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.

Gonadal Degeneration Is Mediated by Apoptotic Processes in the Semelparous Gray Side-Gilled Sea Slug Pleurobranchaea maculata.

AbstractSpecies undergoing postreproductive death experience great changes in their reproductive organs, which are driven by numerous physiological processes. To assess whether apoptotic processes are involved in the dynamics of the reproductive organs of Pleurobranchaea maculata, the gonadal structure of this semelparous side-gilled sea slug was studied using light and scanning electron microscopy. Apoptotic cells at different gonadal developmental stages were detected by in situ TUNEL assay. Apoptosis was primarily focused on spermatogonia during gonadal cell proliferation, probably as a regulatory mechanism that maintains homeostasis in reproductive cells. Visible gonadal degeneration at the end of the reproductive period is accompanied by apoptosis of the basal lamina cells of the acini, suggesting that apoptotic processes are involved in the gonadal degeneration observed in P. maculata.

Tetrodotoxin concentrations in Pleurobranchaea maculata: temporal, spatial and individual variability from New Zealand populations.

Tetrodotoxin (TTX) is a potent neurotoxin that has been identified in a range of phylogenetically unrelated marine and terrestrial organisms. Tetrodotoxin was recently detected in New Zealand in Pleurobranchaea maculata (the grey side-gilled sea slug). From June 2010 to June 2011 wild specimens were collected from 10 locations around New Zealand. At one site (Narrow Neck Beach, Auckland) up to 10 individuals were collected monthly for 6 months. Attempts were also made to rear P. maculata in captivity. Tetrodotoxin was detected in samples from eight of the ten sites. The highest average (368.7 mg kg⁻¹) and maximum (1414.0 mg kg⁻¹) concentrations were measured in samples from Illiomama Rock (Auckland). Of the toxic populations tested there was significant variability in TTX concentrations among individuals, with the highest difference (62 fold) measured at Illiomama Rock. Tetrodotoxin concentrations in samples from Narrow Neck Beach varied temporally, ranging from an average of 184 mg kg⁻¹ in June 2010 to 17.5 mg kg⁻¹ by December 2010. There was no correlation between TTX levels and mass. The highest levels correspond with the egg laying season (June-August) and this, in concert with the detection of high levels of TTX in eggs and early larval stages, suggests that TTX may have a defensive function in P. maculata. Only one larva was successfully reared to full maturation and no TTX was detected.

Development of a non-lethal biopsy technique for estimating total tetrodotoxin concentrations in the grey side-gilled sea slug Pleurobranchaea maculata.

High concentrations of tetrodotoxin (TTX) have been detected in some New Zealand populations of Pleurobranchaea maculata (grey side-gilled sea slug). Within toxic populations there is significant variability in TTX concentrations among individuals, with up to 60-fold differences measured. This variability has led to challenges when conducting controlled laboratory experiments. The current method for assessing TTX concentrations within P. maculata is lethal, thus multiple individuals must be harvested at each sampling point to produce statistically meaningful data. In this study a method was developed for taking approximately 200 mg tissue biopsies using a TemnoEvolution(®) 18G × 11 cm Biopsy Needle inserted transversely into the foot. Correlation between the TTX concentrations in the biopsy sample and total TTX levels and in individual tissues were assessed. Six P. maculata were biopsied twice (nine days apart) and each individual was frozen immediately following the second sampling. Tetrodotoxin concentrations in biopsy samples and in the gonad, stomach, mantle and the remaining combined tissues and fluids were measured using liquid chromatography-mass spectrometry. Based on the proportional weight of the organs/tissues a total TTX concentration for each individual was calculated. There were strong correlations between biopsy TTX concentrations and the total (r(2) = 0.88), stomach (r(2) = 0.92) and gonad (r(2) = 0.83) TTX concentrations. This technique will enable more robust laboratory studies to be undertaken, thereby assisting in understanding TTX kinetics, ecological function and origin within P. maculata.

Ergot alkaloid from the sea slug Pleurobranchus forskalii.

The sea slug Pleurobranchus forskalii is a carnivorous scavenger that is widely distributed in shallow subtidal areas. Very few investigations of the chemical components of this gastropod have been reported. In this study we performed a comprehensive analysis of an extract of the marine mollusc, P. forskalii, collected off Ishigaki Island, Japan. As a result, an alkaloid was isolated from the chloroform extract. Remarkably, the structure elucidation based on the spectral data revealed that it was an ergot alkaloid peptide, ergosinine. Various ergot alkaloids have previously been isolated mainly from terrestrial higher plants or fungi. This is the first report of the isolation of an ergopeptine from marine life, and thus the known geographical extent of ergot alkaloids now includes both terrestrial and aquatic organisms.

Neurochemical and neuroanatomical identification of central pattern generator neuron homologues in Nudipleura molluscs.

Certain invertebrate neurons can be identified by their behavioral functions. However, evolutionary divergence can cause some species to not display particular behaviors, thereby making it impossible to use physiological characteristics related to those behaviors for identifying homologous neurons across species. Therefore, to understand the neural basis of species-specific behavior, it is necessary to identify homologues using characteristics that are independent of physiology. In the Nudipleura mollusc Tritonia diomedea, Cerebral Neuron 2 (C2) was first described as being a member of the swim central pattern generator (CPG). Here we demonstrate that neurochemical markers, in conjunction with previously known neuroanatomical characteristics, allow C2 to be uniquely identified without the aid of electrophysiological measures. Specifically, C2 had three characteristics that, taken together, identified the neuron: 1) a white cell on the dorsal surface of the cerebral ganglion, 2) an axon that projected to the contralateral pedal ganglion and through the pedal commissure, and 3) immunoreactivity for the peptides FMRFamide and Small Cardioactive Peptide B. These same anatomical and neurochemical characteristics also uniquely identified the C2 homologue in Pleurobranchaea californica (called A1), which was previously identified by its analogous role in the Pleurobranchaea swim CPG. Furthermore, these characteristics were used to identify C2 homologues in Melibe leonina, Hermissenda crassicornis, and Flabellina iodinea, species that are phylogenetically closer to Tritonia than Pleurobranchaea, but do not display the same swimming behavior as Tritonia or Pleurobranchaea. These identifications will allow future studies comparing and contrasting the physiological properties of C2 across species that can and cannot produce the type of swimming behavior exhibited by Tritonia.