Journal of Experimental Marine Biology and Ecology 186 (1995) 103-l 15

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

The use of polyclonal antisera and blocking of antibodies in the identification of marine dinoflagellates: species-specific

and clone-specific antisera against Gymnodinium and Alexandrium

Hector Mendoza, Victoria L6pez-Rodas, Sonsoles Gonztiez-Gil,

Angeles Aguilera, Eduardo Costas*

Unidad de Gen&ica. Facultad de Veterinaria, Universidad Complutense, E-28040 Madrid, Spain

Received 5 April 1994; revision received 19 September 1994; accepted 28 September 1994

Abstract

Separate polyclonal antibodies were developed against cell surface antigens of the 4 dinoflagellate species: (a) Gymnodinium catenatum; (b) a morphologically similar non-toxic dinoflagellate G,v- rodinium sp.; (c) Alexandrium minutum; and (d) morphologically similar A. lusitanicum. Block- ing of antibodies to obtain clone-specificity were performed. Positive immunofluorescence reac- tions were visualized with epifluorescence microscopy as well as spectrofluorometry using FITC indirect immunofluorescence assay. Specificity and cross reactivity were tested with cells from laboratory cultures as well as cells from natural samples. Polyclonal antisera were species-specific (for G. catenatum and Gyrodinium sp.) and genus-specific (for Alexandrium). After blocking the antisera were clone-specific. An excellent labelling was obtained with fresh cells and from those preserved in 4% buffered formaldehyde. The blocking of the antibodies shows great promise as a technique to obtain clone-specific antisera.

Keywords: Blocking antibody; Dinoflagellate; Identification; Polyclonal antibody

1. Introduction

Toxin-producing dinoflagellates are receiving increased attention due to the human health risk they pose, and the economic impact they have on fisheries and aquaculture. Generally, morphological criteria are sufficient to classify dinoflagellates to species, and

* Corresponding author.

Elsevier Science B.V.

SSDI 0022-0981(94)00160-X

104 H. Mendoza et ul. // J. Esp. Mar. Biol. Ecol. 186 (1995) 103-115

to identify potentially toxin-producing dinoflagellates. For thecate dinoflagellates, the plate patterns provide useful characters for species determination (Taylor, 1993). Ba- lech (1985, 1989) using detailed morphological analysis, has recognized more than 30 species within the genus Alexandrium. However, the work required to characterize an Alexatzdrium species is detailed (plate dissection) and skilled, and is not simple for routine monitoring programmes. In addition, thecate toxic dinoflagellates such as Al-

exandrium minutum Halim, A. ibericum Balech and A. lusitanicum Balech have dificulties to morphological classification (Balech, 1985, 1989). In athecate dinoflagellates there are more difficulties in morphological identification, e.g. the European populations, formally called Gvrodinium aureolum Hulburt are the same species known as G. na- gasakiense Takayama & Adachi in Japan (Taylor, 1993).

Some morphospecies have proven to be consistently linked to toxicity (A. catenella Whedon & Kofoid has been found to be constantly toxic), but other morphospecies such as A. tamarense (Lebour) Taylor are known to exist in both toxic and non-toxic strains (Taylor, 1993).

In the coastal waters of the Iberian Penninsula, G. catenatum Graham is one of the main dinoflagellates which causes PSP toxin production. A complicating factor for programmes that monitor for toxic phytoplankton is the occurrence in recent years of a new chain-forming athecate red tide dinoflagellate, GJyroditzium sp. which has bloomed in coastal waters of Spain (Fraga et al., 1993). Initially, it was thought to be the toxic G. catenatum, but after more careful observation this Gvrodinium sp. showed some differences, being smaller in size, forming shorter chains, an having bigger and more rounded chloroplasts (Fraga et al., 1993). A detailed analysis showed that Gyrodinium sp. is non-toxic (Fraga et al., 1993). Although it is not very difficult to distinguish them by observing live samples, when fixed it can be very difficult. This fact presents seri- ous problems in monitoring programmes as at times both species appeared at the same time in the same waters, i.e. in Galician waters during summer (Fraga et al., 1993).

Alexundrium minutum is another PSP toxin-producing dinoflagellate which frequently occurs in Atlantic waters of the Iberian Penninsula. At least on two occasions a new dinoflagellate, called A. lusitanicum by Balech (1985, 1989) which is morphologically similar to A. minutum, appeared in Portuguese waters (Sampayo, pers. comm.). Alex- andrium lusitanicum is also a PSP toxin-producing dinoflagellate, but exhibits slight differences in toxin composition with regard to A. minutum (Franc0 et al., 1993).

