Marcel Le Pennec

Oocyte size, a means to evaluate the gametogenic development of the Pacific oyster, Crassostrea gigas (Thunberg)

Abstract This study reports on the results of 38,100 measurements of the oocyte diameters of the Pacific oyster, Crassostrea gigas , in three populations living along the French Atlantic shoreline. The analysis of oocytes diameters and histological examinations of the gonads indicated that C. gigas individuals collected in 1996, 1997 and 1998 in the Bay of Brest (Anse du Roz and Pointe du Château) and Marennes-Oleron showed a clearly defined pattern of seasonal gametogenic development. Comparisons between reproductive stages, sites and years did not reveal significant differences in oocyte diameter (* P >0.05). Considerations from qualitative and quantitative characteristics of this species allowed us to propose a reproductive scale based on oocyte diameter and consisting of four stages: (1) “Early gametogenesis” with an oocyte diameter mean of 8.47±4.6 μm, (2) “Growing stage” with a diameter mean of 21.4±8.4 μm, (3) “Mature stage” with a mean of 36±4.4 μm and (4) “Degenerating stage” with 46±7.3 μm in mean diameter. This scale was generated from a modal distribution analysis (interactive program for fitting mixtures of distributions) and microscopic descriptions of gonad characteristics. The use of this scale for reproductive studies in C. gigas is discussed.

Broodstock conditioning of the oyster Crassostrea gigas: origin and temperature effect

Abstract Broodstock conditioning of the pacific oyster Crassostrea gigas (Thunberg) was examined in the laboratory under controlled conditions. Three experiments were undertaken to determine (i) the effect of the origin on gametogenesis, (ii) the time necessary to obtain the first mature oocytes in standard conditioning procedures, and (iii) the impact of different conditioning temperatures (16, 19, 22 and 25 °C) on gametogenesis. Oocyte size was used to identify mature oocytes. Significant differences among populations were observed in the number of mature oocytes produced. Only the oysters from northern locations (Normandy and Brittany) reached the mature stage by the end of the standard conditioning. Concerning the effect of different temperatures during conditioning, the first mature oocytes were detected (absolute days) by the 27th day at 16 °C, 23rd day at 19 °C, 19th day at 22 °C and 22nd day at 25 °C. This information was applied to fit a logistic model to predict oocyte growth using temperature and time as main affecting parameters; D = D max /(1+ a e − kt ) with D —oocyte diameter at time t (μm), D max —the asymptotic oocyte diameter (μm), a —adjustment parameter, k —slope of the curve during growing stage and t —time (days). The model fit consistently with values achieved at 16, 19 and 22 °C. The results obtained during this study could be used to improve conditioning procedures for C. gigas .

Autumn conditioning of the oyster Crassostrea gigas: a new approach

Gametogenesis, emitted gametes and larval yield (D larvae) of the oyster, Crassostrea gigas (Thunberg), were simultaneously examined in laboratory under two different conditions of temperature and photoperiod. One batch (“natural” cycle, NC) followed the equivalent cycle of average conditions of these parameters measured in La Tremblade (Charente-Maritime, France) during the last 15 years. The second batch (“accelerated” cycle, AC) was maintained under a variation two times faster of these parameters. Three conditioning experiences were performed during October, November and December 1998 with oysters from both NC and AC conditions. “Accelerated individuals” produced oocytes in growing stage from October and mature oocytes were noticed from December. In contrast, oysters from NC produced growing oocytes until January. Animals in AC produced 42% (October), 56% (November) and 96% (December) of mature oocytes after conditioning. Only the “natural” lot conditioned in December showed growing (23%) and mature oocytes (8%). Higher Walne–Mann index (WMI) values were recorded for “accelerated oysters” and significant differences (P<0.05) between treatments were noticed during October and November, suggesting nutrient accumulation before conditioning in the “accelerated” condition. Gamete emission and D larval yield results were equivalent to those reported in literature during early spring for the same species. The effect of temperature and photoperiod is discussed in order to understand their relationship with gametogenesis under these particular experimental conditions. It was demonstrated that internal clocks regulating gametogenesis can be change if stimulating factors (environmental cues) also change.

