Haliotis Tuberculata Research Articles

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010–31 March 2010

This article documents the addition of 228 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anser cygnoides, Apodemus flavicollis, Athene noctua, Cercis canadensis, Glis glis, Gubernatrix cristata, Haliotis tuberculata, Helianthus maximiliani, Laricobius nigrinus, Laricobius rubidus, Neoheligmonella granjoni, Nephrops norvegicus, Oenanthe javanica, Paramuricea clavata, Pyrrhura orcesi and Samanea saman. These loci were cross-tested on the following species: Apodemus sylvaticus, Laricobius laticollis and Laricobius osakensis (a proposed new species currently being described).

Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata

An integrated study of shell formation was initiated covering the entire life cycle of the marine gastropod Haliotis tuberculata. Shell microstructure, chemistry and mineralogy were investigated by polarized microscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX) and infra-red (IR) spectroscopy. SEM images of trochophore and veliger larvae showed the different stages of shell growth from the initial shell field to the late calcified protoconch. Cross-sections revealed the microstructural arrangement of biominerals, showing the progressive mineralization of the organic protoconch prior to metamorphosis. To gain more information on mineralogical composition, EDX analyses and IR spectroscopy were performed along the development stages. The results demonstrated that early protoconch was mostly composed of amorphous calcium carbonate, while veliger stages showed a gradually crystallization under the form of aragonite. Post-metamorphic shell contained two distinct parts, the original protoconch supporting the new juvenile shell characterized by a marked sculptural pattern. The shells from post-larval and juvenile abalones were essentially made of aragonite.

ANTIBIOTIC RESISTANT BACTERIA FROM HALIOTIS TUBERCULATA AND MYTILUS GALLOPROVINCIALIS

The antibiotic resistance (AR) of Gram negative bacteria from Haliotis tuberculata (Ht) and Mytilus galloprovincialis (Mg) was assessed. Essential differences between R profiles of Pseudomonas spp and of other strains was not observed. Strains AR from Ht and Mg was similar.

Haliotis marmorata from Senegal; a sister species of Haliotis tuberculata: Morphological and molecular evidence

Two abalone species were described in Senegal by Linnaeus in 1758: Haliotis tuberculata and Haliotis marmorata. The present study investigates differences at the molecular level by comparing 18S rDNA – ITS1 and mitochondrial sequences, in addition to making a morphological description of 35 OTUs from the Senegalese coast. A comparison was made between the two sympatric sub-species: Haliotis tuberculata tuberculata, present throughout Northern Europe, and H. t. coccinea, present in the Canary Islands and on Northern Atlantic coasts. Although shell morphology cannot be used to easily differentiate Senegalese specimens, the number of open respiratory pores in the shells was significantly lower. Nuclear 18S rDNAs showed 0.6% differences and ITS1 0.37% between H. marmorata and H. tuberculata sub-species. The divergence of the coding genes of mitochondrial DNA ranged from 4.5 to 13.3%, between H. tuberculata and H. marmorata whilst it ranged from 1.2 to 5.5% between H. tuberculata sub-species. These variations are lower for tRNAs. A phylogenetic analysis using three different markers (ITS1, COI and 16S) from Haliotidae revealed a robust cluster for the species complex H. tuberculata and H. marmorata. Seven different haplotypes were found inside the 35 OTUs from Senegal, by using COI, that are unrelated to the defined H. tuberculata sub-species haplotypes. The ancient geographical isolation during the Quaternary period may explain their allopatric speciation. From our results we consider these two species as sister species but, other studies are necessary to verify their status.

