Establishing a protocol to measure tetrodotoxin (TTX) levels in small-bodied newts (genus Lissotriton) in western Europe Rosa Wagstaff This Thesis is Submitted in Fulfilment of the Requirements for the Degree of MSc by Research School of Science, Engineering and Environment University of Salford, Salford, UK March 2021 1 Abstract Tetrodotoxin (TTX) is a defence toxin most commonly found in marine organisms, and amphibians are the only land vertebrates (tetrapods) which are known to produce TTX. However, the origin of TTX in amphibians remains largely unknown, and our knowledge about the existence and distribution of TTX across taxa and populations is very incomplete. The present study summarises our knowledge of TTX in amphibians, and describes a series of experiments to determine whether TTX can be detected in newts (genus Lissotriton and Icthyosaura) across north-eastern Europe. The study was based on eggs and fresh roadkills collected in England, Scotland, Wales and France. The roadkill samples were collected at a site inhabited by both Lissotriton vulgaris and L. helveticus, and species identification was attempted using mtDNA sequencing. An initial set of TTX detection experiments considered all samples, and trialled alternative extraction protocols before employing HPLC/UV/Vis spectrometry. While strong candidate peaks for TTX were identified in some samples, the employed approach was not sufficiently sensitive to unambiguously demonstrate its presence. Three of the adult newts collected from England were further tested for TTX using liquid chromatography and high-resolution mass spectrometry. No tetrodotoxin was detected in any of the newts that were tested. The findings are discussed in light of the observed constraints by the protocols used and should serve as a useful basis for future studies on the presence of TTX in European amphibians. 2 Contents 1. Introduction………………………………………………………………………………….………5 1.1 Animal Defence Mechanisms Against Predators…… …………………….5 1.2 Newts and Salamanders: Order Caudata…………….………………….….…8 1.2.1 Newts of the Western Palearctic...…...........................……..10 1.3 Tetrodotoxin………………………………………………………………………………..14. 1.4 Techniques to Identify and Quantify TTX……………………………………..21. 1.5 Aims and Objectives…………………………………………………………………….29. 2. Materials and Methods………………………………………………………………………..31. 2.1 Fieldwork and Sample Collection……….………………………………………..31. 2.2 Genetic Species Identification……………..…………..…………………..……..34. 2.3 TTX Detection Method Development: Sample Preparation…….……36. 2.4 TTX Detection………………….…………………………………………………………..38. 2.4.1 TTX Standard Formation…..…………………………………..………..38. 2.4.2 HPLC/UV/Vis/MS/FLD Detection…….…………..……………..…..39. 2.4.3 LC-MS Detection…………………………………..………………..……….40. 3. Results…………………………………………………………………………………………..……..42 3.1 Genetic Species Identification………………………………………………………42 3.2 TTX Standard………………………………………………………………………………..42 3.3 TTX Detection……………………..……….…….....……………..………………..……45. 3.4 LC-MS Analysis……………………………………………………………………………..54 57 3 57 4. Discussion.............................................................................................. 57 4.1 Fieldwork and Study Sites............................................................... 57 4.2 Storage and Transportation............................................................ 62 4.3 Sample Preparation, LC-MS and Repeat Experiments.................... 63 4.4 Species Identification...................................................................... 66 4.5 Impacts of Tetrodotoxin.................................................................. 68 4.6 Further Research Opportunities...................................................... 74 4.7 Conclusion....................................................................................... 75 5. References............................................................................................. 77 6. Appendix................................................................................................89 6.1 Gels.................................................................................................. 89 6.2 Sample Preparation Records............................................................91 6.3 HPLC/UV/Vis Results........................................................................93 6.4 Sequences........................................................................................112 4 1. Introduction 1.1 Animal Defence Mechanisms Against Predators Since the first organism evolved to consume another, defence mechanisms have in turn evolved so that prey organisms may evade their predators. Defence mechanisms are seen in all types of organisms, including bacteria, fungi, plants and animals, and the predator-prey relationship has