The first description of the potentially toxic dinoflagellate, Alexandrium minutum in Hunts Bay, Kingston Harbour, Jamaica

Harmful Algae 6 (2007) 29–47 www.elsevier.com/locate/hal

The first description of the potentially toxic dinoflagellate, Alexandrium minutum in Hunts Bay, Kingston Harbour, Jamaica Emma R. Ranston a,*, Dale F. Webber a, Jacob Larsen b a

Department of Life Sciences, University of The West Indies, Mona Campus, Mona, Kingston 7, Jamaica, West Indies b IOC-DANIDA, Science and Communication Centre on Harmful Algae, University of Copenhagen, Øster Farimagsgade 2D, DK-1353 Copenhagen K., Denmark Received 2 February 2006; received in revised form 20 April 2006; accepted 30 May 2006

Abstract The occurrence and morphology of the potentially toxic dinoflagellate species Alexandrium minutum found for the first time in Jamaica, were examined and described by light and scanning electron microscopy. Classical morphological examinations of whole cells, the thecal plate pattern of intact cells and more importantly the structure of individual thecal plates of squashed cells, were conducted in an attempt to positively identify the species. Characteristics such as a tear-drop shaped apical pore plate with a commashaped apical pore and no anterior attachment pore; a narrow sixth precingular plate; a narrow anterior sulcal plate longer than or approximately as long as it is wide; and a posterior sulcal plate wider than long, confirmed the Jamaican species as A. minutum. This dinoflagellate which produces potent neurotoxins responsible for paralytic shellfish poisoning (PSP) in humans in many parts of the World, as well as mass mortality of various marine flora and fauna, was identified in water samples collected during an extensive bloom of the species in the brackish to saline water body of Hunts Bay, an estuarine arm of Kingston Harbour, Jamaica in August 1994. The highest cell concentration was 4.6  105 cells l1, a concentration which far exceeds acceptable concentrations (<103 cells l1) of PSP-toxin producing A. minutum in several countries including: Spain and Denmark. No PSP human symptoms were reported during the bloom; however it was accompanied by a large kill of small pelagic fish extending across a third of the bay. Since then, smaller blooms of A. minutum have occurred with the most recent in February and April 2004. Hunts Bay is an important fishing, shrimping and to some extent oyster/mussel collection area and provides an important source of livelihood and food for many fishermen in nearby fishing communities as well as an important source of food for members of other communities. Although there are no known records of human illness due to PSP in Jamaica, the occurrence and blooming in Jamaican waters of this potentially toxic dinoflagellate, is great cause for concern. # 2006 Elsevier B.V. All rights reserved. Keywords: Alexandrium minutum; First description; Hunts Bay; Kingston Harbour; Jamaica

1. Introduction

* Corresponding author at: P.O. Box 34, Red Hills P.O., St. Andrew, Jamaica, West Indies. Tel.: +876 944 4624; fax: +876 944 4324. E-mail address: [email protected] (E.R. Ranston). 1568-9883/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2006.05.006

Alexandrium minutum was first described in Alexandria, Egypt (Halim, 1960). It has also been found in Europe (Montresor et al., 1990), Asia (Chang et al., 1997), Australia and North America (Hallegraeff et al., 1988, 1991) where it can form blooms in estuarine

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waters (Hallegraeff et al., 1988), eutrophic brackish lagoons (Giacobbe et al., 1996) and aquaculture ponds (Yoshida et al., 2000). A. minutum is one of the at least nine toxic species of the Alexandrium genus, known to be responsible for PSP in many parts of the world including South Australia (Hallegraeff et al., 1988) and France (ErardLe Denn, 1991; Belin, 1993). Dinoflagellates such as A. minutum are therefore receiving increasing attention due to the public health risk they pose and the economic impacts they have on fisheries and aquaculture developments (Hallegraeff et al., 1991). The coastal waters of Jamaica have been the site of several confirmed and unconfirmed red tides over the years, sometimes accompanied by fish kills (Steven, 1966; Goodbody, 1970; Wade, 1971; Simmonds, 1997). Kingston Harbour, the principal port of the island (Fig. 1), has been the major site of several red tides, which according to Goodbody (1970), are due to blooms of various diatoms and dinoflagellates, many of which have not been identified. More recent studies such as Simmonds (1997) have identified the numerically important red tide organisms in Kingston Harbour as Ceratium furca, Trichodesmium spp., Cylindrotheca closterium and Cyclotella sp. Kingston Harbour is an extensive harbour located on the south coast of the island of Jamaica in the Caribbean Sea between 17857.00 and 17857.50 N and 76848.20 and

76848.50 W (Fig. 1). With a total surface area of approximately 51 km2 (Wade et al., 1972), Kingston Harbour has developed as a major international transshipment centre for the Caribbean. The harbour is bounded on the north by Kingston, the larger of the two cities of the island, with a population of about 716,000 (STATIN, 2000). A narrow strip of land, the Palisadoes Spit, forms the southern boundary and at the southwestern corner, the harbour communicates with the Caribbean Sea via an opening approximately 3.2 km wide (Goodbody, 1970). The harbour has been divided into four major regions based on bathymetry; Hunts Bay, the Upper Basin, Inner Harbour and Outer Harbour (Goodbody, 1970) (Fig. 1). In August 1994, an extensive red tide occurred throughout Hunts Bay accompanied by a large kill of small pelagic fish extending across a third of the bay. The organism creating the bloom was found to be a small dinoflagellate that at the time could not be identified to the species level due to lack of expertise. The organism was subsequently identified as A. minutum during preliminary investigations of the bloom cells at an IOC-DANIDA training course on the Taxonomy and Biology of Harmful Marine Microalgae in Denmark in 1996. Ranston (1998) observed that the A. minutum like cells were consistent members of the phytoplankton community of Hunts Bay during a 14-month water

Fig. 1. Map of Kingston Harbour with major features.

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quality study of the area extending from December 1993 to February 1995. In February 2004, another extensive red tide was observed throughout the bay with previously observed A. minutum like cells dominating. A smaller bloom of the species was reported in April 2004; however this occurred outside of Hunts Bay along a section of the northern shoreline of Kingston Harbour. It is not known whether any illnesses were associated with the previous and more recent blooms of this species. Countries such as Jamaica which experience blooms of potentially toxic species and lack historical records of these species can protect and prepare themselves from potential problems through adequate species identification, thus allowing scientists and officials to project potential impacts to public health, aquaculture and coastal community economy (Steidinger et al., 1989). The main aim of this research was thus to confirm the specific identity of the Alexandrium species in Hunts Bay by examination and description of the morphological characteristics of the cells and comparison with classical taxonomical descriptions of Alexandrium, such as Balech (1989, 1995). This paper therefore describes the morphology of A. minutum discovered for the first time in Jamaican coastal waters. 2. Materials and methods 2.1. Study site description Hunts Bay is a relatively shallow, semi-enclosed arm of the Kingston Harbour (Fig. 1), with an average depth of about 2.4 m and an area of approximately 6.5 km2 (Ranston and Webber, 2003). The bay has a soft, black, highly anoxic mud bottom and receives fresh water discharges from two rivers: the Rio Cobre and the Duhaney Rivers, and from two major gullies – the Jew and Sandy Gullies (Fig. 1). The Rio Cobre, one of the largest rivers of Jamaica, drains major sugar cane fields and other agricultural lands to the west of Hunts Bay and receives a number of industrial and municipal discharges (Fig. 2). The Duhaney River and Jew Gully pass through major industrial sectors to the north and north-east of the bay, respectively, and the Sandy Gully drains a very large section of the residential area of the city of Kingston which lies to the north of the harbour (Fig. 1). Near the west-south-western shore of the bay, a small sewage disposal culvert, the Portsmouth Sewage outfall, directs semi-treated sewage into the bay (Fig. 2). With all this nutrient input, the area has been described as one under severe ecological stress,

