Growth Rate, Prey Preference, and Feeding Rate of Evasterias Troschelii

Growth rate, prey preference, and feeding rate of Evasterias troschelii

Carla Di Filippo

A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE REQUIREMENTS FOR

THE DEGREE OF BACHELOR OF SCIENCE IN LAND AND FOOD SYSTEMS

Applied biology program (honours)

FACULTY OF LAND AND FOOD SYSTEMS

THE UNIVERSITY OF BRITISH COLUMBIA

October 2017

We accept this thesis as conforming to the required standard

______

Abstract

The outbreak of sea star wasting disease along the Pacific Northwest in 2013 is associated with a shift in sea star community structure, with the normally abundant Pisaster ochraceus decreasing in abundance relative to Evasterias troschelii in and around Vancouver,

BC. This change in relative abundance could directly affect the abundance and distribution of important prey species such as Mytilus trossulus (mussels) and Balanus glandula (barnacles), with Mytilus observed to exclude and decrease species richness of additional prey species in the intertidal. Previous research indicates that Pisaster prefers mussels over barnacles, however with

Evasterias being understudied, these preferences are unknown. The goal of this research project was to improve our understanding of Evasterias prey preferences, feeding rates, and whether diet affects sea star growth. We conducted a lab experiment using organisms collected from Burrard

Inlet, BC, and provided Evasterias with a diet of mussels, barnacles, or both, and recorded prey consumption and predator growth rate. Additionally, we conducted a feeding rate experiment between Evasterias and Pisaster. Results show the growth rate of Evasterias was higher when mussels were available. However, the proportion of mussels or barnacles consumed did not differ when sea stars were presented with only one or both prey species, suggesting that they did not have a strong prey preference. The number of mussels consumed overall was lower than that of barnacles, but tissue mass consumed was higher in mussels than that of barnacles. The feeding rates between Pisaster and Evasterias showed similarity. Although the strength of dietary preferences of Evasterias and Pisaster may differ, our results suggest that Evasterias may nevertheless play a similar ecological role when it becomes abundant on rocky shores.

Table of contents:

Page

List of Figures …………………………………………………………………………………. 5.

Acknowledgments ……………………………………………………………………………. 6.

Introduction ………………………………………………………………………………….... 7.

1.1. Climate change & disease ……………………………………………………………. 7.

1.2. Role in the intertidal - Pisaster ochraceus & Evasterias troschelii …………………. 8.

1.3. Feeding ecology ……………………………………………………………………… 10.

1.3.i. Prey preference …………………………………………………………………. 11.

1.3.ii. Effects of diet on growth ………………………………………………………. 15.

1.3.iii. Feeding rates …………………………………………………………………... 15.

1.4 Research objectives and hypotheses…………………………………………………. 17.

2. Materials and Methods …………………………………………………………………... 19.

2.1. Animal collection ……………………………………………………………………. 19.

2.2. Feeding experiment 1 – Evasterias …………………………………………………. 20.

2.3. Feeding experiment 2 – Evasterias …………………………………………………. 24.

2.4. Feeding rate experiment – Evasterias & Pisaster ………………………………….. 25.

2.5. Tissue consumption analysis ……………………………………………………….. 25.

2.6. Growth, prey preference, and feeding rate analysis ………………………………. 28.

3. Results …………………………………………………………………………………….. 30.

3.1. Growth of seastars …………………………………………………………………... 30.

3.2. Consumption – Quantity & Prey tissue ……………………………………………. 31.

3.3. Feeding rate ………………………………………………………………………….. 34.

4. Discussion ……………………………………………………………………………….... 37.

4.1. Growth rate ………………………………………………………………………….. 37.

4.2. Tissue consumption …………………………………………………………………. 38.

4.3. Feeding rate ………………………………………………………………………….. 40.

4.4. Applicability to the field – Role of Evasterias as a Pisaster substitute ………….... 41.

4.5. Future changes to the intertidal & Management implications …………………… 42.

5. Literature Cited ………………………………………………………………………….. 45.

List of Figures:

Figure 1. Map of collection sites at Stanley Park, Vancouver, BC, Canada …...... 19.

Figure 2. Laboratory setup of feeding experiments ……...... 20.

Figure 3. The impacts of diet on the cumulative growth rates for Evasterias from feeding

experiments 1 and 2 ……...... 30.

Figure 4.a., b. The daily consumption rate of barnacles and mussels for feeding

experiment 2 ...... 31.

Figure 5. The weekly proportion of barnacles consumed from feeding experiment 2 ...... 32.

Figure 6.a., b. Graphs displaying the relationship between prey size and dry tissue weight for

mussels and barnacles ………………………………………………………………. 33.

Figure 7. Total tissue consumption of mussels and barnacles by Evasterias in

feeding experiment 2 ...... 34.

Figure 8. The daily consumption rate of mussels by Evasterias and Pisaster from

feeding rate experiment ……………………………………………………………... 35.

Figure 9. The preferred mussel sizes by Evasterias and Pisaster from

feeding rate experiment ……………………………………………………………... 36.

Acknowledgments:

I thank my supervisor Dr. Chris Harley for the research opportunity, resources, guidance and support throughout the entirety of this project that otherwise would not have been possible to complete. Colin MacLeod for his help in seawater chemistry, seawater system knowledge, laboratory resources, and overall guidance and support during the tougher times of my writing process. Sharon Kay for her past knowledge and advice on seastar species in the Burrard Inlet.

Cassandra Konecny for her advice and support in creating the map for collection sites. Angela

Stevenson for her advice, feedback, and constant support. The Harley lab for their unbelievable positive presence, acceptance, advice, and willingness to help. Dr. Wayne Goodey for supplies and resources. The Stanley Park Board and Royal Vancouver Yacht Club for allowing me access to the collection sites and organisms. Ocean Leaders for providing me with funding to conduct my research.

1. Introduction:

1.1 Climate change & disease

A changing climate caused by increasing CO2 emissions has contributed to altering surface temperatures on a global scale (Solomon, 2007). Over the past one hundred years, it is estimated that global temperatures have increased by 0.74°C ± 0.18°C, with future predictions indicating a rise of 2°C to 6.4°C by the year 2099 (Sokolov et al., 2009; Solomon, 2007). This increase in global surface temperature will consequently result in an increase in ocean temperature, believed to be an important factor driving disease outbreaks in many marine taxa

(Harvell et al., 1999; Lester et al., 2007; Ward and Lafferty, 2004). Specifically, increased disease frequency and intensity have occurred in vertebrates (mammals, turtles, fish), invertebrates (corals, crustaceans, echinoderms), and plants (seagrasses) (Karvonen et al., 2010;

Lafferty et al., 2004; Ward and Lafferty, 2004). It has been proposed that an explanation for this phenomena is, 1) pathogen growth rates and fitness have a higher probability of increase at higher temperatures, 2) climate change shows an increase range expansion for pathogens, and 3) hosts exhibiting heat stress have an increase in susceptibility to disease (Bates et al., 2009;

Harvell et al., 2002). Some examples of disease outbreak can be observed in coral reefs, with a dramatic global increase in the severity of coral bleaching in 1997 to 1998 coinciding with high

El Nin͂ o temperatures (Harvell et al., 1999). Additionally, a study conducted on the prevalence of infection on two fish farms in northern Finland from 1986 to 2006 found an increase in disease prevalence during summer periods of increased water temperature for some, but not all, diseases observed (Karvonen et al., 2010). The latter example emphasizes that disease prevalence is not only impacted by local environmental conditions, but additionally by the biology of the disease.

