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Cultivation of toxic dinoflagellates - FC0725

Description
R&D

Summary Objectives:-
Provide a regular, reliable and pure supply of DSP and PSP toxin - producing algae, particularly from UK strains, as a source of material for analytical standards and for use in other areas of shellfish toxin research, such as examining the effects of processing on intoxicating tissues.

Key customer purpose:-
Make toxins freely available to the research effort including the development of a non-animal, low cost test for toxins in shellfish, thus helping to fulfil testing requirements arising from the new EC directive on shellfish hygiene.
Objective
2.1 Element 1. Maintenance and isolation of toxic strains by CCAP. 2.1.1 Obtain and maintain more strains of DSP and PSP toxic algae OF EUROPEAN ORIGIN (from both European and N. American collections). This includes any new ALEXANDRIUM strains which may be isolated and made available by Dr Jane Lewis (University of Westminster) in addition to the ones already in culture at DML (strains 2c and 4). In the main, these would be available to the research community. 2.1.2 Attempt to render some of these algae axenic by standard methods i.e. antibiotic treatment and/or micromanipulation). For example, Dr Lewis' strains of ALEXANDRIUM are unialgal but not axenic. Obtaining axenic cultures of algae known to produce PSP and DSP would help resolve the ongoing debate as to whether the toxins are of algal or bacterial origin. Axenic cultures are also essential for physiological and biochemical studies and are essential for such techniques as dialysis culture. This element, together with element 3, would be the responsibility of the Culture Collection of Algae and Protozoa, which will also be closely involved with other elements of the project. 2.2 Element 2: Pilot scale culture 2.2.1 DSP: Produce biomass/toxin from PROROCENTRUM LIMA as required by TRS. TRS will monitor toxin production at various stages of the growth cycle. Great batch-to-batch variability in the yield of okadaic acid (OA) per unit biomass has been found in cultures of P. LIMA grown under identical conditions (age at harvest, temperature, light, medium). It was found that yield of OA/unit biomass did not seem to alter when cultures were limited or replete in inorganic phosphate). A paper published recently (Tomas et al., 1993) demonstrated that in one strain of P. LIMA the amount of toxin per cell was up to 35% greater when organic phosphorus (glycero-phosphate) was the source of toxin. The yield of toxin per unit biomass in the strain of P. LIMA used by DML was about 10% of that reported elsewhere (deFreitas et al. 1993). The work referred to was carried out at the Institute of Marine Biosciences (IMB) of the National Research Council of Canada (NRCC). IMB was contacted and it was ascertained that yields of biomass per litre obtained at IMB and DML were similar and culture methods and conditions were similar. The main differences were a) the strain of P. LIMA used and b) a slightly different seawater enrichment (including glycerophosphate) than that used at DML. Batch-to-batch variability in toxin/unit biomass was also found by IMB. The strain of P. LIMA (an isolate originally from Spain) used at IMB is available from the North East Pacific Culture Collection and this has recently been obtained. We propose to culture this strain in the same medium and as near as possible under the same light regime and temperature as used at IMB. It is proposed to screen a few other strains of P. LIMA of European origin that are available, including the only UK isolate so far obtained. TRS has already run a small-scale trial with DSP toxic PROROCENTRUM LIMA supplied by DML and obtained toxic mussels (124 ug okadaic acid/100 g wet. wt tissue). TRS wishes to produce further batches of toxic mussels and also scallops in order to proceed with experiments on storage, depuration, cooking and processing of DPS positive tissue, and also PSP toxic tissue as soon as this becomes feasible. TRS also proposes in the longer term to feed toxic shellfish tissue to crustacean shellfish and finfish to determine the tissue distribution. With a view to possible expansion of this work a preparatory study is proposed as outlined in element 4. 2.2.2 PSP: Continue scale-up work on culturing of ALEXANDRIUM SPP. The problem is not so much in growing the dinoflagellates as obtaining sufficiently toxic strains. Laboratories already producing algal toxins, such as the Institute for Marine Biosciences, will not release these strains to other laboratories. We shall therefore continue to grow known toxic strains in sufficient quantities for screening by TRS. Any promising candidates will be cultured in larger quantities. TRS will carry out analyses and mouse assays for saxitoxin (ST). Item of information: IMB grows PSP toxic dinoflagellates in 30L batches in carboys using standard media. Cell densities obtained are typically 5000.ml-1 and the most toxic strains produce ca. 5 pg (10-12g) ST/cell. These cultures would therefore potentially yield 25 ug/L of ST. 2.2.3 Semi-preparative scale production of DSP toxins: with a view to producing material for standards (for example DTX-1 and DTX-2 are present in P. LIMA but difficult to obtain as standards). This element is only feasible if TRS is able to collaborate. The protocol for extraction and purification of OA and related toxins from PROROCENTRUM SPP. already exists (deFreitas, A.S.W. et al., 1992). A logical development from this would be to produce radio-isotopically labelled toxin which might, for example, if available in sufficient quantity and combined with an inert physical carrier be used as a tracer in studies of distribution in various organs and tissues after uptake. 2.3 Element 3: To attempt to isolate PROROCENTRUM LIMA from selected sites in the UK As part of the initial MAFF commission (May - October, 1992) Professor John Dodge (Royal Holloway New College) obtained mixed culture of PROROCENTRUM LIMA but was unable to obtain unialgal cultures. He sent these cultures to Dr Jack McLachlan, a Canadian visiting worker at the Plymouth Marine Laboratory who is an expert on the biology of P. LIMA (see Jackson et al. 1993). JM picked out single cells and inoculated them into sterile medium. Very few of these single cells grew. Only one out of a number of tubes sent to CCAP has produced a viable culture; this isolate grows very slowly but different media and growth temperatures are being tried. MAFF has requested that further collections should be made by CCAP made from UK sites as advised by Prof. Dodge, and isolation of PROROCENTRUM LIMA attempted. 2.4 Element 4: Desk study of the feeding habits of crustacean shellfish How crustacean shellfish become intoxicated with algal toxins is not known. Before any future research on depuration can proceed it is first necessary to understand the route by which the toxins are taken up by the crustaceans. One possible route of DSP intoxication in velvet crabs, for example, might be via the gut after feeding on material to which toxic algae, such as PROROCENTRUM LIMA, are attached. A survey of the literature on the feeding habits and on analyses of gut contents of velvet crabs, edible crabs and NEPHROPS would be inexpensive and provide an indication of a possible route of toxin uptake and how toxin might be fed to crustaceans kept in laboratory systems.
Time-Scale and Cost
From: 1993

To: 1994

Cost: £40,053
Contractor / Funded Organisations
Natural Environment Research Council
Keywords
Fish Disease              
Fisheries              
Food Hygiene              
Shellfish              
Fields of Study
Fish Health and Aquaculture