Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land

Marine pufferfish (family Tetraodontidae) are believed to accumulate tetrodotoxin (TTX) mainly in liver and ovary through the food chain by ingesting TTX-bearing organisms such as starfish, gastropods, crustacean, flatworms, ribbonworms, etc. Consequently, it is hypothesized that non-toxic pufferfish can be produced if they are cultured with TTX-free diets in netcages at sea or aquaria on land, where the invasion of TTX-bearing organisms is completely shut off. To confirm this hypothesis, more than 5000 specimens of the pufferfish (“torafugu”, Takifugu rubripes) cultured in such manners for 1–3 years were collected from several locations in Japan during 2001–2004, and toxicity of their livers and some other parts was examined according to the Japanese official mouse assay method for TTX. In addition, typical specimens were submitted to LC/MS analysis. The results showed that all the livers and other parts tested were ‘non-toxic’ in both of the mouse assay (less than 2 MU/g) and LC/MS analysis (less than 0.1 MU/g). Thus, it is undoubtedly confirmed that pufferfish are intoxicated through the food chain, and non-toxic pufferfish can be successfully produced by netcage or land culture. The livers from these fish can be used with safety as a Japanese traditional food “fugu-kimo” (puffer liver).     

Detection of tetrodotoxin-producing Providencia rettgeri T892 in Lagocephalus pufferfish

Tetrodotoxin (TTX) is a potent toxin but it could be used in pharmaceutical field. Identification of TTX producing bacteria in pufferfish is necessary for TTX yield and the pufferfish conservation. In this study, Lagocephalus was collected from Cam Ranh Sea, a central part of Vietnam during spring season. The liver and intestine were incubated in 0.9 % NaCl for TTX detection in pufferfish. To be benefited from the isolation of new TTX producing bacteria, the liver and intestine were incubated in 6.5 % NaCl. The cultures were used to test the toxin and to isolate the bacterial community that could yield TTX. Surprisingly, Providencia rettgeri T892 in intestine could produce TTX identified by biochemical test and 16S rRNA sequencing. This strain was used to test the production of TTX, based on thin layer chromatography (TLC), mouse bioassay and high performance liquid chromatography (HPLC) analysis. The bacterium was optimized for TTX production in media prepared from the meat-washing water of Auxis thazard, Megalaspis cordyla and Decapterus maruadsi. Interestingly, the TTX obtained 0.106 mg/mL and 0.055 mg/mL in medium prepared from A. thazard and M. cordyla, respectively while there was no TTX production detected in medium prepared from D. maruadsi. This paper could contribute to warn to the human health care system about a possible TTX poisoning in some cases related to eating fishes.     

Sources and survival of Listeria monocytogenes on fresh,leafy produce

Listeria monocytogenes is an intracellular human pathogen which enters the body through contaminated food stuffs and is known to contaminate fresh leafy produce such as spinach, lettuce and rocket. Routinely, fresh leafy produce is grown and processed on a large scale before reaching the consumer through various products such as sandwiches and prepared salads. From farm to fork, the fresh leafy produce supply chain (FLPSC) is complex and contains a diverse range of environments where L. monocytogenes is sporadically detected during routine sampling of produce and processing areas. This review describes sources of the bacteria in the FLPSC and outlines the physiological and molecular mechanisms behind its survival in the different environments associated with growing and processing fresh produce. Finally, current methods of source tracking the bacteria in the context of the food supply chain are discussed with emphasis on how these methods can provide additional, valuable information on the risk that L. monocytogenes isolates pose to the consumer.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay.Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays.TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes – BanII, DdeI, and NlaIII – were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.     

