Conopeptide Production through Biosustainable Snail Farming

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

MOLECULAR BIOSCIENCES AND BIOENGINEERING

DECEMBER 2012

By

Jeffrey W. Milisen

Thesis Committee:

Jon-Paul Bingham (Chairperson) Harry Ako Cynthia Hunter

Keywords: Conus striatus venom variability

Student: Jeffrey W. Milisen Student ID#: 1702-1176 Degree: MS Field: Molecular Biosciences and Bioengineering Graduation Date: December 2012

Title: Conopeptide Production through Biosustainable Snail Farming

We certify that we have read this Thesis and that, in our opinion, it is satisfactory in scope and quality as a Thesis for the degree of Master of Science in Molecular Biosciences and Bioengineering.

Thesis Committee:

Names Signatures

Jon-Paul Bingham (Chair) ______

Harry Ako ______

Cynthia Hunter ______

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Acknowledgements

The author would like to take a moment to appreciate a notable few out of the army of supporters who came out during this arduously long scholastic process without whom this work would never have been. First and foremost, a “thank you” is owed to the USDA TSTAR program whose funds kept the snails alive and solvents flowing through the RP-HPLC. Likewise, the infrastructure, teachings and financial support from the University of Hawai‘i and more specifically the College of Tropical Agriculture and Human Resources provided a fertile environment conducive to cutting edge science. Through the 3 years over which this study took place, I found myself indebted to two distinct groups of students from Dr. Bingham’s lab. Those who worked primarily in the biochemical laboratory saved countless weekend RP-HPLC runs from disaster through due diligence while patiently schooling me on my deficiencies in biochemical processes and techniques. Even more, I would like to thank the students assigned to Dr. Bingham’s aquaculture facility who had to work with me directly during this time, not only with regards to this study but also in the foundational design and building of the aquaculture facility. They were often asked to spend a full afternoon from their other responsibilities in my absence to feed my snails, a process that can be excruciating when the snails aren’t terribly hungry. This was in addition to their daily upkeep tasks, which often required precious weekend commitments as well. I know the sacrifices you all made and I am sincerely grateful. For their slimy donations, I’d like to thank Hank Lynch and J. J. Jackson because, try as I might, I’m still the world’s worst shell collector. Without your contributions, I would have been studying ten empty tanks full of seawater. This brings me to the tireless efforts put forth by the inexhaustible Dr. Jon-Paul Bingham who somehow manages to meet with each of his students every week while teaching the university’s flagship biochemistry course and still figures out a way to make himself available for the odd question of protocol. The opportunities this project afforded me along with the guidance and lessons have returned invaluable life experiences that will follow me for the rest of my life. Finally I would like to take a brief moment to show some gratitude to my personal support team. For all the dinners you delivered when I was stuck at school late at night and for providing the at-home logistical support to keep my demeanor and appearance appropriate for the rest of the world, I will always be indebted to Melanie Kosaka. And finally, to my dad who was a single parent wrestling with two maturing sons and now dutifully serves the role of moral supporter in the face of impossible times.

Thank you.

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ABSTRACT

An area of intensive focus and research has been in the study of conopeptides isolated from cone snail venom. These complex peptide venoms contain between 50 and 100 different bioactive substances, many of which can be utilized for a variety of anthropogenic needs. Bioprospecting from this limited marine resource has caused ecological concerns, echoing that selected Conus species should be listed under CITES to avoid their demise. Utilizing captive husbandry techniques to promote biosustainable methods for venom collection, links were established between venom volume and snail size, venom volume and seasonality, venom volume and time in captivity, lunar phase and feed rate and lunar phase and venom diversity to maximize venom peptide diversity and output from cone snails as a valuable pharmacological source.

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Table of Contents Acknowledgements ...... iii ABSTRACT ...... iv Table of Figures ...... vii List of Tables ...... xi INTRODUCTION ...... 1 Hawai‘ian Biogeography ...... 1 The Ecology of Conus striatus in Hawai‘i ...... 1 Cone Snail Anatomy and Physiology ...... 2 Larval Development ...... 3 Feeding Behavior ...... 4 Venom Delivery ...... 5 Other Venoms ...... 6 Intersexual Variations in Venom Composition ...... 7 Conopeptides of Conus striatus ...... 7 Conus Venom Sources ...... 12 Venom Variation in Conus ...... 13 Anthropogenic Uses for Conopeptides ...... 14 Biosustainability ...... 15 Moon Phase ...... 15 Taxonomy of Conus striatus ...... 16 HYPOTHESES ...... 18 General Hypotheses ...... 18 Specific Hypotheses ...... 18 Reproduction ...... 18 Snail Size ...... 19 Sex ...... 19 Death ...... 19 Pill Diet ...... 19 Morphological Variants ...... 20 Phenology-Seasonality ...... 20 Moon Phase ...... 20 Time in Captivity ...... 20 METHODS ...... 21 Housing ...... 21 Milked Venom Collection ...... 21 Pill-Diet Study ...... 21 Sample Preparation ...... 22 RP-HPLC Profiling and Fractionation ...... 22 Mass Spectrometric Analysis ...... 22 Quantitative Analysis ...... 23 Statistical Analysis ...... 23 Define Number of Peaks ...... 23 RESULTS ...... 25 Snail Size ...... 26 Phenology-Seasonality ...... 28 Sex ...... 30

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Moon Phase ...... 32 Time in Captivity ...... 35 Morphological Variants ...... 38 Reproduction ...... 42 Death ...... 44 Pill Diet ...... 46 Other Comparisons ...... 49 DISCUSSION ...... 53 Number of Dominant RP-HPLC Peaks ...... 53 Snail Size ...... 53 Seasonality ...... 54 Sex ...... 54 Moon Phase ...... 55 Time in Captivity ...... 56 Morphological Variants ...... 57 Pregnancy ...... 58 Death ...... 58 Pill Diet ...... 59 Mitigating Ecological Impacts through Cone Snail Husbandry ...... 59 General Notes ...... 60 REFERENCES ...... 62 Posters ...... 68 Presentations ...... 68

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Table of Figures

Figure 1. Gross morphology of Conus striatus includes the apex or protoconch (A), shoulder (B), anal groove (C), aperture (D), and base (E), foot (F), siphon (G), and eye stalks (H). The proboscis (I) and proboscis sheath, or mouth or rhynchodaeum (J), are only visible during feeding...... 3 Figure 2. Cysteine patterns and disulfide connectivity in the six conotoxin superfamilies. Image taken from Conoserver (http://www.conoserver.org/?page=about_conotoxins&bpage=cononames) ...... 9 Figure 3. Three profiles from Conus striatus that demonstrate the discernment of dominant peaks in this analysis. The top profile is an example of a profile with no dominant peaks because it lacks any apparent peptides. The middle profile would be classified as having two peaks. The final profile has four peaks that are all taller than half of the height of the tallest peak...... 24 Figure 4. Snail mass and venom volume output exhibit a strong positive correlation (n=25, Mean Mass=99±37 g, Average Volume=35±12 uL, Mass to Volume Covariance=393.9 r=0.92 p=0). The greatest average volume came from the largest snail in the system, snail 4-1 which had a mass of 211 grams...... 26 Figure 5. The number of picomoles of St4082/κ-conotoxin SIVA did not appear to change in relation to snail mass (r=0.18 p=0.52)...... 27 Figure 6. The concentration of St4082/ κ-conotoxin SIVA (picomoles/uL injected volume) had no correlation to snail mass (r=0.076 p=0.10)...... 27 Figure 7. Average monthly volumetric milking data averaged from the captive Conus striatus population. Statistically significant months included May, June and July 2010 and October 2011. Error bars represent standard deviation...... 28 Figure 8. Feed rate varied insignificantly with seasonality...... 29 Figure 9. Mean monthly amounts of St4082/ κ-conotoxin SIVA milked from select snails during the study period. Very large deviations impede conclusions from these data. Error bars represent standard deviation...... 29 Figure 10. Mean monthly concentrations of St4082/ κ-conotoxin SIVA peaked during the month of October but exhibited a very large degree of deviation. Error bars represent standard deviation...... 30 Figure 11. Compositional diversity of the venoms from known male versus known female snails differed in that males produced more complex venom with a greater number of dominant constituents (whole population mean=1.13±0.26 peaks, p=.056, t-Crit.=2.44). Error bars represent sex-specific standard deviations...... 31 Figure 12. Known females and males in the collection produced differing amounts of St4082/ κ-conotoxin SIVA...... 31 Figure 13. The difference between females and males in the concentration of St4082/ κ- conotoxin SIVA in picomoles per uL injected venom was 45±37 and 150±117 respectively (whole population mean=80±84 pmol St4082, p=.085, t-Crit.=2.4). Error bars represent sex-specific standard deviation...... 32 Figure 14. Conus striatus fed at a statistically similar rate regardless of moon phase (n=25, p=0.89, f=0.2, f-critical=3.5)...... 33

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Figure 15. Snails injected the most complex venoms during the new and full moon phases (Standard deviations= 1.01, 0.95, 1.02, and 1.03 for new, first quarter, last quarter, and full moons respectively. population=25, number of milkings=956, mean=1.4±1.0 peaks, p=0.47)...... 33 Figure 16. The average amount of St4082/ κ-conotoxin SIVA per milking did not vary significantly with moon phase (number of milkings=108, mean=3575±7659 pmol St4082/k-conotoxin SIVA)...... 34 Figure 17. The concentration of St4082/ κ-conotoxin SIVA did not vary significantly with lunar phase (number of milkings=105, mean=114±214 pmol St4082/uL milked venom)...... 34 Figure 18. When the forty weeks’ worth of samples were separated into 10-week bins, the captive snails showed a steady decline in venom production with time in captivity. This could be a result of malnutrition or insufficient husbandry techniques. N=270 samples were placed into 4 bins encompassing 10 weeks each. Standard deviations equaled 13.9, 11.8, 13.5, and 13.0 for weeks 1-10, 11-20, 21-30, and 31-40 respectively...... 35 Figure 19. Another view of the decrease in venom volume through time in captivity. .... 36 Figure 20. Feed probability decreased during the first 30 weekly feeds before abruptly increasing to 85% during the final 10 weeks of the study...... 36 Figure 21. The average number of dominant RP-HPLC peaks observed over the course of the forty-week study stayed relatively constant meaning that the snails neither lost nor gained venom diversity in captivity...... 37 Figure 22. The mean concentration of St4082/ κ-conotoxin SIVA ranged from 35 to 152 pmol/uL venom...... 37 Figure 23. Morphological subspecies determinations of the specimens studied herein. Identifications were based primarily on the concave/convex nature of the spire. In specimens where spire height was inconclusive, the relative diameter (RD=Y/X) was used. Snails 3-1 and 5-2 had neither distinctive spire heights nor relative diameters characteristic of either subspecies...... 39 Figure 24. All Conus striatus specimens that possessed a concave spire also had a slightly wider shell in relation to the shell's width (N=23, p=0.00002, t –Crit=2.08)...... 40 Figure 25. The difference in venom volume between the two subspecies was negligible (Mean=32.6±9.7uL, p=0.54, t-Crit.=2.09)...... 40 Figure 26. The average number of peaks observed from samples collected from C. s. striatus and C. s. oahuensis was very similar, although the variation in diversity was much higher in samples collected from C. s. striatus (Mean=1.43±0.48 peaks, p=.76, t-Crit.=2.09)...... 41 Figure 27. Feeding rates for C. s. striatus and C. s. oahuensis were comparable (mean=0.76±0.068%, p=0.55, t-Crit.=2.08)...... 41 Figure 28. A comparison of the concentration of St4082/κ-conotoxin SIVA in milked venom injected by C. s. striatus versus C. s. oahuensis reveals no difference between the two subspecies (Mean=95±106pmol St4082/uL injected venom, p=0.9, t-Crit.=2.2)...... 42 Figure 29. A temporal look at the mean injection volumes collected from gravid females during the 7 weeks prior and 10 weeks post egg-laying events (T=0 on the x-axis).