Considerable time and effort are required to identify a particular species when its different morphological characteristics are difficult to distinguish under the light mi- croscope. An alternative to microscopy identification is the use of molecular probes that can bind to either internal or external sites on the target species and be visualized using fluorescence or enzyme-linked calorimetric techniques (Anderson, 1993). In this regard the use of ribosomal RNA sequences (Scholin et al., 1993) and FITC-conjugated lectins (Costas et al., 1993a) appear to be promising techniques. Another alternative is the use of immunological procedures, such as antibodies which are being widely used for taxonomic determination of bacteria and protozoa.

Immunochemical techniques are increasingly being used to detect marine phytoplank- ton, generally to identify ultraplanktonic marine coccoid cyanobacteria (Campbell et al., 1983; Campbell & Carpenter 1987; Campbell, 1988) or small size and/or lack of

H. Mendoza et al. /_I. Exp. Mar. Biol. Ecol. 186 (1995) 103-115 105

characteristic morphology phytoplankton (Fliermans & Schmidt 1977; Anderson et al.,

1988; Shapiro et al., 1989a,b). In addition, immunochemical procedures have recently been used to detect nuisance and toxic marine phytoplankton cells (Anderson et al., 1988; Hiroishi et al., 1988; Uchida et al., 1989; Nagasaki et al., 1991; Sako et al., 1992; Vrieling et al., 1993a,b). Recently, Bates et al. (1993) were able to distinguish between domoic-acid-producing and non-toxic forms of the diatom Pseudonitzschia pungens

using immunofluorescence. Indirect immunofluorescence assay is the current immunological procedure used to

identify unicellular algae. In an indirect immunofluorescence assay, cells are first in- cubated with a primary antiserum, and then with a fluorescently tagged secondary antibody directed against the primary antibody, thus rendering the target cells visible using fluorescence microscopy. Polyclonal primary antibodies are obtained by immu- nizing rabbits with whole phytoplankton cells, and are thus directed against cell sur- face antigens (Anderson et al., 1988; Bates et al., 1993; Vrieling et al., 1993a).

If different species have common surface antigens then using polyclonal antisera positive cross reactivity can be obtained (i.e reaction of the antiserum against species not present in the immunization preparation). As an alternative, monoclonal antibodies can be used as primary antibodies (Hiroischi et al., 1988; Uchida et al., 1989; Nagasaki et al., 1991; Sako et al., 1992; Vrieling et al., 1993a).

Since considerable effort and well-appointed laboratories are necessary to obtain monoclonal antibodies, the blocking of antibodies can be the simplest alternative to obtain species-specific or clone-specific antisera. The blocking of the antibodies can be easily performed from polyclonal antiserum by elimination of common antigens by absortion on whole cells from clones or species not present in the immunization prepa- ration. The blocking of the antibodies has been done in many areas of immunology, but has not been used in immunodetection of unicellular algae. They are frequently used in food analysis and their specificity allows detection, i.e. bovine milk in ovine milk or cows milk in ewes milk and cheese (Bernhaver et al., 1983; Rodriguez et al., 1990; Garcia et al., 1991).

This paper presents the results of using an indirect immunofluorescence assay with polyclonal antibodies and the blocking of antibodies to distinguish between the toxic G. catenatum and the non-toxic Gyrodinium sp. as well as between A. minutum and A. lusitanicum

2. Materials and methods

2.1. Immunization cultures

Axenic cultures of G. catenatum Graham (clone Gc21V), Gyrodinium sp. (clone GglV), A. minutum Halim (clone AllV) and A. lusitanicum Balech (Clone AL18V) were grown in K medium (Sigma) at 20 “C and 12: 12 h light-dark photoperiods at a pho- ton flux density of about 50 ~E.m-2.s~’ provided by Cool-White fluorescent lamps (Costas et al., 1993b). Cells where harvested by centrifugation at the mid log of the exponential growth phase (about Day 12) washed in PBS (0.02 M phosphate, 0.15 M ClNa, pH 7.5) and used in the immunization of rabbits.

106

Table 1

H. Mendozcr et al. /J. Exp. Mar. Biol. Ecol. 186 (1995) 103-115

Test for specificity and cross reactivity against phytoplankton cultures

Species

G. cutenum

GclV

Gc9V

Gcl9V

Gc21V

Gyodinium sp. GglV

GgVa2V

GgVa3V

GgVa4V

A. minutum

AllV

Al2V

A. lusitunicum

Apl8V

A. excu+arrr

PelV

Pc2V

A. c@ne

Pa2V

Pa3V

Pa4V

P. lima

Pl5V

P17V

Pl8V

P. triestinum

Pt3V

Pt5V P. micuns

PmL

PmA

P. minimum

PminA

PminL

Tetruselmis suecico

(prasinophyceae) Te40

Chlorellu spp. (Chlorophyceae)

Chl Isochryis galvanu

(Prymnesophyceae) IS0

Oscillatoriu spp.

