Developing a FISH-based Method for Inversion Diagnostics in Culex pipiens Mosquitoes

This project developed an improved protocol for the dissections of imaginal discs from 4th instar Culicine larvae. The design of the protocol and use of visuals have now allowed dissections to be more accessible and streamline the learning process for undergraduate students or new lab members. The new protocol is important for instructional use in laboratories and classrooms with the aim of studying mitotic chromosomes isolated from mosquito larvae. For this project, probes were designed for diagnostics for the Chromosome 1 inversion 1p3” and the Chromosome 2 inversion 2q1 for the field-collected strains Cx. p. pipiens Northfield strain, Cx. p. molsestus Chicago strain, and the genomic standard JHB strain of Cx. quinquefasciatus. Primers were developed and used in PCR and produced products ranging between 3,000 and 4,500kb that are suitable for subsequent labeling by Nick Translation method and visualization of the status of inversions using Fluorescent in situ Hybridization (FISH). Lastly, a FISH-based approach for the detection of the chromosome 1 inversion, 1p3” was optimized for the mitotic chromosomes from the imaginal discs for the standard genomic strain JHB of Cx. quinquefasciatus. Two probes produced clear signals indicating a standard arrangement for this inversion in the chromosome 1 of this strain. This shows that probes developed for FISH-based inversion diagnostics using mitotic chromosomes have the potential for further method development.