Sheltering behavior of the abalone, Haliotis tuberculata L., in artificial and natural seawater: The role of calcium

There is an increased interest worldwide in developing land-based closed systems to cultivate the abalone, Haliotis tuberculata L. Here, we analyzed whether artificial conditions, and particularly the use of artificial seawater, might affect sheltering, a critical behavior of this species. The study was composed of two experiments. In the first, we compared sheltering between two groups of 100 individuals each reared in either natural or artificial seawater. For 15 days, a score was assigned every day to the position occupied by each individual, i.e. “0” when it was out of the hide, “1” when it was on the bottom of the maintenance tank underneath the hide, and “2” when it occupied the inner spot of the hide. Our results show that artificial seawater significantly affects sheltering: only the individuals reared in natural seawater were able to reach the hide and maintain a position in it. Chemical analyses revealed that artificial seawater differed from natural seawater for a relatively low concentration of Ca 2+. In the second experiment we compared H. tuberculata's sheltering behavior in three media, natural, artificial, and calcium-enriched artificial seawater. Sheltering was restored in enriched artificial seawater, which supports the hypothesis that calcium has an effect on this species' behavior. However, calcium concentration both in the artificial and “natural” water showed a progressive decrease with time likely due to the absorption by abalones, which suggests that in closed systems calcium supply is constantly required, even when the used medium is natural seawater.

Function of mucus secretion by lamellose ormer, Haliotis tuberculata lamellosa, in response to starfish predation

Gastropods subjected to predation from asteroids have a number of different defensive mechanisms to prevent and defend against attacks. I investigated the production and function of mucus in the lamellose ormer in response to attacks by the spiny starfish, Marthasterias glacialis (Asteriidae, Asteroidea). Production of mucus from respiratory pores on the shell helped obscure the direction of escape of the gastropod from the starfish. The mucus also had repellent properties against this predator and there was some evidence of it being an intraspecific alarm. The results suggest that mucus secretion is part of a larger stereotyped escape response, in which the mucus has an olfactory interference role.

Structural distortion of biogenic aragonite in strongly textured mollusc shell layers

The stabilisation of strong textures in mollusc shells has for long been a strong drawback towards precise structural determinations of these natural biocomposites. We demonstrate here on several crossed lamellar and nacre layers from two gastropods ( Charonia lampas lampas and Haliotis tuberculata tuberculata) and one bivalve ( Pinctada maxima), that on real specimens (without grinding or specific preparation), the textural information can be used efficiently for precise structural determination of the biogenic aragonite. This is done through the combination of orientation distribution function and cyclic Rietveld refinements on several hundreds to thousands of diffractions diagrams.

Influence of temperature and spawning effort on Haliotis tuberculata mortalities caused by Vibrio harveyi: an example of emerging vibriosis linked to global warming

AbstractSince 1998, Haliotis tuberculata mass mortalities have been occurring regularly in wild abalone populations in France during their reproductive period and in conjunction with seawater summer temperature maxima and Vibrio harveyi presence. To confirm the importance of bacterial exposure, temperature and reproductive status on abalone susceptibility, experimental infections via bath exposure were performed using abalone ranging from immature to reproductively mature. Ripe abalone were more susceptible to the bacterium than immature specimens (P<0.001), and a difference of only 1 °C in temperature had a highly significant impact on the mortalities (P<0.001). The natural mortalities that were surveyed during summer 2007 confirmed that recent epidemic losses of European abalone appeared in conjunction with host reproductive stress, elevated temperatures and presence of the pathogen V. harveyi. In view of the elevation of the mean summer temperatures observed in Brittany and Normandy over the last 25 years, this temperature‐dependent vibriosis represents a new case of emerging disease associated with global warming.

Fatty acid composition of juvenile abalone (Haliotis tuberculata coccinea) fed formulated diets containing various n3 HUFA levels

The effect of feeding different levels of n3 highly unsaturated fatty acids (HUFA) on the fatty acid profile was evaluated in soft tissue from juvenile Haliotis tuberculata coccinea. Five diets were formulated with different oil sources (palm, colza, fish, sunflower, and soybean), containing from 2.52% to 12.33% n3 HUFA, while fresh Ulva rigida was used as reference diet. The different diets were provided to groups of 20 abalone during 120 days in independent triplicates. Though no significant differences in growth (P < 0.05) were registered among all formulated diets, a tendency to accumulate different fatty acids in muscle was observed. The proximate composition of abalone tissue showed that the diet containing 6.8% HUFA resulted in higher lipid accumulation in muscle tissue than that observed in the other treatments. Our results suggest that H. tuberculata coccinea, contrary to to abalone species from colder environments, does not have high HUFA requirements; however, the fatty acid profile of muscle and viscera tissues of abalone may be modulated by the fatty acid content in diets.