(Cyanophyceae)

osc

-

-

Location Antiserum

G. catenutum G~rodinium sp. A. minutum A. lustztanicum

Gc2lV GglV AllV Apl8V

Vigo

Vigo

Vigo

Vigo

Vigo Valencia

Valencia

Valencia

Vigo

Vigo

Lisbon

Vigo

Vigo

Vigo

Vigo

vigo

Vigo

Vigo

Vigo

Vigo

Vigo

Lisbon

Albufera

Algarbe

Lisbon

Vigo

Vigo

Vigo

Madrid

l+

1-t

I+

l+

2+ 2+

2i

2+

_ _

_ _ _

3+

3+

3+

_ I+

l+ _

_ _

_

_ _

_ _

3+

3+

3t

_

l+

_

_

_

_

_

_

_

_

_

_

_

_

Immunologicalcross reactivity is scaled from strong (3 + ) to none (-).

H. Mendoza et ul. 1 J. Exp. Mar. Biol. Ecol. 186 (1995) 103-I I5 107

2.2. Test cells

The dinoflagellates and the other cultured unicellular algal cells used for the immu- nological test are given in Table 1. Cultures were axenically grown as previously de- scribed in Costas et al. (1993b). The test cultures were harvested by centrifuge in both mid-log exponential and stationary phases. The cells were tested either fresh or pre- served in 4% buffered formaldehyde.

In addition, cells fixed in 47; buffered formaldehyde, directly collected by centri- fugation natural seawater samples from Galician shores, N.W. Spain (October 1993) were also tested (Table 2).

2.3. Antisera

Rabbits were immunized to produce separate polyclonal antibodies against cell surface antigens of the four species. 1 X lo6 cells of G. catenatum. 1 x lo6 cells of Gyrodinium sp., 3 x lo6 cells of A. lusitanicum or 3 x lo6 cells of A. minutum were re- moved in 0.5 ml complete adjuvant and subcutaneously injected into New Zealand White rabbits. Two rabbits were used for each species, a schedule of 4 boots was carried

Table 2 Test for specificity and cross reactivity against phytoplankton species from natural samples (N.W. Spain)

representing major classes

Antiserum

G. catenatum

Gc21V

Gyrodinium sp.

GglV

A. minutum

AllV A. lusitanicum

Apl8V

Dinophyceae

G. catenatum 3t

A. minutum

Gyrodinilrm sp. Alexandrium sp.

Dinophysis sp.

Protoperidinium sp.

Prorocentrum sp.

Ceratium sp.

Bacillariophyceae

Chaetoceros sp. -

Thalassiosira sp.

Nitzschia sp.

Rhizosolenia sp. lt”

Skeletonema costatum -

Leptocilindrus damicus -

Asterionella japonica

lt 3+ 3+

lt l+ l+

ltb ltb

2+b 2+h

il Weak labelling on R. afata cells. ’ Weak labelling in C. furca cells.

’ Moderate labelling on broken Chaetoceros sp. cells. Immunological reactivity is scaled from strong (3 + ) to none (-).

108 H. A4endoza et al. ! .I. Ewp. Mar. Biol. Ecol. 186 (1995) 103-l 15

out at 2-wk intervals. When an acceptable titre was achieved, production bleeds were obtained via intracardiatic in anaesthetized rabbits. The blood was allowed to clot (40 min) and was then centrifuged twice to remove red blood cells, yielding 10 ml of serum per bleed. Each serum was tested, divided into 1 ml aliquots and disolved in 50% PBS pH 7.5 and 0.1 sodium azide and stored at -20 “C.

2.4. The blocking qf the untiseru

Different clones of the same species may have common surface antigens. To obtain clone-specific antisera, the antibodies to common antigens were blocked by absorbtion (24 h, 20”) on cell pellets (10’ cells to 100 ~1 antisera) from different clones of the species; i.e. specific antisera against the clone Gc2lv of G. ccztenatum was obtained by successive incubations with cell pellets from the other G. catenatum clones (Gc7b, Gc~v, Gc 19~).

Clone-specific antibodies against clones Gc2lv of G. catenutum, Gc Iv of Gyrodinium sp., Allv of A. minutum and All& of A. Iusitunicum were obtained.

