Copyright by Ivette Yañez 2019

Home , Canthigaster, Canthigaster rivulata, Takifugu, Takifugu xanthopterus

Ivette Yañez

2019

Investigation of genes coding for potential tetrodotoxin binding proteins in Canthigaster

valentini and Taricha granulosa.

Ivette Yañez, B.A.

A Thesis Submitted to the Department of Biology California State University Bakersfield

In Partial Fulfillment for the Degree of Master of Science, Biology

Summer 2019

4 Table of Contents

ACKNOWLEDGEMENTS ...... 6 ABSTRACT ...... 7 KEYWORDS ...... 8 INTRODUCTION ...... 9 INTRODUCTION TO TETRODOTOXIN...... 9 HYPOTHESIS OF TTX ORIGIN IN TOXIC ORGANISMS ...... 12 USES OF TTX...... 14 RESISTANCE OF TTX ...... 15 TTX AS A PHEROMONE ...... 16 PUFFERFISH SAXITOXIN AND TETRODOTOXIN BINDING PROTEINS (PSTBP) ...... 17 RESEARCH PURPOSE …...... 20 MATERIALS AND METHODS...... 27 DISSECTION ...... 27 COMPETITIVE INHIBITION ENZYMATIC IMMUNOASSAY (CIEIA) ...... 27 DNA EXTRACTION...... 27 PCR ANALYSIS ...... 28 CLONING OF CANTHIGASTER VALENTINI AMPLICONS ...... 29 COLONY PCR OF CLONED CANTHIGASTER VALENTINI AMPLICONS ...... 29 SEQUENCING OF CLONED CANTHIGASTER VALENTINI AMPLICONS ...... 29 CONTIG ASSEMBLY OF LARGE PRODUCT (5.1) ...... 30 TARICHA GRANULOSA ...... 30 RESULTS ...... 33 IDENTIFICATION OF TTX IN CANTHIGASTER VALENTINI ...... 33 IDENTIFICATION OF TTX-BP GENES IN CANTHIGASTER VALENTINI ...... 33 INVESTIGATION OF TTX-BP GENES IN TARICHA GRANULOSA ...... 34 INVESTIGATION OF BACTERIA IN TARICHA GRANULOSA ...... 35 DISCUSSION ...... 55 INVESTIGATION OF TTX IN CANTHIGASTER VALENTINI ...... 55 INVESTIGATION OF TTX-BP GENES IN CANTHIGASTER VALENTINI ...... 55 FUTURE WORK IN RESEARCH OF TTX BINDING PROTEINS IN CANTHIGASTER VALENTINI ...... 56 INVESTIGATION OF TTX BINDING PROTEIN GENES IN TARICHA GRANULOSA ...... 57 FUTURE WORK IN RESEARCH OF TTX BINDING PROTEINS IN TARICHA GRANULOSA ...... 58 INVESTIGATION OF BACTERIAL PRESSENCE IN TARICHA GRANULOSA ...... 58 CONCLUSION ...... 60 LITERATURE CITED ...... 61

5 Acknowledgements

I would like to express my gratitude to my thesis advisor and committee chair, Dr. Szick for her guidance and encouragement during these past three years. Support from Dr. Szick has allowed to pursue multiple research projects that have sprouted form my original research objective, thank you.

I would also like to acknowledge and thank the members of my thesis committee Dr.

Stokes and Dr. Francis for their support and guidance during my thesis research. Their patience and guidance have helped me become a better scientist.

I would like to extend my gratitude to the Student Research Scholars Grant, the Grants,

Research and Sponsored Programs (GRaSP), the Graduate Student-Faculty Collaborative

Initiative, and the Graduate Student Center at California State University, Bakersfield for its support of my research through grants and scholarships. Their support also provided me with opportunities to present my work at competitions and prepare for my defense.

I would like to thank Xochilt Ramirez for her contributions to this work as a research assistant. I would also like to thank Linsi Gutierrez and Aaron Baumgardner. This project would not have been possible without their help, support, and friendship.

Quisiera darle agradecimiento a mi familia. El apoyo de parte de mi madre Gloria

Valencia y hermanos, Rubi Contreras, Perla Cantu, y Salvador Yañez Valencia ha hecho toda le diferencia en mi carrera académica. Quisiere también reconocer a mi padre Salvador Yañez

Morales, aunque no pudo estar conmigo durante mis estudios, espero honorar su memoria con lo que he logrado durante estos tres años.

6 Abstract

Tetrodotoxin (TTX) is a powerful neurotoxin that selectively binds to voltage gated sodium channels in excitable tissues causing paralysis, asphyxiation, and death. TTX is found in a variety of unrelated taxa where multiple species have been reported to produce TTX binding proteins. Pufferfish in the Tetraodontidae family (mainly Takifugu spp.) have been demonstrated to accumulate high TTX concentrations however, the presence of TTX is still unknown for many pufferfish species. Canthigaster valentini is a species of pufferfish thought to be toxic but has yet to be assayed for the presence of TTX. Other species such as Taricha granulosa are also capable of accumulating high concentrations of TTX, however, the origin of TTX in this species is still debated. This study has multiple objectives i) determine if TTX is present in C. valentini liver tissues using CIEIA analysis, ii) identify possible TTX binding protein genes in C. valentini using PCR analysis with TTX binding protein gene specific primers, iii) identify possible TTX binding protein genes in T. granulosa using PCR analysis with TTX binding protein gene specific primers, and iv) to identify bacterial flora present on skin, intestine, liver, and ovary tissues using PCR analysis with 16S rDNA specific primers. In this study, the first instance of

TTX identification and quantification in C. valentini is reported. Also, BLAST analysis of amplified products identified gene fragments similar to PSTBPs found in Takifugu spp. and their ancestral TBT-bp2 genes in pufferfish. Here, it is suggested that C. valentini may possess TTX binding proteins similar to those found in Takifugu spp. pufferfish. PCR amplification of T. granulosa DNA using TTX binding protein gene specific primers produced an amplicon of 615 bp however, BLAST analysis did not produce a significant alignment. Although bacterial DNA was amplified from total DNA extracts from T. granulosa tissues, metagenomic analysis did not produce data.

7 Keywords

Tetrodotoxin, Tetrodotoxin binding protein, Taricha granulosa, Canthigaster valentini

8 Introduction

Introduction to Tetrodotoxin

Tetrodotoxin (TTX) is a potent neurotoxin found to be over 1000 times more toxic to humans than cyanide (as reviewed by Lago et al. 2015) (Figure 1A). Its name is derived from the

Tetraodontidae family, that contains pufferfish, the species from which TTX was originally isolated (Suehiro 1994). TTX selectively binds to the outer pore of voltage gated sodium channels that respond to and propagate action potentials passing through excitable tissues (Backx et al. 1992). This small electrical charge normally triggers the opening of the voltage gated sodium channel and allows movement of sodium ions down a concentration gradient into the cell. The influx of sodium ions depolarizes the cell, which produces and allows for propagation of action potentials (as reviewed by Bane et al. 2014). TTX bound to these channels blocks sodium from moving into the cells, thus blocking action potentials from firing in excitable muscle tissues, such as skeletal muscle, limiting life sustaining muscle movements. In addition to paralysis in muscles involved in movement, paralysis also occurs in the diaphragm, which eventually leads to asphyxiation and death (How et al. 2003).

Because of the targeted effects of TTX on the peripheral nervous system and muscles,

TTX has become a prime candidate for therapeutic use in humans. In particular, TTX is a promising contender as a therapeutic agent for pain (Marcil et al. 2006) because of its targeted action against voltage gated sodium channels commonly involved in pain reception (Nieto et al.

2012, Duran-Riveroll and Cembella 2017). Furthermore, rodent pain models have demonstrated that TTX provides pain relief without the undesirable side effects of traditional analgesics such as motor deficit and sedation (Marcil et al. 2006). Additionally, the use of TTX as a tool in neuroscience research provides a strong demand for TTX (reviewed by Chau et al. 2011). TTX can be extracted from various tissues of TTX harboring organisms (Noguchi and Hashimoto

9 1973, Daly et al. 1994), but pufferfish ovaries are preferred (Zhou and Shum 2003). However, this method is unsustainable because of the negative impacts to the populations from which TTX is extracted (reviewed by Chau et al. 2011). Thus, alternative sources of purified TTX are in demand. Although in vitro synthesis of TTX is possible, the unique structural arrangement and rare guanidinium group make the synthesis of TTX a complex and costly endeavor. Given that the exact TTX biosynthetic pathway remains unknown, chemical synthesis of TTX requires multiple steps to attain the complex binding of a single guanidinium moiety to a carbon backbone that is arranged in a cage-like form (reviewed by Chau et al. 2011, Duran-Riveroll and

Cembella 2017) (Figure 1A). Furthermore, efforts to purify or synthesize TTX have resulted in low TTX yields (reviewed by Chau et al. 2011). Because of the challenges of TTX synthesis and purification, there is an emphasis on identifying and exploiting natural TTX biosynthetic pathways. The current efforts to identify the TTX biosynthetic pathways mainly involve the investigation of TTX producing bacteria, the primary TTX producers in many TTX systems. It is also hypothesized that when the biosynthetic pathway is identified, novel enzymes corresponding to the production of the unique guanidinium group and structure of TTX will also be uncovered

(reviewed by Chau et al. 2011).

Apart from bacteria, a wide range of organisms can harbor TTX. Research has demonstrated that TTX is introduced to marine organisms, such as pufferfish and their toxic prey including crabs, starfish, gastropods, flatworms, and toxic pufferfish eggs (Noguchi et al. 2006a, reviewed by Noguchi et al. 2006b, Itoi et al. 2014, Yamada et al. 2017) by bacteria in the food web that in turn colonize the gut of the consumer (Duran-Riveroll and Cembella 2017).

However, organisms may also become toxic through consuming TTX laden food (Kono et al.

2008, Kudo et al. 2017).

10 TTX has been isolated from a diverse group of phylogenetically unrelated organisms such as pufferfish (Table 1) (Noguchi and Arakawa 2008, reviewed by Chau et al. 2011), marine worms, blue ringed octopuses, frogs, salamanders, and newts (Table 2) (reviewed by Chau et al.

