Diversity and biosynthetic potential of culturable microbes associated with toxic marine animals

Tetrodotoxin (TTX) is a neurotoxin that has been reported from taxonomically diverse organisms across 14 different phyla. The biogenic origin of tetrodotoxin is still disputed, however, TTX biosynthesis by host-associated bacteria has been reported. An investigation into the culturable microbial populations from the TTX-associated blue-ringed octopus Hapalochlaena sp. and sea slug Pleurobranchaea maculata revealed a surprisingly high microbial diversity. Although TTX was not detected among the cultured isolates, PCR screening identifiedsome natural product biosynthesis genes putatively involved in its assembly. This study is the first to report on the microbial diversity of culturable communities from H. maculosa and P. maculata and common natural product biosynthesis genes from their microbiota. We also reassess the production of TTX reported from three bacterial strains isolated from the TTX-containing gastropod Nassarius semiplicatus

Investigating diet as the source of tetrodotoxin in Pleurobranchaea maculata

The origin of tetrodotoxin (TTX) is highly debated; researchers have postulated either an endogenous or exogenous source with the host accumulating TTX symbiotically or via food chain transmission. The aim of this study was to determine whether the grey side-gilled sea slug (Pleurobranchaea maculata) could obtain TTX from a dietary source, and to attempt to identify this source through environmental surveys. Eighteen non-toxic P. maculata were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 h, 24 h, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using liquid chromatography-mass spectrometry. Tetrodotoxin was detected in all organs/tissue after 1 h with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). Benthic surveys at sites with dense populations of toxic P. maculata detected very low or no TTX in other organisms. This study demonstrates that P. maculata can accumulate TTX through their diet. However, based on the absence of an identifiable TTX source in the environment, in concert with the extremely high TTX concentrations and short life spans of P. maculata, it is unlikely to be the sole TTX source for this species

Risks for public health related to the presence of tetrodotoxin (TTX) and TTX analogues in marine bivalves and gastropods

Peer reviewedPublisher PD

Intracellular immunohistochemical detection of tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria)

Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring

No evidence for a culturable bacterial tetrodotoxin producer in Pleurobranchaea maculata (Gastropoda: Pleurobranchidae) and Stylochoplana sp. (Platyhelminthes: Polycladida)

Tetrodotoxin (TTX) is a potent neurotoxin found in the tissues of many taxonomically diverse organisms. Its origin has been the topic of much debate, with suggestions including endogenous production, acquisition through diet, and symbiotic bacterial synthesis. Bacterial production of TTX has been reported in isolates from marine biota, but at lower than expected concentrations. In this study, 102 strains were isolated from Pleurobranchaea maculata (Opisthobranchia) and Stylochoplana sp. (Platyhelminthes). Tetrodotoxin production was tested utilizing a recently developed sensitive method to detect the C9 base of TTX via liquid chromatography—mass spectrometry. Bacterial strains were characterized by sequencing a region of the 16S ribosomal RNA gene. To account for the possibility that TTX is produced by a consortium of bacteria, a series of experiments using marine broth spiked with various P. maculata tissues were undertaken. Sixteen unique strains from P. maculata and one from Stylochoplana sp. were isolated, representing eight different genera; Pseudomonadales, Actinomycetales, Oceanospirillales, Thiotrichales, Rhodobacterales, Sphingomonadales, Bacillales, and Vibrionales. Molecular fingerprinting of bacterial communities from broth experiments showed little change over the first four days. No C9 base or TTX was detected in isolates or broth experiments (past day 0), suggesting a culturable microbial source of TTX in P. maculata and Stylochoplana sp. is unlikely

Na(V)1.5 sodium channel window currents contribute to spontaneous firing in olfactory sensory neurons

Olfactory sensory neurons (OSNs) fire spontaneously as well as in response to odor; both forms of firing are physiologically important. We studied voltage-gated Na+ channels in OSNs to assess their role in spontaneous activity. Whole cell patch-clamp recordings from OSNs demonstrated both tetrodotoxin-sensitive and tetrodotoxin-resistant components of Na+ current. RT-PCR showed mRNAs for five of the nine different Na+ channel α-subunits in olfactory tissue; only one was tetrodotoxin resistant, the so-called cardiac subtype NaV1.5. Immunohistochemical analysis indicated that NaV1.5 is present in the apical knob of OSN dendrites but not in the axon. The NaV1.5 channels in OSNs exhibited two important features: 1) a half-inactivation potential near −100 mV, well below the resting potential, and 2) a window current centered near the resting potential. The negative half-inactivation potential renders most NaV1.5 channels in OSNs inactivated at the resting potential, while the window current indicates that the minor fraction of noninactivated NaV1.5 channels have a small probability of opening spontaneously at the resting potential. When the tetrodotoxin-sensitive Na+ channels were blocked by nanomolar tetrodotoxin at the resting potential, spontaneous firing was suppressed as expected. Furthermore, selectively blocking NaV1.5 channels with Zn2+ in the absence of tetrodotoxin also suppressed spontaneous firing, indicating that NaV1.5 channels are required for spontaneous activity despite resting inactivation. We propose that window currents produced by noninactivated NaV1.5 channels are one source of the generator potentials that trigger spontaneous firing, while the upstroke and propagation of action potentials in OSNs are borne by the tetrodotoxin-sensitive Na+ channel subtypes.This work was aided by support from Boston University, the Rocky Mountain Taste and Smell Center Core for Cellular Visualization and Analysis [National Institute on Deafness and Other Communication Disorders (NIDCD) P30 DC-04657; D. Restrepo, principal investigator], and NIDCD Grants DC-04863 to V. Dionne and DC-006070 to D. Restrepo and T. E. Finger. (Boston University; P30 DC-04657 - Rocky Mountain Taste and Smell Center Core for Cellular Visualization and Analysis [National Institute on Deafness and Other Communication Disorders (NIDCD)]; DC-04863 - Rocky Mountain Taste and Smell Center Core for Cellular Visualization and Analysis [National Institute on Deafness and Other Communication Disorders (NIDCD)]; DC-006070 - Rocky Mountain Taste and Smell Center Core for Cellular Visualization and Analysis [National Institute on Deafness and Other Communication Disorders (NIDCD)])https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122723/Accepted manuscrip

Towards the timely detection of toxicants

We address the problem of enhancing the sensitivity of biosensors to the influence of toxicants, with an entropy method of analysis, denoted as CASSANDRA, recently invented for the specific purpose of studying non-stationary time series. We study the specific case where the toxicant is tetrodotoxin. This is a very poisonous substance that yields an abrupt drop of the rate of spike production at t approximatively 170 minutes when the concentration of toxicant is 4 nanomoles. The CASSANDRA algorithm reveals the influence of toxicants thirty minutes prior to the drop in rate at a concentration of toxicant equal to 2 nanomoles. We argue that the success of this method of analysis rests on the adoption of a new perspective of complexity, interpreted as a condition intermediate between the dynamic and the thermodynamic state.Comment: 6 pages and 3 figures. Accepted for publication in the special issue of Chaos Solitons and Fractal dedicated to the conference "Non-stationary Time Series: A Theoretical, Computational and Practical Challenge", Center for Nonlinear Science at University of North Texas, from October 13 to October 19, 2002, Denton, TX (USA

The envenomation of general physiology throughout the last century.

Toxins are the poisonous products of organisms. Toxins serve vital defensive and offensive functions for those that harbor them: stinging scorpions, pesticidal plants, sanguinary snakes, fearless frogs, sliming snails, noxious newts, and smarting spiders. For physiologists, toxins are integral chemical tools that hijack life's fundamental processes with remarkable molecular specificity. Our understanding of electrophysiological phenomena has been transformed time and time again with the help of some terrifying toxins. For this reason, studies of toxin mechanism are an important and enduring facet of The Journal of General Physiology (JGP). This Milestone in Physiology reflects on toxins studied in JGP over its first 100 years, what they have taught us, and what they have yet to reveal

Genetic structure of Pleurobranchaea maculata in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics, The New Zealand Institute for Advanced Study, Massey University, Auckland, New Zealand

The grey side-gilled sea slug (Pleurobranchaea maculata), which is native to the western and south Pacific, is known to contain high concentrations of tetrodotoxin (TTX). P. maculata populations around New Zealand exhibit individual, spatial and temporal differences in TTX concentration, but the origin of TTX in P. maculata is not fully understood. The main goal of my PhD project was to examine the genetic structure and demographic history of P. maculata populations from different regions in New Zealand and to clarify whether there is a correlation between variability in TTX concentrations and genetic structure. A sample of 146 P. maculata individuals were collected from three populations from the north-eastern North Island (Ti Point-TP, Auckland-AKL and Tauranga-TR), one population from the southern North Island (Wellington-WL) and one population from the northern South Island (Nelson-NL). TP, AKL and TR were designated the “Northern cluster”, whereas the WL and NL population were labelled as the “Southern cluster” due to the relative geographical locations of these clusters. Twelve nuclear microsatellite markers that were developed based on shotgun sequence were obtained from the genome of P. maculata. The markers were used to analyse the genetic structure of P. maculata populations. The mitochondrial cytochrome c oxidase subunit I (1153 bp) and cytochrome b (1060 bp) genes were also partially sequenced in P. maculata individuals. The microsatellite data reveal high genetic diversity and lead to the rejection of the hypothesis of panmixia: populations from the Northern cluster are highly connected but significantly differentiated from the Sothern cluster. A weak differentiation was also observed between the WL and NL populations. The two populations correlate with regional variations in TTX concentrations: the Northern cluster populations contain highly toxic individuals, whereas the Southern cluster (WL and NL) populations harbour either slightly toxic or non-toxic populations. The disjunction between the Northern and Southern clusters can be explained by biogeographical barriers specific to New Zealand but also with a stepping stone model. The geographical gap between the sampling locations made it impossible to draw firm conclusions as to the origin of the disjunction. The mtDNA sequence data reveal high haplotype diversity, low nucleotide diversity and a star-shaped haplotype network. These data can be explained by a population expansion dating back to the Pleistocene era. All the sampling locations are significantly differentiated from each other according to mtDNA data. Given that microsatellite and mitochondrial sequences evolve at different rates, incomplete linkage sorting is expected to be completed for mtDNA before, which should be reflected in a more pronounced structure for mtDNA markers where members of the populations have diverged recently. Although this may explain the geographical conflict between the microsatellite and mtDNA data, it is necessary to consider the possibility that the discordance between microsatellite markers and mtDNA may be in part attributable to the relatively small sample size