2.5. Immunojluorescence protocol

The specificity of each polyclonal antisera as well as each blocking antisera was tested by cross reaction against: (a) fresh cells from the algal cultures summarized in Table 1; (b) cells fixed in 49; formaldehyde from the algal cultures summarized in Table 1; (c) cells fixed in 4”; formaldehyde from natural samples (Table 2).

In each case, about 10” cells were collected by centrifuging and rinsed 3 times with PBS (0.02 M phosphate; 0.15 M ClNa; pH 7.5) to remove the culture medium or preservative. The incubation with the primary test antiserum diluted to 1:4 was per- formed at 25 “C for 45 min. Control samples were incubated identically with normal rabbit serum. After rinsing 3 times with 1 ml of PBS, the binding activity of antiserum was estimated using an immunofluorescence assay with a second fluorescein isothio- cynate FITC-conjugated goat anti-rabbit antibody (Serva) employed at 1.6 10e2 pg for 1 h at 25 “C.

After rising 3 times in PBS the samples were examined for: (a) the quality of stain- ing using a Zeiss Axiover 35 epifluorescence microscope with filter set for FITC fluo- rescence. Quality of staining was estimated according to the current procedure follow- ing the scale: 3 + ) bright stain, 100% of cells stained; 2 + ) less bright loo:,, of cells stained; 1 + ) low intesity stain but obviously different from controls and more than 50% of cells stained; - ) non-detectable reaction in more than 500, cells. All tests were read “blind”, i.e. the person reading the test did not know the identity of tested material. All antisera were compared to non-immunized sera controls to eliminate non-specific binding which could be interpreted as (false) positives.

(b) The quantity of binding measured the fluorescence emitted from the FITC- conjugated secondary antibodies bound to dinoflagellate cells by using a Shimadzu spectrofluorometer with 490 nm excitation and 515 nm emission. 10” 2 10’ were ob- seved in each case and three replicates were performed.

1 Oh + 10” cells of each clone/species were treated identically to the experimental cells,

H. Mendoza et al. / J. Exp. Mar. Biol. Ecol. I86 (1995) 103-115 109

but were incubated with a primary antiserum from non-immunized rabbits which served as autofluorescence controls in both quantitative measurements.

3. Results

For A. minutum and A. lusitanium, an antiserum dilution of 1: 1000 was the lowest concentration that produced positive labelling. The G. catenatum and Gyrodinium sp. antisera had a slightly lower titre with labelling at a dilution of 1:600. A working di- lution of 1:lO was used for the specificity and cross-reactivity test.

Fresh cells showed similar labelling intensity to cells preserved in 4% buffered formaldehyde. Frozen antisera showed labelling similar in intensity to fresh antisera. In addition, cells retained their FITC fluorescence for more than 1 month after label- ling, if kept refrigerated at 4 “C in darkness.

Antisera were active against cells at all stages of growth. In addition, cells from mid-log exponential phase cultures showed similar labelling intensity to cells from stationary phase cultures.

Test for specificity and cross reactivity (i.e. reactions of antiserum against antigens present or not present in the immunization preparations, respectively) against phy- toplankton cultures as well as against phytoplankton species from natural samples are shown in Tables 1 and 2, respectively.

Positive antibody labelling was found not only with clones used for the immuniza- tion but also with clones from the same species. Antisera against G. catenatum (Gc2lv) showed specificity at species level. It reached an intense immunofluorescence reaction (3 + ) with G. catenatum cells from cultures. This antisera was unable to differentiate between the different clonal cultures of G. catenatum. It also showed weak binding (1 + ) to Gyrodinium sp. cells from cultures, but it was unable to bind cells from 18 other dinoflagellate cultures nor cells from prasinophyceae, chlorophyceae, cryptophyceae or cyanobacteria cultures examined for cross reactivity. In addition, anti-Gc2lv was able to reach an intense immunofluorescence reaction (3 + ) with G. catenatum cells from natural samples. All the analysed G. catenatum cells (more than 1000) from N.W. Spain seawater samples were intensely bound. On the contrary, no cross reactions were found against the other phytoplankton cells from natural samples representing major classes, with the exception of some Rhizosolenia (Bacillariophyceae) cells which showed a weak labelling (1 + ) by anti-Gc2lv antiserum.