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No obvious changes in volumetric production were noted (n=4, Mean=47±23.8 uL)...... 43 Figure 30. The feed rate decreased for gravid females during the week immediately following egg deposition (n=4, mean=0.81±0.19%)...... 43 Figure 31. No changes were observed in the mean number of dominant peaks in samples collected from female snails around the time of egg deposition (time=0) (n=4, Mean=0.82±0.44 dominant peaks)...... 44 Figure 32. Milked venom volumes for snail 5-1 were relatively steady for the time between capture on 6/17/10 and its natural death on 7/13/11 and accepted food until days before death...... 45 Figure 33. Number of dominant peaks present in the weeks leading up to the natural death of snail 5-1 at week 41...... 45 Figure 34. From 7/6/2011 through 10/17/2011 snail 5-2 failed to feed or milk. Milking resumed on 10/17/2011 for a period of 6 weeks however, the last two milkings prior to the snail's natural death on 12/7/2011, St4082/ κ-conotoxin SIVA production decreased greatly...... 46 Figure 35. When snails were switched to a pill diet, venom production decreased and did not recover even after the snails were put back on a diet of fish...... 47 Figure 36. The number of dominant RP-HPLC peaks sampled from snails given a pill diet increased both while the snails were on the pill and after the snails were switched back to a natural diet of fish...... 47 Figure 37. Snails that were offered a pill diet increased feed success during the pill-diet trial before decreasing feed response after the pill-feed trial ceased...... 48 Figure 38. A pill diet appears to decrease the concentration of St4082/κ-conotoxin SIVA in the population. The snails chosen for the study produced a high concentration of St4082/ κ-conotoxin SIVA at t=0 when compared to the mean of the rest of the community (n=38, mean=177±279 pmol St4082/uL milked venom)...... 48 Figure 39. The number of dominant peaks found in the venom correlated in a strong directly proportional relationship with the concentration of the peptide St4082/ κ- conotoxin SIVA (n=94, mean diversity=1.6±1.15 peaks, mean concentration=122±225 pmol St4082/uL milked venom)...... 49 Figure 40. A display of milkings 1, 10, 20, 30, and 40 from snails in tanks that did lay eggs (right column) versus the same milkings taken from snails in tanks that never produced eggs (left column). Examples of the most and least stable profiles through the 40 week period were chosen from each category. Note that the most stable profiles taken from a snail in a tank that did not produce eggs (snail 7-2) still had a number of peptides that were produced some weeks and not others. The least stable example from a snail in a tank that did produce eggs (snail 6-1) still showed a level of stability in spite of vast changes in concentration for the majority of its time in captivity. Conversely, snail 1-1 which produced the least stable profiles of all of the snails, showed little congruency at all and snail 3-2 consistently produced very similar profiles throughout its stay...... 50 Figure 41. Examples of 1, 10, 20, 30, and 40 week profile series extracted from C. s. oahuensis (snail 4-3) and C. s. striatus (snail 3-3). Note that both snails produced profiles with two strong peaks eluting at 32 and 34 minutes. This level of similarity would not be observed in two different species...... 51

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Figure 42. Shown above are samples taken at weeks 1, 10, 20, 30, and 40 from two different snails. Snail 1-1 (left profiles) was fed pills between weeks 5 and 13 and produced very inconsistent profiles overall. The control period from weeks 1 through 4 was dominated by profiles with 4 main peaks as seen in week 1. The snail stopped producing venom for most of the pill diet period and the switched to producing highly variable profiles for the rest of the 40 week study. This pattern was similar in the other two pill diet animals. Snail 10-3 (right) was selected as an example of a snail fed that was fed exclusively fish yet also produced highly variable venom profiles. Importantly, in spite of its apparent inconsistency, snail 10-3 maintained some level of congruency by always producing a peak eluting at 33 minutes. A pill diet therefore seems to initiate a highly variable series of profiles from which the snail doesn’t recover after 40 weeks, however diet does not explain all highly diverse venom profiles...... 51 Figure 43. Each of the profiles shown represents a snail’s normal, or baseline, profile. Typically, most of the snails produced venom with only 1 or 2 main peaks and a number of smaller ones. Snails 1-1, 5-3 and 9-1 regularly produced highly complex venom profiles with high concentrations of many components. Contrariwise, snail 7-1 routinely produced venom that was completely devoid of conopeptides. For reference, profiles marked with “O” represents baselines that come from C. s. oahuensis while those marked with “S” were taken from specimens of C. s. striatus...... 52

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List of Tables

Table 1. List of all known conopeptides isolated from Conus striatus...... 10 Table 2. List of all known conopeptides isolated from Conus striatus (continued)...... 11 Table 3. Summary of the correlations observed. S=Strong correlation; P=Possible correlation; N=No correlation NA=Not tested ...... 25

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INTRODUCTION

Hawai‘ian Biogeography

Hawai‘i is the most isolated archipelago on earth separated from the nearest landmass of North America by 2,000 miles (3,219 km) (Mueller-Dombois, 1975). The ecological results of this isolation are a high rate of endemism, a lack of overall species diversity and a complete absence of many otherwise common organisms. Hawai‘i’s roughly 1000 species of mollusks compares poorly against the 2500 species found in the Ryukyu Islands (Kay, 1987). However, 21% of the mollusk species and three genera found in Hawai‘i are thought to be found nowhere else (Kay and Palumbi, 1987; Severns, 2011). Some mollusks that never made the oceanic crossing from the Indo-Pacific to Hawai‘i include the giant clams (Tridacnidae) and cuttlefish (Sepiidae) (Kay, 1987). This blend of absenteeism and endemism in the local species assemblage has defined Hawai‘i’s marine ecosystem.

The Ecology of Conus striatus in Hawai‘i

Due to the archipelago’s isolation, the species found in Hawai‘i tend to have a long planktonic larval stage that can disperse over great distances; species with shorter or even lacking a pelagic larval developmental phase occur less frequently (Severns, 2011). At least 33 species of Conus including five endemic species are recorded in Hawai‘i (Kay and Palumbi, 1987; Kohn and Weaver, 1962; Severns, 2011). The habitat of Conus specimens tends more toward algae-bound sand and less live coral (Kohn, 1983). Conus striatus is known to occur in sand beneath rocks at depths ranging from 7- 16 m (Severns, 2011), although many of the specimens used in the current study were found as deep as 50 m. Two species of small reef fish have been found in the alimentary

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tract of Conus striatus including Blenniela gibbifrons and Bathygobius fuscus with other fish species such as Enteromacrodus marmoratus and Kuhlia xenura consumed under laboratory conditions (Kohn, 1956).

Cone Snail Anatomy and Physiology

The shell of a Conus snail starting from the thick end consists of an apex or protoconch, shoulder, anal groove, aperture, and base (Figure 1). A developing Conus veliger starts life with just a protoconch and grows outward from there forming the shell’s shoulder. The anal groove on the posterior end of the aperture is where wastes are excreted. The living tissues of the animal interact with the environment through the aperture with the majority of external organs being situated toward the base. External anatomy of the living animal includes the foot, siphon, and eye stalks (Figure 1). The proboscis and proboscis sheath (mouth) are only visible during feeding, a process that is described in further detail below. During respiration, incurrent water enters the siphon to pass over the gills and main chemosensory organ, the ophradium (Kohn, 1956). Little is known about the visual acuity of cone snails. Marine gastropods possess simple eyecups with peak sensitivity around 470-500nm in wavelength (Cronin, 1986). Important to this study, light detected by the eyes of another gastropod mollusk, Aplysia californica reinforced internal circadian rhythm mechanisms (Abran, Anctil and Ali, 1994). This suggests that other mollusks might maintain a sense of circadian rhythm reinforced by light stimuli.

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Figure 1. Gross morphology of Conus striatus includes the apex or protoconch (A), shoulder (B), anal groove (C), aperture (D), and base (E), foot (F), siphon (G), and eye stalks (H). The proboscis (I) and proboscis sheath, or mouth or rhynchodaeum (J), are only visible during feeding.

Larval Development

Cone snails, like other Prosobranchs, have separate male and female sexes. Females lay 3 to 78 egg capsules containing up to 11,000 eggs per capsule on hard substrata generally between the months of May and August (Kohn, 1959). The size of the eggs correlates inversely with the time spent as pelagic veligers and whether the resulting veligers take on a lecithotrophic or planktotrophic lifestyle (Perron, 1981; Strathmann, 1985). In the case of C. striatus, eggs are 0.25 mm long with the veligers emerging from the egg capsule many days after hatching at a length of 0.5 mm and settle after about 20 days at a length of 1.5 mm (Perron, 1981). Laboratory reared C. striatus veligers grew best when fed an equal mix of Phaeodactylum tricornutum and Isochrysis

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galbana (Perron, 1981). Metamorphosis is marked by settlement on the substrate and resorption of the veligers’ swimming apparatus known as the velum (Perron, 1980; Perron, 1981).

Feeding Behavior

Cone snail species can be divided into three prey types: worm, gastropod, and fish eaters. Those that prey on worms, known as vermivores, may or may not employ an active envenomation strategy. Often the snail will simply engulf the prey with its rhynchodaeum and swallow (Bingham, Mitsunaga and Bergeron, 2010; Wang and Chi, 2004). Others, as in the case of Conus imperialis, will reach toward the prey item with their proboscis. Upon contact, hydrostatic forces inject and detach the venom-laden radular tooth into the prey item. Excess venom can be seen as a white cloud coming from the site of injection (Kohn and Hunter, 2001). The snail hunting cone snails, otherwise known as molluscivores, employ a similar hunting strategy to the actively-envenoming vermivores. When a prey snail is chemically detected, the predator will extend its harpoon-tipped proboscis, orienting the prey such that the proboscis can access the prey item’s flesh. Upon the proboscis contacting the animal’s flesh, the predator releases the radular harpoon while simultaneously injecting venom. Often, the molluscivores will inject the prey multiple times to ensure adequate envenomation (Bingham, et al., 2010). Fish-hunting cone snails, or piscivores, first detect their prey and become stimulated to feed through chemoreception (Kohn, 1956). Prey capture is by one of two mechanisms. First, the snail may use the rhynchodaeum as a net to capture the fish. Envenomation is then done while only after the fish is enclosed within the rostrum. The second method is through active hunting. A chemical trigger incites the snail to become active and begin searching the vicinity using its eye stalks and extended proboscis. The proboscis uses a tactile cue to fire the harpoon. Unlike molluscivores, however, the proboscis holds onto the radula using a sphincter muscle and “reels” the fish in to its mouth (Bingham, et al., 2010; Salisbury, et al., 2010; Stewart and Gilly, 2005).

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It is important to note that snails may not feed exclusively on one prey type. Radular tooth studies suggest that many young piscivorous snails may begin life as vermivores (Nybakken, 1990). Conus californicus has been observed to feed on mollusks, worms, and fishes (Stewart and Gilly, 2005) and may employ a pack hunting strategy to subdue larger prey (Biggs, et al., 2010).

