The Impacts of Putative Ptilosarcus Gurneyi Toxins on Cancer Cell Growth and on Predator Species, Tritonia Tetraquetra

THE IMPACTS OF PUTATIVE PTILOSARCUS GURNEYI TOXINS ON

CANCER CELL GROWTH AND ON PREDATOR SPECIES, TRITONIA

TETRAQUETRA

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A University Thesis Presented to the Faculty

California State University, East Bay

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In Partial Fulfillment

of the Requirements for the Degree

Master of Science in (Biological Sciences)

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Archana Navinbhai Patel

August 2018

Abstract

Orange sea pens (Ptilosarcus gurneyi) are known to have few predators as a result of their chemical defense mechanisms. Very little is known about the sequestered chemicals and their toxic effects, if any, on prey species Tritonia tetraquetra (sea slug). A series of suction electrode nerve recordings were used to observe if exposure of sea pen extract to the Tritonia buccal ganglia and brain would change spike activity. Sea pen extract was also used to test for cytotoxic effects on human epithelial cells (MDA-MB-231) by measuring death rate. Results showed 3 out of

7 of the recordings exhibited increased activity in response to the sea pen extract,

2 out of 7 showed depressed activity in response to the sea pen extract, and 2 out

7 displayed increased activity only during the sea pen extract rinse phase. In response to the sea pen extract, MDA-MB-231 cells were observed to have cytotoxic effects in a dose and time-dependent manner. The 1:2 dilution (filtered sea pen extract) increased cell death by 5 times more than the control (ASW) over the 5-day span. These results support a possible neurophysiological effect on

Tritonia, helping us gain further insight as to why Ptilosarcus gurneyi has so few predators. This may also further substantiate evidence of Tritonia tetraquetra’s resistance to putative toxins as an evolved adaptation.

iii

Acknowledgements

I would first like to thank my thesis committee and the staff/faculty at

CSUEB, for helping me navigate through the masters’ process diligently. I specifically want to mention my primary investigator, Dr. James Murray, who worked with me relentlessly to develop a great neurophysiological research study and encouraged me to see it through. I would also like to thank Dr. Caron

Inouye for her knowledge and comments on toxicity, and Dr. Kenneth Curr for his background on cell culture techniques. Lastly, I’d like to thank my family for always supporting my dreams while shading me from the heat, and giving me refuge through the storm. Dad, Mom, Ace, Bhabs, Calum, and Chief…I love you.

Table of Contents

ABSTRACT ...... iii

LIST OF FIGURES ...... vii

LIST OF TABLES……………………………………………………………………...viii

INTRODUCTION...... 1

OBJECTIVES……………………………………………………………………………..3

METHODS/MATERIALS...... 3

1. COLLECTION...... 3 2. P. gurneyi TOXIN……………………………………………………………….4 3. NEUROPHYSIOLOGICAL RECORDINGS………………………………….4 4. SPIKE DATA ANALYSIS……………………………………………………....6 5. CELL CULTURE………………………………………………………………...7 A. SEPARATING CELLS INTO WELLS……………………………………..7 B. CREATING AND ADDING DILUTIONS TO WELLS…………...……11 C. COUNTING CELLS…………………………………………………..…....12

RESULTS ...... 15

DISCUSSION ...... 44

CONCLUSION ...... 48

REFERENCES ...... 49

List of Figures

Figure 1 Photos of predator/prey species………………………………….….2

Figure 2 Hemocytometer…………………………………………………….…..9

Figure 3 MDA-MB-231 live cells under 10x magnification…………………10

Figure 4 MDA-MB-231 dead cells under 10x magnification..………………14

Figure 5 Spike amp (µV) over spikes/1min/bin; matches prediction……..19

Figure 6 Spike amp (µV) over spikes/1min/bin; matches prediction….….23

Figure 7 Spike amp (µV) over spikes/1min/bin; matches prediction….….27

Figure 8 Spike amp (µV) over spikes/1min/bin; low P2 activity……….…31

Figure 9 Spike amp (µV) over spikes/1min/bin; low P2 activity……….....34

Figure 10 Spike amp (µV) over spikes/1min/bin; high P3 activity…….…...37

Figure 11 Spike amp (µV) over spikes/1min/bin; high P3 activity…….…...41

Figure 12A Growth curve MDA-MB-231 cell concentration…………….……..42

Figure 12B Histogram MDA-MB-231 cell concentration………………….……43

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List of Tables

Table 1 Spike rate per phase (6min-brain)………………………………..17

Table 2 Ratios of spike rate per phase (6min-brain)………..………....…18

Table 3 Spike rate per phase (50min-buccal)…………………………..…20

Table 4 Ratios of spike rate per phase (50min-buccal)……....………...... 21

Table 5 Spike rate per phase (55min-buccal)……………………………..24

Table 6 Ratios of spike rate per phase (55min-buccal)……....………...... 25

Table 7 Spike rate per phase (60min-brain)……………………………....28

Table 8 Ratios of spike rate per phase (60min-brain)………..………...... 29

Table 9 Spike rate per phase (51min-brain)……………………………....32

Table 10 Ratios of spike rate per phase (51min-brain)………..………...... 33

Table 11 Spike rate per phase (30min-buccal)…………………………...... 35

Table 12 Ratios of spike rate per phase (30min-buccal)……....………...... 36

Table 13 Spike rate per phase (41min-brain)……………………………....38

Table 14 Ratios of spike rate per phase (41min-brain)………..………...... 39

viii

Introduction

The orange sea pens, Ptilosarcus gurneyi, are sessile marine cnidarians that have very few predators (Birkeland, 1974) as a result of sequestration of a chemical defense (Wekell, 1978). Their chemical defense has been attributed to two secondary metabolites, ptilosarcone and ptilosarcenone (Wratten, Fenical,

Faulkner, & Wekell, 1977). These diterpenoid compounds behaved similarly to neurotoxins, which target acetylcholinesterase when administered to mice and fish (Wekell, 1978). Since neither mice nor fish are closely related neurophysiologically to T. tetraquetra, for the context of this paper we will refer to the extracted chemical from P. gurneyi, as a sea pen extract.

