Matthieu Garnier

Evidence for the Involvement of Pathogenic Bacteria in Summer Mortalities of the Pacific Oyster Crassostrea gigas

A study was conducted to investigate the involvement of bacteria in oyster mortalities during summer. Moribund and apparently healthy oysters were sampled during mortality events along the French coast and in rearing facilities, usually when temperature reached 19°C or higher, and oysters were in the gonadal maturation phase. Hemolymph samples were aseptically withdrawn and submitted to bacteriological analysis. In healthy oysters, bacteria colonized hemolymph at low concentrations depending on the location. In most moribund oysters, bacteria were present in hemolymph and other tissues. These bacterial populations were more often diverse in oysters originating from the open sea than from facilities where animals were generally infected by a single type of bacterium. Only the dominant colonies were identified by phenotypic and genotypic characters (RFLP of GyrB gene and partial sequence of 16S rRNA gene). They belonged to a limited number of species including Vibrio aestuarianus, members of the V. splendidus group, V. natriegens, V. parahaemolyticus, and Pseudoalteromonas sp. The most frequently encountered species was V. aestuarianus (56% of isolates), which was composed of several strains closely related by their 16S rRNA gene but diverse by their phenotypic characters. They appeared intimately linked to oysters. The species within the V. splendidus group were less prevalent (25% of isolates) and more taxonomically dispersed. A majority of the dominant strains of V. aestuarianus and V. splendidus group injected to oysters induced mortality, whereas others belonging to the same species, particularly those found in mixture, appeared innocuous.

Gametogenic cycle and reproductive effort of the tropical blacklip pearl oyster, Pinctada margaritifera (Bivalvia: Pteriidae), cultivated in Takapoto atoll (French Polynesia)

The gametogenic cycle and the reproductive effort of the blacklip pearl oyster, Pinctada margaritifera, cultivated in Takapoto lagoon were studied for a 1-year period (March 1997-April 1998) by bimonthly observations of gonadal sections, dry tissue weights and gonadal index in a population of pearl oyster composed of three age-groups. Pearl oysters attained sexual maturity in the end of their first year (height ≈ 40 mm), implying that P. margaritiferais a late-maturing species in comparison with other Pteriidae. This species was also confirmed to be a marked protandrous successive hermaphrodite in culture, with 100 % of males at first maturity and 75 % in older pearl oyster (height > 120 mm). The general pattern of gametogenic activity, fairly synchronous in both sexes, was comparable with that of other tropical bivalves: reproduction occurs continuously throughout the year with a maximal activity during the warm season (November-May). No resting period was observed. Quantitative growth data showed that P. margaritifera exhibits an annual synchronised polymodal spawning pattern, with two spawning peaks in age-group I (height ≈ 70 mm) and five in age-groups II (height ≈ 100 mm) and III (height ≈ 120 mm). Spawning was sometimes incomplete, nevertheless a clear relationship between gamete production (PR, g) and size (height H, mm) was obtained: PR = 5.26 × 10 -7 H 2.91 (R_ = 0.99, P < 0.05). Estimation of PR was used to calculate the annual reproductive effort in P. margaritifera. Reproductive effort (%) was similar to those calculated for temperate species and showed a progressive increase with the age of pearl oyster, from 7 % in age-group I to 38 % in age-group III. This study showed that, in a fairly stable tropical environment such as the Takapoto lagoon, P. margaritifera is a multiple spawner, which uses an opportunistic reproductive strategy, allowing investment, all year around, of any surplus energy into gamete production. Surplus energy is ensured by the high pumping rates developed by this non-symbiotic bivalve to succeed in low seston conditions.

Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas.

Eleven Vibrio isolates invading the hemolymph of live and moribund oysters (Crassostrea gigas) collected in the field and from a hatchery in France, were characterized by a polyphasic approach. Phylogenetic analysis of 16S rRNA, gyrB and toxR genes indicated high homogeneity between these strains and the Vibrio aestuarianus type strain (ATCC35048(T)), and confirmed previous 16S rRNA analysis. In contrast, DNA:DNA hybridization was from 61% to 100%, while phenotypic characters and virulence tests showed a large diversity between the strains. Nevertheless, several common characters allowed the isolates to be distinguished from the reference strain. On the basis of several distinct phenotypic characteristics, it is proposed to establish two subspecies within the V. aestuarianus spp. group, V. aestuarianus subsp. aestuarianus [D. Tison, R. Seidler, Vibrio aestuarianus: a new species from estuarine waters and shellfish, Int. J. Syst. Bacteriol. (1983) 699-702] and V. aestuarianus subsp. francensis for these French isolates. The characters that differentiate the new strains from V. aestuarianus subsp. aestuarianus(T) are virulence (positive for 63% of the isolates) and 12:0 fatty acid content. The colonies were smaller and uncoloured, whereas no growth occurred at 35 degrees C or on TCBS, and the strains did not utilize several substrates, including L-serine, alpha-cyclodextrin, D-mannitol, alpha-glycyl-L-aspartic acid, L-threonine and glucose-1-phosphate.

