function in the malaria mosquito Anopheles gambiae. Biol Cell 108:294-305
Hughes, Grant L; Raygoza Garay, Juan Antonio; Koundal, Vikas et al. (2016) Genome Sequence of Stenotrophomonas maltophilia Strain SmAs1, Isolated From the Asian Malaria Mosquito Anopheles stephensi. Genome Announc 4:
Sakamoto, Joyce M; Ng, Terry Fei Fan; Suzuki, Yasutsugu et al. (2016) Bunyaviruses are common in male and female Ixodes scapularis ticks in central Pennsylvania. PeerJ 4:e2324
Hughes, Grant L; Raygoza Garay, Juan Antonio; Koundal, Vikas et al. (2016) Genome Sequences of Staphylococcus hominis Strains ShAs1, ShAs2, and ShAs3, Isolated from the Asian Malaria Mosquito Anopheles stephensi. Genome Announc 4:
Hegde, Shivanand; Rasgon, Jason L; Hughes, Grant L (2015) The microbiome modulates arbovirus transmission in mosquitoes. Curr Opin Virol 15:97-102

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