Location: Livestock Arthropod Pest Research Unit
Project Number: 3094-32000-041-005-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2018
End Date: Jun 30, 2022
Objective:
Isolate and identify the cuticular hydrocarbon signatures from multiple populations of screwworm and related calliphorids towards understanding the chemotaxonomy and develop sex-biased and unbiased attractive baits.
Approach:
Employ two different yet complementary techniques to collect the CHCs. The collection method can greatly impact the compounds represented and their relative abundance, both of which are crucial to physiological, ecological, behavioral and evolutionary insights. The most common methods currently used for hydrocarbon analysis are solvent extraction (usually using an organic solvent such as hexane) and solid-phase microextraction (SPME). Solvent extraction has the advantage of sampling large numbers of insects at once to minimize variation and increase sample abundance. Additionally, solvent extraction can be used to collect compounds deposited on a surface by the insects. On the other hand, SPME has the advantage of being able to sample either headspace compounds or surface compounds and can be completed with little or no damage to a live insect. Therefore, we will utilize both methods to perform the twofold task of whole profile investigation on the primary screwworm, secondary screwworm, and the common green bottle fly in conjunction with species comparisons and assess the appropriateness of each method. The CHCs will be resolved on a high-resolution HP-5 capillary column in a state-of-the art Agilent 5975C Series GC/MS with Triple-Axis Detector. Chemical identity will be determined using NIST 2008 MS library. Final identification will be based on injecting a synthetic standard back onto the GC-MS system to verify the spectral match. This process has been employed by the cooperator's laboratory for multiple systems. The cooperator has over 20 years’ experience in chemical ecology of disease vectors. The MS data will be subjected to Principal Component Analysis (PCA) taking the relative abundance of the determined compounds as loading factors. The major principal components (PCs) explaining most of the variation will be retained for further analysis and are usually graphed into 3D clustering by species and sex by plotting the first three PCs (Figure). Treating males and females of a single species as one cluster and performing a pairwise MANOVA between the groups, we will determine if each of analyzed spp. are significantly separated from each other. Next, we will perform a second pairwise MANOVA on males and females within each species to make intraspecies comparisons between sexes. To identify which spp. have isomorphic or dimorphic CHC profiles. Finally, we will assess the relationship between CHC profiles using hierarchical cluster analysis. This uses similarity to classify samples and draw relationships among them. The overall pattern of relationship in CHS profiles usually resolves phylogenetic relationships. When branches are considered a single cluster beneath a threshold height, distinct clusters are formed revealing the taxonomic status. Similarity between separated clusters is represented by the height at which clusters are joined; increasing height corresponds to a decrease in similarity.