Microalgae, Functional Genomics and Biotechnology

Abstract Microalgae have been studied for decades, but a new wave of research has recently begun as part of the search for renewable and sustainable energy sources. For economic optimization, microalgal biomass is being considered as a whole (main products and co-products) in an overall ‘biorefinery’ concept. Applications of microalgae cover a broad spectrum, including the food and (livestock) feed industries, bioenergy, cosmetics, healthcare and environmental restoration or protection. In the field of biotechnology, the access to genomic data is playing a growing role. As the cost of sequencing strategies has fallen, studies of gene function at the transcript, protein and biosynthesis pathway levels have multiplied. Notably, sequencing and mass spectrometry technologies are used to delineate the pathways of lipid synthesis, which will be valuable for the future application of microalgae in the biotechnology and biofuel industries. Another field making an applied use of genomics is the ‘cell factory’ approach, which uses the cell to manufacture (express) natural or recombinant proteins for diverse purposes. In this chapter, we present a vision of the potential future of genomics in the biotechnology of microalgae from several points of view.

Growth of the black-lip pearl oyster, Pinctada margaritifera, in suspended culture under hydrobiological conditions of Takapoto lagoon (French Polynesia)

Abstract Growth of the black-lip pearl oyster, Pinctada margaritifera var. cumingi, was studied for an annual cycle, from March 1997 to April 1998, in the lagoon of Takapoto atoll (Tuamotu archipelago, French Polynesia). Growth in shell and in tissue were measured every 15 days on three successive age groups of cultivated pearl oysters. At the same time, hydrobiological parameters (temperature, salinity, oxygen concentration, suspended particulate matter), known to have influence on bivalve growth, were followed each week during culture. No seasonal trend was observed in hydrobiological parameters, except for temperature which varied between 26°C and 31°C. The potential food for pearl oysters (particulate organic matter, POM, mg l−1) was slightly concentrated, but always available, so that, in this lagoon environment, no period seemed to be unfavourable to pearl oyster growth. Effectively, growth in shell was regular and shell did not exhibit any annual ring. Nevertheless, as it is often the case for bivalves, shell growth showed a progressive decrease with the age of pearl oyster and followed a classical Von Bertalanffy model: H=160.5 (1−e−0.038 (t−3.73)) with H the shell height (in mm) and t the age (in months). On the other side, growth in tissue did not follow the same pattern than for shell: P. margaritifera exhibited reduced growth rate in tissue during the warm season (November–April) so that a seasonal growth model was more appropriate: Wtissue=6.9/(1+e(5.58–0.208 t−0.435 sin (2π/12 (t−1.427)) with Wtissue, the dry tissue weight (in g) and t the age (in months). Several results concerning growth rates should be of interest for pearl farming. Firstly, the progressive decrease measured in shell growth rate implies, for pearl seeding operations, that the sooner the nucleus is implanted, the greater is the rate of nacreous deposition on this nucleus, and shorter is the time to obtain a marketable pearl for farmers. Secondly, exhaustive comparison, between growth rates obtained in our study and those obtained in other lagoons, tended to demonstrate that there is a small but significant variability in growth between lagoons of the Tuamotu archipelago. Further investigations need to be engaged in order to determine the most suitable sites for pearl farming in French Polynesia. Finally, comparison between growth of P. margaritifera var. cumingi and growth of other pearl oysters showed that P. maxima but also P. margaritifera var. erythraensis would also exhibit fast growth in Polynesian waters and then, would constitute potential candidates for further Polynesian diversification projects.

Adaptation to Cold and Proteomic Responses of the Psychrotrophic Biopreservative Lactococcus piscium Strain CNCM I-4031

ABSTRACT There is considerable interest in the use of psychrotrophic bacteria for food biopreservation and in the understanding of cold adaptation mechanisms. The psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031 was studied for its growth behavior and proteomic responses after cold shock and during cold acclimation. Growth kinetics highlighted the absence of growth latency after cold shock, suggesting a very high promptness in cold adaptation, a behavior that has never been described before for lactic acid bacteria (LAB). A comparative proteomic analysis was applied with two-dimensional gel electrophoresis (2-DE), and upregulated proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both cold shock and cold acclimation triggered the upregulation of proteins involved in general and oxidative stress responses and fatty acid and energetic metabolism. However, 2-DE profiles and upregulated proteins were different under both conditions, suggesting a sequence of steps in cold adaptation. In addition, the major 7-kDa Csp protein was identified in the L. piscium CNCM I-4031 genome but was not cold regulated. The implication of the identified cold shock proteins and cold acclimation proteins in efficient cold adaptation, the possible regulation of a histidyl phosphocarrier protein, and the roles of a constitutive major 7-kDa Csp are discussed.

