First, we explored the transcriptional activity of TEs in East African cichlids of every Great Lake and Nile Tilapia as an outgroup to the radiation. By profiling TE expression in cichlid gonads and throughout embryogenesis, we have found hundreds of expressed TE families and dynamic expression patterns. Of note, we have identified differential expression of TE families according to sex, and a developmental period in early gastrulation where TEs are most highly expressed, consistent with a relaxed period of epigenetic silencing before zygotic production of TE silencing factors.
To define the TE silencing pathways of cichlids, we first identified the cichlid proteins homologous to known TE silencing factors in humans, mouse, and zebrafish, and profiled their expression in gonads and brain. TE silencing factors of the piRNA pathway were expressed in gonads, which harbour germ cells, suggesting that piRNA-driven TE silencing is in place in germ cells, much like in other animals. Conversely, we could not detect consistent expression of piRNA pathway factors in the brain, arguing against a robust piRNA-driven TE silencing pathway in cichlid brains. Interestingly, we have discovered an expansion of piwil1, a key element of the piRNA pathway, in Lake Malawi cichlids. These fishes encode three piwil1 genes, one full-length and two other truncated versions carrying only the Piwi catalytic domain. We have reconstructed the evolutionary history of this expansion and found that the truncated proteins may have retained catalytic activity. The presence of TEs directly flanking the truncated piwil1 genes, but not the full-length copy, suggests that this expansion may have been mediated by TEs.
Also, we profiled small RNAs in the gonads of representative cichlid species of each East African Great Lake, using Nile Tilapia as an outgroup to the radiation. We have identified abundant small RNA populations with length and sequence signatures consistent with piRNAs. We mapped the origins of piRNAs and found that they are produced from TEs and intergenic regions. We further refined this analysis and identified fast evolving piRNA clusters. Overall, these findings strongly indicate that an active and fast evolving piRNA pathway is in place in cichlid gonads to silence TEs.
To understand how cichlids tolerate hybridisation, we have created two reciprocal crosses between two cichlid species of Lake Malawi. We found a degree of embryonic lethality in the F2 hybrid generation of both crosses, consistent with post-zygotic incompatibilities. We are currently dissecting the molecular basis of the embryonic lethality, as well as its accompanying developmental defects.
These results were presented at several international conferences, and local meetings. Publications resulting from this work will be published in preprint repositories (namely BioRxiv) and in peer-reviewed journals (with open access). All mass spectrometry, mRNA- and small RNA-sequencing datasets generated in this study will be made publicly available on online repositories (GEO or SRA). EU funding was acknowledged in all dissemination opportunities so far, and will be further acknowledged in all future dissemination/communications related to this work.
