Conservation Research on Spotted Eagle Rays in Florida and The

CONSERVATION RESEARCH ON SPOTTED EAGLE RAYS

IN FLORIDA AND THE CARIBBEAN SEA

Project Technical Report Submitted to the National Aquarium Conservation Center

26 January 2012

Kim Bassos-Hull Center for Marine Mammal and Sea Turtle Research Mote Marine Laboratory Sarasota, Florida

Peter Hull, Dean Dougherty, and Krystan Wilkinson Division of Marine Operations Mote Marine Laboratory Sarasota, Florida

Robert Hueter, John Morris and John Tyminski Center for Shark Research Mote Marine Laboratory Sarasota, Florida

MOTE MARINE LABORATORY TECHNICAL REPORT NO. 1595

1 CONSERVATION SIGNIFICANCE

The ultimate goal of the Spotted Eagle Ray Conservation Program is to establish a conservation plan for spotted eagle rays (Aetobatus narinari) in the Gulf of Mexico and Caribbean Sea through collaborative research and multinational relationships. The Spotted Eagle Ray Conservation Program was initiated in 2009 through a conservation partnership between Mote Marine Laboratory and the National Aquarium Conservation Center.

This program provides an excellent opportunity to document long-term trends of a near-threatened elasmobranch species as well as provide training opportunities for researchers from other countries to learn sampling techniques and educational outreach possibilities. By engaging Mexican and Cuban research colleagues we hope to address questions of connectivity of spotted eagle ray populations throughout the Gulf of Mexico and Caribbean Sea, and do conservation capacity training and building in those other countries. Our future goals are to monitor fisheries, conduct live-sampling and tagging of individual rays in Mexico, Cuba, and other Gulf of Mexico and Caribbean countries, and further educational outreach by building on our initial years of research along the west coast of Florida. If results of the biological and fisheries research warrant full protection for spotted eagle rays in the Gulf of Mexico, we aspire to be the driving force to make that happen. If not, we will work to ensure that current harvest levels in the Gulf are sustainable.

We hope our project will serve as a model for multinational collaboration in conservation of migratory elasmobranch species; including sharks, skates and other rays.

PROJECT OVERVIEW

In 2009, Mote Marine Laboratory initiated a new conservation research program in collaboration with the National Aquarium Conservation Center on the life history, reproduction, population status, and captive husbandry of the elasmobranch Aetobatus narinari (Euphrasen, 1790), commonly known as the spotted eagle ray. This beautiful large ray is a prized public aquarium animal and is a protected species in Florida state waters. The spotted eagle ray has a worldwide distribution in tropical and warm temperate regions of the Atlantic, Pacific and Indian Oceans and is heavily fished in many regions. Recent genetic studies have shown that Aetobatus does not comprise a single species (Richards et al. 2009), leading to the conclusion that additional knowledge regarding the number of species, their exact distributions and population sizes is imperative for guiding conservation and management efforts.

2 Eagle rays have been targeted in some harpoon fisheries, are sometimes caught as directed catch or bycatch in net fisheries, and have been used as bait in shark fisheries and as meat for human consumption (Cuevas-Zimbrón et al. 2011). Eagle rays are a specialty fishery in Mexico where annual harvest on the Gulf of Mexico coast is estimated to be 200 metric tons (J.C. Perez Jimenez, ECOSUR, Campeche, Mexico, pers. comm.). This would equal approximately 7,350 individuals (avg. wt. est. at 27kg). The sustainability of this fishery is unknown. Growth rates and age to first reproduction are poorly defined for this species as is the reproductive rate. Although eagle rays are fully protected in Florida state waters, it has no protection in U.S. federal waters and is listed as Near Threatened, with a decreasing population trend, in the International Union for the Conservation of Nature (IUCN) Red List of Threatened Species (Kyne et al. 2006, IUCN 2011).

For many elasmobranch species, there is growing concern regarding harvest sustainability and the need for conservation of at-risk species. However, the conservation status of most elasmobranchs is classified as data-deficient (IUCN 2010), which underscores the need for basic biological and ecological studies necessary for rational conservation decisions. For example, accurate or even any dietary data, needed to assess the role of large elasmobranch predators in marine communities, are still lacking for the majority of elasmobranch species (Braccini 2008; Heithaus et al. 2008). Ecological data for the spotted eagle ray are sparse but the species likely plays a key habitat-structuring role in seagrass beds and soft benthos as a result of their feeding activity on macroinfauna and macroepifauna. They are large, mobile marine predators that hunt within multiple types of benthic habitats primarily targeting organisms such as clams, scallops, oysters, shrimp, possibly crabs and other mobile epifauna, which the rays crush with their broad, flat teeth (Silliman and Gruber 1999).

