Many of the gaps in our knowledge of HAB oceanography and
ecology stem from the autecological or species-specific nature of
the phenomena. A common problem in research and monitoring
programs occurs when the HAB species of interest is only a minor
component of the mixed planktonic assemblage. Many potentially
useful measurements are thus not feasible because of the
co-occurrence of numerous organisms and detritus. Autecological
studies must rely on tedious microscope counts to enumerate the
target species, and unlike many other problems in phytoplankton
ecology, bulk or community measurements of water samples such as
chlorophyll or 
C uptake are of little use. Another constraint
arises from the difficulties in adequately identifying and
distinguishing between species or strains which are morphologically
similar. Considerable time and effort are required to identify a
particular species when its distinguishing characteristics are
difficult to discern under the light microscope. Such fine levels
of discrimination are not generally feasible in monitoring programs
or other studies which generate large numbers of samples for cell
enumeration. This situation is encountered frequently in studies
of harmful or toxic algae. For example, the diatom
Pseudonitzschia pungens occurs in two varieties, one toxic and the
other non-toxic, but these cannot be distinguished from each other
using the light microscope [ Smith et al., 1990]. Likewise,
toxic and non-toxic varieties of toxic dinoflagellates are known to
co-occur within a given region [ Yentsch et. al., 1978].
Whether the problem is distinguishing between closely-related strains or enumerating a single species in large numbers of samples, the need for species- or strain-specific ``probes'' is clear---probes which can be used to label only the cells of interest so they can then be detected visually, electronically, or chemically. To date, the two most common probes of this type for harmful algae are antibodies (both polyclonal and monoclonal) that bind to cell surface proteins, and oligonucleotide probes which target nucleic acid sequences inside cells.
Antibody probes are produced by inoculating cells of a target species into animals to stimulate an immune response (e.g. [ Shapiro et al., 1989; Campbell et al., 1989; Anderson et al., 1989]). The animal then produces antibodies in response to the presence of the intact foreign organism or compounds derived from it. Cultured algal cells have been inoculated into laboratory animals and antibodies obtained that are specific for proteins on the outer cell wall of the target algae. Using a simple series of steps, a plankton sample can be treated with the antiserum in such a way that the cells of the target species are labeled with antibodies to which fluorescent compounds are attached [ Shapiro et al., 1989]. Visual detection of the labeling is possible using an epifluorescence microscope (Figure 3a,b). Alternatively, the samples can be processed using a flow cytometer or other instruments that can detect and quantify fluorescence. Assays can also be conducted on filters using fluorescent, chemiluminescent, or colorimetric detection.
Both polyclonal and monoclonal antisera have been developed for HAB species with excellent specificity. For example, a polycolonal antiserum developed for the brown tide chrysophyte Aureococcus anophagefferens is species-specific, showing no cross reactions with 46 phytoplankton cultures representing 5 algal classes [ Anderson et al., 1989]. An even higher level of specificity was demonstrated for the diatom Pseudonitzschia pungens, where a polycolonal antiserum is able to distinguish toxic from non-toxic varieties of the same species [ Bates et al., 1993].
Much of the effort in immunological detection of harmful algal
species has thus far been focused on development and
characterization of antibodies for individual species.
Applications of this probe technology to field populations of
harmful algae are accordingly limited, but will surely increase as
more highly specific antisera become available. Experience with an
antibody for the brown tide organism A. anophagefferens
suggests that antibody-based recognition of target species has a
large role to play in HAB field programs. That antibody has been
used for cell enumeration [ Anderson et al., 1993] and grazing
studies [ Caron et al., 1989], and was recently used to map
the geographic distribution of this species over a large region
[ Anderson et al., 1993]. The latter study was able to detect
A. anophagefferens at extremely low concentrations (10-20
cells ml
), and demonstrated that the species is present in
many areas with no known history of harmful brown tides. This
degree of resolution is noteworthy since A. anophagefferens
is so small and non-descript that normal microscopic identification
and enumeration are highly uncertain at low cell concentrations.
