Development and application of LSU rRNA probes for Karenia brevis in the Gulf of Mexico, USA

The brevetoxin producing dinoflagellate, Karenia brevis, is the target of several monitoring and research programs in the Gulf of Mexico, where it forms extensive and frequently long-lived annual blooms that can cause human intoxication and fish kills, as well as severe economic losses to coastal communities. Rapid, reliable methods for the detection and enumeration of K. brevis cells, as well as their discrimination from morphologically similar species, are valuable tools for managers and scientists alike. Our aim was to produce a species-specific molecular probe that would serve as a tool to facilitate the efficient and reliable detection of K. brevis in the Gulf of Mexico. We sequenced a fragment of the large-subunit ribosomal RNA gene (LSU rDNA) from five K. brevis cultures isolated from the Texas Gulf coast, the Florida Gulf coast, and the Atlantic coast of Florida, and detected no differences among these isolates. A consensus sequence was thus compiled and compared to a previously published sequence from Karenia mikimotoi, the closest known phylogenetic relative to K. brevis, for the purpose of identifying unique K. brevis signature sequences. Fluorescently-labeled (FITC) oligonucleotide probes targeting these regions of the K. brevis LSU rRNA were designed to include at least two base pair differences, as compared to K. mikimotoi. Among seven probes designed, one uniquely identified all K. brevis isolates to the exclusion of all other species tested (Kbprobe-7), including a Gulf of Mexico K. mikimotoi isolate (Sarasota, FL) and several additional Gymnodinium species, as well as other dinoflagellate, diatom, and raphidophyte taxa. Importantly, K. brevis cells in samples taken during a 2001 bloom, fixed with a mixture of modified saline ethanol and 10% formalin, and stored at 4 °C for 7 months were successfully labeled with Kbprobe-7. In addition, preliminary analysis of labeled cells by flow cytometry revealed that K. brevis could be distinguished from K. mikimotoi in solution, suggesting other potential applications of this probe.     

A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia brevis in the Rookery Bay National Estuarine Research Reserve

A DNA hybridization assay was developed in microtiter plate format to detect the presence of toxic dinoflagellates in coastal waters. Simultaneous detection of multiple species was demonstrated using Karenia brevis, Karenia mikimotoi, and Amphidinium carterae. Molecular probes were designed to detect both K. brevis and K. mikimotoi and to distinguish between these two closely related species. The assay was used to detect K. brevis in coastal waters collected from the Rookery Bay National Estuarine Research Reserve. Assay results were verified by species-specific PCR and sequence analysis. The presence/absence of K. brevis was consistent with microscopic observation. Assay sensitivity was sufficient to detect K. brevis in amounts defined by a regional monitoring program as “present” (≤1000 cells/L). The assay yielded quick colorimetric results, used a single hybridization temperature, and conserved the amount of genomic DNA utilized by employing one set of PCR primers. The microplate assay provides a useful tool to quickly screen large sample sets for multiple target organisms.     

Detecting Karenia brevis blooms and algal resuspension in the western Gulf of Mexico with satellite ocean color imagery

Blooms of the toxic dinoflagellate, Karenia brevis, have had detrimental impacts on the coastal Gulf of Mexico for decades. Detection of Karenia brevis blooms uses an ecological approach based on anomalies derived from ocean color imagery. The same anomaly product used in Florida produces frequent false positives on the Texas coast. These failures occurred during wind-driven resuspension events. During these events resuspension of benthic algae significantly increases chlorophyll concentrations in the water, resulting in confusion with normal water column phytoplankton, such as Karenia. A method was developed to separate the resuspended chlorophyll from the water column chlorophyll, decreasing the false positives used with the detection method.     

