Elucidating the Origin of Tetrodotoxin in Pleurobranchaea maculata and Stylochoplana sp.

Tetrodotoxin (TTX) is an extremely potent neurotoxin that acts by selectively targeting voltage gated sodium channels blocking propagation of action potentials. Long believed to be present only in pufferfish, TTX has now been detected in a wide range of phylogenetically unrelated terrestrial and aquatic taxa. Despite decades of research the exact origin of TTX remains a mystery. Current literature supports three hypotheses: endogenous, symbiotic bacteria, or bioaccumulation through a dietary source. In 2009, the opisthobranch Pleurobranchaea maculata (grey side-gilled sea slug) was found to contain high concentrations of TTX in New Zealand. A large collaborative project, of which my research was a major part of, was initiated to explore the origin of TTX in P. maculata. During extensive benthic surveys conducted to identify possible dietary sources of TTX, high concentrations (ave. 380 mg kg-1) were detected in Stylochoplana sp. (Platyhelminthes) from Pilot Bay (Tauranga, New Zealand). Tetrodotoxin concentrations were found to vary temporally, peaking between June and August. The co-occurrence of Stylochoplana sp. and P. maculata in Pilot Bay raised the possibility that Stylochoplana sp. could be a dietary source of TTX for P. maculata. A real-time PCR assay was developed, and detected Stylochoplana sp. in seven out of nineteen P. maculata foreguts. Symbiotic bacterial production of TTX in the tissues of P. maculata and Stylochoplana sp. was also explored. Isolated strains (102; 17 unique strains - identified using 16S rRNA gene analysis) were analyzed using a recently developed method to detect the C9 base of TTX. In addition to enhanced sensitivity, this method has the advantage that it might detect precursor and degradation products. To explore the possibility that TTX is produced by a consortium of bacteria, experiments were undertaken where homogenized tissue was spiked into marine broth and samples were collected over two weeks for toxin and molecular analysis. No C9 base or TTX production was detected in isolates or from bacterial communities, suggesting that a symbiotic microbial source of TTX is unlikely in these organisms. The ability of non-toxic P. maculata to sequester TTX from an environment known to contain toxic populations of the same species was also assessed. Sixteen non-toxic specimens were kept in mesh cages (eight anchored to the benthos and eight suspended 0.5 m above it) for eight weeks and fed a non-toxic food source. Toxin analysis revealed that more ‘benthic’ specimens (4 verses the 2 from suspended specimens) sequestered TTX and were shown to retain higher concentrations (max. 0.79 versus 0.43 mg kg-1). These data suggest a localized microbial source of TTX that is more readily available from the benthos. Diet analysis, utilizing next generation sequencing of toxic and non-toxic P. maculata identified their diet comprised a wide array of organisms, with Thelepus sp. and Plumularia sp. being prevalent in toxic individuals, and further testing of these organisms is suggested. Lastly, immunohistological methods, employing a monoclonal antibody targeting TTX, were conducted with tissues from P. maculata and Stylochoplana sp.. Strong TTX signals were detected in the mantle and oocytes of P. maculata and the ova and pharynx of the Stylochoplana sp.. These data suggest ecological roles for TTX including: defense in adults, protection in progeny, and prey capture in Stylochoplana sp.. A synthesis of the studies presented in this thesis, and those that were conducted as part of the larger project, are also presented and future studies to elucidate the origin of TTX in New Zealand taxa are suggested

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

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

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

The effects of temperature, CO2, and nitrogen source on the growth and physiology of the raphidophytes Heterosigma akashiwo and Chattonella subsalsa

Warner, Mark E.During the past three decades the frequency and duration of harmful algal blooms (HABs) have significantly increased causing major economical losses world wide as well as being identified as the cause of a number of human health illnesses. These impacts have brought harmful algal species to the forefront of research efforts which are focused primarily on the effects of abiotic and biotic factors on algal growth. Less effort has been placed on understanding how changes in environmental parameters may impact harmful algal physiology. The effects of elevated CO2 and temperature were studied using the raphidophytes Chattonella subsalsa and Heterosigma akashiwo isolated from the Delaware Inland Bays (DIB), and a cold water isolate of H. akashiwo from Puget Sound, WA. Neither increases in temperature, CO2, or a combined treatment of elevated temperature and CO2, had any significant effect on the growth rates of all three isolates. However, significant changes in physiology and carbon uptake were seen among temperature/CO2 treatments and between strains. Both isolates of H. akashiwo exhibited changes in light harvesting capabilities in response to temperature, with a decrease in maximum carbon assimilation occurring in the cold water isolate. In contrast both elevated temperature and CO2 altered the physiology of C. subsalsa although changes were not additive. Carbon assimilation increased in response to elevated CO2 while temperature had a greater effect on light harvesting in this species. While growth and cell size did not change across any treatments in these raphidophytes, the large draw down of pCO2 measured suggests that they could possibly be storing carbon, or releasing it as dissolved organic carbon. Thus, while the bloom dynamics of these HAB species will most likely not change with the predicted increases in global atmospheric carbon or temperature, a potential for them to alter phytoplankton community succession through changes in abiotic conditions remains a possibility.University of Delaware, Department of Marine Science and PolicyM.S

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

Depuration of tetrodotoxin and changes in bacterial communities in Pleurobranchea maculata adults and egg masses maintained in captivity

Depuration of tetrodotoxin (TTX) was investigated in adult grey side-gilled sea slugs, Pleurobranchaea maculata, maintained in captivity on a TTX-free diet. Three adults were harvested every 21 days for 126 days, and TTX concentrations were measured in organs/tissues and egg masses. Automated rRNA intergenic spacer analysis (ARISA) was used to investigate bacterial community structure in selected samples. Linear modeling of adult data demonstrated a decline (P < 0.001) in average total TTX concentrations over time. Temporal data obtained from a wild population showed similar depuration rates, indicating that once adults reach a certain size, or sexual maturity, TTX is no longer produced or acquired substantially. Depuration rates differed among organs, with concentrations in the heart declining the fastest. The gonads had the slowest and least significant depuration rate indicating, at most, weak depuration of this tissue. There was a strong correlation (R (2) = 0.66) between TTX concentrations in the first-laid egg masses and total TTX in the corresponding adult. These data suggest that adult P. maculata transfer TTX to their offspring, and presumably that functions as a chemical defense. ARISA data showed a shift in bacterial community structure within 3 weeks of introduction to captivity. Based on the combined data, the exact origin of TTX in P. maculata is unclear, with evidence both in favor and against a dietary source, and endogenous or bacterial production

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 (r2 = 0.88), stomach (r2 = 0.92) and gonad (r2 = 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