FEEDING ADAPTATION of GREEN ABALONE, Haliotis Fulgens, in RELATION to THE

Home , Common periwinkle, Haliotis cracherodii, Haliotis fulgens, Haliotis midae

FEEDING ADAPTATION OF GREEN ABALONE, Haliotis fulgens, IN RELATION TO THE

INVASIVE MACROALGAL SPECIES Sargassum horneri

______

A Thesis

Presented

to the Faculty of

California State University Dominguez Hills

______

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Environmental Science

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Roger Jaquette

Summer 2017

This thesis is dedicated to Leslee, Bill, Jackie and Abigail without whose love and support it

would not have been possible. Thank you for always being there.

ACKNOWLEDGEMENTS

I would like to thank my thesis committee chair, Dr. John Thomlinson, and my thesis committee members, Dr. Brynne Bryan and Dr. Rodrick Hay, for their invaluable assistance and encouragement with this project.

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TABLE OF CONTENTS

PAGE

DEDICATION ...... ii

ACKNOWLEDGEMENTS ...... iii

TABLE OF CONTENTS ...... iv

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

ABSTRACT ...... vii

CHAPTER

1. INTRODUCTION...... 1

2. REVIEW OF LITERATURE ...... 4

California Abalone ...... 4 Green Abalone ...... 5 Abalone Decline...... 6 Abalone Fishery Management in California ...... 9 Abalone Restoration and Recovery Management ...... 11 Sargassum Horneri ...... 13 Sargassum Horneri in North America ...... 15 Sargassum Horneri Removal Efforts...... 16 Non-Indigenous Species ...... 17 Non-Indigenous Seaweeds in California ...... 19 Invasive Seaweed Control by Herbivores ...... 20 Feeding Studies of California Algivorous Marine Species ...... 22

3. METHODOLOGY ...... 26

Species of Study ...... 26 Study Sites ...... 27 Collection ...... 29 Sample Preparation ...... 30 PCR Analysis ...... 30 Statistical Analysis ...... 32

4. RESULTS AND DISCUSSION ...... 34

Results ...... 34 Discussion ...... 40

5. SUMMARY, RECOMMENDATIONS AND CONCLUSIONS ...... 45

Summary ...... 45 Recommendations ...... 46 Conclusions ...... 47

REFERENCES ...... 50

LIST OF TABLES

PAGE

1. Selected Results from Green Abalone Gut Content Samples, 6 May 2016 ...... 36

2. Selected Results from Green Abalone Gut Content Samples, 4 November 2016 ...... 38

LIST OF FIGURES

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1. Range and Median Percent of S. Horneri DNA by Date and Location ...... 39

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ABSTRACT

Sargassum horneri, a non-indigenous species of brown alga from Asia, was first discovered in Southern California waters in 2003. Since then it has spread tenaciously, displacing giant kelp, Macrocystis pyrifera, and other native seaweeds. Abalone have been observed in areas dominated by S. horneri. This study investigates whether green abalone, Haliotis fulgens, have adapted their diet to include the now ubiquitous S. horneri. Green abalone gut content samples taken from S. horneri dominated areas were analyzed using real-time PCR techniques to determine if the animals had recently consumed S. horneri. Data from samples taken toward the end of the alga’s lifecycle in

May 2016 and data from samples taken toward the beginning of its lifecycle in November

2016 indicate S. horneri was being consumed in significant amounts on both collection dates. This would suggest that green abalone have adapted their diet to include significant amounts of S. horneri. 1

CHAPTER 1

INTRODUCTION

The world is becoming a smaller place and more non•indigenous species (NIS) are finding their way into more foreign environments, often causing damage to ecosystems and fisheries (Williams and Grosholz 2008). Sometimes invasive species eradication efforts can eliminate or greatly reduce the impact of the invader (Anderson

2005). However, in many cases the foreign organism will establish itself beyond the point of no return, impact the local ecology, and become a ubiquitous part of its new habitat (Tanner 2013).

In the last 10 years, a new invasive marine macro alga called Sargassum horneri has spread aggressively in Southern California waters. The first reported sighting was in

2003 when a few plants were discovered in the inner harbor of the Port of Long Beach

(Miller et al. 2006). Quick to grow and tenacious, S. horneri often crowds out

Macrocystis pyrifera (giant kelp) and other native algae. The proliferation of S. horneri has been accelerated by recent El Niño events in which M. pyrifera experienced massive die-offs, leaving reef space for S. horneri to take over (Bushing 2014; Marks et al. 2015).

S. horneri eradication efforts have been able to clear small sections of reef but have not been able to significantly reduce the invasive alga.

As of 2015 S. horneri dominates large areas of the Southern California coast; it ranges over 750 kilometers of coastline from Santa Barbara, California to Isla Natividad,

Baja California. It has established itself well beyond any possibility of eradication

(Bushing 2014).

In its native waters in Asia, S. horneri is the dominant plant supporting a rich ecosystem, in a manner that is similar to how M. pyrifera serves as the dominant primary producer for the California kelp-forest ecosystem (Tanner 2013). Among S. horneri native consumers in Asia are several species of abalone. In fact, in areas in Asia where S. horneri has died off, significant restoration efforts have taken place, in large part to safeguard the important local abalone fishery (Pang et al. 2009).

The Southern California abalone fishery had been a significant economic driver for more than a century until it was shut down in 1997 after a major decline as a result of overharvesting, pollution, and disease (CADFW 2005). In recent years, due to decreased supply and increased demand from Asia, the price of abalone has skyrocketed to more than $100 per pound for cleaned, uncooked meat. Successful California abalone recovery would have major economic and ecological benefits. Significant research has been conducted to examine methods of abalone reintroduction in California (Tegner and Butler

1989; Schiel 1993; Saito 1984; Gaffney et al. 1996; Rogers-Bennett and Pearse 1998), but so far, large-scale reintroduction programs have not taken place.

Divers have recently observed seemingly healthy populations of abalones including green abalone, Haliotis fulgens, in areas in which S. horneri has replaced M. pyrifera and other native seaweeds at Catalina Island, California (Bushing 2014). Given

S. horneri’s overwhelming dominance, the question arises as to whether the abalones have been able to include S. horneri as a significant part of their diet. The answer to this question would add to the body of knowledge that could facilitate abalone restoration by

3 directing reintroduction efforts toward areas rich in S. horneri. The purpose of this study is to use qPCR analysis on abalone stomach content samples to determine whether green abalone have incorporated S. horneri as a significant part of their diet.

CHAPTER 2

REVIEW OF LITERATURE

California Abalone

Abalones are snails in the class Gastropoda, along with other snails, whelks and sea slugs. Abalones are in the family Haliotidae and the genus Haliotis, which means sea ear, referring to the shape of the shell (Cox 1962). It is generally agreed that there are seven species of Haliotis inhabiting California waters: red abalone, H. rufescens; pink abalone, H. corrugata; green abalone, H. fulgens; black abalone, H. cracherodii; white abalone, H. sorenseni; pinto abalone, H. kamtschatkana; and flat abalone, H. walallensis.

Taxonomic analysis shows threaded abalone, that was once considered a separate species, is now classified as a pinto abalone (Leighton 2000).

Abalones can be found off the coast of California from the intertidal zone to depths of 60 m, and they can live up to 30 years. Their growth is slow and varies greatly with environmental conditions. California species range in size as adults from 9 cm to over 30 cm (Leighton 2000).

Young juvenile California abalones feed on bacterial and diatom films on rocks.

Older juveniles and adults feed mostly on broken-off pieces of drift macroalgae. In the

Northern Hemisphere they express a preference for kelp and other brown algae. Bull kelp, Nereocytis luetkean, and giant kelp, Macrocystis pyrifera, are important dietary staples of most species of California abalone (Day and Cook 1995; Guest et al. 2007).

Reproductive maturity varies among species from 1 to 5 years. Abalones have

5 distinct male and female sexes. In an event called “synchronous broadcast spawning” they release their eggs and sperm at the same time. A high density of spawners is essential, with fertilization drastically decreased when the animals are more than 2 m apart (Leighton 2000; Haaker et al. 2003).

After they are fertilized, abalone eggs sink to the sea floor and hatch into larvae, which then float back up into the water column for up to a week. The larvae then settle on the bottom and metamorphose into juveniles when they encounter appropriate habitat of rocky substrate encrusted with coralline algae (Haaker et al. 2003). Survival rates of larval and juvenile abalone are very low with survival to the adult stage occurring only occasionally (Cox 1962).

Green Abalone

Green abalone, H. fulgens, are found from Point Conception, California to

Magdalena Bay, Baja California, Mexico in shallow waters from 0-10 m (Price and Tom

2004). They grow to 27 cm but are usually smaller. Characteristically, green abalone feature a brown, frilly-edged shell with flat ribs that run parallel to the animal’s 4–6 open pores. The tentacles are generally olive green. Green abalone are often found in crevices on seafloor characterized by rocky substrate, often covered by grass and algae (Haaker et al. 2003).

