Highly Migratory Fisheries Research by the National Shark Research Consortium (NSRC) 2004-2005

Award Number: NA16FL2813 Semi Annual Progress Report For Period: January 1, 2005 - June 30, 2005

SUBMITTED TO: NOAA

SUBMITTED By: ROBERT E. BUETER, PH.D. MOTE MARINE LABORATORY 1600 KEN THOMPSON PARKWAY SARASOTA,FL 34236 (941) 388-4441

AUGUST 24, 2005

MOTE MARINE LABORATORY TECHNICAL REPORT NO. 1039 SEMI-ANNUAL PERFORMANCE REPORT

Award Number: NA16FL2813

Amount of Award: Federal Share $ 1,787,864

Project Title: Highly Migratory Shark Fisheries Research by the National Shark Research Consortium (NSRC), 2004-2005

Recipient: Mote Marine Laboratory, Sarasota, Florida

Award Period: July 1,2004 to June 30, 2005

Period Covered by this Report: January 1, 2005 to June 30, 2005

Primary Project Tasks:

The following primary tasks were scheduled for this six-month period:

Mote Marine Laboratory Component lao Shark life history, migration and stock structure I b. Relative abundance studies lc. Satellite tag testing and development I d. Freshwater habitat use 1e . Mortality rates of within a coastal nursery area I f. Essential Fish Habitat environmental studies I g. Use of elemental analysis to investigate nursery habitat in bull sharks 1h . Ecosystem and population modeling Ii. Nurse shark reproductive life history, genetics and habitat studies Ij. Whale shark research in the Gulf of Mexico and Caribbean Sea lk. Publications and scientific conferences

1 Moss Landing Marine Laboratories Component

2a. Eastern orth Pacific chondrichthya n life history data matrix 2b. Age, growth, and demographic studies 2c. Age validation studies 2d. Reproductive biology 2e. Feeding ecology 2f. Stable isotope analysis 2g. Habitat associations and nursery grounds 2h. Population genetic studies 2i. Shelf, slope and pelagic surveys 2j. Eastern Pacific shark fisheries analysis 2k. Taxonomic studies

Virginia Institute of Marine Science Component

3a. Relati ve abundance and distribution studies 3b. Age, growth, and demographic studies 3c. Habitat utilization and migration studies 3d. Shark energetic and osmoregulatory studies 3e. Sandbar shark genetic studies 3f. Publications and conferences

University of Florida Component

4a. Fishery independent sampling 4b. Age, growth, and reproduction 4c. Tagging and tracking of bull sharks 4f. Age-structured simulation model for dusky shark 4h. Regional variation in age determination characteristics of vertebrae 4i. Ova rian tissue of Atlantic stingray 4j. International Shark Attack File 4k. Web- and media-based public education 41. Project Shark Awareness 4m. Requests for information 4n. Publications and conferences

2 Summary of Results:

General Project Overview

Significant progress was made during this six-month period of the project. Important advances by the NSRC were made in both field and laboratory approaches to understanding the life history, abundance and environmental biology of shark species important in commercial and recreational fisheries. NSRC researchers and students continued to make significant scientific contributions through publications and conference presentations during the six-month period. There were no major problems experienced with the overall project during the period.

Mote Marine Laboratory Report (R. Hueter, P/I)

Mote's Center for Shark Research (CSR) served as project coordinator. No problems were encountered in the administration of the project. The following technical accomplishments were achieved during the reporting period:

Shark life history, migration and stock structure. From January through June 2005, the Mote CSR captured a total of 602 sharks of 12 species. Of these sharks, 475 were tagged and released. During this same period, 24 recaptured sharks were reported and logged in the CSR database. Of these, the longest at large was a YOY blacktip shark (Careharhinus limbatus) tagged in September of 1996 along the south end of Pine Island Sound, Charlotte Harbor, Florida. It was recaptured in April of 2005 after 3,158 days (8.65 yrs) at liberty, in Boca Grande Pass, Charlotte Harbor, Florida, about 20 nm (37 km) from its original tagging site. The longest minimum at-sea distance traveled in these recaptures was reported from a blacktip shark tagged near Bahia Honda Key (Florida Keys) in January of2002. This shark was recaptured in June of2005 in the St. Joseph Bay area of Florida's panhandle, about 381 nm (706 km) from its tagging site. Three surveys for large coastal sharks were undertaken during the period, two in winter (one in the Florida Keys during February 2005 and one off Sarasota in March 2005) and one in spring (J LIn e 2005) off Sarasota. All surveys were carried out aboard the RIV Eugenie Clark except the March 2005 trip, which utilized the Florida Institute of Oceanography vessel RlV Suneoaster. Each survey consisted of 4-5 days of longline and drurnline sampling. The February survey caught 41 sharks, comprising Ginglymostoma eirratum (26), C. limbatus (11), C. aeronotus (1), C. leueas (1), C. plumbeus (1) and Sphyrna mokarran (1). The March trip caught 39 sharks, comprising C. brevipinna (30), C. aeronotus (5), C. limbatus (2), C. plumbeus (1) and Negaprion brevirostris (1). The June trip caught 63 sharks, comprising G. cirratum (20), C. aeronotus (10), Rhizoprionodon terraenovae (8), C. leueas (7), N. brevirostris (7), S. mokarran (6), C. limbatus (4) and Galeoeerdo euvier (1). In Terra Ceia Bay, Florida, 65 sets using four gear types (rod and reel, gill net, longline, and drumline) were conducted for this project during the period ofthis report. These efforts resulted in

3 the capture of 38 sharks of 4 species including C. limbatus (32), C. acronotus (3), S. tiburo (2) and C. leucas (1). A total of33 ofthese sharks were tagged in released using conventional tags, internal acoustic tags, external acoustic tags, or a combination of two types. To examine the movement patterns and behavioral responses of sharks translocated from one nursery area (Charlotte Harbor, Florida) to another (Terra Ceia Bay, Florida) a series of24 acoustic hydrophones were deployed within Terra Ceia Bay. Hydrophones were deployed in May of2005 and were used to monitor the presence and movements of sharks from both nurseries. A total of9 C. limbatus were successfully transported from Charlotte Harbor to Terra Ceia Bay. All transported individuals were weighed, measured, sexed and tagged with an external dart tag prior to fitting with acoustic transmitters. After transmitters were attached, sharks were released within the study site. Six transported sharks were released in the northern end of the study site in the region most commonly inhabited by resident C. limbatus. The remaining three individuals were released at the southern end ofthe study site distant from local . Six C. limbatus were captured within Terra Ceia Bay and treated as control animals for translocation experiments. Control animals were placed in transport containers for 3 hours (the duration of transport form Charlotte Harbor to Terra Ceia Bay) prior to fitting with acoustic transmitters to mimic the conditions for transport animals. All 15 sharks survived transport and surgery procedures and were released within the study site. Data from acoustic receivers are currently being gathered to examine the movement and behavior patterns of resident and translocated individuals. Due to adverse environmental conditions (hurricanes, red tide events) no additional sharks have been or will be transported during 2005.

Relative abundance studies. During the period ofthis report, no blacktip relative abundance sets in primary nurseries were conducted. We decided to suspend these field activities temporarily due to concerns over high mortality of pups in the nursery as a result of red tide, as well as the direct effects of our own gillnet fishing. Instead, a large-scale analysis of this project's 10-year dataset of three Florida Gulf coast nurseries (Yankeetown, lower Tampa Bay and Pine Island Sound) was conducted and its results presented at the annual American Elasmobranch Society Meeting, July 2005, in Tampa. Overall, 970 quantitative gill nets sets were made between 1995 and 2004 resulting in the capture of8,257 sharks ofthirteen species, of which 3,842 were juvenile blacktips. Recaptures have been reported from 149 of the tagged sharks comprising 5 species. First-year blacktips have demonstrated movements of more than 280 nautical miles after leaving their summer nursery grounds. Recapture data further indicates a pattern of sharks returning to their natal nursery areas the following season and in subsequent years. A general linear model was used to standardize the CPUE data. Significant differences in the juvenile blacktip catch were found between years, nursery areas, and between grids within a nursery area. When looking at the blacktip catch rates over the entire 10- year period, there were no apparent trends that would be indicative of population level changes.

