Abstract
The symbiotic relationship between anemonefish (aka clownfish) and their sea anemone hosts was first discovered over 150 years ago. However, we still do not know how anemonefish are able to live among the sea anemone’s tentacles without being stung. Anemonefish are thought to undergo acclimation, a process by which they gain protection through gradually increasing contact with a potential host sea anemone. The hypothesis for this study is that anemonefish regulate their mucus coats through this acclimation to accommodate sea anemone species with different characteristics. Transcriptome-wide analysis of gene expression was used to reveal cellular pathways that protect the fish from its host. Comparisons were made using gene expression in the skin tissues of the Clark's anemonefish (Amphiprion clarkii) under three treatments: (1) naïve with no host sea anemone, (2) in association with the bubbletip sea anemone (Entacmaea quadricolor), and (3) with the more toxic Haddon’s carpet sea anemone(Stichodactyla haddoni). Gene expression was measured using paired-end RNA-seq through Illumina sequencing yielding about 1 billion reads. After initial assembly, filtering was used to remove duplicate, redundant, and low count transcripts. Using pairwise comparisons we found 4,2 up-regulated and 2 down-regulated, genes differentially expressed between fish with E.quadricolor and naïve fish, 329 up-regulated and 583 down-regulated genes between fish with S.haddoni and naïve fish, and 225 up-regulated and 445 down-regulated genes between fish with S.haddoni and those with E. quadricolor. Low numbers of differentially expressed genes between naïve fish and fish associating with E. quadricolor indicates an innate protection in A. clarkii to that species of anemone host. The high number of differentially expressed genes between fish with S. haddoni and other treatments shows a need for regulation in A. clarkii when associating with this more toxic anemone host. More differentially expressed genes than expected were involved in actin cytoskeletal regulation, a pathway found to be significantly overrepresented in the last comparison. Experimental disruptions of the actin cytoskeleton accelerates the rate of exocytosis including mucin secretion from mouse goblet cells. While there is no simple answer, the differentially expressed genes show that the skin tissues are regulating some proteins involved with the glycosyltransferase group and immune related activities. This research is the first step in looking at the protection mechanism of the Clark’s anemonefish at a transcriptome level and offers a list of candidate genes that can be investigated in more detail for future work on gene expression and possible sequence evolution of the anemonefish/anemone symbiosis.