Evaluation of Toxicity Equivalency Factors of Tetrodotoxin Analogues with a Neuro-2a Cell-Based Assay and Application to Puffer Fish from Greece.

Tetrodotoxin (TTX) is a potent marine neurotoxin involved in poisoning cases, especially through the consumption of puffer fish. Knowledge of the toxicity equivalency factors (TEFs) of TTX analogues is crucial in monitoring programs to estimate the toxicity of samples analyzed with instrumental analysis methods. In this work, TTX analogues were isolated from the liver of a Lagocephalus sceleratus individual caught on South Crete coasts. A cell-based assay (CBA) for TTXs was optimized and applied to the establishment of the TEFs of 5,11-dideoxyTTX, 11-norTTX-6(S)-ol, 11-deoxyTTX and 5,6,11-trideoxyTTX. Results showed that all TTX analogues were less toxic than the parent TTX, their TEFs being in the range of 0.75-0.011. Then, different tissues of three Lagocephalus sceleratus individuals were analyzed with CBA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained TEFs were applied to the TTX analogues' concentrations obtained by LC-MS/MS analysis, providing an indication of the overall toxicity of the sample. Information about the TEFs of TTX analogues is valuable for food safety control, allowing the estimation of the risk of fish products to consumers.

Ciguatoxin-like toxicity distribution in flesh of amberjack (Seriola spp.) and dusky grouper (Epinephelus marginatus).

Ciguatoxins (CTXs) are marine neurotoxins that cause ciguatera poisoning (CP), mainly through the consumption of fish. The distribution of CTXs in fish is known to be unequal. Studies have shown that viscera accumulate more toxins than muscle, but little has been conducted on toxicity distribution in the flesh, which is the main edible part of fish, and the caudal muscle is also most commonly targeted for the monitoring of CTXs in the Canary Islands. At present, whether this sample is representative of the toxicity of an individual is undisclosed. This study aims to assess the distribution of CTXs in fish, considering different muscle samples, the liver, and gonads. To this end, tissues from four amberjacks (Seriola spp.) and four dusky groupers (Epinephelus marginatus), over 16.5 kg and captured in the Canary Islands, were analyzed by neuroblastoma-2a cell-based assay. Flesh samples were collected from the extraocular region (EM), head (HM), and different areas from the fillet (A-D). In the amberjack, the EM was the most toxic muscle (1.510 CTX1B Eq·g-1), followed by far for the caudal section of the fillet (D) (0.906 CTX1B Eq·g-1). In the dusky grouper flesh samples, D and EM showed the highest toxicity (0.279 and 0.273 CTX1B Eq·g-1). In both species, HM was one of the least toxic samples (0.421 and 0.166 CTX1B Eq·g-1). The liver stood out for its high CTX concentration (3.643 and 2.718 CTX1B Eq·g-1), as were the gonads (1.620 and 0.992 CTX1B Eq·g-1). According to these results, the caudal muscle next to the tail is a reliable part for use in determining the toxicity of fish flesh to guarantee its safe consumption. Additionally, the analysis of the liver and gonads could provide further information on doubtful specimens, and be used for CTX monitoring in areas with an unknown prevalence of ciguatera.

Metabolic Hepatic Disorders Caused by Ciguatoxins in Goldfish (Carassius auratus).

Ciguatera poisoning (CP) is a foodborne disease known for centuries; however, little research has been conducted on the effects of ciguatoxins (CTXs) on fish metabolism. The main objective of this study was to assess different hepatic compounds observed in goldfish (Carassius auratus) fed C-CTX1 using nuclear magnetic resonance (NMR)-based metabolomics. Thirteen goldfish were treated with C-CTX1-enriched flesh and sampled on days 1, 8, 15, 29, 36, and 43. On day 43, two individuals, referred to as 'Detox', were isolated until days 102 and 121 to evaluate the possible recovery after returning to a commercial feed. At each sampling, hepatic tissue was weighed to calculate the hepatosomatic index (HSI) and analyzed for the metabolomics study; animals fed toxic flesh showed a higher HSI, even greater in the 'Detox' individuals. Furthermore, altered concentrations of alanine, lactate, taurine, glucose, and glycogen were observed in animals with the toxic diet. These disturbances could be related to an increase in ammonium ion (NH4+) production. An increase in ammonia (NH3) concentration in water was observed in the aquarium where the fish ingested toxic meat compared to the non-toxic aquarium. All these changes may be rationalized by the relationship between CTXs and the glucose-alanine cycle.

Ciguatoxin Detection in Flesh and Liver of Relevant Fish Species from the Canary Islands.

The Canary Islands are a ciguatoxin (CTX) hotspot with an established official monitoring for the detection of CTX in fish flesh from the authorised points of first sale. Fish caught by recreational fishermen are not officially tested and the consumption of toxic viscera or flesh could lead to ciguatera poisoning (CP). The objectives of this study were to determine the presence of CTX-like toxicity in relevant species from this archipelago, compare CTX levels in liver and flesh and examine possible factors involved in their toxicity. Sixty amberjack (Seriola spp.), 27 dusky grouper (Epinephelus marginatus), 11 black moray eels (Muraena helena) and 11 common two-banded seabream (Diplodus vulgaris) were analysed by cell-based assay (CBA) and Caribbean ciguatoxin-1 (C-CTX1) was detected by liquid chromatography mass spectrometry (LC-MS/MS) in all these species. Most of the liver displayed higher CTX levels than flesh and even individuals without detectable CTX in flesh exhibited hepatic toxicity. Black moray eels stand out for the large difference between CTX concentration in both tissues. None of the specimens with non-toxic liver showed toxicity in flesh. This is the first evidence of the presence of C-CTX1 in the common two-banded seabream and the first report of toxicity comparison between liver and muscle from relevant fish species captured in the Canary Islands.

Screening of New Potential Probiotics Strains against Photobacterium damselae Subsp. piscicida for Marine Aquaculture.

On intensive fish farms, 10% of the population dies exclusively from pathogens, and Photobacterium damselae subsp. Piscicida (Ph. damselae subsp. Piscicida), the bacteria causing pasteurellosis in marine aquaculture, is one of the major pathogens involved. The objective of this study was to obtain new probiotic strains against pasteurellosis in order to limit the use of chemotherapy, avoiding the environmental repercussions generated by the abusive use of these products. In this study, 122 strains were isolated from the gills and intestines of different marine fish species and were later evaluated in vitro to demonstrate the production of antagonistic effects, the production of antibacterial substances, adhesion and growth to mucus, resistance to bile and resistance to pH gradients, as well as its harmlessness and the dynamic of expression of immune-related genes by real-time PCR after administration of the potential probiotic in the fish diet. Only 1/122 strains showed excellent results to be considered as a potential probiotic strain and continue its characterization against Ph. damselae subsp. piscicida to determine its protective effect and elucidating in future studies its use as a possible probiotic strain for marine aquaculture.

Accumulation of C-CTX1 in Muscle Tissue of Goldfish (Carassius auratus) by Dietary Experience.

Ciguatoxins (CTXs) are produced by dinoflagellates usually present in tropical and subtropical waters. These toxins are bioaccumulated and transformed in fish causing ciguatera fish poisoning (CFP) in humans. Few trials have been performed to understand how CTXs are incorporated into fish. This study developed an experimental model of goldfish (Carassius auratus) fed flesh contaminated with Caribbean ciguatoxin (C-CTX1). Fourteen goldfish were fed 0.014 ng CTX1B (Eq. g-1 of body weight) daily, and control goldfish received non-toxic flesh. CTX presence was determined by a cell-based assay on days 1, 8, 15, 29, 36, 43, and 84. Toxicity was detected in muscle from the second sampling and then seemed to stabilize at ~0.03 ng CTX1B Eq. g-1. After two weeks, all experimental goldfish developed lethargy and loss of brightness, but only two of them displayed erratic swimming and jerking movements near the sixth sampling. One of these fish had its toxic diet replaced by commercial food for 60 more days; the fish showed recovery signs within the first weeks and no CTX activity was detected. These results indicate that C-CTX1 could accumulate in goldfish muscle tissue and produce toxic symptoms, but also remarked on the detoxification and recovery capacity of this species.

Presence of CTXs in moray eels and dusky groupers in the marine environment of the Canary Islands.

Local population frequently consumes moray eels and dusky groupers from the Canary Islands. These species are top predators and the interactions between them include predation but also, in some cases, collaborative hunting. These fish are well known to cause ciguatera (CFP) outbreaks in several marine areas such as Japan, Hawaii, French Polynesia and Caribe. Groupers have been involved in CFP events in the Canary Islands, however, moray eels have not yet been well studied in this regard. The present research seeks to describe the finding of a black moray in the stomach of a positive dusky grouper during its necropsy, and to clarify the implication of groupers and moray eels in the food webs, accumulating CTXs in the Canarian environment. The study also updates statistics on the presence of toxic groupers in this archipelago. For these purposes, 248 grouper samples from the CFP official control in the Canary Islands (2018-2019) were analysed and 36 moray eels (5 species) were collected under the EuroCigua project and one was obtained during a dusky grouper necropsy. All samples were analysed with the Neuro-2a cell-based assay (CBA) to evidence CTX-like toxicity. Regarding the necropsied grouper and the moray eel found in its stomach content, the LCMS/MS method allowed the identification and quantification of CCTX1 in both fish at similar levels while none of the P-CTXs for which standards were available were detected. Among groupers, 25.4 % displayed CTX-like toxicity with differences between islands. For moray eels 38.9 % showed toxicity, involving 4 species. Black moray exhibited a high proportion of positives (9/12) and a positive correlation was found between CTX-like toxicity quantification and the black moray weight. Regarding the grouper, and the moray eel found in its stomach, the LCMS/MS method allowed the identification and quantification of C-CTX1 in both fish at similar levels. This found suggests a trophic interaction between these species and their role in maintaining CTXs in the Canary waters where local population commonly demand those species for consumption. The island of El Hierro stands out above all the other Canary Islands with the concerning percentage of positive grouper samples and the high CTX toxicity levels obtained in moray eel specimens analysed in this marine area. This is the first report of CTX-like toxicity in flesh of moray eels fished in the Canary archipelago and the confirmation of the presence of C-CTX1 by LCMS/MS in a black moray from this marine area.