Satellite tag testing and development. Tank-testing of satellite tags was not conducted during this period as the research tanks of Mote CSR's MERF (Marine Experimental Research Facility) were in use for other projects. However, field studies using PAT satellite tags continued. Three Wildlife Computers PAT tags deployed on whale sharks (Rhincodon typus) in previous project periods did not report, leading us to consider the possible reasons for satellite tag failure in this case. These reasons include:

4 • Failure of the hardware or software in the tag. • Physical damage of the tag, such as from the shark rubbing it, a fish attacking the tag or some other mechanical insult to the tag. • Fouling of the tag by algae or other encrusting organisms. • Failure of the tag to detach from the shark. • Premature detachment (should result in transmission, however). • Failure of the tag to float after it detaches. • The sharks dive very deep and the RD1500 release mechanism fails, so the tag is crushed by pressure at very deep depths (> 1,000 meters). • The tag detaches and floats up on land before a signal is received by the satellite. • Human interference: someone damages the tag or pulls it off the shark.

We have discussed these possibilities with other researchers and the tag manufacturers. It is not believed that tag attachment is the cause for failure in this case. One possibility that has come to light, as a result of discussions at the International Whale Shark Conference in Perth, Australia in May 2005, is that the tags may be damaged by repetitive deep diving to depths too shallow for release (1,000 m) but deep enough to repeatedly squeeze-and-expand the device, possibly causing tag failure. We are adjusting our tagging protocols appropriately to guard against this and other possibilities for tag failure, and will deploy three new PAT tags on whale sharks in August 2005.

Freshwater habitat use. Research into the use of freshwater habitats by juvenile bull sharks has continued and expanded in 2005. The original 20 acoustic receivers are still in place within the Caloosahatchee River, but an additional three receivers were added in 2005 to better refine shark movement patterns. A collaborative partnership with Florida Wildlife Research Institute (FWRI) staff has resulted in an additional eight receivers deployed beyond the original array. These eight stations are owned and maintained by FWRI staff, but data are being shared by both groups from all receivers. Seven sharks fitted with acoustic transmitters in 2004 remained within the river and are still currently being tracked within this system. At least one ofthese individuals has been recorded as far up river as the dam to Lake Okeechobee (40+ km upstream). An additional 24 young-of-the­ year bull sharks have been fitted with transmitters in 2005. Blood samples were collected from 14 of these individuals to examine blood parameters and help define any physiological stress associated with residence in freshwater conditions. All of these individuals were collected in salinities below 14 ppt. Blood analyses are currently underway. Current data from shark monitoring efforts have not yet been analyzed, but capture locations of sharks and salinity levels within the river suggest that the pattern of movement down river with salinity level is still present. Salinity levels within the entire ri ver system are currently below 5 ppt due to heavy rainfall and high drainage from Lake Okeechobee. Full analysis of this data set will be conducted during the fall of 2005.

Mortality rates ofsharks within a coastal nursery area. In 2005, the collaboration established with the National Marine Fisheries Service Southeast Fisheries Science Center (SEFSC) to monitor the residency and mortality of juvenile sharks within coastal nursery grounds was continued. Work in 2004 included a pilot study to examine mortality rates of sharks within St. Andrew's Sound. The pilot study established that sharks could be monitored within this area, but the occurrence of a hurricane during the 2004 field season prevented any mortality estimation within this popUlation. In May 2005, the acoustic network of 12 Vemco VR2 receivers was deployed and monitoring efforts

5 were continued. Acoustic receivers currently monitor an area of30 km2 within St. Andrews Sound. Based on data from 2004 that showed blacktip sharks (Carcharhinus limbatus) were not resident within the sound, work in 2005 focused on the Atlantic sharpnose shark, Rhizoprionodon terraenovae. To date 21 R. terraenovae have been fitted with acoustic transmitters and are currently being monitored within this study site. Several sharks have left the study site since monitoring began and several individuals have moved into and out of the area. Data are still being collected from this site to define the residency and mortality of this species within the region. Mortality estimation, residency and home range analyses will be examined in late 2005 once data collection for the summer season has been completed.

Essential Fish Habitat environmental studies. Initial CSR studies on pollutant exposure injuvenile sandbar and blacktip sharks from shark EFH on the U.S. east coast indicated that some of these populations (especially on the Atlantic coast) might be exposed to potentially health-threatening levels of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs). In 2005, we began follow-up studies using the bonnethead shark as a surrogate model to determine if elevated concentrations ofOCPs and PCBs in Atlantic coast sharks are a consequence of high levels of pollutant transfer via embryonic yolk reserves. To date, we have analyzed OCP and PCB concentrations in yolk samples from 18 pregnant ( early-stage) female bonnethead sharks from 4 sites on Florida's Gulf coast and found levels of these compounds to be generally low. These data will be compared with that obtained for Atlantic coast bonnetheads, which will be collected in Spring 2006 because collection efforts in Spring 2005 were unsuccessful. We also examined concentrations of30 individual PCB congeners in Atlantic and Gulf blacktip (n = 10) and sandbar sharks (n = 10) to determine ifmore harmful "dioxin-like" PCB congeners constitute a sizeable proportion of overall PCB load. Analysis of these data is currently underway. In addition to our studies on pollutant exposure in coastal sharks, we have had a great deal of success in developing molecular techniques that will enable us to detect physiological responses of OCP exposure in these fishes. To do this, we succeeded in cloning a partial gene sequence of bonnethead shark vitellogenin, a normally "female-specific" protein that is induced in male vertebrates exposed to estrogen-like pollutants ("ecoestrogens"), such as many OCPs. Vitellogenin is commonly used as a biomarker of estrogenic pollutant effects in non-mammalian vertebrates. We have constructed non-radioactive probes for the detection ofvitellogenin RNA in shark liver samples and have developed and validated a Northern Blot assay that will enable us to screen for pollutant effects in this species. To date, we have used this assay to analyze vitellogenin expression in over 20 male bonnethead sharks collected from two Florida estuaries, Charlotte Harbor and Tampa Bay. We were unable to detect the presence of vitellogenin in male sharks from either of these two sites. These results suggest that ecoestrogen levels in these two estuaries may not be high enough to cause physiological alterations in resident shark populations. More recently, we developed and validated a "one-step" reverse transcription-polymerase chain reaction (RT-PCR) assay for shark vitellogenin that is both faster and more sensitive than the Northern Blot protocol and requires 40 times less RNA to conduct. We used this assay to re­ examine vitellogenin expression in samples previously analyzed using the Northern Blot assay. The results of these measurements confirmed our prior findings, i.e., that Tampa Bay and Charlotte Harbor male bonnethead sharks do not exhibit signs of estrogenic pollutant effects. These results

