Gaël Le Pennec

Demonstration of Nutrient Pathway From the Digestive System to Oocytes in the Gonad Intestinal Loop of the Scallop Pecten maximus L.

The mechanism of nutrient transfer from the digestive system to the gonad acini and developing oocytes was investigated in the gonad-intestinal loop system of the queen scallop Pecten maximus L. Ferritin was injected directly into the purged intestine of specimens from the wild. Subsequently, a histochemical reaction and transmission electron microscopy were used to localize ferritin in various cell types. Ferritin was rapidly absorbed by the intestinal epithelium, and then appeared in hemocytes in the surrounding connective tissue. In the hemocytes, ferritin was stored in variously sized inclusions, as well as in the general cytoplasm. In all sections examined for the 12 experimental individuals, hemocytes were always found in association with connective tissue fibers extending from the base of the intestinal epithelium to gonad acini. After 30-min incubation, ferritin appeared inside the acini of all individuals. Ferritin-bearing cells were rarely found in association with male acini or gametes, nor with mature female gametes, but often with developing female gametes. Not all individuals showed the same temporal dynamics of ferritin transport, suggesting that nutrient transfer to oocytes is either not a continuous process, or that among individuals, transfer is not synchronized on short time scales. This is the first demonstration of a pathway of nutrient transfer from the intestine, and more generally the digestive system, to developing oocytes in the Bivalvia.

Induction of glutathione-S-transferases in primary cultured digestive gland acini from the mollusk bivalve Pecten maximus (L.): application of a new cellular model in biomonitoring studies

In the last three decades, marine invertebrates have been used to monitor environmental health conditions and potential pollution, e.g. in the Mussel Watch Program. The whole animal or specific organs are used to determine contamination levels and disturbances. In the present study, a new in vitro cell culture model was validated for pollution monitoring. A commercial species, the scallop Pecten maximus, was tested for the presence and induction of phase II glutathione-S-transferase (GST) enzymes. These activities were monitored for a year, and the results were found to be consistent with those in the literature. Tributyltin, ethylmethane sulfonate and the water-soluble fraction of crude oil were assayed in, in vitro induction studies. A rapid increase of GST activities was observed within 24 h with all compounds tested, and a time– as well as a dose–response was established. This in vitro cell culture model seems suitable for routine use to predict the effects of pollutants on whole organisms within an ecosystem and in fisheries.

Seasonal digestive gland dynamics of the scallop Pecten maximus in the Bay of Brest (France)

Cytological and biochemical changes in the digestive gland of Pecten maximus throughout a one-year period were investigated in the Bay of Brest (France) relative to lipid storage and glycogen and α-amylase concentrations in tubuloacinar terminations. Seasonal variations in both cell structure and biochemical composition were observed. These changes were independent of the tidal cycle, but correspond to phytoplankton abundance. From November to January, cells of digestive acini were hypertrophied due to the high lipid storage. From February to May, these reserves were markedly reduced, cell size decreased, and the acinar lumen was clearly apparent. From May to September, the RNAs transcribing for α-amylase increased, and a correlation was found between digestion events and lipid storage in acinar cells. The relationships between metabolite transfers from digestive gland to gonad and other tissues are described Distinct lipid storage sites appear to be associated with maintenance energy and acute demand energy, such as gametogenesis when adductor muscle reserves have been depleted. The digestive gland may thus function as a relay organ during periods of energetic stress, notably during the first gametogenesis in February and March and for shell growth in early spring.

Acinar primary cell culture from the digestive gland of Pecten maximus (L.): an original model for ecotoxicological purposes

The purpose of this study was to establish an original primary acinar cell culture model for the mollusc bivalve Pecten maximus (L.), and to define its values and limits for subsequent ecotoxicological applications. To prevent microbial contaminations occurring frequently in invertebrate cell cultures, a perfusion of the stomach-digestive gland complex was performed in situ using a sterile salt solution containing broad-range antibiotics. Digestive acini were isolated using a pronase enzyme that was removed by several washings of the acinar suspension, after which their viability and functionality were determined by three different assays: fluorescein diacetate (FDA) de-esterification, 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium (MTT) reduction and neutral red (NR) incorporation describing de-esterification, mitochondrial dehydrogenase and lysosome activity, respectively. The kinetic conditions for these assays were defined beforehand. The results showed that digestive acini could be maintained in vitro both cytologically and functionally for at least 96 h, which is sufficient for many ecotoxicological applications. Preliminary contamination assays, according to the function studied (cell esterases, mitochondrial respiration, lysosomal incorporation), indicated that polycyclic aromatic hydrocarbons had a negative effect on the survival of acini in vitro.