Venom Delivery

The venom delivery system of cone snails is relatively conserved across all species. The apparatus consists of a muscular bulb, tubular gland, radular sac, radula, and proboscis (Marshall, et al., 2002). The purpose of the proximal bulb is not well known but may provide hydrostatic forces to propel the radula forward during injection. The venom gland or duct connects the venom bulb to the pharynx and produces and stores the venom. The varying sections of the gland consist of differing specialized cells. Most of the venom production occurs in the anterior portion of the duct. The duct enters the pharynx just upstream of a y-shaped apparatus known as the radular sac. The long arm of the radular sac produces the radula while the short arm stores the premade radulae. While cone snail species can often be classified based on the morphology of their radular teeth alone, there are four main groupings of radular teeth seemingly determined by food type. Type 1 teeth belong to those species that prey on mollusks such as Conus dalli and Conus pennaceus. Type 2 teeth are known to occur in piscivorous snails such as Conus magus and the focus of this study, Conus striatus. Type 3 teeth belong to vermivores that feed on amphinomid worms and type 4 are found in vermivores from deeper climes (Nybakken, 1990). Some species are known to change the design of their radula as they mature as is the case with two species (Conus ermineus and Conus magus) that are known to change from vermivorous to piscivorous (Nybakken, 1990; Nybakken and Perron, 1988; Rolan and Boyer, 2000). Snails that are known to maintain a similar diet regardless of maturity tend to produce the same type of radula (Nybakken, 1990). This suggests that radula type is dictated by food preference.

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Other Venoms

Two definitions for the word “venom” currently exist. One posits that a venom is, “A secretion (that interferes with physiological processes) produced in a specialized tissue in one animal and delivered to a target animal through the infliction of a wound.” (Fry, et al., 2012). Included in this definition is a phylogenetic component that incorporates the venom delivery system and the injected chemical. A second definition requires satisfaction of two conditions: that the chemical’s role is injected at lethal quantities and that the chemical’s role in fatality of the victim is demonstrated. This definition excludes bee and many spider, scorpion and most cone snail duct toxin components (Kardong, 2012). For the purposes of this paper, I will use the first, broader definition. Venom is a widely used mechanism for both defense and subduing prey in the natural world. Invertebrates such as bees, ants, scorpions, and spiders all have unique mechanisms for venom delivery and envenomation. Fire ants, for example, produce five alkaloids (solenopsin A, solenopsin B, cis-4-tridecenyl, dehydrosolenopsin B, solenopsin C, and cis-6-pentadecyl) that are known to have antibiotic properties while the sting of Australian bull ants has killed numerous people through anaphylaxis (Brown, et al., 2003; Jouvenaz, Blum and MacConnel, 1972). Bees and wasps produce amines, enzymes, and peptides that cause the characteristically painful sting. Many of the peptides act through phospholipase A2 activity, releasing arachidonic acid (Argiolas and Pisano, 1983). Arachnids are well known for their ability to produce potent venom. Theraphosid spiders produce at least 85 different peptides as part of their armament (Herzig and Hodgson, 2009). Scorpion stings can produce symptoms ranging from severe pain to gastrointestinal and/or pancreatic issues to respiratory failure and death. Each scorpion can produce as many as 100 different peptides, but which peptides are expressed varies with geography (Borges, et al., 2006; Newton, et al., 2007; Ruiming, et al., 2010). Mollusks other than cone snails that have venom include cephalopods such as maculotoxin in the blue ringed octopus. Like μ-conotoxins, maculotoxin is a sodium channel blocker for both defense and prey submission. Unlike cone snails, however,

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maculotoxin is produced by symbiotic bacteria living in the salivary gland of the octopus, not the cephalopod itself (Sheumack, et al., 1978). The best-studied venom-producing vertebrates are snakes. This is because crotalids and elapids account for 32,000 deaths annually in Africa alone through severe local and neuronal acting venoms (Currier, et al., 2010). Their venom consists of hundreds of complex proteins and less complex peptides that act in concert to immobilize and begin digestion of prey items (Menezes, et al., 2006). Of importance to this study, snake venom has been shown to vary widely with species, geography, gender, season, habitat, and age (Chippeaux, Williams and White, 1991).

Intersexual Variations in Venom Composition

Intersexual variations in venom are a rare but important phenomenon to study in cone snails, mostly because at present there are no known dimorphisms between male and female snails (Nybakken, 1978). Current cone snail breeding programs are forced to rely on chance to match conspecifics of the opposite sex. Species that have known venomic dimorphisms include snakes (Bitis nasicornis) and spiders (Chippeaux, et al., 1991; Herzig and Hodgson, 2009). In particular, female Bitis nasicornis inject one extra protein not found in male venom. Such a peptide in cone snails would be the only known dimorphism in cone snails.

Conopeptides of Conus striatus

Conopeptides are arranged into large groups called superfamilies (A, M, T, O, I1,

I2, J1, J2, S, D and P) and then into smaller peptide families (Biggs, et al., 2010; Liu, et al., 2012). Superfamily designation is assigned based on numbers and patterns of disulfide bond connectivity between Cysteine residues (Figure 2) and highly conserved regions in the pre- and pro-peptide regions in the gene coding for that peptide. The disulfide bond connectivity dictates the folding structure of peptides. Families are assigned based on the receptor target. C. striatus can produce representative peptides of

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four of the conopeptide superfamilies and six families (Table 1 and Table 2). Furthermore, conopeptides have a high degree of post-translational modifications (PTMs). Common PTMs found in conotoxins include cleavage of the pre-pro regions, C- terminal amidation, glycosylation, and changing out normal L-amino acids for their D- isomers. These changes tend to have little or no effect on the selectivity of the mature peptide but are thought to assist in folding. The first conotoxin identified and characterized from C. striatus was Conopressin-S, a nine-residue peptide that doesn’t fit into any of the aforementioned classifications (Table 1 and Table 2). It was isolated along with a similar Conopressin found in Conus geographus that differed by only two residues. Conopressins mimic vassopressins that are neuro- and co-transmitters in mammalian brains with similar peptides found in much simpler vertebrate ancestors (Cruz, et al., 1987). In addition to Conopressin-S, C. striatus produces 6 conotoxins that don’t fit into any of the above families (Bromocontryphan-S, Contryphan-S, and Con-ikot-ikot; (Jakubowski, Kelley and Sweedler, 2006; Pi, et al., 2006; Walker, et al., 2009)). The A superfamily of conotoxins have 4 or 6 Cysteines depending on the family and include the α- and κ-conotoxins (Santos, et al., 2004). α−Conotoxins, such as SII from C. striatus, target nicotinic acetylcholine receptors. Of note, while most α- conotoxins have two disulfide bonds and a C-terminal amide, SII has 3 and a free C- terminus (Ramilo, et al., 1992). κ-Conotoxins, represented here by SIVA, act on voltage gated potassium channels. κ-Conotoxin SIVA has 4 PTM’s including a glycosylation in position 7, a pyroglutamate in position 1, a 4-trans-hydroxyproline in position 17 and a C-terminal amide (Craig, et al., 1998; Wang, et al., 2003). M-Conotoxins have 6 cysteine residues and thus three disulfide bonds and are represented in C. striatus venom by the μ-conotoxins that block voltage gated sodium channels (Wang, et al., 2006). I-Conotoxins belong to one of two groups based on the amino acids in the signal and pro regions of the precursor consensus sequence and act on reduce voltage gated sodium channels by decreasing the activation threshold (Teichart, Jimenez and Olivera, 2009). The only two I-conotoxins that come from C. striatus were derived genetically (Buczek, et al., 2005). O-Conotoxins also contain 6 cysteine residues and belong to one of two families.

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Ο-conotoxins are voltage gated calcium channel blockers. C. striatus produces SVIA, the smallest known omega conotoxin (Ramilo, et al., 1992). C. striatus δ-conotoxin SVIE was shown to hold the sodium channel in its “on” position, inhibiting its inactivation, although the exact mechanism is as yet unknown (Leipold, et al., 2005). μΟ-Conotoxins are the only recognized conotoxin family not represented in C. striatus venom and act by inhibiting sodium channel activation (Teichart, et al., 2009).

Figure 2. Cysteine patterns and disulfide connectivity in the six conotoxin superfamilies. Image taken from Conoserver (http://www.conoserver.org/?page=about_conotoxins&bpage=cononames)

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Table 1. List of all known conopeptides isolated from Conus striatus.

Family Structure Class Name Amino Acid Sequence Calc. MH References

(Craig, et al., 1998; Santos, et al., 2004; κ SIVA ZKSLVPSVITTCCGYDOGTMCOOCRCTNSC* 3190.29 Wang, et al., 2003) CC - - C - - C - - C - - C ST4082 EKSLVPSVITTCCGYDOGTMCOOCRCTNSC* Genetic Derived SIVB ZKELVPSVITTCCGYDOGTMCOOCRCTNSCOTKOKKO* 4059.8 (Santos, et al., 2004) A SI ICCNPACGPKYSC* 1352.51 (Benie, et al., 2000; Zafaralla, et al., 1988) α SIA YCCHPACGKNFDC* 1454.5 (Myers, et al., 1991) CC - - C - - C SII GCCCNPACGPNYGCGTSCS 1789.51 (Ramilo, et al., 1992) Genetic Derived S1.1 NGCCRNPACESHRC* 1543.56 (Santos, et al., 2004) (Bulaj, et al., 2005; Schroeder, et al., M CC - - C - - C - - CC μ SIIIA ZNCCNGGCSSKWCRDHARCC* 2205.76 2008; Wang, et al., 2006) SIIIB ZNCCNGGCSSKWCKGHARCC* 2119.75 (Schroeder, et al., 2008) 10

A11.2a GCKKDRKPCSYQADCCNCCPIGTCAPSTNWILPGCSTGPFMAR 4573.91 (Buczek, et al., 2005) I C - - C - - CC - - CC - - C - - C Genetic Derived S11.3 CVPPSRYCTRHRPCCRGTCCSGLCRPMCNLWY 3712.52 (Kaas, et al., 2008) SVIB CKLKGQSCRKTSYDCCSGSCGRSGKC* 2737.14 (Nielsen, et al., 1996; Ramilo, et al., 1992) ω SVIA CRSSGSOCGVTSICCGRCYRGKCT* 2493.01 (Ramilo, et al., 1992) (Bulaj, et al., 2005; Kauferstein, Melaun and Mebs, 2005; Leipold, et al., 2005; δ SVIE DGCSSGGTFCGIHOGLCCSEFCFLWCITFID 3328.3 Ramilo, et al., 1992) Genetic Derived Conotoxin-2 AADCIEAGNYCGPTVMKLCCGFCSPYSKICMNYPKN 3887.61 (Kauferstein, et al., 2005) O C - - C - - CC - - C - - C Conotoxin-3 CESYGKPCGIYNDCCNACDPAKKTCT 2780.04 (Kauferstein, et al., 2005) Conotoxin-9 EGCSSGGTFCGIHPGLCCSEFCFLWCITFID 3325.31 (Kauferstein, et al., 2005) Conotoxin-15 CRPSGSPCGVTSICCGRCSRGKCT 2410.98 (Kauferstein, et al., 2005) S6.1 CKAAGKSCSRIAYNCCTGSCRSGKC 2550.05 (Kaas, et al., 2008) S6.2 CRSSGSPCGVTGICCGRCYRGKCT 2446.98 (Kaas, et al., 2008) S6.6 CKGKGAPCRKTMYDCCSGSCGRRGKC 2748.14 (Pi, et al., 2006)

Table 2. List of all known conopeptides isolated from Conus striatus (continued).

