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.     

Reduction of attractiveness to the sea star Asterias forbesi (Desor) by the clam Mercenaria mercenaria (Linnaeus)

Both in field and laboratory choice tests, the sea star, Asterias forbesi (Desor), was attracted to distant upstream clams, Mercenaria mercenaria (Linnaeus). Clams exposed to upstream sea stars were chosen less frequently by downstream sea stars than clams without sea stars upstream. Sea stars neither attracted nor repelled downstream conspecifics.When clams were exposed to upstream sea stars, their oxygen consumption decreased, as did their pumping rate and activity (as measured by number of visible siphons). The former may result from one or both of the latter.It is concluded that clam and sea star sense each other over a distance by chemical cues. The response of the clam is a general lowering of activity which may result in decreased attractiveness to sea star predators. This response may serve as a defensive measure against distance detection by Asterias forbesi.     

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Preliminary appraisal of an innovative hydraulic dredge with vibrating and sorting bottom on clam beds (Chamelea gallina)

To solve some of the efficiency and environmental problems related to the use of the hydraulic dredge in bivalve mollusc fishing, a new experimental gear with vibrating bottom grid and other technical changes has been tested on clam (Chamelea gallina) beds in the Adriatic Sea. Comparative fishing surveys indicate a significantly different selectivity of the vibrating dredge, with respect to a standard gear: in fact, undersize clams are sieved out during the fishing process, and almost no juveniles are hauled. Regarding the product quality, laboratory analyses show that the amount of intervalvar sediment is significantly lower in the catch from the modified dredge, due to a sort of `warning' device. Nevertheless, the larger number of damaged shells suggests that the vibrating grid subjects the clams to a greater mechanical stress than the standard gear. As for the environmental effects, the vibrating bottom is selective for the associated fauna too, as is shown by the higher mean weight of most the by-catch species in the experimental gear. Moreover, the riddling goes on continuously, allowing the immediate release of the sorted-out organisms, which are repositioned in the area of origin, thus avoiding a `contagious' distribution. In conclusion, these positive preliminary results suggest real benefits of this innovative modified dredge, with wide margins for further improvement.     

Assessment of haemic neoplasia in different soft shell clam Mya arenaria populations from eastern Canada by flow cytometry

Diagnosis of haemic neoplasia (HN) in the soft shell clam, Mya arenaria, is often achieved by hematocytology and histology. Since neoplastic cells display tetraploid DNA contents, haemocyte cell cycle analysis was developed for use as a diagnosis tool. The aim of this study was to assess the application of a flow cytometry procedure of cell cycle analysis established for the common cockle, to clams and to evaluate different thresholds of value for the percentage of tetraploid cells for establishing HN disease status of individual clams and clam populations. HN status of six clam populations from eastern Canada was determined. Results of the present study demonstrate a flow cytometry procedure to be useful for HN diagnosis in clams. Individual clams were considered to be affected by HN when presenting at least 20% of haemocytes in S-4N phase; and negative when presenting less that 5% of haemocytes in S-4N phase. As discussed in this paper, intermediate cases represent uncertain diagnoses including either false-negative or false-positive clams, which are difficult to discriminate. At a population level, an additional threshold of 15% for the mean intensity of the disease is proposed, which means having in the population several individual clams presenting more than 20% of their haemocytes in S-4N phase. Based on these thresholds of value, only one population was considered as free of HN disease, and one population was unequivocally affected by HN. For the four other clam populations, further investigations are needed toward development and use of specific and objective biomarkers of HN.     

基于大叶藻成苗率的新型海草播种技术评价

蛤蜊播种技术是一种新型海草播种技术,该技术中,种子通过糯米糊粘在蛤蜊贝壳上,随蛤蜊穴居被埋入底质。为评价蛤蜊和糯米糊对种子成苗率的影响,以菲律宾蛤仔(Ruditapes philippinarum)和大叶藻(Zostera marina)种子为试验对象,设置了直接播撒种子(A1组)、直接埋种(A2组)、包埋糯米后播撒(B1组)、包埋糯米后埋入底质(B2组)、蛤蜊播种(C)5种处理,每种处理设置3个重复,在实验室的水槽中对其进行试验。结果表明:糯米糊对种子萌发有负面影响,但由于蛤蜊的行为使底质具有透气性,减轻了糯米糊对种子萌发的影响,种子成苗率可达到23.2%;海区试验中利用蛤蜊播种的两个样方中的成苗率分别为19.1%和9.9%。试验表明,蛤蜊播种技术适合作为一种经济、有效的播种技术用于海区海草床的建立和修复。     