Use of space and costs/benefits of locomotion strategies in the abalone, Haliotis tuberculata

This study investigates the use of space by the abalone Haliotis tuberculata in an artificial environment. First, we analyzed locomotion and the hiding behaviour of 60 4-year old individuals; then we described their feeding on either dispersed or concentrated algae. The results show that each individual, independently of its sex, adopts one of two locomotion strategies, behaving as either a “wanderer” (W) or a “sedentary” (S). Ws covered longer distances, occupied larger areas, and made more twisted trajectories than Ss. Ws and Ss also differed for their social behaviour (the nearest neighbour distance was longer for Ws), the use of hides (Ws occupied the hides, particularly their inner spots, more often), food recruitment (Ws were quicker in responding to the food odour and foraged for longer time), and competitive ability (Ws more often displaced other individuals from food patches). The costs and benefits associated with these two strategies are discussed.

Construction of a stable GFP-taggedVibrio harveyistrain for bacterial dynamics analysis of abalone infection

Vibrio harveyi is a bacterial marine pathogen that can cause fatal disease in a large range of vertebrates and invertebrates, including the commercially important marine gastropod, Haliotis tuberculata. Since 1997, strains of this bacterium have regularly been causing high mortalities in farmed and wild abalone populations. The way in which the pathogen enters into abalone and the disease transmission mechanisms are thus far unknown. Therefore, a pathogenic strain, ORM4, was green fluorescent protein-tagged and validated both for its growth characteristics and for its virulence as a genuine model for abalone disease. The strain allows V. harveyi quantification by flow cytometry in seawater and in abalone haemolymph as well as the in situ detection of the parasite inside abalone tissues.

Summer immune depression associated with increased susceptibility of the European abalone, Haliotis tuberculata to Vibrio harveyi infection

Haliotis tuberculata mortality outbreaks have occurred in France since 1998 and were attributed to a pathogenic Vibrio harveyi. These mortalities were recorded in September, a month with abalone reproduction and characterised by high seawater temperatures. The importance of gonadal maturation and temperature increase on abalone immunity and susceptibility to V. harveyi infection needed to be clarified. Therefore, an immune survey analyzing a large panel of parameters was performed from June to September 2007 on abalone from the Bay of Brest. The data obtained were put in relation with abalone reproductive status and its susceptibility to V. harveyi. Most parameters showed clear patterns from early to late summer and during gametogenesis, phagocytosis and phenoloxidase activity were reduced, whereas basal reactive oxygen species production and agglutination titres were significantly increased. Total haemocyte counts went up after the partial spawning event at the end of June, and cell complexity diminished. Using a Principal Component Analysis, the “haemolymph profile” was shown to decrease in parallel with spawning and gonadal maturation processes, and reached a minimum just after total spawning. A significant correlation between this “haemolymph profile” and disease susceptibility allowed us to establish for the first time in abalone, a clear concordance between maturation and spawning processes, immune status and abalone susceptibility to V. harveyi.

Reproduction trials under experimental conditions of Mediterranean abalone (Haliotis tuberculata, L. 1758)

The aim of this paper is to show early results obtained during reproduction trials of Haliotis tuberculata under experimental conditions. Laboratory experiments were carried out at the CISS of the University of Messina (Italy). Two hundred adult abalones were collected, separated by sex and put in 20-litre tanks. In order to induce spawning, temperature was gradually increased. Cultures of eggs and sperm were allowed to develop for 4 h. The egg fertilisation, the formation of trocophora as well as the evolution of such larval stage in veliger were achieved. The adhesion to substrate remains up to now to be optimised. The success in obtaining the earlier stages of the life cycle of H. tuberculata is important considering the possible economic effects due to the industrialisation of the production process of this species.