2011). Of these organisms, pufferfish have been extensively studied for their ability to accumulate high concentrations of TTX. Many, but not all, of the pufferfish species in the

Tetraodotinae subfamily are toxic (Hashiguchi et al. 2015), and trade and handling of known toxin harboring pufferfish is strictly regulated due to the dangers of TTX poisoning in humans.

However, not all pufferfish within the Tetraodontidae family have been assayed for the presence of TTX. Due to this gap in knowledge, many pufferfish of unconfirmed toxin status are easily available through local and online aquarium trades. For example, Canthigaster spp. and the toxic

Takifugu spp. belong to the same family but separate subfamilies, the Canthigasterinae and the

Tetraodontinae subfamilies, respectively (Yamanoue et al. 2008, Yamanoue et al. 2009). Much less is known about the Canthigasterinae than the Tetraodontinae subfamily in regard to toxin presence.

Canthigaster valentini is a small tropical reef pufferfish suggested to be toxic, possibly harboring toxins commonly found in the Tetraodontidae family (Gladstone 1987a). C. valentini are small omnivorous puffers distributed throughout the Indo-Pacific Ocean. In nature, C. valentini live in male dominated harems where both males and females are territorial. Each female aggressively protects her territory from neighboring females and bachelor males, which is within a male’s territory (Gladstone 1987b, Gladstone and Westoby 1988). The suspected presence of TTX in C. valentini was deduced from observations of active predator avoidance of

C. valentini and its eggs (Gladstone 1987a). In a feeding study, C. valentini eggs and larvae were unpalatable to predatory fish. In the event that eggs, or larvae were initially mouthed, they were rejected and subsequently avoided (Gladstone 1987a). This avoidance allows for little parental

11 guarding after spawning; C. valentini eggs are deposited and subsequently left unattended with the assumed presence of TTX as their primary protection. This low parental investment possibly allows for energy resources to be invested in territory guarding (Gladstone and Westoby 1988).

Interestingly, the presumed presence of TTX in C. valentini has been suggested to be exploited by a Batesian mimic, Paraluteres prionurus (Caley and Schluter 2002). P. prionurus is a non- toxic reef fish virtually indistinguishable in appearance to C. valentini (Tyler 1966, Caley and

Schluter 2002). This mimicry relationship was tested in predator sensory trials. In these trials, plastic models varying in pattern similarity to C. valentini were also avoided by predatory fish.

This study illustrated how the risk of predation decreased with increased resemblance to C. valentini. The authors argue that the color pattern of C. valentini is associated with TTX in predators and provides an umbrella of protection to mimics and acts as selective pressure towards close resemblance.

Hypotheses of TTX origin in toxic organisms

There are currently two hypotheses on the source of TTX in TTX harboring organisms.

Although evidence supports an exogenous TTX origin in many systems, an endogenous origin is not completely excluded, and a consensus is yet to be agreed upon by the scientific community.

In marine systems, an exogenous origin is strongly supported with bacteria, specifically Vibrio spp. and Pseudomonas spp., as primary TTX producers (Moczydlowski 2013). These TTX producing bacteria are thought to be distributed throughout the food web or embedded on the skin and other tissues of their host, which are then toxified with TTX. TTX producing bacteria have been found throughout aquatic ecosystems including skin, liver, and ovary tissues of TTX harboring organisms (Jal and Khora 2015) (Table 3).

12 Of the organisms that harbor TTX, pufferfish have been the most extensively studied. In pufferfish, as in many marine TTX harboring systems, TTX production is exogenous in origin and is produced by bacteria (Yasumoto et al. 1986a). Research focused on the introduction of

TTX to pufferfish has determined that TTX can be sourced through both symbionts and diet

(Noguchi et al. 1987, Wu et al. 2005a, Noguchi et al. 2006a, Soong and Ventakesh 2006, Kono et al. 2008, Yu et al. 2011, Lago et al. 2015). The bacterial origin of TTX in pufferfish was confirmed through research that found TTX producing bacteria such as Vibrio spp. and

Pseudomonas spp. in pufferfish digestive tracts (Soong and Ventakesh 2006, Lago et al. 2015) and skin (Yotsu et al. 1987). TTX producing bacteria have also been isolated and identified from ovaries (Wu et al. 2005a, Yang et al. 2010), liver (Wu et al. 2005a), and intestines (Noguchi et al. 1987, Wu et al. 2005a, Yu et al. 2011) of toxic pufferfish.

The dietary origin of TTX in pufferfish was shown during independent feeding studies.

Non-toxic pufferfish fed pellets containing homogenized TTX-containing Takifugu poecilonotus liver became toxic after 30 days and remained toxic for at least 240 days after administration

(Kono et al. 2008). In the 240 days after administration, liver TTX concentration decreased while ovary and skin TTX concentration increased. Noguchi et al. (2006a) found that non-toxic pufferfish could be produced when cultured in net cages on sea or land and kept in filtered seawater preventing consumption of toxic prey. However, pufferfish that were cultured in unfiltered sea water with access to the sea floor could become toxic from consuming TTX- harboring prey introduced by the unfiltered sea water (Noguchi et al. 2006a). While the origin of

TTX has been determined in pufferfish, the mechanism of TTX accumulation/production is still under debate for other organisms.

There is support for both hypotheses on the origin of TTX in T. granulosa. The endogenous hypothesis argues that T. granulosa is capable of producing TTX de novo. This

13 hypothesis would require T. granulosa to possess genetic and cellular mechanisms for TTX production. While there is no evidence in the form of genes or proteins involved in TTX synthesis in TTX producing bacteria or TTX harboring organisms, this hypothesis is supported by findings that T. granulosa can increase TTX levels after long term captivity (Hanifin et al.

2002) and regenerate TTX levels after electric shock induced expulsion of TTX (Cardall et al.

2004).

The exogenous hypothesis argues that T. granulosa is not capable of producing TTX de novo and relies on an external source for TTX. This hypothesis is supported by recent research identifying Pseudomonas spp. in T. granulosa skin (a genus known to produce TTX), via 16S rDNA signatures (Szick, personal communication). Moreover, the findings from a feeding study showed that non-toxic Cynops pyrrhogaster newts became toxic when fed bloodworms supplemented with 20 µg of TTX (Kudo et al. 2017), which indicates that newts can sequester

TTX from their diets.

Uses of TTX

Organisms that accumulate TTX, produced by bacteria, can become highly toxic by concentrating TTX in various tissues (Yotsu et al. 1990, Noguchi and Arakawa 2008,

Hashiguchi et al. 2015). As seen in both pufferfish and newts, there are notable benefits for organisms that harbor TTX, the most important one being a defense mechanism against predators. Pufferfish, especially those within the Takifugu genus, can accumulate high concentrations of TTX in skin glands, ovaries, liver, and sometimes intestinal tissues with the location of accumulation differing between species (Hashiguchi et al. 2015). Pufferfish can also provide protection to their offspring by transferring TTX found in ovaries to the eggs (Itoi et al.

2014). The role of TTX in defense against predators was demonstrated in studies focusing on the

14 survivorship of artificially reared Takifugu rubripes larvae. In this research, larvae fed a TTX containing diet had significantly higher rates of survival (up to 74%) than artificially reared T. rubripes not fed a TTX containing diet when exposed to a pond environment containing predators (Lateoldbrax spp.) (Sakakura et al. 2016).

Toxic newts have also used TTX as a form of predator defense (Brodie 1968, Williams et al. 2010). Salamander species like Taricha granulosa (rough-skinned newt) strongly rely on

TTX for defense as they are especially vulnerable to predation due to their slow speed and soft bodies (as reviewed by Hanifin 2010). Research has demonstrated that toxic newts can extend the protection provided by TTX to their offspring with TTX levels in eggs positively correlated with the TTX levels found in the female parent (Hanifin et al. 2003, Gall et al. 2011).

Resistance of TTX

TTX harboring species require mechanisms to prevent autotoxicity. Members of the

Salamandridae family (to which T. granulosa belongs) (Hanifin and Gilly 2015) and

Tetraodontidae family (to which Takifugu spp. belong) have evolved mutations that alter the physical structure of the outer pore in their voltage gated sodium channels, which provide resistance to TTX by inhibiting TTX binding (Venkatesh et al. 2005, Soong and Venkatesh

2006). However, mutations in voltage gated sodium channel genes do not provide an absolute resistance to TTX. TTX resistant organisms can contain multiple mutations in voltage gated sodium channel genes, with each mutation contributing to an increased resistance to TTX.

Although the positions of the mutations may vary, they all result in changes in the structure of the outer vestibule of the voltage gated sodium channels, where TTX binds (Soong and

Venkatesh 2006).

15 Toxic organisms are not the only species to evolve mutations in their voltage gated sodium channels. Predators of these toxic organisms have been shown to also evolve similar mutations. For example, some populations of garter snakes have evolved resistance to TTX in order to consume their toxic prey (T. granulosa) without experiencing adverse effects. Extensive research has documented predation of toxic T. granulosa by garter snakes (Thamnophis spp.)

(Brodie and Brodie 1990, Greene and Feldman 2009). Garter snake populations that prey on toxic newts have been shown to have resistance to TTX (Brodie and Brodie 1990; 1991;

Feldman et al. 2012). Moreover, in garter snakes the degree of resistance to TTX also varies by population (Brodie and Brodie 1990, Soong and Venkatesh 2006).

Increased TTX resistance in garter snakes has been implicated as a catalyst in the coevolutionary arms race between T. granulosa and garter snakes (Brodie et al. 2005). The evolution of mutations that provide resistance to TTX in garter snakes act as selective pressure to an increase in TTX accumulation and resistance in T. granulosa. However, the increase in TTX concentration in T. granulosa acts as selective pressure for the evolution of stronger resistance mutations in garter snake populations. Thus, this arms race results in T. granulosa populations with extreme TTX levels and garter snake populations with high resistance to TTX (Brodie and

Brodie 1990, Brodie et al. 2002, Geffeney et al. 2005, Hanifin et al. 2008).

TTX as a pheromone

In addition to the defensive functions, TTX has also been hypothesized to function as a pheromone during spawning (Matsumura 1995). Spermating male Takifugu niphobles pufferfish were attracted to as little as 15 pM of TTX during maze trials, while females were not shown to be attracted by TTX. The attraction allowed for males to identify TTX released by females

16 during ovulation. This release of TTX is suspected to aid in the formation of schools and migration to breeding grounds during Takifugu niphobles breading seasons (Matsumura 1995).

