Heart rate reduction during voluntary deep diving in free-ranging loggerhead sea turtles.

Air-breathing vertebrates exhibit cardiovascular responses to diving including heart rate reduction (diving bradycardia). Field studies on aquatic mammals and birds have shown that the intensity of bradycardia can vary depending on diving behaviour, such as the depth of dives and dive duration. However, in aquatic reptiles, the variation in heart rate during deep dives under natural conditions has not been fully investigated. In this study, we released five loggerhead sea turtles (Caretta caretta) outfitted with recorders into the sea and recorded their electrocardiogram, depth, water temperature and longitudinal acceleration. After 3 days, the recorders automatically detached from the turtles. The heart rate signals were detected from the electrodes placed on the surface of the plastron. The mean (±s.d.) heart rate of 12.8±4.1 beats min-1 during dives was significantly lower than that of 20.9±4.1 beats min-1 during surface periods. Heart rate during dives varied with dive depth, although it remained lower than that at the surface. When the turtle dived deeper than 140 m, despite the relatively high flipper stroke rate (approximately 19 strokes min-1), the heart rate dropped rapidly to approximately 2 beats min-1 temporarily. The minimum instantaneous heart rate during dives was lower at deeper dive depths. Our results indicate that loggerhead sea turtles show variations in the intensity of diving bradycardia depending on their diving behaviour, similar to that shown by marine mammals and birds.

Loggerhead Sea Turtles as Hosts of Diverse Bacterial and Fungal Communities.

Research on microbial communities associated with wild animals provides a valuable reservoir of knowledge that could be used for enhancing their rehabilitation and conservation. The loggerhead sea turtle (Caretta caretta) is a globally distributed species with its Mediterranean population categorized as least concern according to the IUCN Red List of Threatened Species as a result of robust conservation efforts. In our study, we aimed to further understand their biology in relation to their associated microorganisms. We investigated epi- and endozoic bacterial and endozoic fungal communities of cloaca, oral mucosa, carapace biofilm. Samples obtained from 18 juvenile, subadult, and adult turtles as well as 8 respective enclosures, over a 3-year period, were analysed by amplicon sequencing of 16S rRNA gene and ITS2 region of nuclear ribosomal gene. Our results reveal a trend of decreasing diversity of distal gut bacterial communities with the age of turtles. Notably, Tenacibaculum species show higher relative abundance in juveniles than in adults. Differential abundances of taxa identified as Tenacibaculum, Moraxellaceae, Cardiobacteriaceae, and Campylobacter were observed in both cloacal and oral samples in addition to having distinct microbial compositions with Halioglobus taxa present only in oral samples. Fungal communities in loggerheads' cloaca were diverse and varied significantly among individuals, differing from those of tank water. Our findings expand the known microbial diversity repertoire of loggerhead turtles, highlighting interesting taxa specific to individual body sites. This study provides a comprehensive view of the loggerhead sea turtle bacterial microbiota and marks the first report of distal gut fungal communities that contributes to establishing a baseline understanding of loggerhead sea turtle holobiont.

Citrobacter freundii-associated lesions in stranded loggerhead sea turtles (Caretta caretta).

Citrobacter freundii, an opportunistic bacterial pathogen belonging to the family Enterobacteriaceae, has been sporadically reported in sea turtles. Here, the authors describe 3 unusual lesions associated with C. freundii infection in 3 loggerhead sea turtles stranded on the coast of Gran Canaria Island, Spain. It is possible that these 3 distinct lesions played a major role in the death of these turtles. The first turtle had caseous cholecystitis, a lesion not previously described in sea turtles. The second turtle had large intestinal diverticulitis, a rare condition in loggerheads. The third turtle had bilateral caseous salt gland adenitis. Histologically, numerous gram-negative bacilli were observed at the deepest edge of inflammation in all cases. Pure cultures of C. freundii were obtained from these 3 lesions. Molecular detection of C. freundii DNA in formalin-fixed paraffin-embedded samples from the lesions of the 3 turtles confirmed the microbiological isolation. These cases, in addition to expanding the limited body of knowledge on bacterial infections in sea turtles, highlight the potential pathogenic role of C. freundii in loggerhead turtles.

