Molecular and Bioinformatic Analysis of the Circadian Clock in Phreatichthys andruzzii

Daily cycles of light and temperature imposed by the rotation of the Earth on its axis have had a major impact on the evolution of all living organisms. Fascinating demonstrations of this fact can be seen in extreme environments such as caves or in the deep sea, where some species have evolved in complete isolation from daily light-dark cycles for millions of years, sharing a range of striking physical characters acquired by convergent evolution such as loss of eyes and pigmentation. One fundamental issue is to investigate whether these “hypogean” species still retain a functional circadian clock, which is a highly conserved self-sustaining timing system that allows organisms to anticipate daily environmental changes and is synchronized primarily by light. In this study, we have performed a comparative analysis of the circadian clock between the Somalian cavefish Phreatichthys andruzzii, which has evolved in perpetual darkness, and the model species Danio rerio (the zebrafish) that is evolved under natural daily light-dark cycles. It has been demonstrated that P. andruzzii retains a food-entrainable clock that is synchronized in response to regular feeding time, but does not respond to light-dark cycles. Moreover, under constant conditions, the cavefish clock oscillates with an extremely long period and also lacks normal temperature compensation. Based on these previous results, we started to analyze in detail one specific clock gene that is light-induced in zebrafish, Period2, where we encountered significant mutations in cavefish. We characterized the coding sequence and the genomic structure of this mutated cavefish gene and we analyzed its expression levels in comparison with zebrafish. Subsequently, for the first time in this species we performed a detailed characterization of clock and visual/non-visual photoreceptor genes as part of a complete P. andruzzii genome and transcriptome analysis. Our RNAseq analysis revealed a surprising phenomenon in cavefish: P. andruzzii mRNA sequences present an unusually high level of retained introns, leading to premature stop codons being introduced into the coding sequences of many transcripts. This mechanism may contribute to the aberrant clock phenotype of this species as well as, from a wider perspective, to other fascinating cavefish adaptations to life in constant darkness. We analyzed in detail this aberrant intron splicing phenomenon in two cavefish transcriptomes, from the brain and from a fin-derived cell line. Finally, the creation of a first P. andruzzii genome assembly by DNAseq analysis allowed us to map and characterize the group of visual/non-visual opsins, the main candidate genes involved in the circadian and non- photoreception system. We used this sequence data in order to perform a comparative analysis of the levels of expression of these genes between cavefish and zebrafish. Our results suggest that the loss of photoreceptor function in cavefish may result either from mutations affecting the coding regions of the opsins, as documented in TMT-opsin and Opn4m2, or, alternatively, from mutations affecting the regulation of the expression levels of this group of photoreceptive genes.