A morphological and structural study of the larval shell from the abalone Haliotis tuberculata

The larval shell of the marine gastropod Haliotis tuberculata was investigated by polarised light microscopy, scanning electron microscopy, Raman microspectroscopy and infra-red spectroscopy. Trochophore and veliger larval sections were used for histological examination of the growing shell and each larval stage was related to the shell development and the appearance of calcified formations. We determined the stage of initial calcification by specific staining combined with polarised light examination. The shell of 30-h-old pre-veliger larvae was found to be mineralized, confirming that calcification occurred before larval torsion. Using both infra-red and Raman spectroscopy, we showed that CaCO3 deposition occurred at the pre-veliger stage and that the mineral phase initially deposited was essentially composed of aragonite.

Shell repair process in the green ormer Haliotis tuberculata: A histological and microstructural study

In the present paper, juvenile and adult shells of the green ormer Haliotis tuberculata (‘Oreille de Saint-Pierre’) were perforated in a zone close to the shell edge and the shell repair process was followed at two levels: (1) by observing the histology of the calcifying mantle in the repair zone and (2) by analyzing with SEM the microstructure of the shell repair zone. Histological data clearly show the presence of calcium carbonate granules into the connective tissues, but not in the epithelial cells. This suggests that calcium carbonate granules are synthesized by sub-epithelial cells and actively transported through the epithelium to the repair zone, via a process which may be similar to that described by Mount et al. [Mount, A.S., Wheeler, A.P., Paradkar, R.P., Snider, D., 2004. Hemocyte-mediated shell mineralization in the eastern oyster. Science 304, 297–300]. Furthermore, SEM observations show that the repair zone exhibits different stratified microstructures (spherulitic, thin prismatic, blocklike, sub-nacreous, nacreous, foliated-like), some of which are not continuous ( i. e. lenticular) along the repair zone. This suggests a complex secreting regime of the calcifying mantle and an elaborate geometry of the epithelium involved in shell repair.

Ultrastructural Developmental Cycle of Haplosporidium montforti (Phylum Haplosporidia) in its Farmed Abalone Host, Haliotis tuberculata (Gastropoda)

The sequential developmental cycle of Haplosporidium montforti, a recently described species from farmed abalone Haliotis tuberculata (Gastropoda), was studied. Ornamented and operculated mature spores were electron dense. The nucleus of the uninucleated free cell divided successively, giving rise to multinucleate plasmodia, containing up to 100-120 nuclei. Later, the plasmodia developed into sporonts inside sporocysts with irregular contours. Each of their nuclei gave rise to uninucleate sporoblasts. At the next phase of development, a very irregular membranous group of cisternae began to differentiate in the cytoplasm of each sporoblast, surrounding each nucleus and the adjacent cytoplasm. Each sporoblast differentiated into a spore. This process was characterized by the appearance of dense blisters of amorphous material at the periphery that gradually formed the prespore wall and pre-operculum. Simultaneously, in the endosporoplasm, the spherulosome and several haplosporosomes were formed. During the final phase of the maturation process, the spores became gradually denser, and the endosporoplasmic structures were barely visible.

Morphologic, cytometric and functional characterisation of abalone ( Haliotis tuberculata) haemocytes

This work presents the first detailed microscopic and functional analysis of the haemocytes of an abalone; the European Haliotis tuberculata. It is shown that in contrast to the situation in bivalves, only very few basophilic “granulocytes” could be found and exclusively with a histological stain. Neither flow cytometry, phase contrast observation nor transmission electron microscopy were able to detect any granular cells. The large majority of cells was constituted of “hyalinocytes”, which could be sorted by flow cytometry, for the first time, into small (blast-like) and large cells. This permits a detailed analysis of haemocytes and especially of the lowly represented blast-like cells. The differences in haemolymph cell composition between bivalves and gastropods is reviewed in depth and discussed in view of the new data we present. Most of the abalone haemocytes analysed harbour many vacuoles, large glycogen deposits, lipid inclusions and acidic compartments. However, although the number of these “inclusions” was rather variable in between individual hyalinocytes, these experiments did not allow to discern subpopulations using these criteria, and the population appears more as a “differentiation continuum”. Haemocytes adhere very rapidly and are immunologically active as they quickly phagocytose latex beads and zymozan particles. This study is the first step towards understanding the H. tuberculata immune system by adapting new tools to gastropods and in providing a first detailed morpho-functional study of their haemocytes.