Newts (Taricha torosa) have also been shown to respond to TTX from the skin of adult newts in their environment (Zimmer et al. 2006). Behavioral bioassays using T. torosa larvae showed that TTX from adult newts is responsible for larvae hiding behavior. TTX was identified as signal molecule through behavioral bioassays using skin swab samples, bath water from starved or fed newts, and a TTX standard as a scent sample with non-toxic frog samples as a control. This avoidance response to TTX is important for survival as larvae are cannibalized by adult newts (Zimmer et al. 2006). This research also showed that saxitoxin, a related but different guanidinium neurotoxin, does not produce a hiding response in T. torosa larvae.

Pufferfish Saxitoxin and Tetrodotoxin Binding Protein (PSTBP)

As previously noted, some TTX harboring organisms have mutations in genes that code for voltage gated sodium channels that reduce TTX binding affinity, however, such mutations do not provide complete protection against TTX. Proteins that bind TTX have been identified in a variety of marine organisms including shore crabs (Nagashima et al. 2002), electric eels (Miller et al. 1983), and gastropods (Hwang et al. 2007), but have been studied and characterized most extensively in pufferfish (Takifugu spp.) (Matsui et al. 2000, Yotsu-Yamashita et al. 2001,

Matsumoto et al. 2007). These proteins are hypothesized to assist in the accumulation and transport of TTX while also preventing the host organism from experiencing negative side effects related to the toxin by binding free toxins in the plasma and tissues of toxic organisms

(Hashiguchi et al. 2015).

A protein that was purified by Matsui et al. (2000) from the plasma of the pufferfish

Takifugu niphobles that demonstrated reversible binding affinity to TTX in laboratory binding

17 assays and was named TTX binding protein (TBP). This binding protein and its isoform were later isolated and characterized by Yotsu-Yamashita et al. (2001) and given the name pufferfish saxitoxin and tetrodotoxin binding proteins (PSTBPs) due to their ability to bind both saxitoxin and tetrodotoxin in the plasma of the pufferfish Fugu pardalis. Saxitoxin is a potent guanidinium neurotoxin that is structurally different (Figure 1B) but functionally similar to TTX. Saxitoxin contains two guanidinium groups that also bind to and block voltage gated sodium channels of excitable neurons (Huot et al. 1989). Saxitoxin is the leading toxin causing paralytic shellfish poisoning but is also present in pufferfish. Similar to TTX, saxitoxin is produced and introduced to marine and fresh water ecosystems by cyanobacteria and dinoflagellates (Hackett et al. 2013) through the food web or through a colonizing symbiont (Landsberg et al. 2006). Given the functional similarities between TTX and saxitoxin, it is not surprising that PSTBPs bind to both toxins.

Two highly similar (93%) isoforms, PSTBP1 and PSTBP2, were identified in the pufferfish Takifugu pardalis (Yotsu-Yamashita et al. 2001). Although PSTBP1 and PSTBP2 are highly similar, both isoforms show a distinct affinity to not only TTX, but other toxins commonly harbored by pufferfish. Recombinant PSTBP1 demonstrated a greater affinity to tributyltin (TBT, an organotin toxin) than recombinant PSTBP2 and did not demonstrate any detectable binding affinity to TTX. Tributyltin is a toxin composed of three butyl groups bound to a tin molecule that in high concentrations causes physical changes including masculinization of female fish and behavioral changes in many organisms leading to decline in population growth through toxin accumulation (Shimazaki et al. 2002). Many marine fish, including pufferfish can accumulate high levels of TBT used in boat paints that pollutes marine waters

(Miki et al. 2011). In contrast, recombinant PSTBP2 has weak TBT binding affinity, but strong

18 TTX binding affinity even after denaturation while recombinant PSTBP1 showed no such binding affinity to TTX regardless of structural arrangement (Satone et al. 2017).

Affinity of PSTBPs to TBT is not surprising, as phylogenetic analysis suggests that

PSTBP genes evolved from TBT binding protein genes (TBT-bp). This ancestral relationship is supported by a 47% sequence homology between PSTBPs and TBT-bp (Oba et al. 2007,

Hashiguchi et al. 2015). Phylogenetic analysis also suggests that PSTBP genes evolved specifically in Takifugu through multiple duplications and fusions of TBT-bp genes (Figure 2).

Hashiguchi et al. (2015) proposed that the duplication and divergence of an ancestral TBT-bp gene produced TBT-bp1 and TBT-bp2 genes, where TBT-bp2 genes also duplicated and diverged to produce TBT-bp2a and TBT-bp2b. TBT-bp2a genes duplicated individually and fused to produce PSTBP1 genes. While, TBT-2a and TBT-2b genes duplicated as a unit and fused to produce PSTBP2 genes. The evolution of PSTBPs in Takifugu pufferfish is further supported by a study that examined the presence of TBT-bp and PSTBP genes in multiple pufferfish species including, five TTX harboring Takifugu spp. (T. rubripes, T. pardalis, T. poecilonotus, T. niphobles, and T. snyderi), the toxic Tetradon nigroviridis, and three non-toxic pufferfish

(Lagocephalus wheeleri, L. gloveri, and Sphoeroides pachygaster). TBT-pb genes were found in all nine species of pufferfish studied regardless of toxin presence, whereas PSTBP genes were only present in 5 of the 6 toxic pufferfish species and none of the non-toxic species studied. All five of the toxic Takifugu species had at least two PSTBP genes. However, the single Tetradon nigrovidis (a toxic pufferfish related to Takifugu spp.) did not contain any PSTBP genes, showing that toxin levels are not reliable indicators of PSTBP gene presence while also supporting the hypothesis of PSTBP gene evolution specifically in Takifugu spp. (Hashiguchi et al. 2015). As PSTBP genes have only been identified in Takifugu spp. pufferfish, it is possible that other toxic pufferfish have evolved TTX binding proteins homologous to PSTBPs.

Research purpose

Takifugu spp. pufferfish are the most extensively studied species with regard to TTX due to their ability to accumulate TTX. Although many species and genera of pufferfish can also harbor TTX, much less is known of other genera of pufferfish within the same family as

Takifugu (Noguchi and Arakawa 2008). For example, the pufferfish Canthigaster valentini is suspected to contain TTX but has yet to be assayed for its presence (Gladstone 1987b, Caley and

Schluter 2002). In this study, the presence of TTX in C. valentini was investigated using competitive inhibition enzymatic immunoassay (CIEIA).

Given that a protein capable of binding TTX has been identified in some TTX harboring organisms, including Takifugu spp., Hemigrapsus sanguineus (shore crab), Electrophorus electricus (electric eel), and a variety of gastropods (Miller et al. 1983, Nagashima et al. 2002, and Hwang et al. 2007), it is hypothesized that TTX binding proteins are also present in other

TTX harboring organisms such as non-Takifugu pufferfish. In this study, the presence of TTX binding protein genes was investigated in C. valentini.

Although toxic newts are highly resistant to TTX (Hanifin and Gilly 2015), they are still susceptible to TTX at very high TTX concentrations (Brodie 1968). Other organisms with similar TTX resistance contain binding proteins, that are hypothesized to further contribute to that resistance (Nagashima et al. 2002). Thus, toxic newts including T. granulosa may contain binding protein genes. In this study the presence of TTX binding protein genes will be investigated in T. granulosa.

Furthermore, the origin of TTX in T. granulosa is still under investigation. Previous research was successful in isolating and identifying bacterial species present on newt skin and livers, however it did not provide a complete scope of the bacterial flora present in T. granulosa

20 tissues (Szick, personal communication). In this study, bacterial DNA presence in total DNA extracts from skin, liver, intestine, and ovary tissues will be investigated using metagenomic analysis in order to provide a more complete analysis on the presence of bacteria in T. granulosa,

Table 1. TTX bearing pufferfish within the Tetraodontidae family. Table adapted from Noguchi and Arakawa 2008 and Chau et al. 2011. Order Family Sub family Species Reference Tetraodontiformes Tetraodontidae Tetraodotinae Takifugu xanthopterus Nagashima et al. 2001 Takifugu niphobles Yu et al. 2004 Takifugu rubripes Wu et al. 2005a,b Fugu vermicularisa Lee et al. 2000, Noguchi et al.1987 Fugu pardalisa Yasumoto et al. 1986b Fugu poecilonotusa Yasumoto et al. 1986b, Yotsu et al. 1987 Takifugu snyderi Noguchi and Arakawa 2008 Takifugu porphyreus Noguchi and Arakawa 2008 Takifugu chinensis Noguchi and Arakawa 2008 Takifugu obscurus Noguchi and Arakawa 2008 Takifugu exascurus Noguchi and Arakawa 2008 Takifugu pseudommus Noguchi and Arakawa 2008 Takifugu chrysops Noguchi and Arakawa 2008 Takifugu xanthopeterus Noguchi and Arakawa 2008 Takifugu stictonotus Noguchi and Arakawa 2008 Tetradon albericulatus Noguchi and Arakawa 2008 Pleuranacathus sceleratus Noguchi and Arakawa 2008 Chelonodon patoca Noguchi and Arakawa 2008 Arothron firmamentum Noguchi and Arakawa 2008 Tetradon nigroviridis Noguchi and Arakawa 2008 Lagocephalus lunaris Noguchi and Arakawa 2008 Lagocephalus inermis Noguchi and Arakawa 2008 Takifugu flavidus Noguchi and Arakawa 2008 Tetradon steinadachneri Noguchi and Arakawa 2008 Canthigasterinae Canthigaster rivulata Noguchi and Arakawa 2008 Canthigaster valentini This study a The genus name Fugu is a lesser used synonym for Takifugu.

22 Table 2. Phylogenetic distribution of TTX harboring organisms excluding pufferfish and bacteria. List adapted from Chau et al. 2011.

