Non-random mating within an Island rookery of Hawaiian hawksbill turtles: demographic discontinuity at a small coastline scale.

Hawksbill sea turtles (Eretmochelys imbricata) from the Hawaiian archipelago form a small and genetically isolated population, consisting of only a few tens of individuals breeding annually. Most females nest on the island of Hawai'i, but little is known about the demographics of this rookery. This study used genetic relatedness, inferred from 135 microhaplotype markers, to determine breeding sex-ratios, estimate female nesting frequency and assess relationships between individuals nesting on different beaches. Samples were collected during the 2017 nesting season and final data included 13 nesting females and 1002 unhatched embryos, salvaged from 41 nests, of which 13 had no observed mother. Results show that most females used a single nesting beach laying 1-5 nests each. From female and offspring alleles, the paternal genotypes of 12 breeding males were reconstructed and many showed high relatedness to their mates. Pairwise relatedness of offspring revealed one instance of polygyny but otherwise suggested a 1 : 1 breeding-sex ratio. Relatedness analysis and spatial-autocorrelation of genotypes indicate that turtles from different nesting areas do not regularly interbreed, suggesting that strong natal homing tendencies in both sexes result in non-random mating across the study area. Complexes of nearby nesting beaches also showed unique patterns of inbreeding across loci, further indicating that Hawaiian hawksbill turtles have demographically discontinuous nesting populations separated by only tens of km.

Natal Origin and Spatiotemporal Distribution of Leatherback Turtle (Dermochelys coriacea) Strandings at a Foraging Hotspot in Temperate Waters of the Southwest Atlantic Ocean.

Leatherback turtles migrate long distances between nesting beaches and distant foraging areas worldwide. This study analyzes the genetic diversity, life history stage, spatiotemporal distribution, and associated threats of a foraging aggregation in the Southwest Atlantic Ocean. A total of 242 leatherbacks stranded or bycaught by artisanal fisheries were recorded from 1997 to 2021 in Uruguay, with sizes ranging from 110.0 to 170.0 cm carapace lengths, indicating that the aggregation is composed of large juveniles and adults. Results of Bayesian mixed-stock analysis show that leatherbacks come primarily from the West African rookeries, based on mitochondrial DNA sequences obtained from 59 of the turtles representing seven haplotypes, including a novel one (Dc1.7). The main threat identified in the area is the fisheries bycatch but most of the carcasses observed were badly decomposed. There was significant seasonal and interannual variability in strandings that is likely associated with the availability of prey and the intensity of the fishing effort. Taken together, these findings reinforce the importance of these South American foraging areas for leatherbacks and the need to determine regional habitat use and migratory routes across the broader Atlantic region, in order to develop effective conservation measures to mitigate threats both at nesting beaches and foraging areas.

Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

Origins of juvenile green sea turtles (Chelonia mydas) in the Bahamas: A comparison of recent and historical rookery contributions.

Conservation of green sea turtles (Chelonia mydas) benefits from knowledge of population connectivity across life stages. Green turtles are managed at the level of genetically discrete rookeries, yet individuals from different rookeries mix at foraging grounds; therefore, rookeries may be impacted by processes at foraging grounds. Bimini, Bahamas, hosts an important foraging assemblage, but rookery contributions to this assemblage have never been resolved. We generated mitochondrial DNA sequences for 96 foraging green turtles from Bimini and used Mixed Stock Analysis to determine rookery contributions to this population using 817 and 490 base pair (bp) rookery baseline data. The MSA conducted with 817 bp data indicated that Quintana Roo, Mexico, and Central Eastern Florida contributed most to the Bimini population. The MSA conducted with 490 bp data indicated that Southwest Cuba and Central Eastern Florida contributed the most to Bimini. The results of the second MSA differ from a previous study undertaken with 490 bp data, conducted in Great Inagua, Bahamas, which suggested that Tortuguero, Costa Rica, contributed the most to that foraging assemblage. Large credible intervals in our results do not permit explicit interpretation of individual rookery contributions, but our results do indicate substantial relative differences in rookery contributions to two Bahamian foraging assemblages which may be driven by oceanic currents, rookery sizes, and possibly juvenile natal homing. Our findings may implicate a shift in contributions to the Bahamas over two decades, highlighting the importance of regularly monitoring rookery contributions and resolving regional recruitment patterns to inform conservation.

