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

authors: Salvitti, Lauren R.

subjects: tetrodotoxin

abstract: 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.

text: 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.

year: 2015

title: Mercenaries and the law

authors: Smith, Ron C.

subjects: mercenary

abstract: The International Convention against the Recruitment, Use, Financing and Training of Mercenaries arose from understandable African concerns about the influence of European mercenaries in the continent during decolonisation in the 1960s and early 1970s. These same concerns also gave rise to a vary adverse treatment of the concept ‘mercenary’ in Protocol 1 (1977) to the Geneva Conventions of 1949. However, these events do not justify, in 2004, further anti-mercenary measures and, particularly, they do not require the ratification of the convention against mercenary activity, about which even the existing small number of parties have considerable doubt. As the British Foreign Secretary said in his foreword to an official green paper on the subject, ‘Today’s world is a far cry from the 1960s when private military activity usually meant mercenaries of the rather unsavoury kind involved in post-colonial and neo-colonial conflicts’.

text: Mercenaries and the law The International Convention against the Recruitment, Use, Financing and Training of Mercenaries arose from understandable African concerns about the influence of European mercenaries in the continent during decolonisation in the 1960s and early 1970s. These same concerns also gave rise to a vary adverse treatment of the concept ‘mercenary’ in Protocol 1 (1977) to the Geneva Conventions of 1949. However, these events do not justify, in 2004, further anti-mercenary measures and, particularly, they do not require the ratification of the convention against mercenary activity, about which even the existing small number of parties have considerable doubt. As the British Foreign Secretary said in his foreword to an official green paper on the subject, ‘Today’s world is a far cry from the 1960s when private military activity usually meant mercenaries of the rather unsavoury kind involved in post-colonial and neo-colonial conflicts’.

year: 2004

title: The marine reservoir effect in the Southern Ocean: an evaluation of extant and new R values and their application to archaeological chronologies

authors: Petchey, Fiona

subjects: ∆R

abstract: The last phase of human colonisation of the south-west Pacific occurred around the 12th Century AD amongst the islands of the subtropical and temperate zones of the Southern Ocean (i.e., Norfolk Island, the Kermadec Islands, New Zealand and the Chatham Islands) (Fig. 1). Archaeological evidence indicates that initial colonisation of the region was rapid, possibly taking less than 100 years. Unfortunately, the chronology of these events has been largely reliant on charcoal radiocarbon determinations that have not been successful in delineating these changes. This paper investigates the use of marine shell for radiocarbon dating as an alternative to charcoal, with specific focus on variation in the marine 14C reservoir (specifically the ∆R) of Raoul Island (Kermadec Islands), Norfolk Island and Chatham Island (Fig. 2). Results from known-age, pre-AD 1950 shellfish indicate that ∆R values south of the South Pacific Convergence Zone are low compared to those recorded for islands within the South Pacific generally (Fig. 1) (see Petchey et al.in press). An average ∆R value of –19 ± 13 14C yr is recorded for Raoul Island and –49 ± 10 14C yr for Norfolk Island. These values are attributed to heightened absorption of atmospheric CO2 in this region. Extant published ∆R information from New Zealand (Fig. 2A) also suggests a low average ∆R value for these southern waters, but the range of values indicates that considerable variability is possible because of the complex interplay of currents around the New Zealand coastline. In contrast, results from Chatham Island are more variable and much higher (average ∆R = 134 ± 83 14C yr). Stable oxygen and carbon isotope data support the hypothesis that these ∆R values are caused by upwelling and mixing of 14C-depleted water along the Chatham Rise. Comparison of marine and charcoal 14C determinations from archaeological sites on Raoul Island support, within the limits of the available data, the ∆R values obtained, but further analysis is required to establish the stability of this value over time.

text: The marine reservoir effect in the Southern Ocean: an evaluation of extant and new R values and their application to archaeological chronologies The last phase of human colonisation of the south-west Pacific occurred around the 12th Century AD amongst the islands of the subtropical and temperate zones of the Southern Ocean (i.e., Norfolk Island, the Kermadec Islands, New Zealand and the Chatham Islands) (Fig. 1). Archaeological evidence indicates that initial colonisation of the region was rapid, possibly taking less than 100 years. Unfortunately, the chronology of these events has been largely reliant on charcoal radiocarbon determinations that have not been successful in delineating these changes. This paper investigates the use of marine shell for radiocarbon dating as an alternative to charcoal, with specific focus on variation in the marine 14C reservoir (specifically the ∆R) of Raoul Island (Kermadec Islands), Norfolk Island and Chatham Island (Fig. 2). Results from known-age, pre-AD 1950 shellfish indicate that ∆R values south of the South Pacific Convergence Zone are low compared to those recorded for islands within the South Pacific generally (Fig. 1) (see Petchey et al.in press). An average ∆R value of –19 ± 13 14C yr is recorded for Raoul Island and –49 ± 10 14C yr for Norfolk Island. These values are attributed to heightened absorption of atmospheric CO2 in this region. Extant published ∆R information from New Zealand (Fig. 2A) also suggests a low average ∆R value for these southern waters, but the range of values indicates that considerable variability is possible because of the complex interplay of currents around the New Zealand coastline. In contrast, results from Chatham Island are more variable and much higher (average ∆R = 134 ± 83 14C yr). Stable oxygen and carbon isotope data support the hypothesis that these ∆R values are caused by upwelling and mixing of 14C-depleted water along the Chatham Rise. Comparison of marine and charcoal 14C determinations from archaeological sites on Raoul Island support, within the limits of the available data, the ∆R values obtained, but further analysis is required to establish the stability of this value over time.

year: 2008