Certain species of fish and crabs seem to find the sequestered chemicals unpalatable (Thompson, 1960a; Shapiro et al., 2011), except for seven invertebrates (Birkeland, 1974) including the sea slug, Tritonia tetraquetra

[formerly known as T. diomedea (Martynov, 2006) or T. gilberti (Thompson, 1976)].

T. tetraquetra is a marine mollusk that has no shell and feeds on sea pens (Figure

1) and related species. T. tetraquetra may not be subject to the toxic impacts of P. gurneyi if they developed or evolved a defense that may include the deactivation of the putative toxin in the gut or blood. However, deactivated, a putative neurotoxin (Wekell, 1978) could travel to the brain where it could affect brain activity. Ptilosarcone was proven to inhibit acetylcholinesterase, an enzyme that terminates synaptic transmission by hydrolyzing the neurotransmitter acetylcholine (Wratten et al., 1977). If acetylcholine is inhibited the sending of signals between nerve cells will be accentuated (Dvir, Silman, Harel, Rosenberry, 2

& Sussman, 2010). Acetylcholine has been shown to be excitatory in some synapses of the Tritonia buccal ganglion (Willows, A.O.D., Lloyd, P.E., &

Masinovsky, B.P., 1988). I proposed testing for the presence of neurological responses to P. gurneyi toxins, with the hypothesis that the sea pen extract impacts Tritonia neurologically specifically inhibiting acetylcholinesterase and generating higher frequency spike activity during exposure.

Figure 1: PHOTOS OF PREDATOR/PREY SPECIES

Left most photograph shows the predator species, sea slug, feeding on prey species, sea pen. Center photo shows orange sea pen and right most photograph shows a non-feeding sea slug. (photos, J. Murray)

Shapiro et al., (2011) tested antifeedant effects of aqueous sea pen extracts offered to crabs in samples of artificial diet. The crabs chose to not eat the sea pen-infused samples despite being starved for more than 10 days. The crabs were hungry since they would eat control samples of raw chicken, indicating selective preference and aversion to chemical stimuli. The crab was equally attracted to both samples, as the crab tasted the sea pen-embedded gelatin and control gelatin equally, but it would immediately spit the sea pen samples back out. The 3

sea pen chemicals are evidently unpalatable to this species, but we cannot attribute the crab’s preferences to presence of toxicity without further testing.

They have yet to determine the effect (if any) of this putative toxin beyond the point that crabs didn’t find them very palatable (Shapiro et al., 2011). In addition to the central nervous system recordings, I tested mammalian cell cultures for general toxicity by measuring cell proliferation (Ekwall, Silano, Paganuzzi-

Stammati, & Zucco, 1990), with the hypothesis that the MDA-MB-231 cells will have lower cell counts in comparison to normal cell line growth (control) after sea pen extract exposure, indicating there are cytotoxic effects by interfering with

PI3K/AKT pathways leading to cell apoptosis (Jian, Zhang, Han, & Liu, 2018).

We can learn a lot about the putative toxins’ effect on certain species and perhaps gain further insight as to why this species has so few predators. This may further substantiate evidence of the sea slugs’ resistance to putative toxins as an evolved adaptation.

Objectives

I. Determine if P. gurneyi toxins have any neurophysiological impacts on T. tetraquetra

II. Test for cytoxicity by measuring death rate in human epithelial cells

Methods/Materials

I. Collection

Collections of slugs were performed on SCUBA at Dash Point WA, Tofino

BC, and Whidbey Island WA. T. tetraquetra and P. gurneyi (Figure 2) were held at the University of Washington Friday Harbor marine labs prior to being sent in 4

seawater to California State University East Bay Biology department where they were kept in a tank with artificial sea water (ASW) temperature ranging from

8°C to 10°C.

2. P. gurneyi toxin

Pinna from live P. gurneyi were removed with scissors and frozen whole at –80 °C within 15 min of removal. They were blended using an Osterizer super deluxe blender on high speed for 90 seconds in a 1:10 mixture by weight with artificial seawater (Instant Ocean, 28 ppt). The homogenate was then centrifuged using an International Equipment Company Clinical Centrifuge for four minutes at 1200rpm (~242 g0) to remove solids and the supernatant was frozen at –20°C until needed.

3. Neurophysiological recordings

T. tetraquetra were dissected live via an incision made along the dorsal region, exposing the brain that sat dorsal to the mass and dorsal/posterior of the esophagus. The paired buccal ganglia were then removed which were located ventral/anterior of the esophagus adjacent to the buccal mass. Both buccal ganglia and brain were removed from 7 sea slugs and pinned individually to a

50mm Sylgard (Corning) lined petri dish immersed in 30 ml of artificial seawater

(28 ppt). Suction electrode nerve recordings were collected from the brain for 4 of the experiments and from the buccal ganglia for the remaining 3 with the use of

LabChart software (AD instruments, version 8.0) running Power Lab 26T, two

HumBug 50/60 Hz Noise Eliminators (North Vancouver, BC), and A-M Systems differential AC amplifier model 1700 were used to filter and actively cancel 5

external 60 Hz noise and amplify extracellular recordings (channels 1 and 2 had filters set with low cut off at 100Hz, high cut off at 1kHz, gain set to x1000, and notch filter set to off). Lab Chart was set to a sampling rate of 4kHz for each channel, range was set to ± 100mV, and a low pass filter set to a cut off frequency of 100Hz with a transition width of 10Hz.

Recordings lasted a minimum of three hours total: an hour per each of the three stages. Stage 1 was a baseline of spontaneous activity. During Stage 2,

20ml of P. gurneyi extract (the 1:10 mixture) was added to 30ml of water already in the dish (final dilution 1:25) using a 60mL dual compartment syringe. The syringe was attached to ¼” clear flexible PVC tubing with end placed directly into 50mm Sylgard lined dish (anchored using a tubing clip, which kept tubing from slipping out of dish) where buccal ganglia and brain were pinned. Perfused water temperature was maintained cool by making sure that the ¼” clear flexible

PVC tubing was submersed within the same chilled seawater bath that surrounded the 50 mm dish with the buccal ganglion. The 20ml of perfused fluid was fed by gravity into the chamber with the ganglion over 30seconds. Stage 3 was a rinse stage that involved four cold artificial seawater washes via a large turkey baster holding about 60ml, water being taken from the surrounding seawater bath to maintain similar temperature within 50mm dish (washes helped to eliminate temperature fluctuation due to microscope light heating the sample).