Outcomes from a mechanistic biomarker multi-arm and randomised study of liposomal MTP-PE (Mifamurtide) in metastatic and/or recurrent osteosarcoma (EuroSarc-Memos trial).

The phase III clinical study of adjuvant liposomal muramyl tripeptide (MTP-PE) in resected high-grade osteosarcoma (OS) documented positive results that have been translated into regulatory approval, supporting initial promise for innate immune therapies in OS. There remains, however, no new approved treatment such as MTP-PE for either metastatic or recurrent OS. Whilst the addition of different agents, including liposomal MTP-PE, to surgery for metastatic or recurrent high-grade osteosarcoma has tried to improve response rates, a mechanistic hiatus exists in terms of a detailed understanding the therapeutic strategies required in advanced disease. Here we report a Bayesian designed multi-arm, multi-centre, open-label phase II study with randomisation in patients with metastatic and/or recurrent OS, designed to investigate how patients with OS might respond to liposomal MTP-PE, either given alone or in combination with ifosfamide. Despite the trial closing because of poor recruitment within the allocated funding period, with no objective responses in eight patients, we report the design and feasibility outcomes for patients registered into the trial. We demonstrate the feasibility of the Bayesian design, European collaboration, tissue collection with genomic analysis and serum cytokine characterisation. Further mechanistic investigation of liposomal MTP-PE alone and in combination with other agents remains warranted in metastatic OS.

Spontaneous Isolated Iliopsoas Tendon Tear in Elderly-Case Report and Review of Literature.

Iliopsoas tendon tears are rare. These typically occur in young and can be associated with avulsion fractures of lesser trochanter. We report a case of full thickness rupture of iliopsoas tendon in 87-year-old male without avulsion of the lesser trochanter.

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.

Design, analysis and reporting of multi-arm trials and strategies to address multiple testing.

BACKGROUND: It is unclear how multiple treatment comparisons are managed in the analysis of multi-arm trials, particularly related to reducing type I (false positive) and type II errors (false negative). METHODS: We conducted a cohort study of clinical-trial protocols that were approved by research ethics committees in the UK, Switzerland, Germany and Canada in 2012. We examined the use of multiple-testing procedures to control the overall type I error rate. We created a decision tool to determine the need for multiple-testing procedures. We compared the result of the decision tool to the analysis plan in the protocol. We also compared the pre-specified analysis plans in trial protocols to their publications. RESULTS: Sixty-four protocols for multi-arm trials were identified, of which 50 involved multiple testing. Nine of 50 trials (18%) used a single-step multiple-testing procedures such as a Bonferroni correction and 17 (38%) used an ordered sequence of primary comparisons to control the overall type I error. Based on our decision tool, 45 of 50 protocols (90%) required use of a multiple-testing procedure but only 28 of the 45 (62%) accounted for multiplicity in their analysis or provided a rationale if no multiple-testing procedure was used. We identified 32 protocol-publication pairs, of which 8 planned a global-comparison test and 20 planned a multiple-testing procedure in their trial protocol. However, four of these eight trials (50%) did not use the global-comparison test. Likewise, 3 of the 20 trials (15%) did not perform the multiple-testing procedure in the publication. The sample size of our study was small and we did not have access to statistical-analysis plans for the included trials in our study. CONCLUSIONS: Strategies to reduce type I and type II errors are inconsistently employed in multi-arm trials. Important analytical differences exist between planned analyses in clinical-trial protocols and subsequent publications, which may suggest selective reporting of analyses.

Translating Marine Animal Tracking Data into Conservation Policy and Management.

There have been efforts around the globe to track individuals of many marine species and assess their movements and distribution, with the putative goal of supporting their conservation and management. Determining whether, and how, tracking data have been successfully applied to address real-world conservation issues is, however, difficult. Here, we compile a broad range of case studies from diverse marine taxa to show how tracking data have helped inform conservation policy and management, including reductions in fisheries bycatch and vessel strikes, and the design and administration of marine protected areas and important habitats. Using these examples, we highlight pathways through which the past and future investment in collecting animal tracking data might be better used to achieve tangible conservation benefits.