The rays’ only predators are primarily large coastal sharks, and humans. In some parts of the world, where their shark predators have been severely depleted and the marine ecosystem has been disrupted, large swimming rays such as the spotted eagle ray have been implicated in the decline of shellfish beds. Such cascading ecological effects of worldwide shark depletion are a major theme of much of Mote’s contemporary shark research. Mote researchers have hypothesized that shallow sandbar and seagrass areas in the vicinity of passes may serve as refuges for eagle rays from large sharks, particularly during periods of mating activity or pupping (release of live offspring). Whether they are refuges or not, passes and sandbars appear to be very important areas for spotted eagle ray distribution, meaning that anthropogenic activities that disrupt these areas – specifically, pass dredging and beach renourishment – may constitute threats to the survival of this species in southwest Florida.

Mote’s Center for Shark Research (CSR) and Marine Operations Division conducted monthly aerial surveys and weekly on-water surveys from July 2009 through January 2012, establishing that spotted eagle rays are present in most months of the year in southwest Florida coastal waters. Through January 10, 2012, a total of 319 spotted eagle rays were captured, measured, photo-identified and tagged with passive integrative transponder (PIT) tags in the Sarasota area. Four spotted eagle rays were

3 satellite-tagged with Pop-up Archival Tags (PATs; X-Tag, Microwave Telemetry) to track movements for as long as nine months. Individual rays ranging from newborn size of 42 cm disc width to fully mature animals over 2 m disk width were documented in the area. Aerial surveys observed clusters of the rays from St. Petersburg to Sanibel Island, especially around passes. These observations indicate that this ray is concentrated in southwest Florida waters of the Gulf of Mexico, yet critical habitats for feeding, mating and pupping of this species have not been defined.

In January-February 2010, unusually cold conditions resulted in a mortality event in the vicinity of a Tampa Bay power plant, which serves as a thermal refuge for a number of marine species including elasmobranchs. More than a dozen spotted eagle rays were found dead and emaciated with poor body condition. Four live rays were caught in a similar emaciated condition. Necropsies of dead specimens showed no food in the animals’ stomachs. During the spring of 2010, when the rays were migrating back into the Sarasota Bay area, several of the rays that were caught near Sarasota Bay passes were visibly thin and underweight. Over the summer of 2010, most of the observed rays appeared to be in normal or robust body condition, suggesting the importance of Sarasota Bay feeding grounds for this species.

SUMMARY OF ACTIVITIES & RESULTS

Project activities were conducted to address the following long-term research questions:

• What is the relative abundance, monthly prevalence, and yearly trends of spotted eagle rays along the southwest Florida coast? • What are the local and long-distance movement patterns of these rays in the eastern Gulf of Mexico? • Where do the rays go in winter, and do they return to the same Gulf coast areas? • What is the reproductive cycle of the spotted eagle ray? • Are spotted eagle rays mating and/or pupping in southwest Florida coastal waters? • How do human-related activities such as beach re-nourishment and channel dredging affect critical habitats for the spotted eagle ray?

4 AERIAL SURVEYS OF THE SOUTHWEST FLORIDA COAST

For the past four years, the Mote CSR has conducted aerial surveys to document large coastal sharks along the southwest coast of Florida from Tampa Bay to Sanibel Island (Fig. 1). Typical surveys are conducted once per month when possible during clear weather conditions and take approximately three hours to complete. These surveys are designed to document the location and habitats of large marine vertebrates including marine mammals, sea turtles, teleost fishes, and sharks and rays. Over the past four years (2008-2011), 28 flights have been conducted and a total of 22,150 cownose rays, 2,728 tarpon, 1,067 dolphins, 973 spotted eagle rays, 888 sharks, and 558 manatees have been recorded. Spotted eagle rays are commonly observed on these surveys in the months of May through November (Fig. 2).

Figure 1. Aerial survey ST. PETERSBURG flight path (red line). Total survey area covers ~120 nm from John’s Pass near St. Petersburg to Sanibel Island near Ft. Myers. In February and March of 2010 and 2011, the survey included coverage of the Big Bend Power Plant in Tampa to document the presence of spotted eagle rays in the plant’s warm water discharge in winter.

FT. MYERS

5 Observed SER over 4 years of Aerial Surveys

180

160

140

120

100 2008 2009 80 2010 60 2011 # SER Observed 40

20 * * 0 NF -20 Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec

Figure 2. Number of spotted eagle rays observed on aerial surveys of the southwest Florida coast by month in each year (2008-2011). Bars below the X-axis represent No Flight (NF) for that month; asterisked (*) bars in February and March 2010 were animals recorded only at Big Bend Power Plant in Tampa Bay. In general, there was a decrease in rays recorded in 2010 and 2011 compared with 2008 and 2009, with the exception of the month of October.