Temperature of the seawater bath was kept below 10°C using a pump placed in a chest of ice and freshwater which was connected to an outer acrylic chamber surrounding the inner seawater bath where the petri dish containing the ganglia 6

was partially immersed. Keeping temperature relatively low was important for cell viability while recording from neurons. Buccal ganglia and brain removed from each Tritonia were recorded from simultaneously on different channels, each channel representing recordings from one nerve of the brain or buccal ganglia. The specimen during the experiment was kept in a petri dish filled with seawater slightly immersed in a bath around 5mm deep at a temperature between 8-11°C.

4. Spike Data Analysis

Once action potentials were obtained from the nerves, a maximum of one hour of recording from each of the three stages (stage 1: pre-extract, stage 2: added extract, and stage 3: post-rinse) was selected for analysis review using Lab tutor software (AD Instruments, version 8.0). One hour before the addition of sea pen extract, one hour after 20 ml of sea pen extract exposure, and one hour after the rinse of sea pen extract with 60 ml of ASW was reviewed using spike analysis. Spike analysis allowed for the discrimination of the extracellular neural spike activity by amplitude and duration, to determine if total neuronal activity was higher or lower in response to the extract. The same low-cut off amplitude was used in all three stages of each recording but varied 4µV to 30µV from recording to recording, based on noise exclusion cut-off voltage. If overall spike- firing rate from all of the axons in the nerve were excited there should be an increase in frequency, where as if spike-firing rate has reduced frequency in the same time interval they were on average inhibited. Many cells are spontaneously 7

active even after the brain is removed from the body and we monitored this activity during the three stages of the experiment.

The spike discriminator helps to look at each individual recorded spike, measuring both height and duration. By using the discriminator we selectively excluded recorded spikes making the data more specific to the desired parameters. The choice of threshold for spike detection was modified to each recording because a low threshold could capture many noise events while a high threshold could miss many spikes. A spike height between 0.9µV-300µV, and duration within 1.0-1.2ms were considered to be real action potentials, anything outside of these parameters was considered to be background noise and omitted.

Background noise accounted for noise events such as electrical interference.

After spike analysis, a spike amplitude histogram for each nerve recording was created for the three stages of the experiment (stage 1: pre-extract, stage 2:

20ml extract, and stage 3: post-extract) based on firing rate and time (bin size was set to 2µv). The size of field potential is usually directly proportionate to axon diameter, so the histograms showed activities of multiple motor neurons. By recruiting the smallest motor units first, the amount of fatigue the organism experiences is minimized. Bin size allowed the range of values to be visualized more clearly, by dividing the entire range into a series of spike amplitude ranges, and then counting how many spikes fall into each amplitude range. Ranges can be made smaller or larger by changing the bin width, which were measured in microvolts. The spike histogram allowed us to make observations on specific 8

spike activity per amplitude range, which allowed for comparisons between the three stages of my experiment.

5. Cell Culture

Part A: Separating cells into wells

Cell toxicity tests were run on MDA-MB-231 (ATCC HTB-26) human epithelial cells (derived from metastatic site of mammary gland tissue) to observe the potential effects of the putative toxins extracted from P. gurneyi based on cell death (American Type Culture Collection (ATCC), Manassas, VA). Cell counts were taken over a five-day time span and run in triplicates (five 12-well polystyrene plates with 22mm well diameter) to observe any variation. If the sea pen extract does affect cell death we should see lower cell numbers relative to controls. First, 5mL of culture medium and 3mL of Trypsin-EDTA (filtered using

0.45µm filter which removes any contaminating large particles present during media preparation) were added to the cells in a Nunclon delta surface flask, bringing the total volume of cells to 8mL. Culture medium provides nutrients for the cells, while Trypsin is a proteolytic enzyme that helps to dissociate adherent cells from the vessel. The solution was then pipetted up and down three times using a 1ml pipette to help facilitate breaking up clumps. Next, 0.2mL of MDA-

MB-231 cells were added to 0.1ml of Trypan blue (0.4%) and 0.7mL of PBS brining the total volume to 1mL. Trypan blue is a stain taken up only by dead cells, which makes it easier to count the living cells. PBS is a water-based salt solution containing phosphate that helps to maintain normal osmolarity and a constant pH. The cells were then counted using a hemocytometer (Figure 2), 9

which was first cleaned with 70% ethanol. A cover slip (cleaned with 70% ethanol) was placed on the front face where the rectangular indentation that creates the chamber is exposed, and a micropipettor was used to add 10µL of the cells-Trypan-PBS mixture to each chamber.

Figure 2: HEMOCYTOMETER

Bright-Line Hemocytometer for in vitro diagnostic use for counting blood cells

An inverted microscope (Nikon Eclipse TS100) was then used to count the cells (Figure 3) in all eight-1mm2 quadrants (four in each chamber), using the 10x objective for magnification. A laboratory counter (Clay Adams) was used to keep an accurate count. 10

Figure 3: MDA-MB-231 LIVE CELLS UNDER 10X MAGNIFICATION

Photo pictured shows live MDA-MB-231 cells under 10x magnification using an inverted microscope that were counted to determine cell yield. Arrows point to live cells, which appear bright, located within 1mm2 quadrant within the grid, displayed using the hemocytometer.

All information at this point was used to calculate the dilution factor (DF)

(5), cell concentration (2.1 x 10^07 cells/mL), cell yield (1.6 x 10^07 cells), and initial volume (0.30mL) in order to prepare a master mix that allowed for ~10,000 cells to be in each well.

�� = �� 11

�� (�� )(10!)(��) �� = ��

�� = ��

*Final volume = flask volume – volume of cells added

�2�2 �1 = �1

*10! is a constant that represents the dimension of how much volume the hemocytometer can hold

Once those calculations were complete, it was clear there were enough cells to fill the wells and a master mix was created consisting of 60.7mL DMEM

(Dulbecco’s Modified Eagle’s Medium; growth media for cells) and 0.3mL of

MDA-MB-231 cells bringing the total volume to 61mL. Even though there are only 60 wells, the total volume was calculated for 61mL to account for pipetting error. Using a 10mL pipette, 1mL of the DMEM and cells mixture (~10,000 cells) was transferred to each well (60 total) and plates were placed in the 37°C

(optimal temperature for growth) incubator for 24 hours.

