Ciguatera fish poisoning (CFP) is prevalent around the tropical and sub-tropical latitudes of the world and impacts many Pacific island communities intrinsically linked to the reef system for sustenance and trade. While the genus Gambierdiscus has been linked with CFP, it is commonly found on tropical reef systems in microalgal assemblages with other genera of toxin-producing, epiphytic and/or benthic dinoflagellates – Amphidinium, Coolia, Fukuyoa, Ostreopsis and Prorocentrum. Identifying a biomarker compound that can be used for the early detection of Gambierdiscus blooms, specifically in a mixed microalgal community, is paramount in enabling the development of management and mitigation strategies. Following on from the recent structural elucidation of 44-methylgambierone, its potential to contribute to CFP intoxication events and applicability as a biomarker compound for Gambierdiscus spp. was investigated. The acute toxicity of this secondary metabolite was determined by intraperitoneal injection using mice, which showed it to be of low toxicity, with an LD50between 20 and 38 mg kg−1. The production of 44-methylgambierone by 252 marine microalgal isolates consisting of 90 species from 32 genera across seven classes, was assessed by liquid chromatography-tandem mass spectrometry. It was discovered that the production of this secondary metabolite was ubiquitous to the eight Gambierdiscus species tested, however not all isolates of G. carpenteri, and some species/isolates of Coolia and Fukuyoa.
Ciguatera fish poisoning (CFP) is the most common non-microbial, food-borne illness in the world. It can be extremely debilitating, with symptoms potentially lasting years. Intoxications manifest as a wide array of symptoms including gastrointestinal discomfort (e.g. nausea and diarrhoea), neurological impairment (e.g. parathesia and dysaesthesia) and/or cardiovascular complications (e.g. hypotension and bradycardia) (Friedman et al., 2017; Diogene, 2018). The syndrome is prevalent in the circumtropical regions of the world, including areas of the Pacific Ocean, Indian Ocean, Caribbean Sea and the Gulf of Mexico (Friedman et al., 2017). While the existence of CFP has been known for centuries (Friedman et al., 2008), the true level of incidence is not known. It is estimated that 25,000–50,000 people are affected annually, with epidemiological studies indicating that < 20% of actual cases are reported (ILM, 2014).
Ciguatera fish poisoning is caused by the consumption of reef fish contaminated with ciguatoxins (CTXs) and possibly other compounds, including maitotoxins (MTXs). These compounds are produced by the epiphytic, benthic dinoflagellate genus Gambierdiscus and are some of the most potent non-peptide toxins known (Friedman et al., 2008). In addition to CTXs and MTXs (Rhodes et al., 2014), other bioactive, ladder-shaped polyether secondary metabolites such as gambieric acids (Morohashi et al., 2000), gambierol (Morohashi et al., 1999), gambieroxide (Watanabe et al., 2013) and gambierones (Murray et al., 2019) are produced by Gambierdiscus. However, the role that these compounds play in intoxication events is currently unknown.
Ciguatoxins have been demonstrated to bioaccumulate and biotransform into more toxic analogues as they move up the marine food chain, from herbivorous fish grazing on coral (e.g. parrot fish, Scaridae spp.) or macroalgae (e.g. mullet, Mugil cephalus) (Yasumoto et al., 1971, 1977; Ledreux et al., 2014; Clausing et al., 2018), to the higher trophic level omnivorous (e.g. wrasse, Cheilinus spp.) and carnivorous reef fish species that predate upon them (e.g. Spanish mackerel, Scomberomorus spp.) (Murray et al., 2016; Kohli et al., 2017). While CTXs have been shown to bioaccumulate in fish of all trophic levels, with published estimates of CFP vectors ranging from 60 (Gaboriau et al., 2014) to 90 (Kohli et al., 2015) to over 400 species (FAO, 2014), it is the carnivorous fish species that are most commonly implicated in CFP cases as they are often targeted by commercial and recreational fishers. As a result, carnivorous fish species are responsible for 68% of intoxication events in French Polynesia and 85% in New Caledonia (Caillaud et al., 2010). The role of the additional secondary metabolites produced by Gambierdiscus in intoxication events is currently unknown.
Ciguatera fish poisoning is particularly prolific throughout the tropical and sub-tropical waters of the South Pacific and affects many indigenous island communities intrinsically linked to the reef system for sustenance and trade. In addition, climate change is causing an increase in global ocean temperatures, resulting in an expansion of the sub-tropical latitudes (Rhodes et al., 2020). Consequently, the habitable regions for Gambierdiscus are expanding. These regions now include the New South Wales coastline (Australia), the Rangitāhua/Kermadec Islands (a New Zealand territory) and mainland Aotearoa/New Zealand (Rhodes and Smith, 2019). To date, only one Gambierdiscus species has been reported from New Zealand’s mainland and was reported from a single cell attached to floating macroalgae, Sargassum (Chang, 1996). No culturing was undertaken with the single cell and therefore no chemical analysis nor phylogenic investigation was performed. It is however noted that Pacific-CTX (P-CTX) producing strains of Gambierdiscus polynesiensishave been isolated from the Kermadec Islands (Rhodes et al., 2020).