Phylum Class Order Family Species (Binomial name) Reference Chordata Amphibia Anura Dendrobatidae Colostethus inguinalis Daly et al. 1994 Brachycephalidae Brachycephalus premix Pires et al. 2005 Brachycephalus ephippium Pires et al. 2002 Bufonidae Atelopus oxyrhynchus Mebs and Schmidt 1989 Chordata Salamandridae Notophthalmus viridescens Yotsu-Yamashita and Mebs 2003 Taricha torosa Brown and Mosher 1963 Taricha granulosa Buchwald et al. 1964 Cynops pyrrhogaster Yasumoto et al. 1988 Perciformes Gobliidae Gobius criniger Noguchi and Hashimoto 1973 Mullusca Cephalopoda Octopoda Octopodidae Hapalochlaena maculosa Hwang et al. 1989 Sheumack et al. 1984 Gastropoda Neogastropoda Nassariidae Niotha clathrata Jeon et al. 1984 Nassarius semiplicatus Wang et al. 2008 Muricidae Rapana rapiformis Hwang et al. 1991 Rapana venosa venosa Hwang et al. 1991 Sorbeoconcha Ranellidae Charonia sauliae Narita et al. 1981 Buccinidae Babylonia japonica Noguchi et al. 1981 Neotaeniogloss Naticidae Polinices didyma Shiu et al. 2003 Bursidae Tutufa lissostoma Noguchi et al. 1984 Nemertea Anopla Paleonemertea Cephalothricidae Cephalothrix rufifrons Carroll et al. 2003 Heteronemertea Lineidae Lineus longissimus Carroll et al. 2003 Enchinodermata Stelleroidea Paxillosida Astropectinidae Astropecten latespinosus Maruyama et al.1984 Astropecten polyacanthus Miyazawa et al.1985 Chaetognatha Sagittoidea Aphragmophora Sagittidae Faccisagitta enflata Thuesen and Kogure 1989 Parasagitta elegans Thuesen and Kogure 1989 Pterosagittidae Zonosagitta nagae Thuesen and Kogure 1989 Phargmophora Eukrohniidae Eukrohnia hamata Thuesen and Kogure 1989 Arthopoda Merostomata Xiphosura Cleroidea Carcinoscorpius rotundicauda Dao et al.2009, Kungsuwan et al.1987 Malacostraca Decapoda Carpiliidae Lophozozymus Pictor Tsai et al.1995 Xanthoidea Atergatis Floridius Noguchi et al.1983 Platyhelminthes Tubellaria Polycladida Planoceridae Planocera multitentaculata Miyazawa et al.1986 Dinoflagellata Dinophyceae Conyaulacales Goniodomateceae Alexandrium tamatense Kodama et al.1996

Figure 1. Structure of A) tetrodotoxin, B) saxitoxin, and C) tributyltin molecules. Guanidinium groups are indicated by red circles. (modified from Noguchi and Akarawa 2008, Pearson et al. 2010, and Antizar-Ladislao 2007 respectively)

24 Table 3. TTX producing bacteria found on TTX-harboring organisms. Adapted from Jal and Khora (2015) Year TTX producing bacteria Source of bacterial isolate Reference 1986 Vibrio sp. Xanthid crab, Atergatis floridus Noguchi et al. 1986 1986 Pseudomonas sp. Alga, Jania sp. Yasumoto et al. 1986 1987 Pseudomonas sp. Pufferfish, Fugu poecilonotus Yotsu et al. 1987 1987 Vibrio alginolyticus Pufferfish, Fugu vermicularis vermicularis Noguchi et al. 1987 1987 V. alginolyticus, V. damseia Starfish, Astropecen polycanthus Narita et al. 1987 1987 V. fishcheri Xanthid crab, Atergatis floridus Sugita et al. 1987 1989 Shewanella putrefaciens Pufferfish, Takifugu niphobles Matsui et al. 1989 1989 Alteromonas sp., Bacillus sp., Pseudomonas sp., Vibrio sp. Octopus maculosus Hwang et al. 1989 1990 Listonella pelagia, A. tetraodonis, Shewanella alga Red alga and Pufferfish Simidu et al. 1990 1994 V. alginolyticus, Aeromonas sp. Lined moon shell, Natica lineata Hwang et al. 1994 1995 Aeromonas sp., Pseudomonas sp., Plesiomonas sp., V. alginolyticus, Gastropod, Niotha clathrata Cheng et al. 1995 V. parahaemolyticus 2000 Vibrio sp. Intestine of Fugu vermicularis radiatus Lee et al. 2000 2003 Vibrio sp. Nemertean worm Carroll et al. 2003 2004 Aeromonas molluscorum Bivalve mollus Galbis et al. 2004 2004 Microbacterium arabinogalactanolyticum Ovary of Pufferfish, Takifugu niphobles Yu et al. 2004 2004 Serratia marcescens Skin of Pufferfish, Chelonodon patoca Yu et al. 2004 2005 Bacillus sp., Actinomycete sp. Ovary, liver, intestine of Fugu rubripes Wu et al. 2005a 2005 Nocardiopsis dassonvillei Ovary of Fugu rubripes Wu et al. 2005b 2007 Roseobacter Copepod Pseudocaligus fugu which is present as ectoparasite on Maran et al. 2007 the Pufferfish, Takifugu pardalis 2008 Vibrio, Shewanella, Marinomonas, Tenacibaculum,Aeromonas Digestive gland and muscle of marine gastropod, Nassarius Wang et al. 2008 semiplicatu 2010 Aeromonas Ovary of Pufferfish, Takifugu obscurus Yang et al. 2010 2010 Lysinibacillus fusiformis Liver of Pufferfish, Fugu obscurus Wang et al. 2010 2011 Raoultella terrigena Pufferfish, Takifugu niphobles Yu et al. 2011 2011 Shewanella sp. Ovary of Pufferfish Takifugu oblongus Hein et al. 2011 2012 Shewanella putrefaciens Pufferfish, Lagocephalus lunaris Auawithoothij et al. 2012 2014 Providencia rettgeri Pufferfish, Lagocephalus sp. Tu et al. 2014 2015 Enterobacter cloaca, Rahnella aquatilis Gobyfish, Yongeichthys criniger Wei et al. 2015

Figure 2. Illustration of the series of proposed duplications in and fusion events Takifugu spp. of TBT-bp genes that are hypothesized to have led to the evolution of PSTBP genes found in pufferfish. Genes marked with an asterisk (*) indicate genes that are still known to exist in pufferfish. Modified from Hashiguchi et al. 2015

26 Materials & Methods

Dissection

Two Canthigaster valentini were purchased from an online tropical fish vendor

(AZgardens.com). The fish were euthanized upon arrival by submersion in 1% MS-222. Muscle and liver tissue samples were dissected from both individuals and stored at -80°C.

Two groups of adult Taricha granulosa were euthanized by submersion in 1% MS-222 for at least 30 minutes. Livers were aseptically dissected from three T. granulosa for the investigation of TTX binding protein genes, and were designated as Group 1. Livers, intestines, ovaries, heart, and skin tissue samples were also aseptically dissected from three different T. granulosa newts than mentioned above for metagenomic analysis, and were designated as Group

2. Dissected tissues and euthanized specimens were stored at -80°C.

Competitive Inhibition Enzymatic Immunoassay (CIEIA)

To investigate the presence of TTX in C. valentini, liver samples were analyzed using

CIEIA (Stokes et al. 2012). Liver tissues were chosen over other tissues because in other pufferfish species livers are known to contain high concentrations of TTX. Two C. valetini liver samples were used for the extraction of TTX. Liver samples were weighed and ground in 600 µL of 0.1M acetic acid extraction buffer followed by boiling and centrifugation as described by

Hanifin et al. (2002). Extracted TTX was diluted in 1% BSA-PBS at a 1:1 ratio in order to fall within the detectable range (10 to 500 ng/mL) for this assay. Diluted TTX from each liver was run in triplicate for CIEIA analysis (Stokes et al. 2012). Extracted TTX and diluted samples were stored at -80°C.

DNA extraction

27 DNA was extracted from C. valentini (19-25mg) liver and muscle tissue samples using the DNeasy Blood and Tissue Kit (QIAGEN) following the manufacturers protocol. Extracted

DNA was stored at -20°C.

DNA was extracted from Group 1 liver (~25mg) samples for the investigation of TTX binding protein genes in T. granulosa using DNeasy Blood and Tissue Kit (QIAGEN) following the protocol provided, including the recommended extended lysis incubation of three hours instead of the standard one-hour incubation. DNA was also extracted from Group 2 tissue samples for the investigation of TTX producing bacteria in T. granulosa using DNeasy Blood and Tissue Kit (QIAGEN) with a modified protocol for livers and intestines and the provided protocol for ovary, heart, and skin tissue samples. The modified protocol included a reduced tissue sample (10-19mg), grinding of tissues with a pestle, an extended lysis incubation time

(48hrs), and double wash steps. Extracted DNA was analyzed for purity and quantity using a

Nanodrop One (ThermoFisher). Extracted DNA was stored at -20° C.

PCR analysis

PCR with primers CYTB-FISH (designed for this study) and CYTB3 F/R, targeting the cytochrome B in pufferfish and newts respectively, and extracted C. valentini and T. granulosa

DNA was used to confirm the effectiveness of DNA extractions. To investigate the presence of

TTX binding protein genes and PSTBP genes in C. valentini and Group 1 T. granulosa DNA,

PCR was performed with primers TBP1 F/R, TBP2 F/R, P1/P2 (Yotsu-Yamashita et al. 2001), and PSTBP-5M-1/PSTBP-5M-2 (Hashiguchi et al. 2015).

In order to obtain a single amplicon for each reaction, amplified products from PCR utilizing the TBP1 F/R primers were isolated and purified using the Gel Extraction Kit

(QIAGEN) and re-amplified using TBP1 F/R primers and 5µL of extracted product as template.

Cloning of Canthigaster valentini amplicons

In order to further isolate products amplified from C. valentini DNA using TBP1 F/R primers, re-amplified PCR products were ligated into the pGEM-T Easy Vector (Promega) overnight at +4°C. Ligated samples were transformed into JM109 competent cells (Promega) following the manufacturers protocols. Transformation reactions were plated onto fresh

LB/AMP/x-gal/IPTG plates and incubated overnight at 37°C.

Colony PCR of cloned Canthigaster valentini amplicons

Successfully cloned fragments were selected for colony PCR using white/blue colony screening. A single colony was transferred using a sterile pipette tip and diluted into 30µl of sterile water. PCR using 5µl of each colony dilution as template and TBP1 F/R as primers was used in order to identify the presence and size of cloned fragments. Conditions for PCR with primers TBP1 F/R are listed in Table 4. Plasmids were isolated from each colony from overnight cultures using QIAprep Spin Miniprep Kit (Qiagen) according to manufacturer protocol.

