Abalone Haliotis Tuberculata Research Articles

Development of TaqMan real-time PCR assays for monitoring Vibrio harveyi infection and a plasmid harbored by virulent strains in European abalone Haliotis tuberculata aquaculture

The Gram-negative bacterium Vibrio harveyi is known to be highly pathogenic for the European abalone Haliotis tuberculata, which is a gastronomically important marine gastropod with a high commercial value. Since 1998, some particular bacterial strains are described as implicated in recurrent mortality outbreaks in French farm and field stocks of abalone. Recently, a 9.6kb plasmid named pVCR1, was shown to be harbored by one highly V. harveyi virulent ORM4 strain suggesting its involvement in virulence phenotype. Thus, we have developed in the present study two TaqMan real-time PCR assays allowing to (i) rapidly and specifically detect, by a duplex procedure and in less than 2h, both V. harveyi and the presence of plasmid pVCR1 from unidentified bacterial colony and to (ii) quantify both V. harveyi and the plasmid pVCR1 in the hemolymph of abalone or its surrounding seawater. Quantification curves of V. harveyi or ORM4 strain seeded in hemolymph or artificial sea water samples were equivalent showing excellent qPCR efficacies and detection level as low as 18V. harveyi cell-equivalent genomic DNA in a PCR reaction well. This qPCR allowed us to monitor V. harveyi ORM4 strain in experimentally infected H. tuberculata. These diagnosis assays could provide powerful and useful tools to better understand the epidemiology of vibriosis caused by V. harveyi in different cultured marine species including H. tuberculata.

First steps on technological and genetic improvement of European abalone (<i>Haliotis tuberculata</i>) based on investigations in full-sib families

The European abalone Haliotis tuberculata is of economical importance in Europe, and recently in France where most of the consumed abalones remained wild animals fished on the coast. Recently, the creation of hatcheries allowed the production of cultured animals, and aquaculture is in progress. To optimize selective breeding programs, different studies were performed on adults and on their progenies. First, ten adult families, assumed to be bi-parental and produced in 2007 were analyzed. As these families were developed at the beginning of the hatchery production, the parentage of the individual necessitated to be tested. The progenies parentage assignment was done by using eight microsatellite DNA loci. In fine, the heritability was estimated from the analysis of variance of seven full-sib families which include from 23 to 27 progenies. Heritability estimate was the higher for length, width and weight (0.37, 0.29 and 0.40 respectively). A high correlation was also shown between these heritable traits which can be useful during animal breeding. Shell color traits were also analyzed, using image treatment procedure. Two traits were selected according to the global color of the shell: red color and hue. The distribution of these traits evidenced a polygenic genetic control of shell color and measure of heritability evidenced a high to medium value for red color (Cr) and Hue(H) (0.33 and 0.20 respectively). No correlation was shown between growth and this parameter. In a second time, juveniles families produced in 2010 were studied from parents issued of the first selection. A correlation between size and density at juvenile stage was established but a statistical analysis demonstrated, by using density as co-variable a significant effect of family on size. In these conditions, growth is highly heritable (0.74). This study is a first step toward the use of genetic markers for selection, but also a step toward the breeding improvement of European abalone.

Review of the technical challenges facing aquaculture of the European abalone Haliotis tuberculata in Ireland

Insufficient supply of available market size abalone from the wild stocks has resulted in increasing efforts since the early 1990s to culture this valuable marine shellfish. Despite significant financial investment and the establishment and expansion of farms around Europe, the production of a saleable end product has remained undefined. The technical barriers to producing viable juvenile abalone spat still impair growth of the industry in Ireland. Critical developments required in the industry are identified. Uptake of these developments from research remains slow due to the trial-and-error approach taken by operators. Using technologies and procedures from producing countries and implementing them into Irish culture conditions will aid the development and expansion of the industry.

Identification of Two Carbonic Anhydrases in the Mantle of the European Abalone Haliotis tuberculata (Gastropoda, Haliotidae): Phylogenetic Implications

Carbonic anhydrases (CAs) represent a diversified family of metalloenzymes that reversibly catalyze the hydration of carbon dioxide. They are involved in a wide range of functions, among which is the formation of CaCO(3) skeletons in metazoans. In the shell-forming mantle tissues of mollusks, the location of the CA catalytic activity is elusive and gives birth to contradicting views. In the present paper, using the European abalone Haliotis tuberculata, a key model gastropod in biomineralization studies, we identified and characterized two CAs (htCA1 and htCA2) that are specific of the shell-forming mantle tissue. We analyzed them in a phylogenetic context. Combining various approaches, including proteomics, activity tests, and in silico analyses, we showed that htCA1 is secreted but is not incorporated in the organic matrix of the abalone shell and that htCA2 is transmembrane. Together with previous studies dealing with molluskan CAs, our findings suggest two possible modes of action for shell mineralization: the first mode applies to, for example, the bivalves Unio pictorum and Pinctada fucata, and involves a true CA activity in their shell matrix; the second mode corresponds to, for example, the European abalone, and does not include CA activity in the shell matrix. Our work provides new insight on the diversity of the extracellular macromolecular tools used for shell biomineralization study in mollusks.