Observations en microscopie a epifluorescence de l'ingestion et de la digestion d'algues unicellulaires chez des jeunes larves de Pecten maximus (Pectinidae, Bivalvia)

Abstract The rates of ingestion and digestion of unicellular algae by young bivalve larvae can be measured by epifluorescence microscopy. Applied to the study of the nutrition of larvae of Pecten maximus in experimental rearing, this method provides information regarding the precise moment that larval ingestion occurs, and it is also possible to determine precisely the time interval before the larvae are capable of digesting phytoplankton species such as Pavlova lutheri, Isochrysis galbana, Dunaliella primolecta and Platymonas suecica as a function of temperature. The results show that Pavlova lutheri and Isochrysis galbana are the best algae for the nutrition of Pecten maximus larvae during the first days of development. Ingestion and digestion of Dunaliella primolecta are more difficult while Platymonas suecica , poorly ingested, is not digested.

Influence of plankton concentration on gametogenesis and spawning of the black lip pearl oyster Pinctada margaritifera in Ahe atoll lagoon (Tuamotu Archipelago, French polynesia).

Pearl culture industry represents one of the dominant business sector of French Polynesia. However, it still entirely relies on unpredictable spat collection success. Our aim was to assess the influence of natural plankton concentration fluctuations on maturation and spawning of the black lip pearl oyster Pinctada margaritifera, during a 4 months survey conducted in Ahe atoll lagoon. Plankton concentration was assessed by chlorophyll a extraction and by microscope counts while gonadic index, gonado-visceral dry weights and histology were used to measure pearl oysters reproduction activity. We found that (i) plankton concentration fluctuations were mainly related to wind regime, (ii) gametogenesis rate was mainly related to plankton concentration, (iii) spawning occurred when maximal gonad storage was reached, (iv) plankton concentration was the main spawning synchronizing factor. These results contribute explaining P. margaritifera spat collection variability in French Polynesian atoll lagoon.

Gametogenesis of Atrina maura (Bivalve: Pinnidae) under artificial conditions

Summary Gametogenesis in the Chinese pen-shell bivalve, Atrina maura (Sowerby, 1835) was provoked under artificial conditions in a running water system maintained under constant environmental conditions in semi-closed tanks. The specimens were fed with a diet commonly used in experimental hatcheries. Gametogenesis was described using stereological techniques (based on the oocyte diameters), classical and semi-thin histology procedures, and electron microscopy. Four reproductive stages were recognized based on oocyte size and cytological characteristics of the gonad: early gametogenesis (4–15 μm), growing (16–30 μm), mature (31–45 μm), and degenerating (46–65 μm). During gametogenesis, the auxiliary cells surrounded the oocyte basal region in the growing stage, afterward disappearing. A process of auto- and heterosynthesis characterize the mature stage, and nuclear deterioration and cytoplasmic membrane disintegration define the degenerating stage. The ultrastructural study of gonads showed that the cytological evolutionary process is comparable to those described for other bivalves. Additionally, the use of semi-thin histology permitted observation of oogonia mitosis. The conditioning system used in this experiment allowed the pen-shells to attain gonadal maturity in 35 days. The application of this procedure will permit hatcheries to obtain viable oocytes for larvae culture.

Observations on ingestion and digestion of unicellular algae by Strombus gigas larvae (Mollusca, Gastropoda) using epifluorescence microscopy

Abstract Epifluorescence microscopy was used to observe the ingestion and digestion of unicellular algae in gastropod larvae. Larvae started feeding 8 h after hatching. Three kinds of diet were tested during the period of this study: Isochrysis aff. galbana, Tetraselmis chuii and Isochrysis-Tetraselmis mixture. All the experiments were conducted at 29°C. It was shown that ingestion was faster with Tetraselmis than with Isochrysis. Within 10 min 43% of the larvae had ingested Tetraselmis and no larvae had ingested any Isochrysis. Within 30 min, 60% of the larvae had ingested Tetraselmis while only 25% had ingested Isochrysis. Using a scale based on the quality of fluorescence, it was possible to determine the time interval to digest a diet, which is shorter with Tetraselmis than with Isochrysis. After 8 h the digestion index reached 45% for larvae fed with Tetraselmis while it was only 28% for larvae fed with Isochrysis. Using a quantitative scale based on measurements of the fluorescent surface in each larva, it was demonstrated that the amount of diet ingested is higher with Tetraselmis than with Isochrysis. These results indicate that Tetraselmis chuii forms an adequate diet for young veligers of Strombus gigas.

