Cloning, expression and characterization of metallothionein from the Antarctic clam Laternula elliptica

The genes for two apparent subtypes of metallothionein (MT) isoform were isolated from the Antarctic clam Laternula elliptica. Determination of the nucleotide sequence showed that the gene consists of 222 bp that code a 73-amino acid protein. The comparison between MT cDNA sequences of L. elliptica and other bivalves showed strong homologies on positions of cysteine residues, which are important for their metal binding abilities. The gene for the MT was inserted into a pET vector and overexpressed as a carboxyl terminal extension of glutathionein-S-transferase (GST) in Escherichia coli. After the GST fusion proteins had been purified by glutathione-Sepharose affinity chromatography column and digested with enterokinase, the MT was purified with gel filtration and analyzed for its biochemical properties. Recombinant MTs were reconstituted with Cd, Cu, and Zn, and kinetic studies of the reactions with electrophilic disulphide, DTNB, were investigated to explore their metal binding ability. It is revealed that the Cd-MT and Zn-MT react with DTNB biphasically, and that Zn-MT reacts with DTNB more rapidly, and with a significantly greater pseudo-first-order rate constant. Cu-MT reacts monophasically and releases metal slowly from MT.     

Iron and nitrosative metabolism in the Antarctic mollusc Laternula elliptica

The objective of this work was to study Fe distribution, and oxidative and nitrosative metabolism in Laternula elliptica for physiological analysis and interspecific comparisons. Lipid peroxidation, superoxide dismutase and catalase activity and total Fe content were estimated in the digestive glands (DG) of L. elliptica. The labile Fe pool (LIP) represents the amount of cellular Fe responsible for catalyzing radical-dependent reactions. LIP assessed by the calcein assay, represents 3.5% of the total Fe in L. elliptica. Experimental isolation of ferritin (Ft) was performed. Subunit analyses of the protein by SDS-polyacrilamide gel electrophoresis indicated that the protein was composed of 20.6kDa protein subunits, consistent with the horse spleen Ft and the molecular weight markers, however, a higher molecular mass subunit could appear. The identity of the protein was confirmed by Western blot analysis. The nitrate+nitrite content was 73±7pmol/mg fresh mass (FW). The nitric oxide (NO) content in DG homogenates, assessed by electronic paramagnetic resonance (EPR) spin trapping measurements using the NO trap sodium-N-methyl-D-glucamine dithiocarbamate-Fe at room temperature, was 30±2pmol/mg FW. Nitric oxide synthase-like activity (1.50±0.09pmol/mg FW min) was assessed by measuring NO production by EPR in the presence of L-arginine (L-A) and NADPH. This activity was significantly inhibited by L-A analogs such as Nω-nitro-L-arginine methyl ester hydrochloride (-77%) and Nω-nitro-L-arginine (-62%), or by the lack of added L-A (-55%). The data presented here documented the physiological presence of labile Fe, Ft and highly reactive nitrogen species, and are the first evidence that support the hypothesis that NO being generated in L. elliptica might contribute to restrict oxidative damage by a close link with Fe metabolism.     

Population trajectories for the Antarctic bivalve Laternula elliptica: identifying demographic bottlenecks in differing environmental futures

The world’s oceans are changing, and dramatic shifts have been documented in the Southern Ocean. The consequences of these shifts to coastal benthic organisms are difficult to predict at present, as ocean warming may increase primary production and food resources for benthic consumers, whilst OA may have negative impacts that differentially affect various species and life stages. A model was developed to investigate how different scenarios of change may influence population size of the Antarctic bivalve Laternula elliptica. The model describes potential implications of both pH and temperature change on survivorship and reproductive output of a population of this bivalve species in McMurdo Sound, Ross Sea. Implications of increases and decreases in mortality rate across different life stages of the population (early, mid and late) were assessed. Additionally, effects on energetic resource partitioning and dictating reproductive potential (RP) were also investigated. Significant declines in RP, due to increased basal metabolic demand, were associated with even relatively small changes in temperature and pH, resulting in populations declining to 25 % of the starting equilibrium density within 60 years. As L. elliptica is a pivotal species to the functionality of the Antarctic coastal benthic ecosystem, wide spread repercussions are expected if populations are impacted as the model predicts. Although further model development is required to explore the ecosystem implications of the population decline described in this paper, this work allows a better understanding of the consequences of change as soon as data on the direction and magnitude of the changes affecting Antarctic seas become available.     

