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FISHCAL
Calcium, the backbone of fish culture: Importance in skeletal formation, reproduction and normal physiology

Despite improved methodologies for farming of marine fish species, the viability of egg and larvae is still, in most cases, no more than 20-30%. This is because of high mortalities and/or a high incidence of larvae with skeletal deformities (dystrophies). Often dystrophies are not immediately apparent leading to a wasteful use of food, energy, space and human resources until dystrophic fish are graded and removed. Skeletal deformities arise because of abnormal calcification of cartilaginous tissue and there is evidence that hypercalcaemic factors may be involved. (Whilst hypocalcaemic factors, such as stanniocalcin and calcitonin, have been identified and characterised in fish, until now there has been no recognised hypercalcaemic factor.) Calcium is also important in a range of other physiological processes of practical importance to aquaculture, such as during reproduction when high concentrations of calcium need to be available for vitellogenesis and for growth, which requires both bone remodelling and ossification. The purpose of this project is to determine how calcium homeostasis and availability is maintained during critical stages of the life cycle of the teleost fish, Sparus aurata, an important marine aquaculture species in Southern European countries.

Objectives
One of the objectives of the project is to identify the relative importance of dietary, endogenous and environmental sources of calcium during critical phases of development, growth and reproduction of sea bream during a normal production cycle in the fish farm.
Another objective is to determine, experimentally, the roles of the parathyroid hormone related protein (PTHrP) in complete animal calcium homeostasis. Tissues sensitive to changes in calcium availability, including gut, kidney and gills, will be identified by in vitro and in vivo techniques. This will be carried out at stages in which calcium requirements are high, namely during skeletal development, reproduction and growth. A multidisciplinary approach will be used including molecular techniques to produce recombinant homologous PTHrP for in vivo treatments and production of specific antisera. These will be used to establish specific assays for PTHrP in tissues and plasma.
The third objective will be to study the molecular mechanisms that underpin bone formation in fish and the way in which PTHrP regulates this process. This will involve the identification of factors in vivo that may influence its expression.
Overall, these objectives will provide new information about calcium physiology in sea bream hitherto unavailable. This will allow the identification of causal factors in abnormal bone development in larvae, the mechanism by which calcium is incorporated into vitellogenin, the general requirements of calcium for normally growing fish and the function of PTHrP in these processes.

Progress to Date
One of the main aims of the first reporting period was to produce tools that could be used for the tasks ahead. Among these tools, recombinant PTHrP has been produced using a procariotic expression system. Other tools devised were several antisera for immunocytochemistry, probes for analysis of gene expression and a sensitive radioimmunoassay with which it was possible, for the first time, to measure significant quantities of hormone in plasma and tissues of sea bream.
It has been established that the rate of calcium uptake by sea bream larvae is directly related to fish size and calcium water content. Salinity strongly influences the route of uptake partly through an effect on drinking and PTHrP increased whole body calcium uptake in larvae.
Studies were initiated on a possible relationship between the increase of total blood plasma calcium, estrogen and PTHrP. These include in vivo and in vitro studies that should elucidate how calcium is handled by fish and the relative role of external and internal calcium (stored in bone and scales) in the calcium balance in sea bream.
In order to analyse what genes are being influenced by PTHrP subtracted, cDNA libraries have been produced from larvae treated, or not, with PTHrP. The experiments were successful and an initial micro-array will be produced comprising 2 100 clones (including 45 positive control clones and 26 putative negative controls).

Classified in NON-FOOD PRODUCTS, FISHERIES AND AQUACULTURE

Scientist responsible for the project

Prof. Dr. ADELINO CANARIO
Campus de Gambelas
8005-139 Faro
Portugal - PT

Phone: +351 289800925
Fax: +351 289817079
E-mail: acanario@ualg.pt

References

Project ID QLRT-2000-01465
Organisation UNIVERSIDADE DO ALGARVE
Area 5.1.2
Start date 01 November 2001
Duration (months) 42
Total cost 1 305 875 €
Total EC contribution   1 060 908 €
Status Ongoing
Web address of the project   http://www.ualg.pt/ccmar/fishcal

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