Isolation and Characterization of Natural Microbiota Within

Home , Nudibranch, Sea slug, Soft coral nudibranch, Tochuina, Tritonia (gastropod), Tritoniidae

ISOLATION AND CHARACTERIZATION OF NATURAL MICROBIOTA WITHIN

THE INTESTINAL LINING OF TRITONIA TETRAQUETRA

A University Thesis Presented to the Faculty

California State University, East Bay

In Partial Fulfillment

of the Requirements for the Degree

Master of Science in Biological Sciences

Supraja Kolluri

March 2017

Abstract

Tritonia tetraquetra is a marine nudibranch found in sub-tidal waters in the Northern

Pacific Ocean. Due to the simplicity of the nudibranch brain, T. tetraquetra is continuously used as a model system in neurophysiological studies in connection with animal behavior. However, little to no evidence regarding its digestive system is available. It is well-known that symbiotic relationships between bacteria and marine invertebrates illustrate biological adaptations to life in aquatic environments. This research expands the knowledge of T. tetraquetra physiology by speciating and understanding the symbiotic relationships afforded to T. tetraquetra by the natural microbiota within its intestine. It can be hypothesized that the normal microbiota of the intestinal tract of T. tetraquetra confers protection to the organism due to the presence and binding of bacterial species to the intestinal walls. Also, the main food source for T. tetraquetra is Ptilosarcus gurneyi, a soft coral more commonly known as the orange sea pen, and produces a chemical toxin, ptilosarcenone, which can be assimilated by T. tetraquetra as a defense mechanism against predators.

This study looks at the symbiotic relationships within the intestinal lining of T. tetraquetra and aims to determine categories of species that make up the microflora lining the intestinal tract that helps to protect the nudibranch from invasive species and break down the P. gurneyi toxin. The study uses the intestinal lining of healthy and sickly

T. tetraquetra in order to cultivate, isolate and speciate the natural bacteria that thrive in the organism’s intestine. The project utilized traditional Sanger sequencing to determine

iii the makeup of natural bacterial species within intestinal lining of T. tetraquetra. The 16s rRNA gene enabled identification of twenty species of bacteria within the intestinal lining of the sea slug, including a large number of species from the genera Marinobacter and

Pseudoalteromonas. Interestingly, some of the bacterial populations identified are known to share symbiotic relationships with other marine invertebrates. The microbiota present in the mucosal lining of the slug could provide the organism with secondary metabolites and physical protection due to the bacterial metabolic properties and presence along the surface area of the digestive tract. Understanding the subtleties of relationships amongst these bacterial species, which act as a barrier from invasive bacterial species from taking residence in the intestinal lining, allow for the characterization of the innate immune system of T. tetraquetra.

Three bacterial species were isolated and determined to be potential invasive species within the intestinal lining of sick T. tetraquetra. These bacterial species were not seemingly present within the digestive systems of healthy sea slugs. As such, studying the natural microbiota within the intestinal lining of T. tetraquetra leads to a better understanding of how the sea slug digests foodstuffs and how the microflora inhabiting the intestinal lining confers a natural barrier to infection upon the organism. In addition to the defense provided to the sea slug by the microbiota, the secretion of toxins assimilated from its prey allows T. tetraquetra a chemical defense. This study, using the intestinal lining of T. tetraquetra aims to expand the understanding of endosymbiotic relationships and alter how current models of normal microbiota in marine invertebrates are viewed.

ISOLATION AND CHARACTERIZATION OF NATURAL MICROBIOTA WITHIN

THE INTESTINAL LINING OF TRITONIA TETRAQUETRA

Supraja Kolluri

Approved: Date:

______Kenneth Curr, Ph.D. ______Christopher Baysdorfer, Ph.D. ______Carol Lauzon, Ph.D. ______Claudia Uhde-Stone, Ph.D.

Acknowledgements

I would like to express my gratitude to my advisor, Dr. Kenneth Curr, whose expertise and understanding of cell biology and microbiology added considerably to my graduate experience. I started my graduate degree with a lot of passion, but little idea of what I wanted to do and where I wanted to go. Thank you for honing my skills and constantly driving me to think outside of the box. You got me where I am today. I would also like to thank Dr. Fred Bauzon for providing me with direction, support, and for lending me an ear in times of frustration. I must acknowledge Dr. Jim Murray for his technical knowledge, suggestions, and providing my slug samples, without which my project would not have gotten off the ground floor. In addition, I would like to thank the members of my committee, Dr. Chris Baysdorfer, Dr. Claudia Uhde-Stone, and Dr. Carol

Lauzon for the assistance they gave me throughout my research project. I truly appreciate each one of you.

Finally, a very special thanks goes out to my husband, Bhargav, my son, Ved, and my family: Mom, Dad, Akshay, Aunty, and Uncle. Thank you for dealing with the homework assignments, the missed holidays, the emotional ups and downs, and for standing by me every single step of the way. I doubt that I will ever be able to convey my appreciation fully, but I owe all of you my eternal gratitude. This is really for you.

Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

List of Tables ...... ix

List of Figures ...... x

Chapter 1: Symbiosis and Nudibranchs ...... 1

1.1 Symbiotic Enteric Bacteria in Marine Invertebrates ...... 2

1.2 Introduction to Nudibranchs ...... 4

1.3 The Nudibranch Tritonia tetraquetra...... 5

1.4 Uptake of Cnidarian Toxin by Nudibranchs ...... 7

1.5 Ptilosarcus gurneyi: the Orange Sea Pen ...... 8

1.6 Tritonia tetraquetra and Invasive Species ...... 8

1.7 Significance of Research ...... 9

Chapter 2: Research Hypothesis and Specific Aims ...... 11

Chapter 3: Materials and Methods ...... 13

3.1 Isolation of Genomic DNA ...... 13

3.2 Amplification of Genomic DNA ...... 14

3.3 Sequencing Techniques ...... 15

3.4 Biochemical Profiling of Bacterial Species (data shown in appendix) ...... 16

vii

3.5 Determination of Novel Species not Identified in Healthy Slugs ...... 18

Chapter 4: Isolation and Characterization of Bacterial Species ...... 20

4.1 Isolation and Amplification of Genomic Bacteria ...... 20

4.2 Characterization of Bacterial Species by Sanger Sequencing ...... 22

4.3 Profiling of Bacterial Species by Differential Staining and Biochemical Tests ...... 32

4.4 Bacterial Phylogeny Characterization ...... 33

4.5 Characterization of Invasive Species of T. tetraquetra ...... 37

Chapter 5: Conclusions ...... 41

References ...... 43

Appendix ...... 48

viii

List of Tables

Table 1: Primer Pair used for 16s rRNA gene 20

Table 2: NCBI matches of isolated genomic DNA of bacterial species from anterior intestine 24

Table 3: NCBI matches of isolated genomic DNA of bacterial species from posterior intestine 25

Table 4: NCBI matches of isolated genomic DNA of bacteria found in “sick” T. tetraquetra 38

List of Figures

Figure 1: Adult Tritonia tetraquetra in laboratory settings 6

Figure 2: Gel of genomic DNA of bacterial colonies cultured from T. tetraquetra intestine 21

Figure 3: Sample results of Sanger Sequencing Procedure visualized in Chromas software 23

Figure 4: Bacterial genera identified within the anterior intestine of T. tetraquetra 26

Figure 5: Bacterial genera identified within the posterior intestine of T. tetraquetra 28

Figure 6: Phylogram created from NCBI matches of bacteria from anterior intestine 35

Figure 7: Phylogram created from NCBI matches of bacteria from posterior intestine 36

Chapter 1: Symbiosis and Nudibranchs

Symbiosis is an umbrella term that encompasses a large variety of biological interactions in the plant and animal kingdoms. Symbiotic relationships describe a close bond between two species that allow organisms to survive, sometimes at the cost of one of the species in the relationship. These relationships increase both biological and ecological diversity, and add to the evolutionary ability of certain organisms to survive.

Some symbiotic relationships are obligate, meaning both species in the relationship rely on one another to survive (such as lichens), while other symbionts are facultative, and can survive without the help of the secondary species in the relationship (Hickman et al.,

2004).

Symbiotic interactions are categorized into three broad categories: mutualism, commensalism, or parasitism. In a mutualistic relationship, both parties benefit from one other, either by providing a service or a resource. For example, in pollination, pollen is provided by the flower (a resource), while seed dispersal is provided by the pollinators (a service). In a commensal relationship, one organism benefits while the other organism in the relationship is unaffected (whales and barnacles). In a parasitic relationship, one organism (i.e. a tapeworm) benefits by taking from and/or harming its host (i.e. humans).

Symbionts can also be categorized in two distinct processes: ectosymbiosis and endosymbiosis. Endosymbionts live within their hosts (such as protozoans that live

within termites), using their organs for fuel, while ectosymbionts (for example, lice living on human scalps) live either on or around their hosts.

1.1 Symbiotic Enteric Bacteria in Marine Invertebrates

Endosymbiotic relationships between bacteria and marine invertebrates illustrate biological adaptations to life in aquatic environments, some of which can be very extreme. Chemosynthetic bacteria in some marine invertebrates, such as saltwater clams, aid in detoxification of toxic sulfides and fix carbon for the metabolism of the host

(Stewart and Cavanaugh, 2006). Shipworms, bivalve mollusks of the family Teredinidae, are known to host bacteria that help in the digestion of the wood that shipworms consume

(Betcher et al., 2012). When these bacterial species are grown in vitro, they have been known to produce cellulases that depolymerize oligocellosaccharides, which lends to the conclusion that the bacteria aid in the digestion of wood and other oligocellosaccharides in the shipworm diet (Betcher et al., 2012). Although distribution of bacteria in the intestines within the shipworm was found to vary, there was a marked difference in the abundance of the bacteria Bankia setacea (83.8cells/field with a range of 0-224 cells/field) in the caecum, which is considered to be the beginning of the large intestine; lower amounts of the same bacteria, 0.88 cells/field with a range of 0-14 cells/field, were present in the caecum compared to the rest of intestine (Betcher et al., 2012). As food passes through the caecum before entering the intestine, it could be surmised that there would be more microorganisms in the intestine proper, where more food would be broken down over a longer amount of time.

As stated by King and colleagues in their 2012 paper, species of Vibrio present in the intestines of eastern oysters (Crassostrea virginica) have been found to be important for nutrition acquisition. The stomach and intestines of oysters included Actinobacteria,

Chloroflexi, Firmicutes, Planctomycetes, Proteobacteria, Spartobacteria, Chloroflexi,

Mollicutes, Planctomycetes and Spartobacteria in addition to several species of Vibrio

(King et al., 2012). As in the shipworm study by Betcher and colleagues, the abundance and distribution of bacteria varied greatly between the stomach and intestine. The differences in composition among gut systems could have been due to many variables, such as intestinal anatomy, physiology, diet of the organism and any additional symbiotic relations that may have co-evolved with the organism. Considering this, it could be hypothesized that some functions are conserved across microbacterial environments independent of phylogenetic composition. Hence, the seemingly unique bacterial composition of the eastern oyster intestine could be similar to that of other marine invertebrate species (King et al., 2012).

Symbiotic relationships between bacteria and marine invertebrates are ubiquitous in natural conditions. Although many marine mollusks are known to contain endosymbiotic bacteria, little information has been published regarding symbionts of nudibranchs. Dendrodoris nigra, a nudibranch mollusk of the Indo-West Pacific region, has been found to have symbiotic bacteria in its vestibular gland that may aid in the reproduction of the organism, aiding in the nutrition and care of larvae (Klussmann-Kolb and Brodie, 1999). This may be one of the few cases to date that has led to valuable

information regarding symbiotic bacteria within nudibranchs, yet evidence regarding endosymbionts within the intestines of nudibranchs is still scarce.

1.2 Introduction to Nudibranchs

Nudibranchs are soft-bodied marine gastropod mollusk invertebrates that tend to shed their shells after their larval stage (Thomson, 2009). Often referred to as sea slugs, these organisms can survive in a variety of depths of salt water, but are known as benthic creatures that crawl along the ocean substrate. Nudibranchs vary in their bright colors and body forms, which have evolved as camouflage from predators and are found worldwide.

Although the body forms of nudibranchs can vary greatly between species, all are bilaterally symmetrical, have a simple gut and radula for feeding and are soft-bodied with no shell in their adult forms. They also have dorsal projections known as cerata. These cerata, gill-like appendages, help the organism with gas exchange and act as an exit point for the secretion of waste products (Hickman et al., 2004).

Nudibranchs prey on soft corals, sponges, jellies and even other nudibranchs.

Sponges and jellies are evolutionarily primitive organisms with stinging cells called nematocytes that contain biologically active chemical compounds used for defense from predators (Faulkner and Ghiselin, 1983). As these organisms are the major source of food for most nudibranchs, some species have evolved the ability to assimilate their prey’s stinging cells into the cerata in the posterior portions of their bodies as protection from predators (Frick, 2003). This adaptation of a chemical defense mechanism seems to have left the need for physical protection obsolete, resulting in the loss of the shell in most nudibranch species (Faulkner and Ghiselin, 1983).

1.3 The Nudibranch Tritonia tetraquetra

Tritonia tetraquetra, is a nudibranch mollusk of the Tritoniidae family. It is found in sub-tidal waters (50-750m in depth) in the Northern Pacific Ocean (McLean and

Porter, 1982; McDonald, 1983; Wyeth, 2006). The organism is also known as Tritonia diomedea, before its name was corrected on the basis of taxonomic precedence

(Martynov, 2009). It is characteristically peach in color, but can range from light orange or deep pink depending on its inhabiting waters and diet, pictured below in Figure 1

(photo, J. Murray). The organism has cilia covering the sole of its muscular foot, which it uses to move across the substrate of its environment (Audesirk, 1978).

It has been observed to prey on soft-tissue octocorals such as P. gurneyi, Stylatula elongata, Virgularia sp. and Acanthoptilum spp. However, in its adult form, some groups of T. tetraquetra prey mainly on Ptilosarcus gurneyi, more commonly known as the orange sea pen (Wyeth and Willows, 2006). A species of sea star, Pycnopodia helianthoides, is considered the only predator of the sea slug (Wyeth and Willows, 2006).

This singular population of potential predators is mainly attributed to the sea slug’s escape response, which is 100% effective (Wyeth and Willows, 2006). Like other nudibranchs, T. tetraquetra tends to live among coral beds. The organism relies on its escape swim and tidal currents to help it move away from predators to safer environments. It also relies upon tidal currents to bring odors that guide it towards food

(Wyeth, 2006).

Figure 1: An adult Tritonia tetraquetra in laboratory settings (photo, J. Murray). Cilia on the muscular foot of the slug (white) allow it move across the substrate. The anterior portion contains the radula, which the slug uses to eat its prey.

Tritonia tetraquetra is a very delicate organism that requires specific conditions for optimal growth and propagation. The organism seems to be very susceptible to ocean acidification, with even minor variations in pH affecting its ability to survive (Huynh,

Andrilenas & Murray, 2011). The ability of the adult sea slug to feed on its food source, mainly sea pens, changes as ocean conditions acidify, as does its ability to combat infection as bacterial species in the marine niche evolve (Huynh, Andrilenas & Murray,

2011). For instance, changes in pH can alter the range of species of microbes that thrive in acidic conditions, chancing infection from invasive pathogens not previously encountered. To date, very little is known about marine nudibranchs, and even less is known about the manner in which the organisms are protected against infections.

Due to the simplicity of the nudibranch brain, as well as the large size of its neurons, T. tetraquetra has previously been used as a model system in neurophysiological studies in connection with animal behavior (Wyeth, 2006), as well as numerous other nudibranch studies. Habituation, non-associative learning behaviors based on repeated

stimuli, resembles another form of learning in T. tetraquetra and is of clinical interest in developing anxiolytic drugs used for treatment of anxiety (Frost, Brandon and Van Zyl,

2006). However, little information of any symbiotic relationships afforded to T. tetraquetra by microbacteria within its intestine or defensive chemicals secretions is known.

1.4 Uptake of Cnidarian Toxin by Nudibranchs

It can be inferred that any defenses afforded to the sea slug through symbiotic relationships within its intestinal lining along with the uptake of chemical compounds from its cnidarian prey serve to protect the organism from invasive bacteria, as well as predators. The purported cnidarian toxin, ptilosarcenone, is thought to be released by a contraction of dense muscles surrounding the cnidosac in the cerata of the nudibranchs

(Greenwood and Mariscal, 1984). The composition of the purported sea pen toxin is currently unknown, but can be harmful to certain organisms.

Metabolites and chemicals originally found in sponges have been found in many dorid nudibranch species such as Anisodoris nobilis, Cadlina luteomarginata,

Chromodoris maridadilus, and Hypselodoris ghiselini (Faulkner and Ghiselin, 1983).

Faulkner and Ghiselin’s described how chemical analysis of these nudibranch species showed that they contained the same metabolites as their cnidarian prey. Species that feed upon acidic corals tended to secrete sulfuric acid from their cerata (Faulkner and

Ghiselin, 1983). Some nudibranchs that prey on cnidarians have been known to store nematocysts within cnidophage cells and later, use them for their own defense against predators (Greenwood and Mariscal, 1984). This would mean that sea slug species with

defensive secretions derived from their food sources would be affected if their food sources were not available.

1.5 Ptilosarcus gurneyi: the Orange Sea Pen

The Orange Sea Pen (P. gurneyi) is a soft-bodied octocoral found in the Northern

Pacific Ocean. The organism anchors itself to any available substrate using a specialized basal polyp, but also consists of thousands of individual feeding and pumping polyps that function cohesively (Nurco et al., 2011). The orange sea pen, which is the main food source for T. tetraquetra, contains chemical toxins that are taken up by T. tetraquetra when the sea pen is eaten (Nurco et al., 2011).

Chemical studies have been conducted on the uptake of purported toxin, known as ptilosarcenone, from P. gurneyi to certain species of nudibranchs closely related to T. tetraquetra. One such study that focused on Tritonia gigantea, a close relative of T. tetraquetra, showed that the organism, which also preys on P. gurneyi, actively took in and sequestered ptilosarcenone in its mantle tissues after consuming it (Williams &

Andersen, 1987). A more recent study by Nathan Shapiro in 2012 has shown that T. tetraquetra actively sequesters ptilosarcenone, a purported acetylcholinesterase inhibitor, from its prey into its body tissues, which it then uses as a defense against predators

(Shapiro, 2012).

1.6 Tritonia tetraquetra and Invasive Species

Documented parasitic infections in Tritonia tetraquetra from Eastsound and

Bellingham Bay, Washington, have shown that sea slugs can be afflicted with ringworm, characterized by large yellow spots on the surface layers of the slug (McLean and Porter,

1982). Due to the change in color of the organism from pink to yellow, as well as the appearance of yellow spots, the affliction has been termed “Yellow-Spot Disease.”

Identification of the marine pathogenic fungi, Thraustochytriaceae, was accomplished by electron microscopy (McLean and Porter, 1982). Scales encapsulated each of the cells on the superficial layers of the slugs affected by the parasite (McLean and Porter, 1982).

Each of the cells on the superficial layers affected by the parasite was encapsulated by scales. No symptoms of parasitic infection were found in T. tetraquetra cells that were not encapsulated by these scales (McLean and Porter, 1982).

The study by Mclean and Porter implies that the fungi Thraustochytriaceae may be a primary invader, while other such studies conducted in the octopus, Eledone cirrhosa, demonstrated that the lesions in E. cirrhosa were caused by

Thraustochytriaceae as a secondary invader. This led to the conclusion that the invertebrate nudibranch slug may be an “unnatural host” and infection may have occurred as an accident (McLean and Porter, 1982). The parasite may have infected the slug by entering the surface mucus, and then encapsulating itself within the cells (McLean and

Porter, 1982). It is interesting to note that, to date, there have been no known pathogenic species to invade sea slugs such as T. tetraquetra. This may be due to the lack of information on the organism’s physiology, more specifically, its innate immune system.