Green abalone have traditionally been harvested for their meat as well as their shells. They are known for their beautiful nacre (the mother-of-pearl inner shell), which is used for decorative inlay and jewelry (Leighton 2000). From 1950 to 1995 green

6 abalone accounted for 3.5% of total catch (recreational and commercial) in California.

All harvest of green abalone was halted in 1993 (Price and Tom 2004).

Abalone Decline

Although sharks, large fish, lobster and octopus will routinely feed on juvenile abalone, it is rare for adult abalone to be consumed regularly by anything other than sea otters. Abalones and sea otters co-evolved in the California region with the healthy sea otter population keeping abalone numbers in check (Tegner 1989).

Abalones were a common food source for Native peoples for centuries. Evidence of abalone shells has been found in Native American middens all along the California coast. The relatively small populations of Native American harvesters and their limitation to picking abalone at low tide probably led to only minor effects on the abalone population (Anderson 2003).

Early Asian immigrants harvested California abalones during the mid-to-late

1800s. Commercial abalone fishing was started in the area in the 1850s by Chinese-

Americans. Similar to early Native Americans, the first Chinese fishermen also picked at low tide. But now the harvest was conducted as a commercial fishery at a level that led to a decline in abalone populations in the intertidal zone. The fishery focused on green and black abalones, with a peak catch of 4.1 million pounds in 1879. The intertidal population was considered depleted enough by the end of the century that in 1900 the first abalone regulations were put into place, banning harvest in the intertidal zone

(Haaker et al. 2003; Neumann 2015).

A fundamental shift occurred with the beginning of the use of diving in abalone harvesting. Japanese divers began fishing commercially for abalone in California in 1898

(Croker 1931; Lundy 1997). At first Japanese divers utilized traditional free-diving equipment, but the cold waters of California soon necessitated the use of hard-hat diving and deep-sea dive suits (Lundy 1997). The leap in efficiency of surface-supplied air diving compared to previous methods was immense. It is documented from one group of

Japanese divers working in the Mendocino area that one diver working six hours could average 2,300 red abalone a day (Lundy 1997).

Reports from around the turn of the 20th century underscore how plentiful abalones were in those days. “Abalone [were] found in abundance. It was common to find them in layers of 12 or 13,” said one observer... “At that time the divers used to catch five to six tons a day” (Estes 1977).

Another observer stated, “I have seen the diver send the net up, filled with about fifty green and corrugated (pink) abalones, every six or seven minutes. During his shift below the diver gathered from thirty to forty basketfuls, each containing one hundred pounds of meat and shell, or altogether one and one-half to two tons” (Bonnot 1930).

As a result of this aggressive harvesting, the first size limit requiring all abalone to be 15 inches in circumference was initiated in 1901. Just eight years later a commercial fishing license program was started which led to further regulations such as catch limits and gear restrictions, as well as open/closed areas and seasons (Haaker et al. 2003).

Greatly depleted, the abalone fishery in Southern California was closed in 1913, which had the effect of moving the industry to the northern coast. Records of commercial

8 abalone catch were not kept until 1916, but from 1916 to 1929 the average yearly catch was over 2 million pounds (Cox 1962). From 1916 to 1935 this remaining fishery gradually reached a peak of 3.9 million pounds but declined precipitously to 164,000 pounds in 1942 when many Japanese fishermen were interned during World War II

(Haaker et al. 2003).

Around 1915 recreational abalone fishing first became popular in California and by 1930 sport abalone fishing was thriving. It was reported that at every low tide during open season, “Many hundreds of tourists and ranchers can be seen going over every accessible reef and ledge with a fine-toothed comb. State and county authorities are hard- pressed to enforce the laws on limits and minimum size which are easily broken by thoughtless people” (Croker 1931).

In 1943, the Southern California fishery was reopened to supply food for the wartime effort. Six years later the Northern California fishery was closed due to overharvesting. The Southern California abalone fishery hit a post-war peak of 5.4 million pounds in 1957. However, by the late 1960s the fishery was severely compromised and by 1996 the take had decreased to 229,500 pounds (Haaker et al. 2003). As a result, by

1997 the commercial abalone fishery was closed statewide (Haaker et al. 2003).

A number of other factors which affected the decline in abalone populations included the increased popularity of recreational scuba diving, warm sea temperatures, and withering foot disease. Starting in the mid-1960s, recreational scuba equipment became widely accessible to the general public, and sport fishing for abalones exploded in popularity. Scuba allowed the sport fisherman to take their time and scour every inch

9 of the rocky reef. For example, in Northern California alone recreational fishermen took an average of 533,000 red abalone (about 906 tons) annually from 1985-1989 (Tegner et al. 1996).

Two severe El Niño events occurred in the 1980s and 1990s, which caused extremely warm seas off the California coast. Water nutrient levels decreased and kelp beds withered and died off. With kelp, a mainstay of the abalones’ diet, depleted, the population of abalones, especially in Southern California, was devastated at a time when the population was already under tremendous stress (Anderson 2003).

In addition, a chronic wasting disease called withering syndrome or withering foot syndrome affected California abalones. It was first observed in the Channel Islands in the mid-1980s. Withering syndrome is caused by a bacterium, Candidatua xenohaliotis californiensis, which attacks the lining of the abalones’ digestive tract, interfering with the production of digestive enzymes. As a result, the abalones consume their own body mass to prevent starvation. This reduces the abalones’ ability to adhere to rocks, making them more vulnerable to predation or to starvation (Moore et al. 2002). Withering syndrome outbreaks are thought to be associated with pollution and reduced food supply caused by El Niño kelp die-offs (Davis 1993). While black abalone, H. cracherodii, were the most depleted by the outbreaks, withering syndrome is thought to affect all California species. Studies indicate that some black abalone populations have decreased up to 99% since withering syndrome was first documented (Crosson 2014).

Abalone Fishery Management in California

Beyond the issues of commercial and recreational over-harvesting, El Niño events, and withering syndrome, several management practices contributed to the collapse of abalone populations in California. Early on, all abalone species were managed together as a single fishery. In a practice known as “serial depletion,” as one species of abalone was depleted fishermen would move to another, keeping the total catch relatively stable (Neumann 2015). This would give the appearance of a sustainable fishery while abalone stocks were depleted species-by-species. For example, in Southern California from 1952 to 1968, the decreasing catch of pink abalone was offset by a dramatic increase in the catch of red abalone. Soon, red abalone numbers began to decline due to over-fishing, but this was, in turn, masked by an increase in the catch of green, black and white abalone (Haaker et al. 2003, CADFW 2005; Neumann 2015).

The abalone fishery suffered from several other management problems such as making size limit decisions based on sexual maturity and catch per unit effort. Size limits were set with the idea of the animal reaching sexual maturity before it was of legal size.

This did not take into account that for a number of reasons an abalone might not spawn for several years in a row. This practice allowed the take of many animals that never had the opportunity to reproduce (CADFW 2005). A study done in Southern California in

1989 found that the average abalone only had one successful spawning event in a five- year period (Neumann 2015). Catch per unit effort measures the amount of time required to catch a certain number of individuals. More time per individual would indicate a declining fishery while a stable catch per unit effort is indicative of a sustainable fishery.

Improvements in abalone fishing techniques and diving technology kept catch per unit effort numbers largely unchanged while the actual abalone population steadily declined

(CADFW 2005).

Abalone Restoration and Recovery Management

With the decline of such a commercially important species, the idea of abalone enhancement by seeding, or out-planting, has long been of considerable interest. Seeding has traditionally consisted of releasing small, hatchery-reared juveniles into appropriate ocean habitat. More recently, some experiments have focused on seeding larvae fertilized in containers above water. The free-floating larvae are then taken by divers to the seafloor and “squirted” into natural rock habitats or artificial structures designed to prevent them from floating away and which are conducive to their transition to juveniles.

(Rogers-Bennett and Pearse 1998).

Juvenile abalone seeding has been attempted in many parts of the world. In Japan in 1979 about 10 million juveniles were out-planted. Reported survival rates varied by area from 1 to 80 percent (Tegner and Butler 1989). Large scale juvenile seeding experiments have also taken place in New Zealand, South Africa and British Columbia

(Saito 1984; Tegner and Butler 1989; Schiel 1993; Gaffney et al. 1996). In one study performed in Southern California recapture rates of juvenile red abalone that had been seeded were less than 1 percent (Tegner and Butler 1985), but later genetic work on abalone collected at the previous seeding sites showed similarities to hatchery abalone, suggesting a higher success rate (Gaffney et al. 1996).