6 will be compared with those obtained for sharks from other Florida estuaries (Apalachicola Bay, Florida Bay) and sites on the Atlantic coast, which will be collected during late summer-early fall 2005. Studies on pollutant exposure and effects in juvenile sharks residing in important nursery areas on the U.S. east coast were very productive in the most recent grant period. First, building on earlier studies that revealed high levels of total polychlorinated biphenyls (PCBs) in Atlantic coast sharks, we examined liver concentrations of>30 individual PCB congeners injuvenile sandbar (n = 10, Atlantic coast) and blacktip (n = 10, Atlantic and Gulf) sharks to determine if more toxic, coplanar PCBs constitute the majority of total PCB load in these animals. Second, since the high pesticide and PCB concentrations detected in neonate sharks suggest that chemical transfer via yolk may be an important route of pollutant exposure, we measured liver concentrations of ~30 pesticide/pesticide metabolites and total PCBs in yolk samples from 18 adult female bonnethead sharks collected from 4 sites on Florida's Gulf coast. Pollutant levels in yolk from Atlantic coast bonnetheads will be obtained in Spring 2005 for similar measurements. Analysis of all pollutant data is currently underway. Our efforts to develop biomarkers for pollutant exposure in coastal sharks were also fruitful during this period. Using molecular techniques, we amplified a DNA fragment of the ecoestrogen biomarker vitellogenin from liver samples of estrogen-treated, male bonnethead sharks (n = 2). This DNA fragment was absent in liver of control animals treated with hormone vehicle (n = 2). The putative vitellogenin fragment was cloned and submitted for DNA sequencing. Sequence analysis confirmed the identity of the fragment as vitellogenin. Development and validation of a Northern blot assay for this gene is currently underway. We have also initiated development of a more sensitive and non-lethal bioassay for detecting the effects of pollutant exposure in coastal sharks. This test will examine changes in gene expression in blood cells of bonnethead sharks exposed to estrogen-mimicking pollutants. Since sharks possess nucleated red blood cells, 50 j..lL of blood is all that is required to obtain the minimum amount of RNA needed to conduct the assay. Using blood from the estrogen-treated and control sharks described above, we PCR-amplified, cloned, and identified >100 genes that change expression in response to estrogen exposure. DNA probes for these genes have been developed and will be attached to nitrocellulose membranes in a grid-like fashion to construct a gene array or "DNA chip." We intend to test the chip using blood samples from sharks treated with the estrogenic pollutant, DDE. We will also examine the field applications of this procedure by comparing gene expression in blood samples from Atlantic coast and Gulf bonnethead sharks since our earlier work has demonstrated a six- to ten-fold difference between DDE levels in these animals.

Use ofelemental analysis to investigate nursery habitat in bull sharks. The analysis of elements in fish otoliths have been used to investigate the use of freshwater and estuarine habitats by marine organisms. In particular the ratio between Strontium (Sr) and Calcium (Ca) is a good indicator of changes in habitat use. High levels of Strontium indicate periods in fresh or brackish water. While this methodology has been used in teleosts, it has never been used in elasmobranchs. Additional vertebrae have also been collected to increase the sample size.

7 Ecosystem and population modeling. Studies of shark populations and how they respond to localized fishing were initiated in December 2004. Dr. Sheldon Dudley of the Natal Sharks Board visited MML to work with Dr. Simpfendorfer on this issue. They analyzed data from 14 species of sharks that have been fished since the 1950s in the waters off South Africa. Following the visit by Dr. Dudley work continued on this project, resulting in the submission of a manuscript and two presentations at international conferences (one in South Africa and one in the USA). The results of this work will be used to better understand how shark populations function, and ultimately improve the management of shark fisheries.

Nurse shark reproductive life history, genetics and habitat studies. Five weeks of field investigations into the natural reproductive cycle of the nurse shark, Ginglymostoma cirratum, were conducted in January and June in the Dry Tortugas, Florida by CSR staff during the reporting period. We returned to the Dry Tortugas in January for a thirteen-day trip to download and re-battery the automated listening stations that record the movements of the 10 nurse sharks from the June 2004 breeding popUlation. We discovered that two ofthe five transmittered males had returned to the area in December and January. This was the first evidence that the adult males are in the study area in the winter months. We spent many hours surveying different areas for neonates. A puzzle that remains for us is the location of the habitat for sharks that were born in November and December. We find pregnant females in our study site in October, but so far have been unable to locate their offspring in the Dry Tortugas. Our past and current results have documented adult 2.5-meter sharks mating on shallow grass flats in June and July. In collaboration and with the support of the RlV Tiburon, the donated vessel S/ V Eos and the National Park Service at Fort Jefferson, we spent 18 days on site observing the mating systems and behavior of nurse sharks in this reporting period. Colleagues from Albion College, Michigan, Florida International University, Southern Illinois University and National Geographic Remote Imaging joined us this year. Tagging results 1993-2005: Diver-identifiable tags are used to determine the identity, frequency and time period in which individuals participate in mating and other reproductive activities. Since 1993, we have tagged 242 nurse sharks (103 adults and 139 juveniles) in the Dry Tortugas study popUlation. Eighty different sharks have been recaptured (33.7% recapture rate). Several have been physically recaptured seven or more times. Of the identified adults, 35 were subsequently sighted (visually recaptured or netted) at least once, and some much more frequently. Observations from tagging and natural markings indicate that most adult males visit the study site faithfully every year. Results from our tag studies continue to support our hypothesis that adult females visit the study area to mate in alternate years. On June 20, 1993, we dart-tagged a large adult female using a speargun. It was the third nurse shark tagged in our project in the Dry Tortugas. On June 22 of this year, 12 years and two days later and 50 meters distant, we caught measured and retagged this same 248 cm breeding female. Between 1993 and 2002, we tagged 33 adults with a speargun. Since 2002 we have recaptured 15 of these adults in our study area for a recapture rate of 45.4%. The reproductive significance ofthis philopatric migration and the site fidelity ofboth sexes are unprecedented in any species of recorded shark and underscore the uniqueness of this research situation.

8 Whale shark research in the Gulf ofMexico and Caribbean Sea. This research and conservation project on whale sharks (Rhincodon typus) of the Gulf of Mexico is being conducted by scientists from Mote's CSR in collaboration with Mexican scientists and resource managers with the Comisi6n Nacional de Areas Naturales Protegidas (CONANP) ofthe federal Secretaria de Medio Ambiente y Recursos Naturales (SEMARNAT) in Cancun, Quintana Roo. In Gulf and Caribbean waters off Mexico north ofCancun, a large gathering of whale sharks occurs every spring and summer, from May through September. Our project is the first scientific effort to document the size and characteristics ofthis aggregation and study these animals up close. Our purposes are several: 1) to better understand the basic biology of this largest of shark species in its natural environment; 2) to explore the connectiveness of Gulf, Caribbean and other oceanic habitats in the life history of whale sharks; 3) to develop resource management and ecotourism plans for conservation of this unique species in Mexico. During the reporting period, the data collected in the previous summer's (2004) field trips to the aggregation site were assimilated from various sources, tabulated and analyzed. Dr. Hueter presented project results at the International Whale Shark Conference in Perth, Western Australia in May. At that conference it was learned that the Mexico gathering of whale sharks is special among aggregations around the world in the following ways: 1) the sheer number of animals (several hundred) at Islas ContoylHolbox practically dwarfs those at other sites; 2) the size range of sharks « 3 to > 12 m TL) at ContoylHolbox is larger than seen at most other sites, which typically have mostly small animals; and 3) females are present at ContoylHolbox in numbers of one per every tlu-ee males - most other sites report females as being rare. This raises the speculation that reproductive activity may be happening in the vicinity ofContoylHolbox. Plans were made during the reporting period to expand the scope of on-water studies and aerial surveys of the aggregation in summer 2005. Two field trips by CSR personnel to the study site were planned for August 2005.

Publications and scientific conferences. During the reporting period Mote CSR scientists participated in several international conferences including the International Whale Shark Conference in Australia, a workshop ofthe mCN Shark Specialist Group in the UK, and the American Society of Limnology and Oceanography annual meeting in Salt Lake City, Utah. Dr. Hueter participated in a meeting of the NOAA Highly Migratory Species Advisory Panel in Silver Spring, Maryland in March. During the reporting period CSR scientists had ten peer-reviewed publications appear or in press.

