https://doi.org/10.54718/UGNO761

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Theses and Dissertations

Issuing Body

Mississippi State University

Advisor

King, Jonas

Committee Member

Brown, Ashli

Committee Member

Goddard, Jerome

Committee Member

Popescu, Sorina

Committee Member

Krishnan, Natraj

Date of Degree

12-10-2021

Document Type

Graduate Thesis - Campus Access Only

Major

Molecular Biology

Degree Name

Doctor of Philosophy (Ph.D)

College

College of Agriculture and Life Sciences

Department

College of Agriculture and Life Sciences

Abstract

Hematophagy, or blood-feeding behavior, has independently evolved multiple times in the class Insecta and results in the transmission of vector-borne parasites, bacteria and viruses causing 17% of all infectious diseases and over 700,000 human deaths annually. One of the major challenges in studying the discrete mechanisms involved in host-response to hematophagy or the host seeking behavior of hematophagous insects, is the vast amount of signaling processes and genes that are inextricably tied to these interactions. These experiments used the clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated (Cas) and dsRNA knock-down technologies to examine mechanisms of immunological reactivity and host-seeking behavior. Using Illumina next-generation sequencing, 19,269 unique genes were found to be expressed in the salivary glands of C. lectularius. Of those genes, a total of 10,587 were differentially expressed. There were 6 genes definitively characterized as nitrophorin or nitrophorin-like, which were targeted for RNAi experiments. Silencing two nitrophorin genes, LOC106662976 and LOC106662977, which were the most highly expressed, did not result in any significant changes in feeding behavior or dermal reaction in the host. While RNAi-based knock-down experiments can gauge a proteins function, CRISPR/Cas-based knock-out experiments more definitively assess functionality. The second part of the study is thus focused on performing the first homology directed repair knock-in in Ae. albopictus using a plasmid, Cas9 protein and sgRNA targeting the key olfactory receptor gene, AealbOR4. An injection plasmid was constructed targeting AealbOR4 using standard molecular methods. Preliminary injections of the plasmid yielded no viable embryos. To determine if it is the plasmid or injection components that are lethal to developing Ae. albopictus embryos, further studies are required. Despite this, the development of this technique is a first step to be able to effectively interrogate gene function of an olfactory receptor and its role in host-seeking behavior. Taken together, these studies indicate that both RNAi and CRISPR/Cas-based gene editing technologies can effectively be utilized to answer some of the intricate questions related to insect-host interactions in the future.

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