Correlation Between Microbial Community and Hatching Failure in Loggerhead Sea Turtle Caretta caretta.

Microbial communities provide essential information about host ecology and could be helpful as a tool to improve species conservation efforts. However, microbes can also infect and compromise the host development process and viability. Caretta caretta is the most widespread marine turtle species in the Mediterranean basin and is the only species of sea turtle nesting along the Italian coasts. Little is known about the microbiota composition of the nest of sea turtles and its correlation with hatching failures. In this study, the microbial composition of two nests of C. caretta featuring different rates of hatching success from a nesting beach in Lampedusa (Italy) was analyzed and compared. The bacterial community was determined using culture-dependent methods and next-generation sequencing based on 16S rRNA gene metabarcoding analysis. Our results showed five dominant bacterial phyla (Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, and Firmicutes) and indicated different bacterial families (Pseudomonadaceae and Brucellaceae) as likely causes of hatching failures. Besides, our findings demonstrated the nests' active role in modulating the sand's bacterial communities. This study suggests microbiological analysis could be a valuable tool in monitoring nests to take preventive actions and reduce hatching failures.

Comparative study of polycyclic aromatic hydrocarbons (PAHs) in salt gland and liver of loggerhead turtle Caretta caretta (Linnaeus, Cheloniidae) stranded along the Mediterranean coast, Southern Italy.

The levels of polycyclic aromatic hydrocarbons, PAHs, were determined in the liver and salt gland of 19 loggerhead turtles (Caretta caretta Linnaeus, Cheloniidae) stranded along the coasts of the south Tyrrhenian Sea, Italy, from 2019 to 2021. The 16 EPA's priority PAHs were determined by gas chromatography coupled with mass spectrometry (GC-MS). The average values of ΣPAHs in liver, 139 ± 55.0, were exceptionally high and up to one hundred times those of literature and appeared even more worrying in salt gland, 320 ± 97.6 ng/g, w.w. Naphthalene was the predominant contributor to PAHs richness and accounted for 90.0% and 93.7% of ΣPAHs in the two matrices, highlighting the petrogenic source of exposure. An overall higher bioaccumulation of NAP, more than two-fold, was detected in salt gland and especially in female and adults. Data of PAHs richness highlighted a potential risk of neoplastic disease development and that anthropogenic activities may seriously impair healthy state conditions of C. caretta. populations in south Tyrrhenian Sea.

Repeated Biting by a Mediterranean Loggerhead Sea Turtle.

Mediterranean loggerhead sea turtles (Caretta caretta) are very large. During spawning season, they land on the beaches of Turkey and Greece at night, which are frequently visited by humans, and lay their eggs. Typically, they suck feed in deep water. It is uncommon for C caretta, which have strong mouths and jaws, to attack people. We report a case of a 44-y-old female patient who was attacked by a turtle on the Mediterranean beach in September 2020. She was brought to the emergency department, where she was noted to have a deep cut on the thumb and large bruises on the left side of her waist. The patient was discharged with appropriate wound care, antibacterial drug, and tetanus vaccine. The wound healed within a few months by developing granulation tissue. However, this report draws attention to the fact that, under some circumstances, C caretta can become aggressive toward people swimming off heavily populated beaches.

Bacterial diversity of loggerhead and green turtle eggs from two major nesting beaches from the Turkish coast of the Mediterranean.

This study was conducted during the 2018 nesting season at the Sugözü Beaches (Adana-Turkey) and Göksu Delta (Mersin-Turkey). Egg samples (n = 63) from loggerhead and green turtle nests (n = 43) were collected. Isolated bacteria were initially identified by phenotypic methods and then by MALDI-TOF MS. The bacterial mass spectra were analyzed using Principal Component Analysis. Bacterial isolation was performed for 55 isolates belonging to 12 genera from two major nesting sites. In Sugözü Beaches 62.2% of the bacteria species belonged to Enterobacteriaceae and in Göksu Delta 44.4% of the bacteria species belonged to Morganellaceae. Klebsiella oxytoca and Staphylococcus haemolyticus had not previously been detected in any sea turtle nests. This is the first MALDI-TOF MS study conducted for determination of bacterial variability in loggerhead turtle eggs in Turkey and serves as a reference study for the assessment of bacterial threat in sea turtle nests, enabling the establishment of suitable conservation measures and treatment processes for both sea turtles and nesting sites.