9 Moss Landing Marine Laboratories Report (G. Cailliet, P/I)

Life history database. The life history matrix (LHM) is now available on the PSRC web site for the general public (http://psrc.mlml.calstate.edu) and can be downloaded. The LHM was compiled, organized, and edited using all pertinent regional literature to determine what is known and, more importantly, what is not known about the life history, distribution, and population biology of eastern North Pacific Ocean (ENP) chondrichthyans (including the sharks, rays and chimaeras). The ENP as defined here includes the area ranging from the eastern Bering Sea to the southern tip of the Pacific 8aj a California peninsula. The LHM includes 105 species reported to occur in the ENP. The LHM will be updated periodically as new information becomes available.

Age, growth, and demographic studies. Age determination and validation studies are underway to fill some of the gaps in the life history of poorly studied species. Species currently under investigation include the sandpaper (Bathyraja kincaidii), roughtail (Bathyraja trachura), and California (Raja inornata) skates. To date, vertebrae from 190 sandpaper, 477 roughtail, and 289 California skates have been cleaned and thin-sectioned for age estimation. Age estimates have been calculated for the sandpaper skate with 50% of females maturing at 6.8 years and 50% maturity for males estimated at 7.3 years. The sandpaper skate age and growth study is nearly complete and a Masters thesis is being written. Additional vertebrae for the roughtail and California skates are still being sectioned while those that have already been prepared for ageing are currently being analyzed. Tail thorns are currently being prepared and being examined for use as a non-lethal means to age skates. The growth characteristics using seven different model's is being determined for the California skate. The suitability of neural arches and caudal thorns as alternative ageing structures is being examined. To date, neural arches appear to be poorly calcified and lack patterns that could be consistently interpreted for age estimation. Our analysis suggests that caudal thorns for this species grow at differential rates or are replaced and therefore do not appear to be a suitable ageing structure for this species. A new proj ect studying the age, growth, and demography of four species of Alaskan skates, big (Raja binoeulata), longnose (Raja rhina), Aleutian (Bathyraja aleutica), and Bering (B. interrupta) skates, was initiated in June 2005. These four are part of an emerging fishery for skates in Alaskan waters. Another new project has been initiated to study the age, growth, and demography of the white-spotted chimaera (Hydrolagus coUiei). Demographic analyses of the diamond stingray (Dasyatis dipterura), based on vital rates recently calculated by PSRC personnel, are now completed and a Masters thesis was completed. Results of this study will provide critical baseline information necessary for the effective management of this species in the Mexican Pacific.

Age validation studies. Validation ofthe periodicity of growth band deposition in shark vertebrae and age estimates based on vertebral band counts are being investigated using bomb radiocarbon. This study successfully resolved the issue related to the ageing of the shortfin mako (Isurus oxyrinehus). Using bomb radiocarbon analysis we were able to validate the ageing methodology that

10 assumed annual deposition of one pair of vertebral bands. This portion of the project was carned out as part of a Masters thesis, which is presently being completed. We are continuing to analyze white shark (Carcharodon carcharias) vertebrae for bomb radiocarbon, to validate the age estimation methodology, and stable isotopes (C-13, N-15), to assess trophic level and aid in interpretation of radiocarbon data. We are also continuing radiocarbon age validation studies on northwest Atlantic species, including the sandbar (Carcharhinus plumbeus) and tiger (Galeocerdo cuvier) sharks, using vertebrae from several institutions including the Florida Program for Shark Research at the University of Florida.

Reproduction. Investigations ofthe reproductive biology based on 304 California, 1,179 longnose, 540 roughtail, and 296 sandpaper skates are continuing or nearing completion. The sandpaper skate has been finished and is being written as part of a Masters thesis. The California and longnose skates are nearing completion and will be written by PSRC staff. Analysis and collection ofroughtail skate reproductive data are continuing. A new project examining the reproductive biology offour species of Alaskan skates was initiated in June 2005 . Another new project examining the reproductive biology of the white-spotted chimaera was initiated in the spring 2005. A total of 730 brown (A. brunneus), 104 longnose (A . kampae), and 376 filetail ( xaniurus) has been examined. Information on the distribution and reproduction ofthese catsharks was generated from specimens collected by fishery independent survey cruises from June 2001 through October 2004 between northern Washington and San Diego, California. Longline catches consisted mainly of filetail catsharks, with occasional catch of gravid female brown catsharks. Conversely, trawl catches consisted mainly of species. Brown catsharks were typically found between 300 and 942 m, while longnose catsharks occurred > 1,000 m depth. Filetail were caught between 300 and 550 m depth. Distribution and habitat locations of egg cases of brown and filetail catsharks were determined by trawl surveys and observations of video footage taken by remotely operated vehicle. These egg cases were located in specific sites on areas of high vertical relief at 300 to 400 m depth. Total length at first, 50% and 100% maturity were determined for males and females of all three species. At higher latitudes, brown and filetail catsharks reached sexual maturity at larger sizes. Brown and filetail catsharks reproduce year-round based on the occurrence of gravid females and the lack of seasonal variation in gonadosomatic (GSI) and hepatosomatic indices (BSI) for both males and females. Gravid longnose catshark females were found from July through December. The egg case of the longnose catshark is described and its morphology compared to the egg cases ofthe brown, filetail and other Apristurus species. The catshark study has been written up and completed as a Masters thesis.

Feeding ecology. Dietary analysis on the feeding ecology ofthe sandpaper, California, and longnose skates is continuing. Approximately 493 sandpaper skates have been examined to date with shrimps, euphausids, and mysids being the primary prey items, followed by polychaetes, teleosts (Sebastes spp.), and cephalopods. Dietary analysis of287 California skates is nearing completion. This species feeds mainly on benthic shrimp, crabs, and demersal teleosts. To date, approximately 300 stomachs of longnose skate have been processed with prey items identified. The main prey items appear to be teleosts, crangons, and euphausids.

11 Stable isotope analyses. In addition to the stable isotope analysis in the white shark vertebrae (see above), the feasibility of such analysis is being tested to track the ontogenetic movement patterns of the blue shark (Prionace glauca). Analysis has been performed at the DC Davis Stable Isotope Facility. Vertebrae from eight blue sharks and muscle tissue from 20 specimens from the ENP were obtained from three locations (Southern Baja California, San Diego, and British Columbia) and were examined. Analysis of nitrogen isotopes within vertebrae for all animals in all locations showed that there was little change in nitrogen isotope level (14.87 %0 ± 0.82), indicating that there may be little change in ontogenetic diet changes. Conversely, there were large variations found in carbon isotope values within vertebrae for all blue sharks that may indicate movement throughout life. Changes of2 to 3 %0 in carbon were evident in all of the individuals sampled and showed a cyclic nature that likely reflects patterns of onshore and offshore movement. Future work will include incorporation of the estimated ages of individuals to examine patterns of carbon isotope change within vertebrae as a function of time, and incorporation of muscle tissue samples to elucidate local productivity patterns and hence isotopic composition patterns spatially. Because there are presently no publications on stable isotope composition in shark vertebral centra, our work is unique. To our knowledge the only study of this nature was done by Sterling Peverell in Australia on the Sr/Ca ratios in the sawfish, Pristis microdon, suggesting annual growth zones and ontogenetic habitat changes.

Habitat and nursery ground studies. Habitat association, distribution, and abundance of several chondrichthyan species were initiated during this report period. These include several deepsea skate species and the white-spotted chimaera. During the initial phase of this study two new species of chimaeras were identified using archived video available through the Monterey Bay Aquarium Research Institute (MBARI). Both species may be new to science. A study on the nursery grounds of two catshark species is nearing completion. The field portion of a tracking study on the movement patterns of leopard sharks (Triakis semifasciata) in Elkhorn Slough is complete and data is continuing to be analyzed. A GIS habitat map is being constructed for Elkhorn Slough for use in analyzing the active tracking data. Once these maps are complete, analysis of habitat utilization, rates of movement, tidal movements, and space activity can be initiated. Preliminary analyses indicate that the movement patterns of these sharks appear to be highly influenced by the tides and suggest that they may utilize some areas more intensively than others. A new study on the occurrence of prickly sharks (Echinorhinus cookei) at the head of the Monterey Bay Submarine Canyon has been initiated. This study will attempt to track the movements of this large deepsea shark and determine why this apparently deepsea species moves into shallow water. To date five sharks have been tagged and tracked.