In 1995, 50,000 juvenile abalone averaging 8 mm in shell length were seeded at six sites in Northern California. After one year, divers revisiting the study sites found a total of only 36 abalone of the original 50,000. Even with the knowledge that for every seeded abalone found many more had potentially survived, the effort and expense of this method compared with the outcome is not encouraging (Rogers-Bennett and Pearse

1998).

Another method of seeding is to release larval abalone soon after fertilization into conducive natural or artificial habitat on the seafloor. In a 1994 study in Southern

Australia, larval abalone taken from a land-based hatchery were released in approximate densities of 1,600-120,000 per square meter. Surveys of the plots were taken at 6, 9, 27 and 39 day intervals and then again at 6, 12 and 18 months. Results showed high variation of survival but successfully increased the density of juveniles in the study areas compared to control plots in the short term. After a year, densities were comparable to control plots, but this could be attributed to the natural movement of the animal and the small size of the plots. Researchers concluded that this method was not a labor or cost- effective method of reseeding (Preece et al. 2011).

Releasing juvenile abalones raised in land-based facilities or maintaining broodstock to produce larvae that are later released in the ocean requires high levels of labor and resources. Some current research, especially in California, centers around the potential of deck spawning. Deck spawning is a process by which spawning and fertilization can occur in containers on the deck of a boat immediately after the collection of broodstock. When the process is perfected, the newly fertilized larvae could be

13 outplanted almost immediately, the whole process taking a minimum of time and resources (Reynolds et al. 2015).

Recent efforts have centered around collecting green abalone and experimenting with different methods to induce spawning. Methods for inducing spawning in captive abalone include: raising the ambient temperature of the tank water, the application of ultraviolet light, exposing the abalone to air for 30-60 minutes, and the application of hydrogen peroxide to the water (Food and Agriculture Organization of the UN 1990).

Sargassum horneri

Sargassum horneri is a large member of the Class Phaeophyceae or brown alga in the Order Fucales and the Family Sargassaceae. It was once thought to be a separate species from Sargassum filicinum, but taxonomic study has merged the two (Tanner

2013). In Japan its common name is akamoku, and it is sometimes referred to as devil weed. Native to the warm waters of Japan, Korea and China, S. horneri reaches lengths of around 3 meters and features fern-like blades that branch in a zig-zag pattern. Small air bladders keep the plant upright in the water column (Umezaki 1994; Tanner 2013).

S. horneri typically completes its lifecycle in less than one year. It follows a seasonal cycle with reproduction occurring in winter and spring and the recruits appearing and establishing themselves in summer and fall (Choi et al. 2008).

Plants have both male and female gametes, which enable them to self-fertilize. In addition to reproducing by the dispersal of fertilized gametes, S. horneri can spread by the scattering of fragments broken off the mature plants. Juvenile plants mature rapidly

14 enabling them to out-compete native species for space and light (Tanner 2013).

S. horneri is found from the intertidal region down to depths of 30 m and is most abundant between 3-15 m. It prefers rocky sea bottom similar to that favored by giant kelp, M. pyrifera. S. horneri can tolerate a wide range of light levels and water temperatures ranging from 10-25 C (Umezaki 1994).

In its native ecosystem it is a dominant species. It provides habitat and food for fish and invertebrates and is the primary producer in its habitat (Uwai et al. 2009). S. horneri is an important or even primary food source for commercially harvested species of abalone. It also acts as a biofilter and is able to absorb a significant percentage of the nutrient discharge off the mainland (Tanner 2013). Populations of S. horneri in the northwest Pacific are on the decline due to run-off from on-land development and urbanization. Concern over S. horneri decline is significant enough that much effort has been put into trying to find ways to restore the alga (Terawaki et al. 2003; Tanner 2013).

Between 1978 and 1991, approximately 1400 ha of Sargassum beds were lost along the Japanese coast (Arai et al. 1992). A study published in 2003 examined three different techniques of Sargassum restoration conducted in Japan. First was the construction of gently sloping, shallow-bottom substrata that were conducive to seeding.

The second technique was to use natural (rocks) or artificial (cement) reef material to extend the size of naturally occurring, healthy Sargassum beds. The third technique was the transplantation of Sargassum plants that had been produced in nurseries. All techniques met with success in restoring Sargassum beds, but there were questions as to the sustainability of the techniques given their cost, labor, and time requirements

(Terawaki et al. 2003).

In an experiment conducted in waters off the coast of Jeju City, Korea, 600 concrete blocks (40 x 40 x 15 cm) were placed and planted with artificially-produced recruits of S. horneri and S. fulvellum. These were covered with mesh boxes to protect the young plants from grazing animals such as sea urchin, sea snail, and abalone. Sargassum growth was generally good, with the plants on most blocks growing to over 300 cm after six months (Yoon et al. 2013).

Sargassum horneri in North America

S. horneri was first discovered in California in 2003 by researchers conducting surveys in the inner harbor of the Port of Long Beach. In April of 2006, S. horneri was collected at three locations on the leeward west end of Santa Catalina Island (Tanner

2013). Samples of the Long Beach and Catalina populations were genetically identical to the populations of S. horneri found in the Seto Inland Sea of Japan (Miller et al. 2006). It is thought that S. horneri first arrived in Long Beach in the ballast tanks of cargo ships from Asia and was then carried to Catalina Island by pleasure boats (Tanner 2013).

In the next few years S. horneri spread along the entire Southern California coast, extending into Baja Mexico and the northern Channel Islands (Miller et al. 2006;

Riosmena-Rodríguez et al. 2012; MARINe.gov 2016). Initially, S. horneri had only been found subtidally at depths from 3-19 m. But for the first time in 2009 a survey study found S. horneri located intertidally in Laguna Beach, California (MARINe.gov 2016).

As of 2015, S. horneri ranges 750 km from Santa Barbara, California to Isla Natividad,

Baja California (MARINe.gov 2016).

S. horneri has proven to be a prolific and tenacious reproducer along the

California coast with the ability to spawn as early as four months of age and to continue to reproduce at almost any stage of its lifespan (Tanner 2013). Research shows that S. horneri grows at a rate of about 4.5 percent a day relative to maximum size (Choi et al.

2008). Other research found that 50 percent of the biomass of a mature individual is composed of reproductive tissue (Marks et al. 2015).

As a result, S. horneri has been able to out-compete giant kelp, M. pyrifera, for habitat by monopolizing desirable rocky reef habitat. Once established, S. horneri further inhibits growth by blocking out available sunlight (Marks et al. 2015). In addition, recent warm sea temperatures have led to the reduction of nutrients relied on by kelp, and this has led to significant kelp die-off. An opportunistic species, S. horneri has been able to capitalize on available reef space, and when established it prevents other algae from growing (Bushing 2014).

Sargassum horneri Removal Efforts

There have been several attempts at removal of S. horneri in Southern California.

Early removal efforts consisted of divers removing the alga by hand. These efforts focused on the large adult plants that are easier to pick even though removal of juveniles before they reach reproductive age would be more desirable (Tanner 2013). At first, plants were simply ripped off the rocky substrate by hand and allowed to float away underwater. This was deemed ultimately counterproductive due to the fact that the

17 potentially fertile debris would add to proliferation in the long run at other locations

(Orlowski 2015). As a result, much of the difficulty of S. horneri removal is the transportation of the alga to the surface, hand sorting it for bycatch, and transferring it to land for appropriate disposal (NOAA 2016).

In response to the many difficulties of hand-removal of S. horneri, a device developed by the National Oceanic and Atmospheric Administration (NOAA) referred to as the “Super Sucker” was recently tested in Southern California. Previously used to remove invasive seaweed in Hawaii, this specialized boat-mounted, underwater vacuum was first used by Los Angeles Waterkeeper (LAW) in partnership with NOAA in 2012

(Jacobson 2016). In an experiment off Ship Rock on Catalina Island with members from

NOAA, the University of California Santa Barbara (UCSB), and LAW staff and volunteers, it sucked up about 45 kilograms (100 lbs.) of S. horneri in one day in

September 2013. Although the impact on the amount of S. horneri in the 100 m2 test plot was negligible, the information and experience gained from the study contributed to a larger removal effort in 2015 (Jacobson 2016).

In the winter of 2015, divers with NOAA Fisheries, UCSB, and California State

University Northridge (CSUN), removed over 4.25 metric tons (9,300 lbs) of S. horneri from 14 plots measuring 60 m2 each. These cleared plots will be used for various research projects, focusing on the effectiveness of S. horneri removal and the effect its removal has on the reef ecosystem (NOAA 2016).

Even with improvements in technology, S. horneri removal remains a time, labor, and resource-intensive process. Removal by divers can clear individual sites for research

18 and strategic control efforts but will not have significant impact on the total amount of S. horneri in the region (Tanner 2013; NOAA 2016).