An example of interest showing increased disease frequency and intensity in invertebrates, specifically within echinoderms, are sea stars (class Asteroidea). Sea stars showed extensive mortality during 2013 along the northeastern Pacific Coast, in association with Sea

Star Wasting Disease (SSWD) (Kohl et al., 2016). SSWD causes an increased frequency in body lesions, loss of appendages, behavioural changes, and consequently, death of the individual, observed as rapid degradation (“melting”) (Hewson et al., 2014). Mass die-offs of the sunflower star Pycnopodia helianthoides, provided the first large scale documentation of the outbreak on the coasts of British Columbia, California, and Washington, with other species following soon after (Kohl et al., 2016). The range of present day outbreaks observe to encompass the entire

Pacific coast of North America, from Baja California, MX to Alaska, and USA, impacting at least 20 known species and multiple genera (Kohl et al., 2016). However, not all species documented have been impacted to the same severity in population declines. The blood star,

Henricia levuiscula, is susceptible to the disease but has shown little population decline and is still common throughout the Salish Sea (Kohl et al., 2016). In contrast, the extensively documented ochre star, Pisaster ochraceus, was once very common along the West coast of

North America prior to the outbreak in 2013, but has now become uncommon or absent from many sites within its former range (Kohl et al., 2016). To date, SSWD is one of the largest epidemics in marine ecosystems ever recorded (Menge et al., 2016).

1.2 Role in the intertidal community – Pisaster ochraceus & Evasterias troschelii

The impacts of disease in Asteroidea populations are of particular importance in showing major shifts in ecosystem state and species richness when observed in conjunction with a keystone species (Bates et al., 2009; Monaco et al., 2014). Keystone predators can be defined as

8 consumers that can disproportionally impact their ecosystem relative to their abundance or are capable of removing competitive dominants (Duggins, 1980; Fauth and Resetarits, 1991; Paine,

1966). Pisaster ochraceus (hereafter, Pisaster) has been observed as a quintessential keystone predator for the rocky-intertidal habitat, showing large impacts on intertidal community assemblages through predation on a preferred prey species, mussels, Mytilus californianus

(Menge et al., 2016; Paine, 1966). A study conducted on the range limits of Mytilus californianus in the presence and absence of Pisaster found that upon removal of Pisaster,

Mytilus habitat range expanded, with an increase in vertical distance of 0.85m and 1.93m over several years compared to control sites that showed no expansion (Paine, 1974). Their study location at Mukkaw Bay showed mussels to exclude over 25 species of invertebrates and benthic algae from occupying the same substratum when prey-pressure from Pisaster was absent, indicating the potential enhancement of coexistence and species richness when the keystone predator Pisaster is present (Paine, 1974). This is additionally supported by previous findings that suggest sea stars contribute to determining the lower limits of mussel and barnacle beds, which can cause exclusion of other prey species (Mossop and Bessie, 1921; Paris, 1960).

With a strong decline in Pisaster abundance, the observed benefits to prey coexistence and species richness may be lost unless a species with a similar keystone role can take its place.

Evidence suggests that subordinate predators, such as whelks (Nucella ostrino, N. canaliculate) also found along the coast, could not replicate a similar role on the rocky-intertidal habitat when looking at their populations before the peak of the SSWD outbreak (Cerny-Chipman et al.,

2017). Whelks had a weak effect in limiting mussel range expansion and facilitating prey species recruitment, indicating weak short-term impacts on prey communities after a decline in abundance for the keystone predator Pisaster (Cerny-Chipman et al., 2017). A species that may

9 be better suited as a substitute for Pisaster’s role, also found within Asteroidea, is Evasterias troschelii (hereafter, Evasterias). Evasterias overlaps the habitat range of Pisaster, found along the northeastern Pacific Coast from Alaska to California in Puget Sound, the San Juan Islands, and along the Strait of Juan de Fuca, but has rare occurrence on exposed outer coasts and south of Washington’s Puget Sound (Lambert, 2000; Mauzey et al., 1968; Rogers and Elliott, 2013).

Within British Columbia, both sea stars can be found in the Burrard Inlet, where SSWD is present (“Pacific Rocky Intertidal Monitoring,” 2017). However, even with Evasterias among the 20 species susceptible to SSWD, Evasterias shows decreased susceptibility and transmission compared to that of Pisaster (Kay, 2017). This has resulted in an observable increase in

Evasterias abundance within the Burrard Inlet, and overtime may show a higher abundance than that of the currently dominant Pisaster (Kay, 2017).

1.3 Feeding ecology

With the possibility of Evasterias having a higher abundance and similar keystone effects as Pisaster on prey species, it is important to better understand Evasterias’s basic feeding ecology, which is well understudied (Christensen, 1957; Mauzey et al., 1968; Young, 1984).

Besides feeding behaviour (involving how Evasterias consumes its prey) and diet, our understanding of 1) prey preference, 2) impacts of diet on growth, and 3) feeding rates are not well understood. Factors that can affect these three ecological parameters and should be taken into consideration when understanding a species out in the field are habitat location, prey available for consumption, previous prey experience (including impacts of prior diets and exposure to prey defenses), seasonality, and reproduction (Feder, 1970; Landenberger, 1968;

Mauzey, 1966; Mauzey et al., 1968; Young, 1984). The next section will discuss these factors in

10 full with regards to the three ecological parameters, in hopes of illustrating the similarities or differences in feeding ecology between Pisaster and Evasterias.

1.3.i. Prey preference

According to Mauzey et al., Pisaster and Evasterias are most common on rock or cobble substratum, although they can also be found on sand (1968). Their diet generally consists of barnacles, tunicates, Terebratalia transversa, bivalves, gastropods and polychaetes, but the highest abundance found within the rock or cobble substratum are various mussel and barnacle species, which are observed to make up approximately 96% of these sea stars’ diet (Mauzey et al., 1968). However, between different study sites, a sea star’s preferred prey (defined here as the prey species most commonly consumed) was largely correlated with differences in the abundance of the prey species available (Mauzey et al., 1968). This finding is also supported by observations from Feder on Pisaster, whereby different prey species abundances at various observation sites showed differences in the prey species most commonly consumed (1959). To further expand on the question as to whether Pisaster shows prey preference, a study conducted by Landenberger addressed multiple feeding ecology concepts, one of which described

Pisaster’s prey preference when given the choice to feed on various prey species in a laboratory setting (1968). He found that among the nine species presented (three mussels, three snails, and three chitons), bay mussel (Mytilus edulis) and California mussel (Mytilus californianus) were most preferred, showing the highest percentage eaten when other prey species were provided in equal numbers (Landenberger, 1968). Even when testing for possible confounding factors such as accessibility and density of prey species, Pisaster still shows statistical significance in favourability towards mussels, although selectivity was lower (Landenberger, 1968). From

Mauzey et al., a laboratory experiment conducted on Evasterias showed preference for barnacles

(Balanus glandula) compared to various clam, scallop, tunicate, and chiton species, but without directly including mussels into the choices available, it remains unclear if Evasterias would have a higher preference for mussels similar to that of Pisaster (1968). This is of particular importance as mussels and barnacles are the prey species that make up a large majority of Pisaster and

Evasterias’s diet, and if Evasterias did show similar preference for mussels, it may contribute a similar role as Pisaster in controlling mussel ranges and prey species richness.

With a large abundance of mussels available in the surrounding substratum most common to Pisaster and Evasterias, the choice in mussel preference may be a result of familiarity with the species rather than some inherent preference. Studies conducted by Tinbergen on insectivorous birds addressed the idea of a “search image”, whereby when one prey species becomes highly common, a bird will choose this prey species over others that it encounters, even if the alternatives may be as equally beneficial (Tinbergen and Klomp, 1960). Once the search image is formed, it is observed that prey selectivity is drastically altered, and over a long period of time selectivity observed can exhibit past abundances of prey species (Landenberger, 1968). For sea stars, it is suggested that it is not necessarily a search image that is being formed (implying a visual cue for identification), but rather “olfactory conditioning” in which scent is the primary means of identification (Dale, 1999; Landenberger, 1968). In Landenberger’s experiment, he found that Pisaster did not alter its selectivity when alternative prey were made abundant at various group sizes, with preferences for mussels remaining consistent (1968). However, he does acknowledge that in a laboratory setting, the time period for experiments may be too short to overcome olfactory conditioning from abundance and detect some inherit preference

(Landenberger, 1968). To address this question, Landenberger tested the idea that predators can

12 be conditioned to a specific prey, thereby altering their olfactory conditioning and selectivity

(1968). He presented Pisaster with only turban snails for approximately 3 months, in which after he ran a 3 week experiment with the control containing equal numbers of snails and a less preferred prey species, chitons, and the treatment containing a higher number of snails compared to the hypothesised preferred prey species, mussels (Landenberger, 1968). The control involving chitons showed Pisaster to consume a larger percentage of snails, supporting previous findings.