Patterns of p53, p73 and mortalin gene expression associated with haemocyte polyploidy in the soft-shell clam, Mya arenaria

The molecular mechanisms by which haemocytes of clams are transformed in the course of haemic neoplasia remain by far unknown. The aim of this study was to quantify the expression of p53/p73 and mortalin genes, in relation with the ploidy status of clam haemocytes and to correlate the p53 expression with mortalin expression. For this purpose, soft-shell clams, Mya arenaria, were collected from an endemic zone for neoplasia. The ploidy of haemocytes was assessed for each individual clam by flow cytometry using a propidium iodide protocol, while p53/p73 and mortalin gene expressions were quantified by real-time RT-PCR. Results show that haemocytes of some clams with a moderate percentage (15-50%) of tetraploid cells have a significantly high level of p53 and p73 in comparison with clams belonging to categories with low (<15%) or high levels (>50%) of tetraploid cells, where low levels of expression of these genes were observed. Furthermore, mortalin gene expression is strongly correlated (r² = 0.68, p < 0.01) with p53 gene expression level. This reinforces the hypothesis of a cytoplasmic p53 sequestration mechanism in clam haemic neoplasia. Further studies are needed to confirm these preliminary results and further unravel the molecular pathways involved in this process. Our results are believed to provide phenotypic foundation for such studies to be undertaken.     

Behavioral plasticity of the soft-shell clam, Mya arenaria (L.), in the presence of predators increases survival in the field

Soft-shell clams, Mya arenaria, are sessile, suspension-feeding bivalves that are preyed upon by the exotic green crab, Carcinus maenas. Clams evade crab consumers by burrowing deeper into the sediment after perceiving a threat from a nearby predator. The purpose of this study was to determine the types of signals that M. arenaria use to detect predators and the types of behaviors clams use to avoid being eaten. In a field study, clams increased their burial depth in the presence of green crab predators consuming conspecifics that were caged nearby, and also increased burial depth after artificial tactile stimulation in the laboratory assay. These results indicate that clams can use chemical and mechanical cues to detect potential predatory threats. We performed a field study to examine the difference in survivability of clams that had burrowed deeper into the sediment in response to predators vs. control clams that were burrowed less deeply. Significantly higher survival rates were observed in clams that had initially burrowed more deeply, suggesting that increasing burial depth is a valid predator avoidance strategy. Some bivalves also alter their pumping rates in the presence of predators, making them less apparent and providing more structural defense by covering soft tissue, and we measured pumping time of soft-shell clams in the presence and absence of predators, when burrowing was not an option for escape. Soft-shell clams did not alter their pumping time in the presence of green crab predators, possibly because they employ a burrowing method called “hydraulic” or “jet-propelled” burrowing, where it is necessary for the clam to pump in order to burrow. Chemical signals and tactile cues instigated behavioral changes in M. arenaria, and this change in behavior (increasing burial depth) increased clam survival in the field.     

Differentially expressed genes of the carpet shell clam Ruditapes decussatus against Perkinsus olseni

Suppression–Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a reverse subtraction were carried out to identify up- and down-regulated genes in both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for further investigation into the functional basis of resistance to pathogens, have been detected for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of ESTs with potential similarity with known genes was 97, 42 among them were related with immunity and stress related functions. The pattern of expression of the immune selected genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams and compared with samples from an in vitro infection of clam haemocytes with Perkinsus zoospores. The maximum expression was found 1 h post infection. The complete open reading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined. Our results provide new insights into the molecular basis of host–pathogen interactions in R. decussatus.     

