This research was supported by… Jamie Cassels Undergraduate Research OPSINS in the DARK... Awards (JCURA) Characterizing “light sensitive” genes in the Brazilian eyeless cavefish, Phreatobius cisternarum 1University of Victoria department of Biology 1 2 1 in collaboration with 2Uni Para in Belem. Amy Liu , Patricia Schneider , and , John Taylor Supervised by John Taylor. What are opsins? Discussion Results 1. Many opsins have gone missing relative to the Channel Opsins are light sensitive transmembrane proteins, with 100 Ictalurus punctatus XP 017323575.1 79 Phreatobius Rh1.1 Rh1 Figure 2. Opsins of Zebrafish, Catfish 92 Danio rerio KT008392 Rh1 1 the best-known visual opsinsfound in rod and cone cells 98 Danio rerio KT008393 Rh1 2 Danio rerio KT008391 Exorh Channel Catfish, and 89 Ictalurus punctatus XP 017323342.1 Exorh 2. Present opsins are found mostly transcribed with introns 99 99 97 Phreatobius ExoRh Phreatobius shown in a of the eyes. There are also non-visual opsinsfound in 70 Ictalurus punctatus XP 017352009.1 49 Rh2 100 Danio rerio KT008398 Sws2 phylogenetic tree (missing Rh 3. With one exception, opsins present are incomplete 70 Danio rerio KT008399 Sws1 tissues not normally exposed to light such as the brain, Danio rerio KT008400 Lws1 33 Ictalurus punctatus XP 017352008.1 Lws 1.*).The phylogeny shows, for 100 Phreatobius Lws transcripts 87 Phreatobius Va1 Ictalurus punctatus NP 001187192.1 example, that Phreatobiushas kidney, and heart. Recent studies have reported opsins 61 78 Danio rerio KT008402 Va1 59 Danio rerio KT008403 Va2 4. The opsin sequences in Phreatobius compared to the two 62 Ictalurus punctatus XP 017338415.1 Va2 an Rgr1gene which is sister to 88 81 99 Phreatobius Va2 may have non-light functions such as heat detection in Parapinopsin the Channel Catfish Rgr1and in 98 Danio rerio KT008406 Parietopsin surface fish, do not show evidence of neutral evolution. Ictalurus punctatus XP 017349407.1 Parietopsin 1 2 66 100 98 Phreatobius Parietopsin a clade with the more distantly mammalian sperm and locomotionin larval Drosophila . 100 Danio rerio NM001111164 Opn3 Ictalurus punctatus XP 017319608.1 100 Danio rerio KT008408 Tmt1b related Danio rerioRgr1. 25 91 Ictalurus punctatus XP 017326070.1 Phreatobius Tmt3a OPN3 75 Phreatobiuslacks some opsins We found evidence of as many as 11 opsins that persist through 99 Ictalurus punctatus XP 017350454.1 Danio rerio KT008411 Tmt3a 29 Danio rerio KT008412 Tmt3b 17 compared to Channel Catfish, 84 Ictalurus punctatus XP 017308205.1 evolution in the dark with the Phreatobius transcriptome. A 87 Danio rerio KT008431 Opn4x1 100 Danio rerio KT008432 Opn4x2 and Danio rerio, for example Ictalurus punctatus XP 017343895.1 100 Danio-rerio KT008429 Opn4m2 prominent example, Rhodopsin (Rh1), is a highly expressed full 100 Ictalurus punctatus NP 001187239.1 Rgr2. Neighbour-joining tree Hypothesis OPN4 Ictalurus punctatus XP 017318327.1 100 Danio rerio KT008428 Opn4m1 100 Danio rerio KT008430 Opn4m3 from bootstrap method with length sequence known for light-independent functions such as a 53 Ictalurus punctatus NP 001187193.1 94 To identify specific opsins with important roles other than light sensitivity, 99 Phreatobius Opn4m3 OPN5 1000 replicates and 3 76 43 Danio rerio KT008425 Peropsin role in thermosensorysignaling pathways . 100 Ictalurus punctatus XP 017320539.1 Peropsin we hypothesize these genes would still be expressedin the blind eyeless Phreatobius Peropsin evolutionary distances (P 99 Danio rerio KT008427 Rgr2 77 Ictalurus punctatus XP 017338464.1 distance) using the program subterranean fish (Phreatobius cisternarum) that have evolved in the Danio rerio KT008426 Rgr1 Rgr opsin 100 Ictalurus punctatus XP 017318777.1 100 dark for millions of years. 80 Phreatobius Rgr1 MEGA6. Fragments of various sizes of the other 10 opsins were detected in the transcriptome with varying levels of expression and intron Table 1. Comparison of opsins present in opsin families between three fish species and with +/-generalizing We also compare these genes to opsins of a surface relative, Channel presence and absence of opsins found throughout the Mammalia taxa retention (Table 2). Even though these sequences are incomplete catfish (Ictaluruspunctatus), and Zebrafish (Danio rerio) by characterizing and contain introns, they show evidence against neutral intron/exon boundaries and nucleotide substitutions. molecular evolution and for purifying selection, in which opsins have selective pressure to retain their original function (Table 3). Methods Little is known about intron retention, but several studies have Table 2. Summary of Phreatobiusopsin transcripts in Table 3.Codon-based Test of Neutrality analysis found transcripts with introns regulate translation of the those regards to RNA splicing (Missing Rh 1*). Percent % total between sequences of Phreatobius (PC), Zebrafish 4,5 indicates how much of the respective opsin gene the (DR) and Channel Catfish (IP). dSand dNrepresents transcripts from the same gene that lack introns . If it’s true that PBS script of a blast search using 42 Zebrafish opsins as query sequences against the multiple contigs cover. Besides Rh1.1most of the other synonymous and non-synonymous substitutions per these pieces may negatively regulate opsin translation, it’s Phreatobiustranscriptome. The same was done on a surface relative, the Channel Catfish opsins are incomplete transcripts. 13 out of 18 transcripts site respectively. Negative values show evidence for intriguing that we found them in a fish that lives in the dark. transcriptome as comparison. show evidence of intron retention (IR), while 4 transcripts purifying selection of the gene. Analysis used the Nei- show evidence of both intron splicing and IR. Gojoborimethod with the program MEGA7. This study is the first step in identifying opsins with non light- sensing roles. In future work we hope to analyze and compare our transcripts to the Phreatobiusgenome. References

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