Valerio Matozzo

First evidence of immunomodulation in bivalves under seawater acidification and increased temperature

Water acidification, temperature increases and changes in seawater salinity are predicted to occur in the near future. In such a global climate change (GCC) scenario, there is growing concern for the health status of both wild and farmed organisms. Bivalve molluscs, an important component of coastal marine ecosystems, are at risk. At the immunological level, the ability of an organism to maintain its immunosurveillance unaltered under adverse environmental conditions may enhance its survival capability. To our knowledge, only a few studies have investigated the effects of changing environmental parameters (as predicted in a GCC scenario) on the immune responses of bivalves. In the present study, the effects of both decreased pH values and increased temperature on the important immune parameters of two bivalve species were evaluated for the first time. The clam Chamelea gallina and the mussel Mytilus galloprovincialis, widespread along the coast of the Northwestern Adriatic Sea, were chosen as model organisms. Bivalves were exposed for 7 days to three pH values (8.1, 7.7 and 7.4) at two temperatures (22 and 28°C). Three independent experiments were carried out at salinities of 28, 34 and 40 PSU. The total haemocyte count, Neutral Red uptake, haemolymph lysozyme activity and total protein levels were measured. The results obtained demonstrated that tested experimental conditions affected significantly most of the immune parameters measured in bivalves, even if the variation pattern of haemocyte responses was not always linear. Between the two species, C. gallina appeared more vulnerable to changing pH and temperature than M. galloprovincialis. Overall, this study demonstrated that climate changes can strongly affect haemocyte functionality in bivalves. However, further studies are needed to clarify better the mechanisms of action of changing environmental parameters, both individually and in combination, on bivalve haemocytes.

Can the combination of decreased pH and increased temperature values induce oxidative stress in the clam Chamelea gallina and the mussel Mytilus galloprovincialis

The combined effects of decreased pH and increased temperature values on antioxidant enzyme activities and lipid peroxidation were evaluated for the first time in the clam Chamelea gallina and the mussel Mytilus galloprovincialis, two bivalve species that are widespread along the northwestern coast of the Adriatic Sea. For 7 days, bivalves were exposed to three pH values (8.1, 7.7 and 7.4) at two temperatures (22 and 28 °C). Three independent experiments were carried out at salinity values of 28, 34 and 40 psu. Superoxide dismutase, catalase and glutathione S-transferase activities as well as lipid peroxidation were measured in the gills and digestive gland of the bivalves. The results demonstrated that the experimental conditions significantly influenced the biochemical parameters of the bivalves, although the variation pattern varied depending on the species and tissues analysed.

Immunotoxic effects of organotin compounds in Tapes philippinarum.

One of the most harmful groups of coastal pollutants is the organotin compounds (OTCs) which have severe effects on both aquatic organisms and mammals including humans. The immunotoxic effects of OTCs were studied in the cultivated clam Tapes philippinarum by determining the immunosuppressant role on in vitro yeast phagocytosis at low doses (0.01, 0.05, 0.1 microM). The phagocytic index was significantly reduced in an irreversible non-lethal manner depending on concentration and lipophilic affinity. The order of inhibition was TBT > or = DBT > MBT for butyltins and TPTC > TPTA > or = TPTH for triphenyltins.

Biomarkers for TBT immunotoxicity studies on the cultivated clam Tapes philippinarum (Adams and Reeve, 1850).

The aim of this investigation was to quantify the effects of tributyltin (TBT) on the immune reactivity of haemocytes from the cultivated clam Tapes philippinarum (Adams and Reeve, 1850) using a series of in vitro bioassays. It is known that TBT has adverse effects on cellular immune functions like mobility, phagocytosis and lysosomal enzyme activity. As defining TBT-sensitive immunologic biomarkers in sentinel organisms is important in the field of ecotoxicology, the authors propose three indexes, amoebocytic (A.I.), phagocytic (P.I.), and lysosomal activity (L.A.I.), as sensitive and useful biomarkers to assess environmental risks due to TBT contamination.

