Been there, done that: the role of cuticular hydrocarbons in facilitating polyandry in decorated crickets Carie B. Weddle 1, Christopher G. Hamaker 2, & Scott K. Sakaluk 1 1 Department of Biological Sciences, Illinois State University 2 Department of Chemistry, Illinois State University Females of several animal species have been shown to actively discriminate against previous mates in favor of novel mating partners. However, few studies have addressed the proximate mechanisms through which females recognize previous mates. Female decorated crickets, Gryllodes sigillatus, gain genetic benefits by mating polyandrously, and females preferentially mate with novel mating partners over previous mating partners. Results of a recent study suggest that females “tag” males with their own unique scent at mating, permitting them to recognize, and avoid mating with previous mates in subsequent interactions. Such a mechanism requires that females possess unique chemical signatures that would enable this form of self-referencing. The ubiquitous use of cuticular hydrocarbons (CHCs) as recognition cues among The insects makes them likely candidates for the cues used in individual mate recognition. We sampled 31 females from nine F 16 inbred lines (coefficient of inbreeding: F > 0.75). CHCs were extracted by whole-body immersion in 2 mL hexanes for 10 min. The hexane solvent was evaporated overnight, and CHCs were resuspended in 1 mL hexane with 10 ppm dodecane as internal standard. Samples were analyzed by gas chromatography, using a DB5-HT column with a flame ionization detector: Standardized peak areas at each retention time were analyzed using principal component analysis (PCA). PCA scores were analyzed by discriminant analysis (DA) to determine if CHC profiles differ across genetic lines, and to determine the extent to which the CHC profiles of individuals could correctly predict the membership of each female to a genetic family. Supported by grants from NSF to SKS, Beta Lambda Chapter of Phi Sigma and the Orthopterists’ Society to CBW. Special thanks to Tracie Ivy, Bill Perry, Steve Juliano, Geoff Ower, Mike Neville, Violet Kirk, Kelly Ryan, and Andrew Wadler. Introduction Aim of study Prediction Methods In order for CHCs to provide a reliable means of identifying and discriminating against previous mates, they must posses a sufficient amount of genetic variation to permit females to distinguish their own unique chemical profiles in a complex background of chemical cues. Therefore, female CHC profiles should show low variation within genetic lines, relative to between genetic lines. Conclusions Female CHC profiles show low variation within genetic lines relative to between genetic lines. The distinct CHC profiles across inbred lines suggests a genetic component to these chemical cues. The unique CHCs profiles of females from different genetic backgrounds suggests that CHCs could provide reliable cues for self-referent phenotype matching to recognize previous mates. Cuticular hydrocarbons could provide a proximate mechanism through which females maximize the benefits of polyandry. Results Acknowledgements Photo by Scott Sakaluk Figure 1: Discriminant analysis of cuticular hydrocarbons for females from 9 genetic lines. Despite some overlap, genetic lines separate significantly on the basis of the relative amounts of 42 cuticular hydrocarbon peaks (Wilk ’ s λ = 4 x , F 64, = 436.3, P < ). PCA isolated 8 principle components that explained 86.8 % of the total variation in the observed CHCs. DA yielded 5 discriminant functions that explained 90.3% of the between- group variation in PCA scores. The results revealed significant differences in cuticular hydrocarbons due to genetic family (Figure 1). The predictive model correctly assigned to genetic family 100% of females from four of the nine lines, and 90-97% of the females in the five remaining lines, with a total misclassification rate of only 3.23% (Table 1). Table 1: Classification results of predictive discriminant analysis for CHC profiles of females from 9 inbred lines. Data show the percent of females classified into each of the 9 families. Total misclassification rate of the model was 3.23%. Do cuticular hydrocarbons show sufficient genetic variation to be used as reliable cues for individual mate recognition? We used gas chromatography to analyze variation in CHC profiles for females from nine inbred lines. Photo by Kazuyuki Hashimoto