Hyperoxia alleviates thermal stress in the Antarctic bivalve, Laternula elliptica: evidence for oxygen limited thermal tolerance

Understanding thermal limits and the ability of species to cope with changing temperatures is crucial for a cause and effect understanding of climate effects on organisms and ecosystems. Data available for marine species from various phyla and climates led to the hypothesis that a mismatch between oxygen demand and limited capacity of oxygen supply to tissues is the first mechanism to restrict survival at thermal extremes. Here we show that doubling the oxygen content of the ambient seawater from 160 mmHg partial pressure to 350 mmHg raised the upper temperature limits of the Antarctic marine bivalve Laternula elliptica by about 2.5°C. It reduced the accumulation of the anaerobic end product succinate or of total CO₂ as a sign of respiratory distress. These findings provide further evidence that oxygen supply does limit thermal tolerance in marine animals. As water temperatures rise animals will face a double problem of progressively reduced oxygen solubility in the water and enhanced costs reflected in increased metabolic rates.     

Ocean acidification at high latitudes: potential effects on functioning of the Antarctic bivalve Laternula elliptica

Ocean acidification is a well recognised threat to marine ecosystems. High latitude regions are predicted to be particularly affected due to cold waters and naturally low carbonate saturation levels. This is of concern for organisms utilising calcium carbonate (CaCO₃) to generate shells or skeletons. Studies of potential effects of future levels of pCO₂ on high latitude calcifiers are at present limited, and there is little understanding of their potential to acclimate to these changes. We describe a laboratory experiment to compare physiological and metabolic responses of a key benthic bivalve, Laternula elliptica, at pCO₂ levels of their natural environment (430 µatm, pH 7.99; based on field measurements) with those predicted for 2100 (735 µatm, pH 7.78) and glacial levels (187 µatm, pH 8.32). Adult L. elliptica basal metabolism (oxygen consumption rates) and heat shock protein HSP70 gene expression levels increased in response both to lowering and elevation of pH. Expression of chitin synthase (CHS), a key enzyme involved in synthesis of bivalve shells, was significantly up-regulated in individuals at pH 7.78, indicating L. elliptica were working harder to calcify in seawater undersaturated in aragonite (Ω_Ar = 0.71), the CaCO₃ polymorph of which their shells are comprised. The different response variables were influenced by pH in differing ways, highlighting the importance of assessing a variety of factors to determine the likely impact of pH change. In combination, the results indicate a negative effect of ocean acidification on whole-organism functioning of L. elliptica over relatively short terms (weeks-months) that may be energetically difficult to maintain over longer time periods. Importantly, however, the observed changes in L. elliptica CHS gene expression provides evidence for biological control over the shell formation process, which may enable some degree of adaptation or acclimation to future ocean acidification scenarios.     

Metabolic demand,oxygen supply,and critical temperatures in the Antarctic bivalve Laternula elliptica

Oxygen consumption (Mo(2)), heartbeat rate and form, and circulating hemolymph oxygen content were measured in relation to temperature in the large Antarctic infaunal bivalve Laternula elliptica. After elevations in temperature from 0 degrees to 3 degrees, 6 degrees, and then 9 degrees C, Mo(2) and heartbeat rate rose to new levels, whereas maximum circulating hemolymph oxygen content fell. At 0 degrees C, Mo(2) was 19.6 micromol O(2) h(-1) for a standard animal of 2-g tissue ash-free dry mass, which equates to a 8.95-g tissue dry-mass or 58.4-g tissue wet-mass animal. Elevation of metabolism following temperature change had acute Q(10) values between 4.1 and 5, whereas acclimated figures declined from 3.4 (between 0 degrees and 3 degrees C) to 2.2 (3 degrees -6 degrees C) and 1.9 (6 degrees -9 degrees C). Heartbeat rate showed no acclimation following temperature elevations, with Q(10) values of 3.9, 3.2, and 4.3, respectively. Circulating hemolymph oxygen content declined from 0 degrees to 3 degrees and 6 degrees C but stayed at a constant Po(2) (73-78 mmHg) and constant proportion ( approximately 50%) of the oxygen content of the ambient water. At 9 degrees C, Mo(2) and heartbeat rate both peaked at values 3.3 times those measured at 0 degrees C, which may indicate aerobic scope in this species. After these peaks, both measures declined rapidly over the ensuing 5 d to the lowest measured in the study, and the bivalves began to die. Hemolymph oxygen content fell dramatically at 9 degrees C to values between 2% and 12% of ambient water O(2) content and had a maximum Po(2) of around 20 mmHg. These data indicate an experimental upper lethal temperature of 9 degrees C and a critical temperature, where a long-term switch to anaerobic metabolism probably occurs, of around 6 degrees C for L. elliptica. Concurrent measures of mitochondrial function in the same species had indicated strong thermal sensitivity in proton leakage costs, and our data support the hypothesis that as temperature rises, mitochondrial maintenance costs rapidly outstrip oxygen supply mechanisms in cold stenothermal marine species.     

