Change of hemoagglutinin and hemolysin titers in hemolymph of gastropod molluscs in response to immunization with sheep erythrocytes

This work deals with analysis of changes of the levels of hemoagglutinins (HA) and hemolysins (HL) in hemolymph of three gastropod species,Planorbius corneus, Lymnea stagna-lis, andAchatina fulica, in response to immunization with sheep erythrocytes (ShE). The levels of HA and HL were determined before immunization and 3, 5, 10, and 15 days after the antigen administration. The HA titer was estimated from hemoagglutination reaction, the HL titer was measured spectrophotometrically from release of hemoglobin from ShE. It has been found that after immunization the titer of HA falls by 72–88% for the first 3–5 days, but returns to the initial values or even exceeds them statistically significantly by the10–15th days. The time of the decrease of the HA titers correlates inversely with the body mass of the animals, while the amplitude of this decrease is directly proportional to the body mass. The HL titer either changes parallel to the HA titer (inL. stagnalis) or increases statistically significantly by 60–65% during the entire period of observation (in two other species studied). It is suggested that inP. coneus andA. fulica, HA and HL are different compounds.     

Behavioral aspects of host recognition by the symbiotic water mite Unionicola formosa (Acarina,Unionicolidae)

Summary The occurrence and specificity of host recognition behavior of adult and nymphal Unionicola formosa and the capability of adult mites to recolonize various mussel species were examined. Adult U. formosa aggregated on excised tissue from their host mussel, Anodonta imbecilis, in preference to that of two other species of mussels. Nymphs also exhibited an aggregation response to host tissue. A radioisotope (⁵¹Cr) technique was used to monitor the recolonization behavior of U. formosa. Adult female mites preferentially re-entered A. imbecilis rather than the sympatric mussel A. cataracta. The specificity of this behavior parallels the distribution of this water mite among potential bivalve hosts in the southeastern U.S. Host recognition by U. formosa may contribute to re-establishing contact with a host after accidental separation and probably helps to maintain mite-mussel symbioses. Whether or not larval U. formosa employ similar host recognition behavior while selecting a potential host has not as yet been determined.     

Vibrio–bivalve interactions in health and disease

In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated with the main diseases affecting the worldwide oyster production. Here, we review the current knowledge on Vibrio–bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with their bivalve hosts as well as technical issues limiting the monitoring of these bacteria in bivalve health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defences of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators.     

Studies on bucephalid digeneans parasitising molluscs and fishes in Finland I. Ecological data and experimental studies

Two types of bucephalid cercariae are reported from the bivalve Anodonta anatina in two Finnish lakes. One, Type A, resembles in gross morphology the cercaria of Bucephalus polymorphus, and the other, Type B, resembles the cercaria of Rhipidocotyle campanula. Type A daughter-sporocysts develop more slowly, have a greater cercarial productivity and exhibit a differential diurnal rhythm to that of Type B. Cercariae of Type A have a shorter longevity than Type B and tend to encyst in the fins rather than the gill-arches of fish intermediate hosts. The main definitive host of Type A is pike Esox lucius and, in the case of Type B, perch Perca fluviatilis. Adults of Types A and B are morphologically very similar and both species belong to the genus Rhipidocotyle.     

The effect of season and location on phosphoadenylate concentrations and adenylate energy charge in two species of freshwater clams

Summary Concentrations of phosphoadenylate nucleotides and the adenylate energy charge ((ATP+1/2ADP)/(ATP+ADP+AMP)) have been suggested as sensitive integrating measures of the energy state of organisms. This synoptic study investigated the seasonal and spatial variation of phosphoadenylate concentrations and AEC in two freshwater bivalve molluscs, the paper-shell clam, Anodonta imbecillis and the asian clam, Corbicula fluminea. Concentrations of all three adenylates, as well as the total adenylate concentration and adenylate energy charge of both species varied seasonally. These fluctuations were closely related to reproductive periods in both species. Total adenylate concentrations and ATP concentrations were slightly negatively correlated with shell length in A. imbecillis but the ADP and AMP concentrations and AEC were not significantly correlated with shell length. In C. fluminea the AEC was negatively correlated were positively correlated with shell length. Neither species exhibited significant differences in AEC between two collection locations. When C. fluminea collected from the Savannah River were acclimated and fed in the laboratory their AEC increased significantly.     

