title: Investigating Diet as the Source of Tetrodotoxin in the Grey Side-gilled Sea Slug, Pleurobranchaea maculata

authors: Khor, Serena

subjects: Tetrodotoxin

abstract: Pleurobranchaea maculata (grey-side gilled sea slug) was discovered to contain the neurotoxin tetrodotoxin (TTX) in 2009 after a spate of dog poisoning cases on the beaches of Auckland, New Zealand. One of the great mysteries of TTX is the lack of conclusive evidence about its ultimate origin. Possible sources postulated have included both endogenous and exogenous. Additionally, within a species both toxic and non-toxic strains exist. For example, in New Zealand, P. maculate from the North Island are toxic, whereas P. maculata from the South Island are not. The overarching hypothesis of this Master’s project is that TTX has a dietary origin in P. maculata and that they will preferentially feed on TTX-containing food. This Master’s thesis aimed to test this hypothesis through three distinct aims: (1) To develop a non-lethal biopsy method and determine the feasibility for future research concerning TTX in P. maculata; (2) To investigate whether nontoxic P. maculata can accumulate TTX from a dietary source, and how TTX is distributed through the organism; and (3) To investigate whether P. maculata are attracted to TTX and if this varies depending on the TTX content within P. maculata.
A biopsy method was developed for taking approximately 200 mg tissue biopsies using a TemnoEvolution 18G × 11 cm Biopsy Needle inserted transversely into the foot. Six P. maculata were biopsied twice (nine days apart) and each individual was frozen immediately following the second sampling. Tetrodotoxin concentrations in biopsy samples, gonad, stomach, mantle and the remaining combined tissues and fluids were measured using liquid chromatography-mass spectrometry (LC-MS). 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.

To investigate the accumulation of TTX, eighteen non-toxic P. maculate were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 hr, 24 hrs, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using LC-MS. Tetrodotoxin was detected in all organs/tissue after 1 hr with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). This study demonstrated that P. maculata can accumulate TTX from a dietary source.
To explore whether P. maculata were attracted to TTX, three preference experiments were undertaken; (1) an aquarium zonation experiment, (2) a toxic/non-toxic agar trail experiment and, (3) a direct choice experiment using combinations of toxic/non-toxic agar blocks. A statistically significant preference for TTX was found for toxic P. maculata in the agar trails (P < 0.001) and direct choice experiments (P < 0.001). For the non-toxic P. maculata, a statistically significant preference was only found for the direct choice experiment (P = 0.002).
Collectively these studies demonstrate that diet is a possible source in P. maculata. However, given the absence of identifiable TTX sources in environments where P. maculata are prevalent, in concert with their extremely high TTX concentrations and short life spans, it is unlikely to be the sole source for this species.

text: Investigating Diet as the Source of Tetrodotoxin in the Grey Side-gilled Sea Slug, Pleurobranchaea maculata Pleurobranchaea maculata (grey-side gilled sea slug) was discovered to contain the neurotoxin tetrodotoxin (TTX) in 2009 after a spate of dog poisoning cases on the beaches of Auckland, New Zealand. One of the great mysteries of TTX is the lack of conclusive evidence about its ultimate origin. Possible sources postulated have included both endogenous and exogenous. Additionally, within a species both toxic and non-toxic strains exist. For example, in New Zealand, P. maculate from the North Island are toxic, whereas P. maculata from the South Island are not. The overarching hypothesis of this Master’s project is that TTX has a dietary origin in P. maculata and that they will preferentially feed on TTX-containing food. This Master’s thesis aimed to test this hypothesis through three distinct aims: (1) To develop a non-lethal biopsy method and determine the feasibility for future research concerning TTX in P. maculata; (2) To investigate whether nontoxic P. maculata can accumulate TTX from a dietary source, and how TTX is distributed through the organism; and (3) To investigate whether P. maculata are attracted to TTX and if this varies depending on the TTX content within P. maculata.
A biopsy method was developed for taking approximately 200 mg tissue biopsies using a TemnoEvolution 18G × 11 cm Biopsy Needle inserted transversely into the foot. Six P. maculata were biopsied twice (nine days apart) and each individual was frozen immediately following the second sampling. Tetrodotoxin concentrations in biopsy samples, gonad, stomach, mantle and the remaining combined tissues and fluids were measured using liquid chromatography-mass spectrometry (LC-MS). 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.

