An Investigation of Tree Bat Migration Ecology Using Fatty

AN INVESTIGATION OF TREE BAT MIGRATION ECOLOGY USING FATTY

ACID SIGNATURES

Jeffrey Crawford Clerc

A Thesis Presented to

The Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree

Master of Science in Biology

Committee Membership

Dr. Joseph M. Szewczak, Committee Chair

Ted J. Weller, Committee Member

Dr. Jeffrey B. Schineller, Committee Member

Dr. Daniel Barton, Committee Member

Dr. Richard N. Brown, Committee Member

Dr. Michael Mesler, Graduate Coordinator

May 2015

ABSTRACT

AN INVESTIGATION OF TREE BAT MIGRATION ECOLOGY USING FATTY

ACID SIGNATURES

Jeffrey Crawford Clerc

Bat migration ecology is an important, yet understudied facet of natural history and conservation. We know little about the movement patterns of migrating bats and the physiological demands that bats experience in preparation for and during migration. This has particular relevance to tree bats (Lasiurus spp. and Lasionycteris noctivagans) that make the longest annual migrations across North America and have recently become subject to alarming fatality rates by wind turbines. As the absence of effective methods to study tree bat migration has made conservation efforts challenging, I investigated the potential for a non-lethal lipid extraction method (fine needle adipocyte aspiration). This method uses fatty acid signatures as an intrinsic geo marker potentially capable of answering questions about migration ecology. If fatty acid signatures remain stable throughout migration, they have the potential to indicate an individual’s diet at their origin. Samples taken during the resident period can tell us about the dietary shifts that may take place prior to migration. These samples can indicate the summer residence region of migrants sampled during migratory movement. Of 136 attempts, our lipid extraction method had a 72.79% success rate. I used fatty acid signatures produced from ii

fine needle adipocyte aspiration to compare a group of resident and a group of migrant silver-haired bats. I used a multivariate statistical approach for our analysis and found significantly different fatty acid signatures between residents and migrants. It remains

unclear whether geographic segregation or temporal shifts in diet are driving the

separation of migrants and residents. However, I observed greater fatty acid signature dispersion in migrating individuals, which led to the conclusion that if geographic segregation caused the observed separation, then more than one unique resident group used the migration route where we captured and sampled bats. But if temporal shifts in diet caused the separation, then it may indicate that the bats began to exhibit varied individual dietary preferences during migration. However, the latter seems less likely; we

expect the bats to form the majority of their fat deposits during summer residency, and

we also expect that they would have a net depletion of fat reserves during migration. The

results of this study provide a new non-lethal approach to studying bat migration ecology.

iii

ACKNOWLEDGEMENTS

This project is the culmination of the minds and hearts of many. I am forever grateful to everyone who dedicated his or her time and energy to making this research a reality. I am especially grateful to my advisor, Dr. Joe Szewczak, for not only giving me the opportunity to become his graduate student, but for the way he encouraged me to ask questions that I couldn’t imagine we could ever start to answer. Ted Weller was my primary collaborator on this project. He gave me the opportunity to learn the skills of batting and spent countless hours letting me bounce ideas off of him. I am extremely grateful to have been able to get to know him. I appreciate Dr. Jeffrey Schineller for getting me through the lab analysis and for giving up his lab space and time to me. No person spent more time being hounded by my questions than he did. Dr. Daniel Barton encouraged me to go to graduate school in the first place and was always there to bounce ideas around with through both graduate and undergraduate school. Dr. Rick Brown helped me to navigate the IACUC process and gave critical advice in developing our fat extraction method. My lab mate, Alyson Brokaw, helped me train my field crew, navigate grad school, and has being a great friend. Bern Fahey took me in the field with him and is the nicest dude I know. Skylar Giordano taught me how to hold a bat. Hobo crew: Christen Long, Katelyn Southall, and Craig Zurek dedicated their summer and fall to this project. Christen and Katelyn also dedicated their winter and spring to my project.

They not only made this experience possible but enjoyable as well. The redwood hoary

bat crew: M3, Matt Parker, Matt Lau, Shannon Mendia, Rachel Nypaver, Matthew Scott,

and Cory Andrikopoulos, contributed countless nights of field assistance. Tamar

Danufsky helped me identify where the fat is on tree bats. Thor Holmes helped acquire

carcasses from the vertebrate museum. Leila Harris donated carcasses to the project.

Warren Carter sent me his lab protocol for FAME analysis. David Orluck has been my

best buddy through grad school. Melissa DeSiervo helped me learn the R package Vegan

and geeked out on statistics with me. Tom Rickman was an incredible host in Lassen

National Forest. Danny Yencich edited a draft of my proposal. Darrell Burlison helped

me order gear. Dr. Matthew Hurst dealt with me trying to trouble shoot problems that

didn’t generally exist on the GC-MS. The HSU Biology department provided partial funding for this project. To everyone else who came out in the field and lugged a triple high down the creek or helped me in other ways during my time in Humboldt County, I am grateful.

Most importantly, I am grateful to my family: Bettie and Joe supported me and gave me a place to exhale for the past 8 years, my Dad for being hilarious, my father-in-law for always giving me great advice, and my mom and sister who are always loving and interested in what I am up to. I am thankful for my wife, Laura, who keeps me grounded, in love, and in the moment. No one has sacrificed more than she has for this project. My son, Remy LeBeau, has added width to my life and joy in my soul. This project and life is dedicated to my son, Truman Montgomery, for showing me what it means to be human and for teaching me that all we are guaranteed is this moment.

TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iv

LIST OF FIGURES ...... vii

INTRODUCTION ...... 1

Study Sites ...... 9

Bat Capture ...... 9

Fine Needle Adipocyte Aspiration ...... 10

Lipid Transesterfication, Phase Separation, and GC-MS Analysis ...... 15

Statistical Analysis ...... 16

RESULTS ...... 20

DISCUSSION ...... 27

LITERATURE CITED ...... 33

LIST OF FIGURES

Figure 1. Dorsal view of a hoary bat (Lasiurus cinereus) carcass that was donated to the Humboldt State vertebrate museum after the individual died in captivity at the Humboldt wildlife care center showing the adipose tissue. Black arrow is the needle insertion site. Dotted lines represent graphically where the spine and the hips are located. The solid blue circle represents the location of the pelvis. The solid black line represents where the wall of the body cavity is located……………………………………………………………. 26

Figure 2. A big-brown bat being restrained for fine needle adipocyte aspiration. The person performing the fine needle adipocyte aspiration creates the tent with one hand while using the syringe with the other. The bat is restrained by the medical tape on its legs to the processing surface and the assistant who firmly holds the rest of the bat in the bat bag…………………………………………………………………………………... 27

Figure 3. Average transformed proportion of individual fatty acids in the migrant group (dark bars) and the resident group (light bars). Bars indicate means ± standard error and significance levels calculated by pairwise MANOVA. * P < 0.05, ** P<0.001………31

Figure 4. Nonmetric multidimensional scaling (NDMS) ordinations of transformed fatty acid signature composition data for micro lipid samples taken from a group of silver- haired bat residents captured in Lassen county, CA. (black triangles) and migrants captured in Humboldt county CA. (grey circles). Lines represent the convex hull displayed for both groups……………………………………………………………… 32

Figure 5. Analysis of multivariate homogeneity of group dispersions using transformed fatty acid proportions to calculate the bray-distance principle coordinates and group centroids for a group of silver-haired bat residents captured in Lassen county, CA. (black circles) and migrants captured in Humboldt county CA. (empty triangles). Dotted lines represent the convex hull for each group. Solid lines are a graphical representation of the each individual’s distance to the group centroid………………………………………. 33

Figure 6. Beta flexible cluster analysis with two pruned connections using transformed fatty acid proportions. The cluster analysis shows 3 possible unique resident groups of silver-haired bats using HRSP as a migration route. The largest cluster group represents all of the Lassen county resident individuals as well as some individuals sampled during migration in HRSP. The other two smaller clusters are composed of migrants only and may represent unique resident groups…………………………………..………………. 34

vii

INTRODUCTION

Understanding the ecology of migration is a critical part of conservation, as many

organisms have life histories that require annual seasonal movements (Hansson and

Akesson 2014). Combining spatial and physiological aspects, migration ecology

encompasses a wide range of disciplines. These disciplines, when considered together,

can help predict migratory patterns across the range of a species. Specifically, migration

ecology is interested in answering questions about the origins of migrating individuals,

the number of unique sub-populations being supported by a migration route (uniqueness),

and the limiting factors, such as diet, that shape migration through space and time.

Knowledge about the seasonal movements of bats and the life history traits that propel their migrations is vital to informing bat conservation. Understanding the migration ecology of tree bats (Lasiurus spp. and Lasionycteris noctivagans) is especially important, as they make the longest annual migrations of any bat species (Cryan 2003).

Annual movements are a large part of tree bat life history, as they migrate up to 2000 km from summering grounds to temperate winter residences. Migratory events allow tree bats to avoid hibernation and remain active year – round (Cryan 2003, Flemming and

Eby 2003, Weller and Stricker 2012, McGuire et al. 2012). This can be especially

important to fitness, as reproductive success may rely on an individual’s ability to

encounter mates along migration routes (Cryan et al. 2012, Vonhof and Russel 2015).

In recent years, tree bats have experienced unprecedented mortality rates at wind

energy facilities located along migration routes (Cryan and Barclay 2009). Wind turbines

2 kill thousands of tree bats per year through blunt force trauma from collisions with turbines (Rollins et al. 2012). Pulmonary barotrauma due to rapid changes in pressure near moving turbines may also be a cause of mortalities at wind farms (Baerwald et al.

2008). Conservationists seek ways to mitigate tree bat fatalities at wind energy facilities, but are hampered by limited knowledge of migratory origins/destinations, uniqueness, and the physiological shifts that may occur prior to migration. Knowing the resident origin of a migrating bat can lead to the identification of migration routes and help focus conservation efforts. Uniqueness is important in determining how many unique resident groups are using a migration route. This can help us understand which migration routes support the highest amount of group variation, leading to preservation of diversity. It is equally import to gain information about the physiological shifts that occur prior to migration in order to understand adaptations and trade-offs that tree bats use to propel long distance movements (McGuire et al. 2013). Answering these questions will help guide management efforts by allowing for differential conservation prioritization of certain geographic regions that are important for tree bat migration. However, these questions remain unanswered due to a lack of technology for studying tree bats.