Effects of a parasympathetic blocker on the heart rate of loggerhead sea turtles during voluntary diving.

Diving bradycardia is a reduction in the heart rate mediated by the parasympathetic system during diving. Although diving bradycardia is pronounced in aquatic mammals and birds, the existence of this response in aquatic reptiles, including sea turtles, remains under debate. Using the parasympathetic blocker atropine, we evaluated the involvement of the parasympathetic nervous system in heart rate reduction of loggerhead sea turtles (Caretta caretta) during voluntary diving in tanks. The heart rate of the control group dropped by 40-60% from the pre-dive value at the onset of diving; however, administration of atropine significantly inhibited heart rate reduction (P<0.001). Our results indicate that, similar to mammals and birds, the heart rate reduction in sea turtles while diving is primarily mediated by the parasympathetic nervous system. In conclusion, we suggest that diving bradycardia exists not only in aquatic mammals and birds but also in aquatic reptiles.

Plasmatic B-esterases as potential biomarkers of exposure to marine plastics in loggerhead turtles.

Sea turtles are particularly vulnerable to plastic exposures, and the associated chemical additives, due to their feeding strategies. The species Caretta caretta is a proposed sentinel of plastic pollution worldwide. Thus, there is a need to find adequate biomarkers of plastic exposure through non-invasive protocols for this IUCN protected species. Plasmatic acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and carboxylesterase (CE) which participate in xenobiotic and endogenous metabolic reactions could all serve as biomarkers, as they are responsive to plasticizers and have already proved adequate for identifying organophosphorus esters exposures. Here we measured plasmatic B-esterases in wild specimens captured as accidental by-catch. Measurements were taken in each individual either at entry into the rehabilitation program or immediately before release after a recovery period. For CE measurements, 4 commercial substrates were used as potentially indicative of distinct enzyme isoforms. Increased activity was seen with the butyrate-derived substrates. Plasmatic CE activities were over one order of magnitude higher than AChE and BuChE substrates. Moreover, an in vitro protocol with the inclusion of plastic additives such as tetrabromobisphenol A (TBBPA), bisphenol A and some of its analogues was considered a proxy of enzymatic interactions. A clear inhibition by TBBPA was found when using commercially purified AChE and recombinant CE proteins. Overall, from in vitro and in vivo evidences, CEs in plasma are sensitive and easily measurable and have been shown to significantly increase after turtles have been rehabilitated in rescue centres. Nevertheless, the inclusion of plastic (or plasticizers) characterisation would help to confirm its association with plasmatic enzyme modifications before they can be adopted as biomarkers of plastic contamination.

Marine leech parasitism of sea turtles varies across host species, seasons, and the tumor disease fibropapillomatosis.

Fibropapillomatosis (FP) is a tumorous disease affecting all species of sea turtles and is associated with the pathogen chelonid alphaherpesvirus 5 (ChHV5). Hypothesized ChHV5 vectors include the marine leeches Ozobranchus branchiatus and O. margoi, but data on their associations with FP and ChHV5 are minimal. To establish relationships between leech parasitism, turtle hosts, and FP, we compared green and loggerhead turtles from the Indian River Lagoon (IRL), Florida, USA, in terms of (1) the presence or absence of ChHV5 within associated leeches, (2) the association between leech parasitism and host FP status, and (3) seasonal variation in leech presence. We identified 55 leeches collected from green turtles as O. branchiatus and 22 leeches collected from loggerhead turtles as O. margoi. Of 77 sequenced leeches, 10 O. branchiatus and 5 O. margoi were ChHV5 positive. ChHV5-positive O. branchiatus trended towards coming from FP-positive hosts. Using 12 yr of turtle capture data from the IRL, we found that leech parasitism was significantly correlated with FP and capture month in green turtles but not in loggerhead turtles. These results suggest that O. branchiatus and O. margoi may differ in their ability to transmit ChHV5 or to encounter and remain on FP-positive hosts. Alternatively, potential immunological differences between green and loggerhead turtles may explain the observed relationships. This study is the first to provide robust statistical evidence of an association between leeches and FP, as well as seasonal fluctuations in leech presence, in green turtles but not in loggerhead turtles.