Non-Indigenous Species

It has been estimated that more than 50,000 non-indigenous species (NIS) of animals, plants, and microbes have been introduced into the United States in the last

100 years (Pimentel et al. 2005). These include some 128 crop species that were intentionally introduced but have since become pest species. Marine and estuarine NIS alone have cost the United States over $1 billion per year related to control and removal programs, structural damage, and fisheries production (Williams and Grosholz 2008).

Introduced seaweeds are among the most common marine NIS. It is estimated that there are over 277 significant NIS seaweed species around the world (Schaffelke and

Hewitt 2007; Williams and Smith 2007). An analysis of 69 different publications on the impacts of NIS seaweeds revealed major ecological and evolutionary impacts (Schaffelke and Hewitt 2007). Those impacts included direct and indirect competition with native biota (e.g., for light or substratum), space monopolization, and change in community composition. Common ways in which the community composition was changed by NIS seaweeds include: habitat change, change of ecosystem processes, and genetic effects within species or between species (Schaffelke and Hewitt 2007). Economic and societal impacts of NIS seaweeds were categorized as direct and indirect. Direct impacts are those which relate to costs of loss of ecosystem and impacts on human health, while indirect impacts deal with the costs of management, research, eradication, and public education

(Schaffelke and Hewitt 2007).

Non-native species, including seaweeds, are one of the major components of human-mediated global change (Schaffelke and Hewitt 2007; Williams and Smith 2007).

Key to the success or failure in the spread of NIS seaweed is how the introduced species affects the trophic dynamics of its new community. Native herbivores’ response is an important factor in the success or failure of an introduction. Two hypotheses have been set forth regarding herbivore response to an introduced seaweed. They are the Enemy

Release Hypothesis (ERH) (Darwin 1859; Keane and Crawley 2002) and the Biotic

Resistance Hypothesis (BRH) (Elton 1958; Maron and Vilà 2001; Parker and Hay 2005).

In relation to NIS seaweed, the ERH states that an invader would have an increased likelihood of successful establishment due to native herbivores’ unfamiliarity with the plant and therefore reduced likelihood of their choosing it as a food source. In contrast, the BRH suggests that since NIS seaweed has not co-evolved with local herbivores, it will be more likely to be consumed because of lack of evolved defense mechanisms (Vogt 2010).

Non-Indigenous Seaweeds in California

California waters contain multiple examples of NIS seaweed. Three recent introductions in addition to S. horneri include: S. muticum, Undaria pinnatifida and

Caulerpa taxifolia. The latter two are on the list of the World’s 100 Worst Invasive

Species (Global Invasive Species Database 2016).

S. muticum is a large brown macroalga in the Sargassum genus, which also

20 originates in the northwest Pacific and was most likely transported to California waters in the ballast tanks of cargo vessels. After S. muticum was first found in Southern California in 1970, no significant eradication efforts were attempted. As a consequence it has flourished, becoming a common resident of intertidal and subtidal communities in

Southern California (Ambrose and Nelson 1982).

U. pinnatifida is a fast-growing, non-native kelp from Japan that was first identified in Southern California in 2000 (Silva et al. 2002; Zuccarello et al. 2002). It spreads mostly by attaching to boat hulls. Manual removal efforts have taken place at several harbors on Catalina Island as well as in San Francisco Bay and Monterey Bay

(Reef Check 2011). It is thought that while the species can be somewhat controlled through removal by divers and boat owner education, it is established beyond the point of eradication in California waters (Chapman 2005).

C. taxifolia is a small green alga known for its popularity in aquariums.

Originating in Asian waters, it was first found in Southern California in 2000 (Woodfield and Merkel 2006). Awareness of C. taxifolia as a threat to California waters was high due to the widespread environmental and economic damage it had previously caused in the

Mediterranean (Boudouresque et al. 1996; Anderson 2005). As a result, when it was first detected in California, marine managers were quick to respond. An intensive program was carried out consisting of numerous surveys by scuba divers, hand-removal efforts, and the use of chlorine treatments (Williams and Grosholz 2002; Anderson 2005).

Altogether, about $7 million was spent on the eradication of C. taxifolia. Legislation was also passed banning the plant from the aquarium trade in California (Williams and

Grosholz 2002). As a result of all this effort California was declared free of C. taxifolia as of the end of 2006 (California State Water Resources 2008). The study of responses to

NIS seaweed clearly points to early detection and prompt action as essential components to successful eradication efforts (Reef Check 2011).

Invasive Seaweed Control by Herbivores

The study of herbivores controlling NIS seaweeds is an ongoing field. An attractive alternative to labor-intensive NIS seaweed removal would be consumption by a native or, possibly, introduced herbivore. Studies have been done on C. taxifolia to see if various species of herbivores could eliminate or control the alga (Coquillard et al. 2000;

Anderson 2005).

While C. taxifolia was growing out of control in the Mediterranean, researchers investigated employing either indigenous or exotic reef-grazing species that might be used in a biological control program. Potential species would need to have a number of characteristics: high dietary preference for the target species; the ability to exist at the same temperatures, depths, and other environmental conditions as the target species; and the ability to proliferate and spread (Coquillard et al. 2000; Anderson 2005).

Two native grazers, Lobiger serradifaclci and Oxynoe olivacea, and two exotic species, Elysia subornata and Oxynoe azuropunctata, were studied to gauge their effect on C. taxifolia. Efficiency of C. taxifolia consumption varied by species of these herbivore gastropods, but overall, the approach was not deemed worthy to pursue as a meaningful eradication program. In all cases, consumption of the target species was low.

In the case of L. serradifaclci it led to increased C. taxifolia proliferation because the animal tended to create free-floating fragments during feeding (Coquillard et al. 2000;

Anderson 2005).

Sea urchins are generalist grazers that are able to feed on a wide variety of marine plants. As a result, they have some of the highest potential to significantly impact NIS

(Tomas et al. 2010). A recent study examined the response of Mediterranean sea urchins to four invasive seaweed species: Caulerpa racemosa, Lophocladia lallemandii,

Acrothamnion preissii, and Womersleyella setacea. It explored the potential of the native sea urchin species, Paracentrotus lividus, to consume the NIS and whether the sea urchins could have an impactful effect on plant populations (Tomas et al. 2010).

The study showed three of the four NIS seaweeds were not consumed at all while the fourth, C. racemosa, was readily consumed when the animals had no choice. The study also showed that the sea urchins with a diet of that seaweed did not thrive in terms of performance factors such as growth, reproduction, and size of feeding apparatus. For those reasons, researchers concluded that the sea urchins would not offer significant control of the seaweeds (Tomas et al. 2010).

Outside of the marine environment, there is support for the idea that generalist native herbivores can incorporate exotic plants into their diet and this can contribute to suppressing the spread of NIS (Parker 2006). Data analysis of research on more than 100 terrestrial and fresh water NIS supports that idea (Elton 1958; Parker 2006).

However, multiple studies suggest that marine generalist herbivores (e.g., urchins, abalone, and other snails) tend to avoid exotic seaweeds, offering little impact on the

23 invaders (Boudouresque et al. 1996; Gollan and Wright 2006 for C. taxifolia; Scheibling and Anthony 2001, Scheibling et al. 2008 for Codium fragile; Monteiro et al. 2009 for S. muticum). Notable exceptions include studies on U. pinnatifida and S. muticum in which juvenile stages of the seaweeds were meaningfully suppressed by algivorous native grazers (Thornber et al. 2004; Sjotun et al. 2007).

Feeding Studies of California Algivorous Marine Species

An important consideration in the question of whether green abalone are incorporating the NIS seaweed S. horneri into their diets is the result of previous research on the feeding preferences of abalone and other algivorous species. Due to the fact that S. horneri is a recent arrival in California waters, its acceptance as a food source by green abalone, or by any species of California abalone, has not been widely studied. There have been a few studies that have included S. muticum, another NIS Sargassum species from

Asia (Ambrose and Nelson 1982).

In one study, a wide variety of brown, red, and green algae and surf grasses were offered to experimental groups of green abalone. Thirty marine plant species were ranked into four categories by how readily they were consumed, ranging from 0-3 with 0 indicating rejected and 3 representing readily consumed. S. muticum was ranked a 1, indicating it was ingested in minor amounts. S. agardbianum, a native California

Sargassum, was also ranked a 1. The study described how these two species of

Sargassum were typically consumed only after other, more preferred, food choices were exhausted (Leighton 2000).

In the same study, green abalone growth was measured when the animals were fed an exclusive diet of the same 30 marine plants. Surprisingly, when offered by itself,

S. muticum showed intermediate acceptance and supported a moderate growth rate of 4-

11 percent conversion efficiency. Green abalone accepted a wide variety of marine plants with 21 of the 30 choices consumed in at least minor amounts (Leighton 2000).