Common methods used to study migration in other taxa include GPS tracking, geo-loggers, mark-recapture techniques, genetic studies, and stable isotope analysis

(Robinson et al. 2009). Physiological aspects of migration are difficult to study without destructive sampling to acquire fatty acid samples or other tissues in large enough quantities to satisfy analysis protocols. GPS tracking devices are too heavy to be safely carried by tree bats without interfering with their flight endurance. Low recapture

3 probability of tree bats makes manual recovery of geo loggers difficult or unlikely

(Weller 2007). Low recapture rates have also thwarted attempts to implement mark- recapture studies (Weller 2007). Research using genetic markers to infer population connectivity is ongoing. But because populations mingle and begin mating during and after migration (Druecker 1972), tree bats have low genetic structure due to high gene flow (Moussy et al. 2013, Vonhof and Russell 2015). In addition, the dispersed roosting habits and cryptic pelage of tree bats renders most traditional wildlife study methods ineffective and presents a substantial challenge to studying their migration ecology.

Although no method has fully met this challenge, stable isotope analysis has allowed researchers to use isoscapes, spatially explicit stable isotope ratios on the earth’s surface, to infer migratory origins and migration routes (Cryan et al. 2014, Popa-Lisseanu et al.

2012, Wunder 2010). The stable isotope ratio of a given isoscape is preserved in the inert fur of bats as they acquire a new pelt during the summer molt (Cryan et al. 2004,

Rubenstein and Hobson 2004). Bats can then be assigned back to their origin following capture during migration. Stable isotopes have also proven valuable for determining migration-fueling strategies (Voigt et al. 2012). By measuring the stable isotope ratios of the exhaled breath of bats, it can be determined what molecules of prey items are being oxidized and what molecules are being stored.

Although an established technique, the resolution of stable isotope analysis provides results limited to broad isoscapes that can encompass an area up to hundreds of thousands of square kilometers (Wunder 2010). This makes it difficult to not only discern how many unique resident groups might use a migratory route, but to identify the

4 migratory route itself. And though understanding the fueling strategy of bats is critical, stable isotope analysis cannot tell us what physiological changes occur in response to a particular fueling strategy. However, the use of fatty acid signatures provides an analytical technique that may prove useful in bat migration ecology.

Fatty acid signatures are proportional matrices that comprehensively represent all of the fatty acids present in an individual’s adipose tissue. Researchers have used fatty acid signatures to highlight spatial and temporal variation in diet between unique populations of both seabirds and fishes (Iverson et al. 2007, Wang et al. 2009,

Pethybridge et al. 2014). The premise of using fatty acid signatures in migration ecology follows from the understanding that fatty acids acquired directly by a predator remain mostly metabolically unaltered following the ingestion of the prey providing the fatty acids (Budge et al. 2006). As prey communities across habitat types vary in their fatty acid compositions, so too will the adipose tissues of predators that feed on them. If differences in fatty acid signatures between predators occupying distinct habitats with differing prey resources can be identified, then population assignments can be made on a geographic resolution at the scale of biome/ecosystem. Depending on what body material is sampled, a fatty acid signature can represent an individual’s diet over the course of a few weeks to many months, varying temporally and spatially due to the availability of exogenous fatty acid sources (Budge et al. 2006). For instance, the fatty acid signature of blood would be a reflection of what the individual was feeding on in the short term

(hours) as opposed to the fatty acid signature of a lipid deposit, which would represent the cumulative diet over a longer period of time (Budge et al. 2006). Analyzing fatty acid

5 signatures during the resident period may allow for the investigation of temporal shifts in fatty acid composition, when bats begin to store fat to fuel their migration (McGuire et al. 2012). During migration, fatty acid signatures may be able to be used as intrinsic geo markers that represent the location where individuals acquired their fat reserve during the resident period. As individuals occupying the same resident sites will have similar prey access, their fatty acid signatures will likely be more similar than individuals occupying different resident sites.

For fatty acid signatures to be applied effectively to bat migration ecology, some limitations must be considered to determine if temporal or spatial processes are driving any observed group differences during the migration period. To infer origins from bats collected along migration routes, the fatty acid composition of the adipocyte must remain stable during migration compared to the resident period. If bats feed and have net fat deposition during migration, there is the possibility that the fatty acid signatures may not accurately represent the summer resident site. This is important because fat acquired from feeding is likely not oxidized for immediate use, but stored in the adipose tissue even when the metabolic demands of migration peak (Voigt et al. 2012). However, the reduction of digestive organs and a reduction in fat stores during the migratory period observed in one species of tree bat, hoary bats (Lasiurus cinereus), suggests that tree bats minimize the time spent foraging during migration compared to the resident period, and likely reduce rather than add to their fat stores during migration (McGuire et al. 2013).

The other limitation that must be considered in using fatty acid signatures is the homogeneity of the fat deposit. Because only a portion of lipid material is being used for

analysis, it is important to be sure that lipid samples are representative of the fat deposit

as a whole. Although a homogenous fat deposit has not been confirmed in bat species, the

fat deposit of terrestrial mammals is thought to be homogenous (Budge et al. 2006). This

has been confirmed in mink (Mustela vison), and polar bear (Ursus maritimus) (Layton et

al. 2000, Thiemann et al. 2005). Addressing these issues more directly is beyond the

scope of the current study. Establishing the rate of change to the adipose tissue

composition during resident periods and migratory periods, and verifying the

homogeneity of the fat deposit, is critical to applying fatty acid signatures to bat

migration ecology in the future.