Part B: Creating and adding dilutions to wells

The next day dilutions were prepared using artificial seawater (50mL) and sea pen extract (20mL) that were filtered through a 0.22µm filter into 50mL conical tubes to remove bacteria and sediment. Four 15ml conical tubes were labeled as C (control), 1:50, 1:5, and 1:2 in order of lowest concentration to highest. The 1:2 dilution conical tube consisted of 13mL filtered sea pen extract, the 1:5 dilution conical tube consisted of 1.3mL filtered sea pen extract and

11.7mL filtered artificial seawater, the 1:50 dilution conical tube consisted of 12

1.3mL mix from the 1:5 dilution and 11.7mL of filtered artificial seawater, and the control was 13mL filtered artificial seawater. Due to a miscalculation, two separate batches of dilutions were made to accommodate all five plates since each dilution final volume needed to be 15mL and not 13mL. Next all five plates were removed from the incubator and a Pasteur pipette was used to suction out all the growth media from each well, leaving just the adherent cells. A new

Pasteur pipette must be used for each plate, and should not touch the edges of the glass capsule to avoid cross contamination. The plates were filled from lowest concentration to highest, using a 10mL pipette each well was transferred

1mL of dilution (just enough to cover the cells) and 1mL of DMEM growth media. Plates were then placed in the 37°C incubator for 24 hours. All work was done under the hood in a closed off area of the lab with no reuse of pipettes, as a lack of sterile environment could cause excessive bacterial contamination.

Part C: Counting cells

The next day cells were checked using an inverted microscope to make sure they were still adhered before carrying out aspiration. First the cells were aspirated by using a Pasteur pipette to suction out the master mix. One mL of

PBS was transferred to each well using a 10mL pipette, which helps to remove the media and sera (contains trypsin inhibitor) out of the wells. While the PBS was sitting in wells (5mins), the Trypsin-EDTA (Ethylenediamine tetraacetic acid) was placed in a 37°C water bath to thaw out (normally takes no more than a few minutes). Trypsin helps to dislodge cells from the vessel, while EDTA is a chelator that picks up Mg2+ and Ca2+ ions to disable attachment of cells from 13

surface. As soon as the Trypsin-EDTA thaws out, it must immediately be placed on ice because Trypsin is a non-specific protease and will inactivate itself if it doesn’t remain near its optimal temperature (37°C). After 5minutes, cells were aspirated and 0.5mL of Trypsin-EDTA was transferred to each well (12) using a

2mL pipette. The plate was rocked back and forth a few times to make sure cells were covered, before put back in the incubator at 37°C. After 5 minutes 1mL of complete DMEM was transferred to each well to neutralize the Trypsin-EDTA, and prevent degradation of integral proteins essentially leading to cell death.

Once all dilutions are neutralized, aliquots were made in 12 x 75 mm test tubes consisting of 0.2mL of cells, 0.1ml Trypan Blue, and 0.7mL PBS. A 10µL pipette was used to pipette 5µL of solution from each aliquot into each of the hemocytometer chambers. Live cells were counted from each sample, three separate times, and averaged before calculating cell concentration and yield. Live cells appear as hollow halos, whereas dead cells have broken membranes and will appear dark blue from Trypan blue dye uptake (Figure 4.). Cell counting was repeated each day, for five days, at the same hour (1-2pm). 14

Figure 4: MDA-MB-231 DEAD CELLS UNDER 10X MAGNIFICATION

Photo pictured shows live and dead MDA-MB-231 cells under 10x magnification using an inverted microscope that were counted to determine cell concentration (cells/mL). Black arrow point to live cells, which appear bright, while red arrows point to dead cells, which appear dark blue.

Results

Overall the data indicate that P. gurneyi toxins do have a neurophysiological impact on T. tetraquetra. Figures 5 (brain), 6 (buccal), and 7

(buccal) showed increased spike activity during sea pen extract exposure (P2) in comparison to baseline activity (P1). The increased activity was then seen to decrease between baseline and exposure rate during the sea pen extract rinse phase (P3). Tables 2, 4, and 6 showed excited spike activity from 10-90µV for P2 spikes. The extract exposure ratios (P2/P1) and rinse ratios (P2/P3) were greater than 1 from 10-100µV, indicating there was an increase in P2 (extract exposure) activity in comparison to the P1 (baseline) and P3 (post-rinse).

Figures 8 (brain) and 9 (brain) showed decreased spike activity during sea pen extract exposure (P2) in comparison to baseline activity (P1). The decreased activity was then seen to increase slightly above the baseline during the sea pen extract rinse (P3). Tables 8 and 10 showed inhibited spike activity from 40-60µV and 100-320µV for P2 spikes. The extract exposure ratios (P2/P1) and rinse ratios

(P2/P3) were less than 1 from 10-60µV and 100-320µV, indicating there was a decrease in P2 (extract exposure) activity in comparison to the P1 (baseline) and

P3 (post-rinse).

Figures 10 (buccal) and 11(brain) showed increased spike activity during the sea pen extract rinse phase (P3) only. Tables 12 and 14 showed excited spike activity from 10-80µV for P3 spikes. The rinse ratios (P2/P3) and baseline ratios

(P1/P3) were less than 1 from 10-80µV, indicating there was an increase in P3

(post-rinse) activity in comparison to the P1 (baseline) and P2 (extract exposure). 16

Putative sea pen toxins exerted cytotoxic effects on human epithelial cells in a dose and time-dependent manner. The 1:2 dilution (filtered sea pen extract) had the most effect on increasing cell death each day (Figure 11B) with a final cell concentration of 1.3 x 10^04 cells/mL by the end of day 5 (a 166x reduction from initial cell concentration of 2.1 x 10^06 cells/mL) and the control (filtered artificial sea water) had the least effect on increasing cell death each day (Figure

11B) with a final cell concentration of 6.3 x 10^04 cells/mL by the end of day 5 (a

33x reduction from initial cell concentration of 2.1 x 10^06 cells/mL). The 1:50 and 1:5 dilutions also had moderate effects on increasing cell death in comparison to the 1:2 dilution, with an initial cell concentration of 2.1 x10^06 cells/mL and a final cell concentration of 3.8 x 10^04 for the 1:50 dilution and 3.1 x 10^04 cells/mL for the 1:5 dilution by the end of day 5. ANOVA (p = 0.00826),

Kruskal-Wallis (p = 0.03), and exponential regression (R^2 = 0.689, p = 0.013) data were all significant (p < 0.05). 17

Table 1: The breakdown of spike rates over a 60 minute recording time during pre-extract, extract, and post-extract exposure to brain for a single experiment.

