The Atlantic blue crab (Callinectes sapidus, Rathbun, 1896) is a species of decapod crustacean belonging to the family Portunidae. It is native to the coastal areas and estuaries of the west coast of the Atlantic Ocean, but in recent years has spread to the Mediterranean Sea (Falciai and Minervini, 1992). On the western Atlantic coast, it is a species of high commercial value(Sharov et al., 2003). Its sometimes-excessive fishing has led to an 80% decline in stocks in recent years in places such as the Chesapeake Bay (US East Coast) (Lipcius and Stockhausen, 2002 ). This fact is leading to numerous studies and interested people in the profitability of large-scale farming of this species, which would provide relief to wild populations in most cases (Zmora et al., 2004). In recent years, there has been a growing public concern for animal welfare, although invertebrates do not generally fall under the scope of legislation covering living creatures farmed by humans. Decapod crustaceans are sentient beings, which not only respond to noxious stimuli, but are also capable of feeling pain, discomfort, and stress (de Souza, 2022). In 2021 the UK government officially recognised decapods as sentient animals, and they are now covered by animal welfare laws comparable to those used for mammals. Given that the Atlantic blue crab inhabits sandy and muddy bottoms in which it tends to bury itself (Falciai and Minervini, 1992), we considered that the presence or absence of substrate in the facilities in which these animals can be cultured could be fundamental for the welfare of these animals, as well as contributing to a significant reduction in stress. It has been shown that the presence of substrate in tanks improves behaviour and reduces aggressiveness in other species of portunid Zhu et al., (2022). Therefore, the aim of this study is to demonstrate how the use of substrate can improve the immune response of Atlantic blue crabs when exposed to a pathogenic bacterium.
Materials and methods.
Twenty specimens of Atlantic blue crab (217,5 ± 142 g) were obtained from the fish market ( San Pedro del Pinatar, Murcia, Spain ) and carried to the Marine F acilities at University of Murcia (Spain). The crabs were randomly separated in two groups, each group (n=10) was introduced into a tank with individual compartments for a single crab each one but sharing water and filtration (rack system) with the aim of avoiding fights. O ne of the groups (control) had no substrate at the bottom of the tanks, while the other one had 7 cm of sterile sand. After 30 days of acclimatising, a sample of 2 mL of haemolymph were extracted as initial time (t=0). Then both groups were challenged by bath with Vibrio alginolyticus (35 min, 2 x 106 ufc mL-1) . After this, crabs were sampled after 24, 48, 72 and 96 hours. After each experimental time, 2 mL of haemolymph were extracted from the V right pereiopod and the total haemocyte count and the cell´s percentage populations were determined. In addition, the obtaining of haemocyte lysate supernatant was used to measure the total quantity of protein , as well as the phenoloxidase and the lysozyme activities were measured . The results were expressed as mean ± standard error of the mean. Data were analysed by One-way ANOVA (followed by Tukey tests) to determine differences between experimental groups. The level of significance used was p < 0.05 for all statistical analysis.
Results and discussion.
The percentages of each cell population under basal conditions (time 0) showed that the percentage of granulocytes was the lowest in both groups , followed by the number of hyalinocytes and semigranulocytes . These results are supported by those found by other authors for the Atlantic blue crab(Chung et al., 2015). However, our results showed a change in the cell percentages of the animals challenged with V. alginolyticus . More specifically, a decrease in the percentage of hyaline cells was observed at 24, 48, 72 and 96 hours in the crabs maintained with substrate. In the experimental group without substrate, this decrease was also observed, although it occured at 72 and 96 h . In the case of semigranulocytes , no time-dependent variations were observed in the animals maintained without substrate, although in the crabs maintained in the presence of substrate a significant increase in the percentage of these cells at 24 , 48 and 72
h was observed. Comparing
between experimental groups, a higher proportion of hyalinocytes in the haemolymph of crabs maintained with substrate at 0 and 96 hours were observed compared to the crabs maintained without substrate. In the case of semigranulocytes, the percentage of this cell type was significantly higher in animals without substrate at 24 and 72 h compared to results observed in crabs maintained with substrate. However, no variations were detected in the number of granular cell population between both experimental groups. All these data indicate the great capacity for adaptation and plasticity that these crustaceans can present in the face of a biotic stress factor such as the challenge by pathogenic microorganism. This can be related to the data provided by Du et al. (2012) in the giant freshwater shrimp ( M. rosembergii ) challenged with Spiroplasma , where the cell populations varied over time. T he quantity of proteins in the haemocyte lysate supernatant varied between both experimental groups at 0, 24 and 48 h. R egarding the time, protein concentration increased at 24, 48 and 72 hours in both experimental groups
. These values could be since the pathogen caused a reaction that triggered intracellular antimicrobial protein synthesis at 24 hours, which remained active until 72 hours, at which time the protein synthesis reaction subsided. In relation to the immune-related enzymes, the lysozyme did not show any variation between experimental groups or experimental times. However, the phenoloxidase activity showed a great variability between animals, being undetected at 0 and 24 h in the crabs from both experimental groups. After 48 h of challenge, the phenoloxidase was detected although their activity was stronger at 72 h, being expressed in three crabs of the group without substrate and eight of the group maintained with substrate . Furthermore, the quantity of enzyme per animal was higher in the experimental group maintained with substrate. This fact could indicate that the phenoloxidase is not present in the haemolymph of animals not exposed to any pathogen, which could be released at 48 hours of challenge , being more efficient in the immune response in the case of animals maintained in presence of substrate.
The results obtained could contributed to the knowledge about the immune response and the requirements necessary to improve the animal welfare of brachyurans. Recently, Zhu et al. (2022) reported that the use of substrate significantly benefited South Korean blue crabs (Portunus trituberculatus) , reducing their cannibalism, improving their condition inside the tanks, and resulting in behavioural patterns of interaction with the substrate. Therefore, we could conclude that the substrate may not only act as a factor contributing to animal welfare in animal behaviour, but also that this state may be reflected in a better i mmune response to certain pathogens that often cause huge losses to aquaculture farms based on decapod production. However, there are still many aspects to be known about these animals, the brachyurans, which are essential in ecosystems and of great importance for human consumption.
De Souza Valente, C., Wan , A.H.L., 2021. Vibrio and major commercially important vibriosis diseases in decapod crustaceans. Journal of Invertebrate Pathology 181, 107527.
Du, J., Zhu. , 2012. Flow cytometry studies on the Macrobrachium rosenbergii hemocytes sub-populations and immune responses to novel pathogen spiroplasma MR-1008. Fish and Shellfish Immunology 33, 795-800.
Falciai , L., Minervini , R., 1992. Guida dei crostacei decapodi d’Europa , Scienze Naturali . Muzzio, F. Editor. pp: 132-35.
Zmora , O., Findiesen , A., Stubblefield, J., Frenkel, V., Zohar, Y., 2004. Large-scale juvenile production of the blue crab Callinectes sapidus. Aquaculture 244, 129–139.This research is part of the ThinkInAzul program supported by the MCIN with funding from European Union Next Generation EU (PRTR-C17.I1) and by the Comunidad Autónoma de la Región de Murcia-Fundación Séneca(Spain).