The spotted eagle rays are typically observed in small groups (2-5 animals) or as individuals along the west coast of Florida (Fig. 3). In survey years 2008 and 2009, large groups exceeding 10 and up to 76 animals were observed during our aerial surveys along and inside major passes in the survey area. Conversely, in the survey years 2010 and 2011, there were far less SER sightings and even fewer group sightings (one group exceeding 10 animals was documented in 2010 and none documented in 2011). The reduction of sightings can be seen from year 2008 to 2011 in Fig. 4, where the average number of sightings per flight are graphed for each year. This shows a steady decline in sightings from the peak in 2009 at a total of 97 SER documented for each flight to just 6 individuals documented in 2011. Still the most predictable of these locations for SER sightings has been New Pass, the current site of our tagging project. However, in survey years 2010 and 2011 eagle ray observations have declined in all areas along the west coast of Florida. Only one grouping of SER’s exceeding 10 animals was documented on 20 Oct 10 in Redfish Pass outside of Pine Island Sound as compared to groups of this size observed in Longboat Pass and New Pass in 2009 (Fig 5).

6 Percentage of SER observations

70.0%

61.3% 60.0%

50.0%

40.0% 35.6%

30.0%

20.0%

10.0% 3.1%

0.0% Individuals Groups of 2-10 Groups of 11-76

Figure 3. Percentage of sightings for individuals, small groups not exceeding 10 individuals and large groups of more than 10 individuals as observed during aerial surveys. Majority of observed groups exceeding 10 animals occurred in the summer and fall of 2009.

Average # of SER Observed on each Flight

10 90

9 80 r

8 70 7 60 Flights 6 50 Sightings 5 40 4 30 # of Flights per Year 3

2 20 Average # of SER Sightings per Yea 1 10

0 0 2008 2009 2010 2011

Figure 4. Average number of spotted eagle rays observed on the aerial surveys from 2008 to 2011. Despite fewer flights conducted in 2008 and 2009, the average number of eagle rays recorded those two years far exceeded those of the 2010 and 2011 years. No winter flights were conducted in 2008 and 2009. However, the majority of eagle ray sightings are in the summer and fall seasons.

7 In December 2009, the Mote CSR received reports that an unusually large number of spotted eagle rays was observed near the Tampa Electric Company (TECO) Big Bend Power Plant in Gibsonton on east Tampa Bay (Figs. 1 and 6). This plant provides electrical power to the city of Tampa and discharges warm water in the bay during winter months. The warm plume attracts and entraps a number of marine species including manatees, sharks and rays, and teleosts during the winter. In December 2009, some eagle rays washed up on shore dead and emaciated. Beginning in February 2010, we temporarily expanded our aerial surveys to include the area around the power plant. A total of 16 spotted eagle rays were seen around the plant in February, one was seen in March and none after that until May, when the surveys of the plant were discontinued. Survey year 2011 yielded no SER sightings in the immediate power plant area.

Figure 5. Locations of aggregations of spotted eagle rays comprising 10 or more individuals documented during aerial surveys.

Figure 6. Satellite image of the TECO Big Bend Power Plant showing the plant’s discharge canal. Numerous marine species seek refuge in the warm water inside and around the canal in winter.

In conclusion, over the past four years of aerial surveys the total number of observed eagle rays increased from 223 individuals in 2008 to a peak of 582 in 2009. In the following 2010 and 2011 survey years, we observed a steep decline in SER sightings, with only 117 rays recorded in 2010 during 8 flights and 53 rays in 2011 during 9 flights. This could be related to an unusually harsh and cold winter in 2009- 2010, possibly displacing the rays to other regions with more favorable conditions and abundant food sources; conversely, the rays’ ability to survive through extreme weather conditions may have been compromised and/or the rays suffered direct mortality from the 2009-2010 cold snaps. Surveys will continue in 2012 to document further changes in migration and habitat use patterns.

ON-WATER SURVEYS, SAMPLES, PHOTO-ID AND PIT-TAGGING

On-water surveys to document, measure, sample and tag spotted eagle rays in the vicinity of Mote Marine Laboratory off Sarasota, Florida were initiated in July 2009. By the end of 2011, the field survey team had completed 130 boat-days of effort, and had surveyed more than 7,000 km in the study area (Fig. 6) from 20 July 2009 through 03 November 2011. Number of survey kilometers searching for spotted eagle rays in 2011 was determined by GPS to be 2,637.85 km.

Figure 7. Location of on-water surveys for spotted eagle rays off Sarasota in 2011.