Phase Spike Counts/60min

1. pre-extract 4359

2. extract 8895

3. post-extract 4298

Table 2: Ratio of spikes per phase over a 60 minute recording time, for extract exposure, rinse, and baseline categorized by 10µV increments. Spike counts were summed over 10µV increments then broken up into three ratios, representing extract exposure (P2/P1), rinse exposure (P2/P3), and baseline activity (P1/P3). All calculated values greater than 1 were rounded to the nearest whole number, and values less than 1 to the nearest decimal.

Bin size P1 spikes P2 spikes P3 spikes Extract Rinse Baseline (10µV) exposure (P2/P3) (P1/P3) (P2/P1)

20-30µV 44 71 17 2 4 3

30-40µV 15 30 15 2 2 1

40-50µV 9 21 6 2 3 1

50-60µV 1 11 0.8 9 14 2

60-70µV 2 13 0.03 5 391 72

70-80µV 1 2 0.02 2 95 52

80-90µV 0.1 0.2 0 2 0 0 90- 100µV 0.02 0.1 0 5 0 0 100- 110µV 0 0.1 0 0 0 0 110- 120µV 0 0.1 0 0 0 0 19

20 P1 (counts/min/bin) 15 P2 (counts/min/bin) P3 (counts/min/bin)

Counts/Min/Bin 10

0 0 50 100 150 200 250 300 Spike Amp Baseline

Figure 5: SPIKE AMP (µV) OVER SPIKES/1MIN/BIN; MATCHES PREDICTION Spike amplitude (µV) over spikes/1min/bin for each of the 3 recorded phases over 60 minutes during pre-extract, extract, and post-extract exposure to brain with bin size set at 2µv and noise exclusion cut-off voltage set at 20µV. Data matches prediction, there was an increase in P2 activity from P1, which then decreased during P3. P1 (red) represents the pre-extract phase, P2 (green) represents the sea pen extract exposure phase, and P3 (blue) represents the post-extract phase. 20

Table 3: The breakdown of average spike rates over a 30minute recording time during pre-extract, extract, and post-extract exposure to buccal ganglia for a single experiment.

Phase Spike Counts/30min 1. pre-extract 69711

2. extract 94027

3. post-extract 63564

Table 4: Ratio of spikes per phase over a 30 minute recording time, for extract exposure, rinse, and baseline categorized by 10µV increments. Spike counts were summed over 10µV increments then broken up into three ratios, representing extract exposure (P2/P1), rinse exposure (P2/P3), and baseline activity (P1/P3). All calculated values greater than 1 were rounded to the nearest whole number, and values less than 1 to the nearest decimal.

Bin size P1 spikes P2 spikes P3 spikes Extract Rinse Baseline (10µV) exposure (P2/P3) (P1/P3) (P2/P1)

10-20µV 1889 3127 1597 2 2 1

20-30µV 197 475 330 2 2 0.6

30-40µV 67 91 110 2 0.8 0.6

40-50µV 55 30 33 0.5 0.9 2

50-60µV 17 55 22 3 3 0.8

60-70µV 5 31 13 7 3 0.4

70-80µV 8 6 3 0.8 2 3

80-90µV 15 2 1 0.1 1 11

90-100µV 2 0.4 0.6 0.1 0.6 3 100- 110µV 1 0.2 0.9 0.2 0.2 1 110- 120µV 1 0.2 1 0.1 0.2 1 120- 130µV 0.3 0.4 1 1 0.3 0.2 22

130- 140µV 0.3 0.7 1 3 0.5 0.2 140- 150µV 0.3 1 1 5 1 0.2 150- 160µV 0.1 3 1 19 2 1 160- 170µV 0.1 2 1 23 3 0.1 170- 180µV 0.2 2 2 13 1 0.1 180- 190µV 0.3 2 3 6 0.6 0.1 190- 200µV 0.2 3 4 15 1 0.1

1400

1200

1000

800 P1 (counts/min/bin) 600 P2 (counts/min/bin) P3 (counts/min/bin)

Counts/Min/Bin 400

200

0 0 50 100 150 200 250 300

Spike Amp Baseline (µV) Figure 6: SPIKE AMP (µV) OVER SPIKES/1MIN/BIN; MATCHES PREDICTION Spike amplitude (µV) over spikes/1min/bin for each of the 3 recorded phases over 30 minutes during pre-extract, extract, and post-extract exposure to buccal ganglia with bin size set at 2µv and noise exclusion cut-off voltage set at 8µV. Data matches prediction since there was an increase in P2 activity from P1, which then decreased during P3. P1 (red) represents the pre-extract phase, P2 (green) represents the sea pen extract exposure phase, and P3 (blue) represents the post-extract phase. 24

Table 5: The breakdown of average spike rates over a 41minute recording time during pre-extract, extract, and post-extract exposure to buccal ganglia for a single experiment.

Phase Spike Counts/41min

1. pre-extract 13952

2. extract 17868

3. post-extract 8254

Table 6: Ratio of spikes per phase over a 41 minute recording time, for extract exposure, rinse, and baseline categorized by 10µV increments. Spike counts were summed over 10µV increments then broken up into three ratios, representing extract exposure (P2/P1), rinse exposure (P2/P3), and baseline activity (P1/P3). All calculated values greater than 1 were rounded to the nearest whole number, and values less than 1 to the nearest decimal.