led to various forms of defence mechanisms evolving in prey species. These mechanisms help prey to flee, fight, avoid detection, or they can increase chances of prey rejection. Well known mechanisms include camouflage, mimicry, poison, venom, aposematic colouration, warning signals, as well as various other fight or flight enhancements. Endler (1991) suggested six stages of predation: encounter; detection, identification, approach, subjugation, and consumption. Arbuckle (2015) pointed out that most defence mechanisms will in principle act upon one or more of these phases. Camouflage takes various forms. It can involve either dull or bright colouration that mimics the background that the species is in (Stevens & Merilaita, 2011), or colours that mimic another species that is less likely to be attacked such as another predator, either Batesian or Müllerian (Bates, 1862, cited in Kazemi, Gamberale‐Stille, Wåtz, Wiklund & Leimar, 2018; Müller, 1876). In addition, some species such as octopuses are able to change their colour and texture to match their backgrounds and also mimic shapes (Laschi, 2017). Camouflage that at first appears to make the species stand out, known as dazzle patterns, confuse the observer and cause them to misjudge size, shape, speed and direction. This type of camouflage also enables a herd to blend together, thus making it more difficult for a predator to pick out one individual, as seen in the striped pattern of the zebra (How & 5 Zanker, 2014). A further type of camouflage seen in many animal species involves a darker colour on the dorsal surfaces and a paler colour on the ventral surface. This is known as countershading. Light sources from above create shadows on the underside of animals which help to determine shape. A paler underside offsets this shading to confuse the onlooker (Cuthill et al., 2016). This is seen in prey such as newts, but also predators wishing to avoid detection such as tigers. Aside from visual means of defence, the range of chemical defence mechanisms has received much debate within the scientific community. The definitions of venom and poison appear to change over time, and consequently cause ambiguity. Nelson et al. (2014) performed an extensive literature review and presented a conclusion that defines venom and poison, with the addition of a new term ‘toxungens’ which covers those which do not fit into either the poison or venom category. Venom is described as being deliberately delivered to a target animal via a wound. Poison is passively delivered, i.e. lacks a delivery method and is either ingested, inhaled or absorbed. The new term, ‘toxungens’ includes toxins that are delivered to the target animal with no accompanying wound. These defence mechanisms are well documented throughout the animal kingdom (Fry, 2015). Toxins, as described by Vogt (1970), should adhere to the following criteria: that it is a naturally occurring substance, either an entirely foreign substance to the victim or foreign in the dosage in which it is received by the victim, and that it should have a negative impact on the victim. There are three categories of toxins: biological toxins produced by living organisms, environmental toxins occurring naturally in the environment, and anthropogenic toxins which are man-made (Nelson et al. 2014). For this study, toxin will hereafter refer to biological toxins. A species that possesses a toxin can use this as a poison or venom. 6 However, Nelson et al. (2014) point out it is the relative dose of the toxin which determines the harm that the toxin does. Many substances are toxic in large enough quantities and some highly toxic substances are harmless in very small quantities. They are acquired in two different manners: through biosynthesis, in which the toxin is created by the species itself, or sequestration in which the toxin is up-taken by the species from the environment (Harris & Arbuckle, 2016). Venom is notably found in many marine species, as well as amphibians, reptiles, and insects (Harris & Arbuckle, 2016), although is also found in some mammals (Ligabue-Braun, Verli & Carlini, 2012). Some definitions of venom would also place some plant species as venomous (Maitai, Talalaj, Talalaj & Njoroge, 1981). There are numerous types of venom that are made up of either one or more toxins. The ways in which venoms are delivered are manifold, including stinging, biting, spraying, or injecting through spines or hairs. Venom is used as a defence mechanism as well as predation and occasionally intraspecific competition, whereas poison is used solely for defence (Harris & Arbuckle, 2016). Like venoms, poisons can be made up of one or more toxins. They are common in a vast array of species, famously plants, fungi, insects, reptiles, amphibians and marine