However, the treatment containing mussels showed that with 30 snails and 10 mussels available, the initial conditioning effects had disappeared by the second week, consuming a larger percentage of mussels compared to snails (Landenberger, 1968). This suggests a change in searching behaviour, and that Pisaster’s preference for mussels is not solely driven by the prominence of mussels in the rocky substrate (Landenberger, 1968). This may also be relevant for Evastarias, showing high similarity in habitat preference for rocky substrate and use of chemosensors to detect scent (Dale, 1999; Mauzey et al., 1968).

Alongside the idea that Pisaster and Evasterias have the potential to be influenced by past prey experiences, current prey experiences (involving prey defenses) can also contribute to selectivity and preference. Gastropods, although part of the sea star diet, are consumed in vastly lower numbers than that of mussels and barnacles (Feder, 1959; Mauzey et al., 1968). A possible explanation for this could be the speed at which gastropods can escape and evade sea star predation (Feder, 1959). Sea stars feed by enveloping their prey with their arms, using mechanical force from the longitudinal musculature of their tube feet and possible chemical agent found in their stomachs (proposed to help relax muscle tissue of their prey, although discrepancy is found within the literature) (Christensen, 1957; Feder, 1955). This creates an opening, whereby the eversion of their stomachs on the soft tissue of their prey allows for

13 digestion (Christensen, 1957; Feder, 1955). A study conducted by Christensen found that the mean speed of opening a mussel by Evasterias was approximately 1mm per minute, taking about

20mins to fully open a mussel (1957). This means that a sea stars slow movement and consumption rate can show the speed of gastropods to be unfavourable compared to the more sessile prey of mussels and barnacles. When directly comparing the favoured prey of mussels and barnacles, mussels may show lower favourability due to increased byssus production (a thread filament used for the attachment onto substrate) caused by predatory pressure (Caro et al.,

2008). This increased attachment strength proves challenging for sea stars to orient the hinge of the mussel away from the mouth and have ideal leverage to pull apart the opening of the shell with their tube feet (Caro et al., 2008; Christensen, 1957). This can increase handling time and reduce the profitability of the prey. For barnacles, increased predation pressure can result in phenotypic plasticity, whereby some exhibit a higher frequency of the predator-resistant form

(having a bend at the lateral plates showing a “hood” like structure) that makes the opening less accessible to predators compared to their typical cone shaped form (Lively et al., 2000).

However, the ability for sea stars to evert their stomachs a distance of half their arm length and into openings as little as 0.1mm (possibly increasing the likelihood of bending around the “hood” and reaching the opening)(Christensen, 1957), could show the predator-resistant form of barnacles to be of little use compared to that of the mussels’ increased byssus thread production.

This does not support previous findings suggesting that mussels are of higher preference than barnacles for Pisaster (and hypothesized for Evasterias), however other factors, such as the impacts of prey tissue and energy availability on the growth of sea stars, may play a larger role compared to these mechanisms of prey defense.

1.3.ii. Effects of diet on growth

Field observations on Pisaster in the Monterey Bay, California shows the mean body size of sea stars feeding primarily on mussels to be greater than that of sea stars feeding primarily on acorn barnacles (Feder, 1970). A possible explanation could be that the nutritional (calorie) basis is higher in mussels than that of barnacles, possibly accounted for in the size difference between the prey species (Paine, 1966a). Mussels on average tend to be larger than that of barnacles, showing higher tissue mass available for consumption (Lively et al., 2000; Mcclintock and

Robnett, 1986). This can outweigh the increased handling time associated with orienting, opening, and consuming mussel tissue, and result in higher profitability compared to that of barnacles. Although lab experiments testing the effects of various prey species on the growth of sea stars is understudied, we would hypothesize that sea stars would show similar growth patterns found in the field. Additionally, if Evasterias were to prefer mussels over barnacles and showed similar feeding rates, they could exhibit increased growth rates similar to that of

Pisaster.

1.3.iii. Feeding rates

Feeding rates have been studied for Pisaster when provided a diet of mussels, however our understanding of their feeding rates on barnacles, and the feeding rates of Evasterias on either of these prey species, is lacking. Even with this gap in knowledge, it is important to understand what could impact feeding rates and cause variations in preference between the two prey species, and variations in feeding rates between sea stars.

It has previously been discussed that the handling time would differ between mussels and barnacles, which contributes to the number of prey available for consumption. A food web

15 constructed by Paine on the number of prey items and calories consumed by Pisaster in Mukkaw

Bay, showed that from a diet of chitons, limpets, bivalves, acorn barnacles, and mitella, acorn barnacles comprised the majority of their diet at 63%, while bivalves came next at only 27%

(1966). However, 12% of Pisaster’s calorie intake came from barnacles, while 37%, from mussels (Paine, 1966a). These results suggests higher feeding rates for barnacles than that of mussels, but the contrast in nutritional value could be the driving factor behind the favourability and preference for mussels.

In addition to the differences in tissue availability between prey species, seasonality and reproduction can also show to alter dietary selection and feeding rates. Throughout the year,

Pisaster changes its primary prey consumed depending on the season (Mauzey, 1966). During the summer when Pisaster are scattered individually across the intertidal from approximately plus 5 feet to minus 2 feet tide level, they have a wide exposure to mussels (Mauzey, 1966).

However, during the winter the range of Pisaster shifts to approximately zero to minus 4 feet tide level (out of the range of mussels), exhibiting a clumping behaviour in crevices and other protected areas (Mauzey, 1966). Found in these crevices and tide levels are chitons, which comprise the majority of their diet during this time (Mauzey, 1966). This change in location, aggregation behaviour and prey species availability could all contribute to lower feeding rates observed by Mauzey on various dive expeditions, where less than 5% of individual Pisaster were observed feeding in January and February compared to 60-80% during July and August (1966).

For Evasterias, they may exhibit the same shift in diet, showing similar habitat preferences and possibly changes in tide level during the winter and summer months (Rogers and Elliott, 2013).

This would also lead to decreased feeding rates during the winter months, which is important to consider during laboratory experimentation and the time of year trails are conducted.

A possible explanation for the increase in number of individuals feeding during the summer months could be due to the reproductive cycle of Pisaster. During the summer months

(approximately between May-August) spawning occurs for Pisaster, showing a decrease in gonad index (defined as the gonad mass proportional to body mass) and increase in the number of individuals feeding (Mauzey, 1966). The energy expanded and consequent increase in energy demands during the spawning process could provide one explanation for the higher feeding activity during this time (Mauzey, 1966). To compare this reproductive cycle to that of

Evasterias poses challenging, as there is little documented evidence for their reproductive cycle.

However, with the literature suggesting many similarities between Evasterias and Pisaster (such as habitat range along the coast, substrate preference, diet composition, and internal body structure), one could expect Evasterias to show a similar reproductive cycle, and consequent diet cycle, throughout the year (Christensen, 1957; Kohl et al., 2016; Mauzey et al., 1968; Rogers and

Elliott, 2013).