A more recent study focused on whether four common California kelp forest macroinvertebrate grazers would favor native or NIS seaweeds. It is significant because it is one of the few studies that looks at the attractiveness of S. horneri as a food source of

California kelp forest herbivores. The consumer species were: the purple sea hare Aplysia californica, the marine trochid snail Chlorostoma aureotincta, the striped shore crab

Pachygrapsus crassipes, and the purple sea urchin Strongylocentrotus purpuratus (Vogt

2010).

A two-choice feeding study was conducted in which the four species were offered one native seaweed and one similar, non-native seaweed. Of particular interest were the pairs of Sargassum in which native S. agardbianum was offered as a choice along with either S. muticum or S. horneri. Findings showed that all of the species preferred the native S. agardbianum except P. crassipes, which had a slight preference for the NIS seaweeds S. muticum and S. horneri (Vogt 2010).

A study published in 2012 showed strong evidence that green abalone have the ability to survive on a diet high in Sargassum sp. when more preferred food sources are absent (Mazariegos-Villarreal et al. 2012). The study sampled the stomach content of green abalone before, during, and after an El Niño event, lasting from 1997 to 1998 in

Baja California, Mexico. Results showed that before and after the event abalone fed mainly on M. pyrifera, which has been shown to promote faster growth rates and higher survival rates than other food sources. (Serviere-Zaragoza et al. 2001; Ponce-Díaz et al.

2004). M. pyrifera largely died off during the El Niño and the growth of various

Sargassum species increased. As a result, green abalone increased their consumption of

Sargassum sp. to adapt to the change in available food sources. Before and after the El

Niño event Sagassum sp. was almost non-existent in stomach content samples, but during the event in July of 1998 Sargassum sp. became the second most prevalent food source of the identifiable stomach content and M. pyrifera became undetectable. While the experiment was only able to identify Sargassum sp. down to the genus, it gave a strong indication that abalone are able to survive on Sargassum sp. (Mazariegos-Villarreal et al.

2012).

CHAPTER 3

METHODOLOGY

Species of Study

Green abalone, Haliotis fulgens, was selected as the species of study for several reasons. It was once a commercially important abalone species in California, typically the third most harvested species behind red and pink. According to available records from the

California Department of Fish and Wildlife (CADFW), ranging from 1950 to present, commercial green abalone harvest peaked in 1971 when 1,089,706 lbs was harvested.

That year green abalone accounted for 40 percent of the total abalone harvest (Haaker et al. 2003). Due to its potential commercial significance, it would be a valuable and desirable abalone species to restore.

Another reason green abalone were selected for study is their relative abundance.

Although they are identified by the NOAA Fisheries Service as a “species of concern,” green abalone are more abundant than other abalone species in Southern California

(NOAA Fisheries [date unknown]). CADFW surveys from 2007 to 2009 showed results indicative of a limited green abalone recovery at Santa Catalina Island. The surveys showed an average of 2.4 abalone found per hour of diver search time and in some areas green abalone densities were as high as 1.8 abalone per square meter. In comparison, surveys of the northern Channel Islands found just 0.05 green abalone per hour of diver search time (Lampson et al. 2011). These encouraging Catalina Island numbers showed green abalone to be abundant enough to allow the granting of a Scientific Collecting

Permit for the purpose of this research. Scientific Collecting Permit #1004211504 was issued from May 19, 2015 through May 19, 2016 and was renewed from July 6, 2016 through July 6, 2017.

There are several other factors that make green abalone a desirable target species for recovery actions in Southern California and, therefore, a logical species to study. In recent California studies, green abalone have been found in a wide range of sizes consistent with multiple years of successful recruitment. A CADFW study also showed that green abalone are somewhat resistant to wasting syndrome (WS), a disease that contributed to the collapse of abalone populations in Southern California (Lampson et al.

2011). Increased water temperatures associated with global climate change are thought to be a contributing factor to WS (Lampson et al. 2011). With sea temperatures likely to rise in the future, a species that is more adaptable to warmer water is a better candidate for restoration resources. In addition, green abalone are found in relatively shallow water and prefer the same general depth and bottom type as Sargassum horneri (Leighton 2000).

Study Sites

The three selected study areas were located on Santa Catalina Island, also known as Catalina Island or Catalina. It is an island in the Channel Islands archipelago of

California, located 35 km south-southwest of Los Angeles; it is located within Los

Angeles County. The study areas were located on the mainland-facing, northeast side of

Catalina Island. The study sites were Empire Landing (33°25’44.11” N, 118°26’24.61”

W), Little Gibraltar Point (33°25’19.64” N, 118°24’18.87” W), and Torqua Springs

(33°23’03.26” N, 118°21’35.02” W).

Three major factors determined the location of the study sites. The first major factor related to permitting requirements. Under state law no species of abalone can be taken by any method in Southern California except under a Scientific Collecting Permit issued by the CADFW (CADFW 2016). Permitting requirements stipulated that no animals be taken within the four established marine protected areas on the northeast side of Catalina Island (CADFW [date unknown]). The second factor related to site selection was that the study areas were dominated by the non•indigenous species (NIS) seaweed S. horneri. Traditionally, the dominant plant in those areas would have been giant kelp,

Macrocystis pyrifera. However, the kelp in those areas had most likely died off due to low water nutrient levels caused by recent El Niño events. As a result, the opportunistic

S. horneri appropriated the available rocky reef habitat (Tanner 2013). The third factor is the abundant populations of green abalone at this site. This would make the animals easy for divers to find and the population stable enough to withstand the take of animals needed for this study.

All the study sites have similar underwater topography and bottom composition with plant and animal life typical of the region. The rocky reef structure starts at the waterline and extends down to a depth of approximately 10 m at approximately a 40 degree angle. From there the bottom composition turns to muddy sand and slopes away from the island at about a 20 degree angle. The reef is composed of rocks from cobblestone-sized to large boulders, forming numerous cracks and crevices that are ideal habitat for abalone and other near-shore reef residents. S. horneri was observed to be by

29 far the most common marine plant. Study sites were all located on the leeward side of

Catalina Island with the island acting as a breakwater from ocean weather and waves. As a result, wave action at the sites is generally minimal with calm seas, contributing to an environment that is nearly ideal for plant and animal growth (Leighton 2000).

S. horneri is an annual alga, completing its full lifecycle in less than a year. Two abalone sampling dates were chosen, one at the end and one toward the beginning of the

S. horneri lifecycle. In Southern California waters, S. horneri typically thrives during months with colder water. S. horneri begins its lifecycle in Southern California as ocean temperatures begin to drop in the late fall and early winter. It grows rapidly during winter and reaches maximum length of up to 3 m by late spring or early summer. However, growing time varies widely by location and some individual plants will mature outside of the usual growing season (Tanner 2013; Marks et al. 2015). The first sampling date was

May 6, 2016. At this time the S. horneri was full grown and its total biomass was at its maximum with plant lengths averaging about 2 m. On the second occasion, November 4,

2016, the new recruits were sprouting. The typical length of the young plants ranged from about 10 cm to 30 cm.

Collection

A total of 60 green abalone were collected for this project: 30 on May 6, 2016 and

30 on November 4, 2016. On each date, 10 animals were collected from each of the three study sites using a modified Roving Diver Technique (Schmitt and Sullivan 1996;

Schmitt et al. 2002). Divers entered the water from a vessel at anchor and haphazardly

30 moved along the bottom at depths ranging from 2 m to 10 m. Divers collected one abalone roughly every six minutes to insure that there was space between animals and that the specimens were taken from a variety of aggregations.

Sample Preparation

Samples of the stomach contents of the green abalone were prepared and later sent to the Real-time PCR Research and Diagnostic Core Facility at the University of

California, Davis. After removing the shell, an incision was made in each animal’s stomach/gut, which is located in the upper rear of the shelled body. A fresh razor blade was used on each sample to avoid cross-contamination. Next, a stomach content sample from each animal was scooped into an individual 2 ml polypropylene microtube. The samples were promptly frozen for submission to the PCR facility and shipped using dry ice to insure they remained frozen.

PCR Analysis

Real-time polymerase chain reaction, also known as quantitative polymerase chain reaction (qPCR), was chosen as an accessible and accurate method to determine whether the green abalone gut content samples contained S. horneri (Nejstgaard et al.

2007; Murray et al. 2011).

PCR is a laboratory technique used to amplify small target sections of DNA

(target DNA or amplicon), making thousands to billions of copies of a particular portion of the DNA sequence. Once the amplicon is identified, short fragments of synthesized

DNA called primers are developed, one at the beginning and one at the end of the DNA

31 sequence to be amplified. In this study PCR was used to determine the presence and quantity of a DNA sequence unique to S. horneri in the green abalone gut content samples (Biosystemika [date unknown]; Morley 2014).

