Morphological and cytoenzymatic characterization of haemocytes of the venus clam Chamelea gallina

A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.     

Mechanical disturbance affects haemocyte functionality in the Venus clam Chamelea gallina

The clam Chamelea gallina is fairly common along the western coastline of the Adriatic Sea, where it is subjected to intense fishing. To evaluate possible causes of stress in clam populations, the effects of acute mechanical disturbance on haemocyte functionality were analysed in both laboratory and field experiments. Among the various sources of stress that clams undergo during commercial fishing by hydraulic dredges, water pressure and mechanical sorting were considered, and three increasing levels of stress were applied. A reduction in immunosurveillance was highlighted, concentrations of circulating haemocytes, phagocytic and acid phosphatase activity indices decreased with increasing mechanical stress. The opposite trend shown by the beta-glucuronidase activity index is discussed. The response of clam haemocytes, detected on seasonal bases in two sites, often exhibited peculiar patterns, mostly depending on the reproductive cycle, and were probably influenced by different features of the sea bottom. Although recovery after stress was observed in laboratory experiments, some considerations are made on detrimental effects experienced in the field by under-sized clams, which are first fished and then thrown back into the sea.     

Age related properties of the Adriatic clam Chamelea gallina (L. 1758) hemocytes

The clam Chamelea gallina (L 1758) represents an important shellfish resource along Mediterranean coasts and its progressive depletion has been ascribed both to the overexploitation of stocks and to environmental or anthropic stressors. In this context, the investigation on immune parameters could represent a valid approach to measure the clam homeostasis condition and its possible influence on population dynamics. On this basis, the innate immune system, mainly represented by hemocyte phagocytosis, was investigated in organisms of different size. The results indicated a better phagocytic response in larger clams, strictly related to a greater concentration of granulocytes. A such variation in hemolymph composition appeared not dependent on environmental or endogenous factors, but rather on clam aging.     

Seasonal changes in physiological responses and evaluation of "well-being" in the Venus clam Chamelea gallina from the Northern Adriatic Sea

Chamelea gallina is an infaunal bivalve, widespread in sandy bottoms along Mediterranean coasts. It is an important economic resource for fisheries in the Adriatic, although in recent years over-fishing, and other concurrent factors, have dramatically decreased clam harvesting. In this context, it is of great interest to gain information on seasonal variations in the physiological performance of clams, for an overall evaluation of their well-being. In this study, laboratory experiments were performed to define allometric relationships and effects of temperature on clearance and respiration rates of C. gallina. The mean values of b coefficients were calculated and used to correlate physiological measurements to 'standard' body mass, when seasonally collected clams were analysed. The highest clearance rate (0.42 L h(-1)) was measured in clams collected in July 2000; the highest respiration rate (12.22 micromol O2 h(-1)) was observed in July 2001, leading to a negative scope for growth (-2.8 J h(-1)). The influence of environmental and endogenous factors, mostly reproduction, was discussed. Survival in air and condition indices, showing higher stress conditions in December 2000 and July 2001, were in good agreement with the other physiological measurements. The physiological responses examined in this study appear to be suitable for providing detailed indications on the well-being of C. gallina and may be useful for future studies aimed at eco-sustainable management of the resource.     

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.     

Genotyping of Cryptosporidium Isolates from Chamelea gallina Clams in Italy

Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.     

Genotyping of Cryptosporidium isolates from Chamelea gallina clams in Italy

Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.     

Combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam Ruditapes philippinarum

The combined effects of temperature and salinity on both immune responses and survival in air of the clam, Ruditapes philippinarum, were evaluated for the first time. The animals were kept for 7 days at three differing temperature (5 °C, 15 °C, 30 °C) and salinity values (18 psu, 28 psu, 38 psu), and effects of the resulting 9 experimental conditions on total haemocyte count (THC), Neutral Red uptake (NRU), haemolymph protein concentration, and lysozyme activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. The survival-in-air test was also performed. Two-way ANOVA analysis revealed that temperature influenced significantly THC and NRU, whereas salinity and temperature/salinity interaction affected NRU only. Temperature and salinity did not influence significantly HL and CFH lysozyme activity, as well as haemolymph total protein content. Survival-in-air test is widely used to evaluate general stress conditions in clams. In the present study, temperature and salinity were shown to influence the resistance to air exposure of R. philippinarum. The highest LT₅₀ (air exposure time resulting in 50% mortality) value was recorded in clams kept at 18 psu and 15 °C, whereas the lowest value was observed in clams kept at 28 psu and 30 °C. Overall, results obtained demonstrated that temperature and salinity can affect some functional responses of haemocytes from R. philippinarum, and suggested a better physiological condition for animals kept at 15 °C temperature and 18 psu salinity.     