Metal-based ethanolamine-derived compounds: a note on their synthesis,characterization and bioactivity

Abstract

Metal-based ethanolamines, (L¹)–(L⁴) coordinated with Co(II), Cu(II), Ni(II) and Zn(II) metals in 1:2 (metal:ligand) molar ratio to produce new compounds have been reported. These compounds were screened for their bactericidal/fungicidal activity against a number of bacterial (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Bacillus subtilis) and fungal strains (Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata) alongside against a shrimp species known as Artemia salina. The screening results indicated that metal complexes have significantly higher activity than uncomplexed ligands against one or more bacterial/fungal species due to chelation. The ligand (L⁴) displayed good bacterial and fungal activity as compared to other ligands. The antibacterial results revealed that the Zn(II) complex (16) of (L⁴) was found to be the most active complex and Co(II) complex (14) of the same ligand (L⁴), demonstrated the highest antifungal activity.     

The localization of 4-O-methylglucuronic acid-containing glycosphingolipid on the cell surface of bivalve spermatozoa by use of an immunological method

Specific and high titer antisera against 4-O-methylglucuronic acid-containing glycosphingolipid (Lipid IV) from spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii, were raised in rabbits. The antisera were found to agglutinate spermatozoa of three fresh-water bivalves, H. schlegelii, Anodonta woodiana, and Cristaria plicata (Palaeoheterodonta), but they did not agglutinate those of Corbicula sandai (Heterodonta). The specificity of the agglutination was examined by an inhibition test using various carbohydrates, from which it was concluded that an antigenic determinant is GlcA4Me-Fuc. Immunohistochemical studies indicated that Lipid IV exists on the cell surface of the spermatozoa.     

Adenine and adenosine derivatives of rhodium acetate

Simple 1:1 adducts of the antitumor complex, Rh₂(acetate)₄, have been prepared with Adenine and Adenosine, and the binding site assigned as N₇ of the purine ring. The similarity in properties between these species and those of Rh₂(acetate)₄/DNA solutions indicates unequivocally the same binding site for DNA and the specificity of the reaction is interpreted as due to the presence of favorable H-binding interaction between the exocyclic amino group of adenine and the hydrogen-bond acceptor acetate ligand.     

Studies on bucephalid digeneans parasitising molluscs and fishes in Finland. II. The description of Rhipidocotyle fennica n. sp. and its discrimination by principal components analysis

Rhipidocotyle fennica n. sp. (= Rhipidocotyle Type A of Taskinen et al., 1991) from the intestine of Esox lucius in central Finland is described and compared by means of a principal components analysis (PCA) with R. campanula (= Rhipidocotyle Type B of Taskinen et al., 1991). Its cercaria develops in the bivalve Anodonta anatina and the metacercaria occurs in the skin and fins of Rutilus rutilus. The metacercaria is discriminated from that of R. campanula by PCA and is described along with aspects of the chaetotaxy of the cercaria. The new species is distinguished from R. campanula, R. kovalae, R. papillosa and R. septpapillata.     

Fluorescence studies on the interaction of some ligands with carcinoscorpin,the sialic acid specific lectin,from the horseshoe crab,Carcinoscorpius rotundacauda

The binding affinities of some ligands towards the sialic acid-specific lectin carcinoscorpin, from hemolymph of the horseshoe crabCarcinoscorpius rotundacauda have been determined by protein fluorescence quenching in presence of ligands. Among the ligands studied, the disaccharide O-(N-acetylneuraminyl)-(2→6)-2-acetamido-2-deoxy-D-galactitol has the highest K_a(l.15 × 10⁶ M^-1) for carcinoscorpin. Studies on the effect of pH on Ka values of disaccharide suggests the possible involvement of amino acid residues having pKa values around 6.0 and 9.0 in the binding activity of carcinoscorpin. There were distinct changes in the accessibility of the fluorescent tryptophan residues of carcinoscorpin by ligand-binding as checked through potassium iodide quenching.     