Bin size P1 spikes P2 spikes P3 spikes Extract Rinse Baseline (10µV) exposure (P2/P3) (P1/P3) (P2/P1)

10-20µV 238 278 123 1 2 2

20-30µV 37 29 24 0.8 1 2

30-40µV 14 25 12 2 2 1

40-50µV 25 44 17 2 3 1

50-60µV 3 13 2 4 8 2

60-70µV 6 28 8 5 4 0.8

70-80µV 0.3 4 6 12 0.7 0.1

80-90µV 0.1 1 1 10 1 0.1

90-100µV 0.4 0.5 0.3 1 2 1 100- 110µV 1 0.2 0.4 0.2 0.7 4 110- 120µV 2 0.1 1 0.03 0.1 2 26

120- 130µV 3 0.2 1 0.1 0.3 3 130- 140µV 5 0.2 3 0.1 0.1 2 140- 150µV 1 3 1 3 2 1 150- 160µV 0.2 9 0.1 380 95 1 160- 170µV 0 0.4 0 0 0 1 170- 180µV 0.02 0.05 0 2 0 1 180- 190µV 0 0.02 0 0 0 1 190- 200µV 0 0 0 0 0 0

140

120

100

80 P1 (counts/min/bin) P2 (counts/min/bin) 60 P3 (counts/min/bin) 40 Counts/Min/Bin

0 0 50 100 150 200 250 300 Spike Amp Baseline (µV)

Figure 7: SPIKE AMP (µV) OVER SPIKES/1MIN/BIN; MATCHES PREDICTION Spike amplitude (µV) over spikes/1min/bin for each of the 3 recorded phases over 41 minutes during pre-extract, extract, and post-extract exposure to buccal ganglia with bin size set at 2µv and noise exclusion cut-off voltage set at 10µV. Data matches prediction since there was an increase in P2 activity from P1, which then decreased during P3. P1 (red) represents the pre-extract phase, P2 (green) represents the sea pen extract exposure phase, and P3 (blue) represents the post-extract phase.

Table 7: The breakdown of average spike rates over a 55 minute recording time during pre-extract, extract, and post-extract exposure to brain for a single experiment.

Phase Spike Counts/55min 1. pre-extract 5116

2. extract 4281

3. post-extract 5280

Table 8: Ratio of spikes per phase over a 55 minute recording time, for extract exposure, rinse, and baseline categorized by 10µV increments. Spike counts were summed over 10µV increments then broken up into three ratios, representing extract exposure (P2/P1), rinse exposure (P2/P3), and baseline activity (P1/P3). All calculated values greater than 1 were rounded to the nearest whole number, and values less than 1 to the nearest decimal.

B78in size P1 P2 spikes P3 spikes Extract Rinse Baseline (10µV) spikes exposure (P2/P3) (P1/P3) (P2/P1)

50-60µV 10 23 40 2 0.6 0.3

60-70µV 39 18 28 0.5 0.6 1

70-80µV 3 5 11 2 0.5 0.3

80-90µV 2 3 5 1 0.5 0.4

90-100µV 3 4 10 2 0.4 0.3 100-110µV 1 0.8 4 0.7 0.2 0.3 110-120µV 0 0.3 1 0 0.3 0 120-130µV 1 1 4 0.4 0.1 0.4 130-140µV 0 0 3 0 0 0 140-150µV 0 0 2 0 0 0 150-160µV 0 0 0.8 0 0 0 160-170µV 0 0 2 0 0 0 30

170-180µV 0 0.5 2 0 0.3 0 180-190µV 0 0.2 2 0 0.1 0 190-200µV 0 0.2 3 0 0.1 1 200-210µV 0 0 2 0 0 1 210-220µV 0 0 1 0 0 1 220-230µV 0 0 0.7 0 0 1 230-240µV 0 0 0.2 0 0 1 240-250µV 0 0.3 0.2 0 2 0 250-260µV 0 0.2 0.3 0 0.5 0 260-270µV 0 0 0.3 0 0 0 270-280µV 2 0 1 0 0 2 280-290µV 1 0 0.5 0 0 3 290-300µV 0 0 0.2 0 0 0 300-310µV 0 0.5 0 0 0 0 310-320µV 0 2 0 0 0 0

8 P1 (counts/min/bin) 6 P2 (counts/min/bin) P3 (counts/min/bin) 4 Counts/Min/Bin 2

0 0 50 100 150 200 250 300 350

Spike Amp Baseline (µV) Figure 8: SPIKE AMP (µV) OVER SPIKES/1MIN/BIN; LOW P2 ACTIVITY Spike amplitude (µV) over spikes/1min/bin for each of the 3 recorded phases over 55 minutes during pre-extract, extract, and post-extract exposure to brain with bin size set at 2µv and noise exclusion cut-off voltage set at 30µV. Data doesn’t match prediction since there was a decrease in activity from P1, which then increased during P3. P1 (red) represents the pre-extract phase, P2 (green) represents the sea pen extract exposure phase, and P3 (blue) represents the post-extract phase.

Table 9: The breakdown of average spike rates over a 51 minute recording time during pre-extract, extract, and post-extract exposure to brain for a single experiment.

Phase Spike Counts/51min 1. pre-extract 25649

2. extract 13889

3. post-extract 21180

Table 10: Ratio of spikes per phase over a 51 minute recording time, for extract exposure, rinse, and baseline categorized by 10µV increments. Spike counts were summed over 10µV increments then broken up into three ratios, representing extract exposure (P2/P1), rinse exposure (P2/P3), and baseline activity (P1/P3). All calculated values greater than 1 were rounded to the nearest whole number, and values less than 1 to the nearest decimal.

Bin size P1 spikes P2 spikes P3 spikes Extract Rinse Baseline (10µV) exposure (P2/P3) (P1/P3) (P2/P1)

10-20µV 390 195 350 0.5 0.6 1

20-30µV 86 70 75 0.8 0.9 1

30-40µV 24 7 9 0.3 0.8 7

40-50µV 3 0.4 0.7 0.1 0.6 13

50-60µV 0.2 0.02 0 0.1 0 0 34

250

200

150 P1 (counts/min/bin) 100 P2 (counts/min/bin) P3 (counts/min/bin) Counts/Min/Bin 50

0 0 50 100 150 200 250 300

Spike Amp Baseline (µV)

Figure 9: SPIKE AMP (µV) OVER SPIKES/1MIN/BIN; LOW P2 ACTIVITY Spike amplitude (µV) over spikes/1min/bin for each of the 3 recorded phases over 51 minutes during pre-extract, extract, and post-extract exposure to brain with bin size set at 2µv and noise exclusion cut-off voltage set at 6µV. Data doesn’t match prediction since there was a decrease in activity from P1, which then increased during P3. P1 (red) represents the pre-extract phase, P2 (green) represents the sea pen extract exposure phase, and P3 (blue) represents the post-extract phase. 35

Table 11: The breakdown of average spike rates over a 6 minute recording time during pre-extract, extract, and post- extract exposure to buccal ganglia for a single experiment.