2-DE proteomic analysis of HSP70 in mollusc Chamelea gallina

Bidimensional electrophoresis (2-DE) protocols were adapted on Chamelea gallina digestive glands studies by the analysis of Heat Shock Proteins (HSP) compared with monodimensional electrophoresis (1-DE) results. Because polycyclic aromatic hydrocarbons (PAH) act on HSPs, C. gallina specimens were exposed to 0.5 mg/L of benzo[a]pyrene (B[a]P) for 24 h, 7 and 12 days. Immunoblotting after 1-DE showed a single band of 70 kDa significantly induced after 7 days of B[a]P exposure. After 2-DE, eight major high-resolved spots between 17 and 98 kDa were revealed. Three spots fell within the range of 62–98 kDa and of 5–6 pI, parameters which could include HSP70. Two spots of 77 and 72 kDa, obtained after 2-DE immunoblotting, could correspond to constitutive HSC70 and to inducible HSP70 forms respectively. Changes observed in inducible and in constitutive forms might be related to an adaptation to stress and to a normal protein synthesis capability, respectively. Employment of 2-DE and relationship between HSP70 and HSC70 may be useful to clarify their role in molluscs subjected to stress events.     

Investigation of EROD, CYP1A immunopositive proteins and SOD in haemocytes of Chamelea gallina and their role in response to B[a]P

CYP1A sub-family represents the main form of cytochrome P450 involved in benzo[a]pyrene (B[a]P) detoxification, but there are no clear evidences about its presence in invertebrates. 7-Ethoxy resorufin O-deethylase (EROD) activity is strictly related to CYP1A presence, at the same time P450-dependent oxidative metabolism leads to reactive oxygen species (ROS) production, thought to be an important mechanism of pollutant-mediated toxicity in aquatic organisms. Superoxide dismutases (SODs), EROD and CYP1A activities and/or expressions were detected in haemocytes of pooled clams (Chamelea gallina) and cell-free haemolymph after 24 h, 7 and 12 days of exposure to 0.5 mg/L of B[a]P. After 24 h, B[a]P content was maximum in whole tissues. A 61 kDa band was recognized in haemocytes and cell-free haemolymph by polyclonal anti-fish CYP1A, while 53.5 and 63.8 kDa CYP1A immunopositive proteins were discriminate without differences of expression. Differently, EROD, MnSOD activity/expression and ECSOD expression decreased in haemocytes and haemolymph. C. gallina immune system presents an interesting response dose/time exposure of B[a]P and the 7 days condition highlights the major effects of xenobiotic action. The identification of basal EROD levels supports the possible presence of the CYP1A, never identified in C. gallina and more specifically never isolated in immune cells, as confirmed by CYP1A-immunopositive proteins identification.     

Effects of high temperatures on the ripening responses of bananas to acetylene

Unripe bananas were exposed to 1 ml litre^-1 of acetylene gas in air for different periods of time and different temperatures and then ripened at 20°C. It was found that exposure of fruits to acetylene for 4 h at 20 , 25 and 30°C, or 8 h at 20°C did not initiate ripening. Some fruits which had been exposed to acetylene for 4 h at 35°C or 8 h at 25°C and all fruit exposed for 8 h at 30° and 35°C ripened. These results indicate that bananas became more sensitive to ripening by acetylene as temperatures increased within the range of 20 to 35°C.     

Glycosidases of molluscs. Purification and properties of alpha-L-fucosidase from Chamelea gallina L.

