The effect of the feeding period on larval development was investigated in European sea bass larvae by considering the expression level of some genes involved in morphogenesis. Larvae were fed a control diet except during three different periods (period A: from 8 to 13 d post-hatching (dph); period B: from 13 to 18 dph; period C: from 18 to 23 dph) with two compound diets containing high levels of vitamin A or PUFA. European sea bass morphogenesis was affected by these two dietary nutrients during the early stages of development. The genes involved in morphogenesis could be modulated between 8 and 13 dph, and our results indicated that retinoids and fatty acids influenced two different molecular pathways that in turn implicated two different gene cascades, resulting in two different kinds of malformation. Hypervitaminosis A delayed development, reducing the number of vertebral segments and disturbing bone formation in the cephalic region. These malformations were correlated to an upregulation of retinoic acid receptor g, retinoid X receptor (RXR) a and bone morphogenetic protein (BMP)4. An excess of PUFA accelerated the osteoblast differentiation process through the upregulation of RXRa and BMP4, leading to a supernumerary vertebra. These results suggest that the composition of diets devoted to marine fish larvae has a particularly determining effect before 13 dph on the subsequent development of larvae and juvenile fish.Sea bass larvae: PUFA: Vitamin A: Morphogenesis: Retinoid pathway During the first 3 weeks of life, marine fish larvae undergo major morphological and functional changes to acquire their adult features. From hatching until 7 d post-hatching (dph), the feeding of European sea bass larvae is endogenous. It then becomes mixed until resorption of the vitellus; this generally occurs around 13 dph. At this date, the secretory function of the exocrine pancreas is not fully operational, only becoming efficient around 18 -20 dph. The maturational process of the pancreas is also characterized by a strong decrease in amylase activity between 13 and 20 dph (Zambonino Infante & Cahu, 2001). The maturation of intestinal cells is characterized by a decrease in cytosolic enzyme activity between 18 and 25 dph, whereas the activity of the brush border membrane enzymes increases. During this period of intense functional changes, a huge morphological transformation of the larvae occurs, in particular in the development of the neurocranium and the jaw, the segmentation of the vertebrae that becomes visible around 20 dph, and the settlement of the adult fins between day 27 and day 40 (Barnabé et al. 1976).During the first weeks of development, the maturation processes of the gastrointestinal tract can be influenced by nutritional conditions. Several studies have recently demonstrated that the morphogenesis of marine fish larvae could be perturbed by inappropriate dietary levels of vitamin A (retinol; Haga et al. 2002;Villeneuve et al. 2005a) or PUFA (Cahu et al. 2003). Moreover, the induced skeletal malformations dep...
Marine mollusc shell growth has been widely measured using fluorochrome marking. In order to test the efficiency and reliability of calcein staining on Pinctada margaritifera shells and pearls, the present study examined two administration methods, different concentrations and several immersion times. Immersion in a 150 mg L−1 calcein solution for 12 h to 24 h appeared to be the best method for marking P. margaritifera shells. For pearl marking, injection of a 200 mg L−1 calcein solution into the pearl pouch was the optimal method. Calcein marking was then used to measure the influence of food resource levels on the shell growth. Groups of 23-month-old P. margaritifera were fed at three trophic levels for two months. The two highest food levels tested (6000 cell mL−1 and 15 000 cell mL−1) induced uniform growth between the dorsal and ventral sides of shell, whereas the lowest food level (800 cell mL−1) induced greater growth on the dorsal side. Shell deposits from the ventral side were observed using a scanning electron microscope, revealing that the difference of the trophic level over two months had modified the thickness of the aragonite tablets formed. These results showed that the trophic level is a major factor conditioning P. margaritifera development.
This study aimed to model the food intake of P. margaritifera to examine the relationship between food level and reproductive activity. The effect of microalgae concentration on ingestion rate and assimilation efficiency was studied over a broad concentration range, using a mixture of Isochrysis galbana and Chaetoceros gracilis. Reproductive effort was assessed using three microalgae concentrations of 0.5, 7 and 18 cell lL À1 . Reproductive status was assessed by gonad development index (GDI)the ratio of the gonad surface to the visceral mass surfaceand histological analysis of the gonad based on the presence (continuous or discontinuous) or the absence of gonial cells (GC). Ingestion is a saturating function of seston concentration for bivalves modelled with an adapted Michaelis-Menten function. The maximum ingestion rate of P. margaritifera adults was 193.50 9 10 6 cell h À1 g À1 dw and the half saturation coefficient was 15 cell lL À1 . The concentration of 18 cell lL À1 , supplied for 45 days, induced a significantly higher GDI than the other treatments. GC decreased significantly and even stopped when pearl oysters were under-fed, suggesting that the mitotic process of the germinal stem cells was altered. Differentiation of germinal stem cells therefore appears to be controlled by food availability.
-Cryopreservation is a valuable tool for genetic improvement programs. Several bivalve mollusc species have already been the subject of such programs and the Tahitian black pearl oyster industry is now planning the development of selective breeding for desirable traits in Pinctada margaritifera. The ability to cryopreserve spermatozoa would, therefore, offer significant benefits to the cultured black pearl industry. Spermatozoa were cryopreserved with cryoprotectant agent (CPA) 0.7 M trehalose in 0.8 M Me 2 SO and a two-step freezing process was used: straws were first maintained in nitrogen vapour for 10 minutes, then directly plunged into liquid nitrogen and stored for one week before use. The viability of thawed sperm was 23% lower than that of fresh sperm. When using thawed sperm, therefore, a higher sperm/egg ratio of 100 000:1 was required to reach 80% oocyte fertilization, compared with 100:1 for fresh sperm. Nevertheless, this first demonstration of cryopreserved sperm fertility in black pearl oyster confirms the hatchery applicability of the cryopreservation technique defined here. Monitoring for larval viability during the first 23 days of life revealed no significant differences between the progeny produced with cryopreserved sperm and that produced using fresh sperm.
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