Accumulation of selenium in Ulva sp. and effects on morphology, ultrastructure and antioxidant enzymes and metabolites

The impact of selenium (Se) on Ulva sp., a green macroalga naturally growing in the Venice Lagoon, was investigated. The alga was provided for 10 days with concentrations of selenate (Na(2)SeO(4)) ranging from 0 to 100 μM. Se accumulation in the algal biomass was linearly related to the selenate dose and this relationship was not affected by the high sulfate concentration measured in the seawater. The amount of Se measured in the alga was always relatively low and not hazardous to algal consumers. However, Se induced the formation of hydrogen peroxide (H(2)O(2)) in Ulva sp. and, as a result, the activity of antioxidant enzymes (superoxide dismutase, SOD, and catalase, CAT) and the amount of antioxidant metabolites (phenols, flavonoids and carotenoids) increased, even when selenate was supplied to the macroalga at low concentration (2.5 μM). This indicated that different components of the antioxidant defence system played a pivotal role in overcoming oxidative damage by Se in the macroalga, and explained the lack of morphological and ultrastructural alterations in Ulva sp. exposed to selenate.

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction.

Since it is often difficult to estimate possible adverse effects due to contamination in selected ecosystems, multi-species biomonitoring may provide more information, taking into account different routes of exposure, ecological roles and metabolic capabilities of animals. In this context, we exposed for 7 days the mussel Mytilus galloprovincialis and the crab Carcinus aestuarii to 4-nonylphenol (NP), a well-known xenoestrogen. In mussels (0-0.2 mg NP l(-1)), we measured NP bioaccumulation in soft tissues and vitellogenin (Vg)-like protein levels in digestive glands from both males and females by the alkali-labile phosphate assay (ALP). As no reference data were available for crab exposure, the NP 96-h LC(50) value was previously determined. Then, in sublethally exposed (0-1.0 mg NP l(-1)) male crabs, NP bioaccumulation and Vg levels were measured in hemolymph, gonads and digestive gland. Bioaccumulation of NP increased from 43 to 371 microg g(-1) d.w. in mussels, and from 3.6 to 37 microg g(-1) d.w. in crabs, depending on the NP concentration in water. Dose-dependent Vg-like protein induction was observed in both species, appearing to be related to NP bioaccumulation, although a partial decrease was recorded at the highest concentration tested. A similar trend was observed in both digestive gland and gonad of exposed crabs; Vg increased to a lesser extent, although significantly, in hemolymph. Results demonstrated that NP induces Vg synthesis both in male and female mussels, as well as in male crabs. On the basis of the responsiveness of both species investigated, a multi-species approach is indicated in biomonitoring programmes.

First evidence of cell division in circulating haemocytes from the Manila clam Tapes philippinarum

In the present study, we report on haemocyte distribution, determined by a Coulter Counter, in the clam Tapes philippinarum. In addition, cytoskeleton components of haemocytes were examined using specific probes for F‐actin and α‐tubulin. The mean number of circulating haemocytes was 5 (×106) cells/ml haemolymph. Two main haemocyte populations were found in the haemolymph: small cells, 2–3 μm in diameter and 10–100 fl in volume; and large cells, 6–10 μm in diameter and 150–400 fl in volume. Analysis of the haemocyte cytoskeleton revealed bundles of actin filaments oriented according to the cell major axis, and microtubules radiating from the microtubule‐organizing centre in proximity of the nucleus. Interestingly, mitotic spindles were also found radiating from the microtubule‐organizing centres, located at the spindle poles (centrosomes) of undifferentiated cells. On the basis of both our previous findings regarding circulating stem cells (Cima, F., Matozzo, V., Marin, M.G., Ballarin, L., 2000. Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation. Fish Shellfish Immunol 10, 677‐693) and new information from the present study, we suggest that haemoblasts are able to divide in the haemolymph of T. philippinarum. To our knowledge, this is the first report of mitotic spindles in circulating haemocytes from a bivalve species.

