Objectif
The UE project "GENEPHYS", initiated by the IFREMER laboratory GAP-La Tremblade (Genetique Aquaculture et Pathologie) and CREMA-L'Houmeau (Centre de Recherche en Ecologie marine et Aquaculture), aims to establish the relationships between physiological traits involved in growth (oxygen consumption, absorption efficiency, scope for growth) and their genetic bases (determinism, variability within and among populations) in cupped oyster Crassostrea gigas.
The main objectives are :
1. To express the maximum variability of growth, both at the larval stage and at the adult stage, in a oyster population originating from parents of various origins.
2. To check the persistence over the time, of growth performance and related physiological functions.
3. To explain the observed growth differential by physiological functions studied.
4. To establish whether the growth differential by physiological functions are associated with a genetic basis, using 2 types of markers (allozymes and micro-satellites), and whether these are related to aneuploidy and the identity of missing chromosomes.
5. To estimate, in a second generation, genetic variance (additivity, dominance) and heritability of growth and of related physiological traits. Also to estimate aneuploidy transmission to this generation.
6. To evaluate the possibility of selection for a better assimilation efficiency. The beginning of a genetic map provides the first step for marker-assisted selection and QTL (Quantitative Trait Loci) location.
STATE OF PROGRESS
The 5-year project has now reached the end of its third year. In the first year, efforts were mainly dedicated to the production of the first experimental generation G1 and development of techniques. Year two saw an emphasis on the application of methods for genetic and physiological study on this biological material. Partners initiated a co-ordinated study on a group of common animals. In year three, work on the first experimental population has continued and practical work is reaching an end. Results gathered on the physiological performances and levels of aneuploidy in different animals were used to select parents for the breeding of the second experimental generation in which the levels of these characters will be examined.
Production and management of the G2 generation :
This task was performed in 1998 using animals scored for the stability and efficiency of physiological performance in 1997-1998 and populations displaying different levels of somatic aneuploidy. The first part of the G2 generation is presently being raised under controlled conditions in preparation for growth and physiological study in the coming two reporting periods. It consists of progenies of crosses between combinations of parents of high and low performance. 4 progenies have already been selected for study and individual size measurements begun. G2 animals for the study of inheritance of aneuploidy were bred using parents from populations of contrasting aneuploidy level. 4 progenies available for study issue from crosses between and within groups of high and low aneuploidy.
Recording of growth performances :
The recording of growth performances on the G1 generation was completed this year with the collection of final data from a group of animals grown at Palavas-Etang de Thau. The results indicate differences in growth patterns between this 'semi-natural' environment and that of Bouin where controlled experiments on growth and competition were conducted in the second reporting period. The influence of initial size on growth and final size was re-emphasised but it is clear that in the natural environment, smaller animals may achieve greater size increases relative to their initial size than large animals. Uniform conditions in contrast tend to favour uniform growth between size classes. The difference in results also suggests that genotype x environment interaction occurred, as the genetic material used was similar and that these studies should be followed up by an in depth experiment on individual growth in the natural environment.
Physiological Analysis :
According to the last annual reports two main experiments were performed in 1997 and 1998: temporal stability of physiological traits and characterisation of physiological performances of animals from the G1 generation. At present all the experiments have been finished to schedule. Data from each participant are completely treated.
The development of new continuous or sequential measurement systems for respiration and filtration, have allowed animal activity to be quantified over periods of 24 hours.
The physiological performances of laboratory raised animals were shown to be stable over time (based on 5 experiments over the course of a production cycle of 8 months). This result shows that in production conditions where food is not a limiting factor for growth, animals which perform the best at one period of the year also perform the best during other seasons.
Parents for the 2nd generation were ranked according to an index incorporating the activity and intensity of physiological functions concerning the acquisition and use of energy. The animals with the highest performance have greater absorption and lesser oxygen consumption than those with the lowest performance.
For the first time in this study a linear relationship between oxygen consumption and animal dry weight was observed (p<0.01).
The existence of high variation in absorption could be explained by the existence of two sub-groups which react differently to changes in food availability. If this hypothesis were correct, it would show that acclimation to low food supply can rapidly induce effects on oxygen consumption, while regulation of absorption would be faster or slower depending on the animal.
A similar distinction between G1 animals was found in the digestive enzyme activity study. The division of these animals into two sub-groups based on absorption efficiency data appears to be supported by genetic study. It remains to be shown however, whether the two sub-groups determined in the enzymatic activity study correspond to the two sub-groups distinguished by the non-destructive measurements on physiological functions.
Activities of both leucine aminopepdidase and cathepsin D were higher in the remaining tissues of slower-growers, confirming that the faster whole-body protein synthesis in slower-growers stems from fundamental metabolic differences in non-digestive tissues, and were not directly related to feeding rate.
Development has been completed of a novel assay for cytosolic calcium-dependent proteases will tell us whether differences in whole-body protein turnover derive solely from extracellular lysosomal activity, or whether they also stem from associated differences in basal intracellular metabolism.
