Maria Gabriella Marin

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.

Variations in gross biochemical composition, energy value and condition index of T. philippinarum from the Lagoon of Venice

Abstract Gross biochemical composition, energy value and condition index were investigated in two populations of Tapes philippinarum differently exploited by fishing in the Lagoon of Venice during the period July 2000–July 2001. Proteins, lipids, total carbohydrates and glycogen showed similar patterns in clams from all areas, highlighting the importance of the reproductive cycle and physiological features of the species in determining biochemical composition. The influence of environmental conditions and the effects of different fishing impact could also be observed. Carbohydrates exhibit minima in winter and maxima in spring–summer, while proteins have an irregular trend. Lipid seasonal variations are inversely related to glycogen contents; furthermore, lipids are higher in females than in males. Condition index and total energy content were influenced by both endogenous stress and environmental factors. In particular, the condition index, which gives an indication of the general physiological status of the animals, shows highest values in clams from the control area, not affected by fishing.

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.

Embryotoxicity of butyltin compounds to the sea urchin Paracentrotus lividus

Tributyltin (TBT) has been widely employed in marine anti-fouling paints as a biocide, although it represents a serious risk, particularly in estuarine and coastal water/sediment ecosystems. In this study, the embryotoxic effects of TBT and its degradation products, dibutyltin (DBT) and monobutyltin (MBT), were analyzed during the development of the sea urchin Paracentrotus lividus from post-fertilization to the pluteus stage, to better clarify ecotoxicological impact. The embryotoxicity of butyltins is concentration-dependent and increases proportionally with number of butyl groups. Significant growth reduction was observed at TBT concentrations as low as 0.01 microgram l-1; 1 microgram l-1 was the maximum concentration allowing embryos to reach the pluteus stage at 48 h post-fertilization. Development was blocked at the morula or blastula stage with higher TBT concentrations. DBT and MBT are less toxic: slowed development and a decrease in pluteus size occurred at 10 micrograms l-1 DBT and 0.5 mg l-1 MBT. Effects on both skeletal deposition and blocked embryonic development are suggested to be due to the interference of organotin compounds with intracellular calcium homeostasis.

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.

Air exposure and functionality of Chamelea gallina haemocytes: effects on haematocrit, adhesion, phagocytosis and enzyme contents

The Venus clam Chamelea gallina is fairly common along the western coasts of the Adriatic and is subjected to intense fishing. Since over the last 20 years extensive hypoxic and anoxic conditions have repeatedly damaged this natural resource, we decided to study the effects of anoxic stress on the functionality of clam haemocytes and the consequences on immune responses. Clams, exposed to air, close their valves and tissues become anoxic and metabolism processes switch to anaerobiosis. In these conditions, a significant decrease in the haematocrit value and in the percentage of acid phosphatase-positive haemocytes was observed, while the number of cells with beta-glucuronidase significantly increased after day 1. The above indices generally returned to control values when clams were re-immersed in seawater after 1 day of treatment. Clams exposed to air for 2 days and then re-immersed, attempted to recover in the subsequent 3 days. Animals had fully recovered on day 4. Three-day-exposed clams did not recover. Phagocytic and adhesion indices decreased significantly after the first day of air exposure. The change in frequency of three types of circulating cells (spreading, round, apoptotic) was also monitored.

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.

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.