The experimental part of the transcromic study of lenses from diploid and triploid salmon with varying degrees of cataract was completed. Total RNA was extracted and separated into 18 pools consisting of 9 pools of diploids and 9 pools of triploids with severe, mild or no cataract. Preparation of the cDNA sequencing libraries and sequencing was performed by the Norwegian Sequencing Centre (NSC, Oslo, Norway) using the Illumina TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, USA). The RNA-Seq data was mapped to most recent salmon reference genome (Lien et al 2016) using the Burrows-Wheeler aligner (Li and Durbin 2009). Preliminary analysis of the data using KEGG revealed a high number of differentally regulated metabolic pathways. In particular, the cataractous lenses displayed a high number of differentially expressed genes in pathways associated with focal adhesion, endocytosis, MAPK signalling, and protein processing in endoplasmic reticulum. Analyses of the large data will continue in the future. In addition to the transcriptomic studies, quantitative analyses of the four Aqp0 channels in the lenses of diploid and triploid salmon with varying dregrees of cataract were completed. Densitometric analyses of Western blots using affinity-purified antisera against the salmon Aqp0 channels confirmed that the previously observed downregulation of aqp0 RNA also occurs at the protein level. It is thus possible to conclude that Aqp0 channels are related to cataract in salmon, but it remains unclear whether they are associated with cause or effect. Finally the adhesive properties of the four salmon Aqp0 channels were investigated by heterologously expressing each in adhesion-defieinct mouse fibroblast cells. These experiments revealed that the salmon channels are capable of forming cell-to-cell junctions by preferntially adhering to the same paralog. These data were the first to show the adhesion potential of Aqp0 channels in a non-mammalian vertebrate, and uncover a novel extraocular role of the channels during vertebrate development.
Cataracts represent an occluding of the eye lens due to aggregation of soluble proteins with the result that light is scattered or absorbed and vision is either partially or completely impaired. The disease occurs in all vertebrates and is a leading cause of blindness in humans. In farmed Atlantic salmon, the prevalence of cataract is considered to be unethically high for fish welfare, and can exceed 90% in triploid fish causing poor growth performance, higher risk for infection, and increased mortality. As a result, direct costs associated with the disease are high and a major problem for the aquaculture industry. Previous studies on Atlantic salmon have shown that low dietary histidine affects cataract formation, and gene expression and performance of l enses to osmotic challenges. However, the molecular basis of cataract formation remains poorly understood. The present project aims to acquire this basic knowledge.
Studies on mammals and zebrafish have revealed that regulatory dysfunctions of a subclass of molecular water channels (aquaporin-0, AQP0) are directly related to the aetiology of cataract. In a pilot experiment, we found that gene dosage increased the expression of three Aqp0 paralogues (aqp0a1, -0a2, and -0b) in the lens of triploid Atlantic salmon when compared to diploid fish, yet the expression of some of these paralogues appears to be down-regulated in triploid fish with cataract. In addition, we identified three point-mutations in the aqp0b paralogue that could be involved in the aetiol ogy of cataract. This project therefore aims to provide an in-depth assessment of the physiological roles of aqp0 genes during cataractogenesis in Atlantic salmon, through genomic, SNP mutant, transcriptomic and functional approaches, with the goal of ide ntifying Aqp0-related genetic markers for the selection of fish with high resistance to cataract formation.