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The forkhead transcription factor Foxl2 plays a major role in ovarian development and function in mice and fish, and acts as a female sex-determining gene in goat. Its functional role in the sex determination and gonadal differentiation has not yet been investigated in reptiles. Here, we characterized Foxl2 gene in Chinese soft-shelled turtle Pelodiscus sinensis, exhibiting ZZ/ZW sex chromosomes. Foxl2 exhibited a female-specific embryonic expression pattern throughout the critical sex determination periods in P. sinensis. The expression of Foxl2 was induced at early stage in ZZ embryonic gonads that were feminized by estrogen treatment. Most importantly, Foxl2 knockdown in ZW embryos by RNA interference resulted in female-to-male sex reversal, characterized by obvious masculinization of gonads, significant up-regulation of testicular markers Dmrt1 and Sox9, and remarkable down-regulation of ovarian regulator Cyp19a1. Conversely, gain-of-function study showed that overexpression of Foxl2 in ZZ embryos led to largely feminized genetic males, production of Cyp19a1, and a decline in Dmrt1 and Sox9. These findings demonstrate that Foxl2 is both necessary and sufficient to initiate ovarian differentiation in P. sinensis, thereby acting as a key upstream regulator of the female pathway in a reptilian species.

Despite being the most abundant sea turtle in the world, the olive ridley turtle Lepidochelys olivacea is classified as Vulnerable by the IUCN. There is evidence of congenital malformations in hatchlings, and the associated causes are multifactorial, with both genetic and environmental sources. Santuario Playa Ceuta (SPC) is a sanctuary for the olive ridley, located at the northernmost region of its nesting range in the Mexican Pacific. The objective of this study was to identify and quantify the prevalence and severity of congenital malformations in olive ridley embryos/hatchlings in SPC during the 2017 nesting season. We collected 62907 eggs from 643 relocated nests that were moved to a hatchery, of which 4242 eggs with obvious development did not hatch and were analyzed for this study. Hatching success was 53.9%, with 22.5% of nests (n = 145) and 0.54% of eggs (n = 344) showing embryos or hatchlings with malformations. The nest severity index was 2.4 (range: 1-10) malformed embryos or hatchlings per nest, and the organism severity index was 1.4 (range: 1-7) malformations per malformed embryo or hatchling. Leucism was the most prevalent malformation (34.4%; 170/494 total observed), with the craniofacial region showing the greatest diversity of malformations (17/35 types). Given the geographical position of SPC, extreme environmental conditions (e.g. cold, heat, and dryness) could be one of the main causes of teratogenesis in this species. However, more studies are needed regarding the presence of contaminants, genetic factors, health assessments of nesting females, and malformation rates of nests that remain in situ versus those that are relocated.

Satellite tracking is a key tool for studying sea turtles in the wild. Most tracking has been performed on adult females however, leaving knowledge gaps regarding other population segments, such as adult males. By satellite tracking 12 male green turtles (

As climate-related impacts threaten marine biodiversity globally, it is important to adjust conservation efforts to mitigate the effects of climate change. Translating scientific knowledge into practical management, however, is often complicated due to resource, economic and policy constraints, generating a knowledge-action gap. To develop potential solutions for marine turtle conservation, we explored the perceptions of key actors across 18 countries in the Mediterranean. These actors evaluated their perceived relative importance of 19 adaptation and mitigation measures that could safeguard marine turtles from climate change. Of importance, despite differences in expertise, experience and focal country, the perceptions of researchers and management practitioners largely converged with respect to prioritizing adaptation and mitigation measures. Climate change was considered to have the greatest impacts on offspring sex ratios and suitable nesting sites. The most viable adaptation/mitigation measures were considered to be reducing other pressures that act in parallel to climate change. Ecological effectiveness represented a key determinant for implementing proposed measures, followed by practical applicability, financial cost, and societal cost. This convergence in opinions across actors likely reflects long-standing initiatives in the Mediterranean region towards supporting knowledge exchange in marine turtle conservation. Our results provide important guidance on how to prioritize measures that incorporate climate change in decision-making processes related to the current and future management and protection of marine turtles at the ocean-basin scale, and could be used to guide decisions in other regions globally. Importantly, this study demonstrates a successful example of how interactive processes can be used to fill the knowledge-action gap between research and management., This work was conducted under FutureMares EU project that received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 869300. The Mediterranean Marine Turtle Working Group was established in 2017 and is continuously supported by MedPAN and the National Marine Park of Zakynthos. The work of AC was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “First Call for H.F.R.I. Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment grant” (Project Number: 2340).

