From gross morphology to gill transcriptome in farmed Atlantic salmon (Salmo salar): Lessons from multi-site sampling

Abstract

Sphaerospora molnari is a myxozoan parasite causing skin and gill sphaerosporosis in common carp (Cyprinus carpio) in Central Europe. Prior to spore formation, multicellular proliferative stages of S. molnari circulate for several weeks in the vascular system of its host despite the array of humoral and cellular immune effector mechanisms. Using our laboratory infection model, we aimed to elucidate the kinetics of presporogonic development of S. molnari, while simultaneously analyze the immune responses over a period of 63 days. The obtained results identified two peaks of acute parasitemia on day 28 and 42 respectively. Unexpectedly, the highest parasite load was detected in the liver, a previously unknown localization of S. molnari. In response to the infection, the immune system induced dynamic changes in the expression of pro-and anti-inflammatory cytokines, with a predominant role of IL-10 reaching up to 1456 fold increase compared to control fish. The haematological analysis revealed a steady increase in the number of lymphocytes from day 28 onwards, correlating with the growing number of parasites, and only marginal changes in other populations. Additionally, our data revealed a strong increase in the expression of IgM transcripts and increased number of IgM þ B lymphocytes, which produce specific antibodies recognizing S. molnari antigens in western blot. Strikingly, although the sera of infected fish exhibit potent opsonizing capacity in vitro, S. molnari isolated from the blood of infected fish are not labelled with carp IgM. These findings indicate the presence of so far unknown evasion strategy and questions the importance of S. molnari-specific antibodies in parasite elimination. To our knowledge, this is the first study analyzing the early myxozoan development and immune modulation mechanisms along with innate and adap-tive immune responses of the fish host in a controlled laboratory system, adding important information on host-parasite interaction of early meta-zoans (Cnidaria) with basic vertebrate immune systems. Abstract The gill is a multifunctional organ involved in many physiological processes such as gas exchange, osmotic and ionic regulation, acid-base balance and excretion of nitrogenous waste. Due to its interface with the environment, the gill plays a key role as primary mucosal defence tissue against pathogens and is equipped with the gill-associated lymphoid tissue (GIALT). In recent years, the prevalence of gill damage and gill diseases has increased significantly, leading to the substantial losses in Atlantic salmon aquaculture worldwide. Both the transition from healthy to unhealthy gill phenotypes as well the progression of various gill pathologies such as proliferative gill disease (PGD), amoebic gill disease (AGD) and complex gill disease (CGD) are commonly characterised by inflammation and epithelial cell hyperplasia. Routine monitoring for PGD relies on gross (macroscopic) evaluation of gill health, coupled with histological examination of gill sections. To explore underlying molecular events that are associated with progression of PGD, we examined Atlantic salmon from geographically diverse aquaculture sites in Scotland. Total RNA was extracted from 43 fish presenting low or medium gill PGD scores and analysed by whole transcriptome analysis using RNA-seq to determine the molecular signature of the advanced PGD. For each fish, 20M reads were generated and mapped to the Atlantic salmon genome. Importantly, we showed that the sampling site had greater effect on the gill transcriptome than the actual PGD score, providing support for a complex and multi-factorial aetiology of PGD, with minimal common molecular responses between different sites. Similar pattern was found for histology, agreeing with the outcome of the RNA-seq analysis. In general, both RNA-seq and histology data clustered together based on the origin of samples, suggesting that the PGD scores may inform about the overall progression of gill damage, but not about the underlying pathology.