An Updated Review of Ciguatera Fish Poisoning: Clinical, Epidemiological, Environmental, and Public Health Management.

Ciguatera Fish Poisoning (CFP) is the most frequently reported seafood-toxin illness in the world. It causes substantial human health, social, and economic impacts. The illness produces a complex array of gastrointestinal, neurological and neuropsychological, and cardiovascular symptoms, which may last days, weeks, or months. This paper is a general review of CFP including the human health effects of exposure to ciguatoxins (CTXs), diagnosis, human pathophysiology of CFP, treatment, detection of CTXs in fish, epidemiology of the illness, global dimensions, prevention, future directions, and recommendations for clinicians and patients. It updates and expands upon the previous review of CFP published by Friedman et al. (2008) and addresses new insights and relevant emerging global themes such as climate and environmental change, international market issues, and socioeconomic impacts of CFP. It also provides a proposed universal case definition for CFP designed to account for the variability in symptom presentation across different geographic regions. Information that is important but unchanged since the previous review has been reiterated. This article is intended for a broad audience, including resource and fishery managers, commercial and recreational fishers, public health officials, medical professionals, and other interested parties.

qPCR assays for Alexandrium fundyense and A. ostenfeldii (Dinophyceae) identified from Alaskan waters and a review of species-specific Alexandrium molecular assays.

Paralytic shellfish poisoning (PSP) poses a serious health threat in Alaska and prevents effective utilization of shellfish resources by subsistence and recreational harvesters. Substantial economic losses also affect shellfish growers during PSP events. The toxins responsible for PSP are produced by dinoflagellates in the genus Alexandrium. Despite the persistent threat posed by PSP and the long history of shellfish toxicity research, there is still confusion concerning the Alexandrium species that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandrium species present in Alaska and to develop polymerase chain reaction (PCR) assays for use in screening phytoplankton and sediment samples. Before developing the PCR assays for this study, we evaluated published assays and many were not adequate because of primer dimer formation or because of cross-reactivity. Rather than continue to grapple with the uncertainty and inadequacy of published assays, we developed new assays for the Alexandrium species most likely to be present in Alaska. Only Alexandrium fundyense Group I and A. ostenfeldii were identified from four sampling regions from southeast Alaska to Kodiak Island, indicating that these two species are widely distributed. PCR assays for these two species were converted to quantitative (q)PCR format for use in monitoring programs. During the course of this study, we realized that a systematic evaluation of all published (~150) Alexandrium species-specific assays would be of benefit. Toward this objective, we collated published Alexandrium PCR, qPCR, and in situ hybridization assay primers and probes that targeted the small-subunit (SSU), internal transcribed spacer (ITS/5.8S), or D1-D3 large-subunit (LSU) (SSU/ITS/LSU) ribosomal DNA genes. Each individual primer or probe was screened against the GenBank database and Alexandrium gene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandrium assays that can be recommended for evaluation by the global harmful algal bloom community.

Paralytic shellfish poisoning toxins in butter clams (Saxidomus gigantea) from the Kodiak Archipelago, Alaska.

Consumption of toxic butter clams (Saxidomus gigantea) is the most frequent cause of paralytic shellfish poisoning (PSP) in Alaskan coastal communities. This study examines seasonal variation in total paralytic shellfish toxin concentrations and congener distribution in tissues of butter clams collected in three communities in the Kodiak Islands, Alaska: the City of Kodiak, Ouzinkie and Old Harbor. In response to questions from local harvesters, the efficacy of removing particular clam tissues on total toxin levels was also assessed. Butter clam samples were collected ∼monthly during 2015-2020 in each community to monitor shellfish toxin levels. Results were combined with clam monitoring data collected previously (2013-2015) to document the seasonal distribution of saxitoxin (STX) and its congeners (neosaxitoxin, gonyautoxin) in clam tissues. Seasonally, paralytic shellfish toxin levels in butter clams were highest in summer, declined in winter, but often remained above regulatory limits throughout the year in the three Kodiak communities. Butter clams collected from Ouzinkie (2013-2020) averaged 165 ± 87 µg STX equivalents (Eq.) 100 g - 1, compared to Kodiak 73 ± 54 µg STX Eq. 100 g - 1 and Old Harbor 143 ± 103 µg STX Eq. 100 g - 1. STX accounted for 59-71% of the total toxin concentration in clams at Ouzinkie, Kodiak, and Old Harbor, while neosaxitoxin (neoSTX) accounted for 12-18%. Gonyautoxins (GTXs) represented 31-60% of the total toxin concentration during the seasonal Alexandrium catenella bloom in June-July, with lower percentages in other months. The fraction of total toxin varied among clam tissues: the siphon tip (2-29%), the neck (3-56%), the gut (3-65%) and the body (6-85%). Removal of the siphon tip reduced total toxin content substantially in some samples but had little effect in others. Saxitoxin congeners varied greatly and somewhat unpredictably among clam tissues, and the results indicate removal of specific tissues was not an effective strategy for reducing paralytic shellfish toxin levels in butter clams for safe consumption.

