Introduction
Traditionally the nets used in sea cage farming are made of nylon, a synthetic thermoplastic polymer widely used in fabrics production. As a robust polymer, nylon is not easily degraded (Lewis et al., 2004) , but it can be broken into small sized particles as a result of aging and UV exposure. In the particular case of fishing nets and sea cage nets, biofouling establishment is likely to further potentiate the aging and breakability of nylon.
Net biting is a gilthead sea bream specific behavior long reported by farmers and suggest ed to be related with feeding ratio and the presence of micro- fouling (Glaropoulos et al., 2012; 2013) . Whether sea bream actually ingests net pen debris has not been documented. But if it does, it can be hypothesized whether net pen debris may induce intestinal alterations, directly or indirectly as possible vehicles of toxic chemicals (Oliveira et al., 2013; Peda et al., 2016) .
As an alternative to the traditional netting, a copper-alloy mesh conceived for sea-cages has been recently introduced in the market. Being made of copper, its exposure to micro- fouling (including pathogens) and its susceptibility to predators’ attack is said to be minimized. Although it seems to be promising as an alternative to nylon, it remains to be known whether the mesh aging could lead to copper leaching and whether that could affect fish health.
This study aimed to evaluate the risks coming from the interaction of gilthead seabream with net pens of different materials and aging status by studying: 1) the bacterial community established on the nets with an emphasis on potentially pathogenic genera; 2) the biting behaviour and net erosion in relation to biofouling establishment and 3) the impact of net exposure on fish health status.
Materials and methods
Fish were grown out from 100 to 270g (up to 6Kg/m3, with 12 fish/tank) in a flow-through system, where 12 fiber glass indoor tanks were supplied with unfiltered and untreated seawater withdrawn at 130m from the coastline . The control group was grown in clear tanks, with no net; the NNy group was exposed to a <1-year-old nylon net; the ONy group was exposed to a >5-year- old nylon net; the Cu group was exposed to a new copper net. Nets were placed in the tanks as panels (40x20cm) immersed within the first 35 cm of the water column. Fish were fed once a day, at 12.00, except for Mondays. In each tank, the area surrounding the net panel was videotaped on Mondays (starving day), Tuesdays and Thursdays, in the morning, during the meal-time and in the afternoon, for 10 min, to evaluate the fish-net interaction behavior.
After 125 days, each net panel was hanged up off the water and two pieces of each net were sampled/swabbed for further microbiome analysis. The net panel was then removed off the tank and photographed for damage evaluation and a 10x10cm square was cut off for mobile epifauna counting e identification. Five fish per tank were sampled for blood collection for hematological profile characterization, the plasma was snap-frozen in liquid nitrogen and stored for further analysis of immune status parameters. The liver was excised for further analysis on oxidative status markers. The skin and distal gut were sampled for microbiome analysis. Distal gut sections were sampled also for histopathological qualitative analysis and for the quantification of transcript levels of inflammation-related genes by RT-PCR.
Results and Discussion
The bacterial communities associated with copper and nylon nets used in sea cages were compared using a metataxonomic approach through the high-throughput sequencing of the 16S rRNA V4 hypervariable region. Most differences were found between both nylon nets and copper nets, with less bacterial diversity in copper nets, which confirms its bactericidal effect. However, Tenacibaculum known to harbor potentially pathogenic species, was more abundant in copper nets than in nylon nets. These results bring up the need for monitoring the bacterial communities established on aquaculture gear, particularly on nets and including copper nets, to evaluate whether they may serve as reservoirs for pathogenic bacterial strains.
No mobile epi fauna was observed on copper nets. Mobile epifauna communities were different between old and new nylon nets, being more abundant in the new nylon. Although the behaviour analysis does not suggest clear differences between the 3 net types, the new nylon nets were clearly more damaged at the end of 4 months than the old nylon nets, which is probably related to epifauna abundance that should attract seabream towards the nets.
Concerning possible effects on fish health, although no signs of inflammation were found i n gut histological analysis and no differences were found i n gut microbiota , NNy fish displayed a higher expression of the anti-inflammatory cytokine IL-10 in the distal gut and increased bactericidal activity in plasma when compared to the control fish, as if exposed to a foreign agent. On the other hand, the distal gut of ONy fish displayed higher expression of CD8 which regulates cytotoxic T-cells activity. This group also displayed increased activities of catalase and glutathione S-transferase in their livers, when compared to the control group, while the NNy and Cu groups displayed intermediate levels. Further analysis are being conducted to check for nylon debris detection on fish gills and gut.
Overall, t hese results highlight the importance of sea-cage net material and aging status on the fish-net interactions and associated welfare risks, either due to the biofouling communities that could harbor fish pathogens, but also possibly due to toxicological effects of the net material.
References
Glaropoulos, A., Papadakis, V.M., Papadakis, I.E., Kentouri, M., 2012. Escape-related behavior and coping ability of sea bream due to food supply. Aquaculture International. 20, 965-979.
Glaropoulos, A., Papadakis, V.M., Papadakis, I.E., Georgara, A., Kentouri, M., 2013. Sea bream interactions toward the aquaculture net due to the presence of micro-fouling. Aquaculture International. 22, 1203-1214.
Lewis, P.R., Reynolds, K., Gagg, C., 2004. Forensic Materials Engineering: Case studies. . CRC Press.
Oliveira, M., Ribeiro, A., Hylland, K., Guilhermino, L., 2013. Single and combined effects of microplastics and pyrene on juveniles (0+ group) of the common goby Pomatoschistus microps (Teleostei, Gobiidae). Ecological Indicators. 34, 641-647.
Peda, C., Caccamo, L., Fossi, M.C., Gai, F., Andaloro, F., Genovese, L., Perdichizzi, A., Romeo, T., Maricchiolo, G., 2016. Intestinal alterations in European sea bass Dicentrarchus labrax (Linnaeus, 1758) exposed to microplastics: Preliminary results. Environmental Pollution. 212, 251-256.