Species and population specific gene expression in blood transcriptomes of marine turtles.

BACKGROUND: Transcriptomic data has demonstrated utility to advance the study of physiological diversity and organisms' responses to environmental stressors. However, a lack of genomic resources and challenges associated with collecting high-quality RNA can limit its application for many wild populations. Minimally invasive blood sampling combined with de novo transcriptomic approaches has great potential to alleviate these barriers. Here, we advance these goals for marine turtles by generating high quality de novo blood transcriptome assemblies to characterize functional diversity and compare global transcriptional profiles between tissues, species, and foraging aggregations. RESULTS: We generated high quality blood transcriptome assemblies for hawksbill (Eretmochelys imbricata), loggerhead (Caretta caretta), green (Chelonia mydas), and leatherback (Dermochelys coriacea) turtles. The functional diversity in assembled blood transcriptomes was comparable to those from more traditionally sampled tissues. A total of 31.3% of orthogroups identified were present in all four species, representing a core set of conserved genes expressed in blood and shared across marine turtle species. We observed strong species-specific expression of these genes, as well as distinct transcriptomic profiles between green turtle foraging aggregations that inhabit areas of greater or lesser anthropogenic disturbance. CONCLUSIONS: Obtaining global gene expression data through non-lethal, minimally invasive sampling can greatly expand the applications of RNA-sequencing in protected long-lived species such as marine turtles. The distinct differences in gene expression signatures between species and foraging aggregations provide insight into the functional genomics underlying the diversity in this ancient vertebrate lineage. The transcriptomic resources generated here can be used in further studies examining the evolutionary ecology and anthropogenic impacts on marine turtles.

Persistent organic pollutants in green sea turtles (Chelonia mydas) inhabiting two urbanized Southern California habitats.

Within Southern California, east Pacific green sea turtles (Chelonia mydas) forage year-round, taking advantage of diverse food resources, including seagrass, marine algae, and invertebrates. Assessing persistent organic pollutants (POP) in green turtle aggregations in the Seal Beach National Wildlife Refuge (SBNWR, n = 17) and San Diego Bay (SDB, n = 25) can help quantify contamination risks for these populations. Blood plasma was analyzed for polychlorinated biphenyls (PCBs), organochlorinated pesticides (OCPs), and polybrominated diphenyl ethers (PBDEs). PCBs and body size explained much of the separation of turtles by foraging aggregation in a principal component analysis. Turtles from SDB had significantly (p < 0.001) higher total PCBs than SBNWR turtles. Most PCBs detected in turtles were non-dioxin-like PCB congeners (153, 138, 99) that are associated with neurotoxicity. Recaptured turtles' POP levels changed significantly over time indicating significant variation in POP levels through time and space, even among adjacent foraging locations.

Trace metals in green sea turtles (Chelonia mydas) inhabiting two southern California coastal estuaries.

Foraging aggregations of east Pacific green sea turtles (Chelonia mydas) inhabit the Seal Beach National Wildlife Refuge (SBNWR) and San Diego Bay (SDB), two habitats in southern California, USA, located near urbanized areas. Both juvenile and adult green turtles forage in these areas and exhibit high site fidelity, which potentially exposes green turtles to anthropogenic contaminants. We assessed 21 trace metals (TM) bioaccumulated in green turtle scute and red blood cell (RBC) samples collected from SBNWR (n = 16 turtles) and SDB (n = 20 turtles) using acid digestion and inductively coupled plasma mass spectrometry. Principal component analyses of TM composition indicate that SBNWR and SDB turtles have location-specific contaminant signatures, characterized by differences in cadmium and selenium concentrations: SBNWR turtles had significantly more cadmium and selenium in RBC and more selenium in scute samples, than SDB turtles. Cadmium and selenium concentrations in RBC had a strong positive relationship, regardless of location. SBNWR turtles had higher selenium in RBCs than previously measured in other green turtle populations globally. Due to different retention times in blood vs. scute, these results suggest that SBNWR turtles have high long- and short-term selenium exposure. Turtles from SBNWR and SDB had higher trace metal concentrations than documented in green turtle populations that inhabit non-urbanized areas, supporting the hypothesis that coastal cities can increase trace metal exposure to local green turtles. Our study finds evidence that green turtle TM concentrations can differ between urbanized habitats and that long-term monitoring of these green turtles may be necessary.