Loss of waterborne brevetoxins from exposure to phytoplankton competitors

We tested whether interactions among phytoplankton competitors affect toxin dynamics involving the red tide dinoflagellate Karenia brevis, whose brevetoxins incapacitate and kill coastal wildlife. The addition of a live diatom, Skeletonema costatum, led to decreased concentrations of brevetoxin B (PbTx-2) associated with K. brevis cells in co-culturing experiments and with two of three natural bloom samples containing K. brevis. Similar decreases in PbTx-2 concentration, but not PbTx-3 concentration, occurred when a mixture of brevetoxins (without live K. brevis cells) was exposed to S. costatum, indicating that S. costatum metabolizes waterborne PbTx-2. Liquid chromatography–mass spectrometry (LC–MS) and ELISA analyses indicated that PbTx-2 is probably not transformed into other brevetoxins or into known brevetoxin metabolites, and instead is biotransformed by a previously unrecognized mechanism. Four different S. costatum strains from around the world caused similar loss of PbTx-2, suggesting that evolutionary experience with K. brevis is not a pre-requisite for the ability to metabolize PbTx-2. Additionally, phytoplankton-associated bacteria were found to play no role in the loss of PbTx-2, as bacteria-free S. costatum strains metabolized PbTx-2. Finally, loss of waterborne PbTx-2 caused by exposure to a dinoflagellate, a cryptophyte, and two additional diatom species indicates that this phenomenon is widespread among phytoplankton. Our results unexpectedly suggest that competing phytoplankton species present during K. brevis blooms, and possibly other red tides, could mediate bloom toxicity and therefore ecosystem-level consequences of red tides.     

A handheld NASBA analyzer for the field detection and quantification of Karenia brevis

Blooms of Karenia brevis, the red tide forming dinoflagellate in the Gulf of Mexico, cause a myriad of ecological and economic problems for coastal communities, including massive fish and mammal mortalities, and damage to tourism and fisheries/shellfish harvesting industries. There is a need for accurate detection and prediction of K. brevis blooms, including rapid and inexpensive monitoring of both water and shellfish meats to ensure the safety of shellfish harvested for human consumption. To address this issue, we have developed a protocol for easy field extraction of cellular RNA from water samples and coupled it with a handheld nucleic acid sequence-based amplification (NASBA) sensor that amplifies and detects target mRNA specific to the rbcL gene of K. brevis. This extraction protocol is a modified version of the Qiagen RNeasy Mini Kit spin protocol and requires no specialized equipment or training. Once extracted, the RNA is amplified and detected by NASBA in an in-house designed and produced handheld sensor that provides a real-time fluorescence plotting of the amplification. Both the field RNA extraction protocol and the handheld NASBA analyzer compared favorably to laboratory-based technologies. In duplicate reactions, the amplification curves generated with the handheld detector closely mirrored the curves generated with the bench top Nuclisens EasyQ NASBA analyzer and there was no difference in the sensitivity obtained using the handheld device versus the bench top models. This extraction protocol and detection sensor will be a valuable tool for rapidly monitoring K. brevis in field environments.     

A modified assay to determine hemolytic toxin variability among Karenia clones isolated from the Gulf of Mexico

Karenia brevis (formerly Gymnodinium breve) is a toxic marine dinoflagellate generally restricted to the Gulf of Mexico and is the main causative organism in fish kills, shellfish intoxications and respiratory distress in humans following bloom events. K. mikimotoi is a morphologically similar co-occurring species which is toxic in other parts of the world oceans, but has not been recognized as a major contributor in toxicity of blooms within the Gulf of Mexico. Recently there has been increasing evidence of the simultaneous production of a variety of bioactive compounds in addition to potent neurotoxins (brevetoxin) in Karenia brevis isolates. These compounds are potentially ichthyotoxic and have been shown to cause hemolysis in several bioassays [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519; Kirkpatrick, B., Fleming, L.E., Squicciarini, D., Backer, L.C., Clark, R., Abraham, W., Benson, J., Cheng, Y.S., Johnson, D., Pierce, R., Zaias, J., Bossart, G.D., Baden, D.G., 2004. Literature review of Florida red tide: implications for human health effects. Harmful Algae 3, 99–115]. Presence of hemolytic compounds may therefore add to the overall toxicity levels of bloom events. Current monitoring methods include assays which are highly sensitive in brevetoxin detection and yet may not target other harmful compounds.By adapting protocols developed by Eshbach et al. [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519], Red drum (Sciaenops ocellatus) erythrocytes were used to create a modified bioassay to detect hemolytic activity of crude algal extracts. Red drum was selected because it is endemic to coastal areas throughout the Gulf of Mexico and is sensitive to Karenia blooms, and thus makes this species a valid ecological target. Preliminary data has shown this method is sensitive for use in assessing hemolysis induced by laboratory cultures down to levels of 1 × 10³ cells mL⁻¹. Results showed an unexpectedly high level of hemolytic activity among K. mikimotoi clones, with one Texas strain inducing significantly higher hemolysis compared to Florida K. brevis isolates. Using this approach, future research efforts will examine the difference in production of hemolytic compounds among various Karenia clones.     

