Gill and liver transcript expression changes associated with gill damage in Atlantic salmon (Salmo salar). Gill and liver transcript expression changes associated with gill damage in Atlantic salmon (Salmo salar)

Gills of teleost fish represent a vital multifunctional organ; however, they are subjected to environmental stressors, causing gill damage. Gill damage is associated with significant losses in the Atlantic salmon aquaculture industry. Gill disorders due to environmental stressors are exacerbated by global environmental changes, especially with open-net pen aquaculture (as farmed fish lack the ability to escape those events). The local and systemic response to gill damage, concurrent with several environmental insults, are not well investigated. We performed field sampling to collect gill and liver tissue after several environmental insults. Using a 44K salmonid microarray platform, we aimed to compare the transcriptomes of pristine and moderately damaged gill tissue. The gill damage-associated biomarker genes and associated qPCR assays arising from this study will be valuable in future research aimed at developing therapeutic diets to improve farmed salmon gill health. Overall design: Using a 44K salmonid microarray platform, we aimed to compare the transcriptomes of pristine and moderately damaged gill tissue. The fish were sampled on the 13th and 14th of November 2017. The average weights for all of the collected fish was 1348 grams. The gill samples were collected from the net pen-grown Atlantic salmon located in BC. Fish were randomly netted then euthanized using 400 mg L−1 tricaine-methane sulfonate bath (TMS; Syndel Laboratories, Nanaimo, BC, Canada) after 24 h of fasting. The sampled fish were weighed, fork length-measured, and their gills were scored following the farm’s protocol. Gills with no lesions visible and with single necrotic filament or spot were scored as G0 (i.e., pristine). Gills showing affected area up to 10% of the total gill filament were scored as G1, while G2 was defined as gills having damage affecting ~10% to 25% of the total gill filament. Gills with affected area > 25% of the total fill filament surface are scored as G3. Fish with more than 50% of the total gill filament surface area covered with necrotic tissue were routinely observed but generally in mortalities or very moribund fish and not in healthy fish (B. Milligan, personal communication). Gills were collected for gene expression analyses. Samples around 50-100 mg of each tissue were preserved in 1 mL of RNAlater® solution (Ambion, Inc., Austin, TX, USA) and kept at 4°C overnight, then moved to -20°C until the shipping date to the Ocean Sciences Centre, Memorial University. All the collected samples were subjected for preliminary qPCR study, for sample choice and we aimed to target gills with moderate gill damage. Using a 44K salmonid microarray platform, we aimed to compare the transcriptomes of pristine and moderately damaged gill tissue.

硬骨鱼（teleost fish）的鳃是兼具多种功能的关键器官，然而其极易受到环境胁迫因子的侵袭，进而引发鳃组织损伤。大西洋鲑（Atlantic salmon）养殖产业中，鳃损伤会造成显著的经济损失。全球环境变化进一步加剧了环境胁迫因子诱发的鳃病害，对于开放式网箱养殖（open-net pen aquaculture）而言尤为突出——养殖鱼类无法主动逃离此类胁迫事件。目前，针对鳃损伤以及多种环境胁迫并发情况下的局部与全身应答机制，相关研究尚不够充分。

本研究通过野外采样，在多种环境胁迫事件发生后采集了鳃与肝脏组织。我们采用44K鲑科微阵列（salmonid microarray）平台，旨在比较健康完好与中度损伤的鳃组织转录组。本研究获得的鳃损伤相关生物标志物基因及配套qPCR检测方法，将为后续开发改善养殖鲑鳃健康的治疗性日粮提供重要研究基础。

整体实验设计：本研究采用44K鲑科微阵列平台，旨在比较健康完好与中度损伤的鳃组织转录组。实验鱼于2017年11月13日至14日完成采样，所有采集个体的平均体重为1348克。鳃组织样本采自不列颠哥伦比亚省（BC）的网箱养殖大西洋鲑。实验鱼经随机网捕后，先禁食24小时，随后置于浓度为400 mg L−1的三卡因甲磺酸盐（tricaine-methane sulfonate, TMS；Syndel Laboratories，纳奈莫，不列颠哥伦比亚省，加拿大）溶液中麻醉致死。随后对采样鱼进行称重、叉长测量，并按照养殖场既定规程开展鳃损伤评分：无可见病灶、仅存在单条坏死鳃丝或坏死点的鳃记为G0（即健康完好组）；鳃丝受影响面积不超过总鳃丝10%的记为G1；G2定义为损伤面积占总鳃丝10%~25%的鳃；损伤面积超过总鳃丝表面积25%的鳃记为G3。坏死组织覆盖超过50%总鳃丝表面积的个体通常仅见于死亡或濒死鱼中，健康个体中较为少见（B. Milligan，私人通信）。

采集的鳃组织用于基因表达分析。每份组织取约50~100 mg样本，置于1 mL RNAlater®溶液（Ambion, Inc.，奥斯汀，德克萨斯州，美国）中保存，4℃过夜后转移至-20℃冷藏，直至运往加拿大纪念大学海洋科学中心。所有采集样本均先开展初步qPCR研究以筛选合格样本，目标为获取中度损伤的鳃组织。本研究最终采用44K鲑科微阵列平台，对健康完好与中度损伤的鳃组织转录组进行比较分析。