Evaluation of the toxicity of chemical compounds using digestive acini of the bivalve mollusc Pecten maximus L. maintained alive in vitro

Abstract The digestive gland of bivalve molluscs is a model of choice for experiments in ecotoxicology because of its implication in detoxification processes moreover of its classical functions in digestive phenomena. All physiological deteriorations of this organ, related or not to pollution, can lead to animal death. The recent development of a method allowing digestive acini of Pecten maximus to be maintained alive in vitro for 96 h opens up new research prospects in ecotoxicology. The action of contaminants considered to be cytotoxic or genotoxic in the literature were tested on this model. The results show the high cytotoxicity of ethylmethane sulphonate 80 and 5 mM after 2 h of contact with acini. Other compounds such as 4-nitroquinoline-N-oxide 0.1 mM, cadmium chloride 10−5 M and atrazine 10−4 M, which were weakly toxic after 2 h, became highly toxic after 48 h of contact. Compounds such as 4-nitroquinoline-N-oxide 1 mM, which were not cytotoxic after 2 h, proved to be the most genotoxic of all those tested. Others, such as MMS 1 mM, cadmium chloride 10−4 and 10−5 M, and atrazine 10−5 M, showed an unconfirmed tendency to be genotoxic. The results obtained with this ‘acinus’ model seem more readily transposable to the whole organism than those obtained with ‘isolated cell’ models, in that acini can be considered as digestive glands ‘in miniature’.

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.

Spermatogenesis in the archaic hydrothermal vent bivalve, Bathypecten vulcani, and comparison of spermatozoon ultrastructure with littoral pectinids

Summary Bathypecten vulcani is considered a relict species from the Paleozoic, based on shell characteristics such as the presence of calcite prisms. To date, it is the only pectinid species reported from hydrothermal ecosystems. Histological and ultrastructural studies show that spermatogenesis is identical to that of littoral pectinids. The spermatozoon has a 2.7 μm long pyriform head and a 40 μm flagellum. The four mitochondria of the mid-piece are about 1.2 μm in diameter. The nucleus contains dense chromatin fibres and possesses a wide, shallow (0.1 μm) anterior fossa and a narrow, deeper (0.2 μm) posterior nuclear fossa. Comparison of the ultrastructural characteristics of the spermatozoon of B. vulcani with those of littoral pectinids shows that they can be used as a diagnostic feature of this species. In particular, its acrosome characters will be a useful complement to the shell characters in the study of the phylogenetic position of this species in relation to other pectinids.

Molecular analysis of the seasonal expression of genes coding for different functional markers of the digestive gland of the bivalve mollusk Pecten maximus (L.).

Abstract Digestive gland cells of Pecten maximus accumulate and release lipid storage according to a seasonal cycle. For the first time in molluscs, molecular probes were developed and applied to monitor the lipid accumulation and consumption cycle related to phytoplankton blooms and phenomena of cellular proliferation and apoptosis. The molecular probes consisted of glyceraldehyde-3-phosphate dehydrogenase (GPD), which is involved in the acetylation of fatty acids; aldolase, which favours the formation of pyruvate and dihydroxyacetone; actin, an essential element of the cytoskeleton that disappears during adipocyte cell transformation; and cycline B, an ubiquitous cell cycle protein. α-Amylase, provided by IFREMER-Brest (France), was used to relate these different events to the animals food supply. A positive relation between GPD and aldolase gene expressions was inversely correlated with that of actin, confirming results in mammals. In P. maximus, mRNA transcripts of GPD and aldolase decrease rapidly before gamete emissions whereas those of actin increase rapidly. After gamete emission, the mRNA levels of aldolase, GPD and α-amylase increase, while those of actin decrease. Cycline B mRNA transcripts indicate that the period of digestive cell proliferation is initiated during winter, prior to the release of lipids into the digestive tract and apoptosis.

The seminal receptacle and implications for reproductive processes in the invasive gastropod Crepidula fornicata

The calyptraeid gastropod Crepidula fornicata is the object of considerable research attention, due to its invasive status in the North-Eastern Atlantic, its introduction to habitats throughout the Northern hemisphere, and its scientific interest as a model organism for the study of developmental and reproductive processes in the Metazoa. Since the knowledge concerning the structural foundations for its reproductive processes is surprisingly weak, we investigated the seminal receptacle, a key structure in the reproductive biology of other metazoans, using histology, scanning electron and transmission electron microscopy. The seminal receptacle consists of 9-11 lobes, each subdivided into small, narrow lobules. The inner epithelium of the lobules appears to be highly dynamic, characterised by the perforation and attachment of received spermatozoa, the progressive degeneration of this epithelium, and the concomitant detachment of the spermatozoa. The allocation of spermatozoa to many different lobules, in different phases, may explain the extended reproductive season of C. fornicata, and thereby contribute to its colonizing and invasive success. The same compartmentalisation, as well as the complete covering of the inner epithelium of the lobules by spermatozoa and the large amount of spermatozoan debris in the lumina, suggest that the C. fornicata seminal receptacle may be a site of sperm competition in this polyandrous species.

Spermatozoa and phylogenesis of the pectinid bivalve Flexopecten glaber

Gonad of the Mediterranean pectinid Flexopecten glaber was studied in order to describe the morphology and the ultrastructural characteristics of the mature spermatozoa. The spermatozoon has a 2.5 μm pyriform head and a 40–45 μm flagellum. The four mitochondria of the mid-piece are 0.6 μm in diameter. The nucleus contains dense chromatin fibres and possesses two depressions: a wide (0.19 μm) and deep (0.58 μm) sub-acrosomal one, and the other at the base of the head, less deep (0.36 μm) and less wide (0.12 μm). The objective was to situate this scallop in the phylogenetic diagrams found in the literature concerning the commercial pectinid species.