With this technique the DNA is first separated from the rest of the sample. Then in a process called denaturation the sample is heated to more than 90 °C to separate the double-stranded DNA into two separate strands with one set of nucleotides on each strand. Next, the primers are added to the DNA sample and the tube is cooled, allowing the primers to bond to each end of the amplicon. An enzyme (thermo-stable DNA polymerase) duplicates the short specific amplicon between the primers. At the end of this process two identical copies of all of the original amplicons have been made. The process is repeated multiple times and the amplicon doubles exponentially (Morley

2014).

In qPCR the duplicated target DNA accumulation is measured through a dual- labeled fluorogenic probe (Heid et al. 1996). Amplified DNA is fluorescently labeled, usually with a cyanine-based fluorescent dye. As the DNA duplication process progresses more fluorescence is given off by amplified copies of DNA. The amount of fluorescence released during the amplification process is directly proportional to the number of duplicated amplicons. The level of fluorescence is monitored in real time as the amplification process is repeated. Eventually, the fluorescence can be detected at a point called the quantitation cycle (Cq). A lower number of PCR cycles before attaining the Cq indicates a higher number of target DNA strands in the initial sample (Tse and Capeau

2003; Pohl and Shih 2004; Biosystemika [date unknown]). Essentially, the fewer cycles

32 before the Cq is reached, the more DNA of interest is present in the original sample.

The UC Davis Real-time PCR Research and Diagnostics Core Facility utilized specialized technology, procedures, and equipment to analyze the samples. First, the gut content samples were pulverized and homogenized using a GenoGrinder 2010. These machines are used to grind up a variety of sample types including tissue, plant material, and insects. After pulverization, a BioSprint 96 was used to extract DNA from the other material in the sample. Finally, an AB 7900 HT FAST thermocycler with a laser detection system performed the amplification process while measuring the fluorescence given off by the sample (UC Davis PCR Lab [date unknown]). A PureLink Microbiome

DNA Purification Kit was chosen to extract the green abalone and S. horneri DNA from the rest of the sample material (Thermo Fisher Scientific Inc 2015).

After each round of samples was processed, the qPCR Lab returned various results that were then used in the quantitative analyses. Firstly, the number of cycles before the Cq was reached for both green abalone and S. horneri DNA was reported. A total of 40 cycles were run, so if a sample did not meet the Cq by the 40th cycle it was reported as undetected. Using the Cq numbers from abalone and S. horneri the percentages of S. horneri DNA, H. fulgens DNA, and all other DNA present in the samples were reported.

This study is concerned with the makeup of the green abalone diet. So, the percentage of green abalone DNA in the gut content samples, while necessary for determining the S. horneri DNA percentage, is not relevant to analysis. As a result, the S. horneri DNA percentage in the gut content samples was normalized by the total non-H.

33 fulgens DNA to better represent the percentage of total food item DNA made up by S. horneri. Essentially, by removing H. fulgens DNA from the total DNA profile, what remains is a more accurate representation of the DNA profile of only food items. The normalized S. horneri DNA percentage more clearly reflects the percentage of food DNA comprised by S. horneri.

To normalize the data, the original S. horneri DNA percentage data received from the lab was divided by the S. horneri DNA percentage plus the percentage of all other non-H. fulgens DNA. This normalized data is reflected under the column labeled

Normalized S. horneri DNA Percentage in Tables 1 and 2. All further analysis of S. horneri DNA percentage reflects these normalized results.

Statistical Analysis

Study data were analyzed for each of the three study sites on each of the two collecting dates. First, a Shapiro-Wilk Test for normal distribution was performed on the

S. horneri DNA percentage data. If the data were not normally distributed, a Wilcoxon

Signed-Rank Test would be used to determine whether the median percentage of S. horneri DNA in the abalone gut samples was greater than zero. If the data were normally distributed, a t-test would be used. Analysis then focused on the data from each of the three study sites, using a Kruskal-Wallis Test to determine whether the S. horneri DNA in the abalone gut samples on a given collection date differed among the three study sites.

Finally, A Wilcoxon Rank Sum Test was used to determine if there was a difference in consumption between samples collected in May and samples collected in November.

CHAPTER 4

RESULTS AND DISCUSSION

Results

To calculate the percentage of S. horneri DNA present in the sample, a quantitation cycle number for both H. fulgens and S. horneri DNA was required. Of the

30 samples taken in May, three of the samples showed detectable S. horneri DNA but not detectable H. fulgens DNA. Therefore, a percentage of abalone DNA in the sample could not be reported. As a result, three of the 30 results for percentage of abalone DNA were excluded from analysis even though S. horneri DNA was present in the sample (Table 1).

Of the 30 samples taken in November, one sample showed detectable S. horneri DNA but not detectable H. fulgens DNA. As a consequence, that sample was excluded from analysis (Table 2).

May 6, 2016 Samples

The results from the samples taken on May 6, 2016 were first tested together for normal distribution using a Shapiro-Wilk Test for normality that indicated the results were not normally distributed (Shapiro-Wilk = 0.648, p = <0.0001, n = 27). Given that the data were not normally distributed, the non-parametric Wilcoxon Signed-Rank Test was used to determine that the median percentage of S. horneri DNA in the abalone gut content samples was statistically greater than zero (p = <0.0001, n = 27). Results of a

Kruskal-Wallis Test showed there to be no statistically significant difference among the three study sites (p = 0.353). The mean percentage of S. horneri DNA present in the gut

36 content samples was 13.50 percent, while the median was 0.39 percent. This indicates the higher percentages were atypical. Of the 30 abalone sampled, 24, or 80 percent, had a detectable amount of S. horneri DNA in their gut content sample. The mean number of

PCR cycles run before the Cq was reached for those 24 samples was 31.33, while the median was 30.45 (Table 1).

Table 1. Selected results from green abalone gut content samples collected at Catalina Island, California on 6 May 2016.

Number of PCR % S. horneri Site Cycles Before Cq Percentage of Algae, Abalone, and Other DNA to all non-H. Number (40 Maximum) fulgens DNA Normalized H. S.horneri H. fulgens Other S.horneri S. horneri fulgens DNA % DNA % DNA % DNA % 1 24.98 35.50 30.18% 0.08% 69.75% 30.20% 1 34.67 31.37 0.30% 18.98% 80.72% 0.37% 1 28.33 - * 0.00% * * 1 27.16 35.47 62.75% 0.84% 36.41% 63.28% 1 34.01 32.05 0.20% 4.72% 95.09% 0.21% 1 - 25.20 0.00% * * 0.00% 1 - 28.06 0.00% * * 0.00% 1 30.47 29.72 5.05% 44.39% 50.57% 9.08% 1 38.17 37.67 0.01% 0.13% 99.85% 0.01% 1 30.54 33.85 5.06% 2.60% 92.34% 5.20% 2 38.37 35.91 0.01% 0.53% 99.46% 0.01% 2 30.69 33.45 5.43% 4.13% 90.44% 5.66% 2 24.57 36.93 45.78% 0.03% 54.19% 45.79% 2 - 26.99 0.00% * * 0.00% 2 28.48 31.28 10.30% 6.90% 82.79% 11.06% 2 39.90 32.36 0.01% 16.61% 83.38% 0.01% 2 - 36.49 0.00% * * 0.00% 2 36.38 35.98 0.12% 1.06% 98.83% 0.12% 2 30.43 33.41 6.59% 4.25% 89.17% 6.88% 2 - 32.69 0.00% * * 0.00% 3 28.54 - * 0.00% * * 3 33.57 32.33 0.37% 5.22% 94.41% 0.39% 3 27.26 35.58 20.97% 0.28% 78.75% 21.03% 3 27.92 38.95 19.43% 0.04% 80.53% 19.44% 3 - 31.05 0.00% * * 0.00% 3 27.17 30.82 56.06% 19.53% 24.42% 69.66% 3 28.47 - * * * * 3 39.90 36.19 0.01% 0.57% 99.43% 0.01% 3 33.42 36.43 0.97% 0.70% 98.33% 0.98% 3 28.50 30.34 37.83% 49.68% 12.49% 75.18% Minimum 24.57 25.20 0.00% 0.00% 12.49% 0.00% Maximum 39.90 38.95 62.75% 49.68% 99.85% 75.18% Mean 31.33 33.19 11.39% 7.88% 76.73% 13.50% Median 30.45 32.81 0.37% 1.06% 83.38% 0.39%

* detected but undeterminable Sites: Note: Table created by the author of 1 Empire Landing this thesis. 2 Little Gibraltar Point

3 Torqua Springs

November 4, 2016 Samples

The results from the samples taken on November 4, 2016 were tested in a similar manner to the May results. Again, the samples were found to be not normally distributed

(Shapiro-Wilk = 0.782, p = <0.0001, n = 29). A Wilcoxon Signed-Rank Test indicated that the median percentage of S. horneri DNA in the samples was statistically greater than zero (p = <0.0001, n = 29). Results of a Kruskal-Wallis Test showed there to be no statistically significant difference among the three study sites sampled in November (p =

0.063). The mean percentage of S. horneri DNA present in the samples was 20.20 percent while the median was 14.19 percent. Of the 30 abalone sampled, 28, or 93 percent, had a detectable amount of S. horneri DNA in their gut content samples. The mean number of

PCR cycles run before the Cq was reached for those 28 samples was 30.37, while the median was 29.29 (Table 2).

