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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 #444989

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

Location: Tropical Pest Genetics and Molecular Biology Research Unit

Project Number: 2040-22430-028-032-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Aug 1, 2023
End Date: Sep 29, 2024

The goals of this project 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 medfly males. The primary goals are to determine how fly age and diet formulation impact bacterial establishment.

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 30¿ 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 10^6 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. Flies will be allowed to feed until evaluating establishment at 3d post inoculation and 7d post inoculation. Males (n=7-10 / replicate) will be surface sterilized in 10% bleach and homogenized in saline buffer. 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. In addition to evaluating impacts using S. marcescens strains, work will be conducted to establish protocols for select bacterial strains originally isolated from wild and laboratory populations of medfly. Bacterial plasmids containing fluorescent proteins and antibiotic resistance markers to introduce to naïve bacteria isolated from medfly. 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. Initial experiments will use a sugar-yeast hydrolysate diet, but modification of 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 10^6 CFUs per mL for agar diet. Diet will be mixed, autoclaved, and cooled to 50¿ 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. Fly metabolic parameters in glucose and trehalose will be determined for the flies on each diet and bacterial treatment.