Sequencing of cloned Canthigaster valentini amplicons

Purified plasmids were sequenced via Sanger sequencing at Laragen Inc. (Culver City,

USA) using primers Sp6/T7 and TBP1 F/R. Primers Sp6/T7 bind to the Sp6 and T7 promoter regions within the cloning vector flanking the cloned insert. Sequencing with Sp6/T7 allows for a more complete read of the ligated amplicon. Chromatograms were visualized using Geneious

R11 (https://www.geneious.com) software. Sequences were used to search the NCBI database using the BLASTn and BLASTx algorithms (http://ncbi.nlm.nih.gov/blast) (Altschul et al.

1990). Glycerol stocks (25% [v/v]) of clones were prepared and stored in -80°C.

Contig assembly of large product (5.1)

The insert of a single plasmid (designated as 5.1) was too large to be sequenced in its entirety with TBP1 F/R primers. Thus, primers TBP4 F/R and TBP5 F/R were designed to bind within previously identified sequences and amplify un-sequenced regions (Figure 3). A contig was constructed from the sequences amplified with primers TBP1 F/R, TBP4 F/R, and TBP5

F/R. Primer sequences and their PCR conditions listed in Table 4.

Taricha granulosa

DNA from Group 2 liver, intestine, ovary, and skin tissues was used as template for PCR with primers 8F/1492R targeting 16S rDNA from bacteria present on dissected tissues prior to

DNA extraction. Metagenomic analysis of 40µL of purified DNA from liver, intestine, ovary, and skin samples was conducted by Laragen Inc. (Culver City, USA).

All PCR mixtures contained 0.536µM of each forward and reverse primer set, 1X Gotaq

Green Master Mix (Promega), 5µL of DNA, and nuclease-free water to 28µL total volume. A negative control reaction was also prepared for each set of reactions which included all previously described components except a DNA template. PCR products were analyzed by agarose gel electrophoresis. Agarose gels consisted of 1% (w/v) agarose in TBE buffer and

0.01% of SYBR Safe DNA gel stain (Invitrogen). Primers and their respective PCR conditions are listed in Table 4. A portion (7-25µL) of each reaction was loaded onto agarose gels and electrophorized for 30 minutes to 1 hour at 100-130 volts. Gels were imaged using a Gel Doc

2000 system (Bio-Rad) under UV transillumination.

Table 4. Primers and PCR conditions used in this study. Fragment PCR conditions Primer Sequence (5’-3’) Gene Length Initial Final Source Denaturation Annealing Extension Cycles (bp) denaturation extension 8F AGAGTTTGATCCTGGCTCAG Edwards et al. 1989 16S 1,438 1492R GGTTAC CTTGTTACGACTT rRNA 94° C, 94° C, 53° C, 72° C, 2 35 72° C, Stackenbrandt and 4min 30s 30s min 10min Liesack 1993 CYTB3-F CCTGGTAATAGCCACCGCTT 631 Szick, personal CYTB3-R GTTGGCTACCAGCGATCAGA communication Cytb 94° C, 94° C, 48° C, 72° C, 1 38 72° C, CYTB-FISH F CTACGGCTGACTAATTCG 2min 30s 1min min 8min 590 This study CYTB-FISH R CTGGTTTAATCTGGGC TBP1-F GTTCTACTGCTGATGCTGGCG 388 TBP1-R ACACACGTCTCAAAGAACTTCAC Szick, personal TBP2-F CTAAAGAATAACTCCTGTATGACG communication 377 TBP2-R CGTACATACAGGAGTTATTCTTTAG Possible -- 95° C, 51° C, 72° C, 30 72° C, TBP4-F TGTATAACCTTCAACACCAA TTX-bp 30s 1min 2min 7min TBP4-R ATATTAAACAGTGTAAGGCAG 2000 TBP5-F CCTTGAACAGGAAGAAACGT This study 1326 TBP5-R CCTTGATTGTTTCGGCATAC P1*† GCYCCNGCYCCNGARGA 296/ 830/ -- 95° C, 55° C, 72° C, 30 72° C, Yatsu-Yamashita et *† 30s 1min 2min 7min al. (2001) P2 ACSACBCCYTTYTCYTCCAT PSTBP 836 PSTBP-5M-1* AKCCVTTCDTCTAYGAYGGHGTTK 574 94° C, 98° C, 56° C, 68° C, 30 -- Hashiguchi et al. PSTBP-5M-2* GMAACDCCRTAGAHGAABGGMT 2min 10s 30s 2min (2015) *Degenerate primer codes B= C, G, or T; R= A or G; M= A or C; Y= C or T; D= A, G, or T; V= A, C, or G; H= A, C, or G; N= A, C, G, or T. † Primers P1/P2 produce three amplicon sizes when amplifying PSTBP genes

Figure 3. Illustration of binding sites for primers TBP1 F (5’-GTTCTACTGCTGATGCTGGCG-3’) / TBP1 R (5’- ACACACGTCTCAAAGAACTTCAC-3’), TBP4 F(5’- TGTATAACCTTCAACACCAA-3’) / TBP4 R (5’- ATATTAAACAGTGTAAGGCAG-3’), and TBP5 F(5’-CCTTGAACAGGAAGAAACGT-3’) / TBP5 R (5’- CCTTGATTGTTTCGGCATAC-3’) used to sequence amplicon 5.1 in its entirety. Primers TBP1F/R produced a 2,512 bp amplicon. Primers TBP4 F/R produced a 2,000 bp amplicon. Primers TPB5 F/R produced a 1,326 bp amplicon.

32 Results

Identification of TTX in Canthigaster valentini

In this study, liver samples from two C. valentini pufferfish were used for TTX identification using competitive inhibition enzymatic immunoassay (CIEIA) analysis. CIEIA analysis showed that C. valentini liver 1, that weighed 6.7 mg, contained 60.99 ng of TTX while liver 2, that weighed 27.6 mg, contained 242.74 ng of TTX. When adjusted for weight liver 1 and liver 2 samples contained 8.76 and 9.10 ng of TTX per mg of liver tissue respectively (Table

5).

Identification of TTX-bp genes in Canthigaster valentini

In this study, DNA was extracted from C. valentini tissues to investigate the presence of

TTX binding protein genes. To confirm the effectiveness of DNA extractions, Cyt b genes were amplified in C. valentini DNA using CytB-FISH F/R primers (Figure 4), a negative control did not produce an amplicon (data not shown). PCR analysis was used to identify the presence of potential TTX-bp genes using primers specific to PSTBP genes and C. valentini DNA as template. Primers TBP2 F/R, P1/P2, and PSTBP-5M-1/PSTBP-5M-2 did not produce an amplicon (Figure 5), negative control reactions also did not produce a product (data not shown).

Primers TBP1 F/R resulted in products that varied in size and intensity (Figure 6), the negative control reaction did not yield an amplicon (data not shown).

In order to isolate individual amplicons produced by TBP1 PCR, amplicons E4-E8 were extracted from agarose gels, purified and reamplified using TBP1 F/R primers (Figure 7). This reamplification produced single amplicons of expected size, except in the negative control reaction which did not produce an amplicon (data not shown). PCR products from reamplification of extracted E4-E8 amplicons were cloned using JM109 cells. Colony PCR from

33 selected white colonies showed singular amplicons of varying sizes when amplified with TBP1

F/R primers (Figure 8), except in the negative control reaction, which did not produce an amplicon (data not shown). Plasmids 5.1, 5.2, 7.1, 7.2, 8.1, 8.2, 8.4, 8.5, and 8.6 containing successfully cloned amplicons were isolated and sequenced. BLAST searches using amplicon

5.2, 7.1, 7.2, and 8.5 sequences produced a significant alignment to non TTX binding protein or

TBT binding protein gene sequences (Table 6). BLAST searches for amplicon 8.1, 8.2, and 8.6 did not produce a significant alignment.

The largest amplicon (5.1) produced was 2,512 bp long. Due to current length limitations in Sanger sequencing, primers TBP4 F/R and TBP5 F/R were designed to sequence amplicon 5.1 in its entirety. A contig of the complete 5.1 amplicon sequence was produced from sequences derived from TPB1 F/R, TBP4 F/R, TBP5 F/R, and T7/SP6 (plasmid specific primers that target promoter regions flanking the amplicon insert site on the pGEMT-easy vector) primers (Figure

9). A search with successfully cloned and sequenced inserts, including 5.1 and 8.4 (Figure 10,

Figure 11 respectively), against the NCBI database using BLASTn and BLASTx algorithms

(http://ncbi.nlm.nih.gov/blast) (Altschul et al. 1990) showed that two fragments shared sequence identity with both TBT-bp and PSTBP genes (Figure 12, Figure 13). Amplicon 5.1 aligned to two portions of the PSTBP1 gene in the pufferfish Takifugu rubripes and to one section of the TBT-

2a gene of the pufferfish Takifugu poeilonotus (Figure 14). Amplicon 8.4 aligned with the

PSTBP2 gene in the pufferfish Takifugu snyderi and the TBT-bp2 gene in the pufferfish Takifugu poeilonotus (Figure 15).

Investigation of TTX-bp genes in Taricha granulosa

T. granulosa may contain genes that code for TTX binding proteins; to investigate this

DNA was extracted from T. granulosa livers and used in PCR analysis with PSTBP specific

34 primers. In order to confirm the efficacy of the DNA extraction, Cytochrome B (Cyt b) genes were amplified in Group 1 T. granulosa DNA using CytB3 F/R primers except in the negative control reaction (data not shown) (Figure 16A). To investigate the presence of possible TTX binding protein genes, PCR with T. granulosa DNA was used as template with primers designed to target PSTBP genes. Of the primers used, primers TBP2 F/R, P1/P2, and PSTBP-5M-

1/PSTBP-5M-2 did not produce any amplicons (Figure 17). However, primers TBP1 F/R resulted in multiple products (Figure 18). Of the products amplified, the most intense amplicon

(615 bp) was isolated using a gel extraction kit (QIAGEN gel extraction kit). Isolated amplicons were reamplified resulting in single products using primers TBP1 F/R (Figure 19). Extracted amplicons were sequenced and used to search the NCBI database using the BLASTn and

BLASTx algorithms (http://ncbi.nlm.nih.gov/blast) (Altschul et al. 1990). A search against the

NCBI database did not produce a match for the amplicon sequences (data not shown).