Effects of the antidepressant fluoxetine on the immune parameters and acetylcholinesterase activity of the clam Venerupis philippinarum

Fluoxetine is an antidepressant used worldwide for the treatment of depression and other psychological disorders. The occurrence of fluoxetine in aquatic environments has been demonstrated. However, there is a lack of information about the effects of fluoxetine on non-target species, such as bivalve molluscs. In the present study, the effects of fluoxetine on the immune parameters of the clam Venerupis philippinarum were evaluated for the first time. Clams were exposed to various sublethal concentrations of fluoxetine (0, 1, 5, 25, 125, 625 μg l⁻¹) for 7 days, and the effects on the total haemocyte count (THC), the diameter and volume of haemocytes, haemocyte proliferation, Neutral Red uptake (NRU), and lysozyme activity in cell-free haemolymph (CFH) were evaluated. In addition, acetylcholinesterase (AChE) activity was measured in clam gills as a biomarker of neurotoxicity. A significant increase in THC values was observed in clams exposed to 25 μg l⁻¹ compared with controls, whereas no significant variations were recorded in either the diameter or the volume of haemocytes. Haemocyte proliferation increased significantly in animals exposed to 25, 125 and 625 μg l⁻¹ compared with controls. NRU decreased significantly in the haemocytes of clams exposed to 1 or 5 μg l⁻¹, whereas NRU returned to control values in clams exposed to the highest fluoxetine concentrations tested (25-625 μg l⁻¹). No significant alterations were observed in CFH lysozyme activity, whereas gill AChE activity decreased significantly in clams exposed to 1 or 5 μg l⁻¹. Overall, the obtained results demonstrated that fluoxetine markedly affected immune parameters and AChE activity in clams.

Biomarker responses and contamination levels in crabs (Carcinus aestuarii) from the Lagoon of Venice: an integrated approach in biomonitoring estuarine environments.

An integrated biological-chemical approach is necessary to evaluate correctly the environmental status of bodies of water, as suggested by the EU Water Framework Directive. The shore crab Carcinus aestuarii, sampled in the Lagoon of Venice (NE Italy), was used as a biomonitor species, and the chemical concentrations of 42 organic pollutants (HCHs, PAHs, PCBs, DDTs, PBDEs), biological responses related to neurotoxicity (AChE inhibition), detoxification mechanisms (CYP450 induction) and endocrine alterations (vitellogenin-like protein induction) were measured at the same time. The responsiveness of biomarkers as predictors (or descriptors) of chemical contamination was evaluated by multivariate regression analysis, revealing good predictor potential for the selected biomarkers. Biomonitoring in the Lagoon of Venice revealed a predominance of DDT and PCB compounds, especially near industrial sites or large cities. Endocrine alterations, not always correlated with the presence of measured compounds, were also detected in many areas, suggesting exposure to compounds able to interfere with the crab endocrine system.

The nonsteroidal anti-inflammatory drug, ibuprofen, affects the immune parameters in the clam Ruditapes philippinarum

The effects of ibuprofen (IBU) on the immune parameters of the clam Ruditapes philippinarum were investigated after a 7-day exposure to sublethal IBU concentrations (0, 0 + ethanol, 100, 500, and 1000 μg/L). Total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation and uptake of the vital dye Neutral Red (NR) were measured. The cytotoxicity (assessed by the lactate dehydrogenase assay, LDH) and the capability of IBU to induce DNA fragmentation (indicative of apoptosis) were also investigated. The exposure of clams to the highest IBU concentration significantly reduced their THC, whereas no significant changes were observed in either the diameter or volume of haemocytes. Significant increases in haemocyte proliferation were recorded in clams that were exposed to the two highest tested concentrations of IBU. Exposure of clams to 1000 μg IBU/L significantly reduced NR uptake and increased haemolymph LDH activity. Conversely, IBU did not induce DNA fragmentation in haemocytes. Although the IBU concentrations tested in this study were higher than those generally recorded in aquatic environments, results obtained indicate that exposure of clams to IBU induces significant alterations in the immune parameters and suggest potential immunosuppression in treated clams.