A specificity test of Gglv antiserum showed less labelling (2 + ) than specificity test of Gc2lv antiserum (3 + ), but unquestionably positive reactions (2 + ) were reached by anti-Gglv with all Gyrodinium sp. clones. Also, all the G. catenatum clones were weakly (1 + ) bound, but were unable to bind cells from the other culture species examined for cross reactivity. In addition, anti-Gglv showed a weak immunofluorescence reaction (1 + ) to G. catenatum cells as well as to Gyrodinium sp. cells from natural samples, but no cross reactions were found against other phytoplankton cells from natural samples.

Antisera against A. minutum (Allv) as well as antisera against A. lusitanicum (All&) were unable to differentiate between A. lusitanicum and A. minutum cells. Anti-Allv reached strong immunofluorescent reactions (3 + ) to both A. minutum and A. lusitani-

110 H. Mendoza et al. 1 J. Exp. Mm. Biol. Ecol. 186 (1995) 103-115

cum cells from cultures, and, in a similar way anti-rl. lusitunicum (Al18v) show strong labelling (3 + ) to both A. lusitanicum and A. minutum cells from cultures. In addition, both antisera were able to weakly label A. excavata (clone Pe2v). Anti-Allv was also able to weakly label A. ufJine (clone Pa3v). Both antisera were able to intensely bind to A. minutum cells from natural samples (100 “/:, of A. minutum cells samples were bound; more than 1000 cells sampled). In addition, both antisera showed weak labelling against some Cerutium furca cells. Additionally, both antisera showed a moderate immunofluorescent reaction against broken cells of Chuetoceros sp., but the label ap- peared to be confined to the interior of the cell rather than to the cell surface, and probably thus was a false-positive.

The test for specificity and cross reactivity of blocking antisera is shown in Table 3. It was possible to block the antisera to obtain clone-specific antibodies. However, the intensity of antibody labelling was less than non-purified antisera. A moderate (2 + ) positive labelling was found only with the clones used for the immu- nizations, i.e. purified antibodies against clone Gc2lv only bound cells from clone Gc2lv. Neither cells from other G. cutenutum clones, nor cells from the other analyzed cultures (Table 1) were bound. In the same way no cell from analyzed seawater samples (Table 2) showed a positive immunological reaction.

Spectrofluorometric values of immunological reactions of tests for specificity and cross reactivity of crude antisera as well as blocking antisera are shown in Table 4. These values allow a precise quantification of the immunofluorescence reactions. The quantitative measures agree with semiquantitative data of visual immunofluorescence. Quantitative data indicate that the crude antisera against Gc2lv (G. cutenutum) and

Table 3

Test for specificity and cross reactivity of blocking antisera

Species (clones) Blocking antisera

G. catenutum

Gc7B

Gc9V

Gcl9V

Gc21V

Gyrodinium sp.

GglV GgVa2V

GgVa3V GgVa4V

A. mimmm

AllV A12V

A. lusitanicum

Apl8V

Isolation Anti-Gc2 1 V

Vigo

Vigo _

Vigo _

Vigo 2+

Vigo

Valencia -

Valencia - Valencia -

Vigo

Vigo

Lisbon

Anti-GglV Anti-Al 1V Anti-Ap 18V

2+ _

2+ _

2+

H. Mendoza et al. 1 J. Exp. Mar. Biol. Ecol. 186 (1995) 103-l 15 111

Table 4

Spectrofluorimetric values of specificity and cross reactivity of crude and blocking antisera

Species Crude antisera

G. catenatum

Gc7B

Gc9V

Gcl9V

Gc21V

Gyrodinium sp. GglV

GgVa2V

GgVa3V

GgVa4V

A. minutum

AllV

A12V

A. lusitanicum

Ap18V

G. catenatum

Gc7B

Gc9V

Gcl9V

Gc21V

Gyrodinium sp

GglV

GgVa2V

GgVa3V

GgVa4V

A. minutum

AllV

A12V

A. lusitanicum

Apl8V

G. catenatum Gyrodinium sp. A. minutum A. lusitanicum

Gc21 V GglV A118V AplSV

97 30 16 16

92 26 18 15 91 26 17 16

100 34 12 12

36 100 17 17

39 91 11 13

33 93 13 12

35 87 16 14

14 16 100 87

18 16 97 90

18

Blocking antisera

19 94 100

12

15

18

100

14

10

13

12

12 100

14 16

16 14

16 12

100 10 10 14

11 100

Fluorescence values of each antisera are expressed as a percentage, taking into account 100% of the clone

present in the immunization.