Is senescence inevitable and universal for all living organisms, as evolutionary theories predict? Although evidence generally supports this hypothesis, it has been proposed that certain species, such as turtles and tortoises, may exhibit slow or even negligible senescence—i.e., avoiding the increasing risk of death from gradual deterioration with age. In an extensive comparative study of turtles and tortoises living in zoos and aquariums, we show that ~75% of 52 species exhibit slow or negligible senescence. For ~80% of species, aging rates are lower than those in modern humans. We find that body weight positively relates to adult life expectancy in both sexes, and sexual size dimorphism explains sex differences in longevity. Unlike humans and other species, we show that turtles and tortoises may reduce senescence in response to improvements in environmental conditions.

The tick genus Amblyomma Koch, 1844 (Acari: Ixodidae) has received little attention in Malaysia; therefore, its associated hosts and distribution records are poorly known. In this study, we collected six Amblyomma sp. individuals (two larvae and four adults) that infested a common treeshrew, Tupaia glis (Diard, 1820) (Scandentia: Tupaiidae) caught in a recreational area in Sungai Lembing, Pahang (West Malaysia). The adult female ticks were morphologically identified according to taxonomic keys prior to molecular identification using cytochrome oxidase subunit I (COI) and 16S rDNA genes. The ticks were genetically verified as Amblyomma geoemydae (Cantor, 1847) with 98%–99% similarity to the available GenBank sequences. Neighbor-joining (NJ) trees indicated that A. geoemydae was clearly distinguished from other Amblyomma ticks and this was supported with high bootstrap values. This paper is the first to report A. geoemydae ticks infesting T. glis and provides a new tick-host record from West Malaysia. This information is significant for further investigation, specifically on this tick species as potential vector of tick-borne disease (TBD) agents.

Synopsis There is ample research demonstrating that temperature can have complex effects on biological processes, including the timing of when organisms respond to temperature; some responses occur rapidly while others require an extended exposure time. However, most of what we know about temperature effects comes from studies using constant temperature conditions, which are not reflective of natural, fluctuating temperatures. Species with temperature-dependent sex determination (TSD) present an ideal system to study the temporal aspects of the temperature response because prior research has established a number of temperature-responsive genes involved in TSD, albeit under constant temperatures. To investigate potential differences in timing of sexual development between constant and fluctuating incubation temperatures, we exposed Trachemys scripta embryos to two conditions that produce males (constant 26°C and 26 ± 3°C) and two that produce females (constant 31°C and 31 ± 3°C), and sampled embryonic gonads for gene expression analysisvia qPCR. We analyzed three genes involved in testis differentiation (Kdm6b,Dmrt1, andSox9) and two genes involved in ovary differentiation (Foxl2andCyp19A1). Results show that Kdm6b expression was significantly lower under fluctuating temperatures compared to constant temperatures. Foxl2 and Cyp19A1 expression were also lower under fluctuating temperatures, but not at all stages of development. These results suggest that constant temperatures caused increases in both Foxl2 and Cyp19A1 expression earlier (developmental stage 20) than fluctuating temperatures (stages 22 and 23). Dmrt1 and Sox9 expression did not differ between constant and fluctuating temperatures. These results highlight that not all genes in a temperature-dependent process respond to temperature in the same manner. Whether there are functional consequences of this variation remains to be determined.

The 2019 death of the female Swinhoes soft-shelled turtle (Rafetus swinhoei) individual in China put this species in a dire conservation state. To avoid the loss of genetic information from the female individual and facilitate the protection and preservation of R. swinhoei, we sequenced and assembled its draft chromosome-level genome by combining BGISEQ short reads, Nanopore long reads, and Hi-C sequencing reads. We characterized its potential ZZ/ZW sex determination system. Phylogeny and divergence time analyses indicated that R