1.4 Research objectives and hypotheses

In summary, this study aims to address the basics of Evasterias feeding ecology to better understand its future impacts on the intertidal ecosystem. With the keystone predator Pisaster decreasing in abundance from Sea Star Wasting Disease (SSWD), its absence can lead to alterations in the state of the intertidal with regards to local species richness (Kohl et al., 2016;

Paine, 1974). Without the predation pressure of Pisaster on its preferred prey of mussels, mussels show to outcompete other prey species and decrease local species richness (Paine,

1974). With Evasterias showing similar fundamental ecology (such as habitat and diet) to that of

Pisaster, and a decreased frequency of SSWD resulting in a higher abundance, it may pose an

17 adequate substitute for Pisaster’s role as a keystone predator in the intertidal zone. Therefore, we hypothesize that, 1) Evasterias will show similar prey preference to that of Pisaster (preferring mussels over barnacles), 2) the growth rates will be higher for sea stars on a diet containing mussels compared to barnacles, and 3) the feeding rates of Evasterias on mussels will be similar to that of Pisaster.

2. Materials and Methods:

2.1 Animal collection

We conducted this study in the laboratory at the University of British Columbia, collecting Evasterias, mussels (Mytilus trossulus), and barnacles (Balanus glandula) from three different field sites in Stanley Park, Vancouver, British Columbia, Canada (as shown in Figure

1). We collected animals during the summer of 2017 at low tide, in the months of May for the first feeding experiment, and August for the second feeding experiment. The shore habitat was that of rocky intertidal found in the Burrard Inlet. We collected Evasterias (most commonly found under rocks) at site 1 near the “The Girl In Wet Suite” statue (49°30’27.53”N,

-123°12’61.80”W). We scraped small mussels (~7-30 mm) from the sea wall, also found at site

1. We collected larger mussels (~20-60 mm) from the docks at the Royal Vancouver Yacht Club at site 2 (49°29’58.80”N, -123°127173”W), and we found barnacles on small rocks (~50-100mm in diameter) collected from the Vancouver Harbour just beside the Yacht Club at site 3

(49°29’77.03”N, -123°12’50.20”W).

Figure 1 A map of collection sites (created using ArcMap version 10.4) located in Stanley Park, Vancouver, BC, for Evasterias, mussels, and barnacles. We found Evasterias under rocks from site 1 (49°30’27.53”N, -123°12’61.80”W) along with small mussels (~7-30 mm ) scraped from the seawall. We collected larger mussels (~ 20-60 mm) from site 2, found off of the docks at the Royal Vancouver Yacht Club (49°29’58.80”N, -123°127173”W). We collected barnacles from site 3, found on small rocks (~50-100mm) in the Vancouver Harbour (49°29’77.03”N, -123°12’50.20”W).

2.2 Feeding experiment 1 – Evasterias

Sea stars, mussels, and barnacles were maintained submerged in a laboratory recirculating seawater system (13-14°C). We conducted the feeding experiment during the summer months (May and August), during the time Pisaster shows highest consumption rates

(Mauzey, 1966). Sea stars were starved for thirteen days and placed in 2.5 Gallon treatment tanks one day prior to prey exposure, allowing for acclimatization. We conducted 50% water changes every second day to reduce build up of toxic waste and minimize disturbance within the tanks.

We conducted three treatments to address the question of prey preference. The first includes tanks that have both mussels and barnacles present (represented as “mb”), the second and third treatments contain only one prey species, barnacles (represented as “b”) or mussels (represented as “m”). We had five replicates per treatment, resulting in a sample size of n = 15 tanks (as shown in Figure 2).

Figure 2 Laboratory setup of tank numbering represented in red and treatments represented in blue (mb = both mussels and barnacles available, m = only mussels available, and b = only barnacles available). We had five replicates per treatment, resulting in a sample size of n = 15 tanks.

To randomly assign treatments to tanks, we split the fifteen tanks into five groups of three. Within these three tanks, one will become the m, b, and mb treatments, assigned using a stopwatch (iphone 5c). By randomly starting and stopping the stopwatch, and observing the centiseconds to minimize bias, we assigned treatments based on the centisecond number. If the centisecond landed on a 1, 2, or 3, that tank became the treatment containing only mussels. If it landed on a 4, 5, or 6 it was the treatment containing only barnacles. The remaining numbers of

7, 8, or 9 we assigned to the treatment of having both mussels and barnacles present. If the stopwatch landed on a 0, it was discounted, and started again. When one tank was decided the remaining two were assigned through the same process, but numbers 0, 1, 2, 3, and 4 represented the treatment with the lowest assigned number (m = 1, b = 2, mb = 3), and 5, 6, 7, 8, and 9 represented the treatment with the highest assigned number.

Once assigned, we placed the designated prey species in each tank, which provided an optimal size range to minimize uncontrolled bias on prey size preference. However, any mussels smaller than 10mm were not used, as personal observations showed the sizes of our Evasterias to not consume mussels of that size. Additionally, any mussels containing barnacles on their outer shell were only used if the barnacles could be removed to avoid potential influence on mussel consumption. We placed 20 mussels in treatment m, 2 barnacle rocks (ranging from approximately 40-100 barnacles per rock) for treatment b, and 10 mussels and 1 barnacle rock

(approximately 20-50 barnacles) for treatment mb. Providing 20 mussels and approximately 40-

100 barnacles was justified on the idea of providing them with an overabundance of prey

(avoiding any limitations on feeding rate). Our decision to provide only half of the number of individuals for the mb treatment to show prey preference was based around optimal foraging theory and the optimal diet model (Caraco, 1979; Pyke, 1984). The theory states that when food

21 becomes scarce, an individual will exhibit a more specialist behaviour than that of a generalist, choosing a prey item that provides the highest benefits to maximize fitness (Caraco, 1979; Pyke,

1984). Providing Evasterias with fewer individuals of their preferred prey item and increasing search time could result in a selective pressure towards their optimal prey species (in this case, either mussels or barnacles).

We counted mussels everyday during the weekdays, removing the ones consumed

(showing to be open and completely empty of tissue) and replacing them with ones of a similar or equal size. Upon counting, we removed all mussels from the substrate, causing daily breakage of the byssal threads. As discussed in Gooding and Harley, the absence of byssal attachment may show reduced handling time compared to that of sea stars in the field, feeding primarily on attached mussels (2015). However, with the capability of sea stars to evert their stomachs into the small opening of the shell provided by the byssal threads, and consume the entirety of tissue available without having to detach the mussel from the substrate, the absence of byssal threads should not drastically alter handling time and add any additional bias to our experiment

(Gooding and Harley, 2015). If a mussel contained soft tissue that filled approximately more than 25% of the internal space, we recorded it as not fully consumed and discounted it from analyses. We measured the length of every mussel consumed using a non-digital caliper

(measuring the same side for consistency), starting at the hinge and measuring to the longest part of the shell at the opposite end. For barnacles, we counted the starting number of barnacles presented to the sea stars (pushing lightly on the operculum to distinguish between living and already dead individuals) and recounted the rocks once a week, using a quadrat technique with string to improve accuracy and reduce sampling error. This number could then be compared to later recounts, to determine the number of barnacles consumed over time. Barnacles less than

3mm in diameter across the longest part of their operculum were not included in the counts, as personal observations showed that our size of Evasterias did not frequently consume that size of individual. If the number of barnacles remaining on the rock consisted of 20 individuals or less, the rock was replaced. Conducting a control experiment, we observed that without the presence of sea stars, the barnacle count remained consistent, with minor fluctuations in numbers due to sampling error. This shows that a decrease in barnacles in the treatment tanks is a result of sea star feeding.

We placed two sea stars in every treatment tank (size ranges of 9.7-68.8g and 4.59-

7.79cm arm length) to control for possible illness and loss of replicate with consequent death.

