Location: Tropical Pest Genetics and Molecular Biology Research Unit
Project Number: 2040-30400-003-013-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jun 15, 2026
End Date: Dec 31, 2027
Objective:
Goal 1: Full characterization of the regulation and expression of adult medfly and mexfly odorant receptor systems, both in developmental time and space.
Objectives:
1a) Collect and freeze adult flies and tissues for functional genomic analysis
1b) Process samples for Kinnex IsoSeq full length isoform sequencing and FiberSeq nucleosome regulatory network profiling.
1c) Analyze data to generate novel gene and isoform models for each chemosensory associated gene and associate with regulatory regions driving the expression at specific timepoints, sex and developmental stages.
1d) Identify a list of putative chemosensory associated genes for targeted gene knockout in year 2 and protein structural prediction in year 3 of future suggestions for identification of novel compounds for lures and attractants (if funded).
Approach:
1a) Collect and freeze adult flies and tissues for functional genomic analysis
At USDA-ARS Hilo, we have a constant supply of wild type medfly, and through collaboration with the Guatemala Petapa facility, we will also have access to wild type Mexican fruit fly. All samples will be collected and shipped cryogenically (liquid nitrogen dry shipper) to USDA-Hilo for parallel processing with medfly samples under the same conditions.
Approach: Male and female adult medfly and mexfly will be collected simultaneously from the same collection, either as wild type line at USDA Hilo for medfly or MOSCAMED Petapa for mexfly. Six replicate samples will be collected for each timepoint, each representing a pool of 10 individuals. Virgin adults will be collected as newly emerged (1-2 day old), developing (5-6 day old), and mature (14 day old). In addition, mated adults will be collected from 14-21 day old adults which have mated and egged.
1b) Process samples for Kinnex IsoSeq full length isoform sequencing and FiberSeq nucleosome regulatory network profiling.
For each replicate of individuals, heads will be separated from the rest of the body and processed for synchronized nucleosome regulatory profiling (FiberSeq) from nuclei preps of the samples, and full-length isoform sequencing (Kinnex IsoSeq) from polyadenylated cDNA derived from total RNA from each sample. Sequencing of subsequent libraries will be performed on PacBio Revio platform, targeting 10-20M full length isoform reads for each sample, and approximately 50-100X coverage of WGS FiberSeq for each sample on a Revio 8M sequencing cell. Approximately 12-15 Kinnex IsoSeq samples can be pooled per run and approximately 3-4 FiberSeq samples per run assuming run outputs previously obtained on our sequencing platform in-house.
1c) Analyze data to generate novel gene and isoform models for each chemosensory associated gene and associate with regulatory regions driving the expression at specific timepoints, sex and developmental stages.
Resulting data will be analyzed using the IsoSeq3 pipeline for the Kinnex IsoSeq data and the FiberTools workflow for the FiberSeq data. High quality reference genomes already exist for both medfly and mexfly, and these will be used to map and process the data. An in-house UCSC genome browser instance will be deployed to allow for real time visualization of the data for specific chemosensory genes.
1d) Identify a list of putative chemosensory associated genes for targeted gene knockout in year 2 and protein structural prediction in year 3 of future suggestions for identification of novel compounds for lures and attractants.
Using expression profiles of these high-quality gene annotations, genes associated with olfaction will be identified that have a high likelihood to be impactful towards characterizing new attraction mechanisms in these fly species. These target genes will be targeted for utilization in reverse genetics and computational prediction strategies outlines in future years of this project.