Comparative analysis of two algicidal bacteria active against the red tide dinoflagellate Karenia brevis

The red tide dinoflagellate Karenia brevis blooms annually along the eastern Gulf of Mexico, USA, and is often linked to significant economic losses through massive fish kills, shellfish harvest closures, and the potential threat to humans of neurotoxic shellfish poisonings as well as exposure to aerosolized toxin. As part of an effort to enhance the strategies employed to manage and mitigate these events and their adverse effects, several approaches are being investigated for controlling blooms. Previous studies have established the presence of algicidal bacteria lethal to K. brevis in these waters, and we aim to characterize bacterial–algal interactions, evaluate their role as natural regulators of K. brevis blooms, and ultimately assess possible management applications. Herein, the algicidal activity of a newly isolated Cytophaga/Flavobacterium/Bacteroidetes (CFB)-bacterium, strain S03, and a previously described CFB-bacterium, strain 41-DBG2, was evaluated against various harmful algal bloom (HAB) and non-HAB species (23 total), including multiple clones of K. brevis, to evaluate algal target specificity. Strains S03 and 41-DBG2, which employ direct and indirect modes of algicidal lysis, respectively, killed 20% and 40% of the bacteria-containing isolates tested. Interestingly, no bacteria-free algal cultures were resistant to algicidal attack, whereas susceptibility varied occasionally among bacteria-containing isolates of a single algal taxon originating from either the same or different geographic location. The dynamics of K. brevis culture death appeared to differ according to whether the algicidal bacterium did or did not require direct contact with algal cells, with the former most rapidly affecting K. brevis morphology and causing cell lysis. Both bacterial strains promoted the formation of a small number of cyst-like structures in the K. brevis cultures, possibly analogous to temporary cysts formed by other dinoflagellates exposed to certain types of stress. Results were also consistent with earlier work demonstrating that bacterial assemblages from certain cultures can confer resistance to attack by algicidal bacteria, again indicating the complexity and importance of microbial interactions, and the need to consider carefully the potential for using such bacteria in management activities.     

Use of dissolved inorganic and organic phosphorus by axenic and nonaxenic clones of Karenia brevis and Karenia mikimotoi

Nearly annual blooms of the marine dinoflagellate Karenia brevis, which initiate offshore on the West Florida Shelf in oligotrophic waters, cause widespread environmental and economic damage. The success of K. brevis as a bloom-former is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, although relatively little is known about the ability of K. brevis and the closely related Karenia mikimotoi to use a variety of organic sources of phosphorus, including phosphomonoesters, phosphodiesters, and phosphonates. Through a series of bioassays, this study characterized the ability of axenic and nonaxenic K. brevis and K. mikimotoi clones isolated from Florida waters to use a variety of organic phosphorus compounds as the sole source of phosphorus for growth, comparing this utilization to that of inorganic sources of phosphate. Differing abilities of axenic and nonaxenic K. brevis and K. mikimotoi cultures to use phosphorus from the compounds evaluated were documented. Specifically, growth of axenic cultures was greatest on inorganic phosphorus and was not supported on the phosphomonoester phytate, or generally on phosphodiesters or phosphonates. The nonaxenic cultures were able to use organic compounds that the axenic cultures were not able to use, often after lags in growth, highlighting a potential role of co-associated bacterial communities to transform nutrients to bioavailable forms. Given the ability of K. brevis and K. mikimotoi to use a diverse suite of inorganic and organic phosphorus, bloom mitigation strategies should consider all nutrient forms.     