Gambierdiscus is found attached to macroalgae (e.g. in the Pacific region it favours filamentous red and calcareous green species), coralline turfs, dead corals and volcanic sands around the world (Rhodes et al., 2017a). It is regarded as an opportunistic dinoflagellate that proliferates following damage to the reef system from tropical hurricanes, crown of thorn starfish outbreaks or coral bleaching events (Rongo and van Woesik, 2013; Xu et al., 2016). Adding to the complexity of CFP is that Gambierdiscus is commonly found co-habitating in assemblages with other toxin-producing benthic dinoflagellates from the genera Amphidinium, Coolia, Fukuyoa, Ostreopsisand Prorocentrum (Hachani et al., 2018; Yong et al., 2018; Rhodes and Smith, 2019). These genera of microalgae produce multiple toxic secondary metabolites including palytoxins (Ishii et al., 1997), okadaic acid (Malagoli et al., 2008), dinophysistoxins (Carmody et al., 1996) and amphidinols (Echigoya et al., 2005). While significant research has been conducted on these metabolites to determine their toxicity, they are currently not considered to play a role in CFP events.
The World Health Organisation considers the CFP syndrome a neglected disease worldwide and in 2015, to help promote research activities on CFP, the United Nations Educational, Scientific and Cultural Organisation (UNESCO) formulated a global research strategy (IOC/IPHAB Global Ciguatera Strategy 2015–2019) through its Intergovernmental Oceanographic Panel on Harmful Algal Blooms (IPHAB). This document highlighted several priority research areas, including the urgent need for improved monitoring capabilities for CTXs and MTXs, along with the development of tools for early warning/monitoring of toxic Gambierdiscusblooms (IOC/UNESCO, 2015). One such tool could be the identification of a biomarker compound(s) in the environment, as this would enable the development of better management and mitigation strategies.
Biomarker compounds have been used for epidemiological investigations for decades and in more recent years have been adopted as an early detection method for the planktonic paralytic shellfish poisoning dinoflagellate genus Alexandrium (Chan et al., 2006). However, identifying a biomarker for CFP is complicated as the causative CTXs are produced at very low levels, are analytically difficult to detect and their production has only been confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in one species, G. polynesiensis (Chinain et al., 2010; Rhodes et al., 2014). As a result, there is currently no biomarker compound that can be used to confirm CFP intoxication in humans nor has one been identified for the early detection of toxic Gambierdiscus blooms in the environment (Friedman et al., 2008). In an effort to develop biomarkers for CFP, progress has been made in animal studies using blood samples, however, these are not appropriate in human clinical scenarios (Friedman et al., 2017).
One secondary metabolite that is more easily detectable by LC-MS/MS than CTXs is 44-methylgambierone (Fig. 1). This compound has previously been reported as putative maitotoxin-3 (MTX-3) and was recently structurally characterised from G. australes(Murray et al., 2019) and G. belizeanus (Boente-Juncal et al., 2019). To better understand whether 44-methylgambierone plays a role in CFP intoxication events, its acute toxicity to mice was determined by intraperitoneal injection. To evaluate the potential of 44-methylgambierone to be used as a biomarker for the presence of Gambierdiscus spp. in areas of high CFP risk, this study screened its production in 252 microalgal isolates consisting of 90 species from 32 genera across seven classes using LC-MS/MS.
2. Materials and methods
2.1. Chemicals and reagents
High purity methanol (MeOH) and acetonitrile (MeCN) were obtained from Thermo-Fisher (Fisher-Optima). Purified water (18.2 MΩ) was produced with a Milli-Q system (Millipore, Canada). Ammonium hydroxide (≥ 25%) was from Honeywell Research Chemicals.
2.2. Acute toxicity of 44-methylgambierone by intraperitoneal injection
2.2.1. Quantitative nuclear magnetic resonance spectrometry
44-methylgambierone reference material was produced in-house from a cultured G. australesisolate (CAWD149) using the purification procedure described in Murray et al. (2019). The reference material was dissolved in 0.5 mL of deuterated methanol (CD3OD) and transferred to a 5 mm NMR tube. The millimole level of the reference sample, and subsequently the mg mL-1 concentration, of 44-methylgambierone in the reference solution was determined by quantitative nuclear magnetic resonance spectroscopy (QNMR) using a Bruker AVIII-HD 8000 MHz NMR spectrometer and electronic reference to assess in–vivo concentrations (ERETIC2) QNMR software (Wider and Dreier, 2006). External standard quantification was performed using a dioxane solution (1.58 mg mL−1 in CD3OD). Eight sets of triplicate ERETIC2 QNMR analyses were performed over several days using 90- or 30-degree pulses with 60- or 30 s pulse delays respectively (total n = 24). Quantification was performed using the integrated peak areas of the four methyl proton signals of 44-methylgambierone which occurred at 1.23 (s), 1.21 (s), 1.15 (s) and 1.02 (d) ppm (equivalent to 12 protons) and the dioxane proton signal at 3.68 ppm (equivalent to eight protons). The concentration of the 44-methylgambierone reference solution was determined to be 2.33 ± 0.02 mg mL−1, with a precision of 0.9% relative standard deviation.
Female Swiss albino mice (18–22 g) were bred at AgResearch, Ruakura, New Zealand. The mice were housed individually during the experiments and were allowed unrestricted access to food (Rat and Mouse Cubes, Speciality Feeds Ltd., Glen Forrest, Western Australia) and water. All experiments were approved by the Ruakura Animal Ethics Committee established under the Animal Protection (code of ethical conduct) Regulations Act, 1987 (New Zealand), Project Number 14,320, approval date 2 November 2017.
2.2.3. Toxicity assessment
Acute toxicity was determined using the principles of Organisation for Economic Co-operation and Development (OECD) guideline 425 (OECD, 2006). This guideline employs an up-and-down procedure whereby one animal is dosed and if it survives the dose for the next animal is increased, whereas if it dies, the dose for the next animal is decreased. To determine the LD50, dosing is continued until four live-death reversals have been achieved.
Toxicity was determined by intraperitoneal injection. Each mouse was weighed prior to dosing and the appropriate quantities of test compound calculated to yield the required dose on a mg kg−1 basis. The dose was prepared by taking the appropriate volume of stock solution (pure 44-methylgambierone in 90% aq. MeOH), drying it down under nitrogen and immediately re-dissolving in 1% Tween 60 in normal saline (1 mL) with the aid of sonication. This solution was injected into mice. All dosing was conducted between 8 and 9.30 am to avoid any diurnal variations in response. Mice were monitored intensively during the day of dosing and any that survived were monitored for a 14-day period which included a daily measurement of food consumption and bodyweight. After 14 days, the animals were euthanized by carbon dioxide inhalation and necropsied. The weights of the liver, kidneys, spleen, heart, lungs, stomach (full and empty) and the whole gut were measured and expressed as a percentage of bodyweight.
2.3. Screening of microalgal cultures for 44-methylgambierone production
2.3.1. Microalgal culturing and sample extraction
Microalgal isolates (252 in total) consisted of 90 species from 32 genera across seven classes. Depending on the specific nutritional requirements of each genus, cultures were grown in either 25% f/2, 33% f/2, f/2 (Guillard and Ryther, 1962), GP, 50% GP, K, modified K, L1 (Bigelow, 1985) or metals mix SWII medium (Matsuda et al., 1996; Nishimura et al., 2019) diluted in sterile seawater (autoclaved and filtered; 0.22-µm). Depending on the origin of the isolate, the cultures were grown at either 17 °C (± 2 °C; for temperate isolates) or 25 °C (± 2 °C; for sub-tropical and tropical isolates) with 40–70 µmol m−2 s−1photon irradiance and a 12:12 h light:dark cycle. Isolates were sourced from previous research expeditions; the Cawthron Institute Culture Collection of Microalgae (CICCM); or donated as frozen cell pellets by researchers from French Polynesia, Hong Kong, Spain and Australia. Cultures were harvested in the late exponential or stationary phase and contained at least 1 × 106 cells. The cells were harvested by centrifugation (3200 × g, 4 °C, 10 min), the growth medium decanted, and the resulting cell pellets frozen at −20 °C.
Each cell pellet was extracted twice with 90% aq. MeOH at a ratio of 1 mL per 2 × 105 cells using ultrasonication for 10 min in a 59 kHz water bath (model 160HT, Soniclean Pty, Australia). Cellular debris was pelleted by centrifugation (3200 × g, 4 °C, 5 min) and the supernatant was transferred to another vial before the cell pellets were re-extracted in the same manner. The resulting extract supernatants were pooled to give a final extract equivalent to approximately 1 × 105 cells mL−1. The combined extracts were stored at –20 °C for 24–48 h to precipitate insoluble matrix co-extractives, which were removed using centrifugation (3200 × g, 4 °C, 5 min) prior to analysis. An aliquot of the clarified extract was transferred into a 2 mL glass autosampler vial for analysis by LC-MS/MS using a modified version of the method described in Murray et al. (2018).
2.3.2. Liquid chromatography-tandem mass spectrometry conditions
Analysis was performed on a Waters Xevo TQ-S triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC i-Class with flow-through needle sample manager. Chromatographic separation used a Waters Acquity UPLC BEH phenyl column (1.7-μm, 100 × 2.1 mm column) held at 50 °C. The column was eluted at 0.55 mL min−1 with Milli-Q water (A) and acetonitrile (B) mobile phases, each containing 0.2% (v/v) of a 25% ammonium hydroxide solution. Fresh mobile phases were prepared daily to ensure optimal sensitivity and stable retention times. The initial solvent composition was 5% B with a linear gradient to 50% B from 0 − 2.5 min, ramped up to 95% B by 3 min and held at 95% B until 3.2 min, followed by a linear gradient back to 5% B at 3.5 min. The column was then re-equilibrated with 5% B until 4 min. The autosampler chamber was maintained at 10 °C and the injection volume was 1 μL. The mass spectrometer used an electrospray ionisation source operated in negative-ion mode. Other settings were: capillary voltage 3.0 kV, cone voltage 40 V, source temperature 150 °C, nitrogen gas desolvation flowrate 1000 L h−1 at 600 °C, cone gas 150 L h−1 and the collision cell was operated with 0.15 mL min−1 argon. 44-Methylgambierone was monitored using the following transitions: m/z 1037.6 > 96.8 (Channel 1) and 899.6 > 96.8 (Channel 2), with collision energies of 60 and 48 eV respectively, and using a dwell time of 30 ms.
Data acquisition and processing were performed with TargetLynx software (Waters, Milford, US). 44-Methylgambierone was identified in sample extracts based on the retention time (2.61 min) and a fragment ion ratio of 3:1 (Channel 1 / Channel 2; as determined using reference material). The isolates were analysed qualitatively yielding a result of presence/absence for 44-methylgambierone, with an ‘absent’ result equating to less than 2 ng mL−1 in an extract generated from a cell pellet of 1 × 105 cells mL−1, or 0.02 pg cell−1.
3.1. Acute toxicity of 44-methylgambierone by intraperitoneal injection
Single mice were dosed with 44-methylgambierone at dose rates of 0.89, 4.45, 20 and 38 mg kg−1. The mice dosed at 0.89, 4.45 and 20 mg kg−1 showed no adverse effects on the day of dosing and their movement, behaviour, appearance and respiration appeared normal throughout the 14 days of the study. At necropsy, none of these mice showed any abnormalities and the organ weights, as expressed as% of bodyweight, were all normal. On the day of dosing, the mouse dosed at 38 mg kg−1 also looked normal. However, over the first 24 h period, it was found to have eaten very little and although it was moving normally, its behaviour was slightly abnormal (hunched posture) by day two. At 48 h post-dosing, the mouse was alert and moving normally but it still had a very low food intake. At 56 h post-dosing, the mouse was hunched, and its breathing was laboured. To avoid long-term suffering, this animal was euthanised and necropsied in accord with the requirements of the OECD Humane Endpoints Guidance Document (OECD, 2000). Under OECD guideline 425, this euthanised animal can be considered in the same way as an animal which died during the test. The food intake and bodyweight data suggest that this mouse, dosed with 38 mg kg−1 of 44-methylgambierone, suffered from anorexia and the reduced hepatic and splenic weights observed were consistent with that diagnosis. Necropsy of this mouse showed that the stomach, caecum and intestines contained a dark green, runny material. Due to the limited availability of 44-methylgambierone, a full LD50 determination could not be completed. However, since the mouse dosed at 20 mg kg−1was healthy and death was induced at a dose rate of 38 mg kg−1, the LD50 of 44-methylgambierone sits between 20 and 38 mg kg−1.
3.2. 44-methylgambierone production by common co-habitating benthic microalgae
Isolates from the six main dinoflagellate genera that co-habitate in the Pacific region (Amphidinium, Coolia, Fukuyoa, Gambierdiscus, Ostreopsis and Prorocentrum; 190 isolates in total from 34 species) were collected during previous research expeditions to the Kermadec Islands (Raoul Island, North Meyer Island and Macauley Island), the Cook Islands and the Federated States of Micronesia, or cell pellets were received as gifts from researchers in French Polynesia, Hong Kong, Spain and Australia.
44-Methylgambierone production was ubiquitous to all Gambierdiscus species analysed from Australia, the Cook Islands, French Polynesia, the Kermadec Islands and Micronesia. These species included G. australes, G. caribaeus, G. carpenteri, G. cheloniae, G. honu, G. lapillus, G. pacificus and G. polynesiensis (total 78 isolates; Table 1). However, it is noted that for G. carpenteri, isolates from the Cook Islands and French Polynesia produced this secondary metabolite whereas G. carpenteri isolates from Australia did not (total 5 Australian isolates; Table 1).
Table 1. Summary of the 190 benthic dinoflagellate isolates from 34 species spanning 6 genera tested for 44-methylgambierone production.