Clinical trial results on the use of a recombinant feline interferon-omega to protect Japanese pearl oysters Pinctada fucata martensii from akoya-virus infection

Japanese pearl oysters (akoya oyster: Pinctada fucata martensii) are currently suffering mass mortalities from akoya-virus infection. In the present study, akoya oysters were injected with an anti-viral, recombinant feline interferon-omega) (rFeIFN-omega) in an attempt to confer resistance to this virus. In infectivity experiments, oysters were twice injected with rFeIFN-omega at 1 mega unit kg(-1) of the meat weight. They were challenged with a single inoculation of cultured akoya-virus and held for 20 to 30 d at 25 degrees C. Control oysters received only the viral challenge without rFeIFN-omega administration. In prophylactic experiments, oysters that were given the akoya-virus on Days 1 to 5 after rFeIFN-omega administration showed lower mortalities. Furthermore, the survivors had fewer muscular lesions resulting from the virus infection than the controls. In treatment experiments, the virus was inoculated on Days 1 to 3 before rFeIFN-omega administration. None of the treated oysters died within a 30 d experimental period. Survivors displayed repaired lesions with fibrous tissues that were produced by enhanced agranulocytes in the body musculature. Moreover, rFeIFN-omega) was not toxic to akoya oysters. Thus, rFeIFN-omega administration is efficacious in preventing mortality of akoya oysters with akoya-virus infection.     

Cytological response of hemocytes in the European flat oyster, Ostrea edulis, experimentally exposed to mercury

Molluscs bivalves have been widely used as bioindicators to monitor contamination levels in coastal waters. In addition, many studies have attempted to analyze bivalve organs, considered pollutant-targets, to understand the bio-accumulation process and to characterize the effects of pollutants on the organisms. Here we analyzed the effects of mercury exposure on flat oyster hemocytes. Optical and electronic microscope procedures were used to characterize hemocyte morphology. In addition, cell solutions treated with acridine orange were analyzed by flow cytometry and laser scanning cytometry in order to evaluate the variations of cytoplasmic granules (red fluorescence, ARF) and cell size (green fluorescence, AGF) of hemocyte populations over time. Light and electron microscopical studies enabled us to differentiate four hemocyte subpopulations, agranulocytes (Types I and II) and granulocytes (Types I and II). Slight morphological differences were observed between control and Hg-exposed cells only in granulocytes exposed to Hg for 30 days, where condensed chromatin and partially lysed cytoplasmic regions were detected. Flow and laser scanning cytometry studies allowed us to differentiate three hemocyte populations, agranulocytes (R1) and granulocytes (R2 and R3). The exposure time to Hg increased the average red fluorescence (ARF) of agranulocytes and small granulocytes, while there was no change in large granulocytes, which showed a loss of membrane integrity. In control oysters, the three hemocyte populations showed an increase of ARF after 19 days of exposure although initial values were restored after 30 days. The average green fluorescence (AGF) was more stable than the ARF throughout the experiment. In Hg-exposed oysters, the values of AGF of agranulocytes showed an increase at half Hg-exposure period while the AGF values of large granulocytes decreased throughout the experiment, confirming the instability of these types of cells. The relative percentage of small granulocytes and granulocytes showed time variations in both control and exposed oysters. However, the values of small granulocytes remained constant during the whole experiment. The fact that there were only changes in agranulocytes and large granulocytes suggested a possible relationship between these two types of cells. In a quantitative study, we found a significant linear relationship between the agranulocytes and large granulocytes.     

Damage to cultivated Japanese pearl oysters by oxidative stress that was related to "mass mortality"

Increased blood-DNA breakage was observed in diseased pearl oysters. They showed significant formation of 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA), whereas the oysters that had a low mortality rate from the disease had high activity of superoxide dismutase (SOD) and low amounts of 8-OHdG and MDA. These results suggest that radical damage had occurred only in the diseased pearl oysters with the cytolysis of their haemocytes, which was related to the mass mortality of the Japanese pearl oysters.     

Damage to Cultivated Japanese Pearl Oysters by Oxidative Stress That Was Related to “Mass Mortality”

Increased blood-DNA breakage was observed in diseased pearl oysters. They showed significant formation of 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA), whereas the oysters that had a low mortality rate from the disease had high activity of superoxide dismutase (SOD) and low amounts of 8-OHdG and MDA. These results suggest that radical damage had occurred only in the diseased pearl oysters with the cytolysis of their haemocytes, which was related to the mass mortality of the Japanese pearl oysters.     

