Reproductive cycle of the mangrove oyster Crassostrea rhizophorae (Bivalvia: Ostreidae) in Camamu Bay, Bahia, Brasil

The mangrove oyster Crassostrea rhizophorae is important fishery resource along the entire Brasilian coast with excellent potential for marine culture. The purpose of this paper was to examine the reproductive characteristics of the oyster of the Maraú river estuary in Camamu Bay, Bahia, Brasil. The samples were collected monthly, from September 2006 to August 2007, at two points (I and II) in Camamu Bay. At each site 20 oysters were collected for histological analysis, fixed in Davidson's solution, embedded in paraffin, dehydrated in an ethanol series, sectioned (7 microm thick) and stained with Harris hematoxylin and Eosin (HE). Additionally, 30 oysters were sampled, at each point, for a condition index analysis. The water temperature ranged from 23.5 degrees C to 30 degrees C and the salinity from 15 to 25 ups at Point I (Maraú) and from 25 to 35 at Point II (Tanque Island). The oyster's height ranged from 30 to 92 mm at Point I and from 27 to 102 mm at Point II, with an average of 49.0 mm +/- 9.1 (n = 230) and 49.9 mm +/- 9.9 (n = 237), respectively. Among the sampled oysters at Point I, 59.1% were females, 31.3% males, 1.3% hermaphrodites and 8.2% of the oysters of undetermined sex. At Point II, 66.2% were females, 30.4% males, 0.8% hermaphrodites and 2.5% (n = 237) of undetermined sex. The gonadic stage analysis indicated that the reproduction period of the C. rhizophorae in the Maraú Peninsula was continuous all year, without any regressive phase. The condition index (R) ranged from 8.0% to 17.7%. The peak periods of R coincided with the expressive oyster's percentage in the maturation and liberation gametic stages. The results of these findings will contribute information for the oyster spat collection and to the process installation of the oyster culture in Camamu Bay.     

Effect of probiotic bacteria on survival and growth of Cortez oyster larvae, Crassostrea corteziensis (Bivalvia: Ostreidae)

Disease control problems have major constraints in aquaculture production, and the use of probiotics in larviculture is a valid alternative to antibiotics. This study analyzed the effect of probiotic bacteria on survival and final size of Cortez oyster larvae Crassostrea corteziensis. Two different probiotic concentrations were evaluated, 1 x 10(4) and 1 x 10(5) CFU/ml of Lactic acid bacteria (strain NS61) isolated from Nodipecten subnodosus, and bacilli isolated from the white leg shrimp, Litopenaeus vannamei (Pseudomonas aeruginosa, strain YC58) and C. corteziensis (Burkholderia cepacia, strain Y021). Bacteria were added directly into culture tanks, starting the bioassays from veliger to pediveliger stages as follows: (1) Control, without probiotics; (2) lactic acid bacteria (Lb); (3) bacilli mix (Mb) in a proportion 1:1. Results showed a higher larval survival with Lb and Mb at a dose of 1 x 10(4) CFU/ml compared to the control group. Larvae exposed to Mb at 1 x 10(5) CFU/ml showed higher survival than Lb and control. Larval final size was not significantly increased with the tested probiotics, but larvae treated with Lb at 1 x 10(5) CFU/ml showed less survival rate than those treated at 1 x 10(4) CFU/ml. This study showed the beneficial effect of these probiotics, added individually or mixed in C. corteziensis larvae culture.     

Differential sensitivity to cadmium of key mitochondrial enzymes in the eastern oyster, Crassostrea virginica Gmelin (Bivalvia: Ostreidae)

Combined effects of cadmium (Cd) and temperature on key mitochondrial enzymes [including Complexes I-IV of electron transport chain and Krebs cycle enzymes citrate synthase (CS), and NAD- and NADP-dependent isocitrate dehydrogenases (NAD-IDH and NADP-IDH)] were studied in a marine ectotherm, Crassostrea virginica in order to better understand the mechanisms of Cd-induced impairment of mitochondrial function. Matrix enzymes including CS and isocitrate dehydrogenases were the most sensitive to Cd making Krebs cycle a likely candidate to explain Cd-induced impairment of mitochondrial substrate oxidation. CS and NAD-IDH had IC(50) of 26 and 65 microM at the acclimation temperature (15 degrees C) and 65 (CS) and 1.5 (NAD-IDH) microM at elevated temperature (25 degrees C), respectively. Mitochondrial NADP-IDH was the most sensitive to Cd with IC(50) of 14 and 3.4 microM at 15 degrees and 25 degrees C, respectively. Electron transport chain (ETC) complexes were significantly less sensitive to the direct effects of Cd with IC(50) ranging from 260 to >400 microM. Temperature increase led to a higher sensitivity of mitochondrial enzymes to the inhibitory effects of Cd as indicated by a decline in IC(50) with the exception of Complex III from gills and CS from gills and hepatopancreas. Cd exposure also resulted in a decrease in activation energy of mitochondrial enzymes suggesting that mitochondria from Cd-exposed oysters could exhibit reduced capacity to respond to temperature rise with an adequate increase in the substrate flux. These interactive effects of Cd and temperature on mitochondrial enzymes could negatively affect metabolic performance of oysters and possibly other ectotherms in polluted environments during temperature increase such as expected during the global climate change and/or tidal or seasonal warming in estuarine and coastal waters.     

