An unusual hermaphrodite reproductive trait in the Antarctic brooding bivalve Lissarca miliaris (Philobryidae) from the Scotia Sea, Southern Ocean

The Antarctic marine environment is extreme in its low temperatures and short periods of primary productivity. Invertebrates must therefore adapt to maximise reproductive output where low temperature and limited food slow larval development. Brooding is a common reproductive trait in Antarctic marine bivalves; larval development occurs within the mantle cavity, and larvae are released as fully developed young. Lissarca miliaris is a small, short-lived, shallow-water brooding bivalve of circum-Antarctic distribution and found most abundant in the sub-Antarctic Magellan Region and islands of the Scotia Arc. Here, an unusual hermaphrodite reproductive trait is described for L. miliaris from King George Island (62°14′S, 58°38′W) and Signy Island (60°42′S, 45°36′W), Antarctica, using histological and dissection techniques. Specimens demonstrate simultaneous and sequential hermaphrodite traits; male and female gonads develop simultaneously, but the production of oocytes is reduced while testes are ripe. Functional females are more abundant in specimens above 3 mm shell length, although male reproductive tissue persists and functional males are found in all size classes. The number of previtellogenic oocytes produced by far exceeds the number of oocytes extruded and brooded, which may indicate an ancestral link to a planktotrophic past. Hermaphroditism in L. miliaris maximises reproductive efficiency in a short-lived species, in which the female’s capacity to brood its young is limited, and demonstrates a specialised adaptation to a cold stenothermal and food-limited environment prevailing in the Southern Ocean.     

Lissarca notorcadensis (Bivalvia: Philobryidae) living on Notocidaris sp. (Echinoidea: Cidaridae): Population dynamics in limited space

Summary Population dynamics of the epizoic bivalve Lissarca notorcadensis living on spines of cidaroid sea urchins in the Weddell Sea were investigated. Total production (somatic & gonad) of the suspension feeding bivalve ranged between 16.5 and 487.4 mg AFDM y^–1 per sea urchin. Annual sedimentation rates are not sufficient to maintain the production of the Lissarca sub-populations carried by the sea urchins, and resuspension of organic matter is most likely to be an important food source. The ratio of the number of freshly settled juveniles to the number of embryos brooded is between 0.054 and 0.207 and seems negatively related to the biomass already present, indicating intraspecific competition for space. Interspecific competition for space is caused by the strong preference of L. notorcadensis as well as other epizoa (colonial anthozoans and bryozoans) for the spines located on the aboral hemispere of the sea urchins.AWI Publication No. 572     

Metal and antibiotic resistance in psychrotrophic bacteria associated with the Antarctic sponge Hemigellius pilosus (Kirkpatrick, 1907)

High concentrations of heavy metals have been previously detected in Antarctic sponge tissues, but their effect on the associated bacterial assemblages has been never investigated. Metal tolerance is often linked to antibiotic resistance and can also affect biochemical activities within microbial populations. In the present work, the response to heavy metals and antibiotics, as well as the enzymatic profile, of bacteria associated with the sponge Hemigellius pilosus, was analyzed. Tolerance to mercury, cadmium and zinc (at concentrations between 10 and 10,000 ppm) was tested by the plate diffusion method. Almost all isolates completely tolerated zinc and cadmium up to 1,000 and 2,500 ppm, respectively, whereas complete tolerance to mercury was generally observed at concentrations between 10 and 500 ppm. As bacteria can develop resistance in the growing presence of toxic compounds in the environment, this finding could be related to the concentrations of metals in the sponge tissues. The susceptibility assay to 11 antibiotics revealed that multiple antibiotic resistance was generally exhibited, with gentamicin that inhibited all Antarctic isolates. The comparison of the heavy metal and antibiotic resistance patterns at phylogenetic level revealed some distinctive features, suggesting that the dissemination of heavy metal tolerance and antibiotic resistance may possess great relevance for the population dynamics. Additionally, growth patterns often highly differed among strains in the same species, thus appearing to be more likely strain specific rather than species specific. The enzyme expression by the isolates was not really affected by the heavy metal tolerance they showed, as variation in the enzymatic profiles was observed in strains within the same genus that showed different/similar heavy metal tolerance patterns.     

New perspectives on the dispersal mechanisms of the Antarctic brooding bivalve Mysella charcoti (Lamy, 1906)

. Brooding is a widespread phenomenon among Antarctic bivalves. Although it should represent a handicap to dispersion, many brooding species have achieved a wide distribution in Antarctic and subantarctic waters, suggesting that they have alternative and effective methods of dispersal. Evidence of such an alternative method is presented here for the bivalve Mysella charcoti, unexpectedly found alive and healthy in feces expelled by Notothenia coriiceps (Nototheniidae: Pisces). The finding indicates that the snug-fitting shell of Mysella functions as a barrier to digestive enzymes. Withstanding passage through the digestive tract of fish allows Mysella to be passively dispersed (within the home range of the fish) and colonize new habitats or re-colonize shallow-water substrates severely impacted by ice scours.     