Family Structure Class Name Amino Acid Sequence Calc. MH References

S6.8 DGCSNAGGFCGIHPGLCCSEICLVWCT 2738.05 (Kaas, et al., 2008) S6.10 CTPDDGACAEPVQCCSTFCNPVTNMCIDWLGIGLSRSVL 4111.74 (Pi, et al., 2006) S6.11 CRTWNAPCSFTSQCCFGKCAHHRCIAW 3109.26 (Pi, et al., 2006) Genetic O C - - C - - CC - - C -- C (Lu, et al., 1999; Wen, et al., Derived S03 CKAAGKPCSRIAYNCCTGSCRSGKC* 2559.08 2005; Yan, et al., 2003) (Kauferstein, et al., 2005; Lu, et S04 ATDCIEAGNYCGPTVMKICCGFCSPYSKICMNYPKN 3917.62 al., 1999) (Kauferstein, et al., 2005; Lu, et S05 STSCMEAGSYCGSTTRICCGYCAYFGKKCIDYPSN 3762.47 al., 1999) Bromocontryphan-S GCOWEPWC* 1068.27 (Jakubowski, et al., 2006) (Jakubowski, et al., 2006; Contryphan-S GCOwEPWC* 990.36 Nielsen, et al., 1996)

11 (Cruz, et al., 1987; Gray, Olivera and Cruz, 1988;

Conopressin S [Arg] CIIRNCPRG* 1027.52 Walker, et al., 2009)

Con-ikot-ikot SGPADCCRMKECCTDRVNECLQRYSGREDKFVSFCYQ

-EATVTCGSFNEIVGCCYGYQMCMIRVVKPNSLSGAHEA Unclassified -CKTVSCGNPCA 9425.97 (Walker, et al., 2009)

S18.1 AGLTVCLSENRKRLTCSGLLNMAGSVCCKVDTSCCSSQ 3930.76 (Kaas, et al., 2008) S4.3 QKELVPSKTTTCCGYSPGTMCPSCMCTNTCPPQK 3617.49 (Pi, et al., 2006) Genetic S6.7 CMEAGSYCGSTTRICCGYCAYSASKNVCDYPSN 3500.3 (Pi, et al., 2006) Derived KDRPSLCDLPADSGSGTKAEKRIYYNSARKQCLRFDYTGQGGNENNFRR (Bayrhuber, et al., 2005; Dy, et Conkunitzin-S1 TYDCQRTCLYT 6929.63 al., 2006) ARPKDRPSYCNLPADSGSGTKPEQRIYYNSAKKQCVTFTYNGKGGNGNN Conkunitzin-S2 FSRTNDCRQTCQYPVG 7207.9 (Korukottu, et al., 2007)

Conus Venom Sources

There are five sources of venom available for study: genomic DNA, cDNA, duct venom, milked venom, and radular venom. Genomic DNA work isolates the start and stop codons of the DNA straight from the genome while cDNA methods isolate mRNA. Both methods then use the nucleic acid code to assign corresponding amino acids to build the peptide. One big advantage of these methods is that researchers also derive the highly conserved pre- and pro-regions that help identify a peptide’s family (Olivera, 2002; Yuan, et al., 2007). These methods also help elucidate what the snail is genetically capable of producing in spite of what may not be present in the duct, radular, or milked venom. There are two big disadvantages of these methods. The first is the need to sacrifice the animal for the sample negating the possibility of long-term studies on intra- specimen variation (Chivian, Roberts and Bernstein, 2003; Jakubowski, et al., 2005). Further is the inability of these methods to identify the post-translational modifications that are prevalent in conopeptides. Like nucleic acid sources of venom, extracting the venom stored in the lumen of the duct requires the sacrifice of the animal. Unlike cDNA and genomic DNA methods, duct venom extraction isolates the mature conopeptide complete with post-translational modifications. Importantly, venom components contained in the venom duct change from the anterior to posterior ends of the gland. This is probably a reflection of the differential expression from the different specialized cells contained in each section of the venom duct (Bingham, et al., 1996; Marshall, et al., 2002). Studies comparing milked and duct venom show that milked venom contains cleaner samples with fewer peptides but a higher, more variable concentration of those components necessary for prey capture (Biass, et al., 2009; Bingham, et al., 1996; Jakubowski, et al., 2005). The cleaner milked venom samples lack the cellular debris and unrefined venomic peptides found in duct venom samples (Hopkins, et al., 1995). Furthermore, a study of Conus consors found that 48% of the peptides found in the milked venom were not present in the dissected duct venom, suggesting that venom production is a burden of other organs in addition to the venom duct (Biass, et al., 2009).

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Recent work collected cDNA of three α-conotoxins from the salivary gland of Conus pulicarius that were not found in the venom gland (Biggs, Olivera and Kantor, 2008). The most recently exploited source of venom is taken from the radular teeth through one of two methods. First the teeth can be dissected out of the animal and second, the teeth can be collected and contents extracted post feeding. Venom taken from the radular teeth in Conus californicus is most similar to that of the distal end of the venom duct (Marshall, et al., 2002).

Venom Variation in Conus

The study of the compositional variability of venom has direct implications in biological, biochemical, pharmacological, management of envenomations and snail evolutional studies. Cone snail venoms are known to vary between species (inter- species), within species (intra-species) and over time within the same specimen (intra- specimen). There are approximately 700 species of cone snails, each genetically capable of producing over 1000 venom compounds (Davis, Jones and Lewis, 2009) of which at least 100 peptides are unique to that species (Teichart, et al., 2009). Each species produces a unique set of peptides that can be used for identification much like a fingerprint (Bingham, et al., 1996). Dramatic intra-species variations have been noted across all three Conus feeding clades. For example, only one hydrophobic peptide was found to be common among six dissected Conus textiles (Bingham, et al., 1996). Conus vexillum venom has been shown to vary across geographic scales (Abdel-Rahman, et al., 2011). C. striatus venom output was qualitatively placed into three whole-venom categories based on the principle constituents in the venom and their combined toxinologic effects. The first produced venom primarily composed of s4a (κ-conotoxin SVIA) with the second venom group producing a mix of s4a and s4b. Both s4a and s4b induce a convulsive, tetanic paralysis in their fish victims. The third venom type commonly produced by C. striatus blocks neuromuscular signal transmission inducing a paralytic relaxing of the muscles (Jakubowski, et al., 2005). One study failed to find intra-species variation in the duct

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venom of Conus regius (Braga, et al., 2005). Duct venom as a source has since been found to be much less variable than milked venom (Marshall, et al., 2002). In a similar study conducted on C. striatus, little intraspecific variation was found in the crude duct venom while significant variations were noted in milk venom collected from the same specimens (Jakubowski, et al., 2005). Which of the 1000 potential peptides in a snail’s chemical arsenal get injected and to what concentration is a highly complicated matter that is not fully resolved. In Conus consors, some specimens were found to maintain a relatively stable profile while others varied wildly with little overlap between consecutively collected profiles. Similar profile stability was noted in C. striatus with specimens reacting to stressors but then returning to roughly the original profile (Jakubowski, et al., 2005). Another study, however, started noticing large deviations from the typical species fingerprint after 2-4 months in captivity (Dutertre, et al., 2010). Other factors with which venom constituents may vary include geography, environment, gender, and genetic variation (Abdel-Rahman, et al., 2011). These suggest that the animals maintain some level of regulation over the constituents contained in their venom (Jakubowski, et al., 2005). It has been posited that venom’s primary purpose is likely prey subjugation because shifts in prey preferences among venomous snakes are often followed by deterioration of the venom system, (Fry, et al., 2012). However, because cone snails have swapped prey preference many times through their evolution and even during their developmental stages and maintain their venom delivery systems throughout, conopeptides may have alternate biological uses, lending potential predictability to this apparent variability once other evolutionary purposes are found.

Anthropogenic Uses for Conopeptides

The majority of cone snail research now focuses on utilizing conotoxins for human needs. The most successful example of this is Ziconotide, clinically known as Prialt®. Ziconotide is an analgesic synthetically based on the ω-conotoxin MVIIA, a potent calcium channel blocker found in the venom of Conus magus. Unlike with more

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popular opiates, patients do not develop addiction or tolerance to Ziconotide (Bingham, et al., 2010). Ziconotide is most often used synergistically with other intrathecal analgesics to resolve the full range of symptoms (Saulino, 2009). Other health issues that could be treated with conotoxin synthetics that are currently in preclinical trials include nerve damage from diabetes, epilepsy, and small cell lung cancer (Chivian, et al., 2003; Nelson, 2004). Conotoxins have potential in agricultural pest control, too. Since many conopeptides produced by Conus textile are known to be phyla-specific for gastropod mollusks, synthetic C. textile venom may one day be used to control the highly invasive apple snails without harming the native fish, plant crops, or human consumers (Terlau and Olivera, 2004).

Biosustainability

A recent letter in Science (Chivian, et al., 2003) suggested that the yearly take of cone snails for research purposes to be in the hundreds of thousands of snails. In addition to the research take, the ornamental shell trade imports hundreds of tons of shells into the U.S. every year. The recommendation was for trade in cone snails to be controlled under Appendix 2 of CITES (Chivian, et al., 2003). While the research component of this take was later shown to be an overestimation, the article introduced the idea that cone snails may be at risk of over-collection and researchers should be mindful of their environmental impact (Duda, et al., 2004; Fainzilber, 2004). Similar concerns exist for another gastropod, the Giant keyhole limpet (Megathura crenulata) that produces another biotechnological compound known as Keyhole limpet hemocyanin (California, 2006).

Moon Phase

The primary literature has many examples of moon phase coupling with behavior rhythms of invertebrates. In fact, the largest migration of animals on earth, the diel vertical migration of mesopelagic zooplankton toward the surface, is triggered by the

15

absence of light at dusk and controlled by following a specific isolume, or constant light level as dictated by the moon phase (Alldredge and King, 1980; Tarling, Bucholz and Matthews, 1999). The best documentation of lunar phase/behavior coupling is from a 64- year study on the spawning of Pacific Palolo (Eunice viridis) where the activity, celebrated as Palolo days by the Samoans, occurs in the few days surrounding the third quarter of the moon in October (Naylor, 2001). In many cases, a coupling of animal behavior with moon phase is selected strongly enough that the behavior’s timing has become endogenous and continues under laboratory conditions even when the moon cue is no longer present (Naylor, 2001). More specific to cone snails, shell collectors have noted increased activity around certain moon phases (Harold Jackson, personal communication). Furthermore, of eight egg-laying events witnessed in our laboratory aquaria in conjunction with a related project, all have been within five days of the full moon (unpublished data).

Taxonomy of Conus striatus

Piscivory has evolved at least twice in the genus Conus (Duda and Palumbi, 2004). C. striatus branched off with C. geographus and C. catus from a similar genotypic line as C. ebraeus, C. chaldeus and C. abbreviatus. The most parsimonious mode of dietary shift with respect to cladograms built from genotyping is one from vermivory to piscivory (Duda and Palumbi, 2004). More recent cladograms show C. californicus diverging from the Conus tree, suggesting a more logical shift from generalist feeding to piscivory (Biggs, et al., 2010). A previously cryptic subspecies within C. striatus was recently discovered. The holotype was found off Maile Point, Oahu, Hawai‘i. This diagnosis was based on two main morphological characteristics: spire height and relative shell diameter. Spires in the newly found Conus striatus oahuensis were lower with the spire outline being concave. The relative diameter of C. s. oahuensis is smaller compared to that of C. s. striatus. No genetic or biochemical studies have been performed yet on these related subspecies (Tucker, Tenorio and Chaney, 2011).

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In this study I correlate environmental data and the life-history of captive Conus with the differential expression of conopeptides within their milked venom. Undertaking one of the largest continuous RP-HPLC/UV studies describing the milked venom of C. striatus over a 40 week period I examine variables such as sex, season, ovula, moon phase, snail mass, and morphology and examine their effect on total venom production as measured by milked venom volume, frequency of milking, and chromatographic changes in the milked venom. I provide a clear indication that the diversity of peptides and the production of milked venom can be radically changed by captive cone snail manipulation. An increase in the chemical diversity within individual milked venoms can be induced through environmental manipulation.

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HYPOTHESES

General Hypotheses

• The ecological impacts stemming from cone snail collecting for venomic research can be mitigated through the development of an aquatics laboratory dedicated to the biosustainable supply of venom through the housing, feeding and milking of live cone snails.

• An analysis of milked venom under long-term captivity may reflect snail health and productivity showing the impacts of controlled stresses on milked venom production.

• A comparison between the quantitative analysis of a single milked venom constituent and the lunar cycles, seasonality, feeding, and death of C. striatus may add value to advanced husbandry techniques by maximizing milked venom production.