Phase Spike Counts/6min 1. pre-extract 377

2. extract 354

3. post-extract 758

Table 12: Ratio of spikes per phase over a 6 minute recording time, for extract exposure, rinse, and baseline categorized by 10µV increments. Spike counts were summed over 10µV increments then broken up into three ratios, representing extract exposure (P2/P1), rinse exposure (P2/P3), and baseline activity (P1/P3). All calculated values greater than 1 were rounded to the nearest whole number, and values less than 1 to the nearest decimal.

Bin size P1 spikes P2 spikes P3 spikes Extract Rinse Baseline (10µV) exposure (P2/P3) (P1/P3) (P2/P1)

30-40µV 24 23 67 1 0.3 0.4

40-50µV 36 35 47 1 0.7 0.5

50-60µV 25 20 35 0.8 0.6 0.7

60-70µV 7 1 8 0.1 0.1 0.9

70-80µV 0.3 0.1 2 0.2 0.05 0.1

80-90µV 0.02 0 2 0 0 0.01

90-100µV 0.05 0 2 0 0 0.04 100- 110µV 0.02 0.02 1 1 0.02 0.02 110- 120µV 0.02 0 1 0 0 0.02

20 18 16 14 12 10 P1 (counts/min/bin) 8 P2 (counts/min/bin) P3 (counts/min/bin) 6 Counts/Min/Bin 4 2 0 0 50 100 150 200 250 300 Spike Amp Baseline (µV)

Figure 10: SPIKE AMP (µV) OVER SPIKES/1MIN/BIN; HIGH P3 ACTIVITY Spike amplitude (µV) over spikes/1min/bin for each of the 3 recorded phases over 6 minutes during pre-extract, extract, and post-extract exposure to buccal ganglia with bin size set at 2µv and noise exclusion cut-off voltage set at 20µV. Data doesn’t match prediction since there was only an increase in activity during P3. P1 (red) represents the pre-extract phase, P2 (green) represents the sea pen extract exposure phase, and P3 (blue) represents the post- extract phase. 38

Table 13: The breakdown of average spike rates over a 50 minute recording time during pre-extract, extract, and post-extract exposure to brain for a single experiment.

1. pre-extract 220,015

2. extract 213,704

3. post-extract 401,807

Table 14: Ratio of spikes per phase over a 50 minute recording time, for extract exposure, rinse, and baseline categorized by 10µV increments. Spike counts were summed over 10µV increments then broken up into three ratios, representing extract exposure (P2/P1), rinse exposure (P2/P3), and baseline activity (P1/P3). All calculated values greater than 1 were rounded to the nearest whole number, and values less than 1 to the nearest decimal.

Bin size P1 spikes P2 spikes P3 spikes Extract Rinse Baseline (10µV) exposure (P2/P3) (P1/P3) (P2/P1)

10-20µV 3682 3620 7108 1 0.5 0.5

20-30µV 465 408 786 0.9 0.5 0.6

30-40µV 150 123 170 0.8 0.7 0.9

40-50µV 74 44 92 0.6 0.5 0.8

50-60µV 15 12 22 0.8 0.5 0.7

60-70µV 6 13 23 2 0.6 0.3

70-80µV 6 17 18 3 0.9 0.3

80-90µV 1 21 0.7 21 30 1

90-100µV 0.5 4 0.1 9 40 5 100-110µV 0.14 0.72 0 5 0 0 110-120µV 0.06 0.4 0.2 6 2 0.4 40

120-130µV 0.02 0.3 0.04 17 9 0.5 130-140µV 0.2 1 0.1 5 9 2 140-150µV 0.7 3 0.2 5 17 4 150-160µV 0.3 3 0.1 10 30 1

3500

3000

2500

2000

1500 P1 (counts/min/bin) P2 (counts/min/bin) P3 (counts/min/bin) Counts/Min/Bin 1000

500

0 0 50 100 150 200 250 300

Spike Amp Baseline (µV)

Figure 11: SPIKE AMP (µV) OVER SPIKES/1MIN/BIN; HIGH P3 ACTIVITY Spike amplitude (µV) over spikes/1min/bin for each of the 3 recorded phases over 50 minutes during pre-extract, extract, and post-extract exposure to brain with bin size set at 2µv and noise exclusion cut-off voltage set at 6µV. Data doesn’t match prediction since there was only an increase in activity during P3. P1 (red) represents the pre- extract phase, P2 (green) represents the sea pen extract exposure phase, and P3 (blue) represents the post-extract phase. 42

2.50E+06

2.00E+06

1.50E+06 Control (ASW) 1:50 dilution 1.00E+06 1:5 dilution 1:2 dilution (SPE) 5.00E+05

Cell Concentration (cells/mL) 0.00E+00 0 1 2 3 4 5 6 Time (days)

2.50E+06

2.00E+06 Control (ASW) 1:50 dilution 1.50E+06 1:5 dilution 1:2 dilution (SPE) 1.00E+06

5.00E+05 Cell Concentration (cells/mL) 0.00E+00 0 1 2 3 4 5 Control (ASW) 2.08E+06 5.25E+05 1.25E+05 1.13E+05 7.50E+04 6.25E+04 1:50 dilution 2.08E+06 2.50E+05 7.50E+04 6.25E+04 5.00E+04 3.75E+04 1:5 dilution 2.08E+06 2.25E+05 6.25E+04 5.00E+04 3.75E+04 3.13E+04 1:2 dilution (SPE) 2.08E+06 1.38E+05 3.75E+04 3.13E+04 2.50E+04 1.25E+04 Time (days)

Figure 12: GROWTH CURVE & HISTOGRAM MDA-MB-231 CELL CONCENTRATION A. Growth curve of cell concentrations (cells/mL) of MDA-MB-231 cells following sea pen extract exposure over five-day span B. Histogram of cell concentrations (cells/mL) (+ SD) of MDA-MB-231 cells following sea pen extract exposure over five-day span. Dilutions: Red represents the Control (only ASW), orange represents 1:50 dilution, green represents 1:5 dilution, and blue represents 1:2 dilution (only filtered sea pen extract). 44

Discussion

The production of defensive chemicals may be one of the only effective strategies employed by many organisms to avoid consumption. As a chemical defense Ptilosarcus gurneyi (sea pen) is able to produce a metabolite that is repellent to crabs with suspected toxic properties (Wratten et al., 1977). These metabolites are classified as diterpenes, and after isolation have been identified as ptilosarcone and ptilosarcenone (Wratten et al., 1977). Diterpenes may sometimes act to inhibit acetylcholinesterases (Barbosa et al., 2006), which are enzymes that catalyze the breakdown of the neurotransmitter acetylcholine

(Williams et al., 1987.). Acetylcholine is a chemical with both excitatory and inhibitory functions that alter the speed of nerve cells signaling to one another

(Dvir et al., 2010).