Gglv (Gymdinium sp.) were species-specific. In addition, immunological reaction against the cells from the G. catenatum clone Gc2lv was higher than from the other G. catenatum clones, suggesting a slight clone-specificity. Anti-Gglv also showed slight clone-specificity. On the other hand, crude antisera against Allv (A. minutum) and Apl8v (A. lusitanicum) were unable to distinguish between A. m&turn and A. Iusitani-

112 H. Mendma et ul. 1 J. Exp. Mar. Biol. Ecol. 186 11995) 103-l 15

cum clones. The values of specific reactions with blocking antibodies were more than 5 times higher than the cross-reaction values, demonstrating that it is easy to block a polyclonal antisera to obtain clone specific antibodies.

4. Discussion

Cells from the PSP toxin-producing dinoflagellates G. catenatum, A. minutum and A. lusitanicum were injected into rabbits, which survived and produced antibodies against the toxic cells. The lowest titre found in this study (1:600-1:lOOO) was less than that reported for other phytoplankton, i.e. 1: 12 800 for Aureococcus anophagefirens (Ander- son et al., 1988) but similar to 1:500-1:lOOO reported by Bates et al. (1993) for the toxic diatom Pseudonitzschia pungens. According to Bates et al. (1993) a high working titre (1: 10) was used to sure detection of cross reactivity.

The labelling was excellent in both fresh and buffered-formaldehyde preserved cells in agreement with Bates et al. (1993) who obtained excellent labelling in preserved cells. We did not find the non-specific stain for preserved cells shown by Campbell (1988) and Shapiro et al. (1989b).

Our results show that the toxic PSP dinoflagellate G. catenatum and the morpho- logically similar non-toxic dinoflagellate Gyrodinium sp. can be distinguished visually by an immunofluorescence stain using a crude polyclonal antisera. Since our immuno- logical assay is able to distinguish both species in fixed samples from laboratory cul- tures or seawater (it can be very difficult distinguish both species in fixed samples), this immunological procedure will be of direct interest to monitoring programe in Galicia Rias (N.W. Spain) where both species appear at the same time.

In this study, A. minutum and A. lusitanicum could not be distinguished by crude polyclonal antisera. The classification of A. minutum and A. lusitanicum as a separate species was made by Balech (1985, 1989) based only on morphological characters. There is no total correlation between morphological similarity and immunological se- mejances. The indirect immunofluorescence assays using polyclonal antisera have gen- erally shown genus-specific or species-specific reactions (Anderson et al., 1988; Camp- bell, 1988; Shapiro et al., 1989a,b; Vrieling et al., 1993b). Bates et al. (1993) showed that polyclonal antisera are able to successfully distinguish between two forms of P. pungens f. multiseries, P. australis, P. frandulenta and P. .subcurvata, resembling differences be- tween immunological and morphology.

Protists, including dinoflagellates, present special problems in attempts to apply the biological species concept and in these organisms only morphological or biochemical characters can be used as any discontinuances constitute a “species” in itself (Taylor, 1993).

Generally, morphological characters are used to classify dinoflagellates to species, but increasing in the sophistication of our knowledge of non-morphological features relating to the species problem now requires a new synthesis (Taylor, 1993). On the other hand, the role played by genetic variability within phytoplanktonic species is unclear (Wood & Leatham, 1992).

In this regard, immunological procedures are promising. The weak intraclonal dif-

H. Mendoza et al. /J. Exp. Mar. Biol. Ecol. 186 (1995) 103-l 15 113

ferences detected by spectrofluorometric evaluation suggest that unique and as yet unidentified epitopes exist on the cell surface of each clone. This fact is demonstrated by the blocking of the antisera, to obtain clone-specific reactions. The antibodies for common antigens are eliminated by blocking, and positive immunofluorescence reac- tions are due to the clonal-specific antigens. There are genetic differences among the clones for surface antigens.

There are numerous benefits of an immunochemical assay capable of distinguishing dinoflagellate clones (i.e. to provide a research tool to carry out laboratory experiments on the competitive growth of clones, or population genetic studies in nature). In ad- dition, due to the predominance of asexual reproduction, species definition in di- noflagellates is problematic as many species are able to establish large “clonal families”. There has been a general lack of appreciation by marine biologists of the potential for genetic variability within morphological recognized dinoflagellate species (Taylor, 1993). However, due to clones of ancient divergence they are genetically quite different from one another. The characterization of clones must have an impact on studies of harmful dinoflagellates as reviewed by Taylor (1993). The used of blocking antibodies may be a useful tool to distinguish dinoflagellate clones. This procedure could be an alterna- tive to the use of lectins previously employed in this study (Costas et al., 1993a).