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exhibiting all the typical characteristics of a eutrophic body of water (Wade, 1976), which are known to encourage the development of red tides (Lam and Yip, 1990). Ranston (1998) confirmed increasing eutrophic conditions in Hunts Bay and reported observing obnoxious red tides occasionally accompanied by fish kills, throughout the bay, during routine sampling of the area over a 14-month period. Hunts Bay is characterized by a water column stratified into a fresh to brackish water surface layer, due to freshwater input from the rivers and gullies, and a saline deeper layer. The surface waters are often supersaturated, high in nutrient concentrations, phytoplankton biomass and abundance, while deeper water is characterized by oxygen deficiency, lower nutrient concentrations and lower phytoplankton biomass and abundance (Ranston and Webber, 2003). Hunts Bay used to be a very productive and important fishing and shrimping ground and also served as a nursery area for young commercial species that are caught in the open sea (Goodbody, 1970). The decreasing water quality of the area over the years has contributed to a drastic reduction of the fish and shrimp populations of the bay. At present all that remains is a small shrimping industry and an even smaller fishery for bait and other small fish throughout the bay, resulting in general hardship for fishermen of the surrounding communities. Despite this decline, Hunts Bay is still an important fishing, shrimping and to a lesser extent, oyster/mussel collection area and still functions to some extent as a nursery area for fish and shrimp, as well as a feeding area for dolphins during the beginning of each year. Everyday up to 30 fishing vessels can be seen in the bay engaged in line and net fishing, as well as the setting of fish and crab pots. The small pelagic fish caught in the area are used as bait to catch larger fish out in the open ocean. Some fishermen collect and sell the various species of oysters and mussels that grow on the mangrove roots surrounding the bay. Hunts Bay is thus an important source of livelihood for the many fishermen in the area, an important feeding and nursery area for fish, shrimp and marine mammals, and an important source of food for the surrounding communities. 2.2. Sampling station locations The A. minutum blooms coincided with routine water monitoring exercises being conducted in Hunts Bay in August 1994 and February 2004. Quantitative and qualitative bloom samples were therefore collected at the same stations established for the monitoring

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Fig. 2. Map of Hunts Bay showing its major features and location of sampling stations.

exercises. These stations comprised nine sites (Fig. 2), each with two sampling depths, one in each water layer, (surface, 0 m and deep, 2–5 m) with the exception of station 1, which was too shallow (1 m) for a deeper sampling depth. Each station was strategically positioned such that the general area of the bay was sampled, but more importantly to allow the monitoring of the various fresh water inflows (point sources) to the bay. Five stations (1–3, 5 and 6) were located at the mouth of the fresh water inflows to the bay (Fig. 2). One station (4) was located in the middle of the bay, two stations (7 and 8) were located near an area of mangrove swamp and a small fishing village, respectively, and station 9 was located at the mouth of the bay, just outside the causeway bridge and the point of outflow of Hunts Bay water into Kingston Harbour (Fig. 2).

2.3. Field procedures Measurements of temperature, salinity and dissolved oxygen were made in situ using a Hydrolab Datasonde 4 water quality multiprobe HL002080 at the surface and at depth with simultaneous collection of quantitative and qualitative phytoplankton samples. Quantitative samples of planktonic microalgae constituting the blooms were collected at each sampling site using a 6 l Niskin whole water bottle sampler. Samples were collected just below the surface and at a depth between 2 and 5 m depending on the depth of the sample site. An aliquot of each whole water sample was collected in a 230 ml opaque plastic bottle containing 3 ml of neutral Lugol’s iodine solution for immediate fixing and staining of the microalgal cells for later identification and enumeration (Steidinger, 1979).

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Qualitative concentrated samples (net samples) of phytoplankton were collected by hauling and towing a 20 mm plankton net through the water at each sampling site. A vertical haul was conducted by gently lowering the net to a point just above the seafloor and drawing the net several times up through the water column until the water in the net became unclear or coloured by the concentrated algae. A horizontal surface tow was conducted by allowing the net to sink below the water surface followed by slow towing in a circular path behind the boat for about 2 min. About 25 ml of one of these samples was collected into a bottle and stored in a cool, dark igloo as a live sample until analysis. Each net sample was washed into a 230 ml opaque plastic bottle containing 3 ml of neutral Lugol’s iodine solution, using water in a spray bottle collected from each site. Preserved qualitative and quantitative plankton samples were stored in a cool dark area until analysis was possible.

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Copenhagen University, using the following procedures. Lugol’s preserved net samples of the bloom were filtered directly on to a Nuclepore polycarbonate filter by gravity filtration using a Swinnex filterholder. Gravity filtration was used to thoroughly wash the filtered samples with distilled water in order to get rid of salt in the samples. After washing, samples were chemically dehydrated using acidified 2,2-dimethoxypropane (DMP) (three drops 1N HCl to 25 ml DMP) for a 10 min period. This dehydration process was repeated and followed by critical point drying in which the dehydration liquid in the samples was replaced by liquid carbon dioxide, which was then vaporized. The dried material on the filters was transferred on to a double-sided adhesive carbon disc mounted on an SEM stub. The material was coated with a thin layer of gold– palladium applied by a sputtering process before viewing using a scanning electron microscope. Electron micrographs of the bloom cells were taken using a camera attached to the microscope.