Biology and functional morphology of the pallial organs of the Antarctic bivalve Mysella charcoti (Lamy, 1906) (Galeommatoidea: Lasaeidae)

Mysella charcoti is an Antarctic lasaeid bivalve and the most frequently encountered mollusc in Admiralty Bay, King George Island, South Shetlands. The behaviour of the species in aquaria, combined with analyses of the gross and microscopic morphologies and functioning of the organs in the mantle cavity of living and preserved specimens have allowed an understanding of important aspects of its biology. The role of the foot and its ciliature during the processes of dislodgement and burrowing within the sediment are described. The species is a free-living, shallow-burrower, with a predominantly deposit-feeding habit and derives part of its food from the labile settled organic deposits carried into the mantle cavity by the anterior–posterior current of water. Pedal sweep-feeding was not detected. M. charcoti is the first known lasaeid with ctenidia formed of the descending lamellae of the inner demibranchs only, a feature probably related to its highly specialised brooding habit.     

Microanatomy and ultrastructure of the foot of the infaunal bivalve Tegillarca granosa (Bivalvia: Arcidae)

In this study, the morphology and ultrastructure of the foot of Tegillarca granosa was compared with the bivalves from different habitats. The sediment of habitat of T. granosa is mostly a mixture of sand (68.93%) and mud (24.12%). The foot is wedge-shaped with multiple projections on the surface and covered with ciliary tufts. The epithelial layer is simple and composed of ciliated columnar epithelia and mucous cells. Although the mucous cells are distributed mostly in the epithelial layer, they are developed even in the connective tissues and muscle layers, and the mucous cells mostly contain acidic carboxylated mucosubstances. From the TEM observation, secretory cells are classified into three types. Type A secretory cell has a goblet form and is most widely distributed among the three types. Type B secretory cell has an oval form and the secretory granule has fibrous substance. Type C secretory cell has an elongated elliptic form and membrane-bounded secretory granules. The muscle fiber bundles are composed mainly of smooth muscle fibers. The smooth muscle fibers can be divided into two types. Type A muscle fibers have evenly distributed thick microfilaments between the thin microfilaments of cytoplasm. Type B muscle fiber has cluster of condensed microfilaments in the medulla cytoplasm while the cortical cytoplasm has loose distribution of thin microfilaments.     

The diversity of relationships between Antarctic sponges and diatoms: the case of Mycale acerata Kirkpatrick, 1907 (Porifera,Demospongiae)

The diatom assemblage associated with the Antarctic sponge Mycale acerata was studied through an analysis of the diatom frustule and pigment concentrations in both the sponge ectosome and choanosome. Sponges were sampled weekly from November 2001 to February 2002 at Terra Nova Bay, Antarctica, at a depth of 25–35 m. The most abundant diatoms were Porannulus contentus, Fragilariopsis curta, Thalassiosira cf. gracilis, T. perpusilla and Plagiotropis sp. High abundances of P. contentus were found on the sponge ectosome up to the beginning of November, before the ice melted, while later frustules were incorporated inside, indicating that P. contentus lives epibiontically on M. acerata and represents a potential food source for the sponge. The presence of other diatom species was mainly related to the summer phytoplankton bloom. The sponge incorporates diatoms from the water column and utilises them as a food source, accumulating frustules inside the choanosome. The lack of planktonic diatom frustules at the beginning of the summer indicates that they are expelled or dissolved during the cold season.     

Glycoconjugate histochemistry and nNOS immunolocalization in the mantle and foot epithelia of Tapes philippinarum (bivalve mollusc)

Glycosaminoglycans and NO synthase probably regulate mucous cell secretion in the skin of Tapes philippinarum. We have demonstrated the presence of "protein" cells, "glycogen" cells, "phenol" cells and five types of mucous cells, with different chemical composition of the mucus in the mantle epithelium of T. philippinarum. The foot epithelium contained "protein" cells and two types of mucous cells. Using biotinylated lectins, in the mantle and foot epithelia we have shown specific sites for the following oligosaccharides: alpha-D-glucose, alpha-D-mannose, alpha-L-fucose, alpha-D-1,3-N-acetyl-galactosamine and alpha-N-acetyl-glucosamine. nNOS immunoreactivity in the intraepithelial and intradermal cells and in the mucocytes suggested a regulatory role of NO in mucus secretion, as demonstrated also in other invertebrates.     