1.7 Significance of Research

This study aims to expand the knowledge of T. tetraquetra physiology by characterizing and understanding the symbiotic relationships afforded to the marine sea slug by the plethora of microbiota that line the intestinal wall using traditional sequencing

techniques. With this information, it will be possible to assess the natural health of T. tetraquetra by observing any mutualistic or commensal relationships afforded to the sea slug by probiotic microbes. A sample composition of microbes in the digestive tract of T. tetraquetra were determined to aid in a basic baseline of known and culturable microorganisms of healthy slugs that can be extrapolated to define the bacterial composition of healthy organisms versus non-healthy or geriatric animals.

Understanding the inner workings of T. tetraquetra, a species whose biology thus far remains elusive, will open doorways to understanding other marine species that may have adapted to survive in changing climate conditions in an ever-evolving planet. Little is currently known about the manner in which T. tetraquetra and other such marine invertebrates are protected against infections in changing ecological niches. Because the natural flora residing in the intestinal wall inhibits pathogenic bacteria from establishing an infection, they act as a first line defensive mechanism against infection. The results from this study provide a foundation for future research in endosymbiotic relationships and protection afforded by microbiota alike in marine invertebrates.

Chapter 2: Research Hypothesis and Specific Aims

Due to the little information concerning T. tetraquetra and its intestinal physiology, this research is aimed to expand the knowledge of T. tetraquetra gut physiology by identifying populations of bacteria that reside in the intestinal lining to better understand the symbiotic relationships afforded to T. tetraquetra by the microbiota within its intestinal tract. The normal microbiota of the intestinal tract of T. tetraquetra confers protection to the organism due to the presence and binding of bacterial species to the intestinal walls, preventing pathogenic organisms from taking residence. This study was designed with specific goals in mind:

Specific Aim 1:

Bacterial species were obtained from the intestinal lining from the T. tetraquetra gut, a kind gift from the lab of Dr. James Murray. Isolated cultures were cultivated from the slug’s intestinal tissues, both anterior and posterior regions.

Specific Aim 2:

Isolated bacterial species were characterized as potentially normal microbiota within the digestive tract of T. tetraquetra. The project utilized traditional Sanger sequencing techniques, using fluorescent dNTPs to determine the genetic makeup of natural bacterial species within intestinal lining of T. tetraquetra. Primers specific for the

16s ribosomal rRNA gene were used to determine the differences amongst the cultivated bacteria. Phylogenetic analysis was conducted to determine evolutionary correlations amongst identified species.

Specific Aim 3:

Basic characterization of isolated bacterial populations was conducted to ensure similar metabolic characteristics of bacterial species thought to be within the isolated populations. This data is presented in the appendix, and included testing whether the bacterial species was Gram (+) or Gram (-), and had metabolic similarities within the populations based on several biochemical tests (see appendix).

Specific Aim 4:

Natural immunity of T. tetraquetra was assessed, and invasive species within the organism’s intestines were observed. The composition of microbes within the digestive tract of T. tetraquetra was used to determine a baseline for “healthy” slugs.

Chapter 3: Materials and Methods

3.1 Isolation of Genomic DNA

Tritonia tetraquetra intestine samples were a kind gift from Dr. James Murray

(CSUEB). The slugs were stored in tanks filled with artificial sea water that was created in the laboratory using commercial Instant Ocean. The temperature was held constant between 9-11°C, An ultraviolet (UV) sterilizer was used to keep the water sterile. The salinity level was maintained at 28-31 parts per thousand (ppt). The pH was maintained at a close range between 8-8.2. The conditions were checked periodically and recorded throughout the study, especially when any diseased tissue was noticed.

The intestine of T. tetraquetra was extracted and separated into two portions: anterior and posterior, by measuring the length of the intestine and cutting it in half.

These samples were treated as separate entities and processed in the same manner.

Samples were stored in sterilized artificial sea water made from commercial Instant

Ocean (Spectrum Brands, Blacksburg, VA). The intestine samples were streaked onto

Difco Marine Agar growth media (BD Diagnostic Systems, Sparks, MD), and incubated at 25°C for 24-48 hours. Bacteria were harvested from plates streaked with the anterior and posterior of the intestine of T. tetraquetra. Overnight cultures were prepared from one resulting bacterial colony in 5mL of Difco Marine Broth (BD Diagnostic Systems,

Sparks, MD), and incubated at 25°C for 24-48 hours.

DNA from the overnight cultures was isolated using a phenol-chloroform extraction procedure in order to obtain high quality genomic DNA. The sample was

vortexed for ten seconds at high speed then centrifuged at 1300 rpm for three minutes to make a pellet. The pellet was resuspended in 400uL Tris-EDTA, pH 8.0, and 30µL 1%

SDS with 50µL 1mg/mL Proteinase K being added immediately before incubating at

37°C for one hour. 700µL phenol:chloroform was used to extract the genetic material, which was vortexed for one minute and centrifuged for two minutes at 10,000 rpm. The aqueous layer was transferred to a new microfuge tube, and 350µL chloroform was added. After vortexing for one minute, the mixture was centrifuged for two minutes at

10,000 rpm. The aqueous layer was transferred to a new tube, with 2.5x the volume of cold ethanol added and incubated at room temperature for ten minutes. The DNA was precipitated with 1/10th the volume of 5M sodium acetate pH 5.3, and incubated for 15 minutes at -20°C. It was then centrifuged at full speed for 10 minutes to make a pellet.

Ethanol was removed, and the pellet was rinsed with 70% ethanol for five minutes. The pellet was air-dried overnight, and resuspended in 50uL TE pH 8.0.

3.2 Amplification of Genomic DNA

A 1% agarose gel was run to visualize the DNA isolated. The gels were stained with 0.1mg/mL ethidium bromide for visualization and a New England Biolabs 1KB Plus

Molecular Weight Ladder (NEB, Ipswitch, MA) was used to determine the size of the

DNA band. The concentration of isolated genomic DNA was determined using a nanodrop spectrophotometer (Nanodrop 2000, Thermo Scientific, Wilmington, DE).

Isolated genomic DNA was amplified using PCR with universal primers for the 16s bacterial ribosomal RNA (rRNA) gene. The primer pair used consisted of fairly common universal primers 1492R-27F. Table 1 lists the primers used in this study (Invitrogen,

Grand Island, NY). The PCR conditions used were 94°C for two minutes and then 25 cycles of 94°C for 30 seconds, 53°C for 30 seconds, and 72°C for one minute for denaturation, annealing and extension, respectively, followed by seven minutes of extended extension at 72°C. The samples were stored at 4°C until removed for long-term storage at -20°C. The PCR product was cleaned up before proceeding further using an

ExoSAP-IT for PCR Product Cleanup kit (Affymetrix, Santa Clara, CA) consisting of

1µL Exo-SAP and 3µL PCR product. The thermocycler (Proflex, Life Technologies,

Grand Island, NY) was set at 37°C for 30 minutes, followed by 80°C for 15 minutes.

3.3 Sequencing Techniques

Sequencing was performed on amplified PCR products using Big Dye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Grand Island, NY) and automated DNA sequencing using an AB 3130 Genetic Analyzer (Applied Biosystems, Grand Island,

NY). The conditions used were 96°C for one minute, followed by 25 cycles of 94°C for ten seconds, 50°C for five seconds, and 60°C for four minutes. The temperature was held constant at 4°C following the sequencing protocol. The resulting sequence was compared to known bacterial sequences in the NCBI database using the tBlastx feature and 16s rRNA regions as known constants. Any sequences with over 96% homology were deemed a positive match for T. tetraquetra natural microbiota. A total of 31 samples were sequenced for both regions (anterior and posterior) of the T. tetraquetra intestine.

3.4 Biochemical Profiling of Bacterial Species (data shown in appendix)

Gram staining was conducted on isolated bacterial isolates following standard procedures. Gram-positive samples appeared blue/purple in color, while Gram-negative species appeared red/pink in color.

Biochemical tests were performed on bacterial cultures to obtain additional information regarding the physical and metabolic properties of the bacteria. All biochemical tests were conducted with bacterial colonies/broth over 18-24 hours.

Catalase tests were performed to determine if populations expressed catalase, an enzyme found usually in aerobic growing microorganisms that live in oxygenated environments to neutralize toxic forms of H2O2. Since catalase mediates the breakdown of hydrogen peroxide, H2O2, into oxygen and water, approximately 500µL of a homogenous bacterial broth was applied to a slide and 3% hydrogen peroxide was applied to the sample. Formation of bubbles was deemed a positive reaction.

Oxidase tests were performed to determine if bacterial populations expressed

Cytochrome c oxidases. The oxidase test detects the presence of a functional cytochrome oxidase system that catalyzes the transport of electrons between electron donors in the bacteria and the redox dye-tetramethyl-p-phenylene-diamine. The oxidized reagent forms the colored compound indophenol blue. A sterile cotton swab was dipped into the bacterial broth, and applied to a slide. One drop of oxidase (in vitro Diagnostic-Becton,

Dickinson and Company) was added to the slide. A change in color to dark blue/black was deemed a positive result. No color change was a negative result.

IMViC tests, which included an indole test, a Methyl red test, Voges-Proskauer test, and a citrate test, were used to identify mixed acid fermenting bacteria that yield a stable acid end product and identify bacteria capable of 2,3 butanediol fermentation following mixed-acid fermentation. MR-VP broth was inoculated with pure bacterial culture, and incubated at room temperature for 48 hours. Approximately one mL of broth was transferred to two non-sterile tubes while three drops of Methyl Red were added to one tube, and read immediately for a change in color. Change in color to red was deemed a positive result for acid formation using pyruvate as a substrate. Exactly 15 drops of 5% alpha-naphthol were added to the second tube and mixed, followed by five drops of 40%

KOH to expose the sample to atmospheric oxygen. The samples were read at ten minute intervals for 60 minutes. VP positive samples were red at the 60 minute mark, and VP negative samples were unchanged after 60 minutes. A positive result indicated the presence of an acid fermentation pathway. Citrate tests were performed to determine if bacterial species used citrate as its sole carbon source. Simmon’s Citrate tubes were inoculated with bacteria and incubated at room temperature for 48 hours. Tubes were examined for color change after 48 hours. Color change to blue was deemed a positive result for utilization of citrate. SIM (Sulfur-Indole-Motility) tubes were inoculated with bacteria and incubated for 48 hours at room temperature. The tubes were examined for spreading from the stab line, indole production, and formation of any black precipitate indicative of the presence of hydrogen sulfide gas in the medium. Kovac’s reagent was added to each tube so the depth of the reagent was 2-3mm in the tube. Tubes were examined for formation of a red color in the tubes’ upper layers after addition of Kovac’s

reagents. Change in color from pink to red immediately after adding the reagents was deemed a positive result for the formation of indole from tryptophan. and/or growth about the stab line was deemed a positive result. The SIM medium was also used to test for indole production.