In vitro effects of triclosan and methyl-triclosan on the marine gastropod Haliotis tuberculata

Triclosan (2,4,4′-trichloro-2′-hydroxy-diphenyl ether; TCS) is an antibacterial agent incorporated in a wide variety of household and personal care products. Because of its partial elimination in sewage treatment plants, TCS is commonly detected in natural waters and sediments. Moreover, due to its high hydrophobicity, TCS accumulates in fatty tissues in various aquatic organisms. TCS can be converted into methyl-triclosan (2,4,4′-trichloro-2′-methoxydiphenyl ether; MTCS) after biological methylation. In this study, the acute cytotoxicity of TCS and MTCS in short-term in vitro experiments was assessed on cell cultures from the European abalone Haliotis tuberculata. The results showed that morphology and density of hemocyte are affected from a concentration of 8μM TCS. Using the XTT reduction assay, TCS has been demonstrated to decrease hemocyte metabolism activity in a dose- and time-dependent exposure. The IC50 was evaluated at 6μM for both hemocyte and gill cells after a 24h-incubation with TCS. A significant cytotoxicity of MTCS was also observed from 4μM in 24h-old hemocyte culture. Our results reveal a toxic effect of TCS and MTCS on immune (hemocytes) and/or respiratory cells (gill cells) of the abalone, species living in coastal waters areas and exposed to anthropogenic pollution.

Two different and functional nuclear rDNA genes in the abalone Haliotis tuberculata: tissue differential expression

Analysis of the 18S rDNA sequences of Haliotis tuberculata tuberculata and H. t. coccinea subtaxa identified two different types of 18S rDNA genes and ITS1 regions. These two different genes were also detected in H. marmorata, H. rugosa and H. diversicolor that are separated from H. tuberculata by 5-65 mya. The mean divergence value between type I and type II sequences ranged from 7.25% for 18S to 80% for ITS1. ITS1 type II is homologous with the ITS1 consensus sequences published for many abalone species, whereas ITS1 type I presented only minor homology with a unique database entry for H. iris ITS1. A phylogenetic analysis makes a clear separation between type I and type II ITS1 sequences and supports grouping H. t. tuberculata, H. t. coccinea and H. marmorata together. The two subtaxa do not show any significant differences between the homologous 18S rDNA sequences. A general structure of the ITS1 transcript was proposed, with four major helices for the two types. The two genes were expressed and, for the first time, a putative differential expression of ITS1 type I was detected in the gills, digestive gland and gonads whereas ITS1 type II was expressed in all tissues.

Comparative performances of juvenile abalone ( Haliotis tuberculata coccinea Reeve) fed enriched vs non-enriched macroalgae: Effect on growth and body composition

Abalone Haliotis tuberculata coccinea Reeve (1846), is a target species for diversification of European aquaculture production. Taking into account that sustainable, eco-friendly production methods are to be a part of future expansion of the abalone industry, growth performance of juvenile abalone reared in an integrated culture system was evaluated and compared with that of abalone fed non-enriched macroalgae. Four macroalgae treatments, three monospecific: Ulva rigida (UN), Hypnea spinella (HN) and Gracilaria cornea (GN) and a composite one (MN), were produced out of fishpond wastewater effluents, while other four control treatments consisted of the same species reared in fresh seawater (U; H; G; M). Seaweeds reared in fishpond wastewater effluents increased their protein content from 11–17% to 29–34%. Lipids consisted mainly of saturated fatty acids (SFA) (43–60%), palmitic acid being the most abundant fatty acid (40–47%). Highest EPA percentage was found in red algae H. spinella (6.9%), being ten times higher than that of U. rigida (0.7%). All the algae tested contained very low levels of arachinodic acid (0.1–1.6%) and docosahexaenoic acid (0.5–3%). Protein levels in foot muscle (74–76%) did not differ significantly (P < 0.05) among treatments. Survival was generally high, ranging from 85 to 100%. Weight gain (17–561%) and SGR (0.2–2.3%) were positively related to protein content; whereas, protein efficiency ratio (PER) (0.5–3.7) was negative correlated. PE ratios increased by 82–159% (DW) as a function of the enrichment among the different diets. Food conversion ratio (FCR) (7–188) improved according to the increase in PER. Overall, biofilter-produced macroalgae showed a significantly higher dietary value compared to the control treatments. Similarly, animals fed the mixed diets performed significantly better than those fed a single algal diet. Feeding G. cornea led to the lowest growth performance probably due to the lowest feed intake. The results clearly indicate that H. tuberculata coccinea growout can efficiently take place in an integrated-culture system suggesting that on-farm seaweed-abalone production could be part of future development of the abalone industry in the Canary Islands.