Pharmacokinetics of ketorolac in juvenile loggerhead sea turtles (Caretta caretta) after a single intramuscular injection.

Ketorolac is a non-steroidal anti-inflammatory drug administered as an analgesic in humans. It has analgesic effects comparable to opioids but without adverse effects such as respiratory depression or restrictions because of controlled drug status. We designed this study to examine the potential of ketorolac as an analgesic for sea turtle rehabilitative medicine. Our objective was to determine the pharmacokinetics of a single 0.25 mg/kg intramuscular dose of ketorolac in a population of 16 captive-raised juvenile loggerhead sea turtles (Caretta caretta). A sparse sampling protocol was utilized, and blood samples were collected for 12 hours after administration of ketorolac. Samples were analyzed with high-pressure liquid chromatography (HPLC), and a nonlinear mixed effects model (NLME) was used to determine parameters for the population. With these methods, we identified a long elimination half-life (ßT1/2  = 11.867 hr) but a low maximum concentration (CMAX  = 0.508 µg/mL) and concentrations were below the level proposed to be therapeutic in humans (EC50  = 0.1-0.3 µg/mL) for most of the collection period. We conclude that ketorolac may not be an appropriate long-term analgesic for use in loggerhead sea turtles at this dose; however, it may have some benefit as a short-term analgesic.

Sargassum landings have not compromised nesting of loggerhead and green sea turtles in the Mexican Caribbean.

The Mexican Caribbean is a vital nesting destination for loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles. Since 2015, massive periodical landings of pelagic Sargassum species (sargasso) have affected coastal ecosystems. Dense accumulations of sargasso on the shoreline may preclude access to sea turtles' preferred nesting areas and compromise hatching. In this study, we assess whether the number of nests and hatches of loggerhead and green sea turtles has been affected by the massive influx of sargasso. We compare data from before (2010-2014) and after (2015-2019) the first sargasso event, obtained from the same 17 marine turtle camps, which collectively account for 72.3 km of sampling distance over a 210 km section of shoreline. No differences in preferences on nesting beaches were recorded between periods for the two species. The mean number of nests per kilometer of coastline remained without statistically significant changes between periods in 16 camps and increased significantly in one camp for each species. Overall, the mean annual number of nests per kilometer of beach was 37% higher in the period after massive landings of sargasso began. The mean number of hatchlings increased significantly in one camp for C. mydas and in three for C. caretta. Periodical massive landings of sargasso from 2015 to 2019 do not appear to have compromised nesting and hatching of loggerhead and green sea turtles along the Mexican Caribbean coast.

Some Digenetic Trematodes Found in a Loggerhead Sea Turtle (Caretta Caretta) from Brazil.

This paper reports three recovered species of digeneans from an adult loggerhead sea turtle - Caretta caretta (Testudines, Cheloniidae) in Brazil. These trematodes include Diaschistorchis pandus (Pronocephalidae), Cymatocarpus solearis (Brachycoeliidae) and Rhytidodes gelatinosus (Rhytidodidae) The first two represent new geographic records. A list of helminths reported from the Neotropical region, Gulf of Mexico and USA (Florida) is presented.

Managing fisheries in a world with more sea turtles.

