Introduction:
The gilthead sea bream is an important marine fish for Mediterranean aquaculture. This species has historically been considered resistant to viral nervous necrosis (VNN) , a devastating fish disease affecting several major aquaculture species . However, with the emergence of RGNNV/SJNNV reassortant strains, also sea bream hatcheries are now seriously threatened, as these viral strains severely affect larvae and juveniles. While host response to betanodavirus in adult fish has widely been investigated, little is known regarding the most sensitive early life stages and currently the transcriptomic response of gilthead sea bream to RGNNV/SJNNV reassortant is still limited. Interestingly, early life stages (i.e. larvae younger than 35 days) are susceptible to VNN , while later stages (i.e. larvae older than 35 days) are resistant. This offers a unique opportunity for a comparative approach in order to understand the transcriptomic mechanisms that are triggered in susceptible vs resistant larvae, understand the genes and pathways that are activated in resistant larvae and pinpoint the molecular bases of resistance to RGNNV/SJNNV.
Materials and Methods:
This study reports the first time-course RNA-seq analysis on 21-day old (susceptible) and 35-day old (resistant) sea bream larvae experimentally infected with a RGNNV/SJNNV strain. Infected and mock samples from each developmental stage (i.e. 21 and 35 days) were collected at 6h, 12h, 24h, and 48h post infection (hpi). Four biological replicates, of five pooled larvae each, were analysed at all the time points .
Results:
Results highlighted a large set of significantly regulated genes, especially at 6hpi and 12hpi in 21-day old larvae. Particularly, several heat shock protein encoding transcripts were up-regulated (e.g. hspa5 , dnaj4 , hspa9 , hsc70 ), while many immune genes were down-regulated (e.g. myd88 and irf5 at 6hpi , pik3r1 , stat3 , jak1 , il12b and il6st at 12hpi ). A gene set enrichment analysis (GSEA) was implemented to identify altered pathways/processes. Interestingly, many immune processes were found down-regulated in susceptible larvae and up-regulated in resistant larvae. In particular, the neutrophil degranulation process was down-regulated at 6hpi, 12hpi and 24hpi in susceptible larvae, while it was up-regulated at 6hpi, 12hpi and 24hpi in resistant larvae; the autophagy pathway was down-regulated at 6hpi in susceptible and resistant larvae, while it was up-regulated at 12hpi and 24hpi in resistant larvae only; the peroxisome, an important component of the antiviral immunity, was down-regulated at 6hpi in susceptible larvae, while there was no impairment of this process in resistant larvae; the immune system process was up-regulated at 12hpi and 24hpi in resistant larvae while it was not differentially regulated in susceptible larvae.
Conclusions:
Differently from previous studies in which it was proven that the RGNNV strain is able to induce mortality only in sea bream larvae infected by intramuscular route (while no mortality nor severe NNV signs are observed in older fish), our data confirms the hypothesis that the reassortant RGNNV/SJNNV induces transcriptional changes in the early larval stages of this species putatively by interfering with important immune processes and ultimately leading to the death of infected larvae.