Specific Hypotheses

Series of alternative hypothetical statements can be made from this study involving the comparison of a number of select chemical development indicators (feed rate, number of dominant RP-HPLC peaks, volume, and κ-conotoxin SIVA concentration) with a number of physiological (reproduction, snail size, sex, death, diet, and morphological variance) and phenological (seasonality, moon phase) variables.

Reproduction - Ovum depositing events have a predictable effect on the rate of the gravid snails’ feed rate. - Ovum depositing events correlate with increases or decreases in volumetric venom production. - Ovum depositing events correlate with increases in venom peptide diversity.

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Snail Size - Feed rate will vary with snail mass in a predictable manner congruent with other ectothermic marine species. - Snail mass has a predictable effect on the volume of venom injected. - The number of dominant peaks (defined below) will vary with the mass of the snail. - Larger striated cone snails will produce differing concentrations of κ-conotoxin SIVA than will smaller specimens.

Sex - Male and female snails will feed at different rates. - Male and female snails will tend to inject different volumes of venom. - Male and female snails will produce different venom diversities. - κ-Conotoxin SIVA production will differ in male and female snails.

Death - Snails nearing natural death will cease feeding. - Venom production near natural death will decrease. - Venom diversity will vary in the weeks leading up to snail death. - Differences in production of κ-conotoxin SIVA will be seen in the weeks leading up to death.

Pill Diet - Feed rate will vary in preference to the food item offered. - The snails will alter their injected volumes in response to dietary change. - Venomic diversity will change with dramatic shifts in diet. - The production of venomic peptide κ-Conotoxin SIVA will change with a change in diet

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Morphological Variants - Specimens morphologically identified as Conus striatus oahuensis feed at different rates than those identified as C. s. striatus. - Specimens morphologically identified as C. s. oahuensis will inject differing volumes as those exhibiting characteristics of C. s. striatus. - The two identified subspecies will have differing numbers of dominant peaks indicating differing venomic peptide diversity. - The two subspecies will have varying concentrations of κ-conotoxin SIVA.

Phenology-Seasonality - The snails will feed at varying rates depending on the season. - The snails will inject varying volumes of venom depending on the season. - The snails will change the composition of their venom in response to changes in season. - The snails will inject different amounts of κ-conotoxin SIVA depending on the season.

Moon Phase - The snails will feed at different rates during different phases of the moon. - The snails will inject different volumes of venoms during different moon phases. - The snails’ venom peptide diversity will vary with moon phase. - The snails will inject different concentrations of κ-conotoxin SIVA during different moon phases.

Time in Captivity - C. striatus will change the volume of injected venom with time in captivity. - A snail’s venom composition will change with time in captivity - A snail’s peptide diversity will change with time in captivity. - κ-SIVA concentration in the venom makeup will change with time in captivity.

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METHODS

Housing The cone snails were kept in two modified Aquatic Habitat® Benchtop Systems with added protein fractionators. Each system supported 10 ten-liter aquaria with physical (to 50 micron), biological, and carbon filtration pumped at a rate of six water exchanges per tank per hour.

Milked Venom Collection Twenty-six C. striatus were collected from the reefs around Oahu on SCUBA. Individuals were photographed for unique shell patterns and catalogued in a database. Venom was collected from the snails during their weekly feedings as previously described (Nelson, 2004) for a period of 40 weeks. Two-milliliter eppendorf collection vials were fitted with a latex membrane banded in place over the open end. The collection vial was then banded to the end of a dental tool in preparation for feeding. A swordtail (Xyphophorus sp.) served as the food item. When the excited snail's proboscis extended, the tail of the fish was presented to the snail over the collection vial. The snail then shot its radular tooth through the fish’s tail and into the membrane injecting venom into the eppendorf tube.

Pill-Diet Study The snails in tank 1 were fed a pill diet. A control period encompassed the first two months in captivity when the snails were kept on a diet of fish. Then the snails were fed a pill diet for a period of not more than 12 milkings. Gelatin capsules were filled with 63±0.02 mg crushed Silvercup™ fish pellets and offered in place of fish during weekly milkings. The snails were switched back to a fish diet after the 12 milkings or when they stopped accepting the pill diet, whichever came first.

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Sample Preparation Collected samples were volumetrically measured using an Eppendorf p200 pipette and dried on a speed-vac centrifuge. One hundred microliters of 0.1% trifluoroacetic acid (TFA) in distilled water were added to each tube to dissolve the sample. From these samples, 60 uL were placed in a 150 uL insert within a glass vial that entered the Waters 2695 Separations Module via robotic carousel.

RP-HPLC Profiling and Fractionation

The samples were pumped onto a Phenomenex C18 column (00G-4053-A0 Jupiter 5U C18 300A 250x1.00 mm 5 micron 466646-1) in an aqueous solvent (99.9% v/v distilled water, 0.1% v/v TFA). The concentration of an organic solvent (90% v/v acetonitrile, 9.99% v/v distilled water, 0.01% v/v TFA) was increased at a linear rate of 1% every minute for one hour eluting the total sample from the column. Extracted elutants were analyzed at 214nm on a Waters 996 Photodiode Detector and Waters Millennium 32 (v3.2) software. A known standard sample of κ-conotoxin SIVA will be run on the same gradient.

Mass Spectrometric Analysis Peptide masses of RP-HPLC fractionated peaks were determined on a PE Sciex API 3000 Triple Quadrupole LCMS system using Analyst software. Masses were compared to known masses from Conus striatus in the literature. Peptide mass of the fractionated St4082/ κ-conotoxin SIVA standard peak from above will be determined on a PE Sciex API 3000 Triple Quadrupole LCMS system using Analyst software. This process will yield a molecular mass for the isolated peak to compare with the known target weight of κ-conotoxin SIVA of 4082 Daltons. This also displayed other masses collected in the isolation, verifying the purity of the collected sample.

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Quantitative Analysis The area under the curve of the RP-HPLC profiles obtained in Objective 2 will be calculated from the Waters Millennium 32 (v3.2) software to render the quantity of St4082/ κ-conotoxin SIVA peptide present in each sample. This can then be calculated with the total volume of the sample collected at the time of milking to determine the concentration of St4082/ κ-conotoxin SIVA which were then be compared to the life history events and phenology investigated in Objective 2.

Statistical Analysis

Where applicable, the data were analyzed on StaPlus software using three statistical comparisons; correlations, t-tests, and ANOVA. Statistical significance was defined as any results returned with a p-value less than 0.05. In the case of seasonality correlations, the monthly mean was compared to the total mean. In this case, if the monthly mean lay outside of two standard deviations (~95% confidence) from the total mean, it was deemed significant.

Define Number of Peaks

In an effort to gain a numerical measure for overall peptide diversity, a count of the number of dominant peaks was applied. The most dominant peak was defined as the peak that had the greatest arbitrary unit as determined by RP-HPLC. Secondary dominance was determined as peaks that rose to greater than ½ the height of the dominant peak. The final data was a simple count of the number of primary and secondary dominant RP-HPLC peaks.

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Figure 3. Three profiles from Conus striatus that demonstrate the discernment of dominant peaks in this analysis. The top profile is an example of a profile with no dominant peaks because it lacks any apparent peptides. The middle profile would be classified as having two peaks. The final profile has four peaks that are all taller than half of the height of the tallest peak.

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RESULTS

Of the 31 potential correlations examined, five showed some indication of affecting one of the four aspects of venom production investigated. These were snail mass vs. volume, season vs. volume, lunar phase vs. feed rate, lunar phase vs. venom diversity, and time in captivity vs. volume (Table 3). The ways in which these factors affected the venom were very different. Statistics presented in mean ± standard deviation.

Table 3. Summary of the correlations observed. S=Strong correlation; P=Possible correlation; N=No correlation NA=Not tested

Milked Number of Venom Dominant RP- St4082 Feed Rate Volume HPLC Peaks Concentration Pregnancy P N N NA Snail Size N S N P Sex N N P P Death P N P S Pill Diet S P P S Morphology N N P N Seasonality P P N P Lunar Phase S N P N Time in Cap P S P P S=Strong P-Possible N-Not Present NA=Not Run

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Snail Size

The factor with the greatest influence on the volumetric output of the venom was size with a best-fit slope of 0.3074 and a covariance of 393.9 (n=25) indicating a very strong correlation (Figure 4). However, size did not correlate strongly with our measure of venom diversity (covariance=-3.37 n=25) or rate of feed (covariance=1.25 n=25). The amount of St4082/ κ-conotoxin SIVA had a very strong relationship to the mass of the animal (covariance= 25,885; Figure 5) however the mass of the snail did not vary with the concentration of St4082/ κ-conotoxin SIVA (covariance= 1.44; Figure 6).

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70 y = 0.3074x + 4.7502

60

50

40

30

20

10

Mean Injected Milked Venom Volume (uL) 0 0 50 100 150 200 250 Snail Mass (g)

Figure 4. Snail mass and venom volume output exhibit a strong positive correlation (n=25, Mean Mass=99±37 g, Average Volume=35±12 uL, Mass to Volume Covariance=393.9 r=0.92 p=0). The greatest average volume came from the largest snail in the system, snail 4-1 which had a mass of 211 grams.

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250

200

150

100 Snail Mass (g)

50

0 0.0 2000.0 4000.0 6000.0 8000.0 10000.0 12000.0 14000.0 pmol St4082/ κ-conotoxin SIVA

Figure 5. The number of picomoles of St4082/κ-conotoxin SIVA did not appear to change in relation to snail mass (r=0.18 p=0.52).

250

200

150

100 Snail Mass (g)

50

0 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0 450.0 pmol St4082/uL Injected Venom

Figure 6. The concentration of St4082/ κ-conotoxin SIVA (picomoles/uL injected volume) had no correlation to snail mass (r=0.076 p=0.10).

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Phenology-Seasonality

Snails produced statistically more venom (confidence >95%) in the months of May, June, July and October (Figure 7). While not statistically significant, a similar seasonal cycle with peaks in June, July and December was observed with feed rate (June, July and December - Figure 8). However, the resulting venom exhibited the same level of diversity regardless of season. Amount and concentration of St4082/ κ-conotoxin SIVA, peaked in October but varied to such a degree that no reliable predictability could be discerned (Figure 9 & Figure 10).

70

60

50

40

30 2010 20

Injected Volume (uL) 2011 10

0

Month

Figure 7. Average monthly volumetric milking data averaged from the captive Conus striatus population. Statistically significant months included May, June and July 2010 and October 2011. Error bars represent standard deviation.

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120

100

80

60 2010 2011 40

20 Successful Milking Rate (%) 0

July June May

April April

March March August

January January October October

February February Month December December November November September September

Figure 8. Feed rate varied insignificantly with seasonality.

4.0E+04 3.5E+04 3.0E+04 2.5E+04 2.0E+04 1.5E+04 2010 -conotoxin SIVA 1.0E+04 κ 2011 5.0E+03 0.0E+00 -5.0E+03 pmol St4082/ -1.0E+04 -1.5E+04 Month

Figure 9. Mean monthly amounts of St4082/ κ-conotoxin SIVA milked from select snails during the study period. Very large deviations impede conclusions from these data. Error bars represent standard deviation.

29

1000

800

600

400 2010 2011

Venom 200 -conotoxin SIVA )/uL Milked κ 0 July May June April March August January

-200 October February December November September pmol St4082 (

-400 Month

Figure 10. Mean monthly concentrations of St4082/ κ-conotoxin SIVA peaked during the month of October but exhibited a very large degree of deviation. Error bars represent standard deviation.

