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CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say.

ABSTRACT: Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.

SUBMITTER: Purusothaman DK

PROVIDER: S-EPMC8298393 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: The natural history and potential impact of mosquito-specific flaviviruses on the transmission efficiency of West Nile virus (WNV) is unknown. The objective of this study was to determine whether or not prior infection with Culex flavivirus (CxFV) Izabal altered the vector competence of Cx. quinquefasciatus Say for transmission of a co-circulating strain of West Nile virus (WNV) from Guatemala. METHODS AND FINDINGS: CxFV-negative Culex quinquefasciatus and those infected with CxFV Izabal by intrathoracic inoculation were administered WNV-infectious blood meals. Infection, dissemination, and transmission of WNV were measured by plaque titration on Vero cells of individual mosquito bodies, legs, or saliva, respectively, two weeks following WNV exposure. Additional groups of Cx. quinquefasciatus were intrathoracically inoculated with WNV alone or WNV+CxFV Izabal simultaneously, and saliva collected nine days post inoculation. Growth of WNV in Aedes albopictus C6/36 cells or Cx. quinquefasciatus was not inhibited by prior infection with CxFV Izabal. There was no significant difference in the vector competence of Cx. quinquefasciatus for WNV between mosquitoes uninfected or infected with CxFV Izabal across multiple WNV blood meal titers and two colonies of Cx. quinquefasciatus (p>0.05). However, significantly more Cx. quinquefasciatus from Honduras that were co-inoculated simultaneously with both viruses transmitted WNV than those inoculated with WNV alone (p = 0.0014). Co-inoculated mosquitoes that transmitted WNV also contained CxFV in their saliva, whereas mosquitoes inoculated with CxFV alone did not contain virus in their saliva. CONCLUSIONS: In the sequential infection experiments, prior infection with CxFV Izabal had no significant impact on WNV replication, infection, dissemination, or transmission by Cx. quinquefasciatus, however WNV transmission was enhanced in the Honduras colony when mosquitoes were inoculated simultaneously with both viruses.

Project description:Understanding mechanisms that contribute to viral dissemination in mosquito vectors will contribute to our ability to interfere with the transmission of viral pathogens that impact public health. The expression of genes in two Culex pipiens quinquefasciatus populations from Florida with known differences in vector competence to West Nile virus (WNV) were compared using high throughput sequencing.A total of 15,176 transcripts were combined for comparison of expression differences between the two populations and 118 transcripts were differentially expressed (p < 0.05). The fold change in expression of the differentially expressed genes ranged from -7.5 - 6.13. The more competent population for WNV (Gainesville) over expressed 77 genes and down regulated 44 genes, compared with the less competent population for WNV (Vero Beach). Also, splicing analysis identified 3 transcripts with significantly different splice forms between the two populations. The functional analysis showed that the largest proportion of transcripts was included in the catalytic activity and transporter activity groups except for those in the unknown group. Interestingly, the up- regulated gene set contained most of the catalytic activity function and the down- regulated gene set had a notable proportion of transcripts with transporter activity function. Immune response category was shown in only the down regulated gene set, although those represent a relatively small portion of the function. Several different vitellogenin genes were expressed differentially. Based on the RNAseq data analysis, ovary development was compared across the populations and following WNV infection. There were significant differences among the compared groups.This study suggests that ovary development is correlated to vector competence in two Culex populations in Florida. Both populations control energy allocations to reproduction as a response to WNV. This result provides novel insight into the defense mechanism used by Culex spp. mosquitoes against WNV.

Project description:This study examined pyrethroid resistance intensity and mechanisms in Culex quinquefasciatus (Say) (Diptera: Culicidae) populations from Jigawa, North-West Nigeria. Resistance statuses to permethrin, lambda-cyhalothrin and alphacypermethrin were determined with both WHO and CDC resistance bioassays. Synergist assay was conducted by pre-exposing the populations to Piperonyl butoxide (PBO) using the WHO method. Resistance intensities to 2x, 5x and 10x of diagnostic concentrations were determined with the CDC bottle method. Species analysis and presence of knockdown mutation (Leu-Phe) were done using Polymerase Chain Reaction (PCR). Results showed that Cx. quinquefasciatus was the only Culex spp. present and "Kdr-west" mutation was not detected in all analyzed samples. Using WHO method, Cx. quinquefasciatus resistance to permethrin was detected in Dutse (12.2%) and Kafin-Hausa (77.78%). Lambda-cyhalothrin resistance was recorded only in Kafin-Hausa (83.95%) with resistance suspected in Ringim (90%). Resistance to alphacypermethrin was recorded in all locations. Pre-exposure to PBO led to 100% mortality to alphacypermethrin and lambda-cyhalothrin in Ringim while mortality to permethrin and alphacypermethrin in Dutse increased from 12.2% to 97.5% and 64.37% to 79.52% respectively. Using CDC bottle bioassay, resistance was also recorded in all populations and the result shows a significant positive correlation (R2 = 0.728, p = 0.026) with the result from the WHO bioassay. Results of resistance intensity revealed a very high level of resistance in Kafin-Hausa with susceptibility to lambda-cyhalothrin and alphacypermethrin not achieved at 10x of diagnostic doses. Resistance intensity was also high in Dutse with susceptibility to all insecticides not achieved at 5x of diagnostic doses. Widespread and high intensity of resistance in Cx. quinquefasciatus from North-West Nigeria is a major threat to the control of diseases transmitted by Culex and other mosquito species. It is a challenge that needs to be adequately addressed so as to prevent the failure of pyrethroid-based vector control tools.

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CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say.

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