Environmental Warming and Feminization of One of the Largest Sea Turtle Populations in the World.

Climate change affects species and ecosystems around the globe [1]. The impacts of rising temperature are particularly pertinent in species with temperature-dependent sex determination (TSD), where the sex of an individual is determined by incubation temperature during embryonic development [2]. In sea turtles, the proportion of female hatchlings increases with the incubation temperature. With average global temperature predicted to increase 2.6°C by 2100 [3], many sea turtle populations are in danger of high egg mortality and female-only offspring production. Unfortunately, determining the sex ratios of hatchlings at nesting beaches carries both logistical and ethical complications. However, sex ratio data obtained at foraging grounds provides information on the amalgamation of immature and adult turtles hatched from different nesting beaches over many years. Here, for the first time, we use genetic markers and a mixed-stock analysis (MSA), combined with sex determination through laparoscopy and endocrinology, to link male and female green turtles foraging in the Great Barrier Reef (GBR) to the nesting beach from which they hatched. Our results show a moderate female sex bias (65%-69% female) in turtles originating from the cooler southern GBR nesting beaches, while turtles originating from warmer northern GBR nesting beaches were extremely female-biased (99.1% of juvenile, 99.8% of subadult, and 86.8% of adult-sized turtles). Combining our results with temperature data show that the northern GBR green turtle rookeries have been producing primarily females for more than two decades and that the complete feminization of this population is possible in the near future.

An analytical approach to sparse telemetry data.

Horizontal behavior of highly migratory marine species is difficult to decipher because animals are wide-ranging, spend minimal time at the ocean surface, and utilize remote habitats. Satellite telemetry enables researchers to track individual movements, but population level inferences are rare due to data limitations that result from difficulty of capture and sporadic tag reporting. We introduce a Bayesian modeling framework to address population level questions with satellite telemetry data when data are sparse. We also outline an approach for identifying informative variables for use within the model. We tested our modeling approach using a large telemetry dataset for Shortfin Makos (Isurus oxyrinchus), which allowed us to assess the effects of various degrees of data paucity. First, a permuted Random Forest analysis is implemented to determine which variables are most informative. Next, a generalized additive mixed model is used to help define the relationship of each remaining variable with the response variable. Using jags and rjags for the analysis of Bayesian hierarchical models using Markov Chain Monte Carlo simulation, we then developed a movement model to generate parameter estimates for each of the variables of interest. By randomly reducing the tagging dataset by 25, 50, 75, and 90 percent and recalculating the parameter estimates, we demonstrate that the proposed Bayesian approach can be applied in data-limited situations. We also demonstrate how two commonly used linear mixed models with maximum likelihood estimation (MLE) can be similarly applied. Additionally, we simulate data from known parameter values to test each model's ability to recapture those values. Despite performing similarly, we advocate using the Bayesian over the MLE approach due to the ability for later studies to easily utilize results of past study to inform working models, and the ability to use prior knowledge via informed priors in systems where such information is available.

First Assessment of the Sex Ratio for an East Pacific Green Sea Turtle Foraging Aggregation: Validation and Application of a Testosterone ELISA.

