BITE-SIZED DIVERSITY: CATALOGING CULICOIDES AND DETERMINING HOST ASSOCIATIONS

<b>Project Methods</b><br> Objective One: For DNA extraction,blood engorged Culicoidesare identified andplaced in a 1.5 mL tube with lysis buffer and proteinase labelled with the collection code, collection date, and species identification. Genomic DNA is extracted using DNeasy blood and tissue kit, and extracts are stored at -20°C until processing. Molecular identification uses universal vertebrate (mammals and birds) primers (COI_short_F/COI_short_R and L14841/H15149) for PCR to amplify the COI and cytb genes, respectively. Negative controls (ddH2O) will be included with each PCR run to monitor contamination. If COI amplification fails, cytb will be used instead. To confirm amplification, PCR products will be loaded on a 2% agarose gel stained with SYBR Safe dye. PCR products will then be purified using GeneJET kit, and purified products will be sent for bi-directional Sanger sequencing at Eurofins Genomics. Resulting DNA sequences will be edited and aligned to current reference sequences in GenBank and BOLD using Geneious bioinformatics software. A threshold of >98% host identity is often used to confirm accuracy of host identification, but vertebrate sequences that do not meet this will be identified with the highest matching genus. Results of this portion of the study will be presented at the Entomological Society of America (ESA) and Livestock Insect Workers Conference (LIWC) meetings and submitted for publication in open access, peer-reviewed journals.Objective Two:Nulliparous females and males representing each species collected will be selected as voucher specimens. DNA will be extracted using HotSHOT, a whole body, non-destructive protocol to preserve morphological characteristics for slide-mounting specimens. The final preparation, the extracted DNA, is used for PCR amplification, and the head, wings, legs, and midge body will be slide-mounted. Since Culicoides are so small (1-3 mm) they are mounted on glass microscope slides. Midge bodies will be cleared with warm potassium hydroxide and ethanol, and slide mounted using Permount. For molecular identification, a 658-bp fragment of cytochrome c oxidase subunit 1 (COI) will be amplified using conventional PCR and products will be visualized on a gel electrophoresis. Primers that will be used are LCO1490 and HC02198. The samples will be processed and analyzed in the same manner as mammalian/avian blood meal samples, and sequences will be submitted to GenBank and BOLD. Along with the morphological identification, each slide will have an accession number, which is a unique identifier given to the associated DNA sequences that is linked to database such as GenBank and BOLD, that future researchers can use to compare DNA sequences to the voucher specimens. Voucher specimens will be labeled per accepted curatorial standards.Results will be disseminated in the same manner as objective one.Expected Results: I expect to delineate host-vector relationships based on blood meal analysis. I have collected ~100 blood engorged females so far, and expect to collect more during the 2024 trapping season. Additionally, I will deposit ~15-20 species to the University of Arkansas Insect Museum for future use, based on the number of species I have identified thus far in Arkansas. I will update current genetic references on GenBank and BOLD databases, and add morphologically and molecularly identified voucher specimens.