The Indentation Size Effect on the Micro-Hardness of Sea Mollusc Shell Structures

Abstract The Vickers micro-hardness of three different sea mollusc shell structures has been determined as a function of the indentation load. The following shells from the Adriatic Sea have been investigated: abalone (Haliotis tuberculata), smooth clam (Callista chione), and warty venus (Venus verrucosa). The results show that the measured micro-hardness depends on the load for all of the investigated seashell structures, which indicates the influence of the indentation size effect (ISE). The load dependence of micro-hardness has been analyzed by using the known Meyer's Law, the proportional specimen resistance model (PSR), and the modified proportional specimen resistance model. The best correlation between measured values and used models has been achieved by using the modified PSR model.

Biomineralization markers during early shell formation in the European abalone Haliotis tuberculata, Linnaeus

Larval shell formation was investigated in the European abalone Haliotis tuberculata. Stages of mineralization as well as enzymatic and endocrine biomarkers were monitored throughout larval development, from hatching to post-larval stages. Polarized light microscopy and infrared spectroscopy analyses revealed the presence of crystallized calcium carbonate arranged in aragonite polymorphs from the late trochophore stage. A correlation between the main steps of shell formation and enzymatic activities of alkaline phosphatase and carbonic anhydrase was seen. The variations of these biologic activities were related to the onset of mineralization, the rapid shell growth, and the switch from larval to juvenile shell following metamorphosis. Furthermore, a strong increase in the level of calcitonin gene-related molecules was measured in post-larvae, suggesting that endocrine control takes place after metamorphosis. The changes measured for the three biomineralization markers together with mineralogical analysis allowed us to correlate physiologic mechanisms with early steps of abalone shell formation.

Gene expression patterns of abalone, Haliotistuberculata, during successive infections by the pathogen Vibrioharveyi

Since 1998, episodic mass mortality of the abalone Haliotis tuberculata has been observed along the northern Brittany coast of France caused by a complex interaction among the host, pathogen and environmental factors. In the present study, abalone were submitted to two successive infections with the pathogen Vibrio harveyi under controlled conditions. During the first challenge, infection by V. harveyi resulted in 64% mortality of mature abalone. After a second infection of those surviving the first challenge, only 44% mortality was observed. Physiological variability in the host response appears to be a major determinant in susceptibility to V. harveyi. In order to isolate differentially expressed genes in H. tuberculata challenged with this bacterium, suppression subtractive hybridization (SSH) cDNA libraries were constructed from muscle of moribund abalone (susceptibles), surviving individuals (apparently resistant to the bacterium) and control (unexposed) animals. Of the 1152 clones sequenced, 218 different partial cDNA sequences were obtained and represented 69 known genes. Of these, 65 were identified for the first time in H. tuberculata. Using real-time PCR, a time-course study was conducted on 19 of the genes identified by SSH. A majority of differentially expressed transcripts were down-regulated in susceptible individuals as compared to their resistant counterparts. Bacterial challenge of abalone resulted in the up-regulation of three transcripts (encoding ferritin, heat shock protein HSP84 and fatty acid binding protein FABP) in those that survived exposure to V. harveyi. This study has identified potential candidates for further investigation into the functional basis of resistance and susceptibility to summer vibriosis outbreaks in abalone .

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 &lt; 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.

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.

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.

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).

Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: Transcriptional induction in response to thermal stress in hemocyte primary culture

Heat-shock proteins are a multigene family of proteins whose expression is induced by a variety of stress factors. This work reports the cloning and sequencing of HSP70 and HSP90 cDNAs in the gastropod Haliotis tuberculata. The deduced amino acid sequences of both HSP70 and HSP90 from H. tuberculata shared a high degree of homology with their homologues in other species, including typical eukaryotic HSP70 and HSP90 signature sequences. We examined their transcription expression pattern in abalone hemocytes exposed to thermal stress. Real-time PCR analysis indicated that both HSP70 and HSP90 mRNA were expressed in control animals but rapidly increased after heat-shock.