COMPARATIVE MORPHOLOGY AND MOLECULAR PHYLOGENETIC ANALYSIS OF THREE NEW SPECIES OF THE GENUS KARENIA (DINOPHYCEAE) FROM NEW ZEALAND1

Three new dinoflagellate species, Karenia papilionacea sp. nov., Karenia selliformis sp. nov., and Karenia bidigitata sp. nov., were compared with the toxic species Karenia mikimotoi (Miyake & Kominami ex Oda) G. Hansen & Moestrup, Karenia brevis (Davis) G. Hansen & Moestrup, and Karenia brevisulcata (Chang) G. Hansen & Moestrup using the same fixative. Distinguishing morphological characters for the genus Karenia included a smooth theca and a linear apical groove. The new species can be distinguished on the basis of morphological characters of vegetative cells that include the location and shape of the nucleus; the relative excavation of the hypotheca; the characteristics of apical and sulcal groove extensions on the epitheca; the cellular shape, size, and symmetry; the degree of dorsoventral compression; and the presence of an apical protrusion or carina. Species with pronounced dorsoventral compression swim in a distinctive fluttering motion. An intercingular tubular structure traversing the proximal and distal ends of the cingulum is common to the species of Karenia, Karlodinium micrum (Leadbeater & Dodge) J. Larsen, Gymnodinium pulchellum J. Larsen, and Gyrodinium corsicum Paulmier. Molecular phylogenetic analyses of rDNA sequence alignments show that the new species are phylogenetically distinct but closely related to K. mikimotoi and K. brevis.     

Long-term increase in Karenia brevis abundance along the Southwest Florida Coast

Data collected along the southwest coast of Florida between Tampa Bay and Sanibel Island on the abundance of the toxic dinoflagellate Karenia brevis from 1954 to 2002 were examined for spatial and temporal patterns. K. brevis was found to be approximately 20-fold more abundant within 5 km of the shoreline than 20–30 km offshore. Overall, K. brevis was approximately 13–18-fold more abundant in 1994–2002 than in 1954–1963. In 1954–1963, K. brevis occurred primarily in the fall months. In 1994–2002, it was more abundant not only in the fall, but also in the winter and spring months. It is hypothesized that greater nutrient availability in the ecosystem is the most likely cause of this increase in K. brevis biomass, and the large increase in the human population and its activities in South Florida over the past half century is a major factor.     

A satellite and field portrait of a Karenia mikimotoi bloom off the south coast of Ireland,August 1998

An extensive surface bloom of the dinoflagellate Karenia mikimotoi occurred off southwestern Ireland during August, 1998. The bloom was evident both from remotely sensed satellite ocean colour data and as visibly discoloured water, from the mouth of Bantry Bay around towards Cork, extending some 60 km offshore. The timing of the bloom co-incided with a field survey in the area. This paper compares the surface distributions of chlorophyll and K. mikimotoi concentrations with satellite ocean colour and thermal infra-red sea surface temperature images, from which may be derived the origins of the bloom. It would appear that weak coastal upwelling transported a thermocline population of K. mikimotoi up to the surface in the region of the Fastnet Rock, where it was wind-dispersed eastwards across the northern Celtic Sea.     

Fitness consequences for copepods feeding on a red tide dinoflagellate: deciphering the effects of nutritional value, toxicity, and feeding behavior