Mass mortality of the Japanese pearl oyster Pinctada fucata martensii in relation to water temperature, chlorophyll a and phytoplankton composition

Mass mortalities of the Japanese pearl oyster Pinctada fucata martensii have widely occurred in western Japan since 1994. The causes of these mass mortalities are at present not thoroughly understood. In this study, we investigated oyster survival in relation to some environmental factors such as water temperature, concentration of chlorophyll a and density or composition of phytoplankton. The examined mass mortality occurred from September to December 1998, and the color on the adductor muscle of the oysters was red-brown, suggesting an infectious disease. Oysters that became moribund during the experiment lost weight, while the weight of unaffected oysters increased. The cell density of Nitzschia spp., an inedible algae for the oyster, in Uchiumi Bay increased before and during the mass mortality event. From the results of our study, we hypothesize that P. fucata martensii was weakened by starvation because of the dominance of inedible food and then contracted an infectious disease that resulted in mortality.     

Comparison of Two Pearl Sacs Formed in the Same Recipient Oyster with Different Genetic Background Involved in Yellow Pigmentation in <Emphasis Type="Italic">Pinctada fucata</Emphasis>

Color is one of the most important factors determining the commercial value of pearls. Pinctada fucata is a well-known pearl oyster producing high-quality Akoya pearls. Phenotypic variation in amount of yellow pigmentation produces white and yellowish pearls. It has been reported that polymorphism of yellow pigmentation of Akoya pearls is genetically regulated, but the responsible gene(s) has remained unknown. Here, we prepared pearl sac pairs formed in the same recipient oyster but coming from donor oysters that differ in their color. These two pearl sacs produced pearls with different yellowness even in the same recipient oyster. Yellow tone of produced pearls was consistent with shell nacre color of donor oysters from which mantle grafts were prepared, indicating that donor oysters strongly contribute to the yellow coloration of Akoya pearls. We also conducted comparative RNA-seq analysis and retrieved several candidate genes involved in the pearl coloration. Whole gene expression patterns of pair sacs were not grouped by pearl color they produced, but grouped by recipient oysters in which they were grown, suggesting that the number of genes involved in the yellow coloration is quite small, and that recipient oyster affects gene expression of the majority of genes in the pearl sac.     

Chemical stimuli in coral reefs: how butterflyfishes find their food

Animals use sensory stimuli either to assess and select habitats, mates or food, as well as for communication. The present study aimed to understand the behavioural processes enabling several Chaetodon species (butterflyfishes) to locate one of their food sources (epibionts present on pearl oyster shells) at Rangiroa atoll (French Polynesia). Among the five species tested, our 2-channel choice flume chamber experiments identified three species that were attracted to their food source by chemical stimuli. HPLC experiments showed that pearl oysters and epibionts have specific and unique chemical fingerprints, either one or nine specific peaks, respectively. Overall, chemical stimuli are emitted by both epibionts (used directly by Chaetodon auriga, C. lunula and C. citrinellus) and live pearl oysters (used indirectly by C. auriga and C. lunula) to locate their food source. Biosynthesis of these chemical stimuli could be used to artificially attract butterflyfishes to pearl oyster rearing stations in order to increase the natural cleaning of pearl oyster shells and thus reduce one large cost for this aquaculture.     

Variability of Ribosomal DNA ITS-2 and Its Utility in Detecting Genetic Relatedness of Pearl Oyster

The objective of this study was to detect interspecific and intraspecific genetic variations of the second internal transcribed spacer of ribosomal DNA (ITS-2), and explore the feasibility of using it as a molecular marker phylogenetic analyses and species identification among pearl oysters. ITS-2 sequences of 6 pearl oysters were amplified via polymerase chain reaction. The amplified DNA fragments were about 500 bp, spanning the partial sequences of 5.8S and 28S rRNA genes. The GC contents of all species used in this study were higher than the AT contents. The variations of sequences involved substitutions as well as insertions/deletions and were mainly concentrated in spacer regions. Sequences of about 30-bp in spacer regions showed no variations among 5 Pincatda species. Intraindividual and intraspecific polymorphisms of ITS-2 sequences were detected in some species; the interspecific variability was significantly larger than the variability within species, and the variability at the genus level was higher than that at the species level. Both neighbor-joining and parsimony analyses of ITS-2 sequences revealed the distinguishable species boundary of 6 pearl oysters, and indicated that P. chemnitzi and P. nigra were the closely related species, as were P. maxima and P. margaritifera. The findings revealed that ITS-2 sequences could be an appropriate tool for phylogenetic study of pearl oysters.     