Mixed stock analysis of juvenile green turtles aggregating at two foraging grounds in Fiji reveals major contribution from the American Samoa Management Unit.

In this study we assessed the breeding population, or Management Unit (MU), origin of green turtles (Chelonia mydas) present at Yadua Island and Makogai Island foraging grounds in Fiji, central South Pacific. Based on analysis of mitochondrial (mt) DNA sequences from 150 immature green turtles caught during surveys carried out in 2015-2016, we identified a total of 18 haplotypes, the most common being CmP22.1 (44%) which is a primary haplotype characterizing the American Samoa breeding population. Results of a Bayesian mixed-stock analysis reveals that the two foraging grounds are used by green turtles from the American Samoa MU (72%, Credible Interval (CI): 56-87%), New Caledonia MU (17%, CI: 6-26%) and French Polynesia MU (7%, CI: 0-23%). The prominence of the contribution we found from the American Samoa MU compared to that of French Polynesia, both which have historic telemetry and tagging data showing connectivity with Fijian foraging areas, may reflect the current relative abundance of these two nesting populations and draws attention to a need to update population surveys and identify any significant nesting in Fiji that may have been overlooked.

A versatile Rapture (RAD-Capture) platform for genotyping marine turtles.

Advances in high-throughput sequencing (HTS) technologies coupled with increased interdisciplinary collaboration are rapidly expanding capacity in the scope and scale of wildlife genetic studies. While existing HTS methods can be directly applied to address some evolutionary and ecological questions, certain research goals necessitate tailoring methods to specific study organisms, such as high-throughput genotyping of the same loci that are comparable over large spatial and temporal scales. These needs are particularly common for studies of highly mobile species of conservation concern like marine turtles, where life history traits, limited financial resources and other constraints require affordable, adaptable methods for HTS genotyping to meet a variety of study goals. Here, we present a versatile marine turtle HTS targeted enrichment platform adapted from the recently developed Rapture (RAD-Capture) method specifically designed to meet these research needs. Our results demonstrate consistent enrichment of targeted regions throughout the genome and discovery of candidate variants in all species examined for use in various conservation genetics applications. Accurate species identification confirmed the ability of our platform to genotype over 1,000 multiplexed samples and identified areas for future methodological improvement such as optimization for low initial concentration samples. Finally, analyses within green turtles supported the ability of this platform to identify informative SNPs for stock structure, population assignment and other applications over a broad geographic range of interest to management. This platform provides an additional tool for marine turtle genetic studies and broadens capacity for future large-scale initiatives such as collaborative global marine turtle genetic databases.

Long-term trends in the foraging ecology and habitat use of an endangered species: an isotopic perspective.

Evaluating long-term drivers of foraging ecology and population productivity is crucial for providing ecological baselines and forecasting species responses to future environmental conditions. Here, we examine the trophic ecology and habitat use of North Atlantic leatherback turtles (St. Croix nesting population) and investigate the effects of large-scale oceanographic conditions on leatherback foraging dynamics. We used bulk and compound-specific nitrogen isotope analysis of amino acids (CSIA-AA) to estimate leatherback trophic position (TP) over an 18-year period, compare these estimates with TP estimates from a Pacific leatherback population, and elucidate the pre-nesting habitat use patterns of leatherbacks. Our secondary objective was to use oceanographic indices and nesting information from St. Croix leatherbacks to evaluate relationships between trophic ecology, nesting parameters, and regional environmental conditions measured by the North Atlantic Oscillation (NAO) and Atlantic Multidecadal Oscillation. We found no change in leatherback TP over time and no difference in TP between Atlantic and Pacific leatherbacks, indicating that differences in trophic ecology between populations are an unlikely driver of the population dichotomy between Pacific and Atlantic leatherbacks. Isotope data suggested that St. Croix leatherbacks inhabit multiple oceanic regions prior to nesting, although, like their conspecifics in the Pacific, individuals exhibit fidelity to specific foraging regions. Leatherback nesting parameters were weakly related to the NAO, which may suggest that positive NAO phases benefit St. Croix leatherbacks, potentially through increases in resource availability in their foraging areas. Our data contribute to the understanding of leatherback turtle ecology and potential mechanistic drivers of the dichotomy between populations of this protected species.