Cinnamaldehydes Inhibit Cyclin Dependent Kinase 4/Cyclin D1

A series of cinnamaldehydes was synthesized for the study of inhibitory activity against cyclin dependent kinases (CDKs). A couple of compounds selectively inhibited cyclin D1-CDK4 with an IC50 value of 7-18 microM.

Comparative proteomics reveals proteins impacted by nitrogen deprivation in wild-type and high lipid-accumulating mutant strains of Tisochrysis lutea.

UNLABELLED Understanding microalgal lipid accumulation under nitrogen starvation is of major interest for biomass feedstock, food and biofuel production. Using a domesticated oleaginous algae Tisochrysis lutea, we performed the first comparative proteomic analysis on the wild type strain and a selected lipid over-accumulating mutant. 2-DE analysis was made on these strains cultured in two metabolic conditions, with and without nitrogen deprivation, which revealed significant differences in proteomes according to both strain and nitrogen availability. Mass spectrometry allowed us to identify 37 proteins that were differentially expressed between the two strains, and 17 proteins regulated by nitrogen starvation concomitantly with lipid accumulation. The proteins identified are known to be involved in various metabolic pathways including lipid, carbohydrate, amino acid, energy and pigment metabolisms, photosynthesis, protein translation, stress response and cell division. Four candidates were selected for possible implication in the over-accumulation of lipids during nitrogen starvation. These include the plastid beta-ketoacyl-ACP reductase protein, the coccolith scale associated protein and two glycoside hydrolases involved in biosynthesis of fatty acids, carbon homeostasis and carbohydrate catabolism, respectively. This proteomic study confirms the impact of nitrogen starvation on overall metabolism and provides new perspectives to study the lipid over-accumulation in the prymnesiophyte haptophyte T. lutea. BIOLOGICAL SIGNIFICANCE This paper study consists of the first proteomic analysis on Tisochrysis lutea, a non-model marine microalga of interest for aquaculture and lipids production. Comparative proteomics revealed proteins putatively involved in the up-accumulation of neutral lipids in a mutant strain during nitrogen starvation. The results are of great importance for future works to improve lipid accumulation in microalgae of biotechnological interest for biofuel production. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Comparative Transcriptome of Wild Type and Selected Strains of the Microalgae Tisochrysis lutea Provides Insights into the Genetic Basis, Lipid Metabolism and the Life Cycle

The applied exploitation of microalgae cultures has to date almost exclusively involved the use of wild type strains, deposited over decades in dedicated culture collections. Concomitantly, the concept of improving algae with selection programs for particular specific purposes is slowly emerging. Studying since a decade an economically and ecologically important haptophyte Tisochrysis lutea (Tiso), we took advantage of the availability of wild type (Tiso-Wt) and selected (Tiso-S2M2) strains to conduct a molecular variations study. This endeavour presented substantial challenges: the genome assembly was not yet available, the life cycle unknown and genetic diversity of Tiso-Wt poorly documented. This study brings the first molecular data in order to set up a selection strategy for that microalgae. Following high-throughput Illumina sequencing, transcriptomes of Tiso-Wt and Tiso-S2M2 were de novo assembled and annotated. Genetic diversity between both strains was analyzed and revealed a clear conservation, while a comparison of transcriptomes allowed identification of polymorphisms resulting from the selection program. Of 34,374 transcripts, 291 were differentially expressed and 165 contained positional polymorphisms (SNP, Indel). We focused on lipid over-accumulation of the Tiso-S2M2 strain and 8 candidate genes were identified by combining analysis of positional polymorphism, differential expression levels, selection signature and by study of putative gene function. Moreover, genetic analysis also suggests the existence of a sexual cycle and genetic recombination in Tisochrysis lutea.

Carbon conversion efficiency and population dynamics of a marine algae–bacteria consortium growing on simplified synthetic digestate: First step in a bioprocess coupling algal production and anaerobic digestion

Association of microalgae culture and anaerobic digestion seems a promising technology for sustainable algal biomass and biogas production. The use of digestates for sustaining the growth of microalgae reduces the costs and the environmental impacts associated with the substantial algal nutrient requirements. A natural marine algae-bacteria consortium was selected by growing on a medium containing macro nutrients (ammonia, phosphate and acetate) specific of a digestate, and was submitted to a factorial experimental design with different levels of temperature, light and pH. The microalgal consortium reached a maximum C conversion efficiency (i.e. ratio between carbon content produced and carbon supplied through light photosynthetic C conversion and acetate) of 3.6%. The presence of bacteria increased this maximum C conversion efficiency up to 6.3%. The associated bacterial community was considered beneficial to the total biomass production by recycling the carbon lost during photosynthesis and assimilating organic by-products from anaerobic digestion.