Population genetic studies. Genetic research is continuing on all three thresher shark species; Alopias pelagicus, A. superciliosus, and A. vulpinus. A total of 302 samples were obtained from shark populations in the North and South Pacific, North Atlantic, and southwestern Indian Oceans. So far, sequence data and chromatograms have been received for 263 of those samples. Preliminary analysis indicates that significant population structure exists for all three Alopias species, the scale of which differs depending on the species. This portion of the project is nearing completion and a Masters thesis is being written.

12 Shelf, slope and pelagic surveys. In cooperation with NMFS Northwest Fisheries Science Center (NWFSC) and SCL, PSRC personnel are currently participating and assisting in the annual shelf and slope groundfish surveys in Washington, Oregon, and California. We are continuing to record chondrichthyan landings from monthly NMFS SCL trawl and longline surveys in Monterey Bay. Measurements, wet weights, reproductive tracts, tissue samples, parasites, vertebrae for age and growth estimation, and/or stomachs for feeding analyses have been collected from nearly 5,000 specimens to date. The primary elasmobranchs observed in NMFS SCL groundfish surveys are the big, California, and longnose skates. PSRC personnel are currently participating in the NMFS Alaska Fisheries Science Center (AFSC) 2005 Gulf of Alaska survey. Reproductive data and ve11ebrae for age and growth, and stomachs for diet studies are being collected. The data collected is being developed into new life history projects for at least four species of Alaskan skates.

Chondrichthyan fishery database. A regional fisheries database, incorporating all chondrichthyan species landed in waters of the western continental United States, was expanded during this period. Focus has been directed at acquiring and entering commercial and recreational chondrichthyan fishery landings and regulations from Alaska, California, Oregon, and Washington from 1980 to present. Details were obtained from the Pacific States Marine Fisheries Commission via the RecFin and PacFIN databases, and through state fisheries agencies. Relevant peer-reviewed publications, technical reports, and management/regulatory plans have been acquired and incorporated into the database. State-specific information will be provided for public access through the PSRC website and available later in 2005.

Taxonomic studies. A review and revision of eastern North Pacific skates genus Bathyraja (Family: Arhynchobatidae) is continuing apace. Colleagues at the NWFSC, with assistance from PSRC personnel, are continuing to collect and save skates during their annual slope cruises. Supplemental material is being collected during the AFSC 2005 Gulf of Alaska survey. A revised key to the skate egg cases ofthe eastern North Pacific has been undertaken. It is anticipated that this revised key will complement one recently completed for the eastern Bering Sea by PSRC staff. In addition, specimens from museum collections at the California Academy of Sciences, Los Angeles County Museum Natural History, Smithsonian Natural History Museum, Scripps Institution of Oceanography, and the University of Washington will continue to be examined. PSRC personnel are continuing to consult and collaborate on this project with leading experts at the Shark Research Center, Cape Town, South Africa, CSIRO, Hobart, Tasmania, Australia, and at Texas A & M University. A review of eastern Pacifi c chimaeras has revealed several new species. These new species are being investigated in collaboration with a colleague from Millersville State University.

Publications and conferences. Members of the PSRC attended and presented results from our research at i h Indo-Pacific Fish Conference in Taipei, Taiwan. The PSRC web site monthly "Featured Elasmobranch" that highlights a different species each month continues to attract attention from the general public, especially educators, who visit the web site on a regular basis to find out more about our ongoing research efforts. PSRC staff produced a total of seven papers during this report period that are published, in press, or in review.

13 Virginia Institute of Marine Science Report (1. Musick, PII)

Relative abundance and distribution studies. During the report period, 20 longline sets were made in the Chesapeake Bay and Virginia coastal waters that fished 2, 100 standard hooks and 380 non­ standard hooks (directed toward juvenile sandbar sharks). The catch included a total of226 sharks: 126 sandbar sharks (Carcharhinus plumbeus), 79 smooth dogfish (Mustelus canis), eight spiny dogfish (Squalus acanthias), six dusky sharks (Carcharhinus obscurus), four Atlantic sharpnose sharks (Rhizoprionodon terraenovae), one shortfin mako shark (Isurus oxyrinchus), one spinner shark (Carcharhinus brevipinna), and one scalloped hammerhead shark (Sphyrna lewini). Thus far this season no sandtiger (Carcharias taurus), or tiger sharks (Galeocerdo cuvier) have been captured. The sharks captured were either sampled for age and growth, tagged with VIMS juvenile dart tags, or tagged with NMFS M-type dart tags. All sharks were sampled for DNA analysis. In addition skates and rays of three different species were captured during the same sampling period: 75 cleamose skates (Raja eglanteria), 13 spiny butterfly rays (Gymnura alta vela ), and two cownose rays (Rhinoptera bonasus). VIMS scientists continue to collaborate with scientists from Alaska Department ofFish and Game and Jackson State University to develop techniques using side scan sonar to assess salmon shark stocks in the fjords adjacent to Prince William Sound. Preliminary studies in summer 2000 using sonically tagged sharks (known targets) suggested that an exploratory side scan sonar survey using a line transect design would be possible in selected fjords in summer 2002. This exploratory research was successful and a second acoustic survey during August 2004 was conducted to monitor salmon shark abundance and the presence of prey species (schools of herring or salmon). To further monitor abundance longline sets were made in these areas to capture and tag salmon sharks. During this cruise three salmon sharks were outfitted with thermistor tags to monitor the thermal inertia exhibited by salmon sharks, more specifically to monitor temp changes in the intermuscular rete. A third acoustic and sampling survey trip has been planned to commence in August 2005. To date approximately 300 salmon sharks have been tagged in cooperation with the Alaska Department of Fish and Game on cooperative VIMS cruises.

Age, groHlth, and demography. When VIMS scientists compared growth rates for sandbar sharks before and after stock collapse from overfishing between 1980 and 1992, they found little growth compensation and no change in the age at maturity. Now in 2005 the sandbar shark popUlation is still less than 50% ofthat in 1980 and compensation has had more time to become apparent (growth may be affected over more year classes). VIMS scientists continue to examine sandbar shark growth rates (based on vertebral analysis) collected in 2001-2003 for comparison to published historical rates and those derived from archived vertebrae. VIMS scientists are continuing to work in cooperation with scientists from the University of Hawaii to collect sandbar shark vertebrae from the Hawaiian popUlation which is purported to grow faster, mature younger, and reach a smaller maximum size. Sampling trips were conducted in September 2002, May 2003, September 2003, June 2004, and January 2005. These trips have yielded 160 samples for age and growth studies, stomach content analysis, and genetic analysis. 104 sandbar sharks were tagged, injected with OTC, and released for age validation considerations over the time span. Four tagged sharks were recaptured after periods of 1 week, 6 months, and 9 months. Examination of vertebral centra from these sharks