Since July 2009, a total of 440 net sets to collect rays were attempted, with 304 successful sets resulting in capture of 329 spotted eagle rays (86 individuals captured in 2011; Fig. 7). These rays were each measured, PIT- tagged and photo-documented for spot patterns. Overall, fifteen individuals were recaptured over periods ranging from a few weeks to 13 months and verified through photo-ID of spot patterns and scanning of PIT tags (Fig. 8). One ray was first captured in April 2010 and had a disc width measurement of 129.0 cm and a weight of 29.0 kg (63.9 lbs). When the ray was captured again in May 2011, the disc width had increased to 162.0 cm and weight to 61.5 kg (about 135.6 lbs). Another interesting recapture this year was that of a pup that was first captured on 04 October 2011 and again on 27 October 2011. Over 23 days this ray had grown from 59.0 cm disc width and a weight of 2.8 kg (6.17 lbs) to 61 cm disc width and 3.2 lbs (7.1 lbs). This was the first recapture of a pup giving us an excellent opportunity to observe growth rates during early stages of life. Because the rays leave the Sarasota Bay area in winter months, the fact that these individuals returned in subsequent years indicates a strong possibility of year-to-year philopatry and the importance of this area for these

9 rays. We also believe we have obtained the first growth rates recorded for individual wild, free-ranging spotted eagle rays anywhere in the world.

Figure 8. Capture locations of five spotted eagle rays recaptured in 2011.

Examination of individual rays consisted of taking a suite of measurements and observations of body condition as well as environmental conditions at capture locations (Fig. 9). Environmental conditions were measured and recorded including water temperature, salinity, dissolved oxygen, depth, water visibility, as well as Beaufort state and cloud cover. Animal measurements included disc width, total length (snout tip to

10 pelvic fin tip), distance between spiracles, eyes, and spiracle to snout tip length, and for males, clasper length. Observations were taken of gender, number of barbs, remora presence and scars, tail state (intact, partial, or bob), shark bites, human impacts (hook and line entanglement or boat strikes), overall body condition (thin, normal, robust), and other noticeable anomalies of spot patterns. Fin clips were taken for genetic analysis (Fig. 10) by Anna Sellas (manager of the Center for Genomic Studies at the California Academy of Sciences) and Jennifer Newby (graduate student at College of Charleston). When possible, blood samples were taken for PAH contaminant analyses in Dana Wetzel's ecotoxicology lab at Mote and for Jill Arnold at the National Aquarium to examine blood parameters. Prior to release, the rays were outfitted with internal PIT and external spaghetti tags.

Figure 9. Upper left: anterior dorsal view of spotted eagle ray. Upper right: weighing a live ray on the collecting boat. Lower left: spot patterns are unique and highly variable between individuals, making them excellent candidates for photo-ID. Lower right: collecting blood sample for PAH contaminant and blood parameter analysis.

Figure 10. Upper left: A fin clip is taken from the pelvic fin; Upper right: Fin clip is removed from notcher; Bottom left: Fin clip is then placed into a vial filled with ethanol and shipped for use in genetic analyses; Bottom right: Fin clips taken for genetics are often of similar size and shape to natural notches acquired in the wild.

Genetics: Report from Anna Sellas at California Academy of Sciences

The first step in the genetics portion of this study was to develop a novel set of nuclear microsatellite markers for A. narinari. As both fast evolving and highly variable, microsatellites are the choice maker for studies investigating the structure of recently diverged populations. In collaboration with Dr. Kevin Feldheim of the Field Museum of Natural History, primer pairs to amplify ten variable microsatellite markers were developed and published in March of 2011 (Sellas et al. 2011). This was the first publication reporting on microsatellite markers developed from A. narinari. Since this publication, we have genotyped 195 spotted eagle ray samples using this suite of markers (140 GOM samples from coastal Florida waters and 55 GOM samples from coastal Mexico). Results from preliminary analyses based on data from eight of these ten microsatellite markers indicate a significant amount of differentiation between samples collected off Florida vs. those collected off of Mexico (FST = 0.0047, p < 0.003). Genetic diversity estimates were similar between populations with observed

12 heterozygosity estimates of 0.691 and 0.704 for Florida and Mexico populations respectively. Once final analyses are run, the next step will be writing and submission of a manuscript for scientific publication reporting on the genetic diversity and population structure of spotted eagle rays in the Gulf of Mexico.

Genetics: Report from Jennifer Newby, Graduate Student at College of Charleston

My Master’s thesis work focuses on the spotted eagle rays captured in near-shore waters of Sarasota, FL in the eastern Gulf of Mexico. I am currently investigating population genetic structure at this site as well as looking into social structure among A. narinari that are captured or seen travelling in pairs. I presented a poster on my thesis work to-date this fall at the 2011 College of Charleston Student Research Colloquium of the Graduate Program in Marine Biology. I am employing 10 previously identified microsatellite loci (Sellas, 2011) specific for A. narinari to answer these questions and also to estimate effective population size for this species of conservation concern. For 2012, I will continue work optimizing and multiplexing my A. narinari primers and generating haplotypes for the individuals captured in 2011 to look at genetic and social structure among the captured individuals. Analysis and synthesis of data will be conducted simultaneously with data generation and a presentation of findings will be given at the August 2012 Conference for the American Society of Ichthyologists and Herpetologists.