For decades, fisheries have been managed to limit the accidental capture of vulnerable species and many of these populations are now rebounding. While encouraging from a conservation perspective, as populations of protected species increase so will bycatch, triggering management actions that limit fishing. Here, we show that despite extensive regulations to limit sea turtle bycatch in a coastal gillnet fishery on the eastern United States, the catch per trip of Kemp's ridley has increased by more than 300% and green turtles by more than 650% (2001-2016). These bycatch rates closely track regional indices of turtle abundance, which are a function of increased reproductive output at distant nesting sites and the oceanic dispersal of juveniles to near shore habitats. The regulations imposed to help protect turtles have decreased fishing effort and harvest by more than 50%. Given uncertainty in the population status of sea turtles, however, simply removing protections is unwarranted. Stock-assessment models for sea turtles must be developed to determine what level of mortality can be sustained while balancing continued turtle population growth and fishing opportunity. Implementation of management targets should involve federal and state managers partnering with specific fisheries to develop bycatch reduction plans that are proportional to their impact on turtles.

Fidelity to foraging sites after long migrations.

Patterns of animal movement associated with foraging lie at the heart of many ecological studies and often animals face decisions of staying in an environment they know versus relocating to new sites. The lack of knowledge of new foraging sites means there is risk associated with a decision to relocate (e.g. poor foraging) as well as a potential benefit (e.g. improved foraging). Using a unique long-term satellite tracking dataset for several sea turtle species, combined with capture-mark-recapture data extending over 50 years, we show how, across species, individuals generally maintain tight fidelity to specific foraging sites after extended (up to almost 10,000 km) migration to and from distant breeding sites as well as across many decades. Migrating individuals often travelled through suitable foraging areas en route to their 'home' site and so extended their journeys to maintain foraging site fidelity. We explore the likely mechanistic underpinnings of this trait, which is also seen in some migrating birds, and suggest that individuals will forgo areas of suitable forage encountered en route during migration when they have poor knowledge of the long-term suitability of those sites, making relocation to those sites risky.

Incubation environment and parental identity affect sea turtle development and hatchling phenotype.

For reptiles, the incubation environment experienced by embryos during development plays a major role in many biological processes. The unprecedented rate of climate change makes it critical to understand the effects that the incubation environment has on developing embryos, particularly in imperiled species such as chelonians. Consequently, a number of studies have focused on the effects of different environmental conditions on several developmental processes and hatchling phenotypic traits. In addition to the incubation environment, it is also essential to understand how parental contributions can influence hatchling quality. This is the first study that investigates the effects of parental origin and incubation conditions on sea turtle embryonic development and hatchling phenotype in nests incubating in the field (rather than under controlled laboratory conditions). Here, we used the loggerhead sea turtle (Caretta caretta) to investigate the effects of parental origin (clutch), incubation temperature, and the nest hydric environment on embryonic growth, incubation durations, hatching success, and hatchling phenotype. Our results show that nest moisture and temperature affect embryo mass towards the last third of development, with hatchling size positively correlated with nest moisture content, and maternal origin had a strong impact on hatching success and hatchling size regardless of the incubation conditions. The results from this experiment identify multiple factors that affect turtle embryonic development under field incubation conditions, a fundamental consideration when interpreting the potential impacts of climate change on reptilian development.

A pilot study on molecular diagnosis of Hapalotrema mistroides (Digenea: Spirorchiidae) infection in blood samples of live loggerhead turtles Caretta caretta.

BACKGROUND: Parasites of the family Spirorchiidae cause disease and mortality in marine and freshwater turtles; two species, Hapalotrema mistroides and Neospirorchis sp., are reported in the resident population of loggerhead turtles of the Mediterranean Sea, with the first being the most widespread. In vivo diagnosis of spirorchidiasis can represent a challenge in guaranteeing prompt control and treatment of the disease and is currently limited to copromicroscopy. The aim of this study was the development of a real time PCR assay with TaqMan probe for the detection of H. mistroides infection in the blood of live loggerhead turtles, Caretta caretta, hospitalized in rehabilitation centres. Its potential use for in vivo diagnosis is explored. RESULTS: The developed real time PCR successfully detected H. mistroides DNA from both positive controls and experimental blood samples of live loggerhead sea turtles, showing good specificity, sensitivity and good reaction efficiency. Two out of three turtles which had demonstrated positivity at copromicroscopy also tested positive to this blood assay; DNA of H. mistroides was detected within the blood of one sea turtle, which tested negative for copromicroscopy. CONCLUSIONS: This study describes a specific and rapid molecular assay to detect H. mistroides infection from live sea turtles and highlights for the first time the presence of DNA of this species in turtle blood samples. Since this assay is able to detect low amounts of the parasitic free DNA in blood samples, its application could be helpful for in vivo diagnosis of H. mistroides infection as well as for epidemiological purposes.