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.     

Lipolytic activity of Antarctic cold-adapted marine bacteria (Terra Nova Bay, Ross Sea)

AIMS: The aim of this study was to investigate the lipolytic activity of cold-adapted Antarctic marine bacteria and, furthermore, the combined effect of some environmental factors on this enzymatic process. METHODS AND RESULTS: Strains were assayed for lipolytic activity on a basal medium amended with seven individual fatty acid esters. A significant activity was observed for 148 isolates (95.5% of the total screened). The interactive effect of pH, temperature and NaCl concentration on the substrates was tested for six representative isolates, identified as Pseudoalteromonas, Psychrobacter and Vibrio. Differences between strains according to NaCl and pH tolerances were observed. Only one strain degraded the substrate more efficiently at 4 degrees C than at 15 degrees C. CONCLUSIONS: Our findings demonstrate that the lipolytic activity of Antarctic marine bacteria is rather variable, depending on culture conditions, and occurs in a wide range of salt concentration and pH. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolation and characterization of bacteria that are able to efficiently remove lipids at low temperatures will provide insight into the possibility to use cold-adapted bacteria as a source of exploitable enzymes. Moreover, research on the interactive effects of salt concentration, pH and temperature will be useful to understand the true enzyme potentialities for industrial applications.     

Soluble and membrane-bound acetylcholinesterases in Mytilus galloprovincialis (Pelecypoda: Filibranchia) from the northern Adriatic sea

Three forms of acetylcholinesterase (AChE) were detected in samples of the bivalve mollusc Mytilus galloprovincialis collected in sites of the Adriatic sea. Apart from the origin of the mussels, two spontaneously soluble (SS) AChE occur in the hemolymph and represent about 80% of total activity, perhaps hydrolyzing metabolism-borne choline esters. These hydrophilic enzymes (forms A and B) copurified by affinity chromatography (procainamide-Sepharose gel) and were separated by sucrose gradient centrifugation. They are, respectively, a globular tetramer (11.0-12.0 S) and a dimer (6.0-7.0 S) of catalytic subunits. The third form, also purified from tissue extracts by the same affinity matrix, proved to be an amphiphilic globular dimer (7.0 S) with a phosphatidylinositol tail giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. Such an AChE is likely functional in cholinergic synapses. All three AChE forms show a good substrate specificity and are inactive on butyrylthiocholine. Studies with inhibitors showed low inhibition by eserine and paraoxon, especially on SS forms, high sensitivity to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284c51) and no inhibition with propoxur and diisopropylfluorophosphate (DFP). The ChE forms in M. galloprovincialis are possibly encoded by different genes. Some kinetic features of these enzymes suggest a genetic polymorphism.     

Effects of carbohydrates and leucine on growth of bacteria from Antarctic soils (Casey Station,Wilkes Land)

Growth rates of natural bacterial communities from Antarctic soils are analysed by an epifiuorescence microscopic method using data of microcolonies (colony number and colony areas). Incubations are performed on polycarbonate filters which are put on cellulose pads soaked with soil extracts, different concentrations of naturally occuring carbohydrates, polyols, and leucine. Concentrations of individual substrates were in the range of naturally occuring levels. The results showed that the growth of bacterial microcolonies could best be stimulated with glucose, sucrose, maltose, sorbitol, and mannitol. Leucine stimulated growth to a lower extent than glucose. Data on bacterial biomass production calculated from this approach are discussed in relation to those from tracer techniques carried out with ¹⁴C-labelled glucose from earlier experiments.     

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.     

The functional morphology of the abyssal Limopsis cristata (Arcoida: Limopsidae) with a discussion on the evolution of the more advanced bivalve foot

The bivalve Limopsis cristata pursues a semi‐endobenthic life in abyssal soft sediments. It attaches to particles by up to three byssal threads and filter feeds by inhaling water from posterior and anterior directions. Because of partial burial, however, only the latter is functionally significant. Complex ciliary currents in the mantle cavity concerned with the rejection of unwanted particles keep most material out of the simplified intestine. It is generally considered that the ligament is the constraining force in arcoid evolution. This may be true in part, but the lack of pallial fusions and the retention of anterior and posterior inhalant flows are more powerful limits to radiation in the Arcoida. In the deep sea, the Limopsidae has radiated into many micro‐niches through micro‐morphological adaptations. Loss of the arcoid ‘heel’ has resulted in the union of the separate rejectory currents of the visceral mass and foot, creating a single discharge point in more advanced bivalves. This greatly simplifies the rejectory roles of the visceral mass and foot and is thus of functional and selective advantage.