Blood Analyses: Report from Erin Pulster and Dr Dana Wetzel, Mote Center for Ecotoxicology

Since 2010, blood and serum from spotted eagle rays, along with a variety of their potential prey (i.e., sunray venus, hard clam, cross barred venus, pen shell) were collected in Sarasota Bay to measure baseline levels of PAHs as well as develop models for potential bioaccumulation/magnification due to the recent Deepwater Horizon Oil Spill. The prey items were collected between June and August of 2010 and were stored at -20ºC until analysis. In addition to measuring body burdens, biomarker assays were proposed to evaluate sublethal effects (fertility and immune function) in spotted eagle rays from oil exposure. Blood samples were collected in different tubes depending on analysis and kept cool during transport to the lab for further processing. To date, 64 whole blood samples for PAH analysis and 42 serum samples for biomarker assays have been collected. Blood samples for fertility and immune function assays were immediately centrifuged, serum was then transferred to cryovials and stored at - 80ºC until analysis. Whole blood for PAH analysis was spiked with appropriate surrogate standards and extracted using liquid/liquid extraction methods and stored at - 20ºC until analysis.

Blood Analyses: Research of Jill Arnold, Veterinary Technician at the National Aquarium in Baltimore

Jill Arnold, a veterinary technician at the National Aquarium in Baltimore, participated in field surveys during November 2010 to perfect techniques to collect blood samples in order to establish reference intervals for hematology and plasma chemistry for spotted eagle rays with a goal of 120 samples to meet American Society for Vet Clinical Pathology/Quality Assurance Guidelines (Fig. 11). Mote researchers are continuing to collect these samples for Jill and ship them to the National Aquarium so that she can conduct CBC analyses.

Figure 11. Left: National Aquarium’s Jill Arnold processing blood samples; Upper right: collecting blood from a spotted eagle ray pup; Bottom right: spotted eagle ray blood smear, Hema-3 stain.

14 PHOTO IDENTIFICATION AND BODY CONDITION

The photo-ID catalog we have established with 319 individuals to date consists of head shots and body photos of each individual. These images have been processed into the Interactive Individual Identification System (I³S) spot pattern recognition computer software program (currently used with whale sharks and manta rays) (den Hartog and Reijns, 2007). The I³S program has been successful matching recaptured individual SERs by their spot patterns (Fig. 12).

Figure 12. Images of SER 125 (top and bottom, left) matched with spot pattern markings. Image on right shows spot cloud overlap between the two images.

The recapture of five individuals in 2011 and stability of spot patterns at least over 7-13 months on the head area of these individuals indicates that spotted eagle rays are good candidates for the I³S system and population estimating procedures utilizing mark- recapture from photo identification. Some of the body scars we noted appear to be mostly from remoras (Fig. 13) and but also from shark bites and human interaction (Figs. 14 and 15). A few rays showed signs of human interaction consistent with boat strikes (Fig. 14) or hook and line entanglements (Fig. 14). All recent and healed shark bite wounds and scars are being recorded for captured animals (Fig. 15).

Figure 13. We are further investigating the relationship between remoras and spotted eagle rays. Upper left: spotted eagle ray jumping with remora stuck to its ventral side; Upper right: remora removed from spotted eagle ray; Bottom left: remora scar behind right eye, dorsal side; Bottom right: remora scar under wing tip, ventral side.

Figure 14. Impacts of human interactions include: Upper left: imbedded fish hook; Upper right: healing boat propeller wound; Bottom left: fresh wound from possible hook gash; Bottom right: healed wound from possible hook gash.

Figure 15. Sharks are a natural predator for spotted eagle rays. Upper left: shark bite out of left wing, posterior; Upper right: shark bite out of left pelvic fin, resulting in missing left clasper; Bottom left: fresh shark bite, ventral side, right pelvic fin; Bottom right: shark bite scar, dorsal side, right wing tip.

The on-water team did not observe any spotted eagle rays in the Sarasota Bay study site between late December 2009 and early April 2010. As previously mentioned, 2009-2010 was a record cold winter in Sarasota, with water temperatures reaching as low as 8°C. The staff of the Manatee Viewing Center at the Tampa Electric Company (TECO) Big Bend Power Plant in nearby Tampa Bay reported that spotted eagle rays were washing ashore in February 2010 (Fig. 16). We acquired two fresh ray carcasses from TECO staff on 24 February 2010 and completed a necropsy and determined probable cause of death from emaciation. On 1 March 2010 we captured four rays just outside the thermal basin and all were emaciated and in visibly poor body condition. In total more than a dozen rays were observed dead in the general TECO area in the winter of 2010 (which was reported as unusual by longtime TECO staff).