Determining sex ratios of endangered populations is important for wildlife management, particularly species subject to sex-specific threats or that exhibit temperature-dependent sex determination. Sea turtle sex is determined by incubation temperature and individuals lack external sex-based traits until sexual maturity. Previous research utilized serum/plasma testosterone radioimmunoassays (RIA) to determine sex in immature/juvenile sea turtles. However, there has been a growing application of enzyme-linked immunosorbent assay (ELISA) for wildlife endocrinology studies, but no study on sea turtles has compared the results of ELISA and RIA. This study provides the first sex ratio for a threatened East Pacific green sea turtle (Chelonia mydas) foraging aggregation, a critical step for future management of this species. Here, we validate a testosterone ELISA and compare results between RIA and ELISA of duplicate samples. The ELISA demonstrated excellent correspondence with the RIA for providing testosterone concentrations for sex determination. Neither assay proved reliable for predicting the sex of reproductively active females with increased testosterone production. We then applied ELISA to examine the sex ratio of 69 green turtles foraging in San Diego Bay, California. Of 45 immature turtles sampled, sex could not be determined for three turtles because testosterone concentrations fell between the ranges for either sex (females: 4.1-113.1 pg/mL, males: 198.4-2,613.0 pg/mL) and these turtles were not subsequently recaptured to enable sex determination; using a Bayesian model to predict probabilities of turtle sex we predicted all three 'unknowns' were female (> 0.86). Additionally, the model assigned all turtles with their correct sex (if determined at recapture) with 100% accuracy. Results indicated a female bias (2.83F:1M) among all turtles in the aggregation; when focusing only on putative immature turtles the sex ratio was 3.5F:1M. With appropriate validation, ELISA sexing could be applied to other sea turtle species, and serve as a crucial conservation tool.

Using models of social transmission to examine the spread of longline depredation behavior among sperm whales in the Gulf of Alaska.

Fishing, farming and ranching provide opportunities for predators to prey on resources concentrated by humans, a behavior termed depredation. In the Gulf of Alaska, observations of sperm whales depredating on fish caught on demersal longline gear dates back to the 1970s, with reported incidents increasing in the mid-1990s. Sperm whale depredation provides an opportunity to study the spread of a novel foraging behavior within a population. Data were collected during National Marine Fisheries Service longline surveys using demersal longline gear in waters off Alaska from 1998 to 2010. We evaluated whether observations of depredation fit predictions of social transmission by fitting the temporal and spatial spread of new observations of depredation to the Wave of Advance model. We found a significant, positive relationship between time and the distance of new observations from the diffusion center (r(2) = 0.55, p-value  = 0.003). The data provide circumstantial evidence for social transmission of depredation. We discuss how changes in human activities in the region (fishing methods and regulations) have created a situation in which there is spatial-temporal overlap with foraging sperm whales, likely influencing when and how the behavior spread among the population.

Prediction of fishing effort distributions using boosted regression trees.

Concerns about bycatch of protected species have become a dominant factor shaping fisheries management. However, efforts to mitigate bycatch are often hindered by a lack of data on the distributions of fishing effort and protected species. One approach to overcoming this problem has been to overlay the distribution of past fishing effort with known locations of protected species, often obtained through satellite telemetry and occurrence data, to identify potential bycatch hotspots. This approach, however, generates static bycatch risk maps, calling into question their ability to forecast into the future, particularly when dealing with spatiotemporally dynamic fisheries and highly migratory bycatch species. In this study, we use boosted regression trees to model the spatiotemporal distribution of fishing effort for two distinct fisheries in the North Pacific Ocean, the albacore (Thunnus alalunga) troll fishery and the California drift gillnet fishery that targets swordfish (Xiphias gladius). Our results suggest that it is possible to accurately predict fishing effort using < 10 readily available predictor variables (cross-validated correlations between model predictions and observed data -0.6). Although the two fisheries are quite different in their gears and fishing areas, their respective models had high predictive ability, even when input data sets were restricted to a fraction of the full time series. The implications for conservation and management are encouraging: Across a range of target species, fishing methods, and spatial scales, even a relatively short time series of fisheries data may suffice to accurately predict the location of fishing effort into the future. In combination with species distribution modeling of bycatch species, this approach holds promise as a mitigation tool when observer data are limited. Even in data-rich regions, modeling fishing effort and bycatch may provide more accurate estimates of bycatch risk than partial observer coverage for fisheries and bycatch species that are heavily influenced by dynamic oceanographic conditions.