2.4. Laboratory procedures 2.4.1. Preliminary identification and enumeration procedures On return to the lab sub-samples of the live and preserved planktonic net samples were observed on a microscope slide using a Leitz Wetzlar Dialux 20 EB compound microscope (model no. 020-452.008), in an attempt to identify the dominant species of the bloom. Quantitative determination of the species constituting the bloom was conducted using the Utermo¨hl method (Utermo¨hl, 1958). Lugol’s preserved quantitative samples were gently homogenised by inversion and a 5 ml aliquot of each sample was made up to 10 ml with filtered seawater in a 10 ml settling chamber. The chambers were left to stand overnight to allow settling of the phytoplankton cells before examination. Settled samples were examined using a Leitz Labovert (model no. 020-435.025) inverted microscope. Thirty random fields of view of each settled sample were examined to remove the edge effect in the settling of phytoplankton cells (Sandgren and Robinson, 1984). The phytoplankton in these thirty fields were identified and enumerated at 320 and then each settled sample was fully scanned at 100 to ensure that no phytoplankton species was overlooked. 2.4.2. Confirmatory identification and microscopy procedures 2.4.2.1. Scanning electron microscopy. Cells for electron microscopy were processed at the Electron Microscopy Laboratory of the Botanical Institute,

2.4.2.2. The squash technique. The squashing method (Steidinger, 1979) was used to separate the thecal plates of the bloom cells, allowing each plate to be observed and used to aid in confirming the identity of the species. A drop of a net sample from the bloom was placed on a microscope slide and covered with a coverslip, gently removing any excess seawater with a small piece of blotting paper. The sample was observed using a Leitz Wetzlar Dialux 20 EB compound microscope and a Canon Power Shot G6 digital camera was used to photograph the intact cells at 100 and 400 in an attempt to determine the identity of the cells based on size, shape and general morphological features. A drop of 5% sodium hypochlorite (commercial bleach) solution (1:1 mixture of sodium hypochlorite and distilled water) was placed along a margin of the coverslip and allowed to run under and across by placing a small piece of blotting paper under the opposite coverslip margin. A drop of neutral Lugol’s solution was added in a similar manner in order to stain the thecal plates. The cells were located under the microscope and squashed by applying firm but gentle pressure on the coverslip using a dissecting needle. Individual thecal plates were identified with the aid of descriptive and taxonomic references, with the principal references being Balech (1989, 1995). Thecal plates were viewed at 400 and 1000 with the aid of immersion oil and photographs of these plates were taken to show their morphological characteristics and aid in the confirmation of the identity of the dinoflagellate cells.

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3. Results and discussion 3.1. Qualitative and quantitative analyses The phytoplankton bloom that occurred in August 1994 in Hunts Bay was bright orange in colour and concentrated in the fresh to brackish water surface layer of the bay. The bloom was accompanied by a large kill of small pelagic fish belonging to the clupeid and carangid groups, which extended across approximately 2 km2 of the bay and average salinity, temperature and dissolved oxygen concentrations of 11 ppt, 26.5 8C, and 6.50 mg l1, respectively. Qualitative analysis of the phytoplankton in the bloom samples revealed that the samples were dominated by a dinoflagellate belonging to the genus Alexandrium, which occurred along with a few other dinoflagellate and flagellate species. Preliminary examinations of the Alexandrium cells led to the subsequent identification of the species as A. minutum. Quantitative analyses found that the highest cell concentration of this species in the bloom was 4.6  105 cells l1, a concentration which far exceeds acceptable concentrations of PSP-toxin producing A. minutum in several countries. In the Balearic Islands of Spain, cell concentrations reaching 103 cells l1 result in intensified monitoring and closure of shellfishery areas (Anderson, 1996) while in Denmark, a concentration as low as 500 cells l1 is considered to be the maximum concentration for closing or imposing special restrictions on shellfisheries (Anderson, 1996). 3.2. Species description and identification The genus Alexandrium is notably homogenous with the exception of a few species and lacks conspicuous elements, frequent in other genera such as apical horns and spines, which help to distinguish among species (Balech, 1995). It is therefore necessary to conduct extremely detailed observations in order to identify Alexandrium cells to the species level, a process not necessary for most dinoflagellates. Confirmation of the identity of A. minutum cells in the red tide samples was therefore based on examination of the general cell morphology including characteristics such as size and shape and more importantly, thecal plate pattern of intact cells and the morphology of individual thecal plates of squashed cells. 3.2.1. General morphology Cells of this species were solitary and small in size, a little longer than wide (Fig. 3A), with lengths ranging

from 18 to 31.5 mm (mean length  S.D. = 23.6  2.11 mm) and widths ranging from 15.3 to 23.2 mm (mean width  S.D. = 18.55  1.52 mm). These dimensions are similar to those recorded by Balech (1989), who reported thecae of the species ranging in length from 17 to 29 mm, with most ranging from 21 to 26 mm. Balech (1995) reported that the width of A. minutum equals the length, sometimes being larger but, more often, somewhat smaller, as observed for the Jamaican specimens. Size is a secondary characteristic, as evidenced by the large variations within certain species, however some species such as A. minutum are differentiated by their normally small size (Balech, 1995), as observed for the Jamaican specimens. Shape is an important taxonomic character although it can be considerably altered by environmental conditions, sexual reproductive stages such as zygotes, small changes in position of the specimen, coverslip pressure and adherence of the specimen to the glass (Balech, 1995). The shape of the cells matched descriptions put forward by Balech (1989, 1995) and varied from irregularly oval to elliptical in dorsal view, with roughly equal epitheca and hypotheca in length (Fig. 3B). The epitheca varied in shape from hemielliptical to almost hemispherical in both ventral and dorsal views (Fig. 3B and C), while the shape of the hypotheca ranged from hemielliptical in dorsal view (Fig. 3B) to hemielliptical, with oblique antapical flattening created by the sulcus, in ventral view (Fig. 3C). No spines, or horns were present and plate ornamentation in the form of fairly strong, coarse, irregular reticulations were observed on the hypotheca (Fig. 3D and E). The epitheca lacked any obvious form of ornamentation and was therefore smooth in comparison to the hypotheca (Fig. 3D). Balech (1995) described A. minutum as typically having the beginning of irregular sculpture rather than the distinct, coarse reticulations observed in the Jamaican specimens, but the degree of reticulation was variable, possibly due to external factors such as environmental conditions. He reported strong and true reticulation in the hypotheca and barely perceptible reticulation in the epitheca of Italian specimens of A. minutum from the Gulf of Naples, which was similar to that observed in the Jamaican specimens. Montresor et al., 1990 also described similar heavy reticulation of hypothecal plates in specimens from the Tyrrhenian Sea. Each cell had a median cingulum that was deeply excavated, left-handed, without lists and descending one cingular width (c in Fig. 3F). The cingulum met a

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Fig. 3. General morphology of Alexandrium minutum: (A) Light micrograph (LM) of neutral Lugol’s iodine preserved cells; (B) LM of dorsal view of cell; (C) LM of ventral view of cell; (D) LM of bleached/Lugol’s iodine stained cell showing smooth epithecal plates and reticulated hypothecal plates; (E) SEM view of cell showing reticulated hypotheca and short projections of the sulcal lists (sl); (F) LM of ventral view of cell showing lefthanded, descending cingulum (c) meeting a wide sulcus (s). Scale bars = 20 mm.

wide sulcus with narrow lists on the ventral side of the cell (s in Fig. 3F). These lists were difficult to detect, however some cells when viewed at particular angles showed short projections of the lists beyond the antapex (sl in Fig. 3E), which according to Balech (1995) sometimes appear as false spines.