Nitrate tests were conducted in order to determine if bacterial populations utilized nitrate. Broths were inoculated with high concentrations of bacteria and incubated at room temperature for 24 hours. Each tube was then examined for gas production and/or change in color. If bubbles were produced, the bacteria reduced nitrite to nitrogen gas. In these cases, presence of bubbles indicated the presence of both nitrate reductase and nitrite reductase (nitrates were converted to nitrites, and then reduced to nitrogen gas). If no bubbles were present, eight drops each of Reagent A (consisting of dimethyl- naphthylamine dissolved in acetic acid) and Reagent B (consisting of sulfanilic acid dissolved in acetic acid) were added to each tube and mixed well. If the samples were not red, zinc was added to the tubes, and allowed to stand for ten minutes. If the tube was not red after this step, there was no additional unreduced nitrate present in the tube, and since there was no nitrite present in the medium, either, this meant that ammonia or molecular nitrogen were formed by way of denitrification.

3.5 Determination of Novel Species not Identified in Healthy Slugs

Novel bacteria not identified in healthy Tritonia tetraquetra were conducted in a two-step process: isolation and characterization. The DNA of “sick” or elderly slugs were isolated and sequenced as stated in the speciation procedure. Sick slugs were identified by the change in the color of skin from pinkish-peach to yellow or orange, development

of skin lesions, and/or flaking and sloughing of the skin or cerata. Elderly slugs were identified based on the number of days spent in the laboratory tank from the date of arrival, and the age of the slug on the date of arrival. Sick slugs may not have partook in eating, general activity in the tank and cerata and/or skin had obvious flaking and deteriorating color.

Sequencing results of the bacteria isolated from “sick” slugs were compared to the sequencing results of the bacteria from slugs deemed to be “healthy” to identify any bacterial species that were not the same as the ones found in “healthy” slug species.

These were found to be evidence of invasive bacterial species within in the intestinal lining of T. tetraquetra. Any variations in the bacterial composition of the intestine of the diseased slugs were seen as a departure from the innate immunity of the slug.

Chapter 4: Isolation and Characterization of Bacterial Species

4.1 Isolation and Amplification of Genomic Bacteria

Isolated DNA from single colony cultures resulted in concentrations in the range of 200-400ng/µL (data not shown). DNA was amplified using universal 16s rRNA primers that would yield an amplicon size of 1,456 bp. Annealing parameters for the polymerase chain reaction (PCR) were determined by a temperature gradient based on the melting points (Tm) of the primers.

Table 1: The primers used in this experiment for the 16s rRNA gene. Primer pair 1492R-27F, where M represents A or C, yielded an amplicon that was 1456 bp long (Weisburg et al., 1991).

Universal Amplicon Primer Primer Sequence 5'-3' Size 1492R 5′-TACCTTGTTACGACTT-3’ 1456bp 27F 5’-AGAGTTTGATCMTGGCTCAG-3’

Primers specifically targeting the 16s rRNA gene used in this study are listed in

Table 1. The primer pair used for traditional (Sanger) sequencing was the universal primer pair 1492R-27F (Weisburg et al., 1991). The 16S rRNA gene was chosen for this experiment because it is present in all eubacteria and Archaea, as a multigene family or operon. The 16s rRNA gene is commonly used in phylogenetic studies, as it is known to be a highly evolutionarily conserved sequence. In addition to these highly conserved regions, the 16s rRNA gene has some hypervariable regions that are different for each bacterial species – in essence, a bacterial species-specific signature (Weisberg et al.,

1991). The 16S rRNA gene is also large enough for informatics purposes, which is helpful when creating relationships amongst both known and unknown bacteria.

Figure 2: A 0.8% agarose gel of isolated genomic DNA of single bacterial colonies cultured from the T. tetraquetra intestine. The samples were amplified via standard PCR with primers for the 16s rRNA gene.

Thirty ng of each sample were added in wells 3, 4, 10, 11, 12, 13, and 14. A New England BioLabs (NEB)

1kb DNA Plus marker in well 5 was used for band comparison. A negative control was added to well 6. All amplicons were approximately 1.4kb in length.

Amplified products, using 1492R-27F primer set, were visualized on a 0.8% agarose gel, and the amplicons of the expected size, approximately 1.4 kb size fragment, were obtained (see Figure 2). The agarose gel shown in Figure 2 is representative of the amplified DNA samples used throughout this study. There was some degraded product in the amplified PCR products, which was seen by the streaking within the sample lanes.

Although primer-dimers were seen in some of the samples, they did not affect the concentration, purity of the samples, or downstream processes.

4.2 Characterization of Bacterial Species by Sanger Sequencing

After amplification via PCR, Sanger sequencing was conducted using primers specific for the bacterial 16S rRNA gene. The resulting sequences were between 450 bp and 600 bp in length. The sequences obtained are listed in the appendix. Figure 4 depicts the output of the sequences in Chromas, a software program that allowed visualization of the Sanger sequencing procedure. Each peak indicates the incorporation of a fluorescently-tagged dNTP to the growing sequence. The computerized read of the sequence is listed above the peaks in Figure 3. These bases were checked against the colored peaks of the sequence to ascertain that the computerized read the dNTP incorporated were the same. Bases that appeared in the computerized read incorrectly were corrected and/or removed depending on the whether they were mistaken or extraneous. This ensured that each sequence was of high quality before progressing to steps involving multiple sequence alignments and phylogeny trees.

Figure 3: A sample readout of the results of the Sanger Sequencing Procedure visualized in the Chromas software. Manual changes were made to the sequence as shown by the arrows according to the base pairs indicated by the colored peaks.

Sequences were separated based on bacterial isolates from anterior and posterior portions of the T. tetraquetra intestine and were compared to the 16S rRNA genes of all known bacterial species in the NCBI database, and were determined to be homologically similar to 16S rRNA sequences of twenty bacterial species in ten genera. Since 96%, a high percentage similarity, was used as the cut-off range when determining homology, it is highly probable that the bacterial species identified are present within the intestinal lining of T. tetraquetra.

Tables 2 and 3 list the species matches, the percent similarity, and the number of isolate sequences (samples) that matched each species for the anterior and posterior portions, respectively, of the intestinal lining of T. tetraquetra. The results presented here indicate that the microbial communities from both regions (anterior and posterior) are distinct.

Table 2: NCBI BLAST matches to isolated genomic DNA of bacterial species from the anterior portion of

T. tetraquetra intestine using the bacterial 16S rRNA gene. Ninety six percent homology match was used as a cut-off for match identity. Eleven bacterial species were identified.

Identified Species % Homology E-value No. of Matches (n=18) Marinobacter antarcticus 98% 9.00E-34 2 Marinobacter maritimus 100% 2.00E-167 2 Marinobacter goseogensis 100% 0.00E+00 3 Marinobacter mobilis 96% 1.00E-145 2 Streptococcus oralis 100% 2.00E-117 1 Clostridium argentinense 100% 1.00E-169 1 Clostridium subterminale 100% 0.00E+00 3 Marinobacter halophilus 99% 0.00E+00 1 Photobacterium frigidiphilum 98% 1.00E-179 1 Shewanella corallii 99% 1.00E-108 1 Streptococcus pseudopneumoniae 99% 2.00E-125 1

Of the bacterial genera identified in the anterior portion of the intestinal lining of

T. tetraquetra, the majority of the species were from the genus Marinobacter. Species in this genus have been reported to live in salt water (Yakimov, Timmis and Golyshin,

2007). Some of these species are known to survive within other organisms as symbionts

(Bergey and Holt, 1994; Kim et al., 2007; Yakimov, Timmis and Golyshin, 2007), confirming that these bacteria could be present within the T. tetraquetra intestine, and

may also serve the same functions with the slug as they do in other species (including chemical degradation and absorption of metabolites), though there is not enough information regarding the interpersonal relationships of bacteria within the digestive system to be sure (Yakimov, Timmis and Golyshin, 2007). It is important to consider that these species are most likely only present in the anterior region of the T. tetraquetra intestine because this is where most chemical degradation of foodstuffs would occur.

Table 3: NCBI BLAST matches to the isolated genomic DNA of bacterial species from the posterior portion of T. tetraquetra intestine using the bacterial 16S rRNA gene. Ninety six percent homology match was used as a cut-off for match identity. Nine bacterial species were identified.

No. of Matches Identified Species % Homology E-value (n=13) Thallassospira lucentensis 96% 7.00E-34 3 Bacillus idriensis 100% 2.00E-86 1 Photobacterium indicum 100% 1.00E-133 2 Bacillus subtilis 99% 0.00E+00 1 Bacillus indicus 99% 0.00E+00 2 Pseudoalteromonas haloplanktis 100% 2.00E-126 1 Marinobacter antarcticus 100% 0.00E+00 1 Pseudoalteromonas translucida 100% 2.00E-121 1 Pseudoalteromonas agarivorans 99% 0.00E+00 1

Of the five genera identified within the posterior region of the T. tetraquetra intestine, the majority were in genera Bacillus or Pseudoateromonas. These data are consistent with knowledge that both genera are known to reside in marine water and soil

(Bergey and Holt, 1994). As the sea slug is known to be a benthic creature that resides in

the ocean substrate, it could be probable that the bacteria known to live in the surrounding environment have become part of the T. tetraquetra immune system.

Only one bacterial species, with 100% similarity match to the known bacteria in the NCBI database, was found to be present in both regions: Marinobacter antarcticus.

