Immunological and oxidative stress responses of the bivalve Scrobicularia plana to distinct patterns of heatwaves.

As a consequence of global warming, extreme events, such as marine heatwaves (MHW), have been increasing in frequency and intensity with negative effects on aquatic organisms. This innovative study evaluated for the first time, the immunological and physiological response of the estuarine edible bivalve Scrobicularia plana to different heatwaves, with distinct duration and recovery periods. So, extensive immune (total haemocyte count - THC, haemocyte viability, phagocytosis rate, respiratory oxidative burst of haemocytes, total protein, protease activity, nitric oxide and bactericidal activity of plasma) and oxidative stress (lipid peroxidation - LPO, superoxide dismutase - SOD, catalase - CAT and glutathione-S-Transferase - GST) analyses were performed in an experimental study that tested the impact of heatwaves during 25 days. The survival and condition of S. plana were not affected by the exposure to the extreme events. However, our data suggested that longer heatwaves with shorter recovery periods can be more challenging for the species, since THC and phagocytic activity were most affected under the temperature increase conditions. Regarding the oxidative status, the species increased its SOD activity while MDA production slightly declined to the increase of temperature, protecting the organism from cellular damage. These results indicate that S. plana has a great capacity to adapt to environmental temperature changes, however, the expected higher frequency/duration of heatwaves with climate change trends can cause some debility of the species face to other stressors, which can compromise its success in the future.

Mucosal immune responses in Senegalese sole (Solea senegalensis) juveniles after Tenacibaculum maritimum challenge: A comparative study between ocular and blind sides.

Most pathogens start the process of infection at the mucosal surfaces and therefore the mucosal immune response plays an essential role in the course of the infection. Due to the Senegalese sole (Solea senegalensis Kaup) condition of flatfish, the present comparative study aimed to analyse several immune-related enzymes as well as the bactericidal activity in the skin mucus from ocular and blind sides. For this purpose, Senegalese sole juveniles were bath challenged with a sub-lethal dose of Tenacibaculum maritimum for 24 h and sampled at 1, 2 and 3 weeks. The haematological profile and immune-related parameters were also measured in plasma in order to evaluate the systemic immune response after T. maritimum challenge. Results from this study showed that most parameters tested increased in skin mucus of bath challenged fish compared to unchallenged ones. In contrast, the sub-lethal dose tested did not influence the haematological profile including peripheral numbers the different leucocyte types. No variations were observed in plasma lysozyme, peroxidase, protease and haemolytic complement activities between unchallenged and bath challenged fish. This study suggests that the studied innate immune-related molecules are constitutively present in both skin mucus sides but at different levels. Interestingly, the levels of most parameters measured were higher on the ocular side than on the blind side, possibly due to the higher exposure to invasion by waterborne microorganisms on this side. Therefore, the present study brings some insights regarding local immune responses after bacterial challenge in skin mucus from the ocular and blind sides in one of the most valuable flatfish species in southern Europe.

Dietary tryptophan deficiency and its supplementation compromises inflammatory mechanisms and disease resistance in a teleost fish.

Tryptophan participates on several physiological mechanisms of the neuroendocrine-immune network and plays a critical role in macrophages and lymphocytes function. This study intended to evaluate the modulatory effects of dietary tryptophan on the European seabass (Dicentrarchus labrax) immune status, inflammatory response and disease resistance to Photobacterium damselae piscicida. A tryptophan deficient diet (NTRP); a control diet (CTRL); and two other diets supplemented with tryptophan at 0.13% (TRP13) and 0.17% (TRP17) of feed weight were formulated. Fish were sampled at 2 and 4 weeks of feeding and the remaining were i.p. injected with Phdp (3 × 106 cfu/fish) at 4 weeks and the inflammatory response (at 4, 24, 48 and 72 hours post-infection) as well as survival were evaluated. Results suggest that fish immune status was not altered in a tryptophan deficient scenario whereas in response to an inflammatory insult, plasma cortisol levels increased and the immune cell response was compromised, which translated in a lower disease resistance. When dietary tryptophan was offered 30% above its requirement level, plasma cortisol increased and, in response to bacterial infection, a decrease in lymphocytes, monocytes/macrophages and several immune-related genes was observed, also compromising at some degree fish disease resistance.

