Histopathology of the digestive gland of the bivalve mollusk Crenomytilus grayanus (Dunker, 1853) from southwestern Peter the Great Bay, Sea of Japan

The histomorphology of the digestive gland of the bivalve mollusk Crenomytilus grayanus from Sivuchya Bay, which is located in the southwest of Peter the Great Bay and subjected to the effect of polluted waters of Tumannaya River, was studied. Pathological changes of the digestive tubules, channels, and connective tissue of the gland were recorded in all the mussels studied. The epithelium of the tubules and channels was characteristic with erosive disturbances and by heavy vacuolization of digestive cells; connective tissue of the gland was specified by cells with lipofuscin (granulocytomes) and by foci of cells necrosis and lysis. Nervous fibers running in the gland were swollen in some mollusks. Strongly basophilic spherical formations, presumably one of the development stages of a parasitic plasmodium, were found in the granulocytomes and among vesicular cells of connective tissue of all the mussels. It was concluded that pathological changes in digestive gland of Gray’s mussel might be caused by chronic pollution of the bay and by parasitic invasion.     

Short-term bioaccumulation, circulation and metabolism of estradiol-17β in the oyster Crassostrea gigas

Steroids are active signal transmitters in Vertebrates. These roles have also been hypothesized in other Phyla and endocrine disrupting effects have been reported for different estrogen-like compounds in fishes and some marine invertebrates. As estradiol-17β has shown some physiological activities in the oyster and as estrogens or estrogen-like molecules can be present in water, we have investigated the bioaccumulation and metabolism of this estrogen in vivo in the oyster Crassostrea gigas. When dissolved in seawater, in less than 48 h estradiol-17β concentrated up to 31 times in the soft tissues of the suspension-feeder mollusc. Injected in the adductor muscle, estradiol-17β circulated from muscle to the gonad, the gills, the mantle, the labial palps, and to a lesser extent to the digestive gland. After 2 h, estradiol flow increased specifically towards this gland. Different hypotheses were raised concerning the circulation paths. However, in all cases estradiol metabolism primarily evidenced an in vivo transformation into estrone in the whole oyster and in its digestive gland. This strong 17β-hydroxysteroid-dehydrogenase activity confirms our previous in vitro results. In conclusion, it is proposed that oyster is able to take in charge estradiol as a potential contaminant in seawater. Therefore, its bioaccumulation and transformation into estrone could be studied as potential biomarkers of endocrine disruption. Furthermore, the experimental approach with dissolved steroids in the seawater combined to an anatomical screening appears as an interesting tool to investigate the bivalve endocrinology.     

Distribution of multiple peptidoglycan recognition proteins in the tissues of Pacific oyster, Crassostrea gigas

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors that specifically bind to peptidoglycans, a major component of bacterial cell wall. Generally, PGRPs are responsible for recognition of bacterial invasion in invertebrates. Full length cDNAs of PGRP, designated as CgPGRP-S1S, -S1L, -S2 and -S3, were identified from the Pacific oyster, Crassostrea gigas. Homology and domain searches classified these CgPGRPs as short-type PGRPs for extracellular PGN recognition. Amidase activity was predicted in all CgPGRPs, and defensin-like domains were found in CgPGRP-S1S and -S1L, suggesting that they may also function as antimicrobial proteins. Although phylogenetic analysis indicated that CgPGRPs are closely related to each other, they showed different tissue expression patterns; CgPGRP-S1S in the mantle and the gill, -S1L in the mantle, -S2 in the hemocytes and -S3 in the digestive diverticula. The CgPGRPs seem to survey bacterial invasion in their corresponding expression tissues. This is the first report of the possibility that bivalve mollusks have PGN recognition systems as suggested by the identification of multiple PGRPs distributed in various tissues.     

Storage metabolism in the Pacific oyster (Crassostrea gigas) in relation to summer mortalities and reproductive cycle (west coast of France)

We describe seasonal changes in the biochemical composition of digestive gland, adductor muscle and gonad and surrounding mantle area in Crassostrea gigas from the Western Atlantic coast of France. Seasonality in histology of storage tissues and glycogen storage capacity in isolated vesicular cells were also studied. Proteins, the main muscle components did not contribute to the gametogenetic effort. Glycogen and lipids were stored in the digestive gland, gonad and surrounding mantle area during the wintering period and the gonad and surrounding mantle area represented the main storage compartment supplying the reproductive effort. Gametogenesis in spring and summer was associated with an increase in lipid and protein contents and took place at the expense of glycogen reserves. Histological study of storage tissue in the gonad led us to define four seasonal stages of storage tissue development. In vitro, glycogen storage capacity in isolated vesicular cells was high from November to March and markedly reduced during gametogenesis, decreasing below detectable levels after spawning. This physiological state should be taken into account with relation to summer mortalities occurring in commercial growing areas.     