Fusarium spp. in Loggerhead Sea Turtles (Caretta caretta): From Colonization to Infection.

With the aim of evaluating the presence of Fusarium spp. in sea turtles with and without lesions and assessing the risk factors favoring colonization and/or infection, 74 loggerhead sea turtles (Caretta caretta) admitted to rescue and rehabilitation clinics in Italy were analyzed. The study compared 31 individuals with no apparent macroscopic lesions and 43 individuals with macroscopic lesions. Shell and skin samples were analyzed using Calcofluor white with 10% potassium hydroxide, standard histopathological examination, and fungal cultures. Fusarium spp. were isolated more frequently from animals with superficial lesions (39%) than from those with no macroscopic lesions (16%). Isolates from animals with superficial lesions were Fusarium solani species complex (FSSC) lineages haplotypes 9, 12, and 27 (unnamed lineages), FSSC-2 (Fusarium keratoplasticum), Fusarium oxysporum (27%), and Fusarium brachygibbosum (3%). In contrast, only F. solani haplotypes 9 and 12 were isolated from animals with no macroscopic lesions. The presence of lesions was identified as a risk factor for the occurrence of Fusarium spp. Of the 74 animals, only 7 (9.5%) scored positive on microscopic examination with Calcofluor, and histological examination of those 7 animals revealed necrosis, inflammatory cells, and fungal hyphae in the carapace and skin. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin lesions in sea turtles. Direct examination using Calcofluor and potassium hydroxide was not useful to diagnose the infection. Histopathological examination and fungal culture should be performed to ensure correct treatment and infection control.

Mercury and Organochlorine Pesticides in Tissues of Loggerhead Sea Turtles (Caretta caretta) Stranded Along the Southwestern Mediterranean Coastline (Andalusia, Spain).

Nineteen loggerhead sea turtles (Caretta caretta) stranded along the southwestern Mediterranean coastline (Andalusia) were used in this study. A total of 68 samples of fat (n = 18), liver (n = 15), kidney (n = 13), pectoral muscle (n = 19), and brain (n = 3) were analysed for total mercury (Hg) and organochlorine pesticides [OC: ∑Dichlorodiphenyltrichloroethanes (∑DDT), ∑Hexachlorocyclohexane (∑HCH), ∑Heptachlor, ∑Drins and ∑Endosulfan]. These loggerhead sea turtles showed tissue Hg and OC concentrations similar to or lower than those reported in other studies. Few growth-related variations in Hg or OC levels in relation to straight carapace length were found, probably because the specimens were mostly juveniles. This study will help to fill the gap on spatio-temporal exposure data and ascertain the real world-wide picture of the contamination levels in loggerhead sea turtles.

Activity, not submergence, explains diving heart rates of captive loggerhead sea turtles.

Marine turtles spend their life at sea and can rest on the seafloor for hours. As air-breathers, the breath-hold capacity  of marine turtles is a function of oxygen (O2) stores, O2 consumption during dives and hypoxia tolerance. However, some physiological adaptations to diving observed in mammals are absent in marine turtles. This study examined cardiovascular responses in loggerhead sea turtles, which have even fewer adaptations to diving than other marine turtles, but can dive for extended durations. Heart rates (fH) of eight undisturbed loggerhead turtles in shallow tanks were measured using self-contained ECG data loggers under five conditions: spontaneous dives, resting motionless on the tank bottom, resting in shallow water with their head out of water, feeding on squid, and swimming at the surface between dives. There was no significant difference between resting fH while resting on the bottom of the tank, diving or resting in shallow water with their head out of water. fH rose as soon as turtles began to move and was highest between dives when turtles were swimming at the surface. These results suggest cardiovascular responses in captive loggerhead turtles are driven by activity and apneic fH is not reduced by submergence under these conditions.