Predicting bycatch hotspots for endangered leatherback turtles on longlines in the Pacific Ocean.

Fisheries bycatch is a critical source of mortality for rapidly declining populations of leatherback turtles, Dermochelys coriacea. We integrated use-intensity distributions for 135 satellite-tracked adult turtles with longline fishing effort to estimate predicted bycatch risk over space and time in the Pacific Ocean. Areas of predicted bycatch risk did not overlap for eastern and western Pacific nesting populations, warranting their consideration as distinct management units with respect to fisheries bycatch. For western Pacific nesting populations, we identified several areas of high risk in the north and central Pacific, but greatest risk was adjacent to primary nesting beaches in tropical seas of Indo-Pacific islands, largely confined to several exclusive economic zones under the jurisdiction of national authorities. For eastern Pacific nesting populations, we identified moderate risk associated with migrations to nesting beaches, but the greatest risk was in the South Pacific Gyre, a broad pelagic zone outside national waters where management is currently lacking and may prove difficult to implement. Efforts should focus on these predicted hotspots to develop more targeted management approaches to alleviate leatherback bycatch.

Cost-effectiveness of alternative conservation strategies with application to the Pacific leatherback turtle.

Although holistic conservation addressing all sources of mortality for endangered species or stocks is the preferred conservation strategy, limited budgets require a criterion to prioritize conservation investments. We compared the cost-effectiveness of nesting site and at-sea conservation strategies for Pacific leatherback turtles (Dermochelys coriacea). We sought to determine which conservation strategy or mix of strategies would produce the largest increase in population growth rate per dollar. Alternative strategies included protection of nesters and their eggs at nesting beaches in Indonesia, gear changes, effort restrictions, and caps on turtle takes in the Hawaiian (U.S.A.) longline swordfish fishery, and temporal and area closures in the California (U.S.A.) drift gill net fishery. We used a population model with a biological metric to measure the effects of conservation alternatives. We normalized all effects by cost to prioritize those strategies with the greatest biological effect relative to its economic cost. We used Monte Carlo simulation to address uncertainty in the main variables and to calculate probability distributions for cost-effectiveness measures. Nesting beach protection was the most cost-effective means of achieving increases in leatherback populations. This result creates the possibility of noncompensatory bycatch mitigation, where high-bycatch fisheries invest in protecting nesting beaches. An example of this practice is U.S. processors of longline tuna and California drift gill net fishers that tax themselves to finance low-cost nesting site protection. Under certain conditions, fisheries interventions, such as technologies that reduce leatherback bycatch without substantially decreasing target species catch, can be cost-effective. Reducing bycatch in coastal areas where bycatch is high, particularly adjacent to nesting beaches, may be cost-effective, particularly, if fisheries in the area are small and of little commercial value.

Estimating at-sea mortality of marine turtles from stranding frequencies and drifter experiments.

Strandings of marine megafauna can provide valuable information on cause of death at sea. However, as stranding probabilities are usually very low and highly variable in space and time, interpreting the results can be challenging. We evaluated the magnitude and distribution of at-sea mortality of marine turtles along the Pacific coast of Baja California Sur, México during 2010-11, using a combination of counting stranded animals and drifter experiments. A total of 594 carcasses were found during the study period, with loggerhead (62%) and green turtles (31%) being the most common species. 87% of the strandings occurred in the southern Gulf of Ulloa, a known hotspot of loggerhead distribution in the Eastern Pacific. While only 1.8% of the deaths could be definitively attributed to bycatch (net marks, hooks), seasonal variation in stranding frequencies closely corresponded to the main fishing seasons. Estimated stranding probabilities from drifter experiments varied among sites and trials (0.05-0.8), implying that only a fraction of dead sea turtles can be observed at beaches. Total mortality estimates for 15-day periods around the floater trials were highest for PSL, a beach in the southern Gulf of Ulloa, ranging between 11 sea turtles in October 2011 to 107 in August 2010. Loggerhead turtles were the most numerous, followed by green and olive ridley turtles. Our study showed that drifter trials combined with beach monitoring can provide estimates for death at sea to measure the impact of small-scale fisheries that are notoriously difficult to monitor for by-catch. We also provided recommendations to improve the precision of the mortality estimates for future studies and highlight the importance of estimating impacts of small-scale fisheries on marine megafauna.