One obvious application of antibody probes would be in automated cell enumeration for field programs. As with other field applications, this has not yet been accomplished, however, but is under active investigation using solution and solid support-based formats. A useful instrument in this context is the flow cytometer, which uses a laser beam to excite individual cells in a flow stream, and characterizes their fluorescence and several other optical properties simultaneously. One current obstacle to flow cytometric analysis of immunolabeled cells is that the intensity of positive labeling is often not significantly higher than the background fluorescence of control or unlabeled cells. Signal enhancement is clearly necessary, and several promising approaches are being explored. Another problem is the loss of cells during the multiple steps involved in antibody labeling. Expectations are high that these problems can be overcome, but at this writing, automated applications of immunofluorescent techniques for harmful algae await further technique development.
Another promising probe technology targets particular genes or gene products inside cells using short, synthetic deoxyribose nucleic acid (DNA) segments (termed ``oligonucleotides'') which bind selectively to DNA or ribonucleic acid (RNA) sequences specific for a particular organism. For marine systems, this technique has thus far been used primarily on prokaryotes (e.g. [ DeLong et al., 1989; Amann et al., 1990; Distel et al., 1991]). Work is in its early stages on HAB species, with only two species or genera under investigation---the PSP dinoflagellates in the genus Alexandrium [ Scholin and Anderson, 1993; Scholin, 1992] and the ASP diatoms in Pseudonitzschia [ Scholin et al., 1994].
One of the problems that arises immediately when such work is initiated with HAB species is that little or no sequence information is available. The specificity of a probe will depend on the degree to which a particular sequence is unique, yet that determination requires that sequences of many closely-related or co-occurring algal species be known. Molecular studies of HAB species are still very much in their infancy. If the target gene or gene fragment has not been sequenced previously for the species, genus, or class of interest, it is necessary to establish the sequence database at the outset. This can be both time consuming and expensive.
Many current detection protocols immobilize extracted DNA on a solid surface such as a nitrocellulose or nylon membrane to which a probe is added and allowed to hybridize and establish a double-stranded molecule. Excess unbound probe is washed off, and the hybrid (target + probe) sequences is detected using radioactivity, fluorescence, chemiluminescence, or enzyme-coupled, colorimetric methods. In situ hybridization is also possible, using intact cells that are either immobilized on a microscope slide or suspended in solution. In this format, the probe enters the cell and binds to target sequences, excess probe is washed out, and the complex is detected with fluorescence or radioactivity. As with antibodies, the probe can be directly conjugated to a fluorescent reported molecule such as fluorescein isothiocyanate (FITC), or it can be biotinylated and detected using fluorescent avidin conjugates. (Figure 3c shows an epifluorescent image of an A. fundyense cell labeled with a ribosomal RNA probe. Multiple variations of these detection strategies exist as well.
As with antibodies, much of the effort in oligonucleotide detection of harmful algal species has been focused on development and characterization of probes for individual species, so applications of this technology to field populations of harmful algae are limited to a few unpublished analyses of field samples. Considerable research is thus needed to fully realize the potential that probes have to change the nature of HAB research and monitoring. Nevertheless, it is evident that antibody and oligonucleotide probes have great potential to alter the manner in which research is conducted on harmful algae. They can be valuable in quick, qualitative assays to indicate the presence or absence of a target organism, and they can assist in the identification of harmful species when trained taxonomists are not available. Once the limitations of these methods are better understood, probes will be used for the direct (and automated) enumeration of HAB species, and someday, to assist in physically separating those cells from co-occurring organisms for physiological or toxicological analyses. One can already envision the time when monitoring of HAB species will involve arrays of instrumented buoys that apply these probe techniques in an automated fashion and telemeter the data to shore. It is likely that both antibody and oligonucleotide probes will have roles to play in these detection systems, but there is a great deal of work to be done to reach these goals and to expand the availability of probes to all HAB species. Furthermore, the species-specific methods developed of necessity for HAB studies can have significant benefits to many other areas in biological oceanography where probe technology has yet to be applied. In all of these cases, there is a high probability of commercialization of the probe technology, demonstrating once again that fundamental science can lead to practical applications. This area of HAB research will clearly be active and productive for many years.