Figure 16. Winter mortality event of spotted eagle rays in Tampa Bay in winter 2010 (above) and necropsy on recovered ray from event (right).

Of the 146 Sarasota spotted eagle rays captured in 2010, 21 visibly thin individuals were captured between April and early June. Twenty seven percent of individuals caught during those months in 2010 were classified as "skinny" due to sunken depressions above eyes, behind spiracles, and along the back (Figs. 17 and 18). Reasons for the animals’ poor health during these events might include cold-stressed physiological changes or lack of available food during the cold-affected periods. During 2011 far fewer visibly thin individuals were observed following a warmer winter (Figs. 18 and 19).

Figure 17. SER 113 on 11 May 2010. This animal’s body condition was classified as “skinny” due to areas of sunken depressions around eyes, behind spiracles and along the back.

19 Body Condition of Spotted Eagle Rays during April, May and June 2010-2011

50 2010 40 2011 30 Spotted Eagle Rays Percent of Captured of Captured Percent 20

0 Skinny Normal Robust Body Condition

Figure 18. A higher percentage of “skinny” individuals were observed in 2010 as compared to 2011 during the months of April, May and June.

Average Sea Surface Temperature (°C) by Month for 2010-2011 in New Pass, Sarasota

35.0

30.0

25.0

20.0 2010 2011 15.0

Temperature (°C) 10.0

5.0

0.0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Month

Figure 19. During 2010, wider-ranging sea surface temperature differentials were observed between months compared to 2011.

As summer 2010 progressed and water temperatures warmed, all of the spotted eagle rays we caught were of normal or robust body condition. The numbers of rays observed in our study area (New Pass, Big Pass and Gulf coastal waters), however, were significantly less than in summer 2009, consistent with the aerial survey data. Water temperatures in the passes averaged more than 1°C higher in summer 2010 compared to the same period in summer 2009 (32.4°C compared to 31.2°C). The pass and shallow waters off the beaches may have become too warm for the rays causing them to move offshore into deeper cooler waters, for we received anecdotal reports of rays in the 1-5 mile range offshore during this time. As a result, we expanded our search range in later 2010 to include Longboat Pass to the north and south towards Venice Inlet along the coast. Starting in early September more rays moved back into our coastal study area and were present through early November.

During 2011, TECO staff reported far fewer spotted eagle rays using the thermal discharge basin compared to past years. This reduction in numbers corresponded to what we observed during aerial surveys and boat-based surveys from the mouth of Tampa Bay down though Charlotte Harbor throughout the spring and summer of 2011. Timing of first arrival of SERs was consistent with 2010 as we caught our first ray on 7 April 2011. The majority of the individuals we caught during spring months were larger individuals with disc widths greater than 150 cm. As the summer progressed more individuals in the 120-150 disc width size class were observed and caught.

PUPPING

Between 19 October and 11 November 2010, eleven young-of-the-year spotted eagle rays, ranging in size from 42 cm to 58 cm disc width, were captured. Comparatively, in 2011, the first young-of-year pup was observed in early August. From August 2011 to January 2012, 36 spotted eagle ray pups, ranging in size from 45 cm to 67 cm disc width, were captured (Fig. 20). We hypothesize that the winter presence of spotted eagle ray pups, which were observed as late as December 2011 and into January 2012, was likely due to the mild fall and early winter season. Our consistent observations over the past three years of individuals in this size class has led us to hypothesize that pupping in this species occurs during the fall months in southwest Florida coastal waters.

Figure 20. We hypothesize that pupping occurs for A. narinari during the fall months in southwest Florida coastal waters. Upper left: spotted eagle ray pup; Upper right: pups have been observed ranging in sizes from 41 cm to 67 cm disc width; Bottom left: a bucket and pull spring scale is used to weigh the pups; Bottom right: National Aquarium’s Jill Arnold releasing a spotted eagle ray pup after all measurements have been documented.

22 SATELLITE TAGGING

To study long-term movement patterns and migratory behavior of the spotted eagle ray, four rays were outfitted with 4-9 month satellite archival pop-up tags (X-Tags, Microwave Telemetry) during September and October 2010. Tag attachment methods were initially tested in Mote’s captive facilities on six individuals in 2009, nine individuals in 2010, and one individual in 2011. These rays were caught and held for brief periods (<30 days) in a large research tank of the Mote CSR Marine Experimental Research Facility and then released. During the time in captivity, satellite tag attachment methods were tested, animals were evaluated for PIT tag retention and observations were recorded on the rays’ feeding behavior. While in captivity the rays were fed live hard clams (Mercenaria mercenaria).

