Culex mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people, and separately, poses a conservation threat to insular bird species. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations, yet research in Culex has been lagging compared to other disease vectors, with only a few reports testing the functionality of CRISPR in these species. Here we use this tool to disrupt a set of five pigmentation genes in Culex quinquefasciatus that when altered, lead to visible, homozygous-viable phenotypes. We further validate our approach on two distinct strains of Culex quinquefasciatus that are relevant to potential future public health and bird conservation applications. Lastly, we generate a double-mutant line, demonstrating the possibility of combining multiple such mutations in a single individual. Our work provides a platform of five validated loci that could be used for targeted mutagenesis for research in Culex quinquefasciatus aimed at the development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily-scored transgenesis marker.
Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people, and separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations, yet research in Culex quinquefasciatus has been lagging compared to other disease vectors. Here we use this tool to disrupt a set of five pigmentation genes in Culex quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Culex quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Culex quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily-scored transgenesis marker.
BackgroundExtensive phenotypic plasticity in oysters makes them difficult to identify based on morphology alone, but their identities can be resolved by applying genetic and genomic technologies. In this study, we collected unknown oyster specimens from Hawaiian waters for genetic identification.MethodsWe sequenced two partial gene fragments, mitochondrial 16S ribosomal RNA (16S) and cytochrome c oxidase subunit I (COI), in 48 samples: 27 unidentified oyster specimens collected from two locations on O‘ahu, 13 known specimens from a hatchery in Hilo, Hawai‘i Island, and 8 known specimens from Hilo Bay, Hawai‘i Island.ResultsMolecular data identified approximately 85% of unknown samples as belonging to the Ostrea stentina/aupouria/equestris species complex, a globally distributed group with a history of uncertain and controversial taxonomic status. The remaining unknown samples were the native Dendostrea sandvichensis (G. B. Sowerby II, 1871), and nonnative Crassostrea gigas (Thunberg, 1793), the latter of which is a commercial species that was introduced to Hawai‘I from multiple sources during the 20th century. Phylogenetic analysis placed Hawai‘i Ostrea alongside samples from China, Japan, and New Zealand, grouping them within the recently classified western Pacific O. equestris. Until now, four extant species of true oyster have been documented in Hawai‘i. This study expands the known range of O. equestris by providing the first verification of its occurrence in Hawai‘i.
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