For ease of identification we placed a sea star of slightly larger size than the second individual in the same tank. However, upon later consideration we recognized that the size differences within tanks could possibly impact feeding rates of the individuals (with larger sea stars showing to dominate and consume a more prey than that of a smaller individual) (Gooding and Harley,

2015). We weighed sea stars every two weeks to measure growth rate, using a method of blotting them briefly on paper towel to soak up some of the excess superfluous moisture and placing them directly on a weigh scale to record weight (Mettler Toledo, PL601-S). Additionally, we measured arm lengths using a non-digital caliper, starting at the mouth and measuring to the very tip of the arm. However, we did not consistently measure the same arm over time for each individual, showing inaccurate results for growth rate through this method. Thus, we decided to exclude this data from future analyses. This experiment ran for seven weeks due to time constraints, showing the last weighing of sea stars to occur only one week after the previous weighing.

2.3 Feeding experiment 2 - Evasterias

All methodology is similar to the first experiment, with minor changes to mussel replacement and growth measurements of the sea stars to improve reliability and accuracy of the results. Using new individuals collected in August (with size ranges from 2.7-9.8g and 2.7-4.9cm arm length) sea stars, mussels, and barnacles were maintained submerged in a laboratory recirculating seawater system (17°C, 31.9 ppt salinity, ~7.22-8.3 pH). We used photo- identification rather than size difference to distinguish individuals between tanks and provided similarly sized individuals per tank to remove impacts of sea star size on prey consumption. The sea stars were only starved for one day, due to experimental time constraints, before placement into treatment tanks and exposure to prey species. At the end of each week, we replaced not only the mussels eaten, but additionally all mussels in each tank, as suggested by Landenberger

(1968). This minimizes the possibility of confounding factors influencing the desirability of mussels present (Landenberger, 1968).

For barnacle treatments, it was observed that the weekly recounts would cause fewer disturbances in the tanks (such as hand movements) compared to sea stars in treatment tanks containing mussels (showing daily recounts). To address this, we placed our hands in all tanks and slightly moved the water and barnacle rocks around to create similar levels of daily disturbance observed in that of the treatment tanks involving mussels.

For sea star growth, we found the technique of blotting on paper towel before weighing to vary between researchers and alter results, due to the variation in soaking time of the superfluous moisture found in the sea stars. A suggestion made by Feder that we used in our second experiment was to hold the sea stars out of the water until a slight amount of fluid dripped

(approximately 10 seconds), and place them immediately in a container afterwards (1970). We

24 then weighed the sea star with the container and any remaining fluid, improving consistency of measurements. Lastly, we measured arm lengths using a soft tape measure rather than a caliper, as the twisting and bent position of the arms on occasion would pose difficult to straighten and measure accurately. To avoid the error conducted in the first experiment of failing to consistently remeasure the same arm and obtain accurate growth rates, we used the madreporite as a reference marker to consistently remeasure the same arm throughout the experiment. The duration of this experiment lasted eight weeks, to provide a consistent two-week weighing period.

2.4 Feeding rate experiment – Evasterias & Pisaster

Using new individuals (sizes ranging from 1.8-20.9g and 2.3-6.1cm arm length for

Evasterias, and 4.0-13.1g and 2.3-4.7cm arm length for Pisaster) collected in the month of

October, we starved the sea stars for seven days before placement into treatment tanks. We randomly assigned 10 Pisaster and 10 Evasterias to 20 treatment tanks using a coin toss and placed 20 mussels of various sizes (following the same methodology for mussel selection, counting, and replacement from that of the feeding experiments 1 and 2). We recorded the weights of the sea stars and arm length at the start of the experiment to account for any impacts of sea star size on feeding rate (as larger sea stars tend to have higher consumption rates than that of smaller individuals) (Gooding and Harley, 2015). This experiment ran over the span of five days.

2.5 Tissue consumption analysis

To measure the amount of tissue consumed over the entire experiment, we measured the wet and dry weights and sizes of 20 mussels and 20 barnacles ranging from just under the

25 smallest to just over the largest size presented to the sea stars, to create equations relating size of prey species with available tissue mass. We measured the shell length from the sample mussels

(following the same methodology as described for feeding experiments 1 and 2) and extracted the wet tissue to be placed on a weigh tin and weighed on a scale (Mettler Toledo, PB403-

S/Fact). We then placed the tissue in a drying oven for 24hrs and reweighed the samples on the same weigh scale to obtain dry-weight measurements (providing a measurement of tissue availability without excess fluid weight).

We measured the size of barnacles based on volume rather than the diameter of the longest part of their operculum, as personal observations showed operculum diameter to be a poor indicator of tissue quantity inside. Barnacles with similar sized opercula varied in height and base diameter, showing variation in the volume and resulting space for tissue. To account for this, we assumed barnacles to have a truncated cone shape, and measured the longest and shortest sides of the operculum, the longest and shortest sides of the base, and the height of the barnacle to calculate volume based on this shape. The extraction of tissue from the small shells proved challenging to obtain all pieces of wet tissue from the interior of the walls, so we used an alternative method to that of mussels to calculate tissue quantity. We weighed the shells on the scale without any extraction of tissue (showing a weight for the shell plus tissue inside). We then placed all samples in 20ml falcon tubes and poured approximately 20ml of Old Dutch concentrated bleach (sodium hypochlorite concentration of 3%) to dissolve the organic tissue.

We flipped the tubes upside down and right side up twice to remove any air bubbles sticking to the surface of the shells and left the samples untouched for one hour. After this time, we rinsed the shells in distilled water (pouring out the bleach through fine mesh to keep all the shell pieces inside the tube), and repeated the procedure, leaving the samples for an additional hour. We

26 rinsed the samples and placed them back on the same weigh tins, and into the drying oven for

24hrs. After this time, we observed remnants of tissue on the inside of the larger shells and decided to repeat the bleaching and drying procedure one more time. It was of concern that rebleaching the shells may cause some degradation and alter the weights of the smaller shells showing no remnants of tissue. However, weighing the shells before and after the second round of bleaching shows no difference in weights, indicating that the second round of bleaching caused little to no shell degradation. The weights measured after the second round of bleaching were used to calculate the quantity of dry tissue originally inside the shell. By subtracting the dry weight of the shell plus tissue by the weight of the empty shell, we calculated the total dry weight of tissue available.

We plotted the dry mussel tissue weight by the cubed mussel length and the dry barnacle tissue weight by the calculated barnacle volume to formulate equations relating available tissue mass with prey size. The prey size versus tissue mass equations for barnacles showed a linear relationship, but for mussels this relationship showed a convex curve. When considering

Jensen’s inequality, the theory states that when taking the average between two points on a convex curve, the secant line will lie above the curve, overestimating the average (Jensen, 1906).

To account for Jensen’s inequality when calculating the average mussel size consumed, we considered the allometry of the mussel shell. If we cube the length of the mussel shell, we are provided a volume that does not change the relationship between small and large lengths and gives a linear relationship between mussel size and tissue mass. This allometry alleviates the problem caused by Jensen’s inequality and is thus corrected for in further calculations. If needed, taking the cube root of the transformed mussel length will return the value back to a biologically meaningful measurement. The equations generated are presented below:

1) 푑푟푦 푚푢푠푠푒푙 푡𝑖푠푠푢푒 푤푒𝑖𝑔ℎ푡 (𝑔) = 3.1379(10)−6푥 + 0.0015 (1)

2) 푑푟푦 푏푎푟푛푎푐푙푒 푡𝑖푠푠푢푒 푤푒𝑖𝑔ℎ푡 (𝑔) = 0.0001푥 + 0.0037 (2)

We used these equations to calculate total tissue consumed throughout the experiment to see if differences in tissue mass can contribute to any differences in growth rate between the various treatments.

2.6 Growth, prey preference, and feeding rate analysis

Combining the results from feeding experiments 1 and 2, we will conduct a one-way

ANOVA for comparing differences in the weights measured for growth rates between treatment groups, and a Tukey-Kramer test if significance is shown to distinguish significance between the treatments. We excluded arm lengths from the growth rate analysis as prior literature suggests arm length to be a poor indicator of growth, with high levels of variation in length throughout the day (Feder, 1970). Additionally, throughout the experiment, some sea stars were lost due to

SSWD. However, we still included the growth rates of these individuals in the analyses.

