Development of Molecular Tools for Lepidopteran Functional Genomics in the Plodia Pantry Moth
Open Access DepositedHaving been solely considered as a worldwide pest of stored food products, the pantry moth Plodia interpunctella is gaining traction as a promising laboratory model system for the study of lepidopteran biology. Its biological practicality for transformation via microinjections and the recent availability of a reliable annotated genome assembly have lent itself indispensable for potential functional genetic studies in Lepidoptera. This dissertation is aimed at (1) developing common molecular manipulations in Plodia, such as mutagenesis and transgenesis, (2) reporting their practical efficiency, (3) generating Plodia lines for such techniques, and (4) outlining Plodia rearing procedure.Leveraging the simple phenotype generated by the white pigmentation gene, the optimization of genome editing in Plodia was initiated through the characterization of a 1 bp deletion that causes a coding frameshift. Loss-of-function mutagenesis via CRISPR Non-Homologous End Joining (NHEJ) replicated the spontaneous mutant phenotype, while the complementary CRISPR Homology-Directed Repair (HDR) rescued this mutation back to a wild-type state. In both cases, the CRISPR/Cas9 somatic and germline transformation efficiency were also evaluated. Additionally, the white co-CRISPR strategy was tested along with several developmental genes to explore its reliability as a parental selection marker for streamlining the numbers of crosses and PCR screening of first-generation offspring. Owing to the translucency of eye structures found in Plodia white mutants, random transgenesis mediated by Hyperactive piggyBac transposase (HyPBase) successfully visualized the somatic and germline transformations of EGFP, EYFP, and DsRed fluorescent reporters. This effort also generated a Plodia transgenic line carrying EYFP eye marker and an attP site that was later used as a target for site-specific integration via PhiC31 integrase. Finally, to study the pathway activation in development and immune challenge in Lepidoptera, random transgenesis was also used in an attempt to generate a JAK/STAT reporter in Plodia white mutant, although more optimization or strategy change is required for visualizing the Stat92E activation. With this dissertation, I propose: (1) translucent eye mutation such as white eye is an invaluable asset for early development of molecular methods, such as mutagenesis and transgenesis; (2) CRISPR NHEJ and HDR in Plodia interpunctella provides excellent workflow for routine functional genetics in Lepidoptera; and (3) prioritizing the survival of embryo through careful experimental design and selection of fluorescent markers are crucial in transgenesis.
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