Photosynthesis of Natural Communities Dominated by Cladophora Glomerata and Ulothrix Zonata

Author Institution: The Franz Theodore Stone Institute of Hydrobiology, Pun-in-Bay, Ohi

Specific gene disruption in the major livestock pests cochliomyia hominivorax and lucilia cuprina using CRISPR/Cas9

Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host’s tissues, resulting in severe industry losses. As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, we report a significant addition to the pool of genome manipulation tools through the establishment of efficient CRISPR/Cas9 protocols for the generation of directed and inheritable modifications in the genome of these flies. Site-directed mutations were introduced in the C. hominivorax and L. cuprina yellow genes (ChY and LcY) producing lightly pigmented adults. High rates of somatic mosaicism were induced when embryos were injected with Cas9 ribonucleoprotein complexes (RNPs) pre-assembled with guide RNAs (sgRNAs) at high concentrations. Adult flies carrying disrupted yellow alleles lacked normal pigmentation (brown body phenotype) and efficiently transmitted the mutated alleles to the subsequent generation, allowing the rapid creation of homozygous strains for reverse genetics of candidate loci. We next used our established CRISPR protocol to disrupt the C. hominivorax transformer gene (Chtra). Surviving females carrying mutations in the Chtra locus developed mosaic phenotypes of transformed ovipositors with characteristics of male genitalia while exhibiting abnormal reproductive tissues. The CRISPR protocol described here is a significant improvement on the existing toolkit of molecular methods in calliphorids. Our results also suggest that Cas9-based systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests9930453055FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2017/05432-7We thank Marta Vargas, Rosangela Rodrigues and Yeiny Mudarra for their technical and administrative support. To Pamela Philips and John Welch for helpful discussions during the development of this project. We also thank the COPEG directors, Francisco Pinilla and Vanessa Dellis, for supporting our study at the COPEG and USDA-ARS laboratories inside the biosecurity plant in Pacora, Panama. We are very grateful for the exhaustive laboratory assistance from Nicolas Mendoza, Domitildo Martinez, Rosaura Sanchez, Hermogenes Gonzalez and Amilcar Miranda, at the COPEG and USDA-ARS laboratories and Amy Berger and Scott Harrison at NCSU. We are also thankful to Ana Junqueira for her comments on the FAPESP grant proposal and for providing the draft assembly of the C. hominivorax genome used for sgRNA design against ChY gene, Matthew Bertone for photographs of L. cuprina strains and to the three anonymous referees for their comments and suggestions that improved the final version of our manuscript. This project was supported by a grant from the São Paulo Research Foundation (FAPESP: 2017/05432-7, given to D.F.P), USDA-ARS agreement no. 58-3094-7-015-FN, ARSCOPEG agreement no. 58-6205-4-002-F, and COPEG (grant to M.J.S). D.F.P was also supported by a STRI Short Term Fellowship (project award #4168). USDA is an equal opportunity employer and provide