Genetic Analysis :
The allozyme and microsatellite analysis has been completed on G1. The methods both show that there is an imbalance in parental contribution in the progeny. Microsatellite analysis performed on larvae and juveniles from one set of G1 controlled crosses show that parental contribution is unbalanced from the earliest stages of life, that it changes over time (a feature already exposed in the allozyme study) and that gametic competition occurs during fertilisation. Results of the microsatellite study of G1 adults emphasised that only a limited number of parents used in the crosses made for G1 may be represented in the progeny and therefore that genetic variation is less than the potential suggested by the parent used in a crossing design. Parental effects on growth in the growth experiment in Bouin will be analysed in the coming reporting period.
- Allozyme techniques were applied to the study of heterozygotes in G1 this year. Heterozygote deficiency did not appear to differ consistently with age class, i.e. despite indications from microsatellites that overall variability is reduced, this does not appear to be manifested as a reduction in heterozygotes with increasing age of the experimental population. Deficit of heterozygotes did not appear to be linked with growth slope either but did appear at greater levels for some loci in the progeny of some intra-population crosses more than others.
- Aneuploidy study has advanced with the completion of banding maps for the identification of missing chromosomes in aneuploid cells. Aneuploidy itself has been examined in animals of different size from contrasting populations once again showing that there is a negative relationship between aneuploidy and size and that there is a population (genetic) effect. In the coming reporting period, the untangling of size and genetic effects will be examined using samples of the same size from a series of full-sib families treated for chromosome study this year. The inheritance of aneuploidy will be examined in G2 families issued from crosses made this year between the populations with different levels of this trait.
ACHIEVEMENTS
The experimental studies on the G1 have now been completed.
This year these have yielded:
- Results from the growth experiment at Thau indicating that the influence of initial size is environment dependant and that small oysters can achieve higher specific growth than large ones.
- The confirmation of stability of physiological traits (oxygen consumption and filtration) over a period of 8 months during which the same individuals were measured repeatedly. Different individuals showed different performances and degrees of stability but overall statistics showed animals to be stable.
- The demonstration that faster whole-body protein synthesis in slower-growers stems from fundamental metabolic differences in non-digestive tissues, and is not directly related to feeding rate.
- The support for enzymatic results distinguishing different sub-groups of an experimental sample (digestive enzymes activities, digestive enzyme Michaelis constants) by genetic differences.
- The application of allozymes and microsatellites showing that parental contribution is unbalanced and changes over time.
- The scoring of aneuploidy in populations of different origin and animals of different size confirming the negative correlation of the trait with size and providing further evidence for a genetic basis.
- The creation of G2 progenies for study of physiological traits and aneuploidy.
- The analysis data by each partner individually.
In the context of this study of genetics and physiology of Crassostrea gigas, new techniques have also been developed. This year these include; completion of G and R banding maps for the identification of missing chromosomes in the karyotype, a chromosome specific probe for use in FISH analysis and the assay for cytosolic calcium-dependent proteases for application to protein degradation study.
FUTURE ACTIONS
Genetic and physiological analysis on the G2 generation which will be completed in 1999:
- Study of individual growth performances
- Physiological performances (non-destructive study of filtration and oxygen consumption)
- Protein turn-over and proteolitic activity
- Digestive enzyme activities
- Inheritance of aneuploidy
Work will continue to integrate the G1 data from the different experiments completed in the preceding reporting periods. This should provide important insight into the genetics and physiology of growth in C.gigas.
DESCRIPTION OF THE WORK
This study uses experimental populations of the Pacific cupped oyster, Crassostrea gigas, to examine genetic and physiological mechanisms behind growth patterns in this species. To this end, experimental progenies were produced using oysters from different populations to provide a large genetic base for investigation. The crosses were made principally in 1996, the first year of this five-year project. The resulting generation, G1, has been studied in depth for characters of interest and the possible relationships between genetics and physiology. Work on the project has involved the development of new techniques in different fields of biology in order to examine these traits on the animals. Study of G1 occupied much of the second and third years of the project and has provided a large amount of data, much pertaining to a common group of individuals on which several different experiments were conducted. We are now in a position of synthesis for the data gained for the G1 generation from work done by the 6 different partners and 9 laboratories involved in the project. The G1 generation was also used to produce a G2 generation, which consists of different progenies, which will be used for different investigations. The breeding of a second experimental generation from the first will enable us to gain an idea of inheritance of physiological parameters important for productivity in the species. One set of genitors were selected from G1 animals according to physiological performance and a second were chosen for levels of somatic aneuploidy (missing chromosomes). The G2 was successfully produced in 1998 and studies on this generation will commence in 1999.
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CORDIS classe les projets avec EuroSciVoc, une taxonomie multilingue des domaines scientifiques, grâce à un processus semi-automatique basé sur des techniques TLN. Voir: Le vocabulaire scientifique européen.
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