A Sperm Spawn-Inducing Pheromone in the Silver Lip Pearl Oyster (<Emphasis Type="Italic">Pinctada maxima</Emphasis>)

Pheromones are considered to play an important role in broadcast spawning in aquatic animals, facilitating synchronous release of gametes. In oysters, the sperm has been implicated as a carrier for the spawn-inducing pheromone (SIP). In hatchery conditions, male pearl oysters (Pinctata maxima) can be stimulated to spawn through a variety of approaches (e.g. rapid temperature change), while females can only be induced to spawn through exposure to conspecific sperm, thus limiting development of targeted pairing, required for genetic research and management. The capacity for commercial production and improvement of genetic lines of pearl oysters could be greatly improved with access to a SIP. In this study, we prepared and sequenced crude and semi-purified P. maxima sperm extracts that were used in bioassays to localise the female SIP. We report that the P. maxima SIP is proteinaceous and extrinsically associated with the sperm membrane. Bioactivity from pooled RP-HPLC fractions, but not individual fractions, suggests that the SIP is multi-component. We conclude that crude sperm preparations, as described in this study, can be used as a sperm-free inducer of female P. maxima spawning, which enables for a more efficient approach to genetic breeding.     

圆背角无齿蚌血细胞在体内和体外损伤修复过程中的功能

以圆前角无齿蚌为材料,采用外套膜小片的异体移植与加入血细胞的体外培养,研究了血细胞在体风,唯物 修复及珍珠囊构建中的功能。结果表明人工育珠手术后的损伤修复中,血细胞有形成无颗粒细胞层修复伤口、６诱导表皮细胞迁移和再生并形成珍珠囊的功能,而在体外的组织培养中加入血细胞比不加入血细胞的对照组,能更快地诱导上皮细胞迁移和再生,在整个伤面形成２覆盖层,即形成类似珍珠囊结构。本文首次在国内、外报道了用细胞培     

Identification of chitin in the prismatic layer of the shell and a chitin synthase gene from the Japanese pearl oyster, Pinctada fucata

The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is beta-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell.     

Development of Expressed Sequence Tags from the Pearl Oyster, <Emphasis Type="Italic">Pinctada martensii</Emphasis> Dunker

The pearl oyster, Pinctada martensii, is the primary species used for the aquaculture production of marine pearls in China and Japan. Genetic tools and resources are needed to study the genome of this species and to understand the molecular basis of development, growth, host defense, pearl formation, and other important traits. In this study, we developed a set of expressed sequence tags (ESTs) for P. martensii. We constructed cDNA libraries from adult tissues and sequenced 7,128 ESTs. Clustering analysis identified 788 contigs (covering 5,769 ESTs) and 1,351 singletons, yielding a total of 2,139 unique genes. Of these unique genes, only 935 had significant (E-value ≤ 0.005) hits in GenBank, and the remaining 1,204 (56.3%) were novel. Most of the known genes are related to cellular structure, protein binding, and metabolic processes. Putative host-defense genes (86) were identified including C-type lectin, ferritin, polyubiquitin, proteases, protease inhibitors, scavenger receptors, heat shock proteins, and RAS oncogenes. The EST sequences developed in this study provide a valuable resource for future efforts on gene identification, marker development, and studies on molecular mechanism of host defense in pearl oysters.     

Spat collection studies on pearl oysters Pinctada mazatlanica and Pteria sterna (Bivalvia,Pteriidae) in Bahia de La Paz,South Baja California,Mexico

The present work deals with the temporal and bathymetrical variations of the epifaunal community associated with two species of pearl oysters (Pinctada mazatlanica and Pteria sterna) during a seed collecting season from June to November 1989. A total of 63 items (species, genera and/or families) were recorded; their variations in presence and abundance were followed during three periods (June–July, August–September and October–November). The collectors were examined for different immersion times (2, 4, 6 and 8 weeks) for each period. Community structure was compared through the Brillouin Index, the Morisita Index and the Importance Value.We define the chronology of spatfall for both species of pearl oysters and their bathymetrical distribution. Relationships between these species and the epifaunal community present into the collectors were analysed, searching for possible noxious effects on the survival of juvenile pearl oysters, and identifying index species related with their spatfall. A strategy for starting massive seed collection of both species is established, particularly for P. mazatlanica.     