McGuire et al. (2013) found differences in lipid compositions between migrant and resident hoary bats when comparing individual fatty acids. While it was unclear what might be driving the observed differences, Price et al. (2014), later suggested that it could be due to variation in diet. From this research, many questions remain unanswered and the possibility of applying fatty acids in new ways exists.

McGuire et al. (2013) used destructive methods to sample the lipid material in their study. Destructive sampling makes it difficult to conduct large-scale studies that would be as effective when considering bat migration ecology because robust sample sizes cannot be obtained. As a result, developing a non-destructive method for gaining

access to the adipose tissue is critical to the investigation of differences between migrants

and residents, and for the exploration of further applications of fatty acid signatures.

I had two goals for this study: first, to develop a method for safely extracting

micro lipid samples from live bats in the field, and second, to apply the method to

7 investigate whether fatty acid signatures could reveal seasonal movements of tree bats and help elucidate their migration ecology. My goal led me to predict that I would detect different fatty acid signatures between a unique inland summering population and a population of coastal migrants of unknown origin by using our lipid extraction method coupled with the analysis of fatty acid signatures.

MATERIALS AND METHODS

Study Species

We investigated the long distance migratory tree roosting silver-haired bat (Lasionycteris

noctivagans, 8-14 g mass) as the target species for this investigation. This species ranges

across North America (Cryan 2003), but we sought a field site where we could capture

summering residents and a fall field site where we might expect to capture migrating

individuals, possibly including those from the summer site. Silver-haired bats roost in cavities and crevices preferring forest stands older than 200 years (Perkins and Cross

1988). They are generalist opportunist predators that consume a variety of insects (Kunz

1982). Moths (Lepidoptera) constitute the greatest contribution to diet, but they also eat prey items from 9 other insect families including termites (Isoptera), flies (Diptera), and caddisflies (Trichoptera) (Kunz 1982).

Historical occurrence records suggest that the distribution of silver-haired bats changes seasonally throughout North America. Individuals winter in the Pacific

Northwest and Southwestern United States and then expand their distribution northward and eastward in the spring and summer (Cryan, 2003). Summer sexual segregation is often observed in silver-haired bats (Cryan 2003,Weller and Stricker 2012). This is likely due to the specific roosting requirements that pregnant females have, limiting their summer range to warmer climates (Barclay et al. 1988, Racey and Entwhistle 2000).

We also opportunistically sampled big brown bats (Eptesicus fuscus, 14-17 g

mass). Big brown bats are not considered obligate tree roosting bats, and they make short

seasonal migrations that are typically less than 50 km (Davis et al. 1968). We chose to

sample big brown bats to determine the efficacy of the fat extraction method on a

different species, and to compare our results across species.

Study Sites

We captured bats in Lassen National Forest (NF), Lassen County, California,

USA (Lat: 40.367240, Long: -120.802824) and Humboldt Redwoods State Park (HRSP),

Humboldt County, California, USA (Lat: 40.352316, Long: −123.985523).

It is possible that some Lassen NF summer tree bat residents overwinter there by

hibernating, as it has been documented that in some isolated cases silver-haired bats may

hibernate in caves during the winter (Verts and Carraway 1998, Beer 1956). However, no

large groups of hibernating silver-haired bats have ever been found. This makes it likely,

that most silver-haired bats spending the summer in Lassen NF migrate in fall and

overwinter in more temperate climates. Bull Creek in Humboldt Redwoods State Park

supports a large number of migrating silver-haired bats and hoary bats each fall and spring and also supports a winter resident population of silver-haired bats (Kennedy et al.

2014).

Bat Capture

Resident target species were captured intermittently from 17 July - 13 August,

2014, for a total of 9 occasions with mist nets set across Williard Creek, Lassen National

Forest, Lassen county, California, USA. Presumably migrating target species were

captured from 27 August - 29 October, 2014, for a total of 35 occasions (20 with target

species captures), with mist nets set across Bull Creek, Humboldt Redwoods State Park,

Humboldt County, California, USA (Lat:40.352316, Long: −123.985523).

I used a mixed mist net array based on the landscape features of each capture site

following the mist-netting protocol, bat removal technique, and bat handling techniques

outlined by Kunz et al. (2009). On a given night we used anywhere from 2 to 5 6m, 9m,

or 12m nets. I used up to 3 triple high net systems per night. After extraction from mist

nets, bats were held in cotton bags. All bat handling followed white nose syndrome

decontamination protocol (USFW 2011). Upon capture, we measured forearm length,

weight, sex, reproductive status, and age. I determined age by the degree of ossification

of the phalangeal-metacarpel joints (Brunet-Rossinni and Wilkinson 2009). After the bats were released, we counted the number of guano pellets that they produced and deposited in the cotton holding bag.

Fine Needle Adipocyte Aspiration

Fine needle aspiration is a type of tissue biopsy technique performed routinely in veterinary and human medicine to obtain small biological samples. During a fine needle aspiration procedure, a needle (18 gauge – 23 gauge) is inserted subcutaneously into a

subject and directed to the area of interest. The needle is attached to a 3-10 cc syringe.