Comparisons for daily consumption and proportion consumed will be made only using the data from feeding experiment 2, and only the first four weeks of the experiment. The slight changes in methodology helped control confounding factors and increase accuracy of the results, and the decision to use only the first four weeks was based on many sea stars dying after this time (due to seawater system complications and SSWD), losing multiple replicates for various treatment groups. For daily consumption rates of each prey species, we will conduct a two- sample t-test between the treatments of b and mb, and m and mb, as a test for prey preference.

Additionally, we will compare the proportion of barnacles consumed in treatment b and mb to test for prey preference using another two-sample t-test, to account for the difference in the number of individual prey available that could impact the number of individuals consumed between treatments.

Using the equations (1) and (2), we will calculate the total tissue consumed over the entire eight weeks of feeding experiment 2 to compare if prey preference may be a result of differences in tissue availability between prey species. We will also calculate the amount of dry tissue in the preferred prey size consumed for each prey species to help understand the growth patterns observed for each diet presented.

Lastly, we will analyse the feeding rate experiment using a two-sample t-test to compare daily consumption and mussel size preference between Pisaster and Evasterias, to better understand the similarities or differences between Evasterias and Pisaster feeding ecology.

3. Results:

3.1 Growth of seastars

The one-way ANOVA comparing cumulative growth between the treatments of

barnacles, mussels, and choice shows significance (df = 2, F = 4.23, p = 0.030). A Tukey-Kramer

test shows significance between the treatments with both species available and only barnacles

available, with a p-value of 0.038. All other combinations of treatments were not significant,

although the p-value between mussels and barnacles is close, at 0.070. This trend is supported by

Figure 3, showing barnacles to have the lowest mean cumulative growth rate of 6.80g (sd =

4.69g), while mussels have a higher mean value of 16.75g (sd = 9.42g), and the treatment

containing both species having the highest value only slightly larger than that of mussels, at

18.37g (sd = 9.10g).

a,c

c nu mb er of mu sse ls co nsu Figure 3 The cumulative growth rates of Evasteriasme (representing the combined growth of the two seastars in each tank) from the combined data of feeding experiment 1 and 2. Thed y -axis represents the cumulative growth rate in grams, while the x-axis represents the three treatment levels. “b” representsap the barnacle treatment (mean = 6.80g, sd = 4.69g), “m” represents the mussel treatment (mean = 16.75g, sd = 9.42g), and “mb” represents the treatment where both prey species are available (mean = 18.37g, sd = 9.10g). A one-way ANOVA shows significancepea between treatment levels (df = 2, F = 4.23, p = 0.030), and a Tukey-Kramer test shows the significance to be between “b” andrs “mb” (p = 0.038). ske we 30 d

to the rig ht

3.2 Consumption – Quantity & Prey tissue

A two-sample t-test comparing the number of barnacles consumed between treatment groups, and number of mussels consumed between treatment groups for a four week period shows that there is a significant difference for the number of mussels consumed (df = 8, t = 2.41, p = 0.042), and for the number of barnacles consumed (df = 8, t = 2.86, p = 0.021). The mean values for each treatment are visually represented in Figure 4, with Figure 4.a. showing barnacles to have a higher mean value of sea stars consuming 4.20 individuals per day (sd = 0.72 individuals consumed/day) than that of barnacles when mussels are also present at a mean value of 2.64 individuals per day (sd = 0.99 individuals consumed/day). In Figure 4.b. the number of mussels consumed when it was the only food source is slightly higher at 1.06 individuals per day

(sd = 0.79 individuals consumed/day) compared to when barnacles were also present, at 0.79 individuals per day (sd = 0.21 individuals consumed/day).

(a) (b)

Figure 4 (a) the number of barnacles consumed per day, and (b) the number of mussels consumed per day. The y-axis in both (a) and (b) represents the number of individual prey items eaten in a single day, while the x-axis represents the three treatment groups of “b” = barnacles, “m” = mussels, and “mb” = both prey species available for consumption. The mean values for (a) are 4.20 individuals per day (sd = 0.72 individuals consumed/day) for treatment “b”, and 2.64 individuals per day for treatment “mb” (sd = 0.99 individuals consumed/day). The mean values for (b) are 1.06 individuals per day for treatment “m” (sd = 0.79 individuals consumed/day), and 0.79 individuals per day for treatment “mb” (sd = 0.21 individuals consumed/day). A two- sample t-test shows significance between treatment groups for both (a) and (b).

A two-sample t-test comparing the proportions of barnacles consumed per day shows no significance between treatment groups (df = 8, t = -1.47, p = 0.18). This is visually represented in Figure 5, however there is a slight difference between treatment groups, with the mean proportion of barnacles consumed per day to be slightly higher in the treatment presenting both prey species at 0.25

(sd = 0.069), compared to the diet of only Figure 5 The proportion of barnacles consumed per day, represented on the y-axis, and the two treatment levels of “b” representing barnacles barnacles at 0.33 (sd = 0.10). and “mb” representing both prey species available on the x-axis. The mean value for barnacles is 0.33 (sd = 0.10), and for where both prey We found the average barnacle size species are available it is slightly higher at 0.25 (sd = 0.069). A two- sample t-test shows no significance between treatments. consumed (measured as a volume) to be 203.87mm3 (sd = 228.39mm3), and the average mussel size to be 20.3mm (sd = 8.63mm). Represented in Figure 6.a. and 6. b., we calculated the dry tissue mass for the average mussel size using Eq. (1) to be 0.027g, while using Eq. (2) shows the average barnacle dry tissue mass to be 0.024g. Multiplying the average tissue found in a single barnacle by the average number of barnacles found on each rock, ranging from 50-100mm in diameter, shows roughly each rock to consist of 2.23g of tissue (sd = 1.14g).

0.600 a

0.500

0.400

0.300

0.200 Dry tissue weight (g) tissue Dry weight -6 0.100 y = 3.1379(10) x + 0.0015 R² = 0.94

0.000 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 Cubed mussel length (mm3)

0.100 b 0.090 0.080 0.070 0.060 0.050 0.040

0.030 Dry tissue weight (g) tissue Dry weight 0.020 y = 0.0001x + 0.0037 0.010 R² = 0.88 0.000 0 100 200 300 400 500 600 700 800 900 Barnacle volume (mm3)

Figure 6 Samples from 20 barnacles and 20 mussels of various sizes displaying the relationship between prey size and tissue availability. For both (a) and (b), the y-axis shows the dry tissue weight in grams. The x-axis in (a) shows the cubed length (to account for Jenson’s inequality) of the mussel measured from the hinge to the longest part of the shell (in mm3), while the x-axis in (b) represents the size of the barnacle represented as a volume (in mm3). (a) shows the transformed mussel length to have a linear relationship, with the line of best fit showing y = 3.1379(10)-6x + 0.0015 (R = 0.94), and (b) shows barnacles to have a linear relationship, with the line of best fit showing y = 0.0001x + 0.0037 (R = 0.88).

Multiplying the average tissue mass found in an individual barnacle by the total number of barnacles consumed for each treatment shows that for the duration of the 8 week experiment, a total of 21.38g (sd = 0.55g) of barnacle tissue was consumed in the barnacle treatment. Using the cubed mussel lengths and the equation relating mussel size with tissue weight, we calculated the tissue weight for each mussel length recorded and summed the tissue weights to find a lower total of 10.05g (sd = 0.055g) of mussel tissue was consumed in the mussel treatment (as shown in Figure 7). For the treatment containing both choices, overall tissue consumption for each prey m b species was lower than that of treatments containing an individual prey species, with total barnacle tissue consumption showing 11.09g (sd = 0.35) and mussel tissue showing 6.76g (sd =

0.042).

