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Development and use of Cell Cultures for Commercially Important Aquatic Invertebrates.

Objective

Farmed crustacea and mollusca account for approximately 60% of the worlds consumption of these groups. The organisms represent high value (in economic terms), and are very important to the economies of France, Portugal, and the Far East. Production is likely to increase substantially over the next ten years. Yet, production is blighted by disease and lack of understanding of physiology, leading to high economic losses. This project addressing some of the fundamental issues regarding the biology and pathology of species of commercially important invertebrates. In particular, the development of cell lines will have an immediate impact on the ability to study viruses diseases, which are often suspected to occur but which cannot be confirmed in laboratory studies. Also, well-characterised cell lines will enable the study of exotoxicology, pathology, and disease-control, such as by vaccination. Basic biological aspects, such as the development of monoclonal antibodies, will enable the proper characterisation and screening of species and tissues.

By specifically studying growth factors, e.g. hormones and vitamins, it will be possible to identify growth limiting substances of relevance to whole animal nutrition.

Methods and preliminary results

This project which is a concerted action has just commenced (December 1997) and an inventory of methods available in member laboratories has been compiled. The main techniques use explanted tissue fragments, which are cultured in commercially available media with the osmolarity increased to facilitate culture of marine invertebrate cells. Tissues being investigated at present include Crustacean (Nephrops) gill and hepatopancreas, crustacean (Nephrops and Cancer ) hypodermis and molluscean (Ostrea ) heart . Specific techniques may be made available on application through the coordinator to the individual member laboratory involved in the specific technique of interest.
A major problem with the culture of all these invertebrate tissues is contamination and a focus of the concertation is to exchange methods on control of pathogenic infection. This is particularly important since it is undesirable to use antibiotics in case these affect the response of culturered cells to controlled infection with pathogens and exposure to toxins in the future.

Another problem being considered is that of slow growth at the ambient temperature for culture of these species (below 1 5°C). This means that growth can take several months before useful numbers of cells can be obtained from explanted fragments. Approaches to increase cell division include consideration of hormones and growth factors which have been identified as useful for insect cell culture.

The proposed roles for each partner in the concerted action:

Partner 1 DIT will culture Nephrops - hepatopancreas, brain and gills; and coordinate efforts to optimise media for the growth of the cells by all partners.
Partner 2 Heriot-Watt University will examine the culturing of Peneaus and Nephrops cells, and determine the effect of bacterial pathogens.
Partner 3 University of Montpellier will examine cells for validation; and determine the growth of key viruses.
Partner 4 University of Plymouth will culture cells from the marine decapod Carcinus; and examine differential gene expression following exposure to heavy metals
Partner 5 Trinity College, Dublin will develop systems for culturing sea lice.
Partner 6 University College, Cork will work with molluscan cell cultures.
Partner 7 Universitv of Leon will apply existing successful fish culture systems to the growth of Peneaus and Nephrops cells.
Partner 8 VESO Ltd. will provide facilties for culturing tissues and the whole animal.
Partner 9 Ecoserv Ltd. will measure effects of organic pollutants on Nephrops cultures
Partner 10 Danish Institute of Fisheries will study the effect of bacterial pathogens in cell lines.
Partner 11 Universite of Bretagne will examine the role of bivalve cell cultures for ecotoxicology and marine viruses
Partner 12 Nijmegen will culture freshwater parasites, e.g. Argulus.

The proposed work of the concerted action

1. To co-ordinate research approaches for the development of reproducible organotypic cell cultures for commercially importaIlt aquatic invertebrates, notably Peneaus, Cancer, Ruditapes, Crassostrea and Nephrops spp.

2. To establish standards for the characterisation of the cells types from "1 "

3. To co-ordinate studies to determine the value of these cell cultures and, where relevant, of vertebrate host cultures for the growth of pathogens (bacteria [e.g. Vibrio harveyi ], viruses [baculovirus] and parasites [Bonamia, Caligua ]

4. To evaluate the cell cultures for use in aquatic ecotoxicology, with emphasis on heavy metals and organophosphorus pesticides.

5. To arrange an international workshop to discuss cell culture techniques for aquatic invertebrates

6. To produce a state-of-the-art report on aquatic invertebrate cell culture.

Funding Scheme

CON - Coordination of research actions

Coordinator

Dublin Institute of Technology
Address
40-41 Lower Kevin Street
8 Dublin
Ireland

Participants (11)

Danish Institute for Fisheries Research
Denmark
Address
Soltofts Plads, C/o Technical University Of Denmark, Bygning 221
2800 Lyngby
Ecological Consultancy Services Ltd
Ireland
Address
17 Rathfarnham Road, Terenure
6W Dublin
HERIOT-WATT UNIVERSITY
United Kingdom
Address
Riccarton
EH14 4AS Edinburgh
IFREMER - CNRS - Universite de Montpellier II
France
Address
2, Place Eugene Bataillon
34095 Montpellier
KATHOLIEKE UNIVERSITEIT NIJMEGEN
Netherlands
Address
Toernooiveld 1
Nijmegen
National Centre for Veterinary Contract Research and Commercial Services A/S
Norway
Address
Alhusstrand, Elvalandet
7800 Namsos
THE PROVOST, FELLOWS AND SCHOLARS OF THE COLLEGE OF THE HOLY AND UNDIVIDED TRINITY OF QUEEN ELIZABETH NEAR DUBLIN HEREINAFTER TRINITY COLLEGE DUBLIN
Ireland
Address
East Theatre, Trinity College , College Green
2 Dublin
UNIVERSITY OF PLYMOUTH
United Kingdom
Address
Drake Circus
PL4 8AA Plymouth
Universidad de Leon
Spain
Address
Campus De Vegazana S/n
24101 Leon
Universite de Bretagne Occidentale
France
Address
Avenue Le Gorgeu N° 6
29285 Brest
University College Cork
Ireland
Address
Lee Maltings, Prospect Row
Cork