Annual assessment of the sea urchin (Paracentrotus lividus) humoral innate immune status: Tales from the north Portuguese coast.

Innate immune status of the sea urchin Paracentrotus lividus population from two different rocky shore beaches in the northern Portuguese coast was evaluated for a period of one year. Although some ecological studies regarding the effect of toxics on the immune parameters of the sea urchin were made in Portuguese waters, there is a current lack of knowledge concerning their immune status all over the year. In perspective of a changing ecosystem in these waters due to global warming and colonization of new species, it is important to assess the status of the major species living in the area. In this way, immune parameters such as total protein content, nitric oxide concentration, haemolytic activity, protease activity, lysozyme concentration and bactericidal activity were evaluated in the perivisceral coelomic fluid, and were correlated with the gonadal index of the population and water parameters. Also, the spawning period can upset some immune status parameters, and others such as haemolytic activity and bactericidal activity against Vibrio anguillarum, showed a clear correlation with the gonad maturation status. The knowledge of the basal immune status of the species could serve as ecological indicator of some stress agent or contaminant into the field; also, coelomic fluid is suggested as good quality marker to assess the immune status of sea urchins.

Protein expression profiling in haemocytes and plasma of the Manila clam Ruditapes philippinarum in response to infection with Perkinsus olseni.

The protein expression profiling in clam haemocytes and plasma in response to Perkinsus olseni was addressed. Adult Manila clams from a P. olseni-free bed were experimentally challenged with parasite zoospores to analyse immune response. In another experiment, the effects of longer term infection were assessed in adult clams collected from a P. olseni-affected bed, by comparing moderate to very heavily infected clams with non-infected ones. Haemocyte and plasma proteins were separated by two-dimensional electrophoresis; spot patterns were qualitatively compared between treatments within each experiment and the spots indicating differential protein expression associated with P. olseni challenge or with field infection were processed for protein identification. Fifteen clam proteins (four in haemocytes and eleven in plasma) of which expression was markedly affected by P. olseni were identified. Some of the identified proteins have a well-known role in clam immune response against the parasite, such as lysozyme and lectins. Rho GTPase-activating protein 6 could be a marker of resistance against P. olseni, which should be further studied.

Comparison of protein expression profiles between three Perkinsus spp., protozoan parasites of molluscs, through 2D electrophoresis and mass spectrometry.

The genus Perkinsus includes protozoan parasites of a wide range of marine molluscs worldwide, some of which have been responsible for heavy mollusc mortalities and dramatic economic losses. This study was performed with the aim of increasing the knowledge of Perkinsus spp. proteome. Proteins extracted from in vitro cultured cells of three species of this genus, P. marinus, P. olseni and P. chesapeaki, were analysed using 2D electrophoresis. Four gels from each species were produced. Qualitative and quantitative comparisons among gels were performed with Proteamweaver software. Cluster analysis grouped the four gels of each Perkinsus sp.; furthermore, P. marinus and P. olseni gels were grouped in a cluster different from P. chesapeaki. Around 2000 spots of each species were considered, from which 213 spots were common to the 3 species; P. chesapeaki and P. marinus shared 310 spots, P. chesapeaki and P. olseni shared 315 spots and P. marinus and P. olseni shared 242 spots. A number of spots were exclusive of each Perkinsus species: 1161 spots were exclusive of P. chesapeaki, 1124 of P. olseni and 895 of P. marinus. A total of 84 spots, including common and species-specific ones, were excised from the gels and analysed using MALDI-TOF and nESI-IT (MS/MS) techniques. Forty-two spots were successfully sequenced, from which 28 were annotated, most of them clustered into electron transport, oxidative stress and detoxification, protein synthesis, carbohydrate metabolism, signal transduction, metabolic process and proteolysis.