Mussel tissue consumption 10 Barnacle tissue consumption

5 Total dry tissue mass consumed consumed mass dry Total tissue (g) 0 b mb Treatment

Figure 7 The total tissue consumption by seastars of barnacles and mussels over 8 weeks. The y-axis shows the total dry tissue mass consumed (in grams), while the x-axis shows the treatments of “m” = mussels, “b” = barnacles, and “mb” = both prey species available. The mean value of total mussel tissue consumed in “m” was 10.05g (sd = 0.055g) while tissue consumption was slightly lower in “mb”, at 6.76g (sd = 0.042g). The mean value of total barnacle tissue consumed was much lower than that of mussel tissue, with “b” showing 21.38g (sd = 0.55g), and “mb” with the lowest tissue mass, at 11.09g (sd = 0.35g).

3.3 Feeding rate

The daily number of mussels consumed between Evasterias and Pisaster shows no significance (Two-sample t-test: df = 97, t = 1.65, p = 0.10). However, the data does not fit the

34 assumptions of the test, showing to be not normally distributed (Shapiro-Wilk: p =

1.58x10-08). A histogram plotting the frequency distribution of mussels consumed shows to be skewed to the right, suggesting a ln transformation, or square-root transformation due to the values being counts

(Whitlock and Schluter, 2015). However, both transformations still show a non-normal distribution. The trend observed in Figure 8

Figure 8 The daily consumption of mussels from Evasterias and Pisaster shows the mean number of mussels over a 5 day period. The y-axis shows the number of mussels consumed per day, while the x-axis shows the two species (“e” = Evasterias, and “p” = consumed by Evasterias to be 1.68 Pisaster.) The mean value of mussels consumed by Evasterias is 1.68 individuals per day (sd = 1.49 individuals/day), while for Pisaster it is slightly lower, at 1.22 individuals per day (sd = 1.37 individuals/day). individuals per day (sd = 1.49 individuals/day), while Pisaster shows an almost similar average daily consumption of 1.23 individuals per day (sd = 1.37 individuals/day).

Comparing if there is a difference in size preference of mussels between Evasterias and

Pisaster shows significance (Two-sample t-test: df = 141, t = 4.08, p = 7.41x10-5). However, the data shows the similar problem previously stated for the “daily consumption of mussels” data, whereby the data is not normally distributed. Following the same methods for transformation and corrections (as a histogram plotting the frequency of mussel sizes also appears skewed to the right) shows unsuccessful. The trend observed in Figure 9 shows Evasterias to have a preferred

mussel size similar to that previously stated in the feeding experiment, at 22.17mm (sd =

6.60mm). Pisaster shows a slightly lower preferred mussel size, at 18.38mm (sd = 3.33mm).

Figure 9 The preferred mussel size of Evasterias and Pisaster over a 5 day period. The y-axis shows mussel size preferred while the x-axis shows the species (“e” = Evasterias, and “p” = Pisaster). The mean value of preferred mussels size consumed by Evasterias is 22.17mm (sd = 6.60mm), while for Pisaster it is slightly lower, at 18.39mm (sd = 2.33mm).

4. Discussion:

The growth of Evasterias shows to differ significantly only between a diet of barnacles and a diet consisting of both barnacles and mussels. The overall trend shows Evasterias to have the largest growth rate on a diet consisting of both prey species, then on a diet of only mussels, and the smallest growth rate for the diet of only barnacles. Observing the total number of mussels and barnacles consumed shows that both diets of a single prey species have a larger number of individuals consumed for that prey species compared to the diet consisting of when both prey species are available. However, observing the proportion of barnacles consumed for a diet consisting of only barnacles compared to that of mussels and barnacles, shows no significant difference. The overall tissue consumption for mussels and barnacles shows barnacles to provide more tissue than mussels, even though barnacles contain slightly lower tissue per individual compared to that of mussels. The feeding rate between Evasterias and Pisaster shows no significant difference, showing an almost equal mean value of the daily number of mussels consumed. However, the size preferred between the two species shows Evasterias to prefer slightly larger mussel sizes than that of Pisaster.

4.1 Growth rate

The observations found regarding Evasterias showing a faster growth rate on a diet consisting of mussels compared to that of barnacles (as shown in Figure 3) is supported by prior literature. From the observations conducted by Feder, he found that Pisaster in the Monterey

Bay, California grew larger in size when feeding on a diet primarily consisting of mussels compared to a diet of barnacles (1970). It is interesting to note that in our experiment, the treatments of sea stars exposed to only mussels compared to that of both barnacles and mussels

37 shows almost similar growth rates, suggesting that the sea stars may show a preference in consuming more mussels than barnacles. If preference was shown, we would expect a higher consumption of the favoured prey species and a decrease in consumption of the alternative, resulting in a larger proportion of individuals consumed. However, the proportion of barnacles consumed for a diet consisting of only barnacles to that of having both mussels and barnacles present does not support this idea, with our results showing no significant difference (as illustrated in Figure 5). Thus, there was neither a preference for barnacles or mussels for

Evasterias, contradicting observations found in Mauzey et al. and Landenbergen stating a possible preference for mussels (1968; 1968). An unexplained observation is the slightly higher growth rate in the diet consisting of both prey species. A possible explanation could be the difference in prey defenses and handling time between barnacles and mussels. With the byssal threads of mussels increasing the difficulty for a sea star to provide proper orientation for feeding

(showing increased handling time) (Caro et al., 2008; Christensen, 1957), sea stars may be supplementing their diet with the barnacles in between mussel feedings. This would allow for a more optimal tissue consumption rate, providing an ideal balance between the increased tissue gain (but increased handling time) from mussels, with that of decreased tissue gain (but decreased handling time) from barnacles.

4.2 Tissue consumption

Although differences in prey defense and handling time can provide an explanation for the differences observed in growth rates between various diets, another supporting explanation can be that of the tissue found within each prey species. The total amount of tissue consumed does not support our sea star growth, with mussels showing lower tissue mass consumed than

38 that of barnacles, however experiment methodology, mussel nutrition, and sea star energy expenditure could explain the growth rates observed. Personal observations found sea stars to leave some barnacle tissue behind, possibly showing lower efficiency in prey extraction compared to mussels that were often left with completely empty shells. During calculations, we assumed all barnacle tissue was consumed per individual, which may not accurately represent how sea stars feed on barnacles and over estimate the amount of tissue consumed.

Additionally, the nutrition or digestion of barnacle tissue could be worse than that of mussels. Paine and Menge found barnacle tissue to often be of lower caloric value than that of mussels (Menge, 1972; Paine, 1971). Comparing prey species for the sea star, Leptasterias hexactis, Menge found the mussel, Mytilus edulis to yield 4.27 cal/g per one hour of observation while for the barnacle, Balanus glandula, the caloric yield was approximately three times lower at 1.39 ± 0.47 (Menge, 1972). The study conducted by Paine addressing measurements and application of the caloric value found the mean value of the whole organism of the mussel,

Mytilus californianus, to yield 4600 cal/g, while the whole organism of the barnacle, Balanus cariosus, shows a slightly lower yield of 4520 cal/g (Paine, 1971). This would support our findings of sea stars to show increased growth on a diet of mussels compared to barnacles, even though the average daily consumption of barnacles was higher than that of mussels (as illustrated in Figure 4.a. and 4.b.) Additionally, our results illustrated in Figure 6 of mussels showing higher tissue availability compared to that of barnacles is supported by the findings from Lively et al. and Mcclintock and Robnett, whereby the larger average size of mussels compared to barnacles shows higher tissue biomass available (2000; 1986). This correlates with having a higher number of calories and energy available for consumption that can lead to increased growth for a sea star (Feder, 1970; Paine, 1966b).

Lastly, the energy expenditure during the consumption of prey could vary between prey species. From Figure 4.a. and 4.b. we observed a lower daily consumption of mussels compared to barnacles, suggesting an increased handling time and energy expenditure for mussels. These findings are supported by the prior discussion in the introduction on prey defenses and the potential for barnacle shell shape to be less resistant to predator consumption compared to the shell hinge and byssus threads of mussels (Caro et al., 2008; Christensen, 1957; Lively et al.,

2000). The findings illustrated in Figure 7 provides additional support for the idea that even with the lower number of individual mussels consumed, mussels still provide more tissue per individual than that of a barnacle. This suggests that the increased energy consumption from mussels could compensate for the possible increased energy expenditure in tissue extraction and lead to increased growth.