Despite the sea pen’s chemical defense, the cnidarian provides a major source of food to variant predator species including multiple nudibranchs (e.g.

Diamondback Tritonia sea slug) and asteroids (e.g. Spiny red star). Nudibranchs such as Tritonia tetraquetra [formerly know as T. diomedea (Martynov, 2006)] have seen to begin feeding habits as early as 5 days old (Kempf & Willows, 1977.), which can largely attribute to their role in P. gurneyi having a 97% mortality rate within the first year of growth (Birkeland, 1974.).

Acid secretions were first observed with a single species of opisthobranch,

Pleurobranchus membranceus (Thompson & Slinn, 1959.). Further instances of acid secretions were then recorded amongst variant gastropods, specifically those that lacked external shells as adults (Thompson, 1960a.). It has been proposed they 45

secrete a strong, bitter-tasting acid as a secondary metabolite making them unpalatable to predators (Thompson, 1960). The majority of the secondary metabolites isolated from T.tetraquetra skin extracts to date have been diterpenoids, and are of dietary origin likely biosequestered from its toxic food source, P. gurneyi (Williams et al., 1987; Shapiro thesis, 2012).

Observations of similar chemical defenses between T. tetraquetra and P. gurneyi suggest T. tetraquetra, may have evolved to be physiologically resistant to

P. gurneyi chemical defenses during predation. In the course of this study, sea pen extract exposure did have a mild neurological effect during the recording by increasing or decreasing the spike rate through temporary stimulation. The data taken before and after the sea pen extract exposure was similar in spike rate, confirming an effect since once the extract was rinsed away the neurological activity was seen to return to baseline (Tables 1,3, and 5). Tables 7 and 9 showed a decreased spike rate during sea pen extract exposure, which may be a result of inhibitory neurons being over stimulated. Since recordings were not from the same nerve or specified for each experiment, some nerves might have increased output and others might have decreased. Different nerves likely have axons from different subsets of neurons in the ganglia, so chemicals can interfere with sensory outputs going in and motor outputs going out. Excitation of inhibitory neurons can lead to lowered output because acetylcholine will be blocked disrupting nerve communication. Tables 11 and 13 showed an increase in spike rate during the post-extract phase (P3) only, once the sea pen extract was rinsed away. High P3 activity may be the result of electrical interferences, which can 46

cause increases in measured spike count correlations.

Although the sea pen extract was observed to have a mild neurological effect on T. tetraquetra, much is still to be discovered about the toxicity of P. gurneyi’s chemical defense. MDA-MB-231 human cancer cells are amongst the most popular cells used in cytotoxic studies, particularly because they are adherent and triple negatives. Adherent cells are anchorage-dependent and have a 24-hour doubling time making it quicker to grow and split cells. Triple negative cells lack estrogen receptors, progesterone receptors, and HER2 protein receptors making them very aggressive cancers to treat as they are resistant to hormonal therapies (ECACC, 2017).

In response to the sea pen extract, MDA-MB-231 cells were observed to have cytotoxic effects in a dose and time-dependent manner. The 1:2 dilution

(filtered sea pen extract) increased cell death by 5 times more than the control

(ASW) over the 5-day span. The ANOVA data showed significant results, indicating there was variation between treatments so different treatments do cause different effects. The Kruskal-Wallis data were also significant and used to retest the ANOVA due to lack of normality, because 3 data points aren’t enough to create a bell shape curve. The exponential regression data were significant reinforcing that there were variation between treatments. Cell concentration was observed to be indirectly proportional to sea pen extract concentration.

In a similar study, where cytotoxic effects of putative toxin (extracted with

Ethyl Acetate) from sea pen Virgularia gustaviana were measured on HeLa and

MDA-MB-231 cancer cell lines (Sharifi, Mostafavi, Moradi, Givianrad, & 47

Niknejad, 2017), a 10x decrease in cell viability percentage was seen in comparison to the control suggesting the effect of the P. gurneyi filtered extract on MDA-MB-231 cells was moderate. Observed effects may have been enhanced due to osmolality, which also makes the cytotoxic impacts inconclusive since salt concentration may be the reason cells are dying at a slower rate. Overall the data indicate there may be a potential effect on cell proliferation but more replicates would need to be carried out in order to confirm an actual effect.

Conclusion

Hypothesis (Hyp1) was supported but neither proven to be true or false because recordings times varied from 6mins to 60mins from recording to recording. Also the effect of the sea pen extract wasn’t consistent between recordings so there wasn’t enough information to validate the presence of an evolutionary defense mechanism. Future studies can start by running recordings in a quiet area to help eliminate some background noise, and making sure all internal factors are consistent for each recording (ie: rate of extract exposure, temperature of ASW rinses, temperature in the chamber, etc.). Time consistency was a huge issue as some of the recordings weren’t long enough to analyze data, so making sure each phase is recording for an hour or more would be greatly beneficial in more precise calculations so the system doesn’t underestimate the number of spikes a neuron fires. Lastly recording from the same nerve and specifying which nerve would provide better evidence of any neurological impact when monitoring motor outputs.

Hypothesis (Hyp2) was supported with suggestive data and inferential statistics but neither proven to be true or false because sample size was too small for definitive findings. Also the experiment should be redone with seawater diluted in growth media as control. Future studies can start by replicating the methods and changing the controls, so trials are run in both seawater and non- seawater conditions because we need to see how stable the toxin is outside of seawater. Lastly using purified toxin would also provide better evidence of any neurological impact; during this experiment crude extract was used. 49

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