Environmental factors influencing the distribution and abundance of Alexandrium catenella in Kachemak bay and lower cook inlet, Alaska.

Despite the long history of paralytic shellfish poisoning (PSP) events in Alaska, little is known about the seasonal distribution and abundance of the causative organism, Alexandrium, or the environmental factors that govern toxic bloom development. To address this issue, a five year study (2012-2017) was undertaken in Kachemak Bay and lower Cook Inlet Alaska to determine how the occurrence of Alexandrium catenella, the dominant PSP-causing Alexandrium species, was influenced by temperature, salinity, nutrient concentrations, and other environmental factors. Cell concentrations from 572 surface water samples were estimated using quantitative PCR. Monthly sampling revealed a seasonal pattern of A. catenella bloom development that was positively correlated with water temperature. Prevailing salinity conditions did not significantly affect abundance, nor was nutrient limitation a direct factor. Elevated cell concentrations were detected in 35 samples from Kachemak Bay (100-3050 cell eq. L-1) while a maximum abundance of 67 cell eq. L-1 was detected in samples from lower Cook Inlet sites. Monitoring data showed average water temperatures in Kachemak Bay increased by ∼2 °C over the course of the study and were accompanied by an increase in Alexandrium abundance. Based on these findings, 7-8 °C appears to represent a temperature threshold for significant bloom development in Kachemak Bay, with the greatest risk of shellfish toxicity occurring when temperatures exceed 10-12 °C. The role of temperature is further supported by time series data from the Alaska Coastal Current (station GAK1), which showed that summertime shellfish toxicity events in Kachemak Bay generally followed periods of anomalously high winter water temperatures. These data indicate monitoring changes in water temperatures may be used as an early warning signal for subsequent development of shellfish toxicity in Kachemak Bay.

Ciguatoxicity of Gambierdiscus and Fukuyoa species from the Caribbean and Gulf of Mexico.

Dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa produce ciguatoxins (CTXs), potent neurotoxins that concentrate in fish causing ciguatera fish poisoning (CFP) in humans. While the structures and toxicities of ciguatoxins isolated from fish in the Pacific and Caribbean are known, there are few data on the variation in toxicity between and among species of Gambierdiscus and Fukuyoa. Quantifying the differences in species-specific toxicity is especially important to developing an effective cell-based risk assessment strategy for CFP. This study analyzed the ciguatoxicity of 33 strains representing seven Gambierdiscus and one Fukuyoa species using a cell based Neuro-2a cytotoxicity assay. All strains were isolated from either the Caribbean or Gulf of Mexico. The average toxicity of each species was inversely proportional to growth rate, suggesting an evolutionary trade-off between an investment in growth versus the production of defensive compounds. While there is 2- to 27-fold variation in toxicity within species, there was a 1740-fold difference between the least and most toxic species. Consequently, production of CTX or CTX-like compounds is more dependent on the species present than on the random occurrence of high or low toxicity strains. Seven of the eight species tested (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, Gambierdiscus ribotype 2, G. silvae and F. ruetzleri) exhibited low toxicities, ranging from 0 to 24.5 fg CTX3C equivalents cell-1, relative to G. excentricus, which had a toxicity of 469 fg CTX3C eq. cell-1. Isolates of G. excentricus from other regions have shown similarly high toxicities. If the hypothesis that G. excentricus is the primary source of ciguatoxins in the Atlantic is confirmed, it should be possible to identify areas where CFP risk is greatest by monitoring only G. excentricus abundance using species-specific molecular assays.

Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay.

This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7-40% from log phase growth to late log - early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20-31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60-90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC-MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity.

DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.

Ciguatera fish poisoning and sea surface temperatures in the Caribbean Sea and the West Indies.