Sex

With respective means of 39 uL (±6) and 34 uL (±4), the six known females and four known males produced comparable quantities of venom (two tailed p-value =0.14) and were successfully milked 82% (±0.07) and 80% (±0.06) of the time (two-tailed p- value=0.35) respectively. The mean numbers of dominant peaks varied by more than 0.3 peaks (males=1.3 ±0.18; females=0.975 ±0.21; Figure 11). These values were not significantly different. The amount of St4082/ κ-conotoxin SIVA between males and females was 6226± 5325 pmol and 1821± 1476 pmol respectively (p=0.09; Figure 12). In terms of concentration, males and females respectively produced 150± 117 and 45± 37 pmol St4082/uL (p=0.07; Figure 13).

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1.6

1.4

1.2

1

0.8 Peaks 0.6

0.4

0.2 Average Number of Dominant HPLC 0 Males Females Sex

Figure 11. Compositional diversity of the venoms from known male versus known female snails differed in that males produced more complex venom with a greater number of dominant constituents (whole population mean=1.13±0.26 peaks, p=.056, t-Crit.=2.44). Error bars represent sex-specific standard deviations.

1.4E+04

1.2E+04

1.0E+04

8.0E+03 -conotoxin SIVA κ 6.0E+03

4.0E+03

pmol St4082/ 2.0E+03

0.0E+00 Females Males Sex

Figure 12. Known females and males in the collection produced differing amounts of St4082/ κ-conotoxin SIVA.

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300

250

200

150

100

50 pmol St4082/ul Milked Venom

0 Females Males Sex

Figure 13. The difference between females and males in the concentration of St4082/ κ-conotoxin SIVA in picomoles per uL injected venom was 45±37 and 150±117 respectively (whole population mean=80±84 pmol St4082, p=.085, t-Crit.=2.4). Error bars represent sex-specific standard deviation.

Moon Phase

Moon phase had an effect on both feed rate and venom diversity. During a new moon, 75% (±16%) of snail milkings were successful while successful milkings rose insignificantly during the full moon periods to 79% (±13%; Figure 14). While variability in the data was very high (standard deviations=16, 14, 10, and 12% for new, first quarter, last quarter and full moons, respectively), this trend is corroborated by observational reports from shell collectors who claim to find actively hunting Conus striatus during certain moon phases. The most diverse venom occurred during a new moon (1.47 ±1.01 RP-HPLC peaks), with full moon diversity coming in a close second (1.42 ±1.03 RP-HPLC peaks;

Figure 15). The snails consistently produced an average of 35 uL during the new (±20 uL), first quarter (±18 uL, and last quarter (±16 uL), reducing volumetric output to 34 uL during a full moon (±16 uL). The amount of St4082/ k-conotoxin SIVA in the samples was 3398± 8042 pmol (New), 2500± 4047 pmol (first quarter), 6696± 12580 pmol (last quarter) and 2300± 3902 pmol (Full; Figure 16). These data did not differ significantly. In terms of concentration varying with moon phase, the means of 98± 203 pmol/uL

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venom (New), 91± 145 pmol/uL venom (first quarter), 213± 343 pmol/uL venom (last quarter) and 68±108 pmol/uLvenom (full) did not vary significantly or appear to follow any trends (Figure 17).

95 90 85 80 75 70 65 Milking Probability 60 55 New Moon First Quarter Last Quarter Full Moon Phase

Figure 14. Conus striatus fed at a statistically similar rate regardless of moon phase (n=25, p=0.89, f=0.2, f- critical=3.5).

1.50

1.45

1.40

1.35

1.30

Average Number of RP-HPLC Peaks 1.25 New First Quarter Last Quarter Full Moon Phase Figure 15. Snails injected the most complex venoms during the new and full moon phases (Standard deviations= 1.01, 0.95, 1.02, and 1.03 for new, first quarter, last quarter, and full moons respectively. population=25, number of milkings=956, mean=1.4±1.0 peaks, p=0.47).

33

2.5E+04

2.0E+04

1.5E+04

1.0E+04 -conotoxin SIVA κ 5.0E+03

0.0E+00 New First Quarter Last Quarter Full pmol St4082/ -5.0E+03

-1.0E+04 Moon Phase

Figure 16. The average amount of St4082/ κ-conotoxin SIVA per milking did not vary significantly with moon phase (number of milkings=108, mean=3575±7659 pmol St4082/k-conotoxin SIVA).

600

500

400

300

200

100

0 New First Quarter Last Quarter Full pmol St4082/uL milked venom -100

-200 Moon Phase

Figure 17. The concentration of St4082/ κ-conotoxin SIVA did not vary significantly with lunar phase (number of milkings=105, mean=114±214 pmol St4082/uL milked venom).

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Time in Captivity

Over the course of 40 weeks, the overall venom production was greatest during the first ten weeks post capture and decreased gradually with each 10 weeks after that for a total mean decrease of 5 uL over the 40 week study period (Weeks 1-10=36±13 uL, weeks 11-20=33±14 uL, weeks 21-30=33±12 uL, weeks 31-40=30±14 uL; Figure 18; Figure 19). Temporal changes were noted in feeding probability as well. The snails were brought into the collection feeding at an average rate of 80% (±11%) and declined steadily until weeks 21-30 with a feed rate of 70% (±16) and finally jumping back to an 85% (±9%) rate of feed. The average number of dominant peaks stayed at 1.2 peaks throughout the captive study without significant change (Figure 21). The concentration of St4082// κ-conotoxin SIVA at the time of capture and proceeding 10 weeks was 152± 282 pmol/uL venom before decreasing suddenly to a mean of 35± 65 pmol/uL venom during weeks 21-30. During the last two ten-week bins, the snails averaged 119± 259 and 100± 149 pmol/uL venom for weeks 21-30 and 31-40 respectively (Figure 22).

60

50

40

30

20

10 Average Milked Venom Volume (uL)

0

Weeks 1-10 Week 11-20 Week 21-30 Week 31-40 Time Period in Captivity

Figure 18. When the forty weeks’ worth of samples were separated into 10-week bins, the captive snails showed a steady decline in venom production with time in captivity. This could be a result of malnutrition or insufficient husbandry techniques. N=270 samples were placed into 4 bins encompassing 10 weeks each. Standard deviations equaled 13.9, 11.8, 13.5, and 13.0 for weeks 1-10, 11-20, 21-30, and 31-40 respectively.

35

50 45 40 35 30 25 20 y = -0.119x + 37.374 Covariance=-31 15

Milked Venom Volume (uL) 10 5 0 0 10 20 30 40 50 60 Number of Weeks Weeks in Captivity

Figure 19. Another view of the decrease in venom volume through time in captivity.

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

Milk Success Probability 0.1 0

Weeks 1-10 Week 11-20 Week 21-30 Week 31-40 Time Period in Captivity

Figure 20. Feed probability decreased during the first 30 weekly feeds before abruptly increasing to 85% during the final 10 weeks of the study.

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2.50

2.00

1.50

1.00

0.50 Number of Dominant RP-HPLC Peaks

0.00 Week 1-10 Week 11-20 Week 21-30 Week 31-40 Time Period of Captivity

Figure 21. The average number of dominant RP-HPLC peaks observed over the course of the forty-week study stayed relatively constant meaning that the snails neither lost nor gained venom diversity in captivity.

500

400

300

200

100

0 pmol St4082/uL Milked Venom weeks 1-10 weeks 11-20 weeks 21-30 weeks 31-40 -100

-200 Time Period of Captivity

Figure 22. The mean concentration of St4082/ κ-conotoxin SIVA ranged from 35 to 152 pmol/uL venom.

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Morphological Variants

Based solely on the morphological character of spire height, the collection contained 16 specimens of Conus striatus striatus and 9 specimens of C. s. oahuensis (Figure 23). All of the specimens that had a noticeably larger relative diameter also had a concave spire, both indicative characteristics of C. s. oahuensis. Furthermore, all specimens that had small relative diameters had convex spires, both characteristics of C. s. striatus (Figure 24). With mean volumes of 33.5 ±9.7 uL and 30.5 ±10.2 uL for C. s. striatus and C. s. oahuensis respectively, no significant volumetric differences were noted between the two subspecies (Figure 25). While the number of peaks observed was similar (1.45 and 1.38 for C. s. striatus and C. s. oahuensis respectively), the number of dominant peaks varied much more in samples collected from C. s. striatus (standard deviation=0.55) compared with C. s. oahuensis (standard deviation=0.30). This increase in deviation is in spite of having far more specimens of C. s. striatus (n=16) versus C. s. oahuensis (n=9; Figure 26). Peptide diversity and feeding rate differences between the subspecies C. striatus striatus and C. s. oahuensis, however, were statistically negligible (Figure 25). C. s. striatus fed at a similar rate in comparison to C. s. oahuensis (76 ±7% and 75 ±6% respectively; Figure 27). The concentration of St4082/κ-conotoxin SIVA was similar (97± 103 pmol/uL injected and 89± 130 pmol/uL injected respectively) between C. s. striatus and C. s. oahuensis (Figure 28).

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Figure 23. Morphological subspecies determinations of the specimens studied herein. Identifications were based primarily on the concave/convex nature of the spire. In specimens where spire height was inconclusive, the relative diameter (RD=Y/X) was used. Snails 3-1 and 5-2 had neither distinctive spire heights nor relative diameters characteristic of either subspecies.

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1.6

1.5

1.4

1.3

1.2

1.1 Convex 1 Concave

Shell Width (cm) 0.9

0.8

0.7

0.6 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 3.1 Shell Length (cm)

Figure 24. All Conus striatus specimens that possessed a concave spire also had a slightly wider shell in relation to the shell's width (N=23, p=0.00002, t –Crit=2.08).

50.0 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 Mean Milked Venom Volume (uL) 0.0 C. s. striatus C. s. oahuensis Subspecies Figure 25. The difference in venom volume between the two subspecies was negligible (Mean=32.6±9.7uL, p=0.54, t-Crit.=2.09).

40

2.50

2.00

1.50

Peaks 1.00

0.50 Mean Number of Dominant HPLC 0.00 C. s. striatus C. s. oahuensis Subspecies

Figure 26. The average number of peaks observed from samples collected from C. s. striatus and C. s. oahuensis was very similar, although the variation in diversity was much higher in samples collected from C. s. striatus (Mean=1.43±0.48 peaks, p=.76, t-Crit.=2.09).

Mean Success Rate

0.90 0.80 0.70 0.60 0.50 0.40 0.30

Mean Success Rate 0.20 0.10 0.00 C. s. striatus C. s. oahuensis Subspecies

Figure 27. Feeding rates for C. s. striatus and C. s. oahuensis were comparable (mean=0.76±0.068%, p=0.55, t- Crit.=2.08).

41

250

200

150

100

50

0 C. s. striatus C. s. oahuensis pmol St4082/ul of injected venom -50

-100 Subspecies

Figure 28. A comparison of the concentration of St4082/κ-conotoxin SIVA in milked venom injected by C. s. striatus versus C. s. oahuensis reveals no difference between the two subspecies (Mean=95±106pmol St4082/uL injected venom, p=0.9, t-Crit.=2.2).

Reproduction

Four females laid eggs during this study, allowing data collection on how venom production is affected by the ovum depositing process. No changes in volumetric output were observed during the 17 weeks surrounding egg-laying events (Figure 29) nor were any changes in the number of dominant peaks observed during the study period (Figure 31). Milking rate, on the other hand, decreased dramatically during the week immediately following egg deposition with only one snail accepting the offered food (Figure 30). This week of light appetite was followed by two weeks of 100% milk success then 4 weeks with a feed rate at 75%.

42

100 90 80 70 60 50 40 30 20 10 Mean Milked Venom Volume (uL) 0 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 Number of weeks pre and post egg deposition

Figure 29. A temporal look at the mean injection volumes collected from gravid females during the 7 weeks prior and 10 weeks post egg-laying events (T=0 on the x-axis). No obvious changes in volumetric production were noted (n=4, Mean=47±23.8 uL).