This may be telling of the function of the species within the intestinal lining of the sea slug. As stated by Yakimov and colleagues, some Marinobacter species, such as M. arcticus and M. maritimus, can degrade hydrocarbons (Yakimov, Timmis & Golyshin,

2007). It is possible that M. arcticus works to breakdown foodstuffs and remove contaminants from within the intestinal system of T. tetraquetra.

BACTERIAL GENERA IDENTIFIED WITHIN THE ANTERIOR PORTION OF T. TETRAQUETRA INTESTINAL LINING Shewanella spp. Photobacterium 9% spp. 9%

Marinobacter spp. 46% Clostridium spp. 18%

Streptococcus spp. 18%

Figure 4: Bacterial genera identified from within the anterior portion of intestinal lining of T. tetraquetra.

Species of Marinobacter were found to be the most prevalent within this portion.

Though the sample size was small, there was little overlap in the composition of the microbial communities identified within the anterior and posterior regions of the intestinal lining of T. tetraquetra. While this was not initially hypothesized, the results are consistent with knowledge of the vastly different functions of the anterior and posterior regions of the digestive system: the anterior intestine works to physically break down food, whereas the posterior intestine aids in nutrient absorption.

Betcher et al. (2012) found that the metabolic processes that take place in the anterior part of the digestive system in the five types of Teredinidae studied are vastly different compared to those of the posterior part. The foregut of the shipworm is used for digestion of wood and lignocellulose, while the endosymbionts in the hindgut fix nitrogen and make it usable for their host (Betcher et al., 2012). Although the composition and abundance of the microbial communities within the digestive system of the shipworm and T. tetraquetra may be different due to their foodstuffs, the need for differing microbial communities in the foregut and hindgut might be the same. Digestion and degradation of the orange sea pen, the main food source of T. tetraquetra, may possibly require a number of symbionts working throughout the intestines to degrade the food and glean its nutrients.

BACTERIAL GENERA IDENTIFIED WITHIN THE POSTERIOR PORTION OF T. TETRAQUETRA INTESTINAL LINING

Marinobacter spp. Thallassospira spp. 11% 11%

Photobacterium spp. 11% Pseudoateromo nas spp. 33%

Bacillus spp. 34%

Figure 5: Bacterial genera identified within the posterior portion of intestinal lining of T. tetraquetra.

Species of Baccillus and Pseudoalteromonas were found to be most prevalent within this portion of the intestines.

The composition and abundance of the microbial communities identified within the T. tetraquetra intestinal lining varied between the anterior and posterior regions. The term composition encompasses all of the microbiota identified, whereas the term abundance describes the number of samples that contained each different bacterial culture. Figures 4 and 5 illustrate the composition of the bacterial genera detected within the sea slug’s digestive system. The charts show the relative abundance of each bacterial genus.

It is reasonable to suspect that T. tetraquetra would use microbiota that could aid in the detoxification of its main food source, the sea pen, which has been known to harm other marine species with internalized toxin. After ingestion, the sea slug would need to

digest the sea pen entirely so that nutrients could be taken in, leading to the need for different species of bacteria to perform different functions throughout the digestive tract.

When examining the bacterial populations identified within the intestinal lining of the sea slug, some general patterns seem to arise (see appendix). Most of the bacterial genera identified within the anterior gut were catalase positive, suggesting that these bacteria could protect themselves from the lethal effect of accumulation of hydrogen peroxide as an end product of aerobic carbohydrate metabolism. This information also supports the hypothesis that the bacteria within the foregut are most likely aerobes or facultative aerobes. Moreover, the catalase activity in the bacterial populations identified in the posterior portion of the T. tetraquetra intestine support the hypothesis that enteric bacteria of the sea slug may be either aerobic or facultative aerobes. The populations that showed negative results for the catalase test could have done so due to two possible reasons: (1) the bacteria could have been aerotolerant as opposed to facultative aerobes, meaning that they would only ferment, and use oxygen as a terminal electron acceptor, or

(2) the microbiota are anaerobic and do not contain the catalase enzyme that hydrolyzes hydrogen peroxide.

Data from this study seems to be in line with the results of previous studies that have found symbiotic bacteria within the digestive systems of marine life. Species of

Vibrio and Photobacterium have been associated with marine habitats and many marine organisms such as crustaceans, oysters, and fish (Rungrassamee et al., 2014; Wang, et al.,

2010). Although the bacteria in these genera have frequently been characterized as having a commensalistic relationship with marine animals within their digestive tracts, it

may be that their constant detection in the results of the study by Rungrassamee et al. imply that they are an indigenous bacterial population in the intestines of the giant tiger prawn (P. monodon). Species of Vibrio and Photobacterium have been found to be dominant in all of the barcoded Nexgen libraries of the intestines of the giant tiger prawn, suggesting that these bacterial genera were well-adapted to the conditions (Rungrassamee et al., 2014). However, Vibrio species were not detected within the intestinal lining of T. tetraquetra, though two species of Photobacterium were identified (at 100% similarity to the known sequences in the NCBI database). Again, it is possible that species of Vibrio were present within the T. tetraquetra intestine, but not cultivated, or, more likely, that

Vibrio species are not considered indigenous to the sea slug.

In a study on milkfish from the northern coast of Central Java by Prayitno et al., it was seen that the bacteria inhabiting the gut of the fish comprised of species from the genera Shewanella, Vibrio, and Photobacterium (Prayitno et al., 2015). More interestingly, it was found that the isolates of the S. upenei and S. algae worked to prevent the growth of harmful bacteria within the milkfish intestine, and demonstrated antibacterial activity against pathogenic bacteria of the genus Vibrio (Prayitno et al.,

2015). The presence of Shewanella species within the anterior portion of the T. tetraquetra intestine coupled with the lack of Vibrio species may mean that the

Shewanella bacteria have become indigenous to the T. tetraquetra gut, and use their antibacterial properties to block the binding of virulent bacteria to the intestinal walls.

This could imply that virulent bacteria that enter the sea slug’s digestive system may be

washed out and released with the organism’s waste, without actually taking hold within the intestinal walls.

The results of a similar study conducted on A. japonicus, a sea cucumber that inhabits the south Asian Sea, showed that sulfate-reducing bacteria were the predominant bacteria present in the hindgut of the organism (Fei et al., 2014). It is thought that these sulfate-reducing bacteria work in acetate production and nitrogen fixation for their host

(Fei et al., 2014), which leads to the conclusion that they may provide nutrients for A. japonicus. While no bacteria found within the T. tetraquetra intestines were sulfate- reducing bacteria, it is possible that these species were not cultivated in the laboratory. It would make sense that sulfate-reducing anaerobes would function within the T. tetraquetra intestine to provide nutrients for the sea slug since they obtain energy by oxidizing organic compounds.

Interestingly, two species from the genus known for utilizing fermentation pathways, Streptococcus oralis and Streptococcus pseudopneumoniae, were identified in the anterior portion of the sea slug intestine; more fermenters were identified within the posterior region of the T. tetraquetra intestine. This distinct difference between the two regions of the sea slug intestine brings new insight to the function of the digestive system.

It can be speculated that the microbiota within the foregut are mostly aerobes, while the hindgut contains a mixture of aerobic, facultative aerobic, and aerotolerant anaerobic bacteria.

Species of Bacillus have previously been isolated from the digestive systems of several fish. Fei et al. explain that, in these studies, species of Bacillus were found to inhibit pathogens by producing large quantities of chitinase and protease enzymes (Fei et al., 2014). Hence, they have been used as probiotics to exclude certain bacteria, especially Vibrio (Fei et al., 2014). These results strongly suggest that the microflora within the intestines of marine organisms can be controlled using antibiotic-producing bacteria (Fei et al., 2014). The presence of three species of Bacillus (B. idriensis, B. subtilis, and B. indicus) within the T. tetraquetra intestines may indicate that sea slug is protected against pathogens by the antibacterial properties of Bacillus and possibly other bacterial species within its digestive system. These findings hope to begin to define what the functions of the microbiota within the intestinal lining are, and how they serve their host organism, T. tetraquetra.

While the bacterial genera and species identified in this study surely do not encompass a comprehensive list of bacteria present within the digestive system of T. tetraquetra, these initial findings are helpful in determining a baseline for indigenous microbiota present within the intestinal lining of the sea slug. These results encompass the first step towards more in-depth and detailed research regarding the normal flora that make up the microbiota of T. tetraquetra, and its effect on the immunity of the sea slug.

4.3 Profiling of Bacterial Species by Differential Staining and Biochemical Tests

Pure cultures isolated from T. tetraquetra intestinal lining were subjected to a variety of biochemical tests to begin to examine their metabolic capabilities and support genomic identifications by Sanger sequencing. Each bacterial isolate was subjected to the

same tests: Gram-staining, catalase, oxidase, IMViC (including indole, Methyl Red,

Voges-Proskauer, citrate, and motility tests), and nitrate tests. Although the genus and species of all bacterial isolates could not be identified through these limited tests, the biochemical tests performed on the various populations aided in determining more information regarding the populations’ biochemical nature and potential metabolic makeup. The results of these tests are listed in Tables A1 and A2 in the appendix.

4.4 Bacterial Phylogeny Characterization

The phylogenetic composition of the microbial communities detected within the intestine of T. tetraquetra has not yet been determined. In an effort to do so, the phylogeny of the bacterial species was conducted based on two separate Multiple

Sequence Alignments (MSA): one for the anterior region, and one for the posterior region. Once a phylogram was created based on the evolutionary similarities of the species identified in each region, it was easier to see any relationships amongst the bacterial species.

The purpose of creating the MSA was to identify any common domains and motifs, and to determine any consensus sequences. Before a phylogram was created for these data sets, some editing had to be done, which would allow for clearer phylogenetic trees (Edgar, 2004). The sequences were edited to remove any poorly aligned N and C termini. In this case, any gaps that occurred in the sequences (representing any insertions/deletions in the sequences) were not removed because they may have been distinguishing factors for a certain species.