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.     

Parasites of three commercially exploited bivalve mollusc species of the estuarine region of the Cachoeira river (Ilhéus, Bahia, Brazil)

This paper reports the parasites found in three commercially exploited bivalve molluscs (Mytella guyanensis, Anomalocardia brasiliana and Iphigenia brasiliana) of an estuarine region of Ilhéus, south of Bahia, Brazil (14°48′23′′S; 39°02′47′′W). Samples of 20 individuals of each species were collected fortnightly from August 2005 to August 2006. A total of 1480 individuals was collected and processed by standard histologic techniques; the histologic sections were stained with Harris haematoxylin and eosin and examined with light microscope. The water temperature in the study area varied from 24 to 30.5 °C and the salinity from 0 to 23 ppt. Remarkable differences were found in the parasitic community between the three mollusc species involved in the study, which occupied different habitats in the estuarine region of the Cachoeira river. The following parasites were found: intracellular rickettsia-like colonies in digestive epithelia; intracellular gregarine Nematopsis sp. in gills, mantle, gonad, digestive gland and foot muscle; sporocysts of a Bucephalidae trematode in gonads, mantle, gills, digestive gland and foot; unidentified digenetic metacercariae in digestive gland and gonad; metacestodes of Tylocephalum sp. in connective tissue in the digestive gland and in gonad; and an unidentified metazoan in mantle and intestinal lumen. No significant temporal variation in the prevalence of any parasite was detected, which could be due to the narrow temperature range of the region and the absence of patterns of salinity and rainfall variation through the year. The infestation by sporocyst was the only pathological threat detected for the studied populations because of its potential for host castration. The low infection intensity and/or prevalence of the other parasites and the lack of obvious lesions suggest that there is no other serious pathological risk for the studied mollusc populations.     

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum)

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus.     

Proliferative characteristics of atypical cells in native oysters (Ostrea lurida) from Yaquina Bay, Oregon.

Abnormal, large, possibly neoplastic cells from two Ostrea lurida have been successfully labeled with tritiated thymidine, and thus, for the first time, data are available for analyzing a presumptive invertebrate proliferative disorder. Large numbers of labeled atypical cells (AC) were uniformly distributed throughout the connective tissue (CT) underlying the gills, mantle, stomach, gut, digestive diverticula, kidney, and gonad. Two general types of AC are associated with the proliferative disorder. Both are abnormally large with many features characteristic of undifferentiated cells, and both are probably members of a proliferating compartment. The labeling index (LI) for AC ranged from 1.22% in connective tissue surrounding digestive tubules to 14.17% in CT underlying gill epithelium; the LI for all AC was 4.82%. The data strongly suggest that the AC proliferate at a much greater rate than any other oyster cells subjected to similar treatment.     

Assessment of the state of the Japanese scallopMizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) based on morphological and biochemical parameters

The results of an integrated examination of the state of the scallopMizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) are presented. In mollusks of different ages, shell height was measured; in animals of commercial size (over 100 mm), some size and weight characteristics (annual increment of shell and adductor muscle and soft tissue weight) were determined. The morphology of the digestive gland and gills was studied. In the adductor muscle and digestive gland, the concentration of heavy metals (HMs) (Hg, Cu, Zn, Mn, Pb, Cd, Ni, Co, and Cr) was determined. In the digestive gland, metallothionein and reduced glutathione concentration was also determined, as was the activity of glutathione-dependent enzymes (glutathione peroxidase and glutathione reductase). In scallops collected outside Alekseeva Bight, the linear growth rate and adductor muscle weight were on average 1.3 and 1.7 times greater, respectively, than in those collected in the bight. In scallop organs, numerous histomorphological alterations were revealed: digestive cell vacuolization and hemocyte infiltration of the digestive gland, hyperplasia and vacuolization of the respiratory epithelium, and connective tissue hypertrophy in gill filaments. The biochemical parameters of scallops from Alekseeva Bight differed substantially from those of mollusks collected outside the bight. We conclude that one of the factors negatively affecting the state of theM. yessoensis population in Alekseeva Bight is the contamination of the bight with HMs, especially mercury. This is consistent with the results of chemical analysis of bottom sediments and tissues of two mytilid species,Modiolus kurilensis andCrenomytilus grayanus, specimens of which were collected in the bight together with the scallops [3].     