Characterization of microalgae and associated bacteria collected from shellfish harvesting areas

Marine algal toxins are an important cause of seafood-associated outbreaks. Some marine bacteria living in association with algae are able to produce channel-blocking substances similar to PSP and TTX toxins and a role of these bacteria in the toxicity of dinoflagellates has been hypothesized. The aim of this study was to monitor, over a period of 2 years, areas used in shellfish production in the northern Adriatic Sea, through the determination of phytoplankton and the characterization of bacteria isolated from algae. Toxicity tests on bacterial extracts were performed using in vivo (mouse) and in vitro (cell culture) tests and by HPLC. The Dinophysis genus was detected throughout the year, while the Alexandrium genus was present in winter and spring. Sixteen bacteria isolated from algae, out of 61 bacterial strains tested by in vitro assay, were found to be producers of toxic substances that could block sodium channels in cells. HPLC analysis for the detection of PSP and TTX toxins always gave negative results, but their presence in concentrations undetectable by HPLC, and/or the production of chemically different substances with similar biological action, could not be excluded.     

Identification of Intestinal Bacteria Responsible for Fermentation of Gum Arabic in Pig Model

Acacia spp. produce gum exudates, traditionally called gum arabic or gum acacia, which are widely used in the food industry such as emulsifiers, adhesives, and stabilizers. The traditional gum arabic is highly variable with average molecular weights varying from 300,000–800,000. For this reason a standardized sample was used for the present experiments, based on a specific species of gum arabic (Acacia(sen)SUPER GUM^TM EM2). The literature indicates that gum arabic can be fermented by the intestinal bacteria to short chain fatty acid, particularly propionate. However, the bacteria responsible for the fermentation have not been determined. In this study, we used enrichment culture of pig cecal bacteria from the selected high molecular weight specific gum arabic of (M_W 1.77 × 10⁶). We found Prevotella ruminicola-like bacterium as a predominant bacterium that is most likely to be responsible for fermentation of the gum arabic used to propionate.     

Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na(v)). The skeletal muscle Na(v) (Na(v)1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na(v)1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na(v)1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na(v)1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.     

Convergent adaptation to dangerous prey proceeds through the same first‐step mutation in the garter snake Thamnophis sirtalis

Convergent phenotypes often result from similar underlying genetics, but recent work suggests convergence may also occur in the historical order of substitutions en route to an adaptive outcome. We characterized convergence in the mutational steps to two independent outcomes of tetrodotoxin (TTX) resistance in separate geographic lineages of the common garter snake (Thamnophis sirtalis) that coevolved with toxic newts. Resistance is largely conferred by amino acid changes in the skeletal muscle sodium channel (Na_V1.4) that interfere with TTX‐binding. We sampled variation in Na_V1.4 throughout western North America and found clear evidence that TTX‐resistant changes in both lineages began with the same isoleucine‐valine mutation (I1561V) within the outer pore of Na_V1.4. Other point mutations in the pore, shown to confer much greater resistance, accumulate later in the evolutionary progression and always occur together with the initial I1561V change. A gene tree of Na_V1.4 suggests the I1561V mutations in each lineage are not identical‐by‐decent, but rather they arose independently. Convergence in the evolution of channel resistance is likely the result of shared biases in the two lineages of T. sirtalis—only a few mutational routes can confer TTX resistance while maintaining the conserved function of voltage‐gated sodium channels.     

河豚毒素生态作用研究进展

河豚毒素(tetrodotoxin, TTX)取名于河豚鱼,最早从河豚鱼中分离纯化.自1964年河豚毒素化学结构被阐明以后,河豚毒素研究得到了生物学家、毒理学家、化学家、药理学家的广泛关注.河豚毒素具有许多天然同系物.河豚毒素及其同系物在自然界分布广泛,存在于一系列不同进化水平的海洋生物和少量的两栖动物体内.河豚毒素及其同系物可能具有防御、捕食及信息传递等生态作用,毒素在生物体内的分布与其生态作用密切相关.含有河豚毒素的生物对河豚毒素具有一定的耐受能力,其机制可能与生物体内存在河豚毒素结合蛋白或生物自身具有独特的钠离子通道结构有关.重点针对河豚毒素的生态作用及生物对河豚毒素的耐受机制进行了综述,以期为河豚毒素生态学研究及河豚毒素中毒事件的防范提供科学资料.     