To investigate the accumulation of TTX, eighteen non-toxic P. maculate were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 hr, 24 hrs, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using LC-MS. Tetrodotoxin was detected in all organs/tissue after 1 hr with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). This study demonstrated that P. maculata can accumulate TTX from a dietary source.
To explore whether P. maculata were attracted to TTX, three preference experiments were undertaken; (1) an aquarium zonation experiment, (2) a toxic/non-toxic agar trail experiment and, (3) a direct choice experiment using combinations of toxic/non-toxic agar blocks. A statistically significant preference for TTX was found for toxic P. maculata in the agar trails (P < 0.001) and direct choice experiments (P < 0.001). For the non-toxic P. maculata, a statistically significant preference was only found for the direct choice experiment (P = 0.002).
Collectively these studies demonstrate that diet is a possible source in P. maculata. However, given the absence of identifiable TTX sources in environments where P. maculata are prevalent, in concert with their extremely high TTX concentrations and short life spans, it is unlikely to be the sole source for this species.

year: 2014

title: Submerged Thin Plate Weirs With Unequal Upstream and Downstream Beds

authors: Nalder, Guinevere Vivianne

subjects: thin plate weirs

abstract: This thesis describes a short study to examine the behaviour of submerged flow over a thin plate weir with differing upstream and downstream bed levels i.e. an unequal bed weir as opposed to an equal bed weir where the upstream and downstream beds are at the same level.

As submerged weir flow is a function of downstream conditions, it was thought that a lower downstream bed would make submerged flow over the weir easier, This is turn suggested that;
 	The shape of the upstream head (Hu) vs downstream head (Hd) graph would change, being initially more steep in the unequal bed case.
 	The Froude Number of the approaching flow would be lower for the unequal bed weir than for an equal bed weir at the same submergence.
 	Using one of the existing submerged flow formula would lead to an erroneous calculated value.

A series of measurements was done on two model weirs of different sizes subject to successive levels of submergence. Analysis of the readings of upstream and downstream heads indicated that the difference in bed levels was significant and the three effects above were noted.

The work also suggested a new form of equation to calculate flow over a submerged weir. This was looked at briefly.

text: Submerged Thin Plate Weirs With Unequal Upstream and Downstream Beds This thesis describes a short study to examine the behaviour of submerged flow over a thin plate weir with differing upstream and downstream bed levels i.e. an unequal bed weir as opposed to an equal bed weir where the upstream and downstream beds are at the same level.

As submerged weir flow is a function of downstream conditions, it was thought that a lower downstream bed would make submerged flow over the weir easier, This is turn suggested that;
 	The shape of the upstream head (Hu) vs downstream head (Hd) graph would change, being initially more steep in the unequal bed case.
 	The Froude Number of the approaching flow would be lower for the unequal bed weir than for an equal bed weir at the same submergence.
 	Using one of the existing submerged flow formula would lead to an erroneous calculated value.

A series of measurements was done on two model weirs of different sizes subject to successive levels of submergence. Analysis of the readings of upstream and downstream heads indicated that the difference in bed levels was significant and the three effects above were noted.

The work also suggested a new form of equation to calculate flow over a submerged weir. This was looked at briefly.

year: 2006

title: A history of Māori literacy success

authors: Derby, Melissa

abstract: This article explores literacy in 19th century Māori society, and in particular, this work considers the initial interactions Māori tribal groups had with literacy following contact with British settlers. The overarching purpose of this article is to respond to the increasingly prevalent suggestion that literacy is a Western competency, and that to be concerned with ensuring Māori children are achieving well in reading and writing is an act of colonisation. This work illustrates that these narratives run counter to the history, behaviour, and values of many tribal groups in New Zealand. More specifically, the historical interactions Māori had with literacy illustrate that both the ability and the desire to succeed in reading and writing was a feature of Māori historical practices. Therefore, to ensure high levels of literacy achievement in contemporary times is not a colonising act but rather aligns with Māori history, behaviour, and values.

text: A history of Māori literacy success This article explores literacy in 19th century Māori society, and in particular, this work considers the initial interactions Māori tribal groups had with literacy following contact with British settlers. The overarching purpose of this article is to respond to the increasingly prevalent suggestion that literacy is a Western competency, and that to be concerned with ensuring Māori children are achieving well in reading and writing is an act of colonisation. This work illustrates that these narratives run counter to the history, behaviour, and values of many tribal groups in New Zealand. More specifically, the historical interactions Māori had with literacy illustrate that both the ability and the desire to succeed in reading and writing was a feature of Māori historical practices. Therefore, to ensure high levels of literacy achievement in contemporary times is not a colonising act but rather aligns with Māori history, behaviour, and values.

year: 2021