Cadmium bioaccumulation and detoxification in the gill and digestive gland of the Antarctic bivalve Laternula elliptica

Exposure to a sublethal concentration of cadmium (Cd; 50 microg L(-1)) resulted in significantly increased Cd concentrations in the gill and digestive gland of the Antarctic bivalve Laternula elliptica. Continuous accumulation of Cd in the two organs during the 14-day exposure period was associated with sequestration of Cd to both the soluble cytosolic and insoluble particulate cell fractions. However, the contribution of each cell fraction to Cd sequestration differed between the two organs; in the gill, a larger portion of Cd was associated with the insoluble fraction, while in the digestive gland, both the soluble and insoluble fractions sequestered similar amounts of Cd. Metal-binding components in the insoluble cell fraction were not identified in this study. On the other hand, a metallothionein-like protein (MTLP) was the major Cd-detoxifying component in the soluble cell fraction of the gill and digestive gland. The amount of MTLP increased linearly with exposure time and the amount of Cd accumulated in the tissue, which suggests a potential utility of MTLP as a biomarker for exposure to Cd and possibly other metals.     

Stable isotopes prove shell growth bands in the Antarctic bivalve Laternula elliptica to be formed annually

Growth marks in calcareous shells or skeleton parts of benthic invertebrates are often assumed to be formed annually and hence are used for ageing purposes. For some Antarctic species this assumption has been validated by tagging experiments or direct monitoring of growth throughout the year. Here we use the record of stable isotope ratios (δ¹⁸O and δ¹³C) deposited in shell carbonate of Laternula elliptica from King George Island to demonstrate that shell growth bands are formed annually in this species. Received: 16 July 1995 / Accepted: 27 October 1996     

Physiological plasticity, long term resistance or acclimation to temperature, in the Antarctic bivalve, Laternula elliptica

To further investigate the previously reported limited acclimation capacities of Antarctic marine stenotherms, the Antarctic mud clam, Laternula elliptica (King and Broderip, 1830-1831), was incubated at 3.0°C for 89days. The thermal windows of a suite of biochemical and physiological metrics that characterise tissue aerobic status, were then measured in response to acute temperature elevation (2-2.5°C increase per week). To test if acclimation had occurred at the higher temperature, results were compared with published data, from the preceding year, for L. elliptica which had been incubated at ambient temperature (0.0°C) and then subjected to the same acute temperature treatments. Incubation to 3.0°C led to a temperature induced increase of tissue aerobic status (reduced intracellular cCO(2) with increased O(2) consumption, PLA (phospho-L-arginine) and ATP). At the highest acute temperature (7.5°C) the increase in anaerobic pathways (summed acetate/succinate and propionate) was less after 3.0°C than 0.0°C incubation. No other metric shifted its reaction norm in response to acute temperature elevation and so whole animal acclimation had not occurred, even after 3months at 3.0°C. Combined with the constant mortality throughout the 3.0°C incubation period, these data suggest that the recorded physiological changes were either the early stages of acclimation or, more likely, time limited resistance mechanisms.     