14 supported the use of vertebral centra for aging sandbar sharks in Hawaii. OTC stained vertebral centra and marginal increment analysis support the hypothesis of annuli formation in the winter months. Preliminary results portray much slower growth rates than previously reported by Wass (1973). VIMS scientists continue the process of developing a comprehensive demographic model and stock assessment of the barndoor skate. During this period, the focus of the work has been advancing the methodologies of parameter estimation. Specifically, to use mean lengths of the barndoor skate populations to re-create the fishing mortality history ofthe Georges Bank population. The results ofthis analysis and the results from our earlier work on the life history parameters have been incorporated into these newly developed demographic models. The results of these analyses combined with the observed recovery from NMFS survey indices, suggest that the barndoor skate has a maximum population growth rate (rintrinsic ) of close to 0.5 and is more resilient to fishing pressure than previously believed. A re-analysis of NMFS survey data also indicates that the recovery of barn door skate populations began in the mid 1980's, long before the concern about their extinction began. In addition, over 150 stomach samples were processed in a continuing effort to study the life history of the barndoor skate. Age and growth studies of deep-sea squaloid sharks are ongoing at VIMS. Due to the vulnerability of deep-sea sharks to fishing pressure, this age and growth data will be crucial for the proper management of these long-lived species. VIMS scientists have continued collaboration with Dr. Jose Castro (NMFS, Mote Marine Laboratory) and John Galbraith (NMFS, Northeast Fisheries Science Center) to collect squaloid sharks from the Western North Atlantic and the Gulf of Mexico. Thus far, this collaboration with Dr. Castro and John Galbraith has yielded hundreds of fin spines from 7 different speci es of squaloid sharks to be used for aging. A third deep-water longline trip wi th Dr. Castro is pl anned in the Gulf of Mexico in the fall of 2005. Also, a VIMS scientist will participate in the NMFS Autumn Bottom Trawl Survey in September to sample squaloid sharks captured in their trawl gear. VIMS scientists maintain active participation in the MAR-ECO project (www.mar-eco.no) and continue to work within the MAR-ECO Group to verify the identification of all elasmobranch voucher specimens captured during this cruise. In addition, an annotated list offish species captured in this region along the Mid-Atlantic Ridge is in preparation. This annotated list will represent the sum work of all participants in the MAR-ECO Taxonomy Group and will include any notably rare species, range records and depth records. During the cruise, data was collected (lengths, weight, sex, maturity) for approximately 5500 deep-sea sharks (12 species), and fm spines were collected for age and growth analysis from approximately 500 ofthe squaloid sharks captured (Centrophorus squamosus, Centroscymnus coelolepis, C. crepidator, C. owstoni, Etmopterus princeps, Deania calcea, Centroscyllium fabricii) . Samples were also taken for studies of reproductive biology in a few ofthese squaloid sharks. VIMS scientists will work with Dr. William Hamlett (Indiana University School of Medicine) to process these samples. Specifically, oviducal glands collected from squaloid species will be examined for evidence of sperm storage; also samples were collected from reproductively acti ve female Etmopterid sharks to look for evidence of hi stotrophy in thi s species.

15 Habitat and migration studies. During this period we re-deployed an array of 19 acoustic receiver stations in Wachapreague Inlet. VIMS scientists also deployed a water quality monitoring device (YSI) to monitor the temperature, salinity, and dissolved oxygen at one end ofthe array every fifteen minutes throughout the summer months. The receivers were cleaned, and data was downloaded on a weekly to bi-weekly basis beginning in May when the receivers were deployed to continue throughout the summer until the end of October. During the previous summer, 37 juvenile sandbar sharks were implanted with acoustic transmitters within the array. Two ofthese sharks were caught and killed by a commercial fishermen at the end of last summer, leaving a total of 35 sharks with functional transmitters at the end of the summer nursery season in 2004. During the period of time between the deployment of the array this year through June 30th, 23 of these 35 sharks (68%) had returned to the array. The majority of these sharks returned after June 11th (91 %) from the inlet (68%) with a much smaller percentage of sharks returning from either the southern (14%) inlets or from more northern localities (18%). These sharks will be tracked by the array until their departure in the earl y fall months for their winter migration to the Carolinas. The activity ofthese sharks will be studied as well as the effect of physical parameters including: daylight, tidal cycle, temperature, salinity, and dissolved oxygen concentration on the movements of these sharks. The coastal lagoons of Virginia provide a source of protection and food for many migratory elas mobranchs. Previous studies have examined the importance of these lagoons for sandbar sharks as a nursery. Little has been examined with regards to the rays that inhabit the region. These lagoons have distinct environmental gradients with regards to sediment types and concurrent fauna, temperature regimes and tidal influences. Habitat utilization and movement ofrays should therefore vary according to the how these variables are manifested in the lagoon. We are currently studying the stingrays and butterfly rays that inhabit the region. Specimens are collected using a 22-foot trawl pulled by a 24 foot skiff. Wachapreague and Machipongo inlets and the adjacent lagoons will be sampled monthly beginning in May (before arrival) and ending when sampling indicates the animals have left the lagoons. Sampling is designed to compare deep and shallow regions so to distinguish habitat preferences. Shallow areas are defmed as regions where water depth is less than ten feet. Deep areas are channels throughout the lagoon region. Sediment samples, using a PONAR grab sampler, are taken to further distinguish area preferences. Disc width or total length is recorded for all species of finfish and sex is determined for all elasmobranchs. Age, growth, diet, and reproductive studies will be conducted on the Gymnuridae and Dasyatidae species in the upcoming periods. Currently, stations have been sampled in May and June ofthis period. In May, 24 rays were caught in 48 tows. In June, 35 rays were caught in 60 tows. Nine spiny butterfly rays, ranging from 53 to 207 cm in disc width were caught. Four smooth butterfly rays (Gymnura micrura) were caught with disc widths ranging from 62 to 97 cm. Three bluntnose rays (Dasyatis say ) were caught with disc widths ranging from 22 to 79 cm wide. Fifteen of the rays of interest were caught in shallow regIOns.

Shark energetic and osmoregulatory studies. Plans to study the salinity tolerance and metabolic rates of sandbar sharks at different salinities are currently underway at VIMS' Eastern Shore Lab. Juvenile sandbar sharks present in the Chesapeake Bay nursery area are able to utilize areas within this estuary that have salinities down to approximately 20 ppt. We hypothesize this represents a

16 physiological barrier against predation by larger sharks that are unable to utilize these low salinity waters. In order to test the hypothesis that the animals can successfully maintain life processes at reduced salinities juvenile sandbar sharks are captured by hook and line from the barrier island channels and lagoons off the Eastern Shore of Virginia and maintained at the field station in Wachapreague. The animals are housed in recirculating aquaria in which salinities are decreased incrementally from 33ppt to 20ppt over a 27 day period. Data collected last summer support the expectation that the sharks blood chemistry adapts to cope with the changes and that the animals are able to tolerate salinities down to at least 2Sppt. Work is continuing to examine the animals' abilities to tolerate salinities down to 20ppt and to reject the possibility that blood chemistry changes result from tank effects.

Sandbar shark genetics. VIJ\.1S scientists continue to work on estimating the effective popUlation size of sandbar sharks utilizing the Chesapeake Bay nursery as well as the effective number of female breeders that pup in these nurseries using modem genetic techniques. This study will also examine the possibility of significant loss of genetic variation during the last thirty years of exploitation, by comparing gene frequencies between museum samples collected in the late 70's with samples collected in the last several years. Work is also being done to compare gene frequencies of animals caught in the Chesapeake Bay nurseries with those found in other nurseries on the east coast to examine philopatry and estimate gene flow. Periodicity is being assessed with in nurseries by using highly polymorphic loci to detect YOY kin groups within and between sampling years. In order to accomplish these goals, over 800 fin clips have been taken from juvenile and adult female sandbar sharks in the Eastern Shore and lower Chesapeake Bay nurseries with an additional 2S0 samples from Delaware Bay. Six micro satellite markers have been designed at VIJ\.1S. Cross species amplification of markers have demonstrated the utility ofthese markers for other Carcharhinids. In addition two other microsatellite markers from the literature have been optimized to ensure efficient accurate genetic screening. Two pregnant females and their litters were screened at these eight loci. The first litter contained 11 pups and a minimum of four sires was necessary to explain the observed gene array. The second litter contained seven pups and a minimum of two sires was necessary to explain the observed gene array. In addition a YOY sandbar shark caught in 2003 was identified as the progeny of an adult female caught in the same Eastern Shore Lagoon in 200S using relatedness software. In upcoming periods additional mother/litter groups will be genotyped to examine the prevalence multiple paternity in sandbar sharks throughout the western North Atlantic. Sequencing of the control region will begin in the coming months enabling us to estimate the female effective population size and define the nature of gene flow between this popUlation and others.

