Appraisal of prospective bivalve immunomarkers

Worldwide concern over threats to natural resources and public health has led to increased efforts to monitor and assess environmental conditions. This has stimulated the need for development and application of select biological and ecological measurements, or indicators, that are responsive to environmental stress. Measures of bivalve mollusc defence activities, such as haemocyte density, phagocytic activity, locomotion and production of cytotoxic molecules; and haemolymph constituents, such as agglutinins and lysozyme, have potential as indicators and appear to be responsive to xenobiotic chemical insults in the aquatic environment. However, basic research on the relevance of these measurements in inferring resistance to disease or enhanced survival is currently insufficient, reducing their value as potential biomarkers to address environmental objectives. In addition, variation in defence activities caused by seasonal temperature and reproductive cycling, salinity changes, nutritional status, diseases and parasites, and genetic stocks is high and may limit applicability of bivalve defence-related measurements as indicators. This review examines these sources of variability and their possible implications for interpreting changes in bivalve defence activity as an indicator of stress. Examples of contaminant-induced changes in bivalve defence functions are described.     

DNA damage as a bivalve biomarker

Bivalves have been used in numerous environmental assessment studies, chiefly because they are sessile deposit or suspension feeding organisms found in or near sites of environmental concern, and they can be easily collected, sorted and deployed at sites of interest. Monitoring studies utilizing bivalves currently rely on the comparison of growth, survival, and contaminant bioaccumulation. Data gathered from 'reference' sites are compared with those of populations at assessment sites. These studies require extended periods of exposure, lasting weeks to months, and the use of a well defined population of test organisms of similar size, age, and condition. In many cases time and resources require researchers to restrict their sampling to the organisms on-hand at a particular site without the benefit of any reference data. Therefore more versatile and sensitive assessment methods are needed. Because effects at higher levels of organization such as growth, development, and survival are initiated at the molecular and cellular levels attempts have been made to identify useful biomarkers at these levels. The proposed advantages of molecular/cellular biomarkers are that they will respond to stress predictably and more rapidly, and will be indicative of the mechanisms of toxicity thereby yielding a rudimentary characterization of the contaminant(s) influencing them. In the following communication we will report on past and current developments in the monitoring of DNAdamage as an environmental biomarker.     

Peroxisomes in the digestive diverticula of the bivalve mollusc Nucula sulcata

Summary Electron microscopy of the digestive diverticula of the protobranch bivalve, Nucula sulcata, revealed the presence of peroxisomes in the basal regions of the epithelial cells lining the main and secondary ducts, and in the digestive and secretory (basophil) cells of the tubules. Those in the secretory cells are elongate and somewhat flattened, while those of the other cell types have a spherical form. Two distinct types of nucleoid are normally present within the secretory cell peroxisomes, one compact, crystalline, and finely polytubular, the other comprising isolated secondary tubules arranged in a linear series across the width of the organelle. The peroxisomes of the digestive and duct cells contain coarsely polytubular cores arranged in two clusters orientated more or less at right angles; the duct cell peroxisomes may also contain a second nucleoid in the form of a compact finely polytubular core. Sections incubated in a medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide reveal an electron dense reaction product within the peroxisomes of all the cell types. Catalase is considered to be responsible for the reaction.     

The earliest known estuarine bivalve assemblage, Lower Ordovician of northwestern Argentina

The oldest known estuarine bivalve assemblage is documented from the Lower Ordovician (upper Arenig-lower Llanvirn) Alto del Cóndor Formation, which crops out in the Cordillera Oriental of northwestern Argentina. This unit displays most of the diagnostic sedimentary attributes of estuarine environments. Biotic components include low-diversity trace fossils and a peculiar bivalve fauna consisting of the new genera Konduria, Pseudoredonia, and Pucamya, and the new species Redonia condorensis. This constitutes the earliest known occurrence of bivalves in brackish waters, suggesting that the capability of this clade to colonize estuarine environments developed early in their radiation.     