Molecular cloning and thermal stress-induced expression of a pi-class glutathione S-transferase (GST) in the Antarctic bivalve Laternula elliptica

Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione. The pi-class GST cDNA (leGSTp) was cloned from the cold-adapted Antarctic bivalve Laternula elliptica. We used degenerated primers designed based on highly conserved regions of known mollusk GSTs to amplify the corresponding L. elliptica mRNA. Full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full sequence of the GST cDNA was 1189 bp in length, with a 5' untranslated region (UTR) of 74 bp, a 3' UTR of 485 bp, and an open reading frame of 630 bp encoding 209 amino acid residues with an estimated molecular mass of 23.9 kDa and an estimated isoelectric point of 8.3. Quantitative RT-PCR confirmed basal expression of leGSTp, which was up-regulated upon heat treatment (10 degrees C for different time periods) by a factor of 2.3 (at 24 h) and 2.7 (at 48 h) in the digestive gland and gill tissues, respectively. The recombinant leGSTp expressed in Escherichia coli was purified by affinity chromatography and characterized. The purified leGSTp exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). The recombinant leGSTp had a maximum activity at approximately pH 8.0, and its optimum temperature was 35 degrees C.     

Microbial biosorption of metals: potential in the treatment of metal pollution

The phenomenon of metal biosorption by microorganisms has been thoroughly documented. Although this phenomenon is exhibited by both living and non-living forms of biomass, the purpose of this chapter will be to review biosorption by the latter. In addition, the application of various technological processes required for exploitation of this phenomenon in waste treatment will be examined.     

The annual reproductive pattern of the Antarctic clam, <Emphasis Type="Italic">Laternula elliptica</Emphasis> from Marian Cove,King George Island

The annual reproductive cycle of the Antarctic soft-shelled clam, Laternula elliptica, in Marian Cove, King George Island was studied over a 2-year period from February 1998 to January 2000. Annual changes in the gametogenesis were investigated by measuring the percentage of area occupied by oocytes in a follicle [follicle index (FI)] and the oocyte size. In 1998, the monthly mean FI increased significantly from October to November, peaked in December, and decreased rapidly from December to January. In February and March 1999, degenerated eggs were observed in the spent follicles. Degeneration and resorption of residual eggs by phagocytosis occurred mostly in February and March in both 1998 and 1999, although the resorption process was observed year-round. The histology indicated that complete vitellogenic growth of L. ellpitica at Marian Cove takes at least a year and the clams spawn annually during the austral summer. The ripening and subsequent spawning of clams at Marian Cove in 1998 and 1999 coincided with the algal blooming (September–October 1998 and December and January 1999–2000) suggesting that in coastal Antarctica food supply is a crucial factor that governs gonad maturation and subsequent spawning along with the water temperature.     

Thermal limits of burrowing capacity are linked to oxygen availability and size in the Antarctic clam <Emphasis Type="Italic">Laternula elliptica</Emphasis>

Animal responses to changing environments are most commonly studied in relation to temperature change. The current paradigm for marine ectotherms is that temperature limits are set through oxygen limitation. Oxygen limitation leads to progressive reductions in capacity to perform work or activity, and these are more important and proximate measures of a population’s ability to survive. Here we measured the ability of a large Antarctic clam to rebury when removed from sediment at temperatures between −1.5 and 7.5 °C and at three oxygen concentrations, 10.2, 20.5 and 27.7%. The proportion of the population capable of reburying declined rapidly and linearly with temperature from around 65% at 0 °C to 0% at 6 °C in normoxia (20.5% O₂). Decreasing oxygen to 10.2% reduced temperature limits for successful burial by around 2 °C, and increasing oxygen to 27.7% raised the limits by 1–1.5 °C. There was an interactive effect of body size and temperature on burying: the temperature limits of larger individuals were lower than smaller animals. Similarly, these size limits were increased by increasing oxygen availability. Considering data for all temperatures and oxygen levels, the fastest burying rates occurred at 3 °C, which is 2 °C above the maximum summer temperature at this site.     

Trace metals in barnacles: the significance of trophic transfer

Barnacles have very high accumulated trace metal body concentrations that vary with local trace metal bioavailabilities and represent integrated measures of the supply of bioavailable metals. Pioneering work in Chinese waters in Hong Kong highlighted the potential value of barnacles (particularly Balanus amphitrite) as trace metal biomonitors in coastal waters, identifying differences in local trace metal bioavailabilities over space and time. Work in Hong Kong has also shown that although barnacles have very high rates of trace metal uptake from solution, they also have very high trace metal assimilation efficiencies from the diet. High assimilation efficiencies coupled with high ingestion rates ensure that trophic uptake is by far the dominant trace metal uptake route in barnacles, as verified for cadmium and zinc. Kinetic modelling has shown that low efflux rate constants and high uptake rates from the diet combine to bring about accumulated trace metal concentrations in barnacles that are amongst the