Table 2. Selected results from green abalone gut content samples collected at Catalina Island, California on 4 November 2016. Number of PCR % S. horneri Site Percentage of Algae, Abalone, and Other Cycles Before Cq to all non-H. Number DNA (40 Maximum) fulgens DNA Normalized H. S.horneri H. fulgens Other S.horneri S. horneri fulgens DNA % DNA % DNA % DNA % 1 33.34 32.52 0.47% 4.95% 94.58% 0.49% 1 27.08 39.79 15.57% 0.01% 84.42% 15.57% 1 29.12 34.29 6.72% 0.88% 92.39% 6.78% 1 28.85 35.12 7.46% 0.45% 92.10% 7.49% 1 38.67 33.65 0.01% 2.69% 97.30% 0.01% 1 36.75 38.37 0.03% 0.08% 99.89% 0.03% 1 29.08 36.13 11.74% 0.41% 87.84% 11.79% 1 - 32.34 0.00% * * 0.00% 1 29.34 36.05 5.30% 0.24% 94.46% 5.31% 1 27.48 34.40 49.44% 1.77% 48.79% 50.33% 2 24.97 31.99 17.50% 0.52% 81.97% 17.59% 2 32.28 31.94 1.46% 10.49% 88.05% 1.63% 2 28.12 33.36 15.34% 1.83% 82.82% 15.63% 2 31.57 31.76 2.11% 10.11% 87.77% 2.35% 2 31.03 34.91 4.86% 1.72% 93.43% 4.94% 2 39.60 29.81 0.01% 71.99% 28.00% 0.04% 2 36.26 34.35 0.64% 16.51% 82.85% 0.77% 2 25.87 37.62 19.69% 0.02% 80.29% 19.69% 2 27.04 33.45 36.33% 1.82% 61.85% 37.00% 2 25.07 34.57 86.47% 0.46% 13.07% 86.87% 3 28.32 34.87 29.47% 1.43% 68.82% 29.98% 3 30.07 32.93 12.92% 8.92% 78.16% 14.19% 3 - 33.90 0.00% * * 0.00% 3 27.90 39.39 92.97% 0.14% 6.90% 93.09% 3 29.61 29.71 12.13% 56.62% 31.25% 27.96% 3 38.06 - * 0.00% * * 3 25.98 35.65 38.88% 0.19% 60.93% 38.95% 3 29.81 30.39 19.60% 65.96% 14.44% 57.58% 3 29.94 32.05 12.94% 15.01% 72.05% 15.23% 3 29.23 30.73 17.25% 29.71% 53.04% 24.54% Minimum 24.97 29.81 0.00% 0.00% 13.07% 0.00% Maximum 38.67 39.79 92.97% 71.99% 99.89% 93.08% Mean 30.37 34.01 17.40% 10.89% 69.54% 20.20% Median 29.29 33.90 12.13% 1.75% 81.97% 14.19%

* detected but undeterminable Sites: Note: Table created by the author of 1 Empire Landing this thesis. 2 Little Gibraltar Point

3 Torqua Springs

Comparison of May and November Samples

Finally, analysis of all of the usable data by Wilcoxon Rank Sum Test compared the percentages of abalone DNA found in the gut content samples for May with the percentages found in November. Results show that the sampled animals consumed more

S. horneri in November than in May (p = 0.047). Results of a Wilcoxon Signed-Rank

Test of May and November data analyzed together show the median percentage of S. horneri DNA present in the abalone guts was statistically greater than zero (p = < 0.0001, n = 56). Of all 60 animals tested during the project, 51, or 85 percent, contained detectable amounts of S. horneri in their gut content samples. Figure 1 shows the range and median S. horneri DNA percentage present for all study sites in both May and

November.

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% May November May November May November Empire Landing Little Gibraltar Point Torqua Springs

Figure 1. Range and median percent of S. horneri DNA present in H. fulgens gut content samples by location and collection month in 2016. Note: Table created by the author of this thesis.

Discussion

In recent years, to a large extent S. horneri has replaced M. pyrifera as the dominant seaweed in shallow, rocky reef habitat around Catalina Island and in other coastal areas of Southern California. A major question is whether local green abalone have incorporated S. horneri as a significant part of their diet. Using qPCR analysis on abalone gut content samples, the results of this study indicate that green abalone have, indeed, adapted to S. horneri as an accepted food source.

Analysis of the gut content samples provides a snapshot into what the green

42 abalone had in their digestive system on the day of collection. Research is not available on the speed of abalone digestion but the animals generally feed every night. It can be assumed that gut content samples positive for S. horneri reflect that an animal has fed on the alga in the past few hours or days.

Of the abalone that tested positive for S. horneri DNA, the percentage was as low as 0.01 percent, indicating the animal had eaten only a little S. horneri. In contrast, the percentage of S. horneri DNA in abalone gut content was as high as 75.18 percent in May and 93.08 percent in November. The higher percentages indicate that the animal had purposefully chosen to consume large amounts of S. horneri. Mature abalone generally feed on broken off pieces of drift algae that can be held and manipulated with their mouth parts, but they will feed on attached live-growing seaweed (Day and Cook 1995;

Leighton 2000; Guest et al. 2007). Like many herbivores the physical act of eating plant material consumes a large portion of the animal’s feeding time (Day and Cook 1995).

The results with higher percentages of S. horneri DNA indicate that when an animal had found a suitable piece of the alga, it had chosen to spend its limited nocturnal feeding time consuming the plant.

Of the 27 animals collected in May that had a determinable percent of abalone

DNA in their gut content samples, 8 of the samples (30 percent) showed S. horneri made up more than 10 percent of the total DNA profile in the gut. Of the 29 animals collected in November that had a determinable percent of abalone DNA in their gut content samples, 16 of the samples (55 percent) showed S. horneri made up more than 10 percent of the total DNA profile. These results are consistent with the idea that some of the

43 sampled animals had made a significant meal of S. horneri in the recent past and not just randomly sampled the alga and moved on to other food sources.

At different stages in its lifecycle, S. horneri would offer different advantages and disadvantages to abalone as a food source. At the beginning of the lifecycle the plant tissue would presumably be softer and easier to consume. Less overall biomass would lead to fewer broken off fragments; but the younger, shorter, attached plants would be easier for the abalone to feed on directly. The percentage of abalone that had detectable amounts of S. horneri in their gut content samples was 10 percent greater in November.

Similarly, the median percentage of S. horneri DNA present in the samples was somewhat higher in November. The results of the Wilcoxon Rank Sum Test showed a statistically significant trend that the abalone were consuming more S. horneri in

November with a p value of 0.047. This indicates that abalone are utilizing S. horneri as a food source somewhat more at the beginning of the alga’s life cycle than at the end.

Another factor that may influence the desirability of S. horneri at different stages of its lifecycle is its polyphenol content. Research on a similar Sargassum, S. muticum, shows that phenolic content increases when the plant is reproductively mature (Gorham and Lewey 1984). This is most likely due to the alga producing chemical protection from herbivores and bacteria during the crucial reproductive stage (Plouguerné et al. 2006). If the same fluctuations in polyphenol content are true for S. horneri, the fact that abalone consumption rates are somewhat higher in November suggests the alga is more attractive to the animals when the phenolic levels are lower. Even with less overall biomass S. horneri appears to be abundant enough in November to provide an ample food source.

Comparison to Previous Research

In general, the results of this study are consistent with previous, related research on the feeding preferences of abalone and other algivorous species. Because S. horneri is a recent arrival in California waters, it has not been widely studied. However, a few studies have looked at other species of Sargassum in relation to California abalone diet preference and growth (Ambrose and Nelson 1982).

Abalone appear to be adaptable to a wide variety of food choices. A 1998 study of

H. fulgens showed that both S. agardbianum (native to Southern California) and S. muticum (non-native) were ingested when more preferred food sources were absent

(Leighton 2000). In the same study green abalone were fed an exclusive diet of various marine plants and their growth rates were measured. While S. horneri was not tested, S. muticum (another NIS Sargassum from Asia) showed it was able to support a moderate growth rate of 4-11 percent conversion efficiency. While not a specific experiment focused on S. horneri, the study indicates the likelihood of green abalone to tolerate various species of Sargassum (Leighton 2000).