Investigation of bacteria in Taricha granulosa

PCR and metagenomic analysis of DNA from T. granulosa skin, liver, ovaries, and intestines was used in order to provide a more complete analysis on the presence of bacteria in T. granulosa. PCR amplification of cyt b genes produced amplicons of expected size from all reactions (Figure 16B) except the negative control (data not shown). PCR amplification of DNA extracted from T. granulosa tissue samples using primers 8F and 1492R produced amplicons of expected size (Figure 20) except in the negative control reaction (data not shown). Metagenomic analysis was performed at Laragen Inc. (Culver City, USA) to attempt to identify the bacterial

DNA amplified in newt tissue DNA extractions. However, metagenomic analysis failed to produce sequence data.

Table 5. TTX concentration in C. valentini liver tissue samples assayed with CIEIA. C. valentini specimens were purchased from online aquarium fish vendor and previously not assayed for toxicity. Sample Sample mass assayed (mg) TTX/ sample (ng) TTX/mg liver Liver 1 6.7 60.99 9.10 Liver 2 27.6 242.74 8.79

Table 6. BLAST results for sequences amplified from C. valentini DNA extracted form liver and muscle tissues. C. valentini DNA was amplified using forward primer TBP1 F (5’-GTTCTACTGCTGATGCTGGCG-3’) and the reverse primer TBP1 R (5’- ACACACGTCTCAAAGAACTTCAC-3’), gel extracted, reamplified with primers TBP1 F/R, and cloned into JM109 competent cells using pGEMT- easy vector. Amplicon Sequence Producing Significant Alignment Max E- Identity Genbank accession score value (%) no. PREDICTED: Takifugu rubripes sparc/osteonectin, cwcv and kazal-like 5.2 115 4e-22 96 XM_003961234.2 domains proteoglycan (testican) 2 (spock2), mRNA PREDICTED: Nannospalax galili aconitase 1 7.1 86.1 2e-13 93 XM_008851092.1 (Aco1), mRNA PREDICTED: Takifugu rubripes sparc/osteonectin, cwcv and kazal-like 7.2 91.6 5e-15 98 XM_003961234.2 domains proteoglycan (testican) 2 (spock2), mRNA 8.5 Oryzias latipes strain HSOK chromosome 21 267 2e-67 92 CP020641.1

Figure 4. Agarose gel electrophoresis of Cytb gene amplified in C. valentini DNA. DNA was amplified using the forward primer CytB-Fish F (5’-CTACGGCTGACTAATTCG-3’) and the reverse primer CytB-Fish R (5’-CTGGTTTAATCTGGGC-3’) which produced an amplicon of expected size (590 bp). Lane markers (L1, L2, M1, and M2) represent the C. valentini tissue (L=Liver, M=Muscle) from which DNA was extracted. Gel was visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 5. Gel electrophoresis of PCR using C. valentini DNA isolated from muscle (M1 and M2) and liver (L1 and L2) tissues using primers A) TBP2 F (5’-CTAAAGAATAACTCCTGTATGACG-3’) and TBP2 R (5’-CGTACATACAGGAGTTATTCTTTAG-3’), B) P1 (5’-GCYCCNGCYCCNGARGA-3’) and P2 (5’-ACSACBCCYTTYTCYTCCAT-3’), and C) PSTBP-5M-1(5’- AKCCVTTCDTCTAYGAYGGHGTTK-3’) and PSTBP-5M-2 (5’-GMAACDCCRTAGAHGAABGGMT-3’). Primers failed to produce amplicons. Gels were visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 6. Agarose gel electrophoresis of TBT/PSTBP genes amplified in C. valentini DNA. DNA was amplified using the forward primer TBP1 F (5’-GTTCTACTGCTGATGCTGGCG-3’) and the reverse primer TBP1 R (5’-ACACACGTCTCAAAGAACTTCAC- 3’) which produced amplicons of various sizes. Lane markers (L1, L2, M1, and M2) represent the C. valentini tissue (L=Liver, M=Muscle) from which DNA was extracted. Amplicons extracted from the gel are marked E4-E8. Gel was visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 7. Agarose gel electrophoresis of reamplified amplicons (E4-E8) using forward primer TBP1 F (5’- GTTCTACTGCTGATGCTGGCG-3’) and the reverse primer TBP1 R (5’-ACACACGTCTCAAAGAACTTCAC-3’). Amplicons E4- E8 were amplified using forward primer TBP1 F and reverse primer TBP1 R from C.valentini DNA. Reamplification with primers TBP1 F/R produced bands of expected size corresponding to the original product that was used as template. Gel was visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 8. Agarose gel electrophoresis of colony PCR using primers using forward primer TBP1 F (5’- GTTCTACTGCTGATGCTGGCG-3’) and the reverse primer TBP1 R (5’-ACACACGTCTCAAAGAACTTCAC-3’) and cloned C. valentini amplicons form reamplified E4-E8 extracted amplicons. DNA was originally extracted from C. valentini muscle and liver tissues. Lane markers represent the individual colonies used in colony PCR. Gel was visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 9. Contig aligned from sequence amplified with TTX-binding protein gene primers TBP1, TBP4, and TBP 5 and cloning primers T7 and SP6. Sequence identity represented by the green bar (100%) and consensus sequence shown by the black bar above the green bar. Shorter black bars represent individual sequenced regions used to produce contig with corresponding primer to the left.

> sequence of amplicon 5.1 GTTCTACTGCTGATGCTGGCGGTGCTCGGCACCAGTGCAGCACCGGTTCAAGAAGCATGTCCCAATCTGAACAAGACCCTGACCAAAGCCGACCTGCAGAGGGTGAG TCAGGACCGGTCCAGGCTGGGCAGAACCTCTGATCTTCATTCCTTTGACTCTGCTCCAGGTTGAAGGTGACTGGGTTCTGGTCTGGCTCAACGCTGACGCTAACACA ACTTTTGATGGTTGGAAGAAAATCAAAAGCTCCCTCGTCCATAACAGAGTCCGCTCCGGTGTCATCGACTTCACCGAGAGGAACATGCTCTCGTGAGGCCGTCGTCT TTCCCGGTAGTTCAGTTCCAGGAACCACTAGAGCTGACCTTGTGTGATTTTCAGGGGTAATTTCTGTATAACCTTCAACACCAAAATGACAGCATCCTCTGAGGACC AGGACCTGTTCAGCTACTCCTCTGGCAGTGAGTGTACACACGCACACACACACACACACACACACACACTTTTCTTTGGGTTTGATTCAATTCTATTTAGTTTTATT TACATCGAGTATTTTGCGATAAAATTGTGTCTAGGTGCTTCACAGAGGCAGAGGCCCAGAGGCTGAACCCTCAGGTAGGCACAGTGGCAGGAAGAACTCCCTTGAAC AGGAAGAAACCTTGAGCTAGATCGGGCCCACAAGGAGGAACCGTTCAGCTGAAAGTGGGCTAGGTTAAGGAGGGAGACTGTTAAGTTACATTGCATTTTGGAGGACC CAGAAGCGGAACACAGGAGTGTTTACGGTTTATTTTACACAATTTGAATAAGAACGAAAAACAGGCGATTAACAGAAGGCATTCCACCGGAGGAAAGGGAACAAACT AAAAGCGAGCCGGTGACCTGACGGCCTACGCATTGGGATCGAGGGCTGCGGGCAATTGAATGGAAAACACACATACCACAGGGCAAAGCGACCTCGGGGGAAGAACA CACGGGTTTAGTTCACGGAATTCAGACAGAGTTCATCGAAACATATGTTCCACCGTACCACTGAGCACACAGTACAATCAGACGCCGGTGTGTGCGTGTCGGTTCCT CAAGAAGGTTCACTAATGAAGATCCGGCACAGGTGTGCAGCACCAGCTCACCTCCGGGTCGCCCACGCCCACTGGAAAAAACAAAAACACACACCACAACGCCGCCG GCTCTGCCGATCCCTGACAGGAGACAGGACTCATTAAATCACTTTCTGTTTGCTGTAGCCTCTGACTCAGGCAGCCAGAGCGACTCAGCCTGTCCCTGCGATGAGCT GTCAACAACAACCACAGAGAAACTGAACATCATGCTCACGTGACCCCCGACAACCAACCACACACCACATTCAGCACCGGAACACAACAGCACAACACCTGTAACAA CAGCAACCTGCCTTGCACTCCCCACAAGGAGTACCGAGACCCTTCACAGAACAGGCAACTCGAATCAACATTCAAAAGGGAAAACTGAAGTGACTCTATGCTTGAAG CATAGTCTCAACAGTCGACCAGTCTATACAGAAATCAACAGCTAACTCAACAGCCTCCCCTAATGACTACTTCCTGTATCTTTGGCTTGGGAATGACTTGCCTACTG GAAGTTATCACAACTCTCTCTGGACTGTGTCCAACTGCTGGTGATTTGTCCTGTCCTGAAGTCCATGTCAAGGGCTTCTCCTCGATCATGGGACTTGAGGGCTGGTC CATCCTCAGCTGGATCCAGTTGTGTTTTCTTGTGGTGTTAGCTCTGGGACCATCAAGGAGTCCTCTTCAGGGGGTTGTTTCAGTGGGTCCTTGGACAACTACAGCAC TTGGGTCCATAGGCGCTGGGCCCTAAACTGGGACCAAGTCACTGTTGTAGCGCTTCAACCCGTCATGATGCAGTACCTTGCTGAGTTTTCTTCCCTGGATCTCATAT AATATGGGGCCCCGGTATGCCGAAACAATCAAGGGGCCTATCTCAGTTTTAATTTTCAAAAGTTTGGGAATTCTATTTACACTATGTACACAACCAACAGCTGCAGG AACACAAGTCCGTTGTGCCCTGTAAGGTGTCAATAACAACACTTGAATCTTCGTCAGATGGATGCGCTCACGGGTAGAAAAGTCCACAATAATGGCAGCCTGACACT GGTTGTTGTGGAGGAGACTACATCACAGAACAAGAGATCTTCCTCCTAGAACTTTACCGACGTAGGAGATGTAGGAGATGTCAAAACACTTTCTGTCTGCTGTTGCC TCTGACTCAGGCACAGTGAACAATTCAGGATAACCCTGCGATGAGCCGTGAGCGTCAACAACAACCACAAAGCATCTCACCATCACACCCATGTGACCCGTGACAAC CAATCACAAGCTGCCTTACACTGTTTAATATATTCCTTCTTAAGAAAAGTGTGTTTTGCTTTTCTTTGTGTGTGTGTGTGTGTATCAGGGGTGGAAGAGACGGGAGC CATAATCCAATTTCAAGACAACGCCACGGTGAAGTTCTTTGAGACGTGTGT

Figure 10. Complete nucleotide sequence for the large C. valentini amplicon (5.1) amplified using TBP1 F/R primers, cloned, and sequenced using fragments amplified with primers TBP4 F/R and TBP5 F/R from C. valentini tissues.