Once the needle is directed into the area of interest, negative pressure is placed on the

11 needle by drawing the syringe plunger back. While drawing out the plunger to apply a gentle suction, the needle can be gently redirected multiple times until the desired amount of sample is obtained. The needle is removed from the patient after the negative pressure being applied to the plunger stops. Fine needle aspiration is considered to be the least invasive biopsy technique as it is non-surgical and uses relatively small-gauged needles

(Wu and Burnstein 2004).

We used 13 bat carcasses (9 Tabr, 1 Lano, 2 Anpa) that died at a wildlife rehabilitation center in Sacramento, CA to develop our method of extracting micro lipid samples. I first identified the adipose tissue deposition across all carcasses, and second, determined the best tools for extracting lipids with animal safety in mind. We found the most consistently abundant adipose deposit on the carcasses to be located on the lower lateral abdominal region of the bats (Fig 1). In addition, the lateral and dorsal position of this deposit facilitated restraint and sampling of bats in the field. This enabled us to hold bats dorsal side up, as bats will struggle to resist restraint in a ventral side up position

(Joseph M. Szewczak, pers. Comm., 2014).

For this study we extracted micro lipid samples (< 0.2 mg) from the lower lateral abdominal region using fine needle adipocyte aspiration (FNAA). To ensure the safety of the bat, we required two people to complete the procedure in the field. One person was responsible for securing the bat while the other person obtained the fat sample. To complete the procedure, we used the following equipment: a plastic processing surface, alcohol wipes, medical tape, 23 gauge needles, 3-5 cc syringes, gauze, and a 1 ml micro- reaction vial. Using the cotton bag that the bat was being held in, the bat was situated

12 such that the legs were facing the opening of the bag and the bats wings were closed.

Then, the bag was opened so that the bottom of the body (feet to underneath the rib cage) was outside the bag and the top half of the body (bottom of rib cage to rostrum) was inside the bag (Fig 2). The bat was centered in the bag at this point so that the bag could be used to secure the bat in place by holding the top and bottom of the bag taut on both sides of the bat. The bat was then held securely ventral side down against a small piece of plastic processing surface, using the cloth bag pinched taut on both sides of the bat for restraint.

While the bat was being held on the processing surface, the person performing the

FNAA held one leg at a time and gently extended the leg at a 45 degree angle away from the body placing a piece of medical tape across the knee. In this position, the bat was not able to lift and curl its uropatagium onto itself (Fig 2). The person holding the bat continued to keep the bat secure for the duration of the FNAA. I applied alcohol using an alcohol wipe to the lower lateral abdomen of the bat. After the bat was secure and the lower lateral abdomen was cleaned with isopropyl alcohol, I performed the FNAA.

First, I felt the bat and confirmed that subcutaneous lipids were present in the lower lateral abdominal region. I developed a palpation guideline to identify the adipose deposit and the needle insertion site. I used the midline (spine), pelvis (represented by blue dot in Fig 1), and abdominal wall (shown by dotted lines in Fig 1) as palpation guidelines in the field to help locate the adipose deposit and guide the decision for the best needle insertion site.

With the bat secured to the processing surface, I performed the FNAA by first

placing my pointer finger on the center of the bat’s pelvis. Then, I moved my finger up

the vertebrae approximately 10-15mm and laterally until feeling where the abdominal wall met the adipose deposit. Next I moved my thumb laterally from the center of the pelvis to the hip. I then moved my thumb approximately 4mm towards the anterior to avoid branches of the iliac artery that run near the hip. With the adipose deposit located, I identified the needle insertion site as the point just above my thumb at the base of the adipocyte (Fig 1). If I failed to identify the adipose deposit using this palpation procedure, perhaps due to emaciation or otherwise poor condition of the bat, I did not take a fat sample from that individual. Under these conditions, the bat was immediately released at the capture site. I considered the ability to palpate and target the adipose

deposit prior to needle insertion a requisite skill that I practiced on carcasses prior to

applying it in the field on living specimens.

Once I identified the adipose deposit, I used my free hand to lift and pinch the

skin away from the body creating a “tent” at the needle insertion site (Fig 2). The needle

was then inserted with the bevel up and then rotated 180 degrees such that the bevel was

facing down and directed parallel to the abdominal wall. I then aspirated a lipid sample

less than 0.2mg from the lower lateral flank adipose deposit. I re-directed the needle 6

times or until I could see some lipid in the hub of the needle. Performing the fine needle

aspiration at the lower lateral flank deposit provided the additional benefit of avoiding the

risk of entering the abdominal wall or other body cavity of the bat. The entire process

lasted approximately 90 seconds. While removing the needle, I simultaneously placed

gauze at the needle insertion site, and the person holding the bat took over using the

gauze to keep pressure on the needle insertion site. After holding the gauze in place for

approximately 10 seconds, the medical tape was removed from the legs of the bat, and

the bat was released.

After the FNAA, a small sample was held inside of the needle. To avoid sample

oxidation, the sample was expelled into a PTFE-lined screwcap 1 ml micro-reaction vial

(Wheaton USA) and then placed on dry ice until it was transported to a - 80 C freezer. If no sample was visibly expelled into the micro-reaction vial, I placed approximately 5 drops of distilled water into the micro reaction vial, pulled it into the needle, and then expelled it back into the vial. This was an attempt to flush out any material that may have been stuck on the needle.

During the summer resident and autumn migration periods, we only sampled adult silver-haired bats and big brown bats that were greater than 9 grams and 14 grams respectively. During the summer resident period, we did not sample adult females that were pregnant or lactating.