The edited sequences were then used as input data to create a neighbor-joining phylogram using Phylip. Phylip is a powerful tool with many options for control by the user, which allowed many changes to be computed into the tree, such as bootstrapping and calculating distance based on genes, restriction enzymes, or nucleic acids. Once the sequences were added to Phylip, a Protdist analysis was done with bootstrapping repeated

100 times. Bootstrapping is a method that re-runs the tree a certain number of times. This would mean that the neighbor-joining tree was made 100 times, creating a consensus tree that revealed the relative distance between the sequences using nodes. Each node was assigned a value each time the tree was made, leading to a solid bootstrap value of each node, which could be taken as the confidence level of the node itself. Each node within the neighbor-joining trees in Figures 6-7 was made with a confidence level (bootstrap number) of 98 or higher. With such high confidence levels on these phylograms, it can be assumed that the connections made within these trees are highly accurate.

Figure 6: Neighbor-joining phylogram created using a multiple sequence alignment of the results from the

NCBI BLAST matches to the isolated genomic DNA from the anterior region of the intestinal lining of T. tetraquetra.

A high similarity in the sequences was expected since both the MSA and phylogram were created using the 16s rRNA gene. Any slight differences within the 16s rRNA hypervariable region would lead to the differences in the species detected.

Neighbor-joining phylograms created with the bacterial species identified are depicted in

Figure 6 (anterior) and Figure 7 (posterior). In order to be grouped as they were on the phylogenetic trees, the species must have had some common characteristics or commonalities. These were visible as conserved structural motifs or specific lengths of code when aligned in a multiple sequence alignment. Large sections of similar sequence were found in many of the species, which led to the conclusion that these regions are evolutionarily conserved and most likely code for the same function in each species in which it was present. Structural motifs, such as the C-loop and GNRA tetraloop motif, were seen in a large number of the bacterial species identified. Species with slight

mutations in regions other than these conserved regions were further away from each other on the phylogram, while species with very similar structures mapped as relatives on the phylogram.

Figure 7: Neighbor-joining phylogram created using a multiple sequence alignment of the results from the

NCBI BLAST matches to the isolated genomic DNA from the posterior region of the intestinal lining of T. tetraquetra.

Once the identified microbial communities were mapped, clear connections could be made regarding their phylogenetic and evolutionary relationships. The next step in order to obtain more information and a clearer understanding of the composition of bacterial species within the intestinal lining of T. tetraquetra would be to view the samples under scanning electron microscope (SEM). This would enable the visualization of the morphology and accumulation of the bacterial organisms within the sea slug. SEM studies would allow for a deeper understanding of the composition of the microbiota

within the intestinal lining, and how the presence or absence of certain bacterium affect the survivability of T. tetraquetra.

4.5 Characterization of Invasive Species of T. tetraquetra

T. tetraquetra is a very delicate organism that requires specific conditions for optimal growth and propagation. The wild organisms, retrieved by Dr. James Murray from their natural habitat in the Pacific Ocean (along coast of Washington state), were maintained under laboratory conditions that mimicked their natural ecosystem. Over time, these captive sea slugs did not participate in “normal” feeding habits (personal observation). Some abstained from feeding, while others ate less frequently. Sea slugs were deemed “sick” when a visible change in the color of skin from pinkish-peach to yellow or orange was detected. The genomic DNA from sea slugs deemed “sick” was isolated and sequenced in the same manner as all the other samples in this study. The resulting sequences, which were matched to species in the NCBI database based on the

16s rRNA gene, were then compared to the bacterial species identified in the sections above. The same cut-off, ninety six percent, was used for a similarity match, and any species identified that were different from the ones in the samples from the “healthy” T. tetraquetra intestinal lining were considered to be potentially invasive species.

Table 4: NCBI BLAST matches to the isolated genomic DNA of bacterial species found within the intestinal lining of “sick” T. tetraquetra using the bacterial 16S rRNA gene. Ninety six percent homology match was used as a cut-off for match identity. Three bacterial species were identified to be different from those present in “healthy” sea slugs.

Identified Species % Homology No. of Samples (n=7) Pseudonocardia spinosa 100% 3 Pseudonocardia acacia 100% 2 Pseudonocardia spinosispora 100% 2

The natural microbiota residing in the intestinal wall inhibits pathogenic bacteria from establishing an infection; they act as a first line defensive mechanism against illness. Three species were identified as different from those of the “healthy” T. tetraquetra intestines (listed in Table 4). The three species identified were found to be in the genus Pseudocardia.

While this may be cause to speculate that these are invasive species, it must be considered that T. tetraquetra were outside of their natural environments, and may have developed endosymbiotic relationships with other microbiota that were not present in every slug. For example, bacterial could have been picked up by the T. tetraquetra while living in the aquarium in the laboratory, or even from dying P. gurneyi. Though no real conclusions can be drawn from such a small sample of sick T. tetraquetra, there is reason to believe that isolation of these bacterial species was easier in the “sick” slugs versus in the “healthy” slugs. The results seem to indicate that the three Pseudonocardia species identified (P. spinosa, P. acacia, and P. spinosispora) were established in the intestines of the “sick” slugs, and probably would not be regarded as an indigenous species.

Members of the genus Pseudonocardia have been known to live mutualistically in the cuticles of Acromyrmex leaf-cutter ants, working to provide antibiotic protection from other garden parasites (Zhang et al., 2007). It is possible that these Pseudonocardia species reside within the skin of T. tetraquetra, as they do in the leaf-cutter ants, but were only identified in the “sick” slugs due to sloughing of skin and general worsened health, suggesting that they are not pathogenic, but invasive in “sick” animals. If these species were present within “healthy” slugs, it would presumably add another level of protection for T. tetraquetra from sources of illness in its environment.

Four additional bacterial species were identified by Sanger sequencing in the

“sick” T. tetraquetra, but due to low percent homology, the data was not taken into consideration for this study. Table A3 shows four species within two genera,

Mycoplasma and Pseudomonas, that were identified by NCBI BLAST search using primers for the 16s rRNA gene.

Biochemical tests were not performed on the bacteria identified within the intestines of the “sick” T. tetraquetra, but the properties of these genera bring speculative insight into the differences between the “sick” and “healthy” sea slugs. Early findings indicate that a “sick” intestine contained only fermenters, while a “healthy” intestine contained a mix of fermenters and non-fermenters. Non-fermenters were predominant in the anterior region of the T. tetraquetra intestine, while the fermenters identified were in the posterior region, but the sample size was just too small to draw any assumptions.

While the bacterial species listed in Table 4 surely do not encompass a comprehensive list of microbiota that may be present within the digestive system of a

“sick” sea slug, it is apparent that the bacteria present here are very different when compared to those present in a “healthy” slug. This comparison attempts to establish a proof of concept that sick slugs may play host to invasive bacterial populations due to the slug being sick, could be somewhat pathogenic to the animal, or its presence is just coincidental. Future studies are warranted using expanded culture conditions and media.

Chapter 5: Conclusions

Characterization of microbiota in animal intestines has been central in advancing understanding of the relationship between host and microorganism. While some microbes can be pathogenic to their hosts, other microbial symbionts are beneficial to the development and physiology of their host, playing roles in nutrient absorption, immune response, and epithelial development. In turn, several host factors, such as diet, developmental stage and physiological condition have been identified to affect intestinal bacterial composition. The makeup of the host’s gut and its environment also shape the composition of intestinal bacteria to maintain a relatively stable level of diversity.

This symbiotic relationship study aimed to expand the knowledge of T. tetraquetra physiology by characterizing and understanding the symbiotic relationships afforded to the marine sea slug by the plethora of microbiota that reside within the intestinal lining of the sea slug. With this information, it will be possible to assess the natural health of T. tetraquetra by observing any symbiotic relationships afforded to the sea slug by probiotic microbes. The natural flora residing within the intestinal wall of the sea slug inhibit pathogenic bacteria from establishing an infection, and act as a first line defensive mechanism against infection.

Nudibranch invertebrates are not known to have developed innate immune systems (Huynh, Andrilenas & Murray, 2011), so the implication made here is that T. tetraquetra have developed a protective system through symbiosis with microbiota within its intestinal tract. In accordance with this hypothesis, the samples of cultivated bacteria

from the T. tetraquetra intestines are consistent with bacterial species known to share endosymbiotic relationships with other organisms (Betcher et al., 2012; King et al., 2012;

Stewart and Cavanaugh, 2006). In total, twenty bacterial species were identified within the digestive system of T. tetraquetra. Eleven species were identified in the anterior portion of the intestinal lining, and nine in the posterior portion. It can be surmised that the bacterial species present in each half of the intestine play different roles in the digestion of foodstuffs for the host organism.

Results from differential staining and metabolic tests performed on several similar populations of bacterial isolates (see appendix) helped in the understanding that slug’s intestines would be populated with enteric bacteria with facultative anaerobic metabolism. When these bacterial species are present within the mucosal lining of T. tetraquetra, they would most likely provide the organism with secondary metabolites and physical protection due to their metabolic properties and their presence along the surface area of the digestive tract. As the sea pen, the main food source of T. tetraquetra, expresses nematocysts, it is expected that bacterial species that can detoxify components within the nematocysts would be abundant within the intestinal lining of T. tetraquetra.

These species were not isolated in this study.

The composition of microbes present within the digestive tract of T. tetraquetra was used to determine an initial baseline of known microorganisms of healthy slugs.

Three species were identified in “sick slugs” that were not present in the slugs deemed

“healthy.” These potentially invasive bacterial species influence and alter the physiology and, ultimately, the immunity of the host, T. tetraquetra. Once this information is

understood, it can be extrapolated to define the bacterial composition of healthy organisms versus non-healthy or geriatric animals. This research will contribute greatly by breaking new ground in an area in which little information is available.

Any endosymbiotic relationships afforded to T. tetraquetra by bacteria within its digestive system improve its chances of survival in its environment. Little is currently known regarding how T. tetraquetra and other such marine invertebrates are protected against infections in changing ecological niches. The results from this study provide a direction for future research in endosymbiotic relationships and protection afforded by microbiota alike. Advanced technological protocols (i.e. Ion Torrent Sequencing) would allow the speciation of a greater number of bacterial species than previously possible.

This is because some bacterial species cannot be grown in a laboratory under artificial conditions. Next generation sequencing techniques allow the speciation of bacteria by sequencing the 16s rRNA gene, providing greater insight into the bacterial species.