>Sequence for amplicon 8.4 GTTCTACTGCTGATGCTGGCGGTGCTCGGCACCAGAGCAGCACCGGATCCAGAAGAATGTTCCAGTCTGACCGAGACCCTGACCAAAGCCGACA TGGACAGGGTGAGTCAGGACCGGTCCAGGCCGGGCAGAACCTCTAATCTTCATTCCTTTGCTTCTGCTCCAGGTTGAAGGTGACTGGGTTCTGGT CTGGTCCAGCACGGCAAACGCCACCAACCAAGCCACCGACCTTGATTGGCCCAACCTCATCAGCACCTACGTCCAGATGAGGCTCCACGGTGAC GTCATCACCTTCAAAGAGGAGAGCCGGTTTTTGTAAGCAAAAAGCAGCTGGAGCTTGACCTCCAGGTCCTGGAAAAGCTCTGCTTCTCCTCATGA AACTGCTCTTCTCTGACTTTCAGGGATAAAACCTGCGTTTCCTTCTCCTCCAACCTGTCGGTGACCTCTGAAGACCAGCAGGAGTTCAGTGTAACA TCTGCCAGTCAGTCTCTGCCGGCAAATTCACATTTTTACACACTTCCTGTTGCTGAAAGCTGTTTCTTGTGTGTCAGTGATGGAGAAGGATGGAGT CCAGACATCAGAGGACGACAACGCCAAGGTGAAGTTCTTTGAGACGTGTGT

Figure 11. Complete nucleotide sequence from small C. valentini amplicon (8.4) amplified with primers TBP1 F/R with DNA from C. valentini tissues.

Figure 12. BLASTx alignment of the sequences of cloned amplicon 5.1 amplified using primers TBP1 F/R form C. valentini DNA. Amplicon 5.1 shared sequence identity with TBT-bp gene in Takifugu poecilonotus (left) and PSTBP1 gene in Takifugu rubripes (right).

Figure 13. BLASTx alignment of the sequences of cloned amplicon 8.4 amplified using primers TBP1 F/R form C. valentini DNA. Amplicon 8.4 shared sequence identity with PSTBP gene in Takifugu snyderi (left) and TBT-bp gene in Lagocephalus wheeleri (right).

47 A)PSTBP1 C. valentini 167 VEGDWVLVWLNADANTTFDGWKKIKSSLVHNRVRSGVIDFTERNMLs*grrlsr*fssrN H*S*PCVIFRGNFCITFNTKMTASSEDQDLFSYSSGS 457 T. rubripes 42 VSGDWVLVWYISDNISTSNEWTKLKTSYVEQRVHSGVIRFTERNML------KNNSCMTFKTNMTAGPQGQNTFIYTSGT 115 Conserved V GDWVLVW +D +T + W K+K+S V RV SGVI FTERNML + N C+TF T MTA + Q+ F Y+SG+

C. valentini 167 VEGDWVLVWLNADANTTFDGWKKIKSSLVHNRVRSGVIDFTERNMLs*grrlsr*fssrN H*S*PCVIFRGNFCITFNTKMTASSEDQDLFSYSSGS 457 T. rubripes 214 VSGDWVLVWYISDNISTSNEWTKLKTSYVEQRVHSGVIRFTERNML------KNNSCMTFKTNMTAGPEGQNTFIYTSGT 296 Conserved V GDWVLVW +D +T + W K+K+S V RV SGVI FTERNML + N C+TF T MTA E Q+ F Y+SG+

B) TBT-bp2a C. valentini 19 AVLGTSAAPVQEACPNLNKTLTKADLQRVSQDWVLVWLNADANTTFDGWKKIKSSLVHNRVRSGVIDFTERNMLs*grrlsr*fssrN 346 T. poecilonotus 14 AVLGIRAAPAPEECHNLTKGVTKADVQSVSGDWVLVWSIDENSTISDDWKKLKSSHVELRIHSGVIDYTERNLL------87 Conserved AVLG AAP E C NL K +TKAD+Q VS DWVLVW + +T D WKK+KSS V R+ SGVID+TERN+L

C. valentini 347 H*S*PCVIFRGNFCITFNTKMTASSEDQDLFSYSS 451 T. poecilonotus 88 ------KNNSCMTFKTNMTAGPEGQNTFIYTS 113 Conserved + N C+TF T MTA E Q+ F Y+S

Figure 14. Alignments of amplicon 5.1 amplified and cloned from C. valentini using the forward primer TBP1 F (5’ GTTCTACTGCTGATGCTGGCG 3’) and the reverse primer TBP1 R (5’ ACACACGTCTCAAAGAACTTCAC 3’) matching to A) PSTBP1 (Takifugu rubripes BAR88528.1) and B) TBT-bp2a (Takifugu poeilonotus BAR88522.1). The C. valentini amplicon aligned in two sections of the PSTBP1 gene in T. rubripes. Alignments produced from NCBI BLASTx matches. Sequences conserved in 5.1 and 8.4 alignments are highlighted in yellow while sequences conserved only in 5.1 are highlighted in green.

48 A)PSTBP2 C. valentini 1 VLLLMLAVLGTRAAPDPEECSSLTETLTKADMDRVSQDWVLVWSSTANATNQATDLDW--PNLISTYVQMRLHGDVITFKEESRFL 316 T. snyderi 8 VLLLMLAVFGIRAAPAPEECHNLTKPVTKADVQSVSGDWVLVWS-VANTTER-----WICENLTSSYVEFKLHSDVIEYTERNLFL 87 Conserved VLLLMLAV G RAAP PEEC +LT+ +TKAD+ VS DWVLVWS AN T + W NL S+YV+ +LH DVI + E + FL

C. valentini 399 DKTCVSFSSNLSVTSEDQQEFSVTSASQS-----LPANSHFYTLPVAESCFLCVS 552 T. snyderi 88 GNSCISFYSNLSASTEKQQQFSLNNLQMEEKGVVRPFNDN-GTVKFFETCVDCLS 141 Conserved +C+SF SNLS ++E QQ+FS+ + P N + T+ E+C C+S

B)TBT-bp2 C. valentini 28 GTRAAPDPEECSSLTETLTKADMDRVSQDWVLVWSSTANATNQATDLD-WPNLISTYVQMRLHGDVITFKEESRF 313 L. wheeleri 17 GTRAAPAPEDCPKLTKALTKADLQRVSGDWVLVWSQ--GTSDNATEAELWKKLMSTHAEFRLHSDTIVYRERNLF 89 Conserved GTRAAP PE+C LT+ LTKAD+ RVS DWVLVWS ++ AT+ + W L+ST+ + RLH D I ++E + F

C. valentini 398 RDKTCVSFSSNLSVTSEDQQEFSVTSASQS------LPANSHFYTLPVAESCFLCVS 552 L. wheeleri 90 SENLCISFNANMSLSSENQQEFTITSHSMEENGVVTLLNDNA---TMKFYETCADCLS 144 Conserved + C+SF++N+S++SE+QQEF++TS S L N+ T+ E+C C+S

Figure 15. Alignments of amplicon 8.4 amplified and cloned from C. valentini using the forward primer TBP1 F (5’ GTTCTACTGCTGATGCTGGCG 3’) and the reverse primer TBP1 R (5’ ACACACGTCTCAAAGAACTTCAC 3’) matching to A) PSTBP2 (Takifugu snyderi BAR88538.1) and B) TBT-bp2 (Lagocephalus wheeleri BAR88517.1). Alignments produced from NCBI BLASTx matches. Sequences conserved in 5.1 and 8.4 alignments are highlighted in yellow while sequences conserved only in 8.4 are highlighted in blue.

Figure 16. Agarose gel electrophoresis of Cytb gene amplified in T. granulosa DNA isolated from A) liver tissue. DNA was amplified using the forward primer CytB F (5’-CCTGGTAATAGCCACCGCTT-3’) and the reverse primer CytB R (5’- GTTGGCTACCAGCGATCAGA-3’) which produced an amplicon of expected size (631 bp). Lane markers (1, 2, and 3) represent the newts from which DNA was extracted in Group 1. B) Intestine, ovary, skin, and liver tissues. DNA was amplified using the forward primer CytB F and the reverse primer CytB R. Lane markers represent the newts form which DNA was extracted in Group 2 whith the organ of origin labled above lane markers. Gels were visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 17. Gel electrophoresis of PCR using T. granulosa DNA isolated from liver tissues in Group 1 amplified using primers A) TBP2 F (5’-CTAAAGAATAACTCCTGTATGACG-3’) and TBP2 R (5’-CGTACATACAGGAGTTATTCTTTAG-3’), B) P1 (5’- GCYCCNGCYCCNGARGA-3’) and P2 (5’-ACSACBCCYTTYTCYTCCAT-3’), and C) PSTBP-5M-1(5’- AKCCVTTCDTCTAYGAYGGHGTTK-3’) and PSTBP-5M-2 (5’-GMAACDCCRTAGAHGAABGGMT-3’). Primers failed to produce amplicons. Gels were visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 18. Agarose gel electrophoresis of possible TTX bp genes amplified in T. granulosa DNA isolated from liver tissue. DNA was amplified using the forward primer TBP1 F (5’-GTTCTACTGCTGATGCTGGCG-3’) and the reverse primer TBP1 R (5’- ACACACGTCTCAAAGAACTTCAC-3’) which resulted in products of various sizes. The brightest and sharpest bands (615 bp) were not of expected size (296, 830, or 836 bp). Lane markers (1, 2, and 3) represent the newts from which DNA was extracted. Gel was visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 19. Agarose gel electrophoresis of reamplification of possible TTX bp genes amplified from extracted T. granulosa DNA isolated from liver tissue. Amplicons were extracted from gel electrophoresis of T. granulosa DNA amplified using the forward primer TBP1 F (5’-GTTCTACTGCTGATGCTGGCG-3’) and the reverse primer TBP1 R (5’-ACACACGTCTCAAAGAACTTCAC- 3’) which produced products of various sizes. Lane markers (1, 2, and 3) represent the newts from which DNA was extracted. Gel was visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

Figure 20 . Agarose gel electrophoresis showing the amplification of the 16S rRNA gene of bacterial DNA amplified from total DNA extracts of T. granulosa tissues. DNA was amplified using the forward primer 8F (5’-AGAGTTTGATCCTGGCTCAG-3’), and reverse primer 1492R (5’-GGTTAC CTTGTTACGACTT-3’). The expected amplicon length is 1484 bp. Lane markers represent the newts form which DNA was extracted in Group 2 whith the organ of origin labled above lane markers. Gels were visualized on a 1% (w/v) agarose gel with 0.01% SYBR® Safe.