The political biogeography of migratory marine predators.

During their migrations, marine predators experience varying levels of protection and face many threats as they travel through multiple countries' jurisdictions and across ocean basins. Some populations are declining rapidly. Contributing to such declines is a failure of some international agreements to ensure effective cooperation by the stakeholders responsible for managing species throughout their ranges, including in the high seas, a global commons. Here we use biologging data from marine predators to provide quantitative measures with great potential to inform local, national and international management efforts in the Pacific Ocean. We synthesized a large tracking data set to show how the movements and migratory phenology of 1,648 individuals representing 14 species-from leatherback turtles to white sharks-relate to the geopolitical boundaries of the Pacific Ocean throughout species' annual cycles. Cumulatively, these species visited 86% of Pacific Ocean countries and some spent three-quarters of their annual cycles in the high seas. With our results, we offer answers to questions posed when designing international strategies for managing migratory species.

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.

Natal foraging philopatry in eastern Pacific hawksbill turtles.

The complex processes involved with animal migration have long been a subject of biological interest, and broad-scale movement patterns of many marine turtle populations still remain unresolved. While it is widely accepted that once marine turtles reach sexual maturity they home to natal areas for nesting or reproduction, the role of philopatry to natal areas during other life stages has received less scrutiny, despite widespread evidence across the taxa. Here we report on genetic research that indicates that juvenile hawksbill turtles (Eretmochelys imbricata) in the eastern Pacific Ocean use foraging grounds in the region of their natal beaches, a pattern we term natal foraging philopatry. Our findings confirm that traditional views of natal homing solely for reproduction are incomplete and that many marine turtle species exhibit philopatry to natal areas to forage. Our results have important implications for life-history research and conservation of marine turtles and may extend to other wide-ranging marine vertebrates that demonstrate natal philopatry.

First-line selective internal radiotherapy plus chemotherapy versus chemotherapy alone in patients with liver metastases from colorectal cancer (FOXFIRE, SIRFLOX, and FOXFIRE-Global): a combined analysis of three multicentre, randomised, phase 3 trials.