3.2.2. Thecal plate morphology The plate formula of the Jamaican specimens of A. minutum was determined to be Po, 40 , 600 , 6C, 5000 , 20000 and 8S. This formula was based on visible plates and does not include two very small accessory plates (1 mm long or shorter) that occur in the sulcus and are very

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Fig. 4. Line diagrams showing thecal plate morphology of Jamaican specimens of Alexandrium minutum: (A) ventral view of cell showing the location of the sulcal plates: anterior sulcal plate (S.a.), posterior sulcal plate (S.p.), left anterior sulcal plate (S.s.a.), right anterior sulcal plate (S.d.a.), left posterior sulcal plate (S.s.p.), right posterior sulcal plate (S.d.p.), median anterior sulcal plate (S.m.a.), and median posterior sulcal plate (S.m.p.); (B) apical view of cell showing epithecal plates; (C) antapical view of cell showing hypothecal and some sulcal plates. Scale bar = 10 mm.

difficult to detect (Balech, 1995). Balech (1989, 1995) located at least one of the two accessory plates and therefore reported the formula of A. minutum as Po, 40 , 600 , 6C, 5000 , 20000 and 9–10S. These accessory plates were possibly present, but were not located in the Jamaican specimens, hence the formula Po, 40 , 600 , 6C, 5000 , 20000 and 8S, which is the same as that put forward by Giacobbe and Maimone (1994) who were also unable to detect both accessory plates in specimens of A. minutum from a Mediterranean lagoon. The thecal plates of the Jamaican specimens were typically divided into epithecal plates, hypothecal plates, sulcal plates and cingular plates (Fig. 4A). As the plate formula suggests, the epithecal plates consisted of one apical pore plate (Po), four apical plates (40 ) and six precingular plates (600 ) separated from the hypothecal plates by six cingular plates (6C) lacking much detail (Fig. 4A and B). The hypothecal plates

consisted of five postcingular plates (5000 ) and two antapical plates (20000 ) (Fig. 4C). Eight visible sulcal plates (8S) composed the sulcus located on the ventral side of the cells (Fig. 4A). 3.2.2.1. The epithecal plates. The apical pore plate (Po): the apical pore plate (Po) was approximately 4.8 mm long, teardrop shaped and located at the anterior most point of the epitheca (Fig. 4A) where it was surrounded by four apical plates (10 –40 ) (Figs. 4B and 5A). This plate was convex on the left, concave to straight on the right and tapered ventrally (Fig. 5C and D). The dorsal margin of this plate varied from flattened to slightly convex, while the ventral margin was obliquely truncated in some cells and somewhat pointed in others (Fig. 5C and D). Each apical pore plate had a comma-shaped apical pore or cavity called the foramen (f in Fig. 5C and D).

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Fig. 5. Thecal plate morphology of Alexandrium minutum: (A) LM showing an apical pore plate (Po) surrounded by four apical plates, an indirect connection between the first apical plate 10 and Po via a thread-like extension (arrowhead) and the position of the ventral pore (arrow); (B) LM of a portion of the epitheca showing indirect connection between the 10 and Po (black arrow); (C) LM of Po showing position of the callus (c) and foramen (f); (D) SEM showing structure of the Po including; the callus (c), foramen (f), canopy (d), marginal pores (m) and a direct connection (dc) between the 10 and Po via a short anterior border; (Ea–d) LM showing variations in the morphology of the 10 and different positions of the ventral pore (arrows). Scale bars = 5 mm.

Scanning electron microscopy revealed that in some cells, the foramen was partially covered by a canopy as indicated by (d) in Fig. 5D. On the ventral half of the right margin of the foramen was a thickened area forming a poorly developed callus which is known to contribute to support of the canopy (c in Fig. 5C and D). No anterior attachment pore was present and a number of small marginal pores were observed surrounding the foramen (m in Fig. 5D). Although the Po is rather similar for most Alexandrium species, some have characters that can be used to differentiate amongst the species (Fukuyo, 1985). The Po of the Jamaican specimens had the characters typical of A. minutum described by Balech (1995), particularly the comma-

shaped foramen, poorly developed callus and absence of an anterior attachment pore. The apical plates: four apical plates surrounded the apical pore plate (Figs. 4B and 5A). The first apical plate (10 ) (Figs. 4B and 5A and B) has the most taxonomic value because of its visible and distinguishing characteristics (Balech, 1995) and therefore was used as a taxonomic criterion in the identification of A. minutum in this study. The first apical plate (10 ) was rhomboidal however variations in this shape were observed as shown in Fig. 5Ea–d. The right posterior side of the plate was straight to slightly concave, slanted up to the right anterior side of the plate (Fig. 5Ea–d) and articulated

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with the sixth precingular plate (Figs. 4B and 5A and B). The right anterior side of the 10 was in contact with the fourth apical plate (40 ) (Figs. 4B and 5A and B) and was slightly concave in some cases (Fig. 5Ea), straight in others (Fig. 5Eb) and angular in a few cells (Fig. 5Ec). All 10 had a small ventral pore which in most cells was situated close to the posterior extreme of the anterior right margin, on the suture between the first and fourth apical plates as reported by Balech (1995) (black arrow in Fig. 5A and black arrow in Fig. 5Ea). The presence of the ventral pore is considered to be another important taxonomic characteristic in the identification of A. minutum (Balech, 1995). A small number of cells had the ventral pore located close to the middle of the suture as shown in Fig. 5Eb. A similar observation was made in Vietnamese specimens of A. minutum (Yoshida et al., 2000).

The left posterior side of the 10 was in contact with the first precingular plate (100 ) (Fig. 5B) and varied from straight to gently convex (Fig. 5Ea and b). The left anterior side varied from slightly concave to straight and slanted up to the right (Fig. 5Ea–d). The posterior end of the first apical plate was connected to the anterior sulcal plate (S.a.) as shown in Fig. 6A and was usually truncated in most cells (Fig. 5Ea), while a few cells had a somewhat pointed or conical posterior end (Fig. 5Eb). The most important characteristic of the first apical plate is its position in relation to the Po: disconnected or connected (Balech, 1995). All 10 of the Jamaican specimens were observed to be connected to the Po either indirectly or directly. The anterior end of the 10 in the majority of cells was drawn out into a short to long thread-like extension (Fig. 5Ea–c), which indirectly connected the plate to the ventral margin of the apical

Fig. 6. Thecal plate morphology of Alexandrium minutum: (A) LM of section of the epitheca showing the connection between the first apical plate (10 ) and the anterior sulcal plate (S.a.) and position and morphology of the sixth precingular plate (600 ); (B) LM showing the eight visible plates of the sulcus; (Ca–f) LM showing variations in the morphology of the posterior sulcal plate (S.p.). Scale bars = 10 mm.