Humboldt State University Institutional Animal Care and Use Committee

(Protocol no. 13/14.B.103-A) gave us the approval to capture all of the bats for this study.

The study was conducted under California State Fish and Wildlife scientific collections permit no. 013013.

Lipid Transesterfication, Phase Separation, and GC-MS Analysis

For lipid transesterfication and phase separation, we used sub-microscale in situ

(SMIS) method. For a detailed explanation of the method see Bigalow et al. (2011).

Frozen samples were placed in a centri-vap (Labconco, Kansas City, MO, USA) at 40 ℃

for 3 hours to remove water present in the sample. Afterward, 200 µl of analytical grade

methanolic boron trifluoride (1.3M) (Sigma-Aldrich, St, Louis, MO, USA) I added to each vial, and samples were flushed with nitrogen, and then vortexed for 10 seconds. I then placed the vials in a heat block for 1 hour at 100 ℃. After the vials cooled, I added

250 µl of analytical grade isooctane (Sigma-Aldrich), followed by 500 µl of reagent

grade sodium chloride (Sigma-Aldrich). In between the addition of isooctane and sodium

chloride, I flushed each sample with nitrogen for 10 seconds and vortexed each sample

for 10 seconds. The samples separated after the final vortexing, with the isooctane

containing the fatty acid methyl esters (FAME’s) rising to the top. I then used a 9-inch

glass Pasteur pipette (Fisher Scientific) to transfer the FAME containing isooctane to GC-

MS vials (Varian, Palo Alto, CA, USA). I added sodium sulfate (Agilent, Santa Clara,

CA, USA.) to dry the sample. I then began the GC-MS analysis using 1µl split-less injections of our sample.

I performed the GC-MS analysis on a HP 5890 gas chromatograph and a HP 590 mass spectrometer. I used a 30 m x 0.25 mm x 0.20 µl HP-88 high-resolution polar gas chromatography column (Agilent, USA). Both the injector and detector temperature was set at 250 ℃. The method started by holding an initial temperature of 50 ℃ for 1 min. I

16 then ramped up the temperature rapidly at 45 ℃ per minute to 153℃ where I held the temperature for 2 minutes. I then ramped up the temperature by 5 ℃ per minute until I reached the final temperature of 230℃. I held the temperature at 230℃ for 2 minutes. I identified FAME’s by comparing the retention time of the samples with the retention times of a 28-component external standard (NLEA FAME mix, Restek, USA).

Statistical Analysis

I identified 16 fatty acids present in the samples, but constrained the analysis to 8 long chain fatty acids that had the greatest variance and that were greater than 1% of the total fatty acid proportion. I considered the following fatty acids for this analysis: C16:0,

C16:1, C18:0, C18:1cis, C18:1trans, C18:2, C20:0,C20:1. Since proportional fatty acid signature data are not normal, I transformed the fatty acid proportions for the analysis as recommended by Budge et al. (2006) using the following formula from Aitchinson

(1986): Xtransform = ln(xi/Cr), where Xtransform is the transformed fatty acid data, xi is a given fatty acid in the data-set expressed as a proportion of total fatty acid composition, and Cr is the percentage of a within data-set reference fatty acid. I followed the recommendation of Budge et al. (2006) and used C18:0 as our reference fatty acid.

All statistical analyses were conducted with RStudio (version 0.98.501)(R Core

Team 2012). I used a pairwise multivariate analysis of variance (MANOVA) to assess differences among percent totals of individual fatty acids between migrants and residents.

I used a non-metric multi-dimensional scaling (NMDS) approach to ordinate and graphically represent and assess the Bray-Curtis distances of two groups: migrants and

17 residents. The analysis was completed with the function metaMDS in the R package

Vegan (Oksanen et al. 2013). I calculated the area of the convex hull and the multivariate homogeneity of group dispersions of the two groups around the centroid using the ordihull and betadisper functions in the R package Vegan (Oksanen et al. 2013). I conducted a hierarchical cluster analysis using a generalized weighted average linkage method, beta-flexible cluster analysis, to investigate how many potential unique resident groups were using HRSP as a migration route (Milligan 1989). I performed the cluster analysis with the function agnes in the R package cluster (Maechler et al. 2015). I tested differences between group (migrant and resident) and sex using a non-parametric, permutational multivariate analysis of variance (PERMANOVA). I also tested intra- group differences in sex and sampling period (fixed by week) using PERMANOVA. The analysis was completed with the function adonis in the R package Vegan (Oksanen et al.

2013). I performed a similarity percentage test (SIMPER) to determine the fatty acids most likely driving any observed dissimilarity. The sample size for big-brown bats was small (12 individuals) so we did not include any statistical analyses comparing species.

Figure 1. Dorsal view of a hoary bat (Lasiurus cinereus) carcass that was donated to the Humboldt State Vertebrate Museum after the individual died in captivity at the Humboldt wildlife care center showing the adipose tissue. The black arrow is the needle insertion site. Dotted lines represent graphically where the spine and the hip line is located. The solid blue circle represents the location of the pelvis. The solid black line represents where the wall of the body cavity is located.