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Appendix

List of isolated bacterial samples (by isolate name) and corresponding sequences obtained using primer set 1492R-27F.

>17BE2

GCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATC

CAAAACTGGCGAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGC

GTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGG

TGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTC

AACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGG

GGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACG

>HR11P1

ACTAACTACTTCTGGTGCAATCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCGGGAA

CGTATTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGC

AGACTCCGATCCGGACTACGACGCGTTTTGTGAGATTGGCTCCCCCTCGCGGGTTTGCAGCCC

TCTGTGCGCGCCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACCTGACGTC

ATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCCTAGAGTTCTCAGCCGAACTGCTAGCA

ACTAGGGATAGGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGAC

GACGGCCATGCAGCACCTGTGTCTGAGT

>SK19P2

AGCTCTCATGGTGTTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCT

GATTCGCGATTACTAGCAACTCCGGCTTCATGTAGGCGAGTTTCAGCCTACAATCCGAACTGG

GATTGGTTTTTTAAGTTTAGCTCCACCTCGCGTCTTGCATCTTGTTGTACCAACCATTGTAGCA

CGTGTGTAGCCCTAGACATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCCGGTT

AACCCGGGCAGTCTCACTAGAGTGCTCAACTTAATGGTAGCAACTAATGATAAGGGTTGCGCT

CGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCT

>TANTSK02

GGCTCACCAAGGCAACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACATCGGGACTGAG

ACACGGCCCGAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTG

ATCCAGCCATGCCGCGTGTGTGAAGAAGGCTTTCGGGTTGTAAAGCACTTTCAGTGAGGAGGA

AGGCTTTCAGACTAATACTCTGGAGGATTGACGTCACTCACAGAAGAAGCACCGGCTAACTCC

GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGC

GCGCGTAGGTGGTTGAGTAAGCGAGATGTGAAAGCCCCGGGCTTAACCTGGGAACGGCACTT

CGAACTGCTCGGCTAGAGTGTGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGT

AGATATAGGAAGGAACACCAGTGGCGAAGGCGGCTACCTGGACCAACACTGACACTGAGTGC

>SK15A

CGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTGCTGATCCGCGAATTACTA

GCGATTCCGACTTCATGTAGGCGAGTTGCAGACTACAATCCGAACTGAGACTGGCTTTAAGAG

ATTAGCTTGCCGTCACCGGCTTGCGACTCGTTGTACCAGCCATTGTAGCACGTGTGTAGCCCA

GGTCATAAGGGGCATGATGTTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTC

GCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTA

ACCCAACATCTCACGACACGAGC

>HR22P1

CTTCTGGTGCAGCCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC

ACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTC

CGATCCGGACTACGACGTACTTTGTGGGATTCGCTCACCATCGCTGGTTGGCAGCCCTCTGTAT

ACGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCGTCCCCA

CCTTCCTCCGGTTTATCACCGGCAGTCTCCCTGGAGTTCCCACCATTACGTGCTGGCAACAAGG

ATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCA

TGCAGCACCTGTCTCAGAGTTCCCGAAGGCACTAAACTATCTCTGGACTTTCTGGTGTCA

>15SKE2

GCGCGCGTAACGCTACACATGCAAGTCGTAACAAGGTAACGAAATAGCTTGCTAATTTGCTGA

CGAGCGGCGGACGGGTGAGTAATGCCTGGGAATATGCCTTAGTGTGGGGGATAACTATTGGA

AACGATAGCTAATACCGCATAACGTCTTCGGACCAAAGAGGGGGACCTTCGGGCCTCTCGCG

CTAAGATTAGCCCAGGTGGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAACGATC

CCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACG

GGGGCAGCATGGGATATGCCAATGGGAACCTGATCAGCCACCGGGGTGAAAAGCTTCCGTGA

ATCTTAGCCTGAAGTTGAAGAAATGAGCTTGCGTACGAAAAA

>SK035

ATGCAAGTCGAGCGGTAACAGGAATTAGCTTGCTAATTTGCTGACGAGCGGCGGACGGGTGA

GTAAAGCCTGGGAATATGCCTTAGTGTGGGGGATAACTATTGGAAACGATAGCTAATACCGC

ATAACGTCTTCGGACCAAAGAGGGGGACCTTCGGGCCTCTCGCGCTAAGATTAGCCCAGGTG

GGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAACGATCCCTAGCTGGTCTGAGAGG

TGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGATA

TTGCACAATGGGGAACCCTGATGCAGCCATGCCGCGTGTATG

>ANT031

CTGGTCTGAGAGGATGATCAGCCACATCGGGACTGAGACACGGCCCGAACTCCTACGGGAGG

CAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAG

AAGGCTTTCGGGTTGTAAAGCACTTTCAGTGAGGAGGAAGGCTTTCAGACTAATACTCTGGAG

GATTGACGTCACTCACAGAGAAGCACCGTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGG

GTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTGAGTAAGCGAGAT

GTGAAAGCCCCGGGCTTAACCTGGGAACGGCACTTCGAACTGCTCGGCTAGAGTGGTAGAGG

GTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAA

GGCGGCTACCTGGACCAACACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAG

ATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTT

>SKP24

TTCTGGTGCAATCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCA

CCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCC

GATCCGGACTACGACGCGTTTTGTGAGATTGGCTCCCCCTCGCGGGTTTGCAGCCCTCTGTGC

GCGCCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACCTGACGTCAT

>HRA34

TTCTGGTGCAGCCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCA

CCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCC

GATCCGGACTACGCACGTACTTTGTGGGATTCGCTCACCATCTCTGGTTGGCAGCCCTCTGTAT

ACGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCGTCCCCA

CCTTCCTCCGGTTTATCACCGGCAGTCT

>SKP1127P

CTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCTCAGGGAACGTAT

TCACCGCGTCATTCTGATACGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGAC

TCCAATCCGGACTACGACGCACTTTAAGTGATTCGCTTACTCTCGCGAGTTCGCAGCACTCTGT

ATGCGCCATTGTAGCACGTGTGTAGCCCTACACGTAAGGGCCATGATGACTTGACGTCGTC

>10SKE4

AATCAGTTCCGTTAACATACACATGCAAGTCGTAACAAGGTAACCAGGAGCTTGCTCCCAGGC

GTCGAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCCAGTAGTGGGGGATAGCCCGGG

GAAACCCGGATTAATACCGCATACGCCCTTTTGGGGAAAGCAGGGGACTTCGGACCTTGCGCT

ATTGGATGAGCCTAAGTCGGATTAGCTAGTTGGTGAGGTAATGGCCCACCAAGGCGACGATC

CGTAGCTGGTCTGAGAGGTGATCAGCCACATCGGGACTGAGACACGGCCCGAACTCCTACGG

AGGCAGCAGTGGGGATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGA

AGAAGGTTTCGGGTTGTAAAGCACTTTCAGTGAGGAGGAAGGCTCAAGTAATACTTTGAGGA

TGACGTCACTCACAGAAGAAGCACCGGGTTATCCCGCCCCCCCCCGGAAAAGGAGGGGGAGC

GTTATCGAATTATGGCGAAACCCGGAGGTGTTAATAACAAATGTAAACCCCGGTCACCTGGA

ACGCCTTCAATGTCGTAAGGGGGAAAGGATGGAATTCTGGGGCGTGAAGATAAAAAAGAAGA

CGGGGGGGTTTTTACACTCATGGCGAGTGAAGAGGAAAGATAAGTTGTCCGGAGCAGCGAAA

ATGATTCCTGGATTGACCTAGGGCTTCGCAATGCCCCGGAGAGCCCGGTAAACAATATTCGGC

CCCAACGGACTGGTAATTGCACGAAAACTTCGTTTTGGGATATTCAGAGTGG

>D2POST13

ACTTCGGTTGCAAACTCTCGTGTGTGACGGGCGGTTGTACAAGGCCCGGGAACGTATTCACCG

CGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGCAGGCGAGTTGCAGCCTGCAAT

CCGAACTGAGAATGGTTTTATGGGATTGGCTAAACCTCGCGGTCTCGCAGCCCTTTGTACCAT

CCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCAT

>SK713A

TGGTGCAGCCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCG

TGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGAT

CCGGACTACGCACGTACTTTGTGGGATTCGCTCACCATCTCTGGTTGGCAGCCCTCTGTATACG

CCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATATGACTTGACGTCGTCCCCACCTTC

CTCCGGTTTATCACCGGCAGTCTCCCTGGAGTTCCCACCATTACGTGCTGG

>HRP191

ACTTCTGGTGCAGCCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATT

CACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACT

CCGATCCGGACTACGACGTACTTTGTGGGATTCGCTCACCATCGCTGGTTGGCAGCCCTCTGT

ATACGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCGTCCC

CACCTTCCTCCGGTTTATCACCGGCAGTCTCCCTGGAGTT

>SKANT6

GGAAGTATTCACCGCGGCGTTGCTGATCCGCGAATTACTAGCGATTCCGACTTCATGTAGGCG

AGTTGCAGACTACAATCCGAACTGAGACTGGCTTTAAGAGATTAGCTTGCCGTCACCGGCTTG

CGACTCGTTGTACCAGCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTT

GACGTCATCCCCACCTTCCTCCGG

>SKP11

GCTACTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGT

ATTCACCGCGTCATTCTGATACGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAG

ACTCCAATCCGGAGTACGACGCACTTTAAGTGATTCGCTTACCCTCGCGAGTTC

>SKA14

GCTCCATCAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGG

ATAACTCCGGGAAACCGGAGCTAATACCGGATAGTATCTTGACCGCATGGTTCAAGTTGGAA

AGACGGTTTCGGCTGTCACTTACAGATGGGCCGCGGCGCATTAGCTAGTTGGTGAGGTATGGC

TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACA

CGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACG

GAGCAACGCCGCGTGAGTGATGAAGGTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACA

AGTGCGAGAGTAACTGCTCGCACCTTGACGGTACCTAACCAG

>PSK14

TACTTGCTACTTTGCTGACGAGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTGAGGT

GGGGGACAACAGTTGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTT

CGGCTCTCGCCTTTAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAA

GGCGACGATCCCTAGCTGGTTTGAGAGGAGATCAGCCACACTGGGACTGAGACACGGCCCAG

ACTCCTACGGGAGGCAGCAGTGGGGATATTGCAAATGGGCGCAGCCTGATGCACCATGCCGC

GTGTGTGAGAAGGC

>HR1717

GGAATTAGCTTGCTAATTTGCTGACGAGCGGCGGACGGGTGAGTAATGCCTGGGAATATGCCT

TAGTGTGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACGTCTTCGGACCAAAGA

GGGGGACCTTCGGGCCTCTCGCGCTAAGATTAGCCCAGGTGGGATT

>HR2715

CACATGCAAGTCGAGCGGTAACAGGAATTAGCTTGCTAATTTGCTGACGAGCGGCGGACGGG

TGAGTAAAGCCTGGGAATATGCCTTAGTGTGGGGGATAACTATTGGAAACGATAGCTAATAC

CGCATAACGTCTTCGGACCAAAGAGGGGGACCTTCGGGCCTCTCGCGCTAAGATTAGCCCAG

GTGGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAACGATCCCTAGCTGGTCTGAG

AGGTGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGG

ATATTGCACAAT

>SKP012

GTGCAATCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTG

ACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCC

GGACTACGACGCGTTTTGTGAGATTGGCTCCCCCTCGCGGGTTTGCAGCCCTCTGTGCGCGCC

ATTGTAGCACGTGTGTAG

>SKPOST03

ACTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCTCAGGGAACGT

ATTCACCGCGTCATTCTGATACGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAG

ACTCCAATCCGGACTACGACGCACTTTAAGTGATTCGCTTACTCTCGCGAGTTCGCAGCACTCT

GTATGCGCCATTGTAGCACGTGTGTAGCCCTACACGTAAGGGCCATGATGACTTGACGTCGT

>ANT4517

GGGAACGTATTCACCGCGGCGTTGCTGATCCGCGAATTACTAGCGATTCCGACTTCATGTAGG

CGAGTTGCAGACTACAATCCGAACTGAGACTGGCTTTAAGAGATTAGCTTGCCGTCACCGGCT

TGCGACTCGTTGTACCAGCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGAT

TTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTG

>ANT1517

CTCTTACGGTTACCTCACGGACTTCGGGTTTACCAGCTCTCATGGTGTTGACGGGCGGTGTGTA

CAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCGATTACTAGCAACTCCGGCTTCA

TGTAGGCGAGTTTCAGCCTACAATCCGAACTGGGATTGGTTTTTTAAGTTTAGCTCCACCTCGC

GGTCTTGCATCTTGTTGTACCAACCATTGTAGCACGTGTGTAGCCCTAGACATAAGGGGCATG

ATGATTTGACGTCATCCCCACCTTCCTCCCGGTTAACCCGGGCAGTCTCACTAGAGTGCTCAAC

TTAATGGTAGCAACTAATGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGA

CACGAGCTGACGACA

>POST5172

CGACTTCGGTTGCAAACTCTCGTGTGTGACGGGCGGTTGTACAAGGCCCGGGAACGTATTCAC

CGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGCAGGCGAGTTGCAGCCTGCA

ATCCGAACTGAGAATGGTTTTATGGGATTGGCTAAACCTCGCGGTCTCGCAGCCCTTTGTACC

ATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCAC

CTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGAT

CAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCAT

GCACCAC

>HR17P

TTCTGGTGCAATCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCA

CCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCC

GATCCGGACTACGACGCGTTTTGTGAGATTGGCTCCCCCTCGCGGGTTTGCAGCCCTCTGTGC

GCGCCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACCTGACGTCAT

>A2PSK6

GATTTTTGATCATGGCTCAGATTGAACGCTAGAAAAAAGGCCTAACACATGCAAGTCGAGCG

GTAACAGAAAGTAGCTTGCTACTTTGCTGACGAGCGGCGGACGGGTGAGTAATGCCTGGGAA

TTTGCCCATTCGAGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGG

GAAAGGAGGGGACCTTCGGGCCTTCCGCGAATGGATAAGCCCAGGTGGGATTAGCTAGTAGG

TGAGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGTTCTGAGAGGATGATCAGCCACACTG

GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCG

AAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGC

GAGGAGGAAAGGTTACTGGCTAATATCCAGTAGCTGTGACGTTACTCGC

>PSK13

GTGACGAGCGGCGGACGGGTGAGTAATGCTTGGGAACATGCCTTGAGGTGGGGGACAACAGT

TGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTTT

AGATTGGCCCAAGTGGGATGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAG

CTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG

CAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAG

AAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGG

>P1FSK

CCGTGCTTGGTAACATTCAGTGGTCTGCTCCCTCCGATAAATCTGGATAGCCCACCGGTCTTCG

GGTAAAACCAACTCCCATGGGGGGGACGGGGGGCGTGTACAAGGCCCGGGTAACGTATTCAC

CGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGAACTCGAGTTGCAGAGTGCA

ATCCGAACTGAGATAACTTTTTGGGATTCGCCACTTGTTGCCAAGAAGCAGCCCTCTG

Sequences for bacteria from “sick” slugs obtained using primer set 1492R-27F:

>S17F

CGGCTCGAGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGAC

GACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGATACGGCCCAGACTCC

TACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGGAAGCCTGACGCAGCGACGCCGCG

TGGGGGATGACGGCCTTCGGGTTGTAAA

>SICKT4

TCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCG

ACCGGCCACACTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT

TGCGCAATGGGCGGAAGCCTGACGCAGCGACGCCGCGT

>SICKT3

TCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGC

CTGAGAGGGCGACCGGCCACACTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCA

GTGGGGAATATTGCGCAATGGGCGGAAGCCTGACGCAGCGACGCCGCGTGGGGGATGACGGC

CTTCGGGTTGTAAACCTCTTTCGACCGGGACGAAGAGAGATTGACGGTACCGGTATAAGAA

>SICKT8

TACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGATAC

GGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGGAAGCCTGACGC

AGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCGCTATCGACGAAGCC

>S12F

TGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGTAAGGCCCTTCG

GGGTACACGAGTGGCGAACGGGTGAGTAACACGTGGGTGACCTGCCTCGAGCTCTGGGATAA

GCCTGGGAAACTGGGTCTAATACCGGATATGTCGGCTCGAGATGGGCCCGCGGCCTAGAGGC

AGCAGTGGGGAATATTGCGCAATGGGCGGAAGCCTGACGCAGCGACGCCGCGTGGGGGATGA

CGGCCTTCGGGTTGTAAACCT

Figure A1: Light microscope images of a homogenous sub-cultured sample after completion of the Gram staining procedure. Right: Pseudomonas sample at 1000x, which remained red after the procedure, indicative of a Gram-negative (-) species. Left: negative control sample of Streptococcus at 1000x, which turned blue after the procedure, indicative of a Gram-positive (+) species.

Table A1: Results of biochemical tests on bacterial populations that were most prevalent in the anterior part of the T. tetraquetra intestine. Populations were grouped together based on physical and behavioral similarities.

Voges - Meth Grou Motili Citrat Nitrat Oxida Catal Prosk yl p Gram ty Indole e e se ase auer Red AA n=23 Neg Pos Neg Pos Pos Pos Neg Neg Neg AB n=21 Neg Pos Neg Neg Pos Pos Pos Neg Neg AC Pos, 2 n=9 Neg Pos Neg Pos Pos neg Pos Neg Neg AD n=2 Neg Pos Neg Pos Pos Pos Pos Neg Neg AF n=2 Neg Pos Neg Neg Pos Pos Pos Neg Neg AG n=2 Pos Neg Neg Neg Pos Neg Neg Pos Neg

Although the bacterial species identified were of different cellular and colonial morphologies, five species were Gram-negative (-). While this supported the hypothesis that enteric bacteria may be Gram-negative, it is possible that the growth of bacterial cultures skewed more towards Gram-negative species than Gram-positive ones due to laboratory settings. It is entirely possible that many more Gram-positive species were present within the intestinal lining of the sea slug.

Table A2: Results of biochemical tests on bacterial populations that were most prevalent in the posterior part of the T. tetraquetra intestine. Populations were grouped together based on physical and behavioral similarities.

Voges - Motili Citrat Nitrat Oxida Catala Prosk Methy Group Gram ty Indole e e se se auer l Red PA Neg, 2 n=9 Neg pos Neg Pos Pos Pos Pos Pos Neg PB n=4 Neg Neg Pos Pos Neg Pos Pos Neg Pos PC Neg, 1 n=2 Neg Pos Neg Pos Neg pos Neg Neg Neg PD n=5 Neg Neg Neg Pos Pos Pos Pos Neg Neg PE n=2 Neg Neg Neg Pos Pos Neg Pos Neg Neg PF n=6 Neg Pos Neg Neg Neg Pos Pos Neg Neg PG n=7 Neg Neg Neg Pos Neg Pos Neg Neg Neg PH n=4 Neg Pos Neg Neg Pos Pos Neg Neg Neg PI n=4 Neg Pos Neg Neg Neg Pos Pos Neg Neg PJ n=4 Neg Pos Neg Neg Neg Neg Pos Neg Pos PK n=3 Neg Pos Neg Pos Pos Pos Pos Neg Neg PL n=3 Neg Neg Neg Neg Pos Pos Pos Neg Neg PM n=2 Neg Pos Neg Neg Neg Pos Pos Neg Neg PN n=3 Neg Neg Neg Pos Pos Pos Pos Neg Neg PO n=2 Neg Pos Neg Pos Pos Pos Pos Neg Neg PQ n=2 Pos Neg Neg Neg Pos Neg Neg Pos Neg

Table A3: NCBI BLAST matches to the additional isolated genomic DNA of bacterial species found within the intestinal lining of “sick” T. tetraquetra using the bacterial 16S rRNA gene.

Identified Species % Homology No. of Samples (n=5) Pseudomonas putida 86% 2 Pseudomonas oryzihabitans 86% 1 Mycoplasma elephantis 80% 1 Mycoplasma equigenitalium 80% 1