Xenobiotic biotransformation in the Pacific oyster (Crassostrea gigas)

1. Oyster visceral mass and gill tissues possessed measurable flavin-containing monooxygenase (FMO) activity. 2. FMO activity was confirmed in visceral mass microsomes by oxygen uptake experiments utilizing various nitrogen and sulfur-containing chemicals along with measurement of N,N-dimethylaniline (DMA) N-oxidase and methimazole oxidation activities. DMA N-oxidase and methimazole oxidation activities also were present in gill microsomes. 3. Excluding oyster gill methimazole oxidation, there were no consistent seasonal differences in FMO activity in oyster gill or visceral mass microsomes. 4. Although lacking spectral evidence for cytochrome P-450, a peak at 418 nm was observed along with NADPH-cytochrome c reductase activity in visceral mass and gill microsomes suggesting the presence of a denatured cytochrome P-450 system. 5. NADPH-independent benzo(a)pyrene hydroxylase (BPH) activity was observed in both oyster visceral mass and gill microsomes suggesting a co-oxidation pathway possibly involving a one electron transfer of oxygen from a lipid hydroperoxide.     

Reproductive traits of the freshwater oyster Etheria elliptica (Bivalvia: Etheriidae) in the Pendjari River,Benin: implications for conservation

The African freshwater oyster Etheria elliptica, which is of great economic importance throughout the continent, is facing overharvesting in many fisheries in West Africa. Its reproductive traits (sex ratio, size at sexual maturity, oocyte diameter and fecundity) were studied at four stations located along the Pendjari River, northern Benin, in April 2013. Histological techniques were implemented to identify sex and gonad development stages. Oocyte sizes were measured based on the histological images and mean oocyte diameter was 38 µm. Fecundity, estimated by counting the developing eggs, averaged 106 724 eggs and increased significantly with shell size. The average sex ratio was approximately 1:1. Hermaphrodites were rare. Males reached sexual maturity at 57.6 mm dorsoventral height, earlier than females at 64.7 mm. The majority of the oyster specimens were in ripe-spawning stages, indicating that reproductive activities partly took place towards the end of the dry season. Both mature and immature individuals were subjected to harvesting at all the investigated fishing sites. The mean size of exploited oysters was 60.6 mm, lower than the size of females at maturity. Management strategies must enforce the minimum size of 65 mm required for harvesting, particularly in open access sites where juveniles are mostly harvested.     

First record of parasitism in the mangrove oyster Crassostrea rhizophorae (Bivalvia: Ostreidae) at Jaguaribe River estuary--Ceará, Brazil.

Mangrove oysters Crassostrea rhizophorae were sampled monthly in the estuary of Jaguaribe River, on the east coast of Ceará State, Brazil, between August, 2000 and December, 2001, making up 170 individuals. The water temperature varied from 26 to 30 degrees C and salinity from 21 to 42. The animals' size ranged from 3.4 to 7.2 cm height. Macroscopical and histopathological analyses were carried out in the oysters' tissues. The histological exams showed protozoans and metazoans of genera Nematopsis and Tylocephalum, respectively. Nematopsis prevalence varied from 60 to 100% and it was higher in the gills and mantle. The oocysts presented a mean size of 11.5 microm (+/-1.32) length and 9.1 microm (+/-1.06) width (n = 30), up to 3 oocysts/phagocyte having been observed. Several animals presented focal hemocitical reaction. The percentage of Tylocephalum was 1.7%. In spite of the high infection prevalence by Nematopsis, infected animals did not have their reproductive cycle impaired.     

Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia)

The aim of this study is to describe the early stages of spermatogenesis of the Pacific oyster Crassostrea gigas using both light and electron microscopy. The gonad is formed by gonadal tubules invaginated in a connective tissue constituting a storage tissue. Myoepithelial cells surround each gonadal tubule and are associated with an acellular matrix delimiting the outer part of the tubule, the inner part is composed by intragonadal somatic cells associated with germinal lineage. Two types of spermatogonia are identified, where type I spermatogonia (Spg I) are large, scarce and pale cells leaned against the base of the tubule (nuclear diameter: 5.5+/-0.5 microm). Type II spermatogonia (Spg II) are clustered and dark cells which appear smaller than type I (nuclear diameter: 4.3+/-0.3 microm). The aspect of nuage-like material in cytoplasm is described from pale spermatogonia to primary spermatocytes (nuclear diameter: pachytene 3.6+/-0.3 microm, diplotene 3.4+/-0.3 microm), while no structure related to a chromatoid body was observed in oyster spermatocytes and spermatids.     

