Research paper
Innate immune-gene expression during experimental amyloodiniosis in European seabass (Dicentrarchus labrax)

https://doi.org/10.1016/j.vetimm.2021.110217Get rights and content

Highlights

Abstract

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the causative agent of amyloodiniosis in European seabass (ESB, Dicentrarchus labrax). There is a lack of information about basic molecular immune response mechanisms of ESB during AO infestation. Therefore, to compare gene expression between experimental AO-infested ESB tissues and uninfested ESB tissues (gills and head kidney) RNA-seq was adopted. The RNA-seq revealed multiple differentially expressed genes (DEG), namely 679 upregulated genes and 360 downregulated genes in the gills, and 206 upregulated genes and 170 downregulated genes in head kidney. In gills, genes related to the immune system (perforin, CC1) and protein binding were upregulated. Several genes involved in IFN related pathways were upregulated in the head kidney. Subsequently, to validate the DEG from amyloodiniosis, 26 ESB (mean weight 14 g) per tank in triplicate were bath challenged for 2 h with AO (3.5 × 106/tank; 70 dinospores/mL) under controlled conditions (26−28 °C and 34‰ salinity). As a control group (non-infested), 26 ESB per tank in triplicate were also used. Changes in the expression of innate immune genes in gills and head kidney at 2, 3, 5, 7 and 23 dpi were analysed using real-time PCR. The results indicated that the expression of cytokines (CC1, IL-8) and antimicrobial peptide (Hep) were strongly stimulated and reached a peak at 5 dpi in the early infestation stage, followed by a gradual reduction in the recovery stage (23 dpi). Noticeably, the immunoglobulin (IgM) expression was higher at 23 dpi compared to 7 dpi. Furthermore, in-situ hybridization showed positive signals of CC1 mRNA in AO infested gills compared to the control group. Altogether, chemokines were involved in the immune process under AO infestation and this evidence allows a better understanding of the immune response in European seabass during amyloodiniosis.

Introduction

The ectoparasite dinoflagellate Amyloodinium ocellatum (AO) is one of the most problematic parasites causing disease among brackish and marine water fish, known as marine velvet disease (Brown, 1994). AO causes a parasitic branchitis associated with high mortality and significant economic losses in farming conditions worldwide (Cruz-Lacierda et al., 2004; Fioravanti et al., 2006; Benetti et al., 2008; Saraiva et al., 2011; Soares et al., 2012; Dequito et al., 2015; Gómez and Gast, 2018; Byadgi et al., 2019). This parasite mainly infests the gills, skin, and entire oropharyngeal cavity of almost all species of brackish and marine water fish, including European seabass (Dicentrarchus labrax) (Benetti et al., 2008; Alvarez-Pellitero et al., 1993; Byadgi et al., 2019). Fish surviving the infestation may develop protective immunity, which suggests that the immunoprophylactic control of this disease through vaccination could be feasible (Smith et al., 1994; Cobb et al., 1998; Cecchini et al., 2001). However, the information regarding host responses to A. ocellatum infestation is limited (Byadgi et al., 2019).

Transcriptomics has been used extensively to explore the host response towards fish parasite infestations (Sudhagar et al., 2018). Results from the large yellow croaker (Larimichthys polyactis) after Cryptocaryon irritans infestation indicated enrichment of the Toll-like receptor pathway (TLR), chemokine signalling, complement system and coagulation cascades (Wang et al., 2016). Low, non-lethal infestation by C. irritans enhanced a significant local immune response in large yellow croaker (Larimichthys crocea) and induced immunosuppression (Yin et al., 2016a,b). Similarly, in skin of orange spotted grouper (Epinephelus coioides) affected by C. irritans, a local immune response with intense leukocytes recruitment was observed (Hu et al., 2017). Interestingly, three-spined stickleback (Gasterosteus aculeatus) infested by three different genotypes of the trematode parasite, Diplostomum pseudospathaceum, revealed differential mechanisms by which the host immune system reacts to the immunological threat (Haase et al., 2016 & 2017). Moreover, in large yellow croaker upon infestation with the intestinal myxozoan parasite Enteromyxum scophthalmi an inadequate adaptive immune activation was observed (Robledo et al., 2014). However, during early phase of infestation in turbot (Scophthalmus maximus) by E. scophthalmi an IFN-mediated immune response was recorded (Ronza et al., 2016). During mild natural infestation of Sparicotyle chrysophrii in Gilthead sea bream (Sparus aurata) a strong enrichment of differentially expressed genes in gills, related to apoptosis, inflammation and cell proliferation was observed, whereas inhibition of DEG related to apoptosis, autophagy, platelet activation, signalling and aggregation in the spleen was observed (Piazzon et al., 2019). Ichthyophthirius multifiliis infestation in rainbow trout (Oncorhynchus mykiss) gills triggered an innate immune response by enhancing the Chemokine signalling pathway, platelet activation, Toll-like receptor signalling (TLR) pathway, NOD-like receptor signalling pathway, and Leukocyte transendothelial migration (Syahputra et al., 2019). RNA-Seq-based transcriptome analyses were also employed to study the parasites themselves such as C. irritans (Yin et al., 2016a,b; Mo et al., 2016) and salmon louse Caligus rogercresseyi (Allardo-Escárate et al., 2014), in order to understand the host-parasite antigens interactions and to identify potential vaccine candidates.