EMBRYONIC AND LARVAL DEVELOPMENT OF HALIOTIS TUBERCULATA COCCINEA REEVE: AN INDEXED MICRO-PHOTOGRAPHIC SEQUENCE

First description of the complete embryo and larval development of the Canarian abalone (Haliotis tuberculata coccinea Reeve.) was conducted along 39 stages from fertilization to the appearance of the third tubule on the cephalic tentacles and illustrated in a microphotographic sequence. Eggs obtained by induced spawning with hydrogen peroxide from the GIA captive broodstock were stocked at a density of 10 eggs/mL and kept at 23 ± 0.5C for 62 h until the formation of the third tubule. Live eggs and larvae were continuously observed on a 24 h basis at a 3400 magnification under transmitted light. At each stages, specific morphological features, illustrated by microscopic photographs, were described, as well as the time required for their apparition. Fertilized eggs diameter was 205 ± 8 mm (mean ± SD), whereas length and width of larvae ready to undergo metamorphosis were 216.6± 5.3 mm and 172± 8.8 mm, respectively. Knowledge on the larval morphological development acquired through this study will contribute to the improvement of larval rearing techniques for this abalone species.

CGRP REGULATES THE ACTIVITY OF MANTLE CELLS AND HEMOCYTES IN ABALONE PRIMARY CELL CULTURES (HALIOTIS TUBERCULATA)

Mollusc shell formation requires calcium (Ca ) and bicarbonate (CO3 −) ions, transported through the mantle via the hemolymph, and the secretion of an organic matrix that interacts with the mineral ions to form either the aragonite or calcite polymorph of calcium carbonate (CaCO3). The biomineralization process takes place in the extrapallial space, between inner shell and outer mantle epithelium, and is believed to be under the control of calcium-binding proteins and hormonal factors such as calcitonin-related molecules (CGRP-like peptides). Epithelial cells from the mantle are responsible from the secretion of matrix molecules whereas hemocytes are involved in ion transportation during shell formation and regeneration. To understand the biochemical and cellular events implied in the biomineralization process, we developed primary cell cultures from the nacreous gastropod Haliotis tuberculata. Mantle cells as well as hemocyte cell cultures were successfully maintained in vitro and evaluated for their viability and proliferation using semiautomated assays. The effect of calcitonin-related molecules (h-CGRP) on cellular activity and proliferation was evaluated in those primary cell cultures. The results indicate that human-CGRP modulates the activity of both mantle cells and hemocytes and increases carbonic anhydrase activity in vitro. These results are consistent with those obtained in vivo, supporting the hypothesis that CGRP-like molecules control the activity of target cells involved in the biomineralization process.

Variations of 210Po and 210Pb in various marine organisms from Western English Channel: contribution of 210Po to the radiation dose

Measurements of 210Po were carried out in various marine matrices (mussels, oysters, seaweed, fish, and abalones) and in seawater at several points along the French coast, over a period of 2 years (2003–2005). These measurements contribute to a better knowledge of this element, since few recent data exist for the French coast. Marked seasonal variations have been revealed in some species and there are differences according to the way of life of these species. Activities in mussels ( Mytilus edulis) and oysters ( Crassostrea gigas) are similar and varying between 90 and 600 Bq kg −1 (d.w.). Activities in macroalgae ( Fucus serratus) are lowest, between 4 and 16 Bq kg −1 (d.w.). In oyster, abalone ( Haliotis tuberculata) and fish ( Solea solea, Sparus sp.), the strongest activities are measured in the digestive glands, the gills and the gonads. 210Po/ 210Pb ratios in all cases have values of more than one for all species. From a significant number of measurements, CFs were calculated for seaweed (between 4.6 × 10 3 and 5.0 × 10 3) and for molluscs, with highest CFs (>10 5) found for the digestive gland and gills of the oysters, the digestive gland of the abalones and the liver of fish. Finally, the activities measured have made it possible to estimate the internal dose from chronic exposure due to 210Po received by the marine organisms (0.05 μG h −1 for macroalgae, between 0.70 and 1.5 μG h −1 for mussels and oyster), and the contribution of seafood to the dose received by humans (46–129 μSv y −1).