Guild structure in water mites (Unionicola spp.) inhabiting freshwater mussels: choice,competitive exclusion and sex

Summary Unionicolid water mites inhabit freshwater unionid mussels during the nymphal and adult stages of their life-cycle. Regular sampling of mussels from two sites in St. Mark's River, Fl. established that each of four species of water mite (Unionicola abnormipes, U. fossulata, U. serrata and U. formosa) occurred mainly in one or two of the mussel species available at each site.The role of preference for particular mussel species during host location was assessed for the first three mite species by choice experiments, in which mites were offered different mussel species simultaneously. In five out of six experiments, mites entered normally unused mussels as often as they did normally used ones. Additionally, a sexual difference in choice was found for U. fossulata, with males preferring one mussel species and females showing no preference. One mussel species, (Anodonta imbecilis), normally unused but chosen by mite species during the lab. experiments, is inhabited exclusively by the fourth mite species, U. formosa, in the field. An experiment showed that U. formosa excludes other mite species aggressively from Anodonta imbecilis.The results illustrate the sometimes misleading nature of simple sampling data as an indication of host specificity or host preference in parasites. They suggest also that the population dynamics of some parasites might be more fruitfully compared to unrelated, free-living species than to other parasites.     

Involvement of the adenylyl cyclase signaling system in the action of insulin and mollusk insulin-like peptide

Involvement of the adenylyl cyclase signaling system in the mechanism of action of the mammalian insulin and epidermal growth factor as well as of insulin-like peptide isolated from the bivalve mollusk Anodonta cygnea has been studied. It was shown for the first time that insulin and insulin-like peptide exert in vitro the GTP-dependent stimulating action on the adenylyl cyclase activity. Epidermal growth factor has an analogous effect. Effectiveness of the peptides decreased in the order insulin-like peptide > epidermal growth factor > insulin in the foot smooth muscles of A. cygnea and insulin > epidermal growth factor > insulin-like peptide in the skeletal muscles of rat.     

Sialic acid in hemolymph and affinity purified lectins from two marine bivalves

Sialic acids have been implicated in a variety of complex biological regulatory and signalling events and their functional importance is reflected by their presence in a wide variety of phyla. Potentially they may inhibit intermolecular and intercellular interactions. Lectins that exhibit specificity for sialic acid or sialoglycoconjugates are ubiquitous in the body fluids of invertebrates and this has supported the assumption that these lectins are involved in defense against microbes that express sialic acids on their surfaces. This biological function has also been inferred from the absence of sialic acids in lower invertebrates. However, most invertebrate lectins are heterogeneous and may also bind other ligands. The biological significance of the different carbohydrate specificities are not yet known. We have demonstrated the presence of sialic acids in hemolymph from two marine bivalves, the Pacific oyster Crassostrea gigas (≈15 μg ml⁻¹) and the horse mussel Modiolus modiolus (48–100 μg ml⁻¹) by several different assays. The sialic acid was mostly in free form. Affinity purified lectins from the horse mussel also contained bound sialic acids (2–5 μmol g⁻¹). Oyster hemolymph stimulated the in vitro phagocytosis of bacteria by oyster hemocytes. The stimulation by hemolymph is facilitated by a dialyzable component, that apparently is active irrespective of the binding to sialic acid (BSM). Addition of sialic acid had no significant effect on the in vitro phagocytosis of bacteria by oyster hemocytes.     