Middle and Upper Jurassic bivalve associations from the Iberian Range (Spain)

The four sections richest in bivalves from the Middle and Upper Jurassic of the Iberian Range (Spain) were selected for a quantitative palaeoecological analysis of the bivalve fraction of the macrobenthos. Five bivalve associations and two assemblages were recognized with the help of a Q-mode hierarchical cluster analysis (Ward method). The main environmental factors controlling bivalve associations are thought to be substrate, water energy and distribution of organic matter. The bivalves exhibit a distinct spatial and temporal distribution pattern within the Aragonian Branch of the Iberian Range. Four of the bivalve associations occur in the Upper Oxfordian (Sot de Chera Fm) and one association in the Lower Callovian (Chelva Fm). In the Sot de Chera and Loriguilla formations, the abundance of bivalves decreases from NW to SE i.e., from relatively close to the shore line towards the distal-most part of the carbonate platform. In the Chelva Fm, bivalves are abundant in the Ariño region, interpreted as a palaeogeographic high. The spatial distribution of bivalves might have been largely controlled by the availability of nutrients.     

Spatial heterogeneity in parasite infections at different spatial scales in an intertidal bivalve

Spatial heterogeneities in the abundance of free-living organisms as well as in infection levels of their parasites are a common phenomenon, but knowledge on parasitism in invertebrate intermediate hosts in this respect is scarce. We investigated the spatial pattern of four dominant trematode species which utilize a common intertidal bivalve, the cockle Cerastoderma edule, as second intermediate host in their life cycles. Sampling of cockles from the same cohort at 15 sites in the northern Wadden Sea (North Sea) over a distance of 50 km revealed a conspicuous spatial heterogeneity in infection levels in all four species over the total sample as well as among and within sampling sites. Whereas multiple regression analyses indicated the density of first intermediate upstream hosts to be the strongest determinant of infection levels in cockles, the situation within sites was more complex with no single strong predictor variable. However, host size was positively and host density negatively correlated with infection levels and there was an indication of differential susceptibility of cockle hosts. Small-scale differences in physical properties of the habitat in the form of residual water at low tide resulted in increased infection levels of cockles which we experimentally transferred into pools. A complex interplay of these factors may be responsible for within-site heterogeneities. At larger spatial scales, these factors may be overridden by the strong effect of upstream hosts. In contrast to first intermediate trematode hosts, there was no indication for inter-specific interactions. In other terms, the recruitment of trematodes in second intermediate hosts seems to be largely controlled by pre-settlement processes both among and within host populations.     

Esterase-like and fibronectin-like polypeptides share similar sex-cell-biased patterns in the gonad of hermaphroditic and gonochoric species of bivalve mollusks

The mechanism of sexualization of the tubular gonad in seawater bivalves is unknown, and no information regarding the genes involved in this process is yet available, except for the identification of esterase (Est)-like "male-associated polypeptide" in the male gonad of Mytilus galloprovincialis. Our present work reveals distinct protein profiles specific for the testicular or ovarian portion of the ovotestis of Pecten maximus. Two proteins exhibiting testis- or ovary-dependent enrichment in the ovotestis have been identified and partially characterized as Est-like and fibronectin (Fn)-like polypeptides, respectively. Immunofluorescence has demonstrated a close association between the localization of these polypeptides and the gonad tubule network and interstitial stroma of the ovotestis of P. maximus. We also present evidence of Est-like and Fn-like protein enrichment, respectively, in testicular and ovarian tissue in hermaphroditic, sex-reversal, and gonochoric species of seawater bivalves. Together, the results (1) strongly suggest that sex-cell-biased expression of Est-like and Fn-like polypeptides in gonad tissue is a widespread phenomenon among bivalve mollusks, despite the high diversification of their sexual patterns, (2) confirm and expand our previous demonstration of sex-biased protein expression in M. galloprovincialis, and (3) indicate a direct link between germ cell differentiation and sexual specialization of the bivalve somatic gonad.     

Comparative taphonomy of three bivalve species from a mass shell accumulation in the intertidal regime of North Sea tidal flats

This study comprises a comparative taphonomic analysis of three endobenthic bivalves (Mya arenaria, Cerastoderma edule, and Macoma balthica) derived from a mass accumulation of mainly vertically packed shells from the “Wurster Watt” in Lower Saxony’s Wadden Sea, German Bight. Bulk samples from two transects were analyzed with respect to taxonomic composition, left/right valve presence, counts and weight percentages of taxa, and size-frequency distributions. Taphonomic features including abrasion, fragmentation, encrustation and bioerosion were subjected to a semi-quantitative analysis. Taphonomic results show significant differences with respect to bivalve taxa as well as between transects. Mya arenaria, a large endobenthic bivalve, shows the greatest amount of fragmentation and is often encrusted by balanids and bryozoans. The smaller and more robust Cerastoderma edule has the greatest values for bioerosion especially by the polychaete Polydora ciliata. Macoma balthica, which has the thinnest valves, shows the highest values for abrasion, but low values for the rest of the measured taphonomic features. Taphonomic differences between the taxa, samples and transects are related to (1) the origin of the bivalves (from living populations or exposed colonization horizons), (2) the different size and morphology of the valves (themselves related to mode of life), (3) the taphonomic trajectories of the different bivalves, as well as (4) the varying depositional environment of the two transects.     