Puffer smells tetrodotoxin

Behavioral observation was conducted to test whether olfaction is functional to detect tetrodotoxin (TTX) in tiger puffer Takifugu rubripes using Y-maze. We placed either agarose carrier or one agarose and one agarose containing TTX (200 MU) at each head of the channel of Y-maze. Then three non-toxic hatchery-reared juveniles (body length, 5.6 ± 0.4 cm; n = 18) were released into the Y-maze and pecking behavior to carrier was observed for 3 h. The same procedure was tested for olfactory-ablated juveniles and for juveniles taht received sham operation. Juveniles showed significant selectivity to TTX, except for olfactory-ablated juveniles. These results indicate that pufferfish detects TTX by olfactory organ.     

Osmolarity is an independent trigger of Acanthamoeba castellanii differentiation

Like many yeasts, bacteria, and other sporulating microorganisms, Acanthamoeba castellanii (Neff), a free-living amoeba with pathogenic relatives, differentiates into a dormant form when deprived of nutrients. Acanthamoeba cysts redifferentiate into trophozoites when food is resupplied. We report here that Acanthamoeba encystment is also triggered by elevated osmolarity, and that osmolarity and cell surface receptor binding are synergistic in triggering differentiation. Additions of sodium chloride or glucose to rich growth media were used to produce specific osmolarity increases and similar encystment results were obtained with either additive. Although many organisms, including Acanthamoeba and mammalian cells, have been shown to adapt to hyperosmolar conditions, this is the first demonstration that hyperosmolarity can be a primary differentiation signal. © 1996 Wiley-Liss, Inc.     

Bacteria associated with truffle‐fruiting bodies contribute to truffle aroma

Truffles, symbiotic fungi renown for the captivating aroma of their fruiting bodies, are colonized by a complex bacterial community of unknown function. We characterized the bacterial community of the white truffle Tuber borchii and tested the involvement of its microbiome in the production of sulphur‐containing volatiles. We found that sulphur‐containing volatiles such as thiophene derivatives, characteristic of T. borchii fruiting bodies, resulted from the biotransformation of non‐volatile precursor(s) into volatile compounds by bacteria. The bacterial community of T. borchii was dominated by α‐ and β‐Proteobacteria. Interestingly, all bacteria phyla/classes tested in this study were able to produce thiophene volatiles from T. borchii fruiting body extract, irrespective of their isolation source (truffle or other sources). This indicates that the ability to produce thiophene volatiles might be widespread among bacteria and possibly linked to primary metabolism. Treatment of fruiting bodies with antibacterial agents fully suppressed the production of thiophene volatiles while fungicides had no inhibitory effect. This suggests that during the sexual stage of truffles, thiophene volatiles are exclusively synthesized by bacteria and not by the truffle. At this stage, the origin of thiophenes precursor in T. borchii remains elusive and the involvement of yeasts or other bacteria cannot be excluded.     

A false‐positive food chain error associated with a generic predator gut content ELISA

Conventional prey‐specific gut content ELISA (enzyme‐linked immunosorbent assay) and PCR (polymerase chain reaction) assays are useful for identifying predators of insect pests in nature. However, these assays are prone to yielding certain types of food chain errors. For instance, it is possible that prey remains can pass through the food chain as the result of a secondary predator (hyperpredator) consuming a primary predator that had previously consumed the pest. If so, the pest‐specific assay will falsely identify the secondary predator as the organism providing the biological control services to the ecosystem. Recently, a generic gut content ELISA was designed to detect protein‐marked prey remains. That assay proved to be less costly, more versatile, and more reliable at detecting primary predation events than a prey‐specific PCR assay. This study examines the chances of obtaining a ‘false positive’ food chain error with the generic ELISA. Data revealed that the ELISA was 100% accurate at detecting protein‐marked Lygus hesperus Knight (Hemiptera: Miridae) remains in the guts of two (true) primary predators, Hippodamia convergens Guérin‐Méneville (Coleoptera: Coccinellidae) and Collops vittatus (Say) (Coleoptera: Melyridae). However, there was also a high frequency (70%) false positives associated with hyperpredators, Zelus renardii Kolenati (Hemiptera: Reduviidae), that consumed a primary predator that possessed protein‐marked L. hesperus in its gut. These findings serve to alert researchers that the generic ELISA, like the PCR assay, is susceptible to food chain errors.     