Isolation of sperm bindin from the oyster (Crassostrea gigas)

A method was devised for isolating the insoluble content of the acrosome granule of sperm of the oyster Crassostrea gigas. The method involves the dissolution of the entire cell, except for the acrosome granule, in the detergent sodium lauroyl sarcosinate (sarcosy I). The isolated acrosome granule content is ring-shaped and is 84% protein by weight. SDS-polyacrylamide gel electro-phoresis of this material yields from 1 to 4 bands of 65,000; 53,000; 43,000 and 34,000 apparent molecular weight, all of which stain with the PAS reaction indicating the material is a glycoprotein. The 65,000 molecular weight component is always present, but the presence of the other three bands varies with each preparation. The isolated acrosome granules agglutinate formaldehyde-fixed oyster eggs. A trypsin-generated glycopeptide digest of oyster egg surfaces inhibits the agglutinin activity of the isolated acrosome granules. We propose that the acrosomal glyco-protein material is oyster sperm bindin which functions as the adhesive substance bonding the sperm to the egg during fertilizaion.     

Successful cryopreservation of Pacific oyster (Crassostrea gigas) oocytes

Protocols for cryopreservation of sperm and oocytes would provide the ultimate control over parental crosses in selective breeding programmes. Sperm freezing is routine for many species, but oocyte freezing remains problematic, with virtually zero success in aquatic species to date. This paper describes the development of a successful protocol for cryopreserving high concentrations of Pacific oyster (Crassostrea gigas) oocytes. Ethylene glycol (10%) and dimethyl sulfoxide (15%) were found to be the most effective cryoprotectants resulting in post-thaw fertilization rates of 51.0+/-8.0 and 45.1+/-8.3%, respectively. Propylene glycol was less effective and methanol resulted in zero fertilization post-thaw. The use of Milli-Q water rather than seawater as a base medium significantly improved fertilization (20.4+/-3.0 and 8.7+/-2.2%, respectively) as did the inclusion of a 5 min isothermal hold at -10 or -12 degrees C (35.9+/-5.0 and 31.9+/-4.6%, respectively). The optimal cooling rate post-hold was 0.3 degrees C min(-1), with virtually zero post-thaw fertilization with cooling rates of 3 and 6 degrees C min(-1). Using an optimized protocol, post-thaw fertilization rates for oocytes from eight individual females ranged from 0.8 to 74.5% and D-larval yields from 0.1 to 30.1%. For three individuals, larvae were reared through to spat. Development of D-larvae to eyed larvae and spat was similar for larvae produced from unfrozen (24.8+/-4.1% developed to eyed larvae and 16.5+/-3.2% to spat) and cryopreserved (28.4+/-0.6 and 18.7+/-0.5%, respectively) oocytes. The ability to cryopreserve large quantities of oyster oocytes represents a major advance in cryobiology and selective breeding.     

Fine structure of the spermatozoa of Crassostrea gigas (Mollusca,Bivalvia)

We describe sperm ultrastructure and acrosome differentiation during spermiogenesis in Crassostrea gigas (Mollusca Bivalvia). The sperm cell is a uniflagellated cell of the primitive type. The head region contains a rounded or conical nucleus surmounted by small acrosome. This organelle consists of a membrane-bound acrosomal granule, the contents of which have a homogeneous density, except in the anterior region, which is positive for PTA. The acrosome also surrounds the perforatorium, which includes oriented fibrillar elements: this is the axial body. The middle piece contains four mitochondria encircling two perpendicular centrioles. The distal centriole is provided with a system of mechanical fixation to the plasma membrane, consisting of nine fibers in radial arrangement. The tail flagellum, about 50 m?m long, contains the usual microtubular axoneme. © 1993 Wiley-Liss, Inc.     

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.     

Metabolic adjustments in the oyster Crassostrea gigas according to oxygen level and temperature

The purpose of this study was to examine the responses of the oyster Crassostrea gigas to oxygen levels at subcellular and whole organism levels. Two experiments were carried out. The first experiment was designed to measure the clearance and oxygen consumption rates of oysters exposed at different concentrations of oxygen at 15, 20 and 25°C for 20 h. The goal of this first part was to estimate the hypoxic threshold for oysters below which their metabolism shifts towards anaerobiosis, by estimating the oxygen critical point (PcO₂) at 15, 20 and 25°C. The second experiment was carried out to evaluate the metabolic adaptations to hypoxia for 20 days at three temperatures: 12, 15 and 20°C. The metabolic pathways were characterized by the measurement of the enzymes pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK), the alanine and succinate content and the adenylate energy charge. Respiratory chain functioning was estimated by the measurement of the activity of the electron transport system (ETS). The values of PcO₂ were 3.02±0.15, 3.43±0.20 and 3.28±0.24 mg O₂ l^-1 at 15, 20 and 25°C, respectively. In whole oysters, hypoxia involved the inhibition of PK whatever the temperature, but PEPCK was not stimulated. Succinate accumulated significantly only at 12°C and alanine at 12 and 15°C. A negative relationship between the PK activity and the alanine content was only found in hypoxic oysters. Finally, hypoxia increased significantly the activity of ETS. With high PcO₂ values, the metabolic depression occurred quickly, showing that oysters had a low capacity to regulate their respiration when oxygen availability is reduced, particularly in the summer.     

Characterization of genes involved in ceramide metabolism in the Pacific oyster (Crassostrea gigas)

ABSTRACT: BACKGROUND: The lipid signaling molecule, ceramide, is a key component of the vertebrate stress response, however, there is limited information concerning its role in invertebrate species. In order to identify genes involved in ceramide metabolism in bivalve molluscs, Pacific oyster genomic resources were examined for genes associated with ceramide metabolism and signaling. RESULTS: Several genes were identified including full-length sequences characterized for serine palmitoyltransferase-1, 3-ketodihydrosphingosine reductase, acid ceramidase, and ceramide glucosyltransferase. Genes involved in ceramide synthesis and metabolism are conserved across taxa in both form and function. Expression analysis as assessed by quantitative PCR indicated all genes were expressed at high levels in gill tissue. The role of the ceramide pathway genes in the invertebrate stress response was also explored by measuring expression levels in adult oysters exposed to Vibrio vulnificus. Two genes demonstrated increased expression during the bacterial challenge: a gene involved in hydrolytic breakdown of ceramide (acid ceramidase) and a gene involved in de novo generation of ceramide (3-ketodihydrosphingosine reductase), suggesting a possible role of ceramide in the invertebrate stress and immune responses. CONCLUSIONS: In silico and laboratory results support that Pacific oysters have the basic components of the ceramide metabolism pathway. These results also indicate that ceramide may have analogous functions in vertebrates and invertebrates. The gene expression pattern of acid ceramidase and 3-kethodihydrosphingosine reductase in response to bacterial exposure especially supports that ceramide and sphingolipid metabolism may be involved in the oyster's stress and/or immune responses.     

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.     

太平洋牡蛎酪氨酸酶基因家族的系统发生分析

文章利用生物信息学方法对太平洋牡蛎(Crassostrea gigas Thunberg)酪氨酸酶基因家族的氨基酸序列特征、分类及系统发生进行了分析。结果表明, 太平洋牡蛎酪氨酸酶基因家族在进化过程中存在基因扩张现象, 其主要方式是基因重复。太平洋牡蛎酪氨酸酶可分为3种类型：分泌型 (Type A), 胞内型 (Type B)和具跨膜结构域型 (Type C)。根据太平洋牡蛎酪氨酸酶进化树分析, 发现Type A酪氨酸酶中, tyr18与其他Type A酪氨酸酶分化较大, 可能是较早分化出来的酪氨酸酶; Type B酪氨酸中的tyr2和tyr9以及Type C中的tyr8为较早分化出的酪氨酸酶。系统发生树分析发现太平洋牡蛎酪氨酸酶的聚类受酪氨酸酶类型以及基因位置的影响, 其分泌型酪氨酸酶首先与头足类分泌型酪氨酸酶聚在一起, 然后与线形动物门分泌型酪氨酸酶聚在一起, 与腔肠动物门分泌型酪氨酸酶分化明显。太平洋牡蛎胞内型酪氨酸酶自身分化较大, 总体上与线性动物门、其他软体动物胞内型酪氨酸酶聚为一支, 与扁形动物门、脊索动物门、腔肠动物门胞内型酪氨酸酶分化较大。太平洋牡蛎具跨膜结构域型酪氨酸酶与扁形动物门、环形动物门以及脊索动物门具跨膜结构域型酪氨酸酶分化明显, 与合浦珠母贝具跨膜结构域型酪氨酸酶聚为一支。这表明双壳类的Type C型酪氨酸酶与其他物种的同源酶的进化差异较大。文章首次探讨了太平洋牡蛎酪氨酸酶家族分类、分化及系统发生, 以期对太平洋牡蛎酪氨酸酶基因家族的理论研究和实际应用提供依据。