Comparative study of the sterol composition of the digestive gland and the gonad of Sepia officinalis L. (mollusca,cephalopoda)

• 1.1. The sterol composition of the digestive gland and the gonad of Sepia officinalis L. was investigated by GC and GC-MS.
• 2.2. The same sterols were recognized in both organs, cholesterol being the major component of the sterol mixtures. However, quantitative differences appeared between the sterol composition of the digestive gland and the gonad.
• 3.3. The sterol mixtures of the digestive gland and the gonad of immature and mature females and males of various origins were compared. Quantitative changes in the sterol composition of the gonad were related to sexual maturity whereas the sterol composition of the digestive gland appeared linked to the diet.

     

Regeneration of radiation-damaged digestive tissues in juvenile Pacific oysters (Crassostrea gigas)

Juvenile Pacific oysters, Crassostrea gigas, were irradiated with 16 and 40 krad and their tissues examined histologically. Degenerative syndromes and tissue regeneration processes were determined for the stomach, gut, collecting ducts, and digestive tubules. Following degeneration, tissue regeneration was observed in the digestive tissues of most oysters exposed to 16 krad and in a limited number exposed to 40 krad. Regeneration was first observed in the digestive tubules and subsequently in the stomach, gut, and collecting ducts. Cellular repopulation of the digestive tubules involved epithelialization with large, undifferentiated crypt cells which then differentiated into functional secretory and absorptive cells. Regeneration in the stomach, gut, and collecting ducts was initiated by proliferative islands of small basophilic cells. Mitotic division of those cells and their subsequent differentiation into functional epithelial cells resulted in the rapid restoration and apparent recovery of the affected tissues. The results of these studies indicate that radioresistance of juvenile C. gigas may in part be due to the remarkably efficient regenerative mechanisms involved in replacing injured or lost digestive tissues.     

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.     

Cadmium Accumulation and Metallothionein Biosynthesis in Cadmium-Treated Freshwater Mussel Anodonta woodiana

This study investigated the distribution of cadmium (Cd) and the protein level of metallothionein (MT) and examined the relationship of Cd accumulation and the MT concentration in different tissues of freshwater mussel Anodonta woodiana following Cd treatment. The mussels were exposed to Cd (4.21, 8.43, 16.86, 33.72 and 67.45 mg L^-1) for 24, 48, 72 and 96 h, respectively. After Cd treatment, the gills, mantle, foot, visceral mass and digestive gland tissues were collected for analysis. We found that, in the controls, Cd distributed in all tissues in the concentration order of gills>mantle>foot>visceral mass>digestive gland. Upon Cd treatment, Cd concentration significantly increased in all tissues. The highest Cd accumulation was found in the digestive gland, which was 0.142 mg g^-1 (P<0.05). MT levels in the gills and mantle of the mussels increased significantly (P<0.05), which were in positive correlation with Cd accumulation in the tissues (P<0.05). In conclusion, our results demonstrated a correlation between Cd accumulation and MT up-regulation in gills and mantle of the mussels after Cd treatment. It is suggested that the protein level of MT in gills and mantle of Anodonta woodiana is a good biomarker for Cd contamination.     

Comparison of quantitative gonad maturation scales in a temperate oyster (Crassostrea gigas) and a sub-tropical oyster (Crassostrea corteziensis)

Quantitative scales to evaluate maturity of female gonads were compared for a temperate oyster, Crassostrea gigas and a tropical oyster, Crassostrea corteziensis grown in the same locality. C. gigas had well-defined maturation and spawning periods and a resting phase in winter; in C. corteziensis mature individuals occurred most of the year and there were several spawning peaks. Of the quantitative scales used here, average oocyte diameter and gonad coverage area, much used for C. gigas, and ovary maturity index, less used, were inadequate to distinguish the reproductive pattern of C. corteziensis, since they both skewed the degree of maturation in vitellogenic stages in favor of C. gigas. Maximum oocyte diameter and maximum cytoplasm area were different among species at vitellogenic stages and also in previtellogenic stages. Nucleus:cytoplasm ratio was significantly different in previtellogenic and spawned stages between C. gigas and C. corteziensis. The Index of gonadal development was skewed in favor of C. gigas in postvitellogenic stage. The only scale that was comparable between species was reproductive potential, but it also was one of the most laborious. Other ordinal scales commonly used, such as a visual external evaluation of the gonad, only classified correctly a quarter of the stages.     