The limits of morphometric features for the identification of black-lip pearl oyster (Pinctada margaritifera) larvae

Abstract As with most cultivated bivalves, culture of the Tahitian pearl oyster Pinctada margaritifera is particularly dependent on the natural environment, especially for spat supply. The ability to track in real time the abundance and the development of pearl oyster larvae in the plankton would help optimize spat collection in atolls of French Polynesia. However no identification criteria are available for the larvae of several bivalves species present in the lagoons and it is not yet possible to specifically monitor pearl oyster larvae. The aim of this study is to determine the most pertinent morphological identification criteria, to specifically identify the larvae of P. margaritifera and differentiate them from those of three other abundant species: Pinctada maculata, Crassostrea cuculata and Chama sp. The method of image analysis after photon microscopy was assessed. It allowed automatic measurement of numerous morphometric features that were tested alone or in combination and identification threshold for P. margaritifera larvae were determined by statistical analyses. These results led to a key that allowed correct identification for 77% of P. margaritifera larvae. The hinge diagnosis method under scanning electron microscopy, a prime method for the identification of specific criteria on bivalve larva shells, was also used on larvae of both Pinctada species. The two species could be differentiated precisely because of specific differences in the thickness of their hinge provinculum and the number of denticles it bears. However this approach is too time-consuming and technically demanding to use in real time field studies. This study showed the limitations of image analysis as an identification tool of the P. margaritifera larvae, but proper statistical analyses and especially the decision tree approach could be used to evaluate and efficiently prioritize the choice of the species identification criteria.

Scanning Electron Microscopic Aids for Identification of Larval and Post-Larval Bivalves

ABSTRACT The identification of bivalve larvae and early postlarvae in plankton and benthic samples has long been a challenge, hampering both basic and applied research efforts in marine, estuarine, and freshwater environments. The usefulness of published optical micrographs of the early life-history stages of bivalves is limited because of the great morphological similarity of the imaged articulated shells, particularly at the early (straight-hinge) developmental stages. While a number of techniques have been refined in recent years and show promise for use in routine identifications of larval and post-larval bivalves (e.g., single-step nested multiplex polymerase chain reaction; in situ hybridization protocols through color coding with taxon-specific, dye-labeled DNA probes; coupled fluorescence in situ hybridization and cell sorting; and image analysis techniques using species-specific shell birefringence patterns under polarized light), no adequate comprehensive reference source exists that accurately depicts the morphology and morphometry of the shells of larval and post-larval stages of target bivalve species in a consistent format to assist in identification of such stages. To this end, scanning electron micrograph (SEM) sequences are presented of the disarticulated shell valves of laboratory-reared larval and post-larval stages of 56 species of bivalve molluscs from a wide spectrum of marine, estuarine, and freshwater habitats. Emphasis is placed on the usefulness of the morphology and morphometrics of consistently-oriented, disarticulated shell valves and associated hinge structures in discriminating the early life-history stages of these various bivalve species. Although the scanning electron micrograph sequences presented accurately depict the gross morphologies/ morphometrics and hinge structures of the disarticulated shell valves of the larvae and/or postlarvae of the 56 species of bivalves, it is important to emphasize that a scanning electron microscope is not necessary to observe even fine hinge structures associated with the early ontogenetic stages of these species. Such structures are readily visible using a wide range of optical compound microscopes equipped with high-intensity reflected light sources, although the disarticulated shell valves must be viewed in several planes of focus to discern the often subtle details seen clearly in the scanning electron micrographs. These morphological characters provide researchers with invaluable aids for the routine identification of the early life-history stages of these species isolated from plankton and benthic samples.