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pore plate as indicated by the black arrowhead in Fig. 5A and black arrow in Fig. 5B. Some 10 lacked the extension altogether (Fig. 5Ed) and were directly connected to the apical pore plate by a very short anterior border as indicated by the arrow (dc) in Fig. 5D. Balech (1989) similarly reported both direct and more often indirect connection between the Po and 10 of his A. minutum specimens. Characteristics of the remaining apical plates were of little taxonomic value with the exception of the third apical plate (30 ) for which symmetry or asymmetry has some differential value (Balech, 1995). The 30 of the Jamaican specimens was six sided and appeared to be almost symmetrical as shown in Figs. 4B and 5A. These characters were typical of the 30 of A. minutum cells described by Balech (1989). The precingular plates: surrounding the apical plates and immediately above the cingulum were six precingular plates known as the first, second, third, fourth, fifth and sixth precingulars (100 –600 , respectively) (Fig. 4A and B). The most characteristic and taxonomically valuable of these plates is the sixth precingular (600 ) (Fig. 6A) which can be used to some extent to differentiate between Alexandrium species (Balech, 1995). In the Jamaican specimens, the 600 was small and narrow—longer than wide in comparison to the other precingular plates, with a length/width ratio of about 2:1, typical of A. minutum species, as described by Balech (1989). The right posterior margin of the 600 was straight to slightly convex and articulated with the 500 (Fig. 6A). The right anterior margin was straight to concave and articulated with the fourth apical plate (Fig. 6A). The left posterior margin was arched or slightly concave, to allow for attachment of the convex right margin of the anterior sulcal plate (S.a.) (Fig. 6A), while the left anterior margin was straight or slightly convex to allow for articulation of the 10 . 3.2.2.2. The sulcal plates. All sulcal plates have taxonomic value (Balech, 1995). Each cell had a large ventrally located sulcus which widened slightly posteriorly (Fig. 3F) and has been reported as being composed of ten plates, eight of which were relatively visible (Figs. 4A and 6B) in the Jamaican specimens and two which are very small and were not observed during this study. The eight visible sulcal plates are shown in Fig. 6B and were composed of one anterior sulcal plate (S.a.) and one posterior sulcal plate (S.p.). Two lateral pairs of plates were present with one pair being posterior and composed of the left and right posterior sulcal plates (S.s.p. and S.d.p.). The other pair was anterior

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and composed of the left and right anterior sulcal plates (S.s.a and S.d.a.). Two small median plates known as the median posterior and anterior sulcal plates (S.m.p. and S.m.a.) occurred between the two anterior lateral sulcal plates, one above the other. Of these sulcal plates the most distinctive characters are found in the posterior and anterior sulcal plates (Fukuyo, 1985). The posterior sulcal plate (S.p.): the posterior sulcal plate was somewhat variable in the length/width ratio, usually being wider than long (Fig. 6Ca), however some cells had a S.p. with a length approximately equal to the width as shown in Fig. 6Cb. Most S.p. were somewhat symmetrical with an irregularly thickened anterior margin composed of gently concave right and left regions, which met in the middle of the margin (Fig. 6Ca). In some cells, the left region of the anterior margin was almost straight (Fig. 6Cf) and the right region of the margin in some cases extended down into the plate creating a small indentation as shown in Fig. 6Ca and b. Both the left and right sides of the S.p. varied from straight to slanting to gently convex, with the left side usually shorter than the right side (Fig. 6Ca–f). The posterior margin was generally convex, slanting down toward the left and then curving up towards the anterior margin (Fig. 6Ca–f). The observed variations in the morphology of the S.p. are considered to be important characteristics in the identification of A. minutum and were similar to variations reported by Balech (1989), who particularly noted variation in the length/width ratio and shape of the anterior edge. The surface of the S.p. was irregularly reticulated as was typical of the hypothecal plates and this reticulation varied in pattern from cell to cell (Fig. 6Ca–d and f). Some S.p. had less reticulation and a stria running near the periphery of the left, right and posterior margins as shown in Fig. 6Ce. No attachment pore was present on the S.p. as also reported by both Yuki (1994) and Yoshida et al. (2000) for A. minutum cells from Japan and Vietnam, respectively. The anterior margin of the S.p. articulated with two smaller lateral sulcal plates known as the right and left posterior sulcal plates (S.d.p. and S.s.p.) (Figs. 6B and 7A). The right posterior sulcal plate (S.d.p.): the right posterior sulcal plate (S.d.p.) was longer than wide and had a generally rectangular shape which varied from cell to cell (Fig. 7Ba–e), but fit previous descriptions of this plate by Balech (1989, 1995). The posterior margin of the S.d.p. articulated with the right region of the anterior margin of the posterior sulcal plate (Fig. 7A) and varied from straight to irregularly convex but most

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Fig. 7. Thecal plate morphology of Alexandrium minutum: (A) LM of portion of the sulcus showing the posterior sulcal plate (S.p.), right posterior sulcal plate (S.d.p.) and left posterior sulcal plate (S.s.p.); (Ba–e) LM showing variations in the morphology of the right posterior sulcal plate; (Ca–c) LM showing variations in the morphology of the left posterior sulcal plate. Scale bars = 5 mm.

often was observed to slant to the left and anteriorly (Fig. 7Ba–e). The anterior margin also varied from straight as shown in Fig. 7Bc and d, to slanting gently down towards the left (Fig. 7Ba and b). The external margin of this plate was irregularly convex and often interrupted by small grooves or indentations (Fig. 7Bd). The internal margin varied from straight to gently convex, also interrupted by grooves in some cells as shown in Fig. 7Bd and e and sometimes with a small indentation just before meeting the anterior margin (Fig. 7Bd and e).

The left posterior sulcal plate (S.s.p.): the left posterior sulcal plate (S.s.p.) had an asymmetrical rectangular to square-like shape and was relatively short with a width almost equal to the length in most cases, however some plates were slightly longer than wide. The posterior margin of this plate articulated with the left region of the anterior margin of the S.p. (Fig. 7A) and varied from straight or slightly convex to slanting to the right and anteriorly (Fig. 7Ca–c). The external margin of the S.s.p. was somewhat straight to slightly convex, while the internal margin varied from straight to

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Fig. 8. Thecal plate morphology of Alexandrium minutum: (A) LM showing the articulation of the right anterior sulcal plate (S.d.a.) with the right posterior sulcal plate (S.d.p.); (Ba–b) LM showing variations in the morphology of the right anterior sulcal plate; (C) LM showing the articulation of the left anterior sulcal plate (S.s.a.) with the left posterior sulcal plate (S.s.p.); (D) LM showing a variation in the morphology of the left anterior sulcal plate. Scale bars = 5 mm.