Phytoplankton exhibit a diversity of morphologies, nutritional values, and potential chemical defenses that could affect the feeding and fitness of zooplankton consumers. However, how phytoplankton traits shape plant–herbivore interactions in the marine plankton is not as well understood as for terrestrial or marine macrophytes and their grazers. The occurrence of blooms of marine dinoflagellates such as Karenia brevis suggests that, for uncertain reasons, grazers are unable to capitalize on, or control, this phytoplankton growth—making these systems appealing for testing mechanisms of grazing deterrence. Using the sympatric copepod Acartia tonsa, we conducted a mixed diet feeding experiment to test whether K. brevis is beneficial, toxic, nutritionally inadequate, or behaviorally rejected as food relative to the palatable and nutritionally adequate phytoplankter Rhodomonas lens. On diets rich in K. brevis, copepods experienced decreased survivorship and decreased egg production per female, but the percentage of eggs that hatched was unaffected. Although copepods showed a 6–17% preference for R. lens over K. brevis on some mixed diets, overall high ingestion rates eliminated the possibility that reduced copepod fitness was caused by copepods avoiding K. brevis, leaving nutritional inadequacy and toxicity as remaining hypotheses. Because egg production was dependent on the amount of R. lens consumed regardless of the amount of K. brevis eaten, there was no evidence that fitness costs were caused by K. brevis toxicity. Copepods limited to K. brevis ate 480% as much as those fed only R. lens, suggesting that copepods attempted to compensate for low food quality with increased quantity ingested. Our results indicate that K. brevis is a poor food for A. tonsa, probably due to nutritional inadequacy rather than toxicity, which could affect bloom dynamics in the Gulf of Mexico where these species co-occur.     

Molecular tools separate harmful algal bloom species, Karenia mikimotoi, from different geographical regions into distinct sub-groups

Karenia mikimotoi is a toxic dinoflagellate that is known to form extensive populations in the Eastern North Atlantic and Pacific Oceans that that impacts significantly on recreation and fisheries industries in these areas. Attempts to resolve the complex taxonomy within this “species” have been hampered by the requirement for fine-scale morphological analyses and by the lack of suitable genetic markers. Here we report the use of a novel combination of primer sets designed to facilitate amplification of the rDNA LSU and ITS, and the rbcL genes that can be used to discriminate between K. mikimotoi isolates originating from different geographical regions. We show that isolates from Europe and New Zealand are more closely related to each other than either is to isolates from Japan. Specific PCR-based primers were designed to amplify a region of the rbcL gene for subsequent high resolution analysis of the PCR amplicon melting temperatures. This innovative technique allows us to rapidly discriminate K. mikimotoi from distinct geographic localities and we propose the separation of K. mikimotoi into two distinct sub-species.     

A SURVEY OF THE STEROL COMPOSITION OF THE MARINE DINOFLAGELLATES KARENIA BREVIS,KARENIA MIKIMOTOI,AND KARLODINIUM MICRUM: DISTRIBUTION OF STEROLS WITHIN OTHER MEMBERS OF THE CLASS DINOPHYCEAE1

The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4‐methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (ED) and its 27‐nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi, and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.     

A bacterium that inhibits the growth of Pfiesteria piscicida and other dinoflagellates

Toxic dinoflagellate blooms have increased in estuaries of the east coast of the United States in recent years, and the discovery of Pfiesteria piscicida has brought renewed attention to the problem of harmful algal blooms (HAB) in general. Many bacteria and viruses have been isolated that have algicidal or algistatic effects on phytoplankton, including HAB species. Twenty-two bacterial isolates from the Delaware Inland Bays were screened for algicidal activity. One isolate (Shewanella IRI-160) had a growth-inhibiting effect on all three dinoflagellate species tested, including P. piscicida (potentially toxic zoospores), Prorocentrum minimum, and Gyrodinium uncatenum. This bacterium did not have a negative effect on the growth of any of the other four common estuarine non-dinoflagellate species tested, and in fact had a slight stimulatory effect on a diatom, a prasinophyte, a cryptophyte, and a raphidophyte. Shewanella IRI-160 is the first non-microzooplankton example of a microbe with the ability to control and inhibit the growth of P. piscicida, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of P. piscicida and other dinoflagellates. Such bacteria could also potentially be used as management tools to prevent the proliferation of potentially harmful dinoflagellates in estuaries and coastal waters.     