重金属Cd胁迫对红树蚬的抗氧化酶、消化酶活性和MDA含量的影响

摘要：本文在实验室条件下,观测不同Cd胁迫处理（时间和水平）对红树蚬（Geloina coaxans (Gmelin)）的胃组织中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性、丙二醛(MDA)含量,及淀粉酶、脂肪酶和蛋白酶等3种消化酶活性的影响效应。结果表明,高浓度组（4.00 mg?L-1和8.00 mg?L-1）在较短的暴露时间即出现SOD 活性显著诱导,而低浓度组则需要更长的暴露时间。所有处理组的CAT活性均先受诱导而后抑制,但去除胁迫后低浓度组活性上升,高浓度组则持续下降。低浓度处理组在胁迫初期MDA含量上升,但随后下降至较低水平;高浓度处理组MDA含量上升稍为滞后,但随后上升至较高水平。在消化酶方面,Cd对红树蚬胃组织淀粉酶的影响基本上表现为抑制;在胁迫初期脂肪酶活性受到显著诱导,随着胁迫时间延长酶活性则下降,同时胁迫解除后不同程度恢复;对蛋白酶的影响效应规律性不明显。显著的效应-剂量间相关关系存在于特定时间的SOD活性（1d）,CAT、淀粉酶活性和MDA含量（7d和恢复6d）。本文还探讨了这些指标作为生物标记物应用于监测海洋重金属污染的可能性。 关键词：红树林;红树蚬;Cd胁迫;消化酶;抗氧化酶;脂质过氧化     

Field validation of hsp70 stress proteins as biomarkers in Asian clam (Potamocorbula amurensis): is downregulation an indicator of stress?

The focus of this paper is to consider the applicability of the hsp70 stress protein response as a biomarker in field studies. Stress proteins (or heat shock proteins, hsp) of the hsp70 family are induced by sublethal concentrations of a variety of environmental pollutants. However, few studies have applied these proteins as biomarkers of environmental stress under field conditions. Our laboratory is investigating hsp70 proteins and other responses of Asian clam (Potamocorbula amurensis) as potential biomarkers in laboratory and field studies. Our efforts include two studies presently being conducted in northern San Francisco Bay: (1) monthly collection of clams from four sites along a cadmium contamination gradient; (2) 7 day in situ exposure of clams at two selected sites at Mare Island Naval Shipyard. Here we present results on hsp70 proteins in P. amurensis in field-collected and outplanted clams. Both field projects are ongoing, therefore the results presented here do not represent completed studies; rather, they illustrate a portion of our experience. For this workshop, we illustrate weaknesses and strengths of these proteins as biomarkers, and we underscore where additional work is needed. In field-collected clams (study no. 1), site-specific differences in levels of two hsp70 proteins, hsp70 and hsp76, were measured in May and June 1997. Although an inverse correlation exists between cadmium tissue concentrations and hsp70 protein levels, differences detected may be reflective of a salinity gradient. Results from recent laboratory exposures to cadmium and a range of salinities are discussed. After in situ exposure for 7 days (study no. 2), both hsp70 and hsp76 levels were significantly reduced in clams from site R. However, given a brief heat-shock in the laboratory, hsp70 protein levels were significantly higher in clams from this site than in controls. Results indicate that downregulation as well as upregulation of hsp70 proteins may be indicators of stress in P. amurensis.     

三亚近海诺亚砗磲分布特征及底质对其外套膜颜色的影响

文章调查了三亚近海蜈支洲岛(主要受旅游扰动影响)和三亚珊瑚礁国家级自然保护区(包括亚龙湾、鹿回头半岛和东西瑁岛,主要受非法捕捞影响)的诺亚砗磲(Tridacna noae)分布、密度、种群结构及影响因素,结果显示:(1)亚龙湾和蜈支洲岛海域诺亚砗磲密度最高,分别为2.00×10–2和1.92×10–2 ind./m2,...     

Field validation of hsp70 stress proteins as biomarkers in Asian clam (Potamocorbula amurensis): is downregulation an indicator of stress?

The focus of this paper is to consider the applicability of the hsp70 stress protein response as a biomarker in field studies. Stress proteins (or heat shock proteins, hsp) of the hsp70 family are induced by sublethal concentrations of a variety of environmental pollutants. However, few studies have applied these proteins as biomarkers of environmental stress under field conditions. Our laboratory is investigating hsp70 proteins and other responses of Asian clam (Potamocorbula amurensis) as potential biomarkers in laboratory and field studies. Our efforts include two studies presently being conducted in northern San Francisco Bay: (1) monthly collection of clams from four sites along a cadmium contamination gradient; (2) 7 day in situ exposure of clams at two selected sites at Mare Island Naval Shipyard. Here we present results on hsp70 proteins in P. amurensis in field-collected and outplanted clams. Both field projects are ongoing, therefore the results presented here do not represent completed studies; rather, they illustrate a portion of our experience. For this workshop, we illustrate weaknesses and strengths of these proteins as biomarkers, and we underscore where additional work is needed. In field-collected clams (study no. 1), site-specific differences in levels of two hsp70 proteins, hsp70 and hsp76, were measured in May and June 1997. Although an inverse correlation exists between cadmium tissue concentrations and hsp70 protein levels, differences detected may be reflective of a salinity gradient. Results from recent laboratory exposures to cadmium and a range of salinities are discussed. After in situ exposure for 7 days (study no. 2), both hsp70 and hsp76 levels were significantly reduced in clams from site R. However, given a brief heat-shock in the laboratory, hsp70 protein levels were significantly higher in clams from this site than in controls. Results indicate that downregulation as well as upregulation of hsp70 proteins may be indicators of stress in P. amurensis.     

