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ARS Home » Pacific West Area » Hilo, Hawaii » Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center » Tropical Pest Genetics and Molecular Biology Research Unit » Research » Research Project #444630

Research Project: Optimizing Bacterial Probiotic Establishment for Medfly Sterile Insect Technique

Location: Tropical Pest Genetics and Molecular Biology Research Unit

Project Number: 2040-22430-028-026-I
Project Type: Interagency Reimbursable Agreement

Start Date: Aug 1, 2023
End Date: Nov 30, 2024

The goals of this proposal are to identify the effects of developmental windows and dietary permutations on the delivery and establishment of commensal bacteria in the digestive system of mass reared adult Ceratitis capitata males. The principal objectives are to: 1) determine how fly age impacts the colonization of gut bacteria, 2) evaluate how different diet formulations impact the establishment of gut bacteria, 3) compare different bacterial formulations and delivery strategies on the establishment in gut, and 4) determine optimal concentrations of bacteria for efficient propagation in populations posed for release. We anticipate this research will be a multi-year effort, with FY23 addressing Objs 1&2.

Objective 1: Establishment of bacteria will be determined using a pigmented indicator strain of Serratia marcescens absent in laboratory-reared flies. Cultures will be recovered from glycerol stocks and grown in a 30C incubator shaker for 3-4h prior to inoculation. Bacteria will be enumerated using optical density, and diluted in saline buffer. Bacteria will be administered by addition to a sugar-yeast hydrolysate diet, where 106 colony forming units (CFUs) will be added for each gram of diet and mixed into a slurry. Flies will be reared in a screened enclosure with inoculated diet and water. Assessment of adult age on bacterial establishment will be performed with newly eclosed flies, and flies 24h, 48h, and 72h post eclosion. Determination of establishment will be performed by plating onto non-selective, nutrient rich LB media and CHROMagar™ Serratia media, which is selective for Serratia marcescens. Parallel to culturing, DNA will be extracted from the guts of additional males receiving treatments (n=10), and bacterial communities will be analyzed using PacBio 16S-rRNA sequencing and quantitative PCR. Data will be analyzed using mothur and R. Objective 2: We will modify the consistency and protein-carbohydrate ratio and potential other additives to determine efficiency of bacterial introduction to flies. Diets will be formulated with different sugar-yeast ratios to provide a range that’s entirely (100%) sugar to a protein-biased diet. Adult diets will be formulated as an agar-based diet used prior to fly releases. Diets will be evaluated for both nutritional composition and concentration. Bacteria will be administered at 106 CFUs per mL for agar diet. Diet will be mixed, autoclaved, and cooled to 50C before adding bacteria. We will use newly eclosed and 48h old flies to compare the impact of age, and evaluate resulting microbial populations 7d post-inoculation.