Location: Tropical Pest Genetics and Molecular Biology Research Unit
Project Number: 2040-22430-028-037-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 1, 2024
End Date: Nov 30, 2025
Objective:
The primary purpose of this agreement is to address critical knowledge gaps related to the establishment of bacteria in mass reared medfly (Ceratitis capitata) for sterile insect technique. Prior research has identified that mass-reared medfly exhibit marked differences in gut microbial communities compared to wild flies, and that augmentation of bacteria can lead to improved competitiveness of male flies. To efficiently introduce novel microbial isolates or communities, knowledge of population and community dynamics and the resistance/resiliency of the medfly gut microbiome is necessary. Our goals are to identify how bacterial concentrations and fly rearing density impact the establishment of target isolates. The following years we will work on developing encapsulation and dispersion methods to optimize formulations so that the addition of the lowest concentrations can successfully colonize and propagate in the flies and begin additions of targeted probiotics. Developing these endpoint introduction protocols will help inform how we introduce bacteria for experimental manipulation of gut microbiomes aimed for improving SIT. In this project, we will focus we will focus on how bacterial concentration (titer), diet formulation, fly rearing density, and behaviors impact establishment and retention. The end products will lead to improvements to the SIT process by providing probiotic dissemination strategies.
Approach:
The principal aims of this project are to develop scalable protocols to introduce beneficial bacteria into ongoing SIT processes that would improve the competitive ability of mass reared fruit flies.
Objective 1: Determine bacterial concentration required to establish novel strains in medfly males - Irradiated male medfly pupae will be obtained from the CDFA in Waimanalo, HI. Newly emerged (< 24h old) flies will be used for all assays. A Klebsiella isolate with tetracycline and chloramphenicol resistance will be orally administered to groups of 25 flies in a 32 oz rearing container. Bacteria will be provided by mixing known concentration of isolates with specific volumes of diet. We will use two different diet formulations for this comparison; sucrose and a 3:1 ratio of sucrose and yeast hydrolysate. Bacterial concentrations at the start of the bioassay will range serially from 102 colony forming units (CFUs) to 106 CFUs. Control flies will receive the media without bacteria. Following the initial experiment with Klebsiella, we will repeat the experiment with additional isolates (at least seven) at the lowest dose determined to be successful in the first experiment. Bacteria will be administered through both sucrose and sucrose:yeast combinations. Flies (< 24h old) will be inoculated for three days, then allowed to feed on respective diet for another seven. Flies will be plated on paired nutrient rich media with and without appropriate antibiotics.
Objective 2: Determine how low concentrations of bacteria establish at different fly rearing densities -Klebsiella will be grown and inoculated in newly emerged medfly adults as described in Obj. 1. Three doses will be used, first the lowest as determined in Obj. 1 along with a high (108) and moderate dose that is in between low and high dose. Bacteria will be added to 20g of sucrose-yeast hydrolysate diet, and provided in a cage of insects along with agar blocks made of sugar and water. Cages will be standardized (21 cm3). Flies will once again be obtained from the CDFA facility in Waimanalo, HI. Irradiated adult medfly pupae will be held in cages at varying densities, with numbers being 100, 250, 500, and 1000 flies in each cage. Flies will be administered the treatments for four days, after which 20 flies from each cage will be randomly selected and their colonies will be enumerated as described above with appropriate media and antibiotic.
Objective 3: Evaluate medfly preference to diet inoculated with bacteria - Approximately 100 irradiated adult flies in a 40 × 40 × 60 cm cage will be provided a choice of diet (20g of sucrose-yeast hydrolysate) containing bacteria or no bacteria. Each diet will be weighed prior to placing in the cage. The number of flies that land on each food dish in one hr will be recorded and the amount of food consumed will be measured. The experiment will be repeated at least 5 times. Twenty irradiated adult flies will be placed in a 16 oz plastic cup containing diet with or without bacteria. The flies will feed in the cups for 48 hr, which will result in regurgitant and fecal matter on the walls of the cup and attraction to the cup will be determined.