Previous studies have indicated that Interleukin-1 (IL-1) and Tumor Necrosis Factor α (Tnf-α) were activated in infested ESB reared in an aquaponics system (Nozzi et al., 2016). Experimental infestation of AO in yellowtail (Seriola lalandi) enhanced the TLR22 expression and involved in response to AO infestation (Reyes-Becerril et al., 2015). Moreover, natural outbreaks of AO in ESB resulted in pronounced and sustained inflammation (il-8, cc1, and cox-2) involving many novel molecules (Hepcidin) at the site of parasite attachment. Moreover, some of the genes related to pro-inflammation such as TNF-α and IL1β were down regulated, and this may be a result of a transient process. Therefore, this recent work highlighted the immediate local immune responses of ESB to natural AO infestation (Byadgi et al., 2019). However, further studies are needed to understand the time course expression of these upregulated immune genes under laboratory experimental infestation, in order to describe the physiological status of ESB during AO infestation and the subsequent recovery processes.

Therefore, the objectives of this study were to evaluate immune gene expression in gills and head kidney after AO infestation using RNA-seq, to evaluate the most differentially expressed genes in AO infested ESB and to investigate the chemokine cc1 mRNA using in-situ hybridization (mRNA FISH) in order to survey the involvement of cc1 against AO in the gills of ESB. Altogether, this study will provide a more comprehensive understanding of the roles of ESB immune genes during AO infestation and recovery.

Section snippets

Ethics statement

All the experiments included in the present study have been carried out in the facilities (fish stabularium ID 5E7A0) of Department of Agricultural, Food, Environmental and Animal Sciences (University of Udine), as authorized by the Italian Ministry of Health (decree n 14/2018-UT, 12/11/2018). The animal care and protocols adopted adhere to the Directive 2010/63/EU of the European Parliament, implemented at a national level by the D.L. n. 26 of 4 March 2014.

Fish and parasite origin for experimental infestations

AO-naïve ESB (mean weight 14 g)

Assembly and sequence description

Reads from control (n = 3) and infested (n = 5) tissues (gills and head kidney) were analyzed via Illumina sequencing in a paired-end 2 × 150-nt run. This generated, on average, 23,321,464 raw reads per sample with a GC content of 52 % (Supplementary Table 1). On average, 92.82 % of the raw reads (21,646,982 reads) mapped to the European seabass (D. labrax) reference genome (Supplementary Table 2). The raw FastQ file has been deposited in the National Center for Biotechnology Information (NCBI)

Discussion

The purpose of the present investigation is to provide general insights on how host and parasite interact. In our previous study dealing with ESB response after natural AO infection (Byadgi et al., 2019) we observed that none out of four genes codifying for molecules related to adaptive immunity (mhc i, mhc ii and igm) show upregulation in gills and head kidney. This phenomenon could be due to the nature (characteristics) of the host species and to the infection dynamics (intended as mode of

Conclusions

In this study, the immune mechanisms in ESB gills and head kidney after infestation by AO, indicated partial overview of the mucosal response. Gills showed a higher number of DEG compared to head kidney, indicating the importance of the mucosal immune response at the site of AO attachment. Several immune genes were altered after AO infestation, such as chemokine cc1; multiple genes of the interferon-mediated immune response were upregulated. This points to a recruitment of immune cells towards

Acknowledgements

The present study was performed under the European Union support: Horizon 2020 project Parafishcontrol (grant agreement No. 634429) and Aquaexcel research and innovation programme (grant agreement No. 652831). The output reflects only the authors view, and the European Union cannot be held responsible for any use that may be made of the information contained herein. The authors wish to thanks the technicians Pierluigi Bagatella and Carla Calligaro for their lab assistance and Prof. Emilio

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    current affilition: [email protected] Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

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