A study by Mazariegos-Villarreal et al. (2012) also supports the premise that green abalone can sustain themselves on a diet high in Sargassum sp. The study showed that when giant kelp had been replaced by various species of Sargassum due to an El

Niño event, abalone gut content samples contained high levels of Sargassum sp.

(Mazariegos-Villarreal et al. 2012). The method of gut content analysis (microscopy) was only able to identify Sargassum sp. down to the genus level. Still, its results are consistent with the results of this study, which uses a more precise method of gut content

45 analysis to test specifically for S. horneri.

A Japanese study published in 1986 focused on the suitability of various marine algae to promote growth in juveniles of a local species of abalone (Uki et al. 1986). One purpose of the study was to find beneficial food sources for hatchery-reared abalone that were to be seeded later into the ocean. The commercially important species, Haliotis discus, was used in the study. Groups of juveniles were fed an exclusive diet of 57 different marine algae. Their shell growth and total weight were recorded over the 30-35 day study duration. The algae species were given a grade of dietary value from A to D, with A indicating the quickest growth and D indicating the slowest. S. horneri was given a grade of B, indicating a healthy rate of growth. Interestingly, S. horneri was rated the highest of any species of Sargassum tested, supporting the quickest growth rate. The other Sargassum species, S. ringgoldianum and S. muticum, received a dietary value of C and S. yezoense received a dietary value of D (Uki et al. 1986). These results are interesting when combined with the findings of the current project. While one study focuses on growth rates of H. discus juveniles, this study focuses on stomach content of mature H. fulgens. It is possible that S. horneri, readily eaten by green abalone, could be a high-grade food source for that species.

CHAPTER 5

SUMMARY, RECOMMENDATIONS AND CONCLUSIONS

Summary

This study was initially inspired by informal observations of numerous large adult green abalone, H. fulgens, in areas overgrown with S. horneri. A review of the literature indicated that in its native waters in Asia, S. horneri is a preferred food source for local abalone (Terawaki 2003; Uwai et al. 2009). This information led to the question of whether Southern California abalone are able to take significant nourishment from this

NIS seaweed.

It was determined that DNA analysis of gut content samples would provide confirmation that the sampled abalone had consumed S. horneri in the recent past. Two sampling dates would provide information about any seasonal differences in consumption. Using a modified Roving Diver Technique, 30 animals were collected on each sampling date. On each date 10 abalone were collected from each of three sampling sites. The samples of gut content were prepared, frozen and sent to the UC Davis Real- time PCR Research and Diagnostic Core Facility for analysis. After analysis, the lab reported percentages of the total DNA in the sample from S. horneri, from H. fulgens, and from all other sources in the samples (Table 1, Table 2).

In brief, the data showed that the sampled animals had in fact recently consumed

S. horneri in statistically significant amounts. Gut samples from animals collected during the end of the growing season, in May 2016, showed that 24 out of 30 animals had

47 recently consumed at least some S. horneri. Of those samples, the mean percentage of S. horneri DNA in the gut content sample was 13.50 percent, while the median percentage was 0.39 percent.

Gut samples from animals collected during the beginning of the growing season, in November 2016 showed that 28 out of 30 animals had recently consumed at least some

S. horneri. Of those samples the mean percentage of S. horneri DNA was 20.20 percent, while the median percentage was 14.19 percent. From the data it appears that green abalone consume significant amounts of S. horneri at both the beginning and end of the alga’s lifecycle with an increase in consumption shown toward the beginning.

Recommendations

This study is a step in understanding how California abalone have adapted to the

NIS seaweed S. horneri. Given the new reality of the abundance of S. horneri, the ultimate goal of this line of study would be to encourage reintroduction efforts that would lead to a viable abalone fishery.

Resources for abalone seeding are limited. As a consequence, evidence that abalone can thrive in S. horneri dominated areas would show that seeding in those areas was not a waste of resources. There are several recommendations for further study that would inform future abalone reseeding efforts.

Recommendations for further study:

 Repeat the process of qPCR analysis of abalone gut content samples incorporating more seaweed species and a larger sample size. S. horneri dominance has led to changes in the floral profile of the shallow, rocky reef habitat. It would be useful to have further data about what seaweed is being consumed and in what

percentages by California abalone. It would be desirable to repeat the study with more gut content samples from a greater number of animals. In addition, a survey of the relative abundance of potential food sources in the area would better determine if abalone are seeking out S. horneri or just consuming it because few other choices are available.

 A growth study of juvenile and mature California abalone fed an exclusive diet of S. horneri. Knowledge of growth rates of abalone fed nothing but S. horneri would be a vital step to knowing if abalone reintroduction would be appropriate in areas overgrown by the seaweed. In the past there have been feeding studies that measured California abalone growth when fed a variety of seaweeds, but none have incorporated S. horneri (Ambrose and Nelson 1982; Leighton 2000).

 A comprehensive survey of California abalone populations in areas affected by S. horneri compared to similar but unaffected areas. Informal observations have shown healthy populations of abalone in S. horneri dominated areas. A scientifically sound comparison of populations of juvenile and adult animals in the two different areas would better reveal if abalone are thriving in S. horneri dominated areas.

 Continued research into abalone deck spawning techniques. Deck spawning allows for quick and efficient creation of fertilized abalone larvae (Reynolds et al. 2015). When this process is perfected it would facilitate effective and relatively inexpensive reseeding efforts.

 A multi-year experiment examining reseeded abalone survival rates in S. horneri dominated areas. Ideally the study would incorporate both seeded larvae and juveniles. Surveys would be conducted at test plots over the course of months and years to determine if the reseeding efforts were, indeed, increasing the abalone population in the study areas. This would be a key test to evaluate if reseeding was tenable in areas affected by S. horneri.

Conclusions

The results of this study indicate that green abalone are eating S. horneri in significant amounts. S. horneri was detected in 87 percent of the 60 H. fulgens gut samples. S. horneri has largely supplanted giant kelp as the primary producer in countless hectares of shallow rocky reef environment in Southern California. It has changed the

49 ecosystem where many species of California abalone live. It is not known what the future holds for the status of kelp forests in Southern California. However, it is clear that S. horneri will be plentiful into the future.

The return of a healthy abalone population in Southern California would have vast ecological and economic benefits. How can abalone restoration move forward given the new reality of the dominance of S. horneri in Southern California waters?

For better or for worse, economics is the engine that drives progress in many areas of endeavor. If abalone restoration can be tied to commercial efforts it will be advanced quicker than if left up to limited public resources. After further study of the question of whether abalone can thrive in S. horneri dominated areas, the practice of sea ranching in those areas may provide a viable means of abalone recovery in Southern

California.

Sea ranching is a practice where juvenile animals are released into the natural environment and allowed to grow without containment structures. The environment provides the animals with habitat and food with no additional feeding or care required.

For sea ranching of shellfish such as abalone, a lease for a specific portion of seafloor would be granted to the ranching company (Bell et al. 2008). The lease would give exclusive rights to the company to harvest abalone from that particular area.

A successful example of sea ranching comes from the geoduck clam farming industry in the Pacific Northwest. Ranchers secure a lease to a certain area of public tidelands for a specific number of years, then go about increasing the geoduck population on their lease holdings. Ranchers harvest geoducks from the wild and spawn them in on-

50 land hatcheries. Once the larvae have settled to the bottom and grown for several weeks, they are put in floating seed nurseries in the waters of Puget Sound. After a year the half- dollar-sized clams are planted into the mud in the leased tidelands. There they grow until ready for harvest (Le 2015). This practice has benefits for increasing geoduck populations in surrounding waters. As the geoducks reach reproductive maturity they spawn naturally and the larvae seed the entire local area (Dumbauld et al. 2009).

Sea ranching of abalone in Southern California could have similar benefits.

Ranchers would seed their lease holdings to insure a profit in future years and increase the overall abalone population. Advances in deck spawning methods could make abalone seeding less resource intensive and efficient enough to be economically viable. Abundant abalone populations in leased areas would lead to increased larval dissemination to neighboring areas. This could in turn lead to the restoration of healthy abalone populations in the surrounding waters of Southern California and Baja Mexico. In time abalone populations could increase enough to reopen both traditional commercial and recreational fisheries.

Throughout the centuries native California kelp forest habitat has produced a rich and vibrant underwater ecosystem in the region. Human-mediated and natural influences have led to a decline in the productivity of this system. The proliferation of S. horneri is just one of the many factors. This project indicates that green abalone have adapted their diet to incorporate S. horneri. After further research, abalone reintroduction in S. horneri dominated areas might become an accepted practice.

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