Impact of seston characteristics on qualitative particle selection sites and efficiencies in the pseudolamellibranch bivalve Crassostrea gigas

To date, knowledge of the qualitative particle selection sites and conditions in the widely-distributed bivalve Crassostrea gigas is incomplete, having focussed either on heterogeneous particles, or on particles intentionally too large to enter the gill principal filament tracts. We used endoscope-directed sampling and the intact diatom-empty, cleaned frustule approach to unambiguously establish qualitative selection sites and the influence of seston quality (varying proportions of intact diatoms and empty, cleaned frustules) and quantity (particle loads) on the degree of qualitative selection. Normally-feeding oysters were presented test mixtures of the naturally-occurring Actinoptychus senarius (small enough to enter the gill principal filaments), and the potential selection sites (gill: dorsal and ventral collecting tracts; labial palps: anteriorly-deposited pseudofaeces), were sampled for comparison with the proportions and concentrations of the ambient medium. Qualitative selection was demonstrated at both the gills and labial palps. Gill selection efficiency was shown to be directly proportional to seston quality and quantity, using a technique independant of pseudofeces mucus content. The oyster gill is thus able to increase ingested food quality when environmental food quality is low and / or when seston concentrations are high, which is typical of oyster habitats. Palp selection efficiency was directly proportional to seston quality, but at the highest concentration tested, no qualitative selection was observed on the labial palps, probably due to overload on these smaller organs. The partial functional redundancy of these key processing organs in heterorhabdic species such as oysters and scallops may enhance their success in high-turbidity habitats.     

Incorporation and modification of dietary fatty acids in gill polar lipids by two bivalve species Crassostrea gigas and Ruditapes philippinarum

Two bivalve species Crassostrea gigas and Ruditapes philippinarum were fed eight weeks with three mono-specific algae diets: T-Isochrysis galbana, Tetraselmis suecica, Chaetoceros calcitrans, selected on the basis of their polyunsaturated fatty acid (PUFA) composition. The incorporation and the modification of dietary fatty acids in C. gigas and R. philippinarum gill lipids were analysed and compared. Essential PUFA (20:4n-6, 20:5n-3 and 22:6n-3) and non-methylene interrupted PUFAs (known to be synthesised from monounsaturated precursors) contents of gill polar lipid of both species were greatly influenced by the dietary conditioning. Interestingly, oysters and clams responded differentially to the mono-specific diets. Oysters maintained higher 20:5n-3 level and higher 22:2j/22:i and n-7/n-9 ratio in gill polar lipids than clams. To better discriminate dietary and species influences on the fatty acid composition, a Principal Component Analysis followed by a MANOVA on the two most explicative components was performed. These statistical analyses showed that difference in fatty acid compositions attributable to species were just as significant as the diet inputs. The differences of gill fatty acid compositions between oysters and clams are speculated to result of an intrinsic species characteristic and perhaps of a group characteristic: Fillibranch vs. Eulamellibranch.     

Experimentally induced endosymbiont loss and re-acquirement in the hydrothermal vent bivalve Bathymodiolus azoricus

Invertebrates harbouring endosymbiotic chemoautotroph bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. In these species mechanisms of symbiont transmission are likely to be key elements of dispersal strategies that remained partially unresolved because the early life stages are not available for developmental studies. To study cessation and re-establishment of symbiosis in the host gill a laboratory experiment was conducted over 45 days in a controlled set-up (LabHorta) that endeavour re-creation of the hydrothermal vent chemical environment. Our animal model was the vent bivalve Bathymodiolus azoricus from the Menez Gwen vent site of the Mid Atlantic Ridge (MAR). Animals were exposed to conditions lacking inorganic S supply for 30 days, which is vital for their symbionts, and then re-acclimatized in sulphide-supplied seawater for an additional 15 days.Gradual disappearance of bacteria from the symbiont-bearing gill cells was observed in animals kept in seawater free of dissolved sulphide for up to 30 days, and was evidenced by histological, ultrastructural observations and Polymerase Chain Reaction tests. Following re-acclimatisation in S-supplied seawater, proliferation of sulphur-bacteria in the gill bacteriocytes confirms the functionality of our sulfide-feeding system in supporting chemoautotrophic symbionts. It may also indicate a horizontal endosymbiont acquisition, i.e. from the environment to the host by means of phagocytosis-like mechanism involving special “pit-like” structures on the apical cell membrane.The present work reports the first laboratory set-up successfully used to maintain the hydrothermal vent bivalve B. azoricus for prolonged periods of time by supplying inorganic sulphur as an energy source for its bacterial endosymbionts. Survival of symbiont bacteria is a critical factor influencing the host physiology and thus the methods reported here represent great potential for future studies of host-symbiont dynamics and for post-capture experimental investigations.     