Publications and conferences. During this period two VIJ\.1S researchers attended the Southern Division of the American Fisheries Society meeting and presented papers. One VIJ\.1S researcher attended the ARGOS International Users and Manufacturers Meeting and presented a poster. One VIJ\.1S researcher attended the MAR-ECO Field Phase Workshop in Lisbon, Portugal and presented a paper. This VIMS researcher has a manuscript in preparation with Norwegian coauthors to describe the distribution of fishes captured by longline along this portion of the Mid-Atlantic Ridge.

17 Uni versity of Florida Report (G. Burgess, PIT)

Fishery independent surveys. Fishery independent long-line sampling was conducted aboard leased commercial shark fishing boats in the Spring of 2005, after the winter commercial shark fishing season closed. FPSR scientists were at sea 15 days in March, April, and June in the southeastern Gulf of Mexico and in the Atlantic offthe coasts of the Florida peninsula. A total of 410 sharks were caught on these sampling cruises. The dominant species in the catch were blacktip (114), blacknose (69), Atlantic sharpnose (61), and sandbar (38). Some of these sharks were tagged and released alive; others, primarily those that arrived at the boat dead or moribund, were necropsied and completely "worked up" to provide tissues and materials for biological studies by FPSR scientists and NSRC collaborators. Data and specimens derived from our fishery independent surveys and from our separately funded Commercial Shark Fishery Observer Program have contributed to our ongoing biological studies of age, growth, and reproduction (see items 4b and 4g). We have continued to collect biological samples for collaborators in NMFS and other NSRC institutions. We have provided bull shark muscle samples to scientists in state agencies studying heavy metal concentrations in fish tissues. Additionally we have collected shark vertebrae and reproductive tissues for ongoing life history projects, some of which are collaborative efforts with NMFS Panama City and Pascagoula labs.

Age and growth. We have been examining age and growth characteristics in the scalloped and the great hammerhead sharks in cooperation with NMFS scientists. During this reporting period, we have completed the scalloped hammerhead age and growth project and are currently finishing writing of the manuscript. We anticipate submission of the scalloped hammerhead manuscript during the next reporting period. We plan to finish great hammerhead vertebrae preparation and analysis during the next reporting period. Our preliminary analyses have indicated thus far that scalloped and great hammerheads exhibit different growth rates, with the great hammerhead appearing to grow much quicker. Our oldest scalloped hammerhead shark aged was 30 years. We are continuing the collection and processing of tiger shark vertebrae for a life history study on this species.

Tagging and tracking bull sharks. During the reporting period a total of 30 days were spent in the Indian Ri ver Lagoon (IRL) sampling with longlines and rod and reel, carrying out tracking activities, and maintaining the automated tracking system. Four immature female bull sharks ranging from 72 to 104 cm TL were captured. Three sharks were tagged and actively tracked for periods of 12-15 hours; one shark being relocated and tracked over one week after it was initially tagged. One neonate bull shark expired during tagging and was given to the Florida Fish and Wildlife Research Institute in Melbourne so various tissues could be utilized in biocontaminant, age and growth, and tooth morphology studies. An additional 14 bull sharks were observed free-swimming in lagoonal waters and their habitat characteristics recorded. The array of 19 passive listening stations was maintained the Indian River Lagoon System in conjunction with the Dynamac Corporation at the Kennedy Space Center. Additional bull shark catch datasets have been compiled from various state agencies and universities to bolster our data on the distribution of this species in the IRL. Tagging

18 and tracking activities will continue through the next reporting period. The manual tracking portion of this study will be completed during 2005.

Fatty acid analysis. In a previous reporting period we initiated an experiment dealing with the effect of diet on fatty acid composition of stingrays. This study serves as a validation model for other studies dealing with commercially important sharks. This project was completed during the last reporting period. Our result indicate that fatty acid analysis may not be useful in predicting elasmobranch diets contrary to what has been shown in other vertebrates. Statistical analyses showed that prey fatty acid profiles were not reflected in the storage tissues of Atlantic stingrays after a month of feeding. The failure of stingray storage tissues to shift in fatty acid composition toward the prey item may be related to the lower metabolisms in elasmobranchs. Two manuscripts are being written for this project and we anticipate completing during the next reporting period. This study was conducted with the cooperation of collaborating scientists from MML's Center for Shark Research and the Physiological Ecology and Bioenergetics Lab at the University of Central Florida.

Bomb radiocarbon ageing sandbar sharks. During the last reporting period, 12 sandbar shark vertebral samples were processed in collaboration with NSRC scientists at Moss Landing Marine Lab and scientists at the NMFS lab in Narragansett, RI. Radiocarbon analysis was performed at the Center for Accelerator Mass Spectrometry at Lawrence Livermore National Lab. Putative birth dates for the sharks examined, based on vertebral growth bands, ranged from 1950 to 1980, spanning the time from expected "Prebomb" to "Postbomb" levels of radiocarbon. Delta 14C values ranged from - 115 to + 122. Although postbomb levels were generally higher than prebomb levels, the temporal pattern was not clear. For example, two sharks showed declines in 14C levels in vertebral bands deposited between the early- and the late-1960s, when levels should have been increasing according to the underlying hypothesis. It is not yet known if this decline is due to reworking of collagen carbon in the cartilage or due to changes in diet that took the sharks out of the radiocarbon enriched food chain. Additional study is anticipated, although it is apparent that this technique may not be useful for validation of vertebral growth marks in ageing the sandbar shark.

Dusky shark population modeling. During this reporting period, data collection and research concerning the popUlation viability of the dusky shark has continued. Two statistical models selected for this project have been run with preliminary data. Coordination has continued with colleagues outside the FPSR, who are using similar data with different statistical models. This coordination has included sharing data, modeling skills, and preliminary results. Selection of biological parameters has continued and data sets have been updated to include an additional year's data.

Reproduction, gestation. We have continued collecting reproductive tissues from scalloped and great hammerhead sharks, tiger sharks, sandbar sharks, and blacknose sharks. We have begun analyzing blacknose shark reproduction in collaboration with NMFS Panama City and Pascagoula labs. We anticipate finishing gross observations of female blacknose shark tissues and beginning male blacknose shark analysis during the next reporting period. We will also begin analyzing sandbar shark and hammerhead shark tissues. We anticipate beginning histological work before the