Identification of distinct movement patterns in Pacific leatherback turtle populations influenced by ocean conditions.

Interactions with fisheries are believed to be a major cause of mortality for adult leatherback turtles (Dermochelys coriacea), which is of particular concern in the Pacific Ocean, where they have been rapidly declining. In order to identify where these interactions are occurring and how they may be reduced, it is essential first to understand the movements and behavior of leatherback turtles. There are two regional nesting populations in the East Pacific (EP) and West Pacific (WP), comprising multiple nesting sites. We synthesized tracking data from the two populations and compared their movement patterns. A switching state-space model was applied to 135 Argos satellite tracks to account for observation error, and to distinguish between migratory and area-restricted search behaviors. The tracking data, from the largest leatherback data set ever assembled, indicated that there was a high degree of spatial segregation between EP and WP leatherbacks. Area-restricted search behavior mainly occurred in the southeast Pacific for the EP leatherbacks, whereas the WP leatherbacks had several different search areas in the California Current, central North Pacific, South China Sea, off eastern Indonesia, and off southeastern Australia. We also extracted remotely sensed oceanographic data and applied a generalized linear mixed model to determine if leatherbacks exhibited different behavior in relation to environmental variables. For the WP population, the probability of area-restricted search behavior was positively correlated with chlorophyll-a concentration. This response was less strong in the EP population, but these turtles had a higher probability of search behavior where there was greater Ekman upwelling, which may increase the transport of nutrients and consequently prey availability. These divergent responses to oceanographic conditions have implications for leatherback vulnerability to fisheries interactions and to the effects of climate change. The occurrence of leatherback turtles within both coastal and pelagic areas means they have a high risk of exposure to many different fisheries, which may be very distant from their nesting sites. The EP leatherbacks have more limited foraging grounds than the WP leatherbacks, which could make them more susceptible to any temperature or prey changes that occur in response to climate change.

Stable isotope tracking of endangered sea turtles: validation with satellite telemetry and δ15N analysis of amino acids.

Effective conservation strategies for highly migratory species must incorporate information about long-distance movements and locations of high-use foraging areas. However, the inherent challenges of directly monitoring these factors call for creative research approaches and innovative application of existing tools. Highly migratory marine species, such as marine turtles, regularly travel hundreds or thousands of kilometers between breeding and feeding areas, but identification of migratory routes and habitat use patterns remains elusive. Here we use satellite telemetry in combination with compound-specific isotope analysis of amino acids to confirm that insights from bulk tissue stable isotope analysis can reveal divergent migratory strategies and within-population segregation of foraging groups of critically endangered leatherback sea turtles (Dermochelys coriacea) across the Pacific Ocean. Among the 78 turtles studied, we found a distinct dichotomy in δ(15)N values of bulk skin, with distinct "low δ(15)N" and "high δ(15)N" groups. δ(15)N analysis of amino acids confirmed that this disparity resulted from isotopic differences at the base of the food chain and not from differences in trophic position between the two groups. Satellite tracking of 13 individuals indicated that their bulk skin δ(15)N value was linked to the particular foraging region of each turtle. These findings confirm that prevailing marine isoscapes of foraging areas can be reflected in the isotopic compositions of marine turtle body tissues sampled at nesting beaches. We use a Bayesian mixture model to show that between 82 and 100% of the 78 skin-sampled turtles could be assigned with confidence to either the eastern Pacific or western Pacific, with 33 to 66% of all turtles foraging in the eastern Pacific. Our forensic approach validates the use of stable isotopes to depict leatherback turtle movements over broad spatial ranges and is timely for establishing wise conservation efforts in light of this species' imminent risk of extinction in the Pacific.