slightly concave (Fig. 7Ca–c). The anterior margin of the plate in most cells had a straight left side and a right side, which curved down to the right as shown in Fig. 7Ca and b. In a few cases the left side of the plate was not straight, but curved down to the right as shown in Fig. 7Cc. These slight variations in morphology matched the descriptions and drawings of this plate by Balech (1989, 1995). The right anterior sulcal plate (S.d.a.): attached to the anterior margin of the S.d.p. was another sulcal plate known as the right anterior sulcal plate (S.d.a) (Figs. 6B and 8A). This plate was triangular in shape, which varied somewhat from cell to cell as shown in Fig. 8Ba and b. The S.d.a. was usually longer than wide, with a straight and horizontal posterior margin (Fig. 8Ba and b). The right side of the plate was in some cells slightly concave (Fig. 8Ba) while in others it was deeply concave as shown in Fig. 8A and Bb. The edge of the left anterior side was thickened and in

most cases strongly curved towards the right and posteriorly (Fig. 8A and Bb). Like the left posterior sulcal plate, variations in the morphology of this plate were minor and fit descriptions presented by Balech (1989, 1995). The left anterior sulcal plate (S.s.a.): attached to the left side of the anterior margin of the S.s.p. was the left anterior sulcal plate (S.s.a.) (Fig. 8C). This plate was longer than wide and somewhat rhomboidal in shape, with a wide posterior region and narrow, pointed anterior region (Fig. 8C and D). The posterior margin of the S.s.a. had a relatively straight right side which articulated with the left posterior sulcal plate (S.s.p.) as shown in Fig. 8C and a left side (sometimes shorter or longer than the right side) inclined up towards the left (white arrowheads in Fig. 8C and D). The internal margin of the plate was slightly concave and vertical in some cells as shown in Fig. 8C, while in others this side was inclined to the right (Fig. 8D). The external anterior

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Fig. 9. Thecal plate morphology of Alexandrium minutum: (A) LM showing the articulation of the median posterior sulcal plate (S.m.p.) with the left posterior sulcal plate (S.s.p.); (B) LM showing the connection between the S.m.p. (arrowhead) and the left anterior sulcal plate (S.s.a.); (C) LM showing the morphology of the S.m.p.; (D) LM showing the morphology of the median anterior sulcal plate (S.m.a.); (Ea–e) LM showing variations in the morphology of the anterior sulcal plate and the position of the unciform apophysis (ua). Scale bars = 5 mm.

side was gently curved towards the right, ending in a tapered, pointed anterior region (Fig. 8C and D). Variation in the morphology of this sulcal plate was the least when compared to that of the other sulcal plates, however observations matched those of Balech (1995). The median posterior and anterior sulcal plates (S.m.p. and S.m.a.): between the left and right anterior sulcal plates were two small, narrow median plates

known as the median posterior and median anterior sulcal plates (S.m.p. and S.m.a., respectively) (Fig. 6B). The S.m.p. was longer than wide and somewhat pentagonal in shape (Fig. 9C). The posterior margin of this plate was straight to slightly convex and inclined up towards the left and articulated with the right anterior margin of the S.s.p. (Fig. 9A). The external margin of the S.m.p. was slightly concave and connected to the

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Fig. 10. Thecal plate morphology of Alexandrium minutum: (A) LM showing the hypothecal plates; (Ba–d) LM showing variations in the morphology of the first postcingular plate (1000 ); (Ca–d) LM showing variations in the morphology of the fifth postcingular plate (5000 ). Scale bars = 5 mm.

left anterior sulcal plate (S.s.a.) (Fig. 9B). The internal margin started straight and slanted up to the right, followed by another straight side which was inclined to the left as shown in Fig. 9C. The anterior margin of this plate was also straight and horizontal, articulating with the posterior margin of the median anterior sulcal plate (Fig. 4A). The median anterior sulcal plate (S.m.a.) like the S.m.p, was narrow and elongated but smaller than the S.m.p. (Fig. 9D). The posterior margin of the S.m.a. was straight where the plate joined the S.m.p. The posterior internal margin of the plate curved up to the right forming a rounded anterior region as shown in Fig. 9D which articulated with the unciform apophysis of the anterior sulcal plate (Figs. 4A and 9Ed). The anterior external margin of the plate was also straight and almost

vertical and followed by a slightly concave side that was inclined up to the right (Fig. 9D). Location and identification of these two median plates was difficult as they were fairly small and hard to locate in an intact sulcus. The morphology of these plates does however fit that of previous descriptions (Balech, 1995). The anterior sulcal plate (S.a.): the morphology of the eighth plate of the sulcus, the anterior sulcal plate (S.a.) (Fig. 9Ea–e) is very important in the identification of A. minutum and according to Balech the characteristics of this plate have greater taxonomic value than those of the posterior sulcal plate. The anterior margin of the S.a. did not indent the epitheca (Fig. 4A) and varied from straight to oblique to slightly convex (Fig. 9Ea–e) and articulated with the

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Fig. 11. Thecal plate morphology of Alexandrium minutum: (Aa–f) LM showing variations in the morphology of the first antapical plate (10000 ). Scale bars = 5 mm.

first apical plate (Fig. 6A) and the first antapical plate (Fig. 4a). The posterior margin was interrupted by a fairly round, deep posterior sinus, giving rise to two posterior arms or branches. The left branch was narrow with a posterior margin, which articulated with the left anterior sulcal plate, and a left margin which varied from straight to slanting, to slightly concave or convex (Fig. 9Ea–e) and articulated with one of the plates of the cingulum. The right branch also known as the unciform apophysis (ua in Fig. 9Ed), was thinner than or equal to the left branch in width, with a straight, slanting or slightly convex margin (Fig. 9Ea–e). This unciform apophysis articulated with the rounded anterior region of the median anterior sulcal plate, which almost closed the posterior sinus of the anterior sulcal plate. The posterior and anterior accessory sulcal plates (S.ac.p. and S.ac.a.): the posterior and anterior accessory sulcal plates observed in previous studies of A. minutum (Balech, 1989, 1995; Yuki, 1994) were not observed during this study. This could suggest the lack of these plates in the Jamaican specimens, however

their extremely small size of 1 mm long or shorter (Balech, 1989), could have resulted in them being overlooked, if present, and hence the presence or absence of these two accessory plates in the Jamaican specimens is inconclusive. 3.2.2.3. The hypothecal plates. The postcingular plates: immediately below the cingulum were the five postcingular plates of the hypotheca known as the first, second, third, fourth and fifth precingular plates (1000 – 5000 , respectively) (Fig. 4A and C). Of these five plates the fourth postcingular (4000 ) was the largest, as well as the largest of the hypothecal plates (Fig. 10A). The most characteristic postcingular plates that aided with the identification of the species were the first (1000 ) and fifth (5000 ) postcingular plates (Figs. 4C and 10A and Ba–Cd) which both matched the description of these plates provided by Balech (1989, 1995). The 1000 was short and somewhat elongated or wide, with a relatively straight posterior margin slanted down towards the left (Fig. 10Ba–d). The anterior margin

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Fig. 12. Thecal plate morphology of Alexandrium minutum: (Aa–e) LM showing variations in the morphology of the second antapical plate (20000 ). Scale bars = 10 mm.