Metal-based carboxamide-derived compounds endowed with antibacterial and antifungal activity

Abstract

A series of three bioactive thiourea (carboxamide) derivatives, N-(dipropylcarbamothioyl)-thiophene-2-carboxamide (L¹), N-(dipropylcarbamothioyl)-5-methylthiophene-2-carboxamide (L²) and 5-bromo-N-(dipropylcarbamothioyl)furan-2-carboxamide (L³) and their cobalt(II), copper(II), nickel(II) and zinc(II) complexes (1)–(12) have been synthesized and characterized by their IR,¹H-NMR spectroscopy, mass spectrometry and elemental analysis data. The Crystal structure of one of the ligand, N-(dipropylcarbamothioyl)thiophene-2-carboxamide (L¹) and its nickel(II) and copper(II) complexes were determined from single crystal X-ray diffraction data. All the ligands and metal(II) complexes have been subjected to in vitro antibacterial and antifungal activity against six bacterial species (Escherichia coli. Shigella flexneri. Pseudomonas aeruginosa. Salmonella typhi. Staphylococcus aureus and Bacillus subtilis) and for antifungal activity against six fungal strains (Trichophyton longifusus. Candida albicans. Aspergillus flavus. Microsporum canis. Fusarium solani and Candida glabrata). The in vitro antibacterial and antifungal bioactivity data showed the metal(II) complexes to be more potent than the parent ligands against one or more bacterial and fungal strains.     

Identification of Mytilus spp. and Pecten maximus in Irish Waters by Standard PCR of the 18S rDNA Gene and Multiplex PCR of the 16S rDNA Gene

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.     

Antimicrobial peptides in oyster hemolymph: The bacterial connection

We have explored antimicrobial compounds in oyster hemolymph and purified four active peptides with molecular masses of 4464, 3158, 655 and 636 Da. While no exploitable structural elements were obtained for the former three, a partial amino acid sequence (X-P-P-X-X-I-V) was obtained for the latter, named Cg-636. Due to both its low MM and the presence of exotic amino acid residue (X), we suspected a bacterial origin and tracked cultivable hemolymph-resident bacteria of oyster for their antimicrobial abilities. Supernatants of 224 hemolymph resident bacteria coming from 60 oysters were screened against 10 target bacteria including aquaculture pathogens. Around 2% (5 strains) revealed antimicrobial activities. They belong to Pseudoalteromonas and Vibrio genera. Two closely related strains named hCg-6 and hCg-42 have been shown to produce Bacteriocin-Like Inhibitory Substances (BLIS) even in oyster hemolymph. We report herein first BLIS-producing bacteria isolated from bivalve hemolymph. These results strongly suggest that hemolymph resident bacteria may prevent pathogen establishment and pave the way for considering a role of resident bacteria into bivalve defense.     

The relations between mites of the genus Unionicola and the mussels Anodonta and Unio

The locations in mussels of four species of watermites, Unionicola aculeata, U. bonzi, U. intermedia and U. ypsilophora and their degrees of parasitism have been compared. Unionicola aculeata and U. bonzi do not live parasitic and need Anodonta anatina and Unio pictorum respectively as a shelter for their transformation stages. Unionicola intermedia and U. ypsilophora live parasitic on the gills of Anodonta anatina and A. cygnea respectively. Every female of Unionicola ypsilophora has a territory in the mussel, as contrasted with U. intermedia, many females of which are found together. The consequences for the population density are discussed.Zoological Laboratory, University of Amsterdam     

Reactive Oxygen Species in Unstimulated Hemocytes of the Pacific Oyster Crassostrea gigas: A Mitochondrial Involvement

The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ_m) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ_m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ_m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.     

Respiration Rate and Species-Specific Lifespan in Freshwater Bivalves of Margaritiferidae and Unionidae Families

We studied changes in the respiration rate in five species of freshwater bivalves as a function of animal age and size. The species-specific lifespan was calculated on the basis of the obtained data: Rubner's constant (Ru) and lifespan at standard temperature 20°C (T ₂₀). The longest and shortest lifespan among the studied mollusks was specific for the pearl mussel Margaritifera margaritifera(Ru= 37 kJ and T ₂₀= 36 years) and the freshwater mussel Anodonta anatina(Ru= 8 kJ and T ₂₀= 8 years), respectively.     

Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl‐tRNA synthetase enzymes

Orthogonal aminoacyl‐tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM‐based molecular docking and free energy calculations were used to evaluate rational design of specific protein–ligand interactions for aminoacyl‐tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p‐benzoyl‐L ‐phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man‐α‐O‐Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. Proteins 2010. © 2010 Wiley‐Liss, Inc.