The degradation of Karenia brevis toxins utilizing ozonated seawater

Florida red tides impose both an economic and health impact on the state. The purpose of this research was to examine the effectiveness of ozone to reduce the numbers of Florida red tide organism (Karenia brevis Davis) and its associated toxins in an artificial seawater environment. The results obtained in this experiment showed an approximate 1.25 log₁₀ unit reduction in the major toxin groups recovered after 10 min of ozone exposure (approximately 135 mg). In initial trials, K. brevis toxins were extracted and reintroduced into an artificial seawater (ASW) media. Subsequent experiments exposed whole cell K. brevis culture to ozone treatment. Samples from both experiments displayed approximately 1.10 log₁₀ unit reduction in total toxin and an approximate 1.25 log₁₀ unit reduction in three of the six major toxins associated with K. brevis (PxTx-1, -2, -9). The reduction in toxin concentration, as measured by high performance liquid chromatography (HPLC) analysis, displayed a positive correlation with the reduction of toxicity as determined by a fish (Cyprinodon variegatus) bioassay. Despite large total doses of ozone applied, as compared to levels that might be found at a commercial ozonation facility, some toxins were still recoverable by HPLC after ozone treatment.     

Removal of harmful algal cells (Karenia brevis) and toxins from seawater culture by clay flocculation

Harmful algal blooms (HABs) occur worldwide causing serious threat to marine life, and to public health through seafood-borne illnesses and exposure to toxin-containing marine aerosol. This study was undertaken to assess the ability of phosphatic clay to remove the toxic dinoflagellate, Karenia brevis, and the potent neurotoxins (brevetoxins) produced by this species. Results showed that the addition of an aqueous slurry of 0.75 g (dry weight) clay to 3 l of K. brevis culture, containing 5×10⁶ and 10×10⁶ cells/l, removed 97±4% of brevetoxins from the water column within 4 h after the addition of clay. Clay flocculation of extra-cellular brevetoxins, released from cells ruptured (lyzed) by ultrasonication, removed 70±10% of the toxins. Addition of the chemical flocculant, polyaluminum chloride (PAC), removed all of the extra-cellular toxins. A 14 day study was undertaken to observe the fate of brevetoxins associated with clay flocculation of viable K. brevis cells. At 24 h following the clay addition, 90±18% of the toxins were removed from the water column, along with 85±4% of the cells. The toxin content of clay diminished from 208±13 μg at Day 1, to 121±21 μg at Day 14, indicating that the phosphatic clay retained about 58% of the toxins throughout the 14-day period. These studies showed the utility of natural clay as a means of reducing adverse effects from HABs, including removal of dissolved toxins, in the water column, although considerable work clearly remains before this approach can be used on natural blooms in open waters.     

Detection of Karenia brevis blooms on the west Florida shelf using in situ backscattering and fluorescence data

Using shipboard data collected from the central west Florida shelf (WFS) between 2000 and 2001, an optical classification algorithm was developed to differentiate toxic Karenia brevis blooms (>10⁴ cells l⁻¹) from other waters (including non-blooms and blooms of other phytoplankton species). The identification of K. brevis blooms is based on two criteria: (1) chlorophyll a concentration ≥1.5 mg m⁻³ and (2) chlorophyll-specific particulate backscattering at 550 nm ≤ 0.0045 m² mg⁻¹. The classification criteria yielded an overall accuracy of 99% in identifying both K. brevis blooms and other waters from 194 cruise stations. The algorithm was validated using an independent dataset collected from both the central and south WFS between 2005 and 2006. After excluding data from estuarine and post-hurricane turbid waters, an overall accuracy of 94% was achieved with 86% of all K. brevis bloom data points identified successfully. Satisfactory algorithm performance (88% overall accuracy) was also achieved when using underway chlorophyll fluorescence and backscattering data collected during a repeated alongshore transect between Tampa Bay and Florida Bay in 2005 and 2006. These results suggest that it may be possible to use presently available, commercial optical backscattering instrumentation on autonomous platforms (e.g. moorings, gliders, and AUVs) for rapid and timely detection and monitoring of K. brevis blooms on the WFS.     

Development of microsatellite markers in the marine phytoplankton Karenia mikimotoi (Dinophyceae)

The marine phytoplankton, Karenia mikimotoi, causes severe red tides which are associated with mass mortality of marine fish, and have expanded their distributions in the coastal waters of western Japan. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. Here we developed 12 polymorphic microsatellite markers from K. mikimotoi. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 5 to 23, and the estimate of gene diversity was from 0.551 to 0.933 across the 12 microsatellites. We consider these loci potentially useful for detailing the genetic structure and gene flow among K. mikimotoi populations.