54 Discussion

Investigation of TTX in Canthigaster valentini

Canthigaster valentini is a tropical reef pufferfish previously thought to be toxic (Gladstone

1987b). In a previous study, Caley and Schluter (2002) attributed its unpalatability and the presence of a Batesian mimic to the presence of TTX, although the toxin was not identified. In this study, TTX was identified and quantified for the first time in C. valentini using CIEIA

(Stokes et al. 2012). Broader conclusions regarding the TTX levels of wild C. valentini populations cannot be made due to the small sample size and commercial origin of specimens.

Thus, further research is required to confidently provide a scope on the variability in TTX levels in wild C. valentini populations. Nonetheless, conclusions can be made on the availability of toxic pufferfish in the aquarium fish trade as toxic C. valentini pufferfish were purchased from an online vendor for this study. When considering the evidence of highly toxic pufferfish declining in toxicity over long captivity (Noguchi et al. 2006), it is likely that freshly caught C. valentini specimens have higher TTX concentrations. However, comparison of the TTX levels between the specimens used in this study and wild C. valentini populations cannot be made because this is the first reported instance of TTX in C. valentini and the specific origin of the specimens used in this study is unknown.

Investigation of TTX-bp genes in Canthigaster valentini

Due to the identification of TTX in C. valentini liver tissues and evidence that other species of toxic pufferfish are known to produce TTX binding proteins, PSTBP specific primers were used to determine if possible PSTBP genes are present in C. valentini. PSTBP specific primers amplified two fragments with sequence identity similar to PSTBP and TBT-bp genes in other species, indicating that toxin binding protein genes are present in C. valentini. However, because

55 of limitations in primer specificity the complete gene was not amplified or sequenced. Although genetic analysis of PSTBP genes suggested that the genes likely evolved specifically within the

Takifugu genus (Hashiguchi et al. 2015), toxin binding protein genes may have evolved independently within other pufferfish genera such as Canthigaster. If toxin binding protein genes in Canthigaster spp. evolved from TBT-bp genes independently from Takifugu PSTBPs, the toxin binding proteins in Canthigaster spp. may share similar domains important to toxin binding in PSTBPs. Although, the PSTBP domains that involve toxin binding have not yet been determined, several regions from C. valentini 5.1 and 8.4 alignments have 100% identity to

PSTBP1 and PSTBP2 (Figure 14-15 highlighted in yellow),100% identity to PSTBP2 in only 5.1 alignments (Figure 14 highlighted in green), or high conservation in only 8.4 alignments (Figure

14 highlighted in blue). If protein products from 5.1 and 8.4 alignments function in a similar manner to PSTBP1 and PSTBP2 proteins, the differences observed may be important for differential binding to TTX. Thus, further research is required to identify the complete gene present in C. valentini.

Future work in research of TTX binding proteins in Canthigaster valentini

In this study, only a portion of potential TTX binding protein genes were identified in C. valentini using PCR with PSTBP specific primers. mRNA analysis can be used to further identify the genes that the amplified sequences belong to. Techniques like 5’ rapid amplification of cDNA ends (RACE) using primers designed from the known sequences identified in this study may amplify a more complete mRNA and cDNA from TTX-bp genes in C. valentini.

Complete cDNA sequences may provide sufficient genetic information assist in the distinction between TBT-bp, PSTBP, or possible novel TTX-bp genes in C. valentini.

56 Investigation of TTX binding protein genes in Taricha granulosa

Multiple TTX harboring organisms are capable of producing proteins that bind to TTX

(Miller et al. 1983, Matsui et al. 2000, Yotsu-Yamashita et al. 2001, Nagashima et al. 2002,

Hwang et al. 2007, Matsumoto et al. 2010). Given that T. granulosa can accumulate high TTX concentrations, rough skinned newts may contain genes that code for proteins capable of binding

TTX. To investigate the presence of toxin binding protein genes, DNA was extracted from T. granulosa livers and used for PCR with primers targeting known PSTBP genes from other organisms. Primers designed from PSTBP genes were used because of the absence of other TTX binding protein amino acid sequences in the NCBI database. In this study PSTBP specific primers amplified a fragment of 615 bp from T. granulosa DNA. However, BLAST analysis did not identify any similar sequences in the NCBI database to the 615 bp T. granulosa amplicon.

The absence of similar sequences can be attributed to a lack of salamander, including T. granulosa, genome sequences in the database. Furthermore, if T. granulosa does cotain TTX-bp genes a common ancestral TTX-bp gene is unlikely as pufferfish and newts are distantly related.

A convergent evolution event in the two subjects for a toxin binding protein is a likelier explanation. If convergent evolution of toxin binding proteins did occur, toxin specific domains may be shared between the two proteins while the rest of the protein unrelated to toxin binding would be too dissimilar to produce a match in the NCBI Database.

Although the techniques used in this study for identification of TTX binding protein genes in

T. granulosa were unsuccessful, other methods could aid in the identification of these proteins.

For example, TTX binding proteins have been isolated in the hemolymph the shore crab,

Hemigrapsus sanguineus, using a series of gel filtration steps (Nagashima et al. 2002). A TTX binding protein was also identified from homogenized tissues of five gastropods using similar filtration methods to Nagashima et al. (2002) (Hwang et al. 2007).

57 The identification of PSTBPs in pufferfish also preceded the identification of PSTBP genes.

A TTX binding protein was isolated from Takifugu niphobles blood using gel filtration and column filtration followed by SDS-PAGE and mass spectrometry (Matsui et al. 2000). PSTBPs were sequenced a year later from proteins isolated from pufferfish plasma using various gel and column filtration methods. The isolated protein was assayed for binding affinity to saxitoxin and tetrodotoxin. Primers designed from the amino acid sequences of the isolated protein were used to obtain the complete gene sequence (Yotsu-Yamashita et al. 2001).

Future work in research of TTX binding proteins in Taricha granulosa

As the investigation of PSTBP genes in T. granulosa provided inconclusive results, future work can focus on the isolation and identification of proteins capable of binding TTX from toxic specimens. Considering that TTX binding proteins have been isolated from pufferfish plasma and crab hemolymph (Nagashima et al. 2002), TTX binding proteins may be present in the blood plasma of T. granulosa. To investigate the presence of TTX binding proteins in the plasma of T. granulosa, plasma from toxic specimens can be subjected to gel filtration techniques similar to those that isolated TTX binding proteins in other toxic species (Miller et al. 1983, Matsui et al.

2000, Yotsu-Yamashita et al. 2001, Nagashima et al. 2002). However, TTX affinity assays should also be conducted in order to confirm the binding capabilities of any isolated proteins.

Investigation of bacterial presence in T. granulosa

The rough-skinned newt, Taricha granulosa, can accumulate high TTX levels (Hanifin 1999,

Stokes et al. 2015). However, the mechanism for toxin accumulation in T. granulosa is yet to be identified (Hanifin et al. 2002, Cardall et al. 2004). There are several lines of evidence that support both an endogenous origin and an exogenous origin TTX in T. granulosa. Research

58 supporting the exogenous origin hypothesis includes research demonstrating the presence of some known TTX producing bacteria genera on the skin and liver of toxic T. granulosa (Szick, personal communication). To further investigate the presence and distribution of bacteria throughout T. granulosa tissues, total DNA was extracted from skin, liver, intestine, and ovarian tissues. In this study, bacterial 16S rDNA was amplified in total DNA extracts from T. granulosa tissue. This finding is consistent with previous research that identified bacteria (some of which are known TTX producing species) on the skin and livers of T. granulosa (Szick, personal communication). The presence of bacteria on T. granulosa tissues is also consistent with research focusing on the identification of bacteria in other TTX harboring organisms (Yotsu et al. 1987, Wu et al. 2005a). TTX producing bacteria have been identified and isolated from a variety of tissues from TTX harboring pufferfish such as ovaries (Wu et al. 2005a, Yang et al.

2010), liver (Wu et al. 2005a), and intestines (Noguchi et al. 1987, Wu et al. 2005a, Yu et al.

2011).

Although bacterial DNA could be amplified through PCR, bacteria were not identified from total DNA extracts using metagenomic analysis. Metagenomic analysis was used in this study because it could provide a more complete description of the microbiota within the tissues of T. granulosa. Failure of metagenomic analysis could be due to a number of factors such as low bacterial DNA density as well as a high newt to bacterial DNA ratio. As there are no techniques available to separate bacterial DNA from newt DNA, interference of newt DNA in metagenomic analysis cannot be ruled out. However, there are techniques that may be favorable in identifying bacterial DNA amplified from T. granulosa samples. Cloning of amplified 16S rDNA was also considered for this study but was not selected due to possible bias against DNA species of lower frequency. Although cloning may express a frequency bias, it may allow for the identification of most abundant bacterial DNA present in total DNA extracts of newt tissues.

Conclusion

This study recorded the first instance of TTX identification and quantification in C. valentini. Liver samples from two C. valentini specimens showed TTX levels of 8.79 and 9.10 ng TTX / mg liver. This study also identified sequences similar to PSTBPs and TBT-bps genes from C. valentini DNA, indicating the presence of possible TTX-bp genes.

This study also focused on identifying TTX-bp genes in T. granulosa. Although individual amplicons suspected to belong to TTX-bp genes were produced, the amplicon sequences did not identify any similar sequence within the NCBI Database. This study also attempted to identify bacteria present in the tissues of T. granulosa though metagenomic analysis. Although bacterial DNA was amplified, bacterial species were not identified.

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