Background Transcriptomic methods can be used to elucidate genes and pathways responsible for phenotypic differences between populations. Asellus aquaticus is a freshwater isopod crustacean with surface- and cave-dwelling ecomorphs that differ greatly in multiple phenotypes including pigmentation and eye size. Multiple genetic resources have been generated for this species, but the genes and pathways responsible for cave-specific characteristics have not yet been identified. Our goal was to generate transcriptomic resources in tandem with taking advantage of the species’ ability to interbreed and generate hybrid individuals. Results We generated transcriptomes of the Rakov Škocjan surface population and the Rak Channel of Planina Cave population that combined Illumina short-read assemblies and PacBio Iso-seq long-read sequences. We investigated differential expression at two different embryonic time points as well as allele-specific expression of F1 hybrids between cave and surface individuals. RNAseq of F2 hybrids, as well as genotyping of a backcross, allowed for positional information of multiple candidate genes from the differential expression and allele-specific analyses. Conclusions As expected, genes involved in phototransduction and ommochrome synthesis were under-expressed in the cave samples as compared to the surface samples. Allele-specific expression analysis of F1 hybrids identified genes with cave-biased (cave allele has higher mRNA levels than the surface allele) and surface-biased expression (surface allele has higher mRNA levels than the cave allele). RNAseq of F2 hybrids allowed for multiple genes to be placed to previously mapped genomic regions responsible for eye and pigmentation phenotypes. In the future, these transcriptomic resources will guide prioritization of candidates for functional analysis.

Novel phenotypes can come about through a variety of mechanisms including standing genetic variation from a founding population. Cave animals are an excellent system in which to study the evolution of novel phenotypes such as loss of pigmentation and eyes. Asellus aquaticus is a freshwater isopod crustacean found in Europe and has both a surface and a cave ecomorph which vary in multiple phenotypic traits. An orange eye phenotype was previously revealed by F2 crosses and backcrosses to the cave parent within two examined Slovenian cave populations. Complete loss of pigmentation, both in eye and body, is epistatic to the orange eye phenotype and therefore the orange eye phenotype is hidden within the cave populations. Our goal was to investigate the origin of the orange eye alleles within the Slovenian cave populations by examining A. aquaticus individuals from Slovenian and Romanian surface populations and Asellus aquaticus infernus individuals from a Romanian cave population. We found orange eye individuals present in lab raised surface populations of A. aquaticus from both Slovenia and Romania. Using a mapping approach with crosses between individuals of two surface populations, we found that the region known to be responsible for the orange eye phenotype within the two previously examined Slovenian cave populations was also responsible within both the Slovenian and the Romanian surface populations. Complementation crosses between orange eye Slovenian and orange eye Romanian surface individuals suggest that the same gene is responsible for the orange eye phenotype in both surface populations. Additionally, we observed a low frequency phenotype of eye loss in crosses generated between the two surface populations and also in the Romanian surface population. Finally, in a cave population from Romania, A. aquaticus infernus, we found that the same region is also responsible for the orange eye phenotype as the Slovenian cave populations and the Slovenian and Romanian surface populations. Therefore, we present evidence that variation present in the cave populations could originate from standing variation present in the surface populations and/or transgressive hybridization of different surface phylogenetic lineages rather than de novo mutations.