Relationship between biofouling and growth of the pearl oyster pinctada fucata (Gould) in Kuwait,Arabian Gulf

Results are presented from a three-year investigation of the relationship between accumulations of marine fouling organisms and growth of the pearl oyster Pinctada fucata (Gould). Estimates are provided of the diversity of the foulers, and data on certain hydrological features of the experimental site are also given.There was an inverse correlation between growth of the pearl oyster and diversity of the biofouling assemblages, whatever depth. Growth curves did not follow seasonal variations in the environmental factors very exactly. It was also observed that the polychaete, Polydora vulgaris Mohammad, preferred the oysters planted on the bottom to those suspended near the surface. Bottom oysters suffered the highest mortality, but definite evidence of a relationship between mortality and infestation by the polychaete was not apparent. Percentages of both infestation and mortality were higher among the fouled oysters than those cleaned periodically.     

Morphology and mobility of oyster hemocytes: evidence for seasonal variations.

Hemocytes of Crassostrea virginica were video recorded and tracked to determine their locomotive rates and to assign these rates to Wright-stained morphological variants. From 24 oysters examined in January, February, March, and May, 1571 hemocytes were video recorded, identified, and their rate of locomotion (ROL) measured. Granulocytes (three types) and agranulocytes (one lymphoid and three nonlymphoid types) were recognized. Focusing on 15 oysters in March and May, 20,318 hemocytes were counted from duplicate slides to verify the classification and to show that predominant hemocytes vary greatly between samples and among individual oysters, yet population differences can be detected. Measured rates of locomotion indicate that the granulocyte subpopulation moved significantly faster (3.3 microns/min) than the agranulocyte subpopulation (0.7 microns/min) because most (81%) agranulocytes were not mobile. Of the mobile hemocytes, granulocytes were also significantly faster (4.8 microns/min vs. 3.5 microns/min, P less than 0.0001), and basophilic granulocytes (BASOs) were the most active and abundant cell type. Examination of monthly percentages of cells and ROL indicates, however, that granulocyte dominance and ROL are not invariable. Granulocyte percentages of more than 60% in January, February, and March decreased to 32% in May, and BASO dominance was reduced to 15%. Further, percentages of mobile granulocytes decreased from greater than 65% in January, February, and March to 50% in May. ROL for all cells decreased from greater than 2.3 microns/min in these months to 1.0 microns/min in May. The fewer mobile hemocytes tracked in May had significantly (P less than .05) lower average ROL (4.0 microns/min) than those in January and March (4.7 microns/min each). Agranulocytes increased in May due to an increase in nonlymphoid cells.     

Control of biofouling on pearl oysters Pinctada imbricata using wax and Chinese herbs

Abstract

Biofouling poses severe challenges to pearl oyster Pinctada imbricata culture in China, and controlling it is both labor- and capital-intensive. The antifouling properties of wax, and wax mixed with Chinese herbs, sprayed onto pearl oyster shell surfaces during peak biofouling seasons were evaluated. Pearl oysters coated with three wax treatments (plain wax, Chinaberry seed extract, Chinese honeylocust fruit extract) and a control (no treatment), were cultured in nets for up to 60?days. Mortality rate, fouling organism and pearl-oyster weights, and shell height are reported for individual oysters on each of six sampling dates. With the exception of oysters submerged for 12?days, all oysters were significantly affected by treatment type and submersion duration. Fouling weight increased more rapidly over time in the control-treatment oysters. Wax-based coatings deterred fouling-organism settlement on oysters for at least 2?months during the intensive fouling season, reducing mortality and not adversely effecting growth.     

A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France).

Juvenile oysters Crassostrea gigas cultured in the Bay of Morlaix (France) have suffered unexplained summer mortalities for over a decade. In the present study, we tested the hypothesis that a bacterial pathogen could be responsible for this phenomenon. A first attempt failed to isolate a bacterial pathogen from moribund or weak oysters. Only non-pathogenic, probably opportunistic, bacteria were isolated. As an alternative approach, we focused on oysters presenting reduced stress-response capacities (determined by circulating noradrenaline measurements), a characteristic of juvenile oysters entering an early phase of the disease. Cultures of bacterial isolates on TCBS plates revealed that a Vibrio strain was present in diseased oysters and scarce or absent in healthy oysters. Experimental infections indicated that this Vibrio can cause mortalities of juvenile oysters when injected at concentrations ranging from 10(4) to 10(8) CFU oyster(-1). Similarly to the summer mortality disease, the Vibrio isolate caused higher mortalities at higher temperatures; apparently, it could not be transmitted horizontally, it did not affect adult oysters and it induced stress-response dysfunctions in juvenile oysters. Phenotypic and genotypic characterizations identified the pathogen as Vibrio splendidus. Taken together, the present results satisfy Koch's postulate and suggest that this bacterial strain is probably responsible for the juvenile oyster summer mortalities in the Bay of Morlaix.