Location: Tropical Crop and Commodity Protection Research
Project Number: 2040-22430-027-011-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 1, 2024
End Date: Jul 31, 2025
Objective:
The specific objectives of the second-year project are to:
(1) Isolate and identify gut bacteria of wild OFF females.
(2) Identify bacterial isolates and their growth phase most attractive to female OFF.
(3) Identify bacterial volatiles attractive to female OFF.
Approach:
Objective 1: Isolate and identify gut bacteria of wild OFF females
Wild OFF are obtained from tropical almond fruits collected from infested tropical almond fruits. Emerged adult flies are provided with a mixture of honey and yeast hydrolysate (1:1 w/w) and water ad libitum for maturation and egg development. Tissues of flies from tropical almond are dissected using general aseptic dissection procedures from gut and ovipositor. Gut and ovipositor tissues from each fly is transferred separately into individual aseptic centrifuge tube containing 1 ml of DNA/RNA Shield. The homogenized gut and ovipositor tissues are subjected to serial dilution, and 10 µl of the final dilution is plated onto LB agar plates. Individual bacterial colonies are selected based on their morphological characteristics and sub-cultured onto fresh LB agar plates. DNA from bacteria are extracted using a ZymoBIOMICS DNA Extraction kit on a KingFisher Flex Magnetic Particle Processor. 16S-rRNA barcoding is conducted using the amplification of V4 sub-region of the 16S SSU rRNA. Individual sequence data of bacterial isolates are blasted using NCBI blast to identify the bacterial isolates.
Objective 2: Identify bacterial isolates and their growth phase most attractive to female OFF
To determine attractive bacterial isolates, the bacterial isolates are centrifuged to collect bacterial cells. The cells are inoculated into a 150 ml flask containing 50 ml of LB medium. Bacterial volatiles emitted from the flask are directed using Teflon tubing into a fly trap constructed from a 1-liter food container. To determine attractive growth stages of an attractive bacteria, growth phase curve is determined using a spectrometer equipped with a Peltier system based on optical density of bacterial cells at 600 nm.
Objective 3: Identify bacterial volatiles attractive to female OFF
ARS-Hilo will rear B. dorsalis on artificial diet for GC-EAD and behavioral assays. First, headspace volatiles will be collected from selected bacterial isolate cultures at most attractive growth stage. Then the extracts will be subjected to GC–EAD analysis to determine EAD-active bacterial volatiles that can be detected by OFF antennae. EAD-active chemicals will be identified using GC-MS based on library matches and retention time and mass spectra of authentic chemicals. Synthetic blends (full blends) will be prepared based on ratio of the EAD-active compounds from an attractive bacterial. For laboratory behavioral assays, the blend will be released from a rubber septum. For field trapping experiments, the blend will be released from a 4 ml vial with 3 mm hole as neat chemicals.