On the contrary, immunofluorescence reactions were not influenced by the physi- ological stage of the cells. Mid-log exponentially growing cultures have shown similar immunofluorescence as stationary phase cultures. In addition, since laboratory cultures contained cells in all the phases of their cell division cycle, and since all the cells from cultures were bound by their specific antisera, then antiserun is directed against cells at all stages of growth. The same is true for Emiliuna huxleyi (Prymnesiophyceae) (Shapiro et al., 1989a).

Immunological tests may advance the pace of harmful algal research and monitor- ing. They are promising in the identification, separation and enumeration of cells, but biological problems such as cross reactivity and physiological variability, and techni- cal problems, i.e. signal strength, autofluorescence and loss of cell during processing must be resolved. It is clear that the rich tapestry of immunology must resolve the technical problems. But with respect to biological problems, a lot of work is necessary, due to antigenic variability among species: i.e. most of genus and species have com- mon cell surface antigens and consequently antiserum are genus-specific or species- specific, but in the other cases, crude polyclonal antisera are able to differentiate be- tween forms of the same species (Bates et al., 1993). The blocking of antibodies is an easy procedure to eliminate common cell surface antigens and increase the specificity of polyclonal antisera.

Acknowledgements

We are indebted to I. Bravo, S. Fraga and B. Reguera form IEO Vigo (Spain) and M.A. Sampayo from INP Lisboa (Portugal) for their help and for providing the cul- tures. This work was supported by Fundacion Rambn Areces and DGYCIT.

114 H. Mendozu et al. /J. Exp. Mur. Biol. Ecol. 186 11995) 103-I 15

References

Anderson, D.M., 1993. Identification of harmful algal species using molecular probes. Sixikme Conference

Internationale sur le Phytoplancton Toxiquc, Nantes, France, 18-22 Octobrc.

Anderson, D.M., D. Kulis & E. Cosper. 1988. Immunofluoresccnt detection of the brown tide organism,

Aureococcus anophagefferem. In, Novelplunktorl blooms: causes und impacts of recurrent brown tides atzd other

unusuul blooms, edited by E. Cosper, V. Bricelj & E. Carpenter, Springer-Verlag, New York, pp. 213-

228.

Balcch, E., 1985. The genus Alexandnum or Gon_vaulrrx of the trrrnurensis group. In, Toxic dinoflagellotes.

edited by D. Anderson, A. White and D. Baden, Elsevier Science Publishing, New York, pp. 33-38.

Balech, E., 1989. Redescription of Alexandrium minutum Halim (Dinophyceae) type species of the genus

Alexandrium. Phycologia, Vol. 28, pp. 206-211.

Bates. S.S., C. LCger, B. Keafer & D.M. Anderson, 1993. Discrimination between domoic-acid producing

and nontoxic forms of the diatom Pseudonitschiapungeifs using immunofluorescence. Mar. Ecnl. Prog. Ser.,

Vol. 100, pp. 185-195.

Bernhauer, H., S. Baudner & 0. Giinter, 1983. lmmnological detection of proteins of cow milk in sheep or

goat milk and cheese with a specific immonoglobulin. 2. Lehensm. Unters. Forsch, Vol. 177, pp. 8-10.

Campbell, L., 1988. Identification of marine chroococcoid cyanobacterta by immunofluorescenco. In, fm-

munochemiccrf upproaches IO coastal. estuurine and oceanographic questions, edited by C. Yentsch, F. Mague

& P. Heran, Springer-Verlag, New York, pp. 208-229.

Campbell, L. & E. Carpenter, 1987. Characterization of phytoerythrin-containing Synechococcu.~ spp. popu-

lations by immunofluorescence. J. Plankton Res., Vol. 9, pp. 1167-l 18 1. Campbell, L., E. Carpenter & V. Iacono, 1983. Identification and enumeration of marine chroococcoid

cyanobacteria by immunofluorescence. Appl. Environ. MicrobioI., Vol. 46, pp. 553-559.

Costas, E., E. Gonzalez-Chavarri, A. Aguilera, S. Gonzalez-Gil & V. Lopez-Rodas, 1993a. Use of lcctins to recognize and differentiate unicellular algae. Bot. Mar., Vol. 36, pp. 1-4.

Costas, E., S. Gonzalez-Gil, A. Aguilera & V. Lopez-Rodas, 1993b. An apparent growth factor modula- tion of marine dinoflagellatc excystment. J. Exp. Mctr. Bioi. Ecol., Vol. 166, pp. 241-249.

Fliermans, C. & E. Schmidt, 1977. Immunofluoresccnce for autoecological study of a unicellular bluegreen

alga. J. Phycol.. Vol. 13, pp. 364-368.