4.3 Feeding rate

Our results show Evasterias to have a similar number of mussels consumed daily compared to that of Pisaster (as shown in Figure 8), however with the data appearing to not be normally distributed, even after transformations, the results from the statistical analysis may be biased due to a small sample size. Even with this in mind, this preliminary trend still shows promise in Evasterias showing similar feeding rates to that of Pisaster. When comparing if

Evasterias has a similar size preference to that of Pisaster, the results illustrated in Figure 9 suggest that Evasterias has a preference for slightly larger mussels (at an average size of

22.17mm compared to that of 18.39mm for Pisaster). However, this could be on account of the few larger individuals in the Evasterias treatment groups, as an increase in body size has also been shown to correlate with an increase in prey size preference (Gooding and Harley, 2015).

Comparing Evasterias body size with mussel sizes consumed, to Figure 1 in Gooding and

Harley shows similar values of mussel sizes consumed to that of Pisaster. Thus, the slight variation in mussel size preference observed could a be a result of body size, and we would expect that for the larger population of Evasterias in the Burrard Inlet, they too would consume mussels of similar size to that of Pisaster.

4.4 Applicability to the field - Role of Evasterias as a Pisaster substitute

With Evasterias showing similarities in growth to diet, preferred prey size of mussels, and feeding rates to that of Pisaster, all results suggest that Evasterias could substitute Pisaster for the role as a keystone predator with the changing abundance. However, the lack in prey preference for mussels could be a determining factor in fully taking on this role if they are unable to show the same predation pressures. To address predation pressure, it is important to recognize the differences in results associated with laboratory experiments to that of the field. Feder compares field observations for Pisaster feeding in four different areas, to that of the relative abundance of prey species available (Feder, 1959). Prey preference (defined here as the largest number of individuals consumed) was often in relation with the prey abundance, showing higher preference for the highest prey species abundance (Feder, 1959). Most often in the rocky- intertidal, these prey species showed to be mussels and barnacles (Feder, 1959; Mauzey et al.,

1968). Although experiments conducted by Landenberger support the idea that when given a choice, Pisaster would choose mussels more often than any other prey species presented

(regardless of abundance or previous prey conditioning), Feder’s findings on prey abundance cannot be disregarded for its effects in the field on prey choice (Feder, 1959; Landenberger,

1968). Even though from our findings Evasterias did not show preference between barnacles and

41 mussels, their similarity to Pisaster would suggest that Evasterias’s prey preference would still show some variation in consumption relating to prey abundance for various sites. This would give us some predications as to their possible impacts on the location regarding prey species abundance, and possible cascading effects on prey species richness. To understand the full extent as to whether Evasterias could exhibit the same predation pressures as Pisaster on mussels, further investigation into prey choice needs to be conducted. Comparing the proportion of barnacles consumed between treatment types is not the best method for concluding a distinct prey choice, as the number of barnacles for each treatment differed, biasing the proportion consumed in the “mb” treatment that has less available to start with. Additionally, our previous discussion on the impacts of handling time on the number of individuals consumed could show that for the treatment consisting of both prey species available, sea stars are able to supplement their diet with barnacles in addition to their possibly preferred prey species, mussels. A future experiment to be conducted to tease apart Evasterias’s prey preference is that of a y-maze choice experiment, similar to that of ones conducted on sea stars for chemosensory preferences

(Castilla, 1972; Castilla and Crisp, 1973; Slattery et al., 1997). A y-maze involves two arms, or paths, for an animal to choose from that consist of an item of interest at either end. In the case of

Evasterias, providing barnacles in one arm and mussels in the other can allow for clearer results relating to a distinct choice, and thus, prey preference.

4.5 Future changes to the intertidal & Management implications

The potential problem if Evasterias cannot fill the role of a keystone predator and control mussel ranges can be addressed on a local and global scale. On a local scale, subordinate predators and other species of sea stars may need consideration in their suitability to control

42 mussel ranges, to prevent a drastic change in intertidal species richness. A possible subordinate predator found in the intertidal that could also fill this role are whelks, documented to also feed on mussels. However, whelks show a weak effect in limiting mussel range expansions in the absence of Pisaster (Cerny-Chipman et al., 2017). When considering other Asteroids found in similar tidal ranges as that of Evasterias and Pisaster, the blood star, Henricia leviscula

(intertidal species), and leather star, Dermasterias imbricate (subtidal species) show potential to be possible substitutes (Kohl et al., 2016; “Sea Star Wasting Syndrome | MARINe,” n.d.). These two species appear to have lower infection rates to Sea Star Wasting Disease (SSWD) compared to that of Pisaster, potentially showing increased future abundances. However, Henricia is primarily a planktonic feeder, with its diet consisting of plankton and sponges (Mauzey et al.,

1968). Dermasterias, although occasionally found in the intertidal, feeds on a range of invertebrates (such as sponges, tunicates, holothurian (sea cucumbers), chitons, anemones, hydroids, ectoprocts, and various sea stars), and algae (Mauzey et al., 1968). Neither diet of these two species consist of mussels and could help regulate mussel ranges in the absence of Pisaster.

Without Pisaster, mussels have the potential to exclude 25 species of invertebrates and benthic algae from inhabiting the same substratum, potentially decreasing species richness in the intertidal zone and causing large populations of mussels further down in the intertidal (Paine,

1974). Therefore, on a local scale, there may be no adequate substitute for Pisaster and its role as a keystone predator in the intertidal, and the structure of the intertidal may face permanent alterations unless Evasterias proves to be an adequate substitute. Further investigation is needed into the prey preference of Evasterias to come to a finalized conclusion on this matter.

When viewing the problem of losing Pisaster as a keystone predator in the intertidal on a global scale, the issue of SSWD still remains as the driving force behind the declines in Pisaster

43 abundance. An increase in temperature has been linked to increased susceptibility in Asteroid species, making global climate change of key importance in disease frequency (Kohl et al., 2016;

Menge et al., 2016). A rise in CO2 emissions since the industrial revolution has caused global temperatures to increase by 0.74°C ± 0.18°C, and are estimated to rise by another 2°C to 6.4°C by 2099 (Feely et al., 2004; Sokolov et al., 2009; Solomon, 2007). The pH of ocean surface water has decreased by 0.1 units also during the industrial revolution, with a predicted decrease to 0.3 units by 2100 and 0.7 units by 2250 (Caldeira and Wickett, 2003; Feely et al., 2004). In other marine animals, increasing temperatures has been linked to an increase in disease susceptibility, with specific observations for Pisaster being prevalent in SSWD (Harvell et al.,

1999; Karvonen et al., 2010; Kohl et al., 2016). Additionally, with sea stars as calcifying organisms, the decrease in pH causing acidic conditions makes it increasingly difficult for them to maintain their calcium carbonate skeletons (Orr et al., 2005). This acidification has the potential to contribute to SSWD, by creating increased stress on maintaining homeostasis in body temperature and calcification rates, observed to contribute to increased disease susceptibility (Bates et al., 2009; Harvell et al., 2002). Thus, on a global scale, climate change is a large contributor to increasing SSWD in Pisaster, causing a cascade of effects including the loss of this species as keystone predator in the intertidal ecosystem. This will lead to the possibility of a large exclusion of certain invertebrate species due to the dominant competitive pressures of mussels (Paine, 1974). Any management strategies to manage the disease through an aid or cure, would only provide a short-term solution to a growing, future, problem. The intertidal ecosystem is yet another example illustrating the impacts of climate change, and if climate change cannot be regulated and controlled, it will result in the loss in species richness and diversity within this tidal range.

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