Ciguatera Fish Poisoning (CFP) is a global food safety issue caused by the consumption of reef fish contaminated with ciguatoxins (CTXs) and possibly maitotoxins (MTXs) [1
]. Between 25,000 and 50,000 people from South Pacific communities are affected annually, and epidemiological studies indicate that ≤20% of actual cases are reported (www.ciguatera-online.com
). The poisoning is considered a neglected disease world-wide, and there is an urgent need for research to improve monitoring of CTXs to aid the understanding and management of the syndrome. To stimulate research activities into CFP, the Intergovernmental Oceanographic Commission of UNESCO’s Harmful Algal Bloom programme (http://hab.ioc unesco.org
) has developed an ‘IOC/IPHAB Global Ciguatera Strategy 2015–2019’.
CFP occurs throughout the tropical and sub-tropical waters of the South Pacific Ocean and affects many of the indigenous populations that inhabit the islands, both populated and remote [3
]. It is caused by CTX- and MTX- producing dinoflagellate species in the genus Gambierdiscus
Adachi & Fukuyo and Fukuyoa
Gómez, Qiu, Lopes & Lin [7
]. The perception is that CTXs linked to CFP are bio-magnified and bio-transformed up the food chain, to the higher trophic omnivorous and carnivorous reef fish species often targeted by both commercial and recreational fishers [8
]. This bio-transformation converts the algal-derived toxins into more toxic forms, creating a complex suite of compounds. CTXs are large, extremely lipophilic, ladder-shaped polyether marine toxins that are odourless, tasteless, heat stable, lipid soluble, and resistant to gastric degradation. While MTX is among the most potent marine toxins identified to date, it has been primarily found in the digestive tract of fish rather than bio-accumulating in the flesh. The oral potency is much lower than the i.p. toxicity, which suggests it may only play a role in CFP cases if these tissues are consumed. Intoxication manifests as a wide array of gastrointestinal, neurological, and/or cardiovascular symptoms. While fatalities are uncommon, there is no reliable treatment or antidote, and therefore chronic illness cases provide most of the data for epidemiological assessments [10
may be found living epiphytically on macroalgae and corals, or attached to the benthos, and are globally distributed in tropical to warm-temperate environments [11
]. Global warming has resulted in an expansion of the sub-tropical latitudes and subsequently the habitable regions for Gambierdiscus
are expanding [13
]. These now include the New South Wales (NSW) coastline, Australia [14
], and the northern tip of New Zealand, although Gambierdiscus/Fukuyoa
species isolated in New Zealand so far have been non-toxic [15
]. If climate change and the associated warming seas continue to rise, the habitable range may encompass more of both New Zealand’s and Australia’s coastline, with potentially negative impacts. The Pacific region is being impacted by increasing temperatures, largely caused by carbon emissions generated in the northern hemisphere; low lying atolls are being particularly hard hit. The temperature trends are well documented in a New South Wales Government (Australia) release (http://climatechange.environment.nsw.gov.au/About-climate-change-in-NSW/Evidence-of-climate-change/Observed-Australian-climate-change
), and a New Zealand government report [17
In order to provide information on the risk that Gambierdiscus
) species pose to consumers of potentially contaminated reef fish, microalgae isolates have been collected from the Cook Islands, Kermadec Islands, New Zealand, and New South Wales, Australia (Table 1
), and their toxicity assessed. The assessment includes the newly described species, G. cheloniae
] and G. honu
] (both of which are maintained in the Cawthron Institute Culture Collection of Micro-algae (CICCM)). These organisms have been cultured; extracts of the cultures have been analysed for CTXs and MTX-1 and -3, and they have been examined for acute toxic effects in mice.
The extracts of all the Gambierdiscus and Fukuyoa species induced anorexia in mice, both by intraperitoneal injection and gavage, and the macroscopic changes observed in the animals at necropsy were confined to the gastrointestinal tract. It is possible that the extracts inhibited the normal passage of food through the gastrointestinal tract. This would be consistent with the observation that significant amounts of food were present in the stomachs of the anorexic mice, even though they had eaten little or no food for up to 3 days. The presence of gas in the stomach of these animals could possibly be due to the fermentation of their stomach contents. Inhibition of intestinal peristalsis would also explain the presence of material in the duodenum and upper jejunum (which are almost empty in normal animals), and the excessive amount of food-derived material in the caeca of some test animals. Similarly, the presence of hard, dry pellets in the large intestine of anorexic mice could reflect an unusually high degree of water absorption from pre-faecal material, due to prolonged residence time in the intestine.
