Introgressing pheromone qtl between species: towards an evolutionary understanding of differentiation in sexual communication

As a first step toward understanding how noctuid moths evolve species-specific pheromone communication systems, we hybridized and backcrossed two closely related moth species, Heliothis virescens (Hv) and H. subflexa (Hs), which differ qualitatively and quantitatively in their multi-component sex pheromone blends. We used amplified fragment length polymorphism (AFLP) marker-based mapping of backcross families to determine which of the 30 autosomes in these moths contained quantitative trait loci (QTL) controlling the percentages of specific chemical components in the pheromone blends. In two previous backcrosses to Hs, we found a strong depressive effect of Hv-chromosome 22 on the percentage of three acetate components in the pheromone gland. These acetates are present in Hs and absent in Hv. Here, we describe how we introgressed Hv-chromosome 22 into the genomic background of Hs. Selection for Hv-chromosome 22 started from backcross 3 (BC₃) females. All females that had Hv-chromosome 22 and a low percentage of acetates (< 3% of the total amount of pheromone components present) were backcrossed to Hs males. In BC₅ to BC₈, we determined whether Hv-chromosome 22 was present by a) running only the primer pairs that would yield the markers for that chromosome, and/or b) determining the relative percentages of acetates in the pheromone glands. Either or both genotype and phenotype were used as a criterion to continue to backcross these females to Hs males. In BC₉, we confirmed the isolation of Hv-chromosome 22 in the Hs genomic background, and backcrossed the males to Hs females to eliminate the Hv-sex chromosome as well as mitochondrial DNA. The pheromone composition was determined in BC₃, BC₅, and BC₁₁ females with and without Hv-chromosome 22. All backcross females with Hv-chromosome 22 contained significantly less acetates than females without this chromosome. In addition, BC₃ females with Hv-chromosome 22 contained significantly more Z11-16:OH than BC₃ females without Hv-chromosome 22. However, in BC₅ and BC₁₁ females, the correlation between Z11-16:OH and Hv-chromosome 22 was lost, suggesting that there are separate QTL for the acetates and for Z11-16:OH, and that the relative amount of the alcohol component is only affected in epistasis with other (minor) QTL. Now that we have succeeded in isolating the chromosome that has a major effect on acetate production, we can test in behavioral experiments whether the presence of acetates may have been a driving force for a shift in pheromone composition. Such tests are necessary to move towards an evolutionary understanding of the differentiation in sexual communication in Heliothis spp. moths.