Ecological aspects of a silicified bivalve fauna from the Silurian of Gotland

The silicified Wenlockian (Silurian) bivalve fauna from MÖllbos, Gotland, is part of a life assemblage. The vast number of shells show unusual phenomena, e.g. shell repair, pearl and tumour formation, etc. A number of shells contain epibionts and bored, round holes. Presumptive predators of the bivalve community are discussed. Size-frequency distribution of the two most abundant species possibly reflects age classes. The fauna, comprising eleven species, is dominated by deposit-feeders (90 %). They exhibit niche diversification, including at least three different feeding levels within the sediment.     

Xenobiotic-induced apoptosis: significance and potential application as a general biomarker of response

The process of apoptosis, often coined programmed cell death, involves cell injury induced by a variety of stimuli including xenobiotics and is morphologically, biochemically, and physiologically distinct from necrosis. Apoptotic death is characterized by cellular changes such as cytoplasm shrinkage, chromatin condensation, and plasma membrane asymmetry. This form of cell suicide is appealing as a general biomarker of response in that it is expressed in multiple cell systems (e.g. immune, neuronal, hepatal, intestinal, dermal, reproductive), is conserved phylogenetically (e.g. fish, rodents, birds, sheep, amphibians, roundworms, plants, humans), is modulated by environmentally relevant levels of chemical contaminants, and indicates a state of stress of the organism. Further, apoptosis is useful as a biomarker as it serves as a molecular control point and hence may provide mechanistic information on xenobiotic stress. Studies reviewed here suggest that apoptosis is a sensitive and early indicator of acute and chronic chemical stress, loss of cellular function and structure, and organismal health. Examples are provided of the application of this methodology in studies of health of lake trout (Salvelinus namaycush) in the Laurentian Great Lakes.     

Xenobiotic-induced apoptosis: significance and potential application as a general biomarker of response

The process of apoptosis, often coined programmed cell death, involves cell injury induced by a variety of stimuli including xenobiotics and is morphologically, biochemically, and physiologically distinct from necrosis. Apoptotic death is characterized by cellular changes such as cytoplasm shrinkage, chromatin condensation, and plasma membrane asymmetry. This form of cell suicide is appealing as a general biomarker of response in that it is expressed in multiple cell systems (e.g. immune, neuronal, hepatal, intestinal, dermal, reproductive), is conserved phylogenetically (e.g. fish, rodents, birds, sheep, amphibians, roundworms, plants, humans), is modulated by environmentally relevant levels of chemical contaminants, and indicates a state of stress of the organism. Further, apoptosis is useful as a biomarker as it serves as a molecular control point and hence may provide mechanistic information on xenobiotic stress. Studies reviewed here suggest that apoptosis is a sensitive and early indicator of acute and chronic chemical stress, loss of cellular function and structure, and organismal health. Examples are provided of the application of this methodology in studies of health of lake trout (Salvelinus namaycush) in the Laurentian Great Lakes.     

Linking long-term toxicity of xeno-chemicals with short-term biological adaptation

Understanding the mechanisms of long-term toxicities of chemicals is challenging. The present review discusses evidence suggesting that the biological adaptation to acute xenobiotic exposure could lead in the long run to toxic side effects. Upon acute exposure, hydrophobic xenobiotics are sequestered in the adipose tissue, which consequently protects other organs. However, this could also lead to the persistence of these xenochemicals and to a chronic low level internal exposure. The intrinsic properties of the xenobiotic detection and metabolism systems could also account for long-term toxicity. Indeed, hydrophobic xenochemicals are metabolized into more hydrophilic compounds; the first step of this pathway consists in the “activation” of the parent compound into a more reactive intermediate by cytochromes P450 activity. Those intermediates can be extremely reactive with DNA and proteins and thus could lead to toxic side effects that may become significant over time. Furthermore, recent evidence suggests that xenobiotic receptors also display endogenous functions. It is likely that repeated exposure to xenobiotics disrupts those endogenous functions with possibly dire cellular consequences. Altogether, The hypothesis presented here proposes that one mechanism for long-term toxicity stems from cumulative side effects due to the repeated activity of adaptive pathways triggered by acute intoxication.     