BACKGROUND: Data suggest selective internal radiotherapy (SIRT) in third-line or subsequent therapy for metastatic colorectal cancer has clinical benefit in patients with colorectal liver metastases with liver-dominant disease after chemotherapy. The FOXFIRE, SIRFLOX, and FOXFIRE-Global randomised studies evaluated the efficacy of combining first-line chemotherapy with SIRT using yttrium-90 resin microspheres in patients with metastatic colorectal cancer with liver metastases. The studies were designed for combined analysis of overall survival. METHODS: FOXFIRE, SIRFLOX, and FOXFIRE-Global were randomised, phase 3 trials done in hospitals and specialist liver centres in 14 countries worldwide (Australia, Belgium, France, Germany, Israel, Italy, New Zealand, Portugal, South Korea, Singapore, Spain, Taiwan, the UK, and the USA). Chemotherapy-naive patients with metastatic colorectal cancer (WHO performance status 0 or 1) with liver metastases not suitable for curative resection or ablation were randomly assigned (1:1) to either oxaliplatin-based chemotherapy (FOLFOX: leucovorin, fluorouracil, and oxaliplatin) or FOLFOX plus single treatment SIRT concurrent with cycle 1 or 2 of chemotherapy. In FOXFIRE, FOLFOX chemotherapy was OxMdG (oxaliplatin modified de Gramont chemotherapy; 85 mg/m2 oxaliplatin infusion over 2 h, L-leucovorin 175 mg or D,L-leucovorin 350 mg infusion over 2 h, and 400 mg/m2 bolus fluorouracil followed by a 2400 mg/m2 continuous fluorouracil infusion over 46 h). In SIRFLOX and FOXFIRE-Global, FOLFOX chemotherapy was modified FOLFOX6 (85 mg/m2 oxaliplatin infusion over 2 h, 200 mg leucovorin, and 400 mg/m2 bolus fluorouracil followed by a 2400 mg/m2 continuous fluorouracil infusion over 46 h). Randomisation was done by central minimisation with four factors: presence of extrahepatic metastases, tumour involvement of the liver, planned use of a biological agent, and investigational centre. Participants and investigators were not masked to treatment. The primary endpoint was overall survival, analysed in the intention-to-treat population, using a two-stage meta-analysis of pooled individual patient data. All three trials have completed 2 years of follow-up. FOXFIRE is registered with the ISRCTN registry, number ISRCTN83867919. SIRFLOX and FOXFIRE-Global are registered with ClinicalTrials.gov, numbers NCT00724503 (SIRFLOX) and NCT01721954 (FOXFIRE-Global). FINDINGS: Between Oct 11, 2006, and Dec 23, 2014, 549 patients were randomly assigned to FOLFOX alone and 554 patients were assigned FOLFOX plus SIRT. Median follow-up was 43·3 months (IQR 31·6-58·4). There were 411 (75%) deaths in 549 patients in the FOLFOX alone group and 433 (78%) deaths in 554 patients in the FOLFOX plus SIRT group. There was no difference in overall survival (hazard ratio [HR] 1·04, 95% CI 0·90-1·19; p=0·61). The median survival time in the FOLFOX plus SIRT group was 22·6 months (95% CI 21·0-24·5) compared with 23·3 months (21·8-24·7) in the FOLFOX alone group. In the safety population containing patients who received at least one dose of study treatment, as treated, the most common grade 3-4 adverse event was neutropenia (137 [24%] of 571 patients receiving FOLFOX alone vs 186 (37%) of 507 patients receiving FOLFOX plus SIRT). Serious adverse events of any grade occurred in 244 (43%) of 571 patients receiving FOLFOX alone and 274 (54%) of 507 patients receiving FOLFOX plus SIRT. 10 patients in the FOLFOX plus SIRT group and 11 patients in the FOLFOX alone group died due to an adverse event; eight treatment-related deaths occurred in the FOLFOX plus SIRT group and three treatment-related deaths occurred in the FOLFOX alone group. INTERPRETATION: Addition of SIRT to first-line FOLFOX chemotherapy for patients with liver-only and liver-dominant metastatic colorectal cancer did not improve overall survival compared with that for FOLFOX alone. Therefore, early use of SIRT in combination with chemotherapy in unselected patients with metastatic colorectal cancer cannot be recommended. To further define the role of SIRT in metastatic colorectal cancer, careful patient selection and studies investigating the role of SIRT as consolidation therapy after chemotherapy are needed. FUNDING: Bobby Moore Fund of Cancer Research UK, Sirtex Medical.

Assessing Fukushima-Derived Radiocesium in Migratory Pacific Predators.

The 2011 release of Fukushima-derived radionuclides into the Pacific Ocean made migratory sharks, teleosts, and marine mammals a source of speculation and anxiety regarding radiocesium (134+137Cs) contamination, despite a lack of actual radiocesium measurements for these taxa. We measured radiocesium in a diverse suite of large predators from the North Pacific Ocean and report no detectable (i.e., ≥ 0.1 Bq kg-1 dry wt) Fukushima-derived 134Cs in all samples, except in one olive ridley sea turtle (Lepidochelys olivacea) with trace levels (0.1 Bq kg-1). Levels of 137Cs varied within and across taxa, but were generally consistent with pre-Fukushima levels and were lower than naturally occurring 40K by one to one to two orders of magnitude. Predator size had a weaker effect on 137Cs and 40K levels than tissue lipid content. Predator stable isotope values (δ13C and δ15N) were used to infer recent migration patterns, and showed that predators in the central, eastern, and western Pacific should not be assumed to accumulate detectable levels of radiocesium a priori. Nondetection of 134Cs and low levels of 137Cs in diverse marine megafauna far from Fukushima confirms negligible increases in radiocesium, with levels comparable to those prior to the release from Fukushima. Reported levels can inform recently developed models of cesium transport and bioaccumulation in marine species.