varied from gently convex to straight to slightly concave in some cases (Fig. 10Ba–d). The external margin varied from straight to slightly convex while the internal margin slanted to the right, up towards the anterior margin, giving rise to a pointed triangular region (Fig. 10Ba–d). This internal margin was reinforced and articulated with the left anterior sulcal plate (Fig. 4A). The 5000 was higher than the first, similarly wide, with reticulations which varied from strong to weak (Fig. 10Ca–d). The posterior margin articulated with the posterior sulcal plate (Fig. 4C) and was observed to be short, inclined up to the left and varied from straight to jagged (Fig. 10Ca–d). The external margin varied from straight to convex and slanted up towards the right as shown in Fig. 10Ca–d. The anterior margin of the fifth postcingular was gently convex in most cells and straight in some as shown in Fig. 10Cb. The internal margin of this plate also showed variations from straight to gently concave to jagged and somewhat angular (Fig. 10Ca–d). This side was also reinforced where the plate articulated with the right posterior sulcal plate. The antapical plates: the final plates of the hypotheca were the two plates known as the first and second antapical plates (10000 and 20000 , respectively) (Figs. 4C and 10A). Of all the hypothecal plates the two antapical plates provide the most valuable information with regards to the identification of the A. minutum

species (Balech, 1995) and like the previous hypothecal plates matched the descriptions of Balech (1989, 1995). The first antapical 10000 was pentagonal with a relatively straight to slightly convex posterior margin (Fig. 11Aa–f). The left margin was generally angular, slanting up to the left then back towards the right (Fig. 11Aa–f). The right margin was reinforced where it articulated with the posterior and left posterior sulcal plate (Fig. 4C) and in most cases started out straight and vertical then became gently concave towards the anterior margin (Fig. 11Aa–d). In some cells the right margin of the 10000 remained more or less straight (Fig. 11Af) or inclined to the left approaching the anterior margin (Fig. 11Ae). The anterior margin was very short and straight (Fig. 11Aa–f) and articulated with the left anterior sulcal plate (Fig. 4A). Reticulation of the 10000 varied from strong (Fig. 11Aa–e) to very weak as shown in Fig. 11Af. The second antapical 20000 was somewhat pentagonal and varied from being longer than wide to wider than long, with strong reticulation in most cases (Fig. 12Aa–e). The posterior margin was straight to gently curved to the left (Fig. 12Aa–e) and connected to the third postcingular 3000 (Fig. 4C). The right side was convex and connected to the fourth postcingular 4000 (Fig. 4C) and varied from short to long (Fig. 12Aa–e). The left side slanted up to the left, was gently convex,

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connected to the second postcingular 2000 (Fig. 4C) and also varied from short to long (Fig. 12Aa–e). The left side of the anterior margin was straight, slanting to the right and connected to the first antapical 10000 (Figs. 4C and 12Aa–e). This side was followed by a concave right side, which connected, to the posterior sulcal plate (Figs. 4C and 12Aa–e). 4. Conclusion The reported observations on the morphology and specifically the plate patterns of the Jamaican specimens of A. minutum were in good agreement with classical descriptions of A. minutum presented by Balech (1989, 1995) and do not present substantial differences in plate tabulation and morphology. A. minutum is characterized by its small size, the rhomboidal first apical 10 which is directly or, more often, indirectly connected to the comma shaped Po, a ventral pore which is situated in the posterior half of the right upper side of 10 , the narrow S.a. as long as wide or longer than wide, the short, wider than long S.p., the narrow precingular 600 and the constant lack of anterior and posterior attachment pores (Balech, 1995). These are all characters, which are defined as typical of A. minutum and are used to distinguish this species from other species of Alexandrium, and were characters possessed by the Jamaican specimens. These specimens showed some slight variations from the typical description of the species, however these are not considered to be significant. A distinctly reticulated hypotheca as seen in the Jamaican specimens is not considered to be typical (Balech, 1995), but A. minutum cells from Italy also had strong reticulations suggesting high variability in the appearance of the hypothecal surface possibly due to environmental conditions (Balech, 1995). The ventral pore of the Jamaican specimens was not always near the posterior margin of the 10 , but sometimes in the middle. Such a variation was also described by Yoshida et al. (2000) for A. minutum specimens from Vietnam. To date no studies of the toxic potential of the Jamaican specimens of A. minutum have been conducted, however all Alexandrium species should be regarded as potentially toxic until proven otherwise. The fact that a massive fish kill accompanied the bloom suggests the possibility that the Jamaican specimens of A. minutum are toxic. The fish kill may have been as a result of oxygen depletion during the bloom, however the possibility exists that death could have occurred due to close contact with, or direct ingestion of paralytic shellfish toxin producing A. minutum cells, or ingestion of zooplankton which had

fed on the cells (White, 1981). The relationships between fish kills, paralytic shellfish toxins and the marine food web have been reviewed by White (1984). Marine fish are in general very sensitive to paralytic shellfish toxins although some species can accumulate and retain the toxins without being affected (Geraci et al., 1989). With potentially toxic A. minutum being a consistent and sometimes dominant member of the phytoplankton community of Hunts Bay, and with concentrations far exceeding the acceptable limits in other countries, the continual use of this bay as a source of food is cause for great concern. Fishermen of surrounding communities fish for finfish, crabs, shrimp and shellfish in Hunts Bay everyday, including during red tide occurrences. Bait fish caught every morning in the bay are used to catch larger fish out at sea, compounding the potential for toxic events. There are no known cases of PSP in Jamaica, but the potential for its occurrence is ever present as long as A. minutum continues to occur and bloom in Hunts Bay and possibly other Jamaican coastal waters that are used as food sources by humans and other animals. PSP is appearing in regions where it has never been known (Kao, 1993) and Jamaica is certainly one of these regions. With no historical data on this species and lack of expertise on PSP events, such an occurrence could have detrimental effects on commercial fisheries, public health and the economies of local areas in the country. Acknowledgements We gratefully acknowledge the IOC-DANIDA (Intergovernmental Oceanographic Commission, Danish Agency for International Development Aid) Science and Communication Centre on Harmful Algae for funding this study and providing literature and training at workshops on the identification of harmful marine microalgae at the University of Copenhagen. Thanks to staff of the Botanical Institute, University of Copenhagen for their assistance with the electron microscope observations and photography. Thanks also to The Department of Life Sciences, University of the West Indies, Mona Campus in Jamaica, for use of inverted and compound microscopes and the Port Royal Marine Laboratory in Jamaica for providing boat transport and accommodation during the field work phase of both the 1994 and 2004 exercises.[SS] References Anderson, P., 1996. Design and implementation of some harmful algal monitoring systems. In: IOC Technical Series No. 44. UNESCO, Paris 102pp.

