Across the continuum of the process of speciation is the formation of incompatibilities between reproductive and/or ecologically isolated populations. The biology and ecology of cactophilic Drosophila has facilitated their use in investigating the evolution of reproductive incompatibilities.

Normally a conspecific mating in D. mojavensis results in the formation of a reaction mass in the lower reproductive tract of females that tends to dissolve within a few hours postcopulation. In the case of a heterospecific mating with a male D. arizonae, the reaction mass can last for days or never dissolve effectively sterilizing the female by blocking the oviposition of eggs. In addition, and possibly more significant, a large fraction of eggs failed to get fertilized. We have previously observed that upon a heterospecific mating, even within 15 minutes postcopulation, the normal postmating transcriptional response is highly perturbed (Bono et al 2011). It was also in this study that we identified the first known instance of male derived transcripts being passed along via the ejaculate to the female. Such surprising finding may have significant implications for understanding how postcopulatory interactions can result in incompatibilities. In a follow-up study we have shown how the loci of these male-derived transcripts, some of which are sperm-coating proteins, are under strong divergent positive selection between D. mojavensis and D. arizonae, with some differences found in predicted functional residues (Bono et al 2015). We have observed that the transcriptional response (both differential expression and alternative splicing) is significantly affected in heterospecifically mated D. mojavensis and D. arizonae in both the head (Diaz et al. 2021) as well as in lower reproductive tracts (Diaz et al. 2022). Furthermore, in lower reproductive tracts, we observed that mating responsive genes that have diverged more in expression also have more disrupted transcriptional profiles in heterospecifically mated females (Diaz et al. 2022).

In D. melanogaster, it has been shown that male-derived exosomes are transferred via the ejaculate and can fuse to female epithelial tissues (Corrigan et al., 2014). These exosomes might be the potential vehicle for the male-derived transcripts in D. mojavensis, which post-fusion with female epithelial tissue might be actively translated. In our current NIH award (with my colleague Dr. Jeremy Bono at UCCS) we are further assessing the role of these male-derived transcripts in postmating-prezygotic (PMPZ) incompatibilities. Additionally, we have created knockouts via NHEJ using CRISPR-Cas9 of candidate reproductive incompatibility genes and begun to assess their phenotypic role.