20 RESULTS

I performed fine needle adipocyte aspiration on 121 silver-haired bats and 15 big- brown bats. Of 136 total, 99 (72.79 %) fine needle adipocyte aspirations resulted in micro lipid biopsies from which I derived interpretable fatty acid signatures. I could not interpret fatty acid signatures from 22 (16.17 %) attempts because they lacked sufficient lipids for detection during analysis. Seventeen of these samples did not produce any visible lipids when expelled into the micro-reaction vials. I believe that the other 5 samples that did not produce interpretable results were likely composed of connective tissues instead of pure lipids. I eliminated an additional 15 (11.02 percent) samples because they had visible contamination with blood when expelled into the micro-reaction vial for storage. Samples that had blood in them yielded mixed results, but some that were composed of more blood than lipid produced GC-MS results with spurious fatty acid compositions compared with the samples that had no blood present. In the absence of any documentation on the effect of having blood mixed into the sample, we dropped all 15 samples that had blood visibly present.

C16:0, C18:1, and C18:2 accounted for on average 0.82 ± 0.32 of the proportion of fatty acids in the resident group and C16:0, C16:1, and C18:1trans accounted for on average 0.82 ± 0.32% of the proportion of fatty acids in the migrant group. I found differences in the proportion of all individual fatty acids between residents and migrants except for C18:1trans and C20:0 (Fig 3). The greatest differences between residents and migrants were in C16:0 (residents: 0.42 ± 0.16, migrants: 0.28 ± 0.11, F1,83 = 32.00,

21 P<0.0001) and C18:0 (residents: 0.06 ± 0.03, migrants: 0.02 ± 0.01, F1,83 = 53.656,

P<0.0001).

Using NMDS ordination, I observed distinct fatty acid signatures between

migrant and resident silver-haired bats (Fig 4). The 2-dimensional stress for the NMDS

ordination was 0.11 indicating a good fit for our model, and our resulting graphic is a

good 2-dimensional representation of our data. The migrant group convex hull was 3

times the size of the resident group (resident: area= 23.17 %, migrant: area = 73.78%)

(Fig 4). The results of the multivariate homogeneity of group dispersion show that the

migrant group had a larger dispersion than the resident group when the significance level

is alpha = 0.10 (F1,83 = 3.710, P=0.057) (Fig 5). The hierarchical cluster analysis

produced 3 unique groups. Individuals captured in Lassen are represented in 1 group and

individuals captured in Humboldt are represented in all 3 groups (Fig 6).

Using PERMANOVA analysis, I statistically confirmed our observation of group variation (F.model1,83 = 19.373, P < 0.0001) and found no effects of sex (F.model1,83 =

0.302, P = 0.8518). I did not detect any intra-group sex related differences in fatty acids

among residents (F.model1,24 = 1.149, P= 0.32) or migrants (F.model1,57 = 0.51, P= 0.698) in our PERMANOVA analysis. Furthermore, I did not detect any intra-group sampling period variation among residents (F.model2,23 = 0.67, P = 0.65) or migrants(F.model8,50 =

0.8953, P = 0.57).

The result of the SIMPER analysis showed C16:0, C16:1 and C18:1trans to be the

fatty acids that are most likely responsible for driving the difference between residents

and migrants. Cumulatively, C16:0, C16:1, and C18:1trans contributed 72.59 % of the

22 Bray-Curtis dissimilarity between migrants and residents. Furthermore, these fatty acids are three of the most abundant fatty acids consistently identified across individuals.

26 each individual’s distance to the group centroid.

27 DISCUSSION

In this study, I developed a method for extracting micro lipid samples from live

bats in the field and for applying the use of fatty acid signatures to investigate aspects of

silver haired bat migration ecology.

The results on the efficacy of fine needle adipocyte aspiration (FNAA) on silver- haired bats and big brown bats demonstrate that this method can be used in the field successfully. However, opportunities remain to improve the method to reduce sample degradation due to oxidation and to increase the overall success rate above 72.79%. To reduce the potential for sample oxidation, users should directly expel field samples into

1ml micro reaction vials. Then user should fill the vials with approximately 0.2 ml of chloroform (CHCl3) containing 0.01% butylated hydroxytoluene (BHT) before storage on dry ice (field) and placement in a -20℃ freezer. This suggestion follows the recommended method for tissue storage described by Budge et al. (2006). Doing this will reduce the chance for sample degradation that can produce misleading results.

Using CHCl3 may also result in an increased FNAA success rate, as it will work as a better solvent to flush the needle if lipids are not visibly expelled. In the field, when I observed no visible lipid material in the micro reaction vials, I used distilled water to flush the needle. This was done in an attempt to recover any lipids that were adhered to the needle wall. Flushing the needle in 18 cases resulted in 1 recovered sample. The other

17 yielded no interpretable data because no lipids were recovered. Using CHCl3 to flush the needle may result in an increased lipid recovery rate, when lipids are in fact present,

28 because the CHCl3 will act as a better solvent. One concern though for using CHCl3

instead of distilled water to flush the needle is the potential for sample contamination if

the CHCl3 is drawn into the syringe or hub of the needle where it may come in contact

with plastics.

I supported McGuire et al.’s (2013) results that tree bat migrants and residents can exhibit different proportions of individual fatty acids, yet the driving force of these differences remains unknown. It is unclear if the difference is driven by geographic isolation (i.e. differential prey availability across unique resident habitats), temporal

shifts in diet between resident periods and migration, or a combination of both

geographic and temporal effects. I would expect to see temporal shifts in diet leading to

changes in fatty acid signatures over the course of a resident period, but it is unknown

how rapidly individual fatty acid composition changes over time. Furthermore, it is

unlikely that the rate of change in fatty acid composition is constant (Wang et al. 2009).