Feeding behavior and differential absorption of biochemical components by the infaunal bivalve Mulinia edulis and the epibenthic Mytilus chilensis in response to changes in food regimes

We measured changes in the feeding rate and food absorption efficiency of two suspension feeding bivalves, cross-trasplanted between habitats with special emphasis on their capacity for differential absorption of biochemical components from their food supply. Mulinia edulis were moved from the intertidal zone to the subtidal zone, and Mytilus chilensis from the subtidal to the intertidal zone for a period of 7 days, and then compared with animal that had not been transplanted. Experimentally prepared diets similar to those available in the two different environments were offered to the bivalves, and their rates of feeding and differential uptake of biochemical components were determined and statistically compared. The two species did not achieve complete acclimation of their feeding behaviour during the transplant period since the highest ingestion rates for biochemical components occurred under dietary conditions that reflected their habitats of origin. Absorption efficiency showed greater acclimation than the other physiological parameters measured, indicating the capacity of these species to modulate their enzymatic-digestive activity depending on food composition. We conclude that both Mytilus and Mulinia have a certain degree of physiological plasticity in their feeding behaviour and assimilatory balance of biochemical components, being greater in Mytilus. When both species encounter ambient food conditions characteristic of their normal habitats, they show maximum values of food absorption, while under conditions where their typical diets are exchanged (Mytilus in intertidal and Mulinia in subtidal), the energy absorbed declines in each, but in ways very different between the two species. Thus, Mytilus exposed to high concentrations of low quality seston reduced the energy absorbed by 31.7% compared to its normal habitat, while Mulinia exposed to low concentrations of high-quality food reduced their energy absorption by 64%.     

Purification of full-length human Pregnane and Xenobiotic Receptor： polyclonal antibody preparation for immunological characterization

Pregnane and Xenobiotic Receptor （PXR; or Steroid and Xenobiotic Receptor, SXR）, a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body＇s homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21（DE3） cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.     

Comparative study of the morphology of the gland opening area among Grassatores harvestmen (Arachnida,Opiliones, Laniatores)

Arachnids of the order Opiliones (harvestmen), which includes around 6000 specìes, have a pair of scent glands that open at the sides of the body, producing substances used as defence. Several types of behavioural, morphological and chemical defensive mechanisms have been identified in the order as a whole, although some of these tactics were restricted to particular groups. Only around 60 species have been studied from this perspective so far, more than half of which belong to the largest harvestman family within the order Laniatores, the Gonyleptidae, and have only recently been studied in an evolutionary perspective, showing the usefulness of defensive characters in taxonomy and evolutionary biology. Within Laniatores, the Grassatores clade includes the Gonyleptidae and 20 additional families, mostly poorly or not previously studied. We describe the morphology of the structures involved in fluid displacement during chemical defence in 15 of these families (data on two additional families are available from the literature) and discuss the evolution of such traits based on an available phylogenetic hypothesis of relationships within Grassatores, using the representatives of Triaenonychidae (a non‐Grassatores family of Laniatores) for comparison. We conclude that most non‐gonyleptoid Grassatores share (maybe plesiomorphically) a series of characteristics, mostly strongly different from what is observed within the gonyleptoids, and that smaller groups seem to share diagnostic features related to chemical defence, as is the case of stygnids, cosmetids and triaenonychines, and especially of manaosbiids and cranaids, whose defensive morphologies largely resemble those of derived gonyleptids. The following main synapomorphies were detected: (a) Grassatores: the presence of a deep and well‐defined descending channel; (b) Samooidea+Zalmoxoidea+Assamioidea+Gonyleptoidea: lateral pegs along the lateral channel; (c) Samooidea+Zalmoxoidea: deep channels forming an H on the dorsal scute; (d) Gonyleptoidea: ozopore cutting dorsally (reversing in Agoristenidae and Stygnidae to a laterally placed oval ozopore), a wide and smooth lateral channel, reversing to a lateral channel whose bottom is covered with either small plates (Agoristenidae) or high tubercles (Stygnidae), and apophyses of coxa II close to or covering the ozopore.