Three methods of satellite tag attachment were developed and tested in the captive rays (Figs. 21 and 22): 1) a “through-wing” method in which a short piece of monofilament line was inserted dorso-ventrally through the caudal part of the animal’s pectoral fin and affixed in place with neoprene and plastic disks; 2) a “tail-band” method in which a plastic band encircled the thick part of the tail just rostral to the dorsal fin; and 3) a "through-tail" method in which a short piece of monofilament was inserted and wrapped through the tail between the dorsal fin and insertion of the barbs. All three methods were designed so that the attachment would corrode over time and eventually release from the animal, after the satellite tag had popped up. Each method has its pluses and minuses, with an important factor being the relatively thin and delicate skin of eagle rays becoming abraded with any foreign object touching its surface.

Figure 21. Spotted eagle ray satellite tag attachment methods tested and deployed in 2010: “through-wing” (left) and “tail-band” (right).

Figure 22. A new satellite tag attachment method tested on a captive ray in 2011: “through-tail.”

We deployed two tags using the through-wing method and two using the tail-band method in 2010 (Table 1). Two tags were set to pop up in 120 days, one in 180 days and one in 270 days. All tags were deployed on rays caught in the vicinity of New Pass, Sarasota.

DW Date Attachment Programmed Expected Tag’s Date of Pop-up (cm) Sex Maturity Attached Method Pop-up Time Pop-up Date Outcome 1st Trans Dist. (km) Location 150 F Unknown 9/28/2010 Through-wing 120 days 1/26/2011 Not heard from Did not report but 150 F Unknown 10/7/2010 Tail-band 120 days 2/4/2011 was recovered 151 F Unknown 10/14/2010 Through-wing 270 days 7/11/2011 Not heard from 9 km w of 168 M Mature 10/19/2010 Tail-band 180 days 4/17/2011 Reported early 11/22/2010 91 Redfish Pass

Table 1. X-Tag (Microwave Telemetry) pop-up satellite archival tags deployed on spotted eagle rays in September-October 2010.

24 Two out of the four X-Tags failed to report and one tag came off the ray after less than one day of attachment, and washed up on a beach where it was recovered. On the other hand, a tag set to pop up on 17 April 2011 prematurely popped up and reported on 22 November 2010, just over one month from tagging and release. The tag began transmitting 91 km minimum at-sea distance south down the coast from the tagging site, about 9 km offshore from Redfish Pass near Captiva Island (Fig. 23).

Figure 23. Tagging and pop-up sites for satellite tag on 168 cm DW mature male spotted eagle ray tagged on 19 October 2010.

The detached satellite tag transmitted its archived data for a period of 22 days while floating in the Gulf waters off Captiva and Sanibel Islands. The text data were compiled and sent off to Microwave Telemetry for processing and preliminary analysis. It was determined that the early detachment occurred because the tag’s constant depth release mechanism had been triggered. This failsafe device, which is set to operate after the first 30 days of tag deployment, is a means of ensuring data recovery in the event of a mortality to the study animal.

The short duration and limited horizontal movements of this animal’s track did not enable us to estimate the ray’s most probable track on a finer scale from the raw light- based geolocations. The depth profile of this animal revealed regular vertical movements from the surface to depths exceeding 13 m during the first 4 days after tagging (Fig. 24). The pattern abruptly changed after that and subsequent vertical movements were greatly reduced, with no surface time recorded. At present we cannot be certain of the reason for this apparent change in behavior. The tag-recorded ambient water temperature and a record of the local mean air temperature revealed that a strong cold front moved through the area beginning 4 November 2010 (Fig. 24).

25 Because we know so little about the over-wintering habits of spotted eagle rays in subtropical climates, it is possible that this ray responded to the subsequent drop in water temperature and altered its behavior by remaining at or near the bottom for this extended period of time. It is also possible, however, that the abrupt change in behavior observed on 23 October 2010 represents a predation event on the study animal that resulted in the severing of the ray’s tail (with the attached tag) or an outright mortality. If either of these were the case, the depth changes subsequent to 23 October 2010 would likely be due to tidal fluctuations and/or a shifting of the carcass along the bottom by water currents (this is particularly plausible if a predation event occurred in a high energy area like Redfish Pass).

Possible mortality?

Cold front

30 days

Figure 24. Depth-temperature profiles for satellite-tagged ray. Data are recorded at 15-min intervals. Upper: entire 34-day track with mean air temperature of the area. Lower: close-up of the first 4 days demonstrating regular vertical movements. Tag’s constant depth programming was set to release if a 2 m range was maintained for 4 days in a row, after an initial delay of 30 days after deployment.