E.R. Ranston et al. / Harmful Algae 6 (2007) 29–47 Balech, E., 1989. Redescription of Alexandrium minutum Halim (dinophyceae) type species of the genus Alexandrium. Phycologia 28, 206–211. Balech, E., 1995. The Genus Alexandrium Halim (Dinoflagellata). Sherkin Island Marine Station, Sherkin Island Co., Cork, Ireland, 151 pp. Belin, C., 1993. Distribution of Dinophysis spp. and Alexandrium minutum along French coasts since 1984 and their DSP and PSP toxicity levels. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Phytoplankton Blooms in the Sea. Elsevier, Amsterdam, pp. 469–474. Chang, F.H., Anderson, D.M., Kulis, D.M., Till, D.G., 1997. Toxin production of Alexandrium minutum (Dinophyceae) from the Bay of Plenty, New Zealand. Toxicon 35, 393–409. Erard-Le Denn, E., 1991. Recent occurrence of red tide dinoflagellate Alexandrium minutum Halim from the north western coasts of France. In: Park, J.S., Kim, H.J. (Eds.), Recent Approaches on Red Tides. Department of Oceanography and Marine Resources, National Fisheries Research & Development Agency, Republic of Korea, pp. 85–98. Fukuyo, Y., 1985. Morphology of Protogonyaulax tamarensis (Lebour) Taylor and Protogonyaulax catenella (Whedon and Kofoid) Taylor from Japanese waters. Bull. Mar. Sci. 37, 529–537. Geraci, J.A., Anderson, D.M., Timperi, R.J., St. Aubin, D.J., Early, G.A., Prescott, J.A., Mayo, C.A., 1989. Humpback whales (Megaptera novaeangliae) fatally poisoned by dinoflagellate toxin. Can. J. Fish. Aquat. Sci. 46, 1895–1898. Giacobbe, M.G., Maimone, G., 1994. First report of Alexandrium minutum Halim in a Mediterranean lagoon. Cryptogamie, Algol 15, 47–52. Giacobbe, M.G., Oliva, F.D., Maimone, G., 1996. Environmental factors and seasonal occurrence of the dinoflagellate Alexandrium minutum, a PSP potential producer, in a Mediterranean lagoon. Est. Coast. Shelf Sci. 42, 539–549. Goodbody, I.M., 1970. The biology of Kingston harbour. J. Sci. Res. Counc., Jamaica 1, 10–34. Halim, Y., 1960. Alexandrium minutum nov. g. nov. sp. dinoflagellate provocant des ‘‘eaux rouges’’ Vie et Milieu 11, 102–105. Hallegraeff, G.M., Steffensen, D.A., Wetherbee, R., 1988. Three estuarine Australian dinoflagellates that can produce paralytic shellfish toxins. J. Plankton Res. 10, 533–541. Hallegraeff, G.M., Bolch, S.I., Blackburn, S.I., Oshima, Y., 1991. Species of the toxigenic dinoflagellate genus Alexandrium in South-eastern Australian waters. Botanica Marina 34, 575–587. Kao, C.Y., 1993. Paralytic shellfish poisoning. In: Falconer, I.R. (Ed.), Algal Toxins in Seafood and Drinking Water. Academic Press, London, pp. 75–86. Lam, C.W.Y., Yip, S.S.Y., 1990. A three-month red tide event in Hong Kong. In: Graneli, E., Sundstro¨m, B., Edler, L., Anderson, D.M. (Eds.), Toxic Marine Phytoplankton: Proceedings of the Fourth International Conference on Toxic Marine Phytoplankton, Lund, Sweden, June 26–30. Elsevier, New York, pp. 481–486. Montresor, M., Marino, D., Zingone, A., Dafnis, G., 1990. Three Alexandrium species from coastal Tyrrhenian waters (Mediterra-

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nean Sea). In: Graneli, E., Sundstro¨m, B., Edler, L., Anderson, D.M. (Eds.), Toxic Marine Phytoplankton: Proceedings of the Fourth International Conference on Toxic Marine Phytoplankton, Lund, Sweden, June 26–30. Elsevier, New York, pp. 82–87. Ranston, E.R., 1998. The Phytoplankton Community and Water Quality of a Highly Eutrophic Bay: Hunts Bay, Kingston Harbour, Jamaica. MPhil. Thesis. University of the West Indies, Mona, Jamaica, 249 pp. Ranston, E.R., Webber, D.F., 2003. Phytoplankton distribution in a highly eutrophic estuarine bay, Hunts Bay, Kingston Harbour, Jamaica. Bull. Mar. Sci. 73, 307–324. Sandgren, C.D., Robinson, J.V., 1984. A stratified sampling approach to compensating for non-random sedimentation of phytoplankton cells in inverted microscope settling chambers. Br. Phycol. J. 19, 67–72. Simmonds, R.A., 1997. The Phytoplankton Community and Water Quality of a Eutrophic Embayment; Kingston Harbor, Jamaica. MPhil. Thesis. University of the West Indies, Mona, Jamaica, 214 pp. STATIN, 2000. Demographic Statistics 2000. Statistical Institute of Jamaica, STATIN Press, Kingston, 63 pp. Steidinger, K.A., 1979. Collection, enumeration and identification, of free-living dinoflagellates. In: Taylor, D.L., Selinger, H.H. (Eds.), Toxic Dinoflagellate Blooms. Elsevier, North Holland, Inc., pp. 435–442. Steidinger, K., Babcock, C., Mahmoudi, B., Tomas, C., Truby, E., 1989. Conservative taxonomic characters in toxic dinoflagellate species identification. In: Okaichi, T., Anderson, D.M., Nemoto, T. (Eds.), Red Tides: Biology, Environmental Science and Toxicology. Elsevier, Amsterdam, pp. 285–288. Steven, D.M., 1966. Characteristics of a red water bloom in Kingston Harbour, Jamaica, West Indies. J. Mar. Res. 24, 113–123. Utermo¨hl, H., 1958. Zur vervollkommung der qualitativen phytoplankton methodisk. Mitteilungen Internationale Veroingung fur Theoretische und Sgeurandt Limnologie 9, 1–38. Wade, B.A., 1971. Marine pollution problems in Jamaica. Mar. Poll. Bull. 2, 1. Wade, B.A., 1976. The Pollution Ecology of Kingston Harbour, Jamaica. Volume 1. Scientific Report of the U.W.I./Kingston Harbour Research Project, 142 pp. Wade, B.A., Antonio, L., Mahon, R., 1972. Increasing organic pollution in Kingston Harbour. Mar. Poll. Bull. 3, 106–110. White, A.W., 1981. Marine zooplankton can accumulate and retain dinoflagellate toxins and cause fish kills. Limnol. Oceanogr. 26, 103–109. White, A.W., 1984. Paralytic shellfish toxins and finfish. In: Ragelis, E.P. (Ed.), Seafood Toxins, ACS Symposium Series No. 262, ACS Washington, DC, pp. 171–180. Yoshida, M., Ogata, T., Van, T.C., Matsuoka, K., Fukuyo, Y., Hoi, N.C., Kodama, M., 2000. The first finding of toxic dinoflagellate Alexandrium minutum in Vietnam. Fish. Sci. 66, 177–179. Yuki, K., 1994. First report of Alexandrium minutum Halim (Dinophyceae) from Japan. Jpn. J. Phycol. 42, 425–430.