An alpha-L-fucosidase had been purified approximately 300-fold from the liver (hepatopancreas) of the marine mollusc Chamelea gallina L. (= Venus gallina L.). During the different steps of the purification procedure it was difficult to remove the contaminant N-acetylglucosaminidase activity; but, after electrofocusing, a final preparation free of this and other glycosidades present in the crude extract was obtained. The purified enzyme has a broad specificity; it hydrolyzes p-nitrophenyl alpha-L-fucoside and natural substrates such as oligosaccharides containing fucosidic residues with alpha 1--2, alpha 1--3 and alpha 1--4 linkages; also it hydrolyzes fucose-containing glycopeptides, such as thyroglobulin glycopeptide, and glycoproteins as procine submaxillary mucin (previously rendered free of sialic acid). The enzyme has a pH optimum of 5.2 +/- 0.2, with a Km of 7 X 10(-5) M using p-nitrophenyl L-fucoside as substrate. It is inhibited by Hg2+ and some sugars, and activated by CN-, Zn2+, Ca2+ and EDTA. It shows two peaks by isoelectric focusing (at 6.3 and 6.6). The molecular weight of the alpha-L-fucosidase by gel filtration was over 2000000.     

Oxidative burst in hard clam (Mercenaria mercenaria) haemocytes

Haemocytes of bivalve molluscs are known to be responsible for many immunological functions, including recognition, phagocytosis, and killing or elimination of invading microorganisms, such as potentially infective bacteria and parasites. In many bivalves, killing of microorganisms engulfed by haemocytes is accomplished by a sudden release of reactive oxygen species (ROS) within the haemocytes; this response is referred to as an oxidative burst. Previous studies have failed to detect oxidative burst in haemocytes of the hard clam (northern quahog), Mercenaria mercenaria. In the present study, we applied a widely used chemical probe for ROS detection in haemocytes, dichlorofluorescin-diacetate (DCFH-DA), to haemocytes from this clam species and used flow cytometry to quantify fluorescence in individual haemocytes. Oxidation of DCFH-DA to the fluorescent product, DCF, within unstimulated haemocytes indicated that ROS were clearly produced in these cells. Two activators of oxidative burst, zymosan and bacterial extracellular products, which have been applied successfully to haemocytes in other species, stimulated large increases in ROS production in hard clam haemocytes. Furthermore, two inhibitors of ROS production, W-13 and diphenylene iodinium (DPI), significantly suppressed ROS production by haemocytes. Nitric oxide synthase inhibitors, NMMA and L-NIO, did not suppress ROS production, indicating that the observed oxidation of DCFH-DA is not mediated by nitric oxide. These results show unequivocally that haemocyte oxidative burst is active in M. mercenaria and, therefore, is a likely mechanism in host response to pathogens and parasites.     

Fine structural study of the spermatogenic cycle in Pitar rudis and Chamelea gallina (Mollusca,Bivalvia, Veneridae)

A comparative ultrastructural study of spermatogenesis was performed in the bivalve molluscs Pitar rudis and Chamelea gallina (Veneridae) from Turkey. Sertoli cells appeared to be rich in glycogen, lipid droplets and germ-cell phagolysosomes. Premeiotic cells exhibited nuage and a flagellum, with the Golgi complex and the rough endoplasmic reticulum originating proacrosomal vesicles during the pachytene stage. In round spermatids, the acrosomal vesicle migrated linked to the plasma membrane. In P. rudis, the acrosomal vesicle base formed a thin expansion that attached to the nuclear apex and was associated with development of the perforatorium. The cap-shaped acrosomal vesicle then differentiated into external and internal regions, and also into a small apical light region, although some cells exhibited an apical extension of the external component. On the contrary, two lateroapical light pouches developed in C. gallina. During spermiogenesis, chromatin became fibrillar and then condensed while the nucleus turned conical shaped in P. rudis or slightly curved in C. gallina. In P. rudis, the midpiece contained glycogen and four mitochondria, although five mitochondria were sometimes observed, whereas in C. gallina the midpiece contained four mitochondria. Comparison with other members of Veneroida shows a common ectaquasperm type, but novel findings in acrosome biogenesis.     