19 end of the year. Additionally, we have continued to collect liver samples from pregnant sharks and their embryos to examine fatty acid transfer from the mother to the pups during gestation. The goal ofthis study is to determine what fatty acids are important during gestation. Our DNA collection is expanding and we have samples from many litters for examining multiple paternity in several species of sharks. We anticipate starting examination of mUltiple paternity in the sandbar shark during the next reporting period. For blacknose sharks, we have increased the male and female sample sizes from mature individuals for the months of April, May, June, and July through independent sampling research cruises. Thus far we have measured egg size, nidamental gland widths, and gonadosomatic indices. We anticipate that over the course ofthis fiscal year we be able to increase the sampling size for all months that are deficient. Preliminary analyses of monthly data suggests that blacknose sharks exhibits an annual reproductive cycle with pupping occurring in late May and early June followed closely by ovulation in late June early July. For the blacknose size at maturity study, FPSR scientists have increased the samples of males (from 52 to 62) and females (from 80 to 91) of varying sizes and stages of reproductive maturity. Our goals for the next reporting period will be threefold: 1) we will continue to obtain both female and male specimens that are 75 cm or less in fork length; 2) we will begin analyzing the morphological changes associated with male and female reproductive tracts in order to measure the onset of sexual maturity; and 3) initiate histological examination of testicular spermatogenic stages and oviducal gland contents. During the reporting period, we have initiated a new study on the reproductive biology ofthe roundel skate (Raja texana). To date, FPSR scientists have collected 77 males and 105 females of varying sizes and stages of reproductive maturity. From each specimen, the vertebrae, jaw, and reproductive organs have been removed. In the laboratory, 63 male and 90 females skates have been further processed in the following manner: 1) the vertebrae have been cut into bow-tied sections for ageing; 2) morphological measurements of the reproductive tract and associated organs have been completed; 3) histological examination oftesticular reproductive stages have been initiated; and 4) jaws have been cleaned and processed in order to determine if sexual dental dimorphism is present and what significance this phenomenon may have in mating. We anticipate all size classes will be sufficiently sampled in the next reporting period. Although our long-term goal is to examine the annual reproductive cycle for this species, our current reliance on samples taken as bycatch from the seasonal butterfish trawl fishery will make it difficult to attain this goal without fishery independent sampling. FPSR scientist James Sulikowski and collaborators at the University of New Hampshire are using steroid hormone analyses as a new and potentially innovative means to assess reproductive maturity and seasonal cycles in two exploited skate species in New England waters. Currently, blood samples have been collected from 79 male and 81 females smooth skates (Malacoraja senta) and from 40 male and 48 female thorny skates (Amblyraja radiata). The extraction of estradiol, progesterone, and testosterone has been completed for A. radiata and is in the initial stages for M. senta. It is anticipated that all steroid hormones will be extracted by the and the quantification of each hormone within the plasma will be completed by the end of the next reporting period.

20 Variation in vertebrae. During a previous reporting period we initiated a study to investigate differences in vertebral characteristics (size, shape, and growth band deposition) in different regions of the vertebral column in two species of sharks (sandbar and blacktip) caught in the commercial fishery. This project was completed during this reporting period. Our results indicate that vertebral morphological characteristics vary between the cervical and trunk regions but growth ring counts do not. We found no significant differences between adjacent vertebrae within a region. Our results were presented at a recent scientific meeting and a manuscript has been submitted for peer review.

Atlantic stingray ovarian development. There has been no progress on this topic during the reporting period due to difficulties in collecting female stingrays during the critical time periods. In addition, the graduate student charged with canying out this study withdrew from school during the previous reporting period and we have yet to identify a suitable substitute. We do not expect to continue with this project during the next reporting period unless .a suitable candidate can be recruited.

ISAF. A total of72 new case files were entered into the ISAF database during this time period and a total of 65 cases were updated with new information. A University of Florida "University Scholars" student completed and presented her project on identifying the tidal range and lunar cycle for all attacks in the USA. Another student enrolled in the Geographic Information Systems (GIS) Applications Specialist program at Sir Sandford Fleming College in Lindsay, Ontario, completed his project on applying GIS technology to attack data. Collaboration continues with colleagues working on the Australian Shark Attack File (ASF), and integration ofthe information from the ASF into the ISAF has been initiated. Over the course ofthe next few years, similar collaboration and integration will be undertaken with the Brazilian Shark Attack File, South African Shark Attack File, and Californian Shark Attack File. ISAF staff also began working alongside Museum Information Technology staff to redesign the format of the ISAF computer database, which will greatly improve its functionality. Requests for information from the public, media and scientific communities were continuously provided. The ISAF web pages continued to be the most heavily visited of all pages on the FLMNH web site.

Public education and web site. The FLMNH Ichthyology Division web pages, including the FPSR web pages, received 102,443,536 "hits", 3,342,324 visits, and 1,695,566 unique visitors during the reporting period. Materials relating to elasmobranchs on the site have been expanded and improved. New elasmobranch species accounts have been added to the "Biological Profiles" section. To date, we have a total of 81 elasmobranch profiles available online. The image gallery has been expanded with submissions from new photographers. The "In The News" shark pages are updated daily with the latest news releases on research and conservation. The IUCN Shark Specialist Group (SSG) and AES web sites have been continually upgraded and expanded. This includes news stories for each IUCN SSG region as well as features on the acti vities of this group. Maintenance ofthe AES web site consists primarily of posting new meeting infornlation including abstracts, as well as updating the membership directory, posting meeting minutes, and adding AES newsletters as they become available. Over the next reporting period, the web site will continually be expanded with the addition of new information in appropriate sections.

21 Project Shark Awareness. Project Shark Awareness, a professional development workshop for environmental and science educators, is a recent addition to our education initiatives. It is a classroom-based outreach program for educating school teachers and other educators about the myths and realities of shark behavior, biology, and conservation. The program consists ofworkshops and training sessions augmented by teaching support materials. A teaching kit consisting of a PowerPoint presentation, teaching guide, handouts, methods to evaluate student learning (quiz, puzzles, question sheets), and evaluation sheets have been developed and is available to each workshop participant. In addition, sharkjaws, teeth, skin, and embryos are including in the teaching kits, providing exciting hands-on learning opportunities for students. A reference list is also included for instructors and students who are interested in further pursuing topics related to sharks and rays. Teaching materials have been revised and improved during this reporting period, including the addition of more student activities as well as a pre- and post-workshop evaluation for workshop participants. During the next reporting period, the sawfish component of Project Shark Awareness will be greatly expanded to include detailed information regarding the biology and conservation status of sawfishes as well as the addition of biological profiles, listing documents, and resources. A companion web page within the FLMNH education web site is available online to accompany Project Shark Awareness materials. This web page includes all teaching materials as well as a new interactive shark identification key. During this reporting period, two Project Shark Awareness educator workshops were held. The first workshop was held in February in conjunction with Florida Sea Grant and 4-H as in-service training for extension agents. The second workshop took place on May 25-26 and was funded in part by the Disney Wildlife Conservation Fund. Participants included 17 educators from all regions within the state of Florida. During the upcoming reporting period, there will be at least three more Project Shark Awareness workshops.

Requests for information. The staff of the FPSR, especially those involved with the International Shark Attack File, have been busy responding to requests for information during the reporting period. We have handled 901 individual correspondences (email, phone, or postal mail) with various organizations, news agencies, scientists, and lay individuals asking for information and analysis on matters related to sharks and rays, especially shark attacks and related issues, shark ecology and biology, and shark conservation and fisheries. These requests have come from all over the world. FPSR staff members have also responded to numerous requests for interviews and for information on sharks from the general public, especially students. We have provided tissues, specimens, and information to collaborating scientists working in many different organizations as follows: NMFS labs in Panama City and Pascagoula, NMFS Office of Highly Migratory Species, Dynamac Corporation at Kennedy Space Center, Florida Fish and Wildlife Commission, Wildlife Conservation Society, National Estuarine Reserve Program, and collaborators in the NSRC program at the Center for Shark Research, MML. Two NMFS departments, the Protected Resource Division and the Highly Migratory Species Division, routinely request data and information, and these requests are addressed. We also provided technical support and tissue samples to support ongoing studies on various aspects of shark biology by scientists in South Africa, Rhode Island, and Florida.

22 Publications, presentations and conferences. During the reporting period we have published three papers and have four papers in press in peer reviewed journals. One additional manuscript has been submitted for pUblication. We have given seven oral or poster presentations to professional audiences at four conferences or meetings. FPSR staff members have participated in six workshops related to elasmobranch research, education, and conservation. At these workshops we have contributed twenty individual presentations or demonstrations.

Respectfully submitted,

Robert E. Hueter, Ph.D. Project Coordinator & Director, Center for Shark Research, Mote Marine Laboratory August 24, 2005

23