This makes it difficult to model the rate, as it is possible that rapid changes occur during

the resident period followed by a more static rate during migration. Future research

comparing multiple groups of residents from unique geographic locations will provide a

critical next step and perhaps disentangle the spatial and temporal effects on fatty acid

signatures.

The result of the PERMANOVA confirms that the migrant and resident groups

have distinct fatty acid signatures. Despite this distinction, we found a considerable

number of individuals from the migrant group overlapping in fatty acid composition with

individuals from the resident group (Fig 4). If the group difference is driven solely by

29 geographic segregation, then we expect individuals from the migrant group, those having overlapping fatty acid signatures with the resident group, to have a closely related origin.

Individuals from the migrant group, that do not overlap with the resident group may have a different origin, leading to the conclusion that at least two unique resident populations used HRSP as a migratory route. Our beta flexible cluster analysis suggests that there may be three unique resident groups using HRSP during migration (Fig 6). Additional support for geographic driven separation comes from the fact that the area of the convex hull and the dispersion of the migrant group were greater than the resident group. Further research is needed to determine if the dispersion observed for the residents is typical across multiple unique resident groups. If it is, then it is likely that dispersions that exceed that of the typical resident group may result from the presence of multiple resident groups using a common migration route.

The observed group difference may be driven by geographic segregation of resident groups. FNAA and fatty acid signatures have the potential to help determine resident origins and group uniqueness during migration. If samples are collected during the resident period from multiple unique groups, fatty acid signatures of individuals captured during migration may be able to be used in resident group assignment. This way of applying fatty acid signatures may be especially useful when coupled with stable isotope analysis, such as a triple isotope approach (Popa-Lisseanu et al. 2012), where stable isotope measurements of hydrogen, carbon, and nitrogen are used as signatures to cluster groups of migrating bats and assign origins.

Previous studies have used stable isotope analysis to produce migratory origin

30 resolution on a broad geographic scale. Coupling stable isotope analysis with fatty acid signatures may allow researchers to increase the resolution achieved with stable isotope analysis from a broad geographic scale based on global isoscapes to a narrow geographic scale based on ecosystems. It remains unclear as to what exact scale of resolution may exist and how well unique resident groups will cluster along habitat gradients. But the potential for the use of fatty acid signatures, as a complementary technique in migration ecology, is great. For instance, multiple unique resident groups that exist within the same geographic isoscape may be detected if they occur in varied ecosystems. Conversely, unique resident groups may converge on indiscernible fatty acid signatures, yet stable isotope analysis may enable detectable differences. In this way, coupling the two techniques may provide a better methodology than either method alone, providing a stronger approach to investigating origins and group uniqueness in bat migration ecology.

If the temporal factors alone cause the separation of the observed groups, then we can conclude that this resulted from a shift in dietary preference (or prey availability) during migration and that the turnover rate in lipid composition can be rapid during migration. However, this seems less likely as we expect the bats to deposit the majority of their fat during summer residency and to have a net depletion of fat reserves during migration (McGuire et al. 2013). The increased dispersion observed in the data during migration could also result from more individual preference in diet along migration routes. If the observed differences in fatty acid signatures between the migrant and resident groups are driven by temporal shifts in diet, then the use of fatty acid signatures may not provide an effective intrinsic geo marker for modeling patterns of movement.

31 This would lead to fatty acid signatures only being useful as a way to track changes in

diet over time or to determine the aquatic and terrestrial contribution to overall energetics

during resident and migrant periods (Brownstein et al. 2011).

Both temporal and geographic effects likely contribute to the differences in fatty

acid signatures in tree bats. Temporal differences are likely detectable through the

resident periods when foraging is greater and the adipose deposit is being accumulated in

preparation for migration (McGuire et al. 2013). Geographic differences are likely

detectable immediately prior to and during migration because it is unlikely that rapid

changes in the fatty acid signatures would occur during migration based on decreases in

feeding and the net depletion of the adipose deposit (McGuire et al. 2013). However,

more research is needed to determine the process by which the observed group separation

is driven. Future studies should focus on two primary objectives: 1) The rate at which

fatty acid signatures in a single group change through the resident period and 2) the

sampling of multiple unique summer resident groups to determine the scale of resolution

that fatty acid signatures can obtain.

In this investigation, I developed the methodology of a field ready micro lipid extraction method, fine needle adipocyte aspiration, which can apply broadly to bat ecology. I then used fatty acid signatures to investigate the potential use of fatty acid signatures in answering questions about migration ecology. Using fine needle adipocyte aspiration and fatty acid signatures may prove to be a useful technique in determining the origins, uniqueness, and physiological constraints of migratory tree bats. If research, by way of these techniques, can be implemented, it will be a far-reaching step in bat

32 conservation, given the increase in wind energy facilities across North America. It is feasible that in the future, our understanding of migration routes and uniqueness will lead to informed decisions about placement of wind energy facilities and the optimal timing of shutdowns. This will allow for safe passage of the highest diversity of tree bats. Critical to bat conservation is the continuing development of additional techniques allowing researchers to increase understanding of migratory bat ecology and natural history.

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