1.20

1.00

0.80

0.60

Mean Feed Rate 0.40

0.20

0.00 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 Weeks before/after egg-laying

Figure 30. The feed rate decreased for gravid females during the week immediately following egg deposition (n=4, mean=0.81±0.19%).

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2.5

2

1.5

1

0.5

0

Mean number of dominant HPLC peaks -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

-0.5 Weeks before and after egg laying event

Figure 31. No changes were observed in the mean number of dominant peaks in samples collected from female snails around the time of egg deposition (time=0) (n=4, Mean=0.82±0.44 dominant peaks). Death

Only two snails died during this 40 week study. Snail 9-3’s death was a direct and acute result of laboratory assistant neglect, so any changes in its venom composition in the weeks prior were unrelated to its demise and not included in this analysis. On the other hand, snail 5-1 was found dead in its tank of presumably natural causes on 12/13/2011. The snail milked and maintained a steady feed rate in the weeks immediately prior to its death. Mean venom volume for snail 5-1 was 34 ±8.9 uL, and didn’t change significantly in the weeks prior to death (Figure 32). While the snail’s venom profiles oscillated between 1 and 3 dominant peaks for the first 18 weeks in captivity, it leveled off after that reliably producing 1 dominant peak every week until the week immediately preceding its death when it produced 2. The concentration of St4082/κ-conotoxin SIVA in samples collected from snail 5-1 in the weeks leading up to its death was relatively steady until November 11 or one month prior to death, at which point St4082/κ-conotoxin SIVA production decreased dramatically from 73 pmol/uL to 11.5 pmol/uL to a final milking containing only 8.7 pmol/uL (Figure 34).

44

70

60

50

40

30

20 Milked Venom Volume (uL) 10

0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Number of Weeks

Figure 32. Milked venom volumes for snail 5-1 were relatively steady for the time between capture on 6/17/10 and its natural death on 7/13/11 and accepted food until days before death.

3.5

3

2.5

2

1.5 Milked Venom 1

0.5 Number of Dominant HPLC Peaks in 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Number of Weeks in Captivity

Figure 33. Number of dominant peaks present in the weeks leading up to the natural death of snail 5-1 at week 41.

45

140.0

120.0

100.0

80.0

60.0

40.0

20.0 pmol St4082/uL Milked Venom 0.0 6/6/11 8/5/11 6/26/11 7/16/11 8/25/11 9/14/11 10/4/11 12/3/11 10/24/11 11/13/11 12/23/11 Date

Figure 34. From 7/6/2011 through 10/17/2011 snail 5-2 failed to feed or milk. Milking resumed on 10/17/2011 for a period of 6 weeks however, the last two milkings prior to the snail's natural death on 12/7/2011, St4082/ κ- conotoxin SIVA production decreased greatly.

Pill Diet

The three snails that were offered a pill diet averaged 4 uL less injected volume than the overall community pre-pill diet. The snails that were offered pills in lieu of fish decreased venom production from 32±9.7 to 23±11.7 uL per feeding or a factor of 29%. The pill diet snails never recovered their original fish-diet milking volumes even after switching back to a diet of fish. Feed response of pill-fed snails increased slightly during the pill-feed trial from 75% to 76%. After the snails were switched back to a diet of fish, however, feed success plummeted to 67%. For comparison, average feed success for the rest of the community was 77%. The number of dominant peaks was observed to increase (from 1.7 ±0.95 to 1.9 ±1.56 peaks) when offered a pill diet and stay at an elevated state (1.88 ±1.34) after the snails were once again fed fish. The venom diversity for the snails in the pill diet study remained greater than the community average of 1.34 ±0.93 regardless of treatment. Prior to being offered the pill diet, the snails produced increased levels of St4082/ κ-conotoxin SIVA (406± 334 pmol/uL venom) that decreased

46

once the pill diet began (299± 405 pmol/uL) and decreased further after the pill diet ceased (134± 244 pmol/uL; Figure 38). For comparison, the community’s mean concentration was 75± 155 pmol/uL.

60

50

40

30

20

10 Average Milked Venom Volume (uL) 0 Pre-Pills On Pills Post Pills Community Treatment Figure 35. When snails were switched to a pill diet, venom production decreased and did not recover even after the snails were put back on a diet of fish.

4

3.5

3

2.5

2

1.5

1 the Milked Venom

0.5

0 Number of Dominant RP-HPLC Peaks in Pre Pill On Pill Post Pill Community Treatment

Figure 36. The number of dominant RP-HPLC peaks sampled from snails given a pill diet increased both while the snails were on the pill and after the snails were switched back to a natural diet of fish.

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0.8 0.78 0.76 0.74 0.72 0.7 0.68 0.66 0.64 Milking Success Probability 0.62 0.6 Pre-Pills On Pills Post Pills Community Treatment

Figure 37. Snails that were offered a pill diet increased feed success during the pill-diet trial before decreasing feed response after the pill-feed trial ceased.

800.0

700.0

600.0

500.0

400.0

300.0

200.0

100.0

0.0 pmol St4082/uL Milked Venom Pre Pill On Pill Post Pill Community -100.0

-200.0 Treatment

Figure 38. A pill diet appears to decrease the concentration of St4082/κ-conotoxin SIVA in the population. The snails chosen for the study produced a high concentration of St4082/ κ-conotoxin SIVA at t=0 when compared to the mean of the rest of the community (n=38, mean=177±279 pmol St4082/uL milked venom).

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Other Comparisons

Milking probability was found to hold no relation to volumetric output (covariance= 0.48), St4082/ κ-conotoxin SIVA concentration (covariance= -3.39), or dominant number of peaks (covariance= -0.008). Volumetric output had no relation to St4082/ κ-conotoxin SIVA concentration (covariance= 5.0) or dominant number of peaks (covariance= -1.20; no figure). The concentration of St4082/ κ-conotoxin SIVA did correlate strongly in a direct relationship with the number of observed dominant peaks (covariance= 112.7; Figure 39).

4.5 4 3.5 3 y = 0.0022x + 1.2801 2.5 2 1.5 1 0.5 0 Number of Dominant HPLC peaks 0 200 400 600 800 1000 1200 1400 pmol St4082/uL milked venom

Figure 39. The number of dominant peaks found in the venom correlated in a strong directly proportional relationship with the concentration of the peptide St4082/ κ-conotoxin SIVA (n=94, mean diversity=1.6±1.15 peaks, mean concentration=122±225 pmol St4082/uL milked venom).

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Figure 40. A display of milkings 1, 10, 20, 30, and 40 from snails in tanks that did lay eggs (right column) versus the same milkings taken from snails in tanks that never produced eggs (left column). Examples of the most and least stable profiles through the 40 week period were chosen from each category. Note that the most stable profiles taken from a snail in a tank that did not produce eggs (snail 7-2) still had a number of peptides that were produced some weeks and not others. The least stable example from a snail in a tank that did produce eggs (snail 6-1) still showed a level of stability in spite of vast changes in concentration for the majority of its time in captivity. Conversely, snail 1-1 which produced the least stable profiles of all of the snails, showed little congruency at all and snail 3-2 consistently produced very similar profiles throughout its stay.

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Figure 41. Examples of 1, 10, 20, 30, and 40 week profile series extracted from C. s. oahuensis (snail 4-3) and C. s. striatus (snail 3-3). Note that both snails produced profiles with two strong peaks eluting at 32 and 34 minutes. This level of similarity would not be observed in two different species.

Figure 42. Shown above are samples taken at weeks 1, 10, 20, 30, and 40 from two different snails. Snail 1-1 (left profiles) was fed pills between weeks 5 and 13 and produced very inconsistent profiles overall. The control period from weeks 1 through 4 was dominated by profiles with 4 main peaks as seen in week 1. The snail stopped producing venom for most of the pill diet period and the switched to producing highly variable profiles for the rest of the 40 week study. This pattern was similar in the other two pill diet animals. Snail 10-3 (right) was selected as an example of a snail fed that was fed exclusively fish yet also produced highly variable venom profiles. Importantly, in spite of its apparent inconsistency, snail 10-3 maintained some level of congruency by always producing a peak eluting at 33 minutes. A pill diet therefore seems to initiate a highly variable series of profiles from which the snail doesn’t recover after 40 weeks, however diet does not explain all highly diverse venom profiles.

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Figure 43. Each of the profiles shown represents a snail’s normal, or baseline, profile. Typically, most of the snails produced venom with only 1 or 2 main peaks and a number of smaller ones. Snails 1-1, 5-3 and 9-1 regularly produced highly complex venom profiles with high concentrations of many components. Contrariwise, snail 7-1 routinely produced venom that was completely devoid of conopeptides. For reference, profiles marked with “O” represents baselines that come from C. s. oahuensis while those marked with “S” were taken from specimens of C. s. striatus.

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DISCUSSION

Number of Dominant RP-HPLC Peaks

Before launching into a discussion that utilizes a novel measure of milked venom peptide diversity, it is worthwhile to consider the validity of looking at the number of dominant RP-HPLC peaks. To be certain it is a crude measure designed to provide a quick survey of the milked venom. It does not consider the identities or characterizations of the individual peptides present, but simply the number. It has proved useful here in detecting shifts in production that would have gone unnoticed using the volumetric, feed response and St4082 measures of change in that the snails in this study seemed to produce more peaks when under duress. For example, when snail 5-1 died, the only change in production prior to its death was a slight increase in the number of dominant RP-HPLC peaks it produced. An increase in the variability of number of dominant RP- HPLC peaks coincided with the pill diet. After the pill diet was stopped, production of an elevated number of RP-HPLC peaks continued. Thus, the measure of the number of dominant RP-HPLC peaks seems to be both a valid and useful indicator of change in a venom sample.

Snail Size

The snails milked and fed at similar rates regardless of size. Most young animals tend to feed at a higher rate due to a growth-driven metabolic need. With the study population of snails ranging from 52 to 211 grams, this study population was comprised of only sexually mature animals and lacked representatives of post-settled larva and sub- adults. Furthermore, being sessile invertebrates that are far less mobile than their prey, it may be in the snails’ best interest to take advantage of feeding situations as opportunity allows regardless of metabolic need. The fact that large snails tended to produce more

53

milked venom volume with a higher amount of St4082 is probably a product of a proportionally larger venom gland and hydrostatic bulb.

Seasonality

The snails produced significantly more milked venom during the months of May, June, and July, a period that coincides with a peak in feed rate. This correlation illustrates an active time of year for the snails, concurrent with their known egg-laying season. However, two clutches of eggs were laid in December 2011 correlating with an increased feed rate but a low in milked venom volume and St4082 concentration. As this was the snails’ second year in captivity where natural day-length and food availability fluctuations may dictate seasonal timing, it is possible that the snails’ internal clock lost track of their normal breeding season. Potential causes behind other peaks in production are more cryptic. For example, an increase in venom volume coincides with an increased concentration of St4082 in October of both 2010 and 2011. A second peak in St4082 concentration occurs at the same time as a peak in feeding success in December 2010, but not 2011. A longer-term study involving more animals would help sort the anomalies from the trends in venom production.

Sex

As yet, no evidence for sexual dimorphisms based on morphological traits exists for cone snails. This study sought to explore the possibility that the sexes may be separated based on chemical composition instead. While the mean number of RP-HPLC peaks, milked venom volume, St4082 concentration and feed rates from male and female snails were found to be statistically insignificant, there were some non-significant compositional differences that are worth exploring. The average number of RP-HPLC peaks differed by an average 0.3 peaks between the known males and females. Furthermore, differences in St4082 concentration were an average 100 pmol/uL. Admittedly, the methodology of waiting for the animals to lay eggs is not ideal. Some

54

animals have been shown to maintain living sperm for years. If that were the case with C. striatus, many of the assumed males in this study may in fact be female. Furthermore, a larger population of known male and female animals would have yielded greater statistical resolution, but as yet no methods exist for such a determination short of dissection. Further research into milked venom compositional variations including sexual confirmation through genetic approaches may yield a dimorphism, aiding in future venomic and captive breeding efforts. While no valid sexual differences were found, snails that were placed in tanks with both males and females present as evidenced by the appearance of eggs at some point in the study, tended to produce more stable milked venom profiles from week to week (Figure 40) when compared to milkings taken from snails that did not produce eggs. This phenomenon possibly indicates a level of comfort gained by pairing males with females.