Candidatus Xenohaliotis californiensis and Haplosporidium montforti associated with mortalities of abalone Haliotis tuberculata cultured in Europe

Haliotis tuberculata experimentally grown in Galicia (NW Spain) and originated from Ireland began to experience mortalities during the late spring of 2004. Diseased abalone presented dark foot pigmentation, loss of surface adherence and limited ability for right themselves after they were set on their backs. Histopathological analyses showed the presence of Candidatus Xenohaliotis californiensis, a bacterial pathogen from the family Rickettsiaceae, in the gastrointestinal epithelial cells of diseased abalone. To our knowledge, this is the first report of Candidatus Xenohaliotis californiensis in H. tuberculata cultured in Europe. The presence of several stages of development of a new protozoan parasite belonging to the phylum Haplosporidia, Haplosporidium montforti, described by us in a separate paper, was also detected in the connective tissue of gill, digestive gland and foot muscle. Complete mortality (100% of the stock) was reached during spring and summer. PCR analyses showed that these two pathogens were already present in juveniles from Ireland before their introduction in Galician waters. Juveniles imported from France were also infected with both pathogens. In adult abalone collected from natural beds of the surroundings of the aquaculture facilities, only the pathogen Candidatus Xenohaliotis californiensis was detected by PCR, but not by histology.

Shell disease: abnormal conchiolin deposit in the abalone Haliotis tuberculata

Shell disease in the abalone Haliotis tuberculata L. is characterized by a conchiolin deposit on the inner surface of the shell. The gross clinical signs appear similar to the Brown Ring Disease (BRD) of clams. BRD has been extensively described in clams and is known to be responsible for severe mortalities and the collapse of the clam aquaculture industry in western France. In the clam, it was found to be caused by the infection of the mantle by Vibrio tapetis. Brown protein deposits have been observed in various abalone species around the world; some of these have been associated with a fungal infection in New Zealand, but the ones described here are similar to bacterial infections observed in clams. Larger animals appeared to be more affected by the disease, and a positive correlation of the number of successive infections found in the shells with the level of infestation of the shell by borers suggests that boring polychaetes and sponges may be vectors of the disease, or that the parasite infestation may increase the susceptibility of the animal to this infection. There is no evidence, however, that this infection causes mortality in abalone.

Ultrastructural description of new Rickettsia-like organisms in the commercial abalone Haliotis tuberculata (Gastropoda: Haliotidae) from the NW of Spain

Rickettsia-like organisms (RLOs) were found in the commercially farmed abalone Haliotis tuberculata in the northwestern region of the Atlantic Coast of Spain and are described from light and transmission electron microscopy observations. The RLOs measured approximately 1.6 x 0.9 microm and were found in intracytoplasmic, spherical to ellipsoidal vacuoles (up to 8 microm) in the epithelial cells of the digestive diverticulae. The morphological ultrastructure of these organisms was typically prokaryotic, including a plasmalemma and a thin Gram-negative type cell wall. Several ultrastructural changes were observed in the epithelial cells of the host containing the RLOs. The nuclei became pycnotic and several basophilic dense inclusions appeared in the cytoplasm. In addition, the host cell appeared lysed and was ruptured in advanced stages of infection. It was impossible to ascertain whether the RLOs are responsible for this disease, as a haplosporidian infection was also present. We can only conclude that the presence of RLOs simultaneously with a haplosporidian parasite may contribute to the mortality of the abalone host.

Suitability of three red macroalgae as a feed for the abalone Haliotis tuberculata coccinea Reeve

A 60-day feeding trial was conducted to assess the suitability of three red algae, Hynea spinella, Hynea musciformis and Gracilaria cornea, as potential feed for the culture of juvenile abalone, Haliotis tuberculata coccinea R. Seaweeds were reared in a biofiltration unit with fishpond waste water effluents. The three algal species were found to contain high protein contents which would be related to its production under the high nitrogen culture conditions of the biofilter system. Protein and carbohydrate contents were highest in H. musciformis and lowest in G. cornea. Survival rates of juvenile abalone were very good, regardless of the algae fed. Feed intake of H. spinella was highest, followed by H. musciformis. Growth rates of abalone were within the range obtained under commercial conditions, final shell length and weight being significantly highest in animals fed H. spinella and lowest in those fed G. cornea. Feeding G. cornea lead to the lowest growth performance due to the lowest feed intake, whereas feed conversion ratios were significantly highest for H. musciformis and protein efficiency ratios were higher for both H. spinella and G. cornea. This study suggested the good potential of any of the three red seaweeds tested—successfully produced by the biofilter system, their nutritional composition being similar to other macroalgae used as feed for abalone and matching the abalone protein and lipid requirements—hence promoting growth and survival. Nevertheless, the biofilter produced macroalgae H. spinella showed the highest dietary value for juvenile of H. tuberculata coccinea.