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.     

The effects of water depth and density on the growth of a unionid clam*

SUMMARY. 1. Unionid clams from Narrow Lake, Alberta, were collected to quantify the natural variation in growth, to assess the natural variation in abundance, age and size distribution, and growth with water depth in the lake, and to conduct in situ experiments to directly test the effects of water depth (temperature) and clam abundance on clam growth. 2. The unionid clam, Anodonta grandis simpsoniana, showed wide variation in length at a given age. There were no significant differences in growth between clams collected at 1,3, 5, and 7m depths in the lake despite marked differences in water temperature. The wide variation in clam biomass within each depth zone may have masked possible effects of water depth. 3. The effect of water depth and variation in clam density on clam growth was tested directly by stocking clams into small enclosures at densities equivalent to 50, 150, 250, 350 and 450g m-2 (live weight) at each of 1, 3, 5 and 7 m depths in Narrow Lake (each depth and abundance treatment in triplicate). A uniform sandy substrate was used in all enclosures to eliminate any possible effect of substrate type on growth. 4. Mortality was negligible (0.9%) during the experiment. Clam density had no significant effect on clam growth which suggests that clam growth was not food limited in the lake. 5. Clams reared at 7 m grew more slowly than clams reared at 1, 3 and 5 m. Clams reared at 5 m grew more slowly than clams reared at 1 and 3m. Growth of clams reared at 1 and 3m did not differ. These differences in growth were strongly correlated with the measured differences in water temperature between depths. 6. Migration between depths probably accounts for the lack of a depth effect on clams growing in the natural habitat.     

Megapitaria squalida (Bivalvia: Veneridae) fishery landings and temperature relationship in Bahía de la Paz, México

The clam Megapitaria squalida is a fishing resource with increasing importance in Northwestern Mexico. Nevertheless, this fishery has shown important variations that could be related to environmental factors. To assess this, monthly landings of M. squalida were analyzed during 2002-2005 for Bahía de La Paz, B.C.S., and were related with monthly time series of sea surface temperature, derived from MODIS-Aqua Sensor. The results showed a positive and significant relationship between sea surface temperature and clam landings. The likely impact of anomalous conditions of sea temperature on this resource is discussed.     

Enzyme characterisation of the circulating haemocytes of the carpet shell clam,Ruditapes decussatus(Mollusca:bivalvia)

The occurrence of a number of lysosomal enzymes (Proteases, glycosidases, phosphatases, and esterases) inRuditapes decussatushaemocytes was demonstrated by cytochemical and colorimetric techniques. The levels of 18 enzymes tested monthly varied through the study period (18 months), although they did not conform to a seasonal pattern of variation. No important effect of clam age on enzyme activity levels of haemocytes was detected. In those cytochemical assays in which distinction between granulocytes and hyalinocytes was possible, lysosomal enzymes were only found in granulocytes. Phosphatase was detected inside cytoplasmic granules of granulocytes, suggesting the granules to be lysosomes. NADPH oxidase was not detected in clam haemocytes, which is consistent with the absence of oxidative metabolism coupled with phagocytosis in haemocytes of this clam species. Levels of lysozyme detected inside haemocytes were higher than in serum.     

