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Cultivation of toxic dinoflagellates - FC0725
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.
2.1 Element 1. Maintenance and isolation of toxic strains by
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
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
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
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
Contractor / Funded Organisations
Natural Environment Research Council
Fields of Study
Fish Health and Aquaculture