Physiological and biochemical responses to caffeine and microplastics in Mytilus galloprovincialis.

Caffeine (Caff) is one of the most widely used substances in the human diet and a well-recognized drug. Its input into surface waters is remarkable, but biological effects on aquatic organisms are unclear, particularly in combination with pollutants of suspected modulatory activity, like microplastics (MP). The aim of this study was to reveal the impact on the marine mussel Mytilus galloprovincialis (Lamark, 1819) of Caff (20.0 µg L-1) in the environmentally relevant combination (Mix) with MP 1 mg L-1 (size 35-50 µm) after the exposure for 14 days. Untreated and exposed to Caff and MP separately groups were also examined. Cell viability and cell volume regulation in hemocytes and digestive cells, as well as the indexes of oxidative stress, glutathione (GSH/GSSG) and metallothioneins levels, and caspase-3 activity in digestive gland were assessed. MP and Mix reduced Mn-superoxide dismutase, catalase, and glutathione S-transferase activities and level of lipid peroxidation, but increased the digestive gland cell viability, GSH/GSSG ratio (by 1.4-1.5-fold), metallothioneins level and their Zn content, while Caff did not affect oxidative stress indexes and metallothionein-related Zn chelation. Protein carbonyls were not targeted in all exposures. The distinguishing feature of the Caff group was the decline (2-fold) in caspase-3 activity and low cell viability. The multi-stress effect of Mix was shown by the worsening of the volume regulation of digestive cells and confirmed by discriminant analysis of biochemical indexes. The special capabilities of M. galloprovincialis as a sentinel organism make it an excellent bio-indicator reflecting the multi-stress effects in sub-chronic exposures to potentially harmful substances. The identification of the modulation of individual effects in combined exposure increases the need to base monitoring programs on studies of multi-stress effects in sub-chronic exposures.

Immunohistochemical characterization of Toll-like receptor 2 in gut epithelial cells and macrophages of goldfish Carassius auratus fed with a high-cholesterol diet.

Toll-like receptors (TLRs) are a group of pattern recognition molecules that play a crucial role in innate immunity. The structural conservation of the archaic TLR system suggests that the regulation of the immune response might be similar in fish and mammals. Several TLRs (TLR-1, -2, and -4) are expressed by activated macrophages, "foam cells" in human atherosclerotic lesions. To date, 20 different TLRs were identified in more than a dozen different fish species. In this study we found that feeding goldfish, Carrassius auratus, a high-cholesterol diet (HCD) resulted macrophage foam cell formation in the intestinal tissues. The expression of TLR2 has been found in foam cells and in the cytoplasm of enterocytes, however the staining was more intense at the apical surface of polarized intestinal epithelial cells and in the lamina propria. In the intestinal epithelial cells and in the lamina propria cells of the control fish the TLR2 was expressed at low levels. The intestinal epithelium is directly involved in the mucosal immune response through its expression of proinflammatory genes, release of inflammatory cytokines, and recruitment of inflammatory cells.

Impact of date palm fruits extracts and probiotic enriched diet on antioxidant status, innate immune response and immune-related gene expression of European seabass (Dicentrarchus labrax).

The application of additives in the diet as plants or extracts of plants as natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics and immunoprophylactics. The aim of the current study was to evaluate the potential effects of dietary supplementation of date palm fruit extracts alone or in combination with Pdp11 probiotic on serum antioxidant status, on the humoral and cellular innate immune status, as well as, on the expression levels of some immune-related genes in head-kidney and gut of European sea bass (Dicentrarchus labrax) after 2 and 4 weeks of administration. This study showed for the first time in European sea bass an immunostimulation in several of the parameters evaluated in fish fed with date palm fruits extracts enriched diet or fed with this substance in combination with Pdp 11 probiotic, mainly after 4 weeks of treatment. In the same way, dietary supplementation of mixture diet has positive effects on the expression levels of immune-related genes, chiefly in head-kidney of Dicentrarchus labrax. Therefore, the combination of both could be considered of great interest as potential additives for farmed fish.

Different effects of cGMP and cAMP in the intestine of the European eel, Anguilla anguilla.

The regulation of salt absorption in the sea water eel intestine was studied by evaluating the effects of theophylline, 8 Br cyclic adenosine monophosphate, 8 Br cyclic guanosine monophosphate, atriopeptin III, porcine vasoactive intestinal peptide and prostaglandin E1 on the short-circuit current, the transepithelial voltage difference and conductance and on the dilution potentials. It was shown that theophylline increased the transepithelial conductance and reduced the magnitude of the dilution potentials, indicating that the drug increases the anion conductance of the tight junctions. In addition its inhibitory effect on short-circuit current and transepithelial voltage difference suggests that theophylline also affects the transcellular transport mechanisms. It was shown that 8 Br cyclic guanosine monophosphate and 8 Br cyclic adenosine monophosphate affect transcellular mechanisms underlying C1- transport since both compounds reduced short-circuit current and transepithelial voltage difference; however, cyclic adenosine monophosphate is less effective since unlike cyclic guanosine monophosphate, even at maximal concentration, it was not able to completely abolish transepithelial voltage difference and short-circuit current. The effects of cyclic guanosine monophosphate and cyclic adenosine monophosphate were not additive even if cyclic guanosine monophosphate may produce further inhibition of ion transport in 8 Br cyclic adenosine monophosphate-treated tissues. In addition, cyclic guanosine monophosphate but not cyclic adenosine monophosphate reduced the magnitude of the dilution potentials, suggesting that cyclic guanosine monophosphate acts also on the paracellular pathway. Rat atriopeptin III, a peptide known to increase cyclic guanosine monophosphate cellular levels, behaved like 8 Br cyclic guanosine monophosphate since it lowered the dilution potentials and reduced short-circuit current and transepithelial voltage difference to near zero values, suggesting that the hormone modulates both paracellular and transcellular transport mechanisms, probably acting on the Na-K-2Cl cotransport. Agents acting via cyclic adenosine monophosphate, like porcine Basoactive intenstinal peptide and prostaglandin, behaved like 8 Br cyclic adenosine monophosphate. They were less effective in inhibiting ion transport and did not interfere with the paracellular pathway.