Arsenic and Arsenic Species in Cultured Oyster (Crassostrea gigas and C. corteziensis) from Coastal Lagoons of the SE Gulf of California, Mexico

The aim of this study was to evaluate the bioavailability of arsenic (As) through cultured oyster Crassostrea gigas and Crassostrea corteziensis from four coastal lagoons (SE Gulf of California). Organisms were collected in two seasons (rainy and dry season), and they were analyzed for total arsenic and chemical speciation of this element. The concentrations of As in oyster soft tissue fluctuated between 5.44 and 9.56 μg/g for rainy season and 6.46 and 8.33 μg/g for dry season (dry weight) in C. gigas. In C. corteziensis, the As concentrations were <5 μg/g for both seasons (dry weight). Arsenic speciation indicated arsenobetaine as the major arseno-compound accounting for 43.2–76.3 % of total content of As. Lower contributions were obtained for non-extractable As (11.3–17.5 %) and other molecules such as arsenocholine and methyl-arsonate (<5 %). Inorganic arsenic was detectable in only two samples, at concentrations lower than <0.1 μg/g. These As data are the first generated for these mollusks in NW Mexico and indicate that C. gigas and C. corteziensis farmed in this area are safe for human consumption in terms of arseno-compounds.     

Stress and Stress-Induced Neuroendocrine Changes Increase the Susceptibility of Juvenile Oysters (Crassostrea gigas) to Vibrio splendidus

Oysters are permanently exposed to various microbes, and their defense system is continuously solicited to prevent accumulation of invading and pathogenic organisms. Therefore, impairment of the animal's defense system usually results in mass mortalities in cultured oyster stocks or increased bacterial loads in food products intended for human consumption. In the present study, experiments were conducted to examine the effects of stress on the juvenile oyster's resistance to the oyster pathogen Vibrio splendidus. Oysters (Crassostrea gigas) were challenged with a low dose of a pathogenic V. splendidus strain and subjected to a mechanical stress 3 days later. Both mortality and V. splendidus loads increased in stressed oysters, whereas they remained low in unstressed animals. Injection of noradrenaline or adrenocorticotropic hormone, two key components of the oyster neuroendocrine stress response system, also caused higher mortality and increased accumulation of V. splendidus in challenged oysters. These results suggest that the physiological changes imposed by stress, or stress hormones, influenced host-pathogen interactions in oysters and increased juvenile C. gigas vulnerability to Vibrio splendidus.     

Reproduction and lipids in the sub-Antarctic limpet Nacella (Patinigera) macquariensis Finlay, 1927

The reproductive cycle of the sub-Antarctic limpet, Nacella (Patinigera) macquariensis Finlay, 1927 is described from monthly gonad indices and slide smears of gonadal tissue. Two populations were sampled, from the eulittoral zone and at ≈4 m depth into the sublittoral zone. Gametogenesis started 2 months earlier in the eulittoral zone population; the breeding period, however, was sufficiently long to overlap in the two populations. This phase difference in reproductive cycles is discussed with respect to possible genetic divergence and correlation between timing within the reproductive cycle and environmental factors. Lipid was a prominent component of both the digestive gland and the gonad, but not of the foot. Changes in lipid levels during the reproductive cycle indicated that the digestive gland stored lipids which were transferred to the gonad during gametogenesis.     

Growth and reproduction of the bay scallop, Argopecten irradians (Lamarck) at its southern distributional limit

Growth and reproduction in a Florida population of bay scallop, Argopecten irradians (Lamarck), existing at the southern limit of its distribution, was monitored over a 3-yr period. Somatic growth accompanied increasing water temperature between May and August, when maximum mantle, digestive gland, and adductor muscle weights and indexes were observed. From July through September, reproductive development was correlated with a decline in adductor muscle weight and index. Maximum gonad weight, gonad index, and oocyte diameter were found in late September and early October. Spawning commenced at this time in conjunction with decreasing water temperature.In comparison to more northern populations, the reproductive cycle of the Florida bay scallop occurs later in the year and is characterized by a higher gametogenic initiation temperature, a decreased maximum gonad index and decreased oocyte diameter, and a shift in reproductive energy resources from digestive gland (available food) to adductor muscle (pre-stored reserves). Increased metabolic rate associated with higher temperature and coupled with decreased food supply results in the Florida bay scallop having less energy available for reproduction, ultimately limiting its southern distribution.