Ciguatera fish poisoning (CFP) is a circumtropical disease caused by ingestion of a variety of reef fish that bioaccumulate algal toxins. Distribution and abundance of the organisms that produce these toxins, chiefly dinoflagellates of the genus Gambierdiscus, are reported to correlate positively with water temperature. Consequently, there is growing concern that increasing temperatures associated with climate change could increase the incidence of CFP. This concern prompted experiments on the growth rates of six Gambierdiscus species at temperatures between 18 degrees C and 33 degrees C and the examination of sea surface temperatures in the Caribbean and West Indies for areas that could sustain rapid Gambierdiscus growth rates year-round. The thermal optimum for five of six Gambierdiscus species tested was >/=29 degrees C. Long-term SST data from the southern Gulf of Mexico indicate the number of days with sea surface temperatures >/=29 degrees C has nearly doubled (44 to 86) in the last three decades. To determine how the sea surface temperatures and Gambierdiscus growth data correlate with CFP incidences in the Caribbean, a literature review and a uniform, region-wide survey (1996-2006) of CFP cases were conducted. The highest CFP incidence rates were in the eastern Caribbean where water temperatures are warmest and least variable.

Characteristic profiles of Ciguatera toxins in different strains of Gambierdiscus spp.

Ciguatera fish poisoning characterizes the intoxication caused by consumption of fish from tropical and subtropical areas, which have accumulated ciguatoxins (CTXs). The observed pattern of ciguatoxins in fish highly depends on the marine region and the causative organisms. It is evident that differences exist between ciguatoxins produced by certain strains of the dinoflagellate Gambierdiscus toxicus and other Gambierdiscus spp. In this context cultured strains purchased from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) and strains from Vietnam were analyzed. Besides, lyophilized samples of several Gambierdiscus spp. from the National Oceanic and Atmospheric Administration (NOAA), USA and lyophilized samples of G. toxicus from Vietnam were analyzed. The latter has been cultured at different salinities. We observed differences between the toxin ratios of the analogues in the strain from Vietnam depending on the salinity. The CTX profiles of the Vietnamese samples were compared with cultures of Gambierdiscus spp. from CCMP and the National Oceanic and Atmospheric Administration (NOAA) resulting in an overview of toxins in cultures from different regions. Hence, it was obvious that the strain from Vietnam forms a characteristic CTX profile which is not directly comparable to CTX pattern observed in other tropical marine regions.

Global distribution of ciguatera causing dinoflagellates in the genus Gambierdiscus.

Dinoflagellates in the genus Gambierdiscus produce toxins that bioaccumulate in tropical and sub-tropical fishes causing ciguatera fish poisoning (CFP). Little is known about the diversity and distribution of Gambierdiscus species, the degree to which individual species vary in toxicity, and the role each plays in causing CFP. This paper presents the first global distribution of Gambierdiscus species. Phylogenetic analyses of the existing isolates indicate that five species are endemic to the Atlantic (including the Caribbean/West Indies and Gulf of Mexico), five are endemic to the tropical Pacific, and that two species, Gambierdiscus carpenteri and Gambierdiscus caribaeus are globally distributed. The differences in Gambierdiscus species composition in the Atlantic and Pacific correlated with structural differences in the ciguatoxins reported from Atlantic and Pacific fish. This correlation supports the hypothesis that Gambierdiscus species in each region produce different toxin suites. A literature survey indicated a >100-fold variation in toxicity among species compared with a 2 to 9-fold within species variation due to changing growth conditions. These observations suggest that CFP events are driven more by inherent differences in species toxicity than by environmental modulation. How variations in species toxicity may affect the development of an early warning system for CFP is discussed.

PROROCENTRUM LEVIS, A NEW BENTHIC SPECIES (DINOPHYCEAE) FROM A MANGROVE ISLAND, TWIN CAYS, BELIZE(1).

As part of a long-term study of benthic dinoflagellates from the Belizean barrier reef system, we report a new species: Prorocentrum levis M. A. Faust, Kibler, Vandersea, P. A. Tester et Litaker sp. nov. P. levis cells are oval in valve view and range in size from 40 to 44 µm long and 37 to 40 µm wide. Each valve surface is smooth, with 221-238 valve pores and 99-130 marginal pores. These pores are uniformly small and range in diameter from 0.13 to 0.19 µm. Asexual reproduction in P. levis is atypical, occurring within a hyaline envelope, and produces long branching chains of adherent cells. A phylogenetic analysis of SSU rDNA indicated that of the Prorocentrum species sequenced so far, P. levis was most closely related to P. concavum. P. levis produces okadaic acid and dinophysis toxin-2 (DTX2). Further, SEM observations and SSU rDNA sequence for P. belizeanum M. A. Faust, which was isolated at the same time, are also presented.