Fraga, S., I. Bravo, M. Delgado, J.M. Franc0 & M. Zapata, 1993. Differences bctwccn two chain forming,

athecate, red tide dinoflagellates: Gymnodinium carenutum Graham and Gyrodinium sp. SixiCme Confer-

cncc Internationale sur le Phytoplancton Toxique, Nantcs, France, 18-22 Octobrc.

France, J.M., S. Fraga, M. Zapata, I. Bravo, F.P. Vila & I. Ramilo, 1993. Comparision between different

strains of genus Alexundrium of the minutum group. Sixieme Conference Internationale sur le Phytoplanc-

ton Toxique, Nantes, France, 18-22 Octobre.

Garcia, T., R. Martin, E. Rodriguez, J. Azcona, B. Sanz & P. Hernandcz. 1991. Detection of bovine milk in ovine milk by a sandwich enzyme-linked immunosorbent assay (ELISA). J. Food Protect., Vol. 54,

pp. 366-369.

Hiroishi, S., A. Uchida, K. Nagasaki & Y. Ishida, 1988. A new method for identification of inter- and

intra-species of the red tide algae Charonellu antiqua and ChcrtoneNa marina (Raphidophyceae) by means

of monoclonal antibodies. J. Phycol., Vol. 24, pp. 442-444.

Nagasaki, K., A. Uchida & Y. Ishida, 1991. A monoclonal antibody which recognizes the cell surface of

red tide alga Gymnodinium nagasakiense. Nippon Siusan Gakk, Vol. 57, pp. 121 l- 1214. Rodriguez, E., R. Martin, P. Garcia, P. Hernandez & B. Sanz, 1990. Detection of cow milk in ewcs’milk

and cheese by an indirect enzyme-linked immunosorbent assay (ELISA). J. Dairy Rev.. Vol. 57, pp. 197-

205.

Sako, Y.. C. Kim&Y. Ishida, 1992. Mend&an inheritance of paralytic shellfish poisoning toxin in the marine

dinoflagellatcs Alexnndrium cutenella. Biosci. Biotech. Biochem., Vol. 5, pp. 692-694.

Scholin, C.A., G.M. Hallegracff & D.M. Anderson, 1993. Molecular evolution and global dispersal of the Alexcmdrium tumrrrense/Cu/eneNu species complex. Sixiemc Confi&ncc Internationale Sur Ic Phytoplanc-

ton Toxique, Names, France, 19-22 Octobre.

Shapiro, L., L. Campbell & E.M. Haugcn, 1989a. Immunochcmical recognition of phytoplankton species. Mar. Ecol. Prog. Ser.. Vol. 57. pp. 219-224.

H. Mendoza et al. / J. Exp. Mar. Biol. Ecol. 186 (1995) 103-l I5 115

Shapiro, L.E., E. Haugen, M. Keller, R. Bidigare, L. Campbell & R. Guillar, 1989b. Taxonomic affinities

of marine coccoid ultraplankton: a comparision of immunochemical surface antigen cross-reaction and

HLPC chloroplast pigment signatures. J. Phycol., Vol. 25, pp. 794-797.

Taylor, F., 1993. The species problem and its impact on harmful phytoplankton studies. In, Toxic phy- toplankton blooms in the sea, edited by T.J. Smayda & Y. Shimizu, Elsevier Science Publishers, New York,

pp. 81-86.

Uchida, A., K. Nagasaki, S. Hiroishi & Y. Ishida, 1989. The application of monoclonal antibodies to an

identification of Chattonella marina and Chatonella antiqua. Nippon Susan Gakk, Vol. 55, pp. 721-725.

Vrieling, E., A. Draaijer, W. Van Zeijl, L. Peperzak, W. Gieskes & M. Veenhuis, 1993a. The effect of la-

beling intensity, estimated by real-time confocal laser scanning microscopy, on flow cytometric appearance

and identification of immunochemiacl labeled marine dinoflagel1ates.J. Phycol., Vol. 29, pp. 180-188.

Vrieling, E., W. Gieskes, F. Colinj, J. Hofstraat, L. Peperzak & M. Veenhuis, 1993b. Immunochemical

identification of toxic marine algae: first result with Prorocentrum micans as test organism. In, Toxic phy- toplankton blooms in the sea, edited by T.J. Smayda & Y. Shimizu, Elsevier Science Publishers, Amster-

dam, pp. 925-93 1. Wood, A.M. & T. Leatham, 1992. The species concept in phytoplankton ecology. J. Phycol., Vol. 28,

pp. 723-729.