26 As a result of these disappointing attempts at satellite tagging spotted eagle rays, we have turned our attention to refining the tag attachment method. The challenge with these rays includes: 1) their leaping behavior, which can dislodge tags and/or damage antennas; 2) their shallow habitat, which allows them the opportunity to rub on tags on the bottom, rocks, etc.; and 3) the delicate nature of spotted eagle ray skin, which is not as tolerant as that of other elasmobranch species for tagging applications. To at least address concerns #1 and 3, we are adapting a “through-tail” method of attaching the tags that has the potential of working much more effectively. This method was developed by colleagues at Nova Southeastern University and involves passing a thick monofilament line through the meaty part of the tail, down and around the vertebral column, then back up and out of the tail. The ends of this loop are then used to make a new loop that is attached to the X-Tag. Early testing has yielded promising results, and we currently have three more X-Tags in inventory or production to be used to tag spotted eagle rays with this other method.

27 EDUCATION AND OUTREACH

Raising public awareness and enhancing conservation outreach and education about spotted eagle rays is an essential objective of the Spotted Eagle Ray Conservation Program. During 2010 and 2011, more than 14 high school and 7 college interns participated in hands-on field research training with spotted eagle rays as well as helping with data entry and photo-id (Figs. 25 and 26). Also during 2010-2011, with the help of Mote's high school interns, we conducted more than 200 visitor surveys at Mote's public aquarium and at TECO's Manatee Viewing Center (where spotted eagle rays use the same thermal basin as manatees in winter months) to access knowledge about spotted eagle rays to create effective outreach materials.

Several of these outreach products including posters, coloring sheets, word search, bean-bag toss game, photo-id-the-ray, stuffed puppets, and identify-the-body-parts were used at environmental festivals with overwhelming popularity and success to raise awareness about this species (Fig. 25). Our education outreach goals for 2012 are to make these materials available more widespread and create a website on spotted eagle rays on Mote's website (www.mote.org). As part of a recent grant proposal we submitted to Save Our Seas Foundation, we would like to make these materials bilingual (Spanish versions) and take them to Mexico and Cuba (areas with artisanal fisheries for spotted eagle rays) for use in classroom and festival settings. We also plan to engage fishermen in Mexico and Cuba by inviting them to informal public talks and exchanges about the biology and life history of spotted eagle rays.

During October 2011, we worked collaboratively with the California Academy of Sciences to produce a short, three-minute "Science In Action" video on the spotted eagle ray project, highlighting the genetics work that CAS researchers are doing with this project. This video is played on the public floor of CAS to thousands of visitors each year as part of an exhibit called Science in Action and is viewable online at:

http://www.calacademy.org/sciencetoday/spotted-eagle-rays

A short video program focusing on the National Aquarium’s sponsorship and involvement in this project will be available shortly. In the future we anticipate that project results will be highlighted through conservation exhibits at Mote Aquarium and at the National Aquarium and on Mote's website (www.mote.org), and will be used for policy recommendations on the state, national and international levels dealing with habitat protection, management of protected species, and sustainable use of fishery resources.

Figure 25. (Upper left) Mote high school interns participate in hands-on field research with spotted eagle rays. (Upper right) Mote high school interns create a spotted eagle ray bean bag toss game (theme is to toss the sticky remora on the ray). (Bottom left and right) Educational outreach booth at Mote's World Ocean Day in June 2011 highlighting the educational products created by Mote's High School Interns.

Figure 26. Mote's college intern program offers an intensive 3-4 month hands-on internship program. College interns in the Spotted Eagle Ray Conservation Program got to participate in many aspects of the field research as well as data entry and analysis, photo-id, video production and educational outreach.

30 MANUSCRIPTS, PRESENTATIONS AND GRANT PROPOSALS

One manuscript from this project has been published in Conservation Genetics Resources entitled "Isolation and characterization of polymorphic microsatellite markers from the spotted eagle ray (Aetobatus narinari)." Three other manuscripts are in progress reporting on fieldwork from 2009-2011, genetic analysis comparing samples from Florida and Mexico, and satellite tag data.

Four presentations were made on the project in 2010 and 2011, one by Robert Hueter and Kim Bassos-Hull to the National Aquarium Conservation Center Board at Mote Marine Laboratory, two by Ms. Bassos-Hull to TECO staff and volunteers and to the Sarasota Bay National Estuary Program, and one at the National Aquarium in Baltimore to aquarium staff and volunteers. In addition to the funding received from the NACC and Georgia Aquarium during 2010 and 2011, we submitted six other proposals to fund portions of this work (Florida Fish and Wildlife Conservation Commission, not awarded; Save Our Seas Foundation not awarded in 2011, pending in 2012; Mohamed bin Zayed Species Conservation Fund, not awarded in 2011 and 2012, and SeaWorld and Busch Gardens Conservation Fund, pending in 2012. We plan to submit further proposals for small grants (25K or less) to the following; PADI Foundation (will submit January 2012), Georgia Aquarium (will submit January 2012), Disney Wildlife Conservation Fund (will submit March 2012).

31 REFERENCES

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