The acute toxicities of all but one of the extracts by gavage were much lower than those by intraperitoneal injection (Table 3
). The differences in toxicity were particularly pronounced with extracts of G. pacificus
, G. honu
, and G. cheloniae
, and rather less with extracts of G. australes
. Because of the limited amounts of the extracts of the single samples of G. carpenteri
and F. paulensis
), only a limited amount of testing was possible. No effects were seen after oral administration of these extracts at 16 or 63 times the lethal dose by intraperitoneal injection, and it is likely that the ratios between the two parameters are considerably higher than this. The relative toxicity of G. polynesiensis
(CAWD 212) was very different to that of the other extracts, being only 1.7 times less toxic by gavage than by intraperitoneal injection. This observation is consistent with the unique presence of ciguatoxins in this extract. Since the initial publication regarding the production of CTXs by CAWD 212 [22
], the respective profile and concentration of CTXs per cell has changed significantly. However, as has been presented in this manuscript, the overall toxicity of the algal extract remains high. This suggests that other compounds also contribute to the observed toxicity of the extract. The exact cause for the profile change has yet to be determined; however, publications documenting the effect of epiphytic allopathic bacteria on the growth and toxin production of Gambierdiscus
may provide the starting point [23
]. Studies with purified ciguatoxins have shown that median lethal doses by gavage are similar to those by intraperitoneal injection [25
]. Furthermore, although the extract of G. polynesiensis
induced the same gastrointestinal changes as the other extracts, it was the only one to cause hypersalivation, which is a characteristic symptom of intoxication by ciguatoxins [26
There was no association between levels of MTX-3 and acute toxicity. The extreme example is G. cheloniae CAWD 232, which contained only 0.4 peak area/cell of MTX-3, but was more toxic than G. pacificus CAWD 227, which contained 18 peak area/cells, suggesting that CAWD 232 contains a toxin or toxins other than those quantified in the present study.
In a recent in vitro study [27
], extracts of 13 Gambierdiscus
strains were examined for the presence of CTXs using a neuroblastoma 2a cytotoxicity test, and for MTXs using an erythrocyte lysis test. The results differed from the results by LC-MS/MS analyses presented in this study, as extracts of the G. australis
strains analysed by the bioassays suggested low CTXs as well as MTXs. The presence of MTXs was indicated in a strain of G. carpenteri
from Hawaii, and MTXs have also been detected in G. carpenteri
in strains from Australia, the Cook Islands, and French Polynesia (Dr. Tim Harwood, Cawthron Institute, unpubl. data) (although, analyses carried out in this study showed no MTX-3 in the isolate from New South Wales, Australia). The in vitro assays indicated the highest concentration of CTXs in a strain of G. excentricus
. In vivo toxicity studies with extracts of this organism would be of interest.
Because of the likely spread of Gambierdiscus from beyond their present range, it is important to assess which, if any, species are likely to cause adverse effects in humans if taken up by seafood. In this study, we have examined extracts of 17 isolates (from 6 Gambierdiscus and 1 Fukuyoa species) for their acute toxic effects in mice, by intraperitoneal injection and by gavage. All the extracts were of similar toxicity; however, all but one was considerably less toxic by oral administration (the most relevant route of administration in this situation), since this will be the route by which humans will be exposed to the toxins contained in these organisms. The relatively low oral toxicity of extracts of G. pacificus, G. honu, G. cheloniae, G. carpenteri, and F. paulensis suggests that these species may be of less concern than G. polynesiensis, which was highly toxic by oral administration.
All the isolates of G. australes
contained MTX-1, but only G. polynesiensis
produced ciguatoxins (P-CTX-3B, P-CTX-3C, P-CTX-4A, and P-CTX-4B), with P-CTX-3B representing the dominant analogue (approximately 65% of Total CTX). All isolates, except G. carpenteri,
produced MTX-3. Monitoring for Gambierdiscus
species is difficult, due to morphological similarities between most species under the light microscope. Therefore, molecular tools are likely to be the way forward for the differentiation of toxic from non-toxic species in sea water samples, and work continues on understanding and targeting the toxin gene [7
The results of this study suggest that the MTX(s) present in the Gambierdiscus and Fukuyoa species that were examined are of relatively low oral toxicity, and work is in progress to isolate and purify these MTXs in order to facilitate detailed toxicological examination.
In the far north of New Zealand, where Gambierdiscus
has been reported, and where the related F. paulensis
], a watching brief will be kept in order to determine whether the risk of ciguatera fish poisoning increases with warming seas.