Moon Phase

Lunar phase plays an important role in the life of a snail. Snails milked on and around the full moon had greater milking success and produced relatively diverse venom profile when compared to other phases. If milking success correlates with activity, this means that the snails are most active during a full moon, a statement further backed up by our unpublished egg laying data. Snails milked during the new moon fed relatively reluctantly but produced the most diverse milked venoms. These new moon data are somewhat surprising given that the diversity aligns well with milking rate during all other phases. Perhaps the snail uses the new moon to rest and produce venom, resulting in milkings that consist of unfinished peptides or milked venom production byproducts. Whatever the case may be the change in milked venom probably reflects a change in lifestyle during certain moon phases. Lunar phase is a particularly interesting factor to investigate in a laboratory setting with no visual cues for the snails to check. Notably, all six Conus striatus egg laying events and three Conus textile events took place within the 5 days following the

55

full moon. Three of the Conus striatus events took place after over a year in captivity. As noted above, the snails seemed to lose track of appropriate breeding season after a year in captivity and started laying eggs in December, a full 5 months outside of the recorded norm. Therefore is seems that the snails are getting some kind of non-visual lunar reinforcement without any seasonal stimuli.

Time in Captivity

One of the more important logistical focuses of this study pertained to the captive production of milked venom components from captive Conus specimens. The specimens in this study decreased milked venom volumetric output by an average 5 uL between the initial ten milkings post capture and the last ten milkings in the forty-week study. This is probably due to insufficient husbandry techniques and more specifically, nutrition. Diet studies of wild specimens so far have mostly observed various species of marine goby and blenny in the dietary tracts of Conus striatus, neither of which was offered to the captive specimens presented here. Furthermore, dietary studies have neglected to comment on feeding frequency and food item size. For the purposes of this study we chose to offer one fish on a convenient weekly basis. If the snails feed more frequently in the wild, insufficient nutrition may be to blame for decreased venom production. Conversely, diminishing milked venom volumes might also occur if the venom gland was unable to keep up with an accelerated rate of captive feeding. Ideally, the snails should be feeding 100% of the time. Our overall feeding success rate was only 78% indicating that some aspect of the feeding was off. Our less than ideal feed rate may indicate that the snails were simply overfed and not hungry. Finally, the food items offered in this study varied wildly in size based solely on the available supply from captive breeders. Thus a decrease in milked venom production may be a result of small food items not sufficiently satiating the snails’ needs. A proposed study on the diet of Conus striatus should look at the correlation between feeding and milked venom production from three aspects: feeding preference, frequency and meal size. The evolutionary forces driving the variability of milked venom in Conus striatus

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are still a mystery. We have seen that the milked venom varies to some degree with size, sex, season, and moon phase. All of these factors probably affect the overall metabolism, so it is possible that milked venom varies most with the snail’s activity or metabolic level. However, the fact that the milked venom volume decreased with time in a captive, stable environment is evidence that activity level is not the whole story. This wide range of peptides in C. striatus’ chemical arsenal may help it prey on a wide range of species so it may exploit new environments. Perhaps the decrease in milked venom volume noted over time in the lab was a slow adaptation to a captive diet. This would be further backed by the radical variations in composition observed in the snails that were fed a pill diet. Future work should look at the degree of variability in composition of generalist feeding snails, such as Conus californicus that feeds on fish, worms and snails, in comparison to more specific feeding snails such as Conus obscurus that only feeds on fish. If adaptability is the driving force behind milked venom variation, a generalist snail should have more variable venom to take advantage of a more diverse menu.

Morphological Variants

The study that determined the validity of Conus striatus oahuensis was unable to find any examples of shells with morphological characteristics representative of both subspecies (Tucker, et al., 2011). The current study, which reviewed more specimens (n=25) than were examined in the former paper, found none as well, lending credibility to the morphological observations leading to the subspecies determination. However, since separate species rarely if ever express the same peptide, one would expect that two subspecies, presumably in the process of speciating, would produce at least some differing venom peptides. I was unable to find any venom parameters on which the two subspecies differed. Thus, the differences between the two variants seem to be morphology alone and not related to venom production.

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Pregnancy

The idea that a gravid snail will produce different milked venom starts to examine the purpose of venom production. If the reason for venom is prey capture and egg- bearing snails need to eat as much as other snails, then venom production should not be much different between the two groups. In fact, gravid female snails maintain steady milked venom production before, during and after ovum depositing in terms of both milked venom volume produced and milked venom diversity. One reason a female snail might want to alter milked venom production is if the venom took on more of a defensive role. Parenting is common in cone snails. A pair of adults, for example, usually accompanies egg clutches found in the wild. “Egg guarding,” defined here as exposing the proboscis in the presence of a disturbance, has been observed in the lab during an unrelated study in both Conus textile and Conus striatus. However, with no milked venom variables altering in response to egg-laying events, there is no evidence to suggest that the snails are tailor making their milked venom to suit these special circumstances.

Death

Two snails died during this study. Acute hypoxia was the likely cause of the first (snail 9-3) when the snails’ intake water valves were left off overnight. The snail lingered for only a few hours the next day before it was euthanized. No milkings were obtained while the snail was in the process of dying. The second (snail 5-1) produced venom until its last feeding that took place two weeks prior to expiration when St4082 concentration decreased precipitously and never recovered and the number of dominant RP-HPLC peaks doubled. Aside from the skipped feeding one week prior to death, handlers noticed no indications that the animal was suffering from any lasting maladies. With mollusk disease being a poorly studied field and few symptoms from which to draw conclusions, no cause of death can be determined from the current evidence. Whatever the malady, the venom showed no signs of illness until three weeks prior to death.

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Pill Diet

This study suggests that changing the snails’ diet from fish to fish pills irreversibly altered the snails’ physiology. They produced less overall milked venom, fed with less regularity, and produced less of the peptide St4082; the milked venom that was produced was more diverse in peptides and was much more variable but less consistent (Figure 42). One possibility for this change is that this response mimics the stimuli of moving into a new environment with novel food availability. The stressed snails may limit costly venom production to conserve nutritional resources while producing a more diverse arsenal of toxic peptides with which to assault new prey. Based on pre-pill diet data, the snails selected to take part in this study injected less milked venom volume with elevated concentrations of St4082 and fed with slightly less regularity than the rest of the population. Either the snails may have been adjusting to their captive situation at the start of the pill study or naturally produced anomalous venom. In hindsight, different snails with more stable milked venom profiles should have been utilized for this diet study. Furthermore, all of the snails given a pill diet were in the same tank because it was thought to be easier from a logistical perspective, but this presented a potential bias that wasn’t considered in the planning stages of this study. For future pill diet studies, more thought should be given to randomizing the feeding regime.

Mitigating Ecological Impacts through Cone Snail Husbandry

In the 18 months encompassing this study of biosustainable milked venom collection, we sampled 1,217 milkings totaling 41 mL of Conus striatus venom from 27 snails. Two of the twenty-seven snails died during this time. The same number of samples using traditional venom collection methods would have sacrificed 1,217 wild snails. Wild snails may have lacked the chemical diversity and complexity of their milked venom because they had not been exposed to the stresses of captivity.

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General Notes

In a related study, some specimens of Conus consor frequently failed to include conopeptides in their injected venom, injecting simply fluid comparable in volume to a normal sting (Dutertre, et al., 2010). This phenomenon seemed to show up over time in captivity and may be the result of nutrient conservation or another unknown property of venom production. Similar venom-less injections represented 21% of our total sample pool. Unlike the former study, the blank injections were not correlated with extended stays in captivity. In many cases, snails would produce a single blank injection one week followed by many weeks of normal, venomous injections. In the case of snail 8-1, the milking at T=0 was a blank injection, after which it started producing normal peptide- containing samples after. Therefore, in the case of Conus striatus, blank injections do not seem to be a response to captive diets. Thus the mechanism behind the production of blank injections is as yet unknown and requires further study for a better understanding. A study comparing milked and duct venom found only 73 peptides (48% of milked venom components) to be common between milked and duct venom (Biass, et al., 2009). After comparing 19 venom ducts to 67 milkings collected over 3 months from 6 live specimens, they suggested that the 48% of milk venom components not represented in the duct might come from organs such as the salivary gland. A look at the baseline profiles from this study (Figure 43) suggests that 6 live specimens is too small of a sample to characterize the total venom capacity of a species and significantly underrepresents the percentage of peptides produced by organs other than the venom duct. This initial look into the external factors that affect milked venom production was intended to gain at best a basic understanding of venom variation in Conus striatus. Nonetheless, the following are educated recommendations for feed times and animal choice based on experiences in this study. Optimal milking success can be expected from fresh-caught snails offered fish during the full moon in the months of June and December. Maximal volumetric milked venom output occurred with large snails fed a diet of fish within the first ten weeks of captivity during the months of May, June, July and October. Maximal venom diversity and St4082 production coincided with large male

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snails fed an altered diet during the new and full moons in October. Furthermore, it was shown that morphological variations do not always translate into milked venom differences. Many changes in milked venom production could not be accounted for leaving ample variables for follow up studies. However, the methods of milk venom collection outlined herein are appropriate for this kind of work with regard to both experimental rigor and environmental responsibility.

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Posters

Milisen, Jeffrey, Jon-Paul Bingham. “Maximizing Conopeptide Production through Improved Snail Farming.” University of Hawai‘i College of Tropical Agriculture and Human Resources Symopsium. Manoa, HI. (March, 2012)

Milisen, Jeffrey, Elizabeth Mahi, and Jon-Paul Bingham. “Pavlovian Conovenomics: Venom Variation in Conus striatus.” University of Hawai‘i College of Tropical Agriculture and Human Resources Symposium. Manoa, HI. (March 2011)

Norschow, Alec, Jeff Milisen, Jon-Paul Bingham. “Developmental Analysis through Protein Quantification of Conus striatus veligers.” University of Hawai‘i College of Tropical Agriculture and Human Resources Symposium. Manoa, HI. (March 2011)

Milisen, Jeffrey, Alec Nordschow, Aileen Maldonado, and Jon-Paul Bingham. “Aquaculture and Protein Quantification of Conus striatus veliger.” University of Hawai‘i College of Tropical Agriculture and Human Resources Symposium. Manoa, HI. (March, 2010)

Leong, Jessica, Joycelyn Chun, Jeffrey Milisen, Jason Biggs, Cabrini Rivera, Majdouline LeRoy, and Jon-Paul Bingham. “Venom differentiation within the milked venom of Conus striatus.” University of Hawai‘i College of Tropical Agriculture and Human Resources Symposium. Manoa, HI. (March, 2010)

Presentations

Milisen, Jeffrey. “Conopeptide Production through Biosustainable Snail Farming.” Molecular Biosciences and Bioengineering Master Thesis Defense. Honolulu, HI. (October, 2012)

Milisen, Jeffrey. “Conopeptide Production through Biosustainable Snail Farming.” Molecular Biosciences and Bioengineering Master Thesis Proposal Seminar. Honolulu, HI. (January, 2012)

Milisen, Jeffrey. “Aquaculture of Conus striatus: An ongoing study.” Hawai‘ian Malacological Society Key Speaker. Honolulu, HI. (June, 2010)

Mentored: Norschow, Alec. “Developmental analysis through protein quantification of Conus striatus eggs.” Marine Option Program Symposium. Hilo, HI. (April, 2010) -Best Research Award.

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