Morphological characterization and functional immune response of the carpet shell clam (Ruditapes decussatus) haemocytes after bacterial stimulation

The morphology and functionality of Ruditapes decussatus haemocytes have been characterized by light microscopy and flow cytometry, leading to the identification of three different cellular subpopulations. Granulocytes were the largest cells, the hyalinocytes were smaller and contained fewer granules and the intermediate cells showed a size similar to hyalinocytes and a higher number of granules. The phagocytosis of different particles and the associated production of oxygen radicals were measured by flow cytometric methods. Granulocytes were the most active cells, followed by the intermediate cells and hyalinocytes. The effect of stimulation of haemocytes with lipopolysaccharide (LPS), with a heat inactivated bacterial mixture or with the infection of Vibrio splendidus on the cell viability and the expression of selected immune-related genes were studied. While significant low levels of damaged cells were registered in LPS-stimulated cells, the treatment with dead bacteria or V. splendidus reduced cell viability 1 h, 3 h and 6 h after treatment. The stimulation of haemocytes with LPS and dead bacteria induced changes in the expression of defender against cell death (DAD-1), thrombin, prosaposin, inhibitor of apoptosis (IAP), factor B and C3 complement component.     

Effects of predator confusion on functional responses

A number of experiments have addressed how increases in nitrogen availability increase the productivity and decrease the diversity of plant communities. We lack, however, a rigorous mechanistic understanding of how changes in abundance of particular species combine to produce changes in community productivity and diversity. Single experiments cannot provide insight into this issue because each species occurs only once per experiment, and each experiment is conducted in only one location; thus, it is impossible from single experiments to determine whether responses of particular species are consistent across environments or dependent on the particular environmental context in which the experiment was conducted. To address this issue, we assembled a dataset of 20 herbaceous species that were each represented in at least 6 different fertilization experiments and tested whether responses were general across experiments. Of the 20 species, one consistently increased in relative abundance and five consistently decreased across replicate experiments. A partially-overlapping group of 8 species displayed responses to nitrogen that varied predictably among experiments as a function of geographic location, neighboring species, or a handful of other community characteristics (ANPP, precipitation, species richness, relative abundance of focal species in control plots, and community composition). Thus, despite modest replication and a limited number of predictor variables, we were able to identify consistent patterns in response of 10 out of 20 species across multiple experiments. We conclude that the responses of individual species to nitrogen addition are often predictable, but that in most cases these responses are functions of the abiotic or biotic environment. Thus, a rigorous understanding of how plant species respond to nitrogen addition will have to consider not only the traits of individual plant species, but also aspects of the communities in which those plants live.     

Molecular mechanisms governing plant responses to high temperatures

The increased prevalence of high temperatures(HTs) around the world is a major global concern, as they dramatically affect agronomic productivity. Upon HT exposure, plants sense the temperature change and initiate cellular and metabolic responses that enable them to adapt to their new environmental conditions.Decoding the mechanisms by which plants cope with HT will facilitate the development of molecular markers to enable the production of plants with improved thermotolerance. In recent decades, genetic, physiological, molecular, and biochemical studies have revealed a number of vital cellular components and processes involved in thermoresponsive growth and the acquisition of thermotolerance in plants. This review summarizes the major mechanisms involved in plant HT responses, with a special focus on recent discoveries related to plant thermosensing, heat stress signaling, and HT-regulated gene expression networks that promote plant adaptation to elevated environmental temperatures.     

Effects of low light and high temperature on pediveligers of the fluted giant clam Tridacna squamosa

ABSTRACT

This paper examines the responses of the fluted giant clam Tridacna squamosa pediveligers to elevated temperature and reduced light levels. In a light reduction experiment, a total of 104,000 T. squamosa pediveligers were exposed to four different levels of shading for approximately one month. The most heavily shaded treatment, at 0.4% of ambient light, had significantly lower survival than the other groups, which all received 1% or more of ambient light. In a second experiment, for approximately two weeks 13,000 T. squamosa pediveligers were divided among three treatments: one at ambient temperature averaging 29.5 °C, and two with elevated temperatures averaging 32.2 °C and 34.8 °C. The elevated temperature treatments resulted in near total mortality. The highest temperature survived by any pediveliger was 32.8 °C. Our results indicate a potential synergetic effect, with turbidity causing giant clam pediveligers to settle in shallower water―where they will likely be exposed to higher temperatures.