DIET AND FORAGING BEHAVIOR OF SEA OTTERS IN SOUTHEAST ALASKA

Abstract: Direct observations of feeding sea otters ( Enhydra lutris ) at 11 sites in southeast Alaska showed infaunal clams to be the primary prey utilized by otters throughout the region. Foraging dive times associated with clam and sea urchin prey were significantly longer than those for more easily captured prey (crabs and mussels). Dive times and surface intervals were also generally correlated with water depth or apparent difficulty in obtaining buried prey. Male otters, which fed more extensively on clams than females, made significantly longer foraging dives than females. Foraging success remained high, even at sites where prey numbers were found to be very low during a related study. The very deeply burrowing geoduck clam ( Panope abrupta ), while common at several otter feeding sites, was rarely captured by otters. These results, combined with those of a companion study on prey numbers, indicate that butter clams ( Saxidomus giganteus ) account for the majority of the sea otter diet in southeast Alaska, and that sea urchins may represent relatively short-term prey in comparison to infaunal bivalves in regions where both prey types co-exist. Furthermore, the importance of butter clams in the sea otter diet and the tendency for this bivalve to retain chronically high levels of paralytic shellfish poisoning toxins in southeast Alaska increases the probability that toxic phytoplankton blooms influence sea otter distribution in this region.     

Parasitism and fitness of invaders: oligochaete Chaetogaster limnaei produces gill damage and increased respiration rates in freshwater Asian clams

Biological interactions between invasive and resident species are perceived as a key factor mediating impacts in invaded ecosystems, although symbiotic interactions have received relatively little attention. To evaluate the association between the invasive clam Corbicula fluminea and its oligochaete symbiont Chaetogaster limnaei, we conducted laboratory assessments of prevalence and intensity of infection, external and internal macroscopic alterations, and histological analyses of the gills of uninfected and infected clams collected from the shores of the Río de la Plata estuary in Argentina. To evaluate potential ramifications of physical alterations on physiological traits and host fitness, we conducted respiration experiments on the same clams. Worm prevalence was higher in smaller than large clams. While no macroscopical alterations were detected, several histopathological changes were observed in the gills of infected clams. Infected clams had significantly higher metabolic rates than uninfected conspecifics and smaller clams had higher metabolic rates than larger conspecifics. Increased host metabolic rates, even at very low infections intensities, were likely a compensatory response to the stress caused by gill damaged inflicted by the oligochaetes. The relationship between C. limnaei and invasive C. fluminea in Río de la Plata best fits as parasitism.

     

Exposure to anoxia of the clam, Chamelea gallina: II: Modulation of superoxide dismutase activity and expression in haemocytes

In order to investigate the influence of anoxic stress on haemocyte immune response, specimens of Chamelea gallina were exposed to 24 and 48 h anoxia. To evaluate recovery capacity, clams were maintained, at the end of the anoxic phase, for 24 h in reoxygenated seawater. In this paper, activity and expression of the antioxidant enzyme superoxide dismutase (SOD) were studied on haemocyte lysate and haemolymph. Reported results have shown that the anoxic stress changed strongly the response of C. gallina blood cells. Indeed, at the end of the anoxic phase in both experiments (24 and 48 h of anoxia exposure), SOD activity in haemocyte lysate decreased significantly with respect to the control, likely because of a decreasing superoxide anion generation in anoxia. Expression analyses were coherent with activity values.In the first experiment (24 h anoxia), reoxygenation determined an increase in activity of both Cu/Zn-SOD and Mn-SOD, but with values that remained significantly lower than those of the controls. It seems that after the applied anoxic stress, 24 h of recovery is not sufficient to restore pre-anoxic conditions. In the second experiment (48 h anoxia), SOD isoforms showed a different response during the recovery of animals. Cu/Zn-SOD activity dropped below the values showed by haemocytes of anoxic bivalves, while Mn-SOD activity values exceeded significantly those of controls. The different haemocyte response could be probably due to a further stress suffered by the clams because of a massive spawning during the reoxygenation phase. Therefore, the high values of activity shown by Mn-SOD during the recovery are likely to be due to the high inducibility of this isoform.In Cu/Zn-SOD expression analyses, two immunoreactive bands were highlighted in both experiments. The former (apparent molecular weight of 16 kDa) corresponds to the expression of SOD1 and the latter (apparent molecular weight of 28-30 kDa) could be attributed to EC-SOD (SOD3), a Cu/Zn-SOD isoform located in extracellular ambient and identified both in vertebrates and invertebrates. The strong SOD3 expression during anoxia exposure and the further spawning stress (second experiment) testified its inducibility in C. gallina haemocytes and haemolymph in response to stressful conditions.