Interaction of substituted guanidines with the tetrodotoxin-binding component in Electrophorus electricus.

In efforts to understand the molecular properties of ion channels in biomembranes, we have investigated the interaction of substituted guanidines with the Na⁺ channel site in membranes isolated from Electrophorus electricus. This interaction was measured by equilibrium competitive binding studies with [${}^3\text{H}$]tetrodotoxin ([${}^3\text{H}$]TTX); TTX has been shown to bind specifically to the Na⁺ channel in electrically excitable membranes. Although guanidine and small substituted guanidines such as methylguanidine or aminoguanidine competed with [${}^3\text{H}$]TTX for the membrane binding site, the apparent K_I values for these derivatives were nearly seven orders of magnitude higher than the K_d for TTX. On the other hand, the binding of the guanidines was considerably enhanced by introducing a substituent aromatic ring or aliphatic chain. Detailed analysis of the binding of aliphatic guanidines of varying chain length clearly demonstrated the contribution made by hydrophobic interactions. These results suggest that the channel site may include a hydrophobic region in close proximity to the carboxylate previously postulated to be involved in TTX binding.     

Molecular diversity of skin mucus lectins in fish

Among lectins in the skin mucus of fish, primary structures of four different types of lectin have been determined. Congerin from the conger eel Conger myriaster and AJL-1 from the Japanese eel Anguilla japonica were identified as galectin, characterized by its specific binding to β-galactoside. Eel has additionally a unique lectin, AJL-2, which has a highly conserved sequence of C-type lectins but displays Ca²⁺-independent activity. This is rational because the lectin exerts its function on the cutaneous surface, which is exposed to a Ca²⁺ scarce environment when the eel is in fresh water. The third type lectin is pufflectin, a mannose specific lectin in the skin mucus of pufferfish Takifugu rubripes. This lectin showed no sequence similarity with any known animal lectins but, surprisingly, shares sequence homology with mannose-binding lectins of monocotyledonous plants. The fourth lectin was found in the ponyfish Leiognathus nuchalis and exhibits homology with rhamnose-binding lectins known in eggs of some fish species. These lectins, except ponyfish lectin, showed agglutination of certain bacteria. In addition, pufflectin was found to bind to a parasitic trematode, Heterobothrium okamotoi. Taken together, these results demonstrate that skin mucus lectins in fish have wide molecular diversity.     

Mannitol production by heterofermentative <Emphasis Type="BoldItalic">Lactobacillus reuteri</Emphasis> CRL 1101 and <Emphasis Type="BoldItalic">Lactobacillus fermentum</Emphasis> CRL 573 in free and controlled pH batch fermentations

Certain lactic acid bacteria, especially heterofermentative strains, are capable to produce mannitol under adequate culture conditions. In this study, mannitol production by Lactobacillus reuteri CRL 1101 and Lactobacillus fermentum CRL 573 in modified MRS medium containing a mixture of fructose and glucose in a 6.5:1.0 ratio was investigated during batch fermentations with free pH and constant pH 6.0 and 5.0. Mannitol production and yields were higher under constant pH conditions compared with fermentations with free pH, the increase being more pronounced in the case of the L. fermentum strain. Maximum mannitol production and yields from fructose for L. reuteri CRL 1101 (122 mM and 75.7 mol%, respectively) and L. fermentum CRL 573 (312 mM and 93.5 mol%, respectively) were found at pH 5.0. Interestingly, depending on the pH conditions, fructose was used only as an alternative external electron acceptor or as both electron acceptor and energy source in the case of the L. reuteri strain. In contrast, L. fermentum CRL 573 used fructose both as electron acceptor and carbon source simultaneously, independently of the pH value, which strongly affected mannitol production by this strain. Studies on the metabolism of these relevant mannitol-producing lactobacilli provide important knowledge to either produce mannitol to be used as food additive or to produce it in situ during fermented food production.     

Genetic basis of tetrodotoxin resistance in pufferfishes

Tetrodotoxin (TTX) is a highly potent neurotoxin that selectively binds to the outer vestibule of voltage-gated sodium channels. Pufferfishes accumulate extremely high concentrations of TTX without any adverse effect. A nonaromatic amino acid (Asn) residue present in domain I of the pufferfish, Takifugu pardalis, Na v1.4 channel has been implicated in the TTX resistance of pufferfishes . However, the effect of this residue on TTX sensitivity has not been investigated, and it is not known if this residue is conserved in all pufferfishes. We have investigated the genetic basis of TTX resistance in pufferfishes by comparing the sodium channels from two pufferfishes (Takifugu rubripes [fugu] and Tetraodon nigroviridis) and the TTX-sensitive zebrafish. Although all three fishes contain duplicate copies of Na v1.4 channels (Na v1.4a and Na v1.4b), several substitutions were found in the TTX binding outer vestibule of the two pufferfish channels. Electrophysiological studies showed that the nonaromatic residue (Asn in fugu and Cys in Tetraodon) in domain I of Na v1.4a channels confers TTX resistance. The Glu-to-Asp mutation in domain II of Tetraodon channel Na v1.4b is similar to that in the saxitoxin- and TTX-resistant Na+ channels of softshell clams . Besides helping to deter predators, TTX resistance enables pufferfishes to selectively feed on TTX-bearing organisms.     

Inactivation of Salmonella serovars by Pseudomonas chlororaphis and Pseudomonas fluorescens strains on tomatoes

Salmonella enterica and its serovars have been associated with pathogen contamination of tomatoes with numerous outbreaks of salmonellosis. To improve food safety, pathogen control is of immediate concern. The aim of this research was to assess the populations of natural microflora (aerobic mesophilic bacteria, lactic acid bacteria, yeasts and moulds and Pseudomonas species) on tomatoes, and evaluate the efficacy of Pseudomonas fluorescens (Pf) and Pseudomonas chlororaphis (Pc) for inactivation of Salmonella on tomatoes. Microflora were determined on sanitised and unsanitised produce and enumerated on Plate Count Agar, de Man, Rogosa and Sharpe medium, Potato Dextrose Agar and Pseudomonas Agar F media. The efficacy of Pc and Pf for inactivation of S. enterica serovars Montevideo, Typhimurium and Poona was determined on spot-inoculated tomato stem scars. The effects of storage time on bacterial populations were also investigated. On unsanitised tomatoes, lactic acid bacteria, Pseudomonas sp., aerobic mesophilic bacteria and yeasts and moulds ranged from 3.31–4.84, 3.93–4.77, 4.09–4.80 and 3.83–4.67 log CFU/g of produce, respectively. The microflora were similar at 0 and 24 storage hours on sanitised produce. The suppression of Salmonella Montevideo by P. chlororaphis and P. fluorescens on tomatoes ranged from 0.51 to 2.00 log CFU/g of produce. On Salmonella Montevideo and S. Typhimurium, the suppressive effects ranged from 0.51 to 0.95 and 0.46 to 2.00 log CFU/g of produce, respectively. The pathogen suppressive effects may be attributed to competition ability of Pseudomonas relative to Salmonella strains. Pseudomonas strains may be effective against Salmonella strains as a post-harvest application, but strain synergy is required to optimise pathogen reductions.     

Carnivore identity mediates the effects of light and nutrients on aquatic food‐chain efficiency

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