Evolutionary distinctiveness of fatty acid and polyketide synthesis in eukaryotes

© 2016 International Society for Microbial Ecology All rights reserved. Fatty acids, which are essential cell membrane constituents and fuel storage molecules, are thought to share a common evolutionary origin with polyketide toxins in eukaryotes. While fatty acids are primary metabolic products, polyketide toxins are secondary metabolites that are involved in ecologically relevant processes, such as chemical defence, and produce the adverse effects of harmful algal blooms. Selection pressures on such compounds may be different, resulting in differing evolutionary histories. Surprisingly, some studies of dinoflagellates have suggested that the same enzymes may catalyse these processes. Here we show the presence and evolutionary distinctiveness of genes encoding six key enzymes essential for fatty acid production in 13 eukaryotic lineages for which no previous sequence data were available (alveolates: dinoflagellates, Vitrella, Chromera; stramenopiles: bolidophytes, chrysophytes, pelagophytes, raphidophytes, dictyochophytes, pinguiophytes, xanthophytes; Rhizaria: chlorarachniophytes, haplosporida; euglenids) and 8 other lineages (apicomplexans, bacillariophytes, synurophytes, cryptophytes, haptophytes, chlorophyceans, prasinophytes, trebouxiophytes). The phylogeny of fatty acid synthase genes reflects the evolutionary history of the organism, indicating selection to maintain conserved functionality. In contrast, polyketide synthase gene families are highly expanded in dinoflagellates and haptophytes, suggesting relaxed constraints in their evolutionary history, while completely absent from some protist lineages. This demonstrates a vast potential for the production of bioactive polyketide compounds in some lineages of microbial eukaryotes, indicating that the evolution of these compounds may have played an important role in their ecological success

Evaluation of seafood toxicity in the Australes archipelago (French Polynesia) using the neuroblastoma cell-based assay

Ciguatera fish poisoning (CFP), a disease caused by consuming fish that have accumulated ciguatoxins (CTXs) in their tissue, is regarded as the most prevalent form of intoxication in French Polynesia. Recently, the Australes, one of the least affected archipelago until the early 1980s, has shown a dramatic increase in its incidence rates in 2009 with unusual CFP cases. In the present work, potential health hazards associated with the proliferation of various marine phytoplankton species and the consumption of fish and marine invertebrates highly popular among local population were assessed in three Australes islands: Raivavae, Rurutu and Rapa. Extracts from the marine dinoflagellates Gambierdiscus, Ostreospis and mat-forming cyanobacteria as well as fish, giant clams and sea urchin samples were examined for the presence of CTXs and palytoxin (PLTX) by using the neuroblastoma cell-based assay (CBA-N2a). Cytotoxic responses observed with both standards (Pacific CTX-3C and PLTX) and targeted marine products indicate that CBA-N2a is a robust screening tool, with high sensitivity and good repeatability and reproducibility. In Rurutu and Raivavae islands, our main findings concern the presence of CTX-like compounds in giant clams and sea urchins, suggesting a second bio-accumulation route for CFP toxins in the ciguatera food chain. In Rapa, the potential CFP risk from Gambierdiscus bloom and fish was confirmed for the first time, with levels of CTXs found above the consumer advisory level of 0.01ng Pacific CTX-1Bg(-1) of flesh in three fish samples. However, despite the presence of trace level of PLTX in Ostreopsis natural assemblages of Rapa, no sign of PLTX accumulation is yet observed in tested fish samples. Because this multi-toxinic context is likely to emerge in most French Polynesian islands, CBA-N2a shows great potential for future applications in the algal- and toxin-based field monitoring programmes currently on hand locally

Use of folk tests to detect ciguateric fish : a scientific evaluation of their effectiveness in Raivavae Island (Australes, French Polynesia)

Ciguatera fish poisoning is a seafood intoxication commonly afflicting island communities in the Pacific. These populations, which are strongly dependent on fish resources, have developed over centuries various strategies to decrease the risk of intoxication, including the use of folk tests to detect ciguateric fish. This study aims to evaluate the effectiveness of two folk tests commonly used in Raivavae Island (Australes, French Polynesia): the rigor mortis test (RMT) and the bleeding test (BT). A total of 107 fish were collected in Raivavae Lagoon, among which 80 were tested by five testers using the RMT versus 107 tested by four testers using BT. First, the performance between testers was compared. Second, the efficiency of these tests was compared with toxicity data obtained via the receptor binding assay (RBA) by assessing various parameter's values such as sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV). Comparisons of outcomes between folk tests and RBA analyses were considered: tests used separately or in a parallel versus the series approach by each tester. The overall efficiency of the RMT and BT tests was also evaluated when the judgments of all testers were pooled. The results demonstrate that efficiencies varied between testers with one showing the best scores in detecting toxic fish: 55% with RMT and 69.2% with BT. BT gave the best results in detecting toxic fish as compared with RMT, giving also better agreement between testers. If high NPV and Se values were to be privileged, the data also suggest that the best way to limit cases of intoxication would be to use RMT and BT tests in a parallel approach. The use of traditional knowledge and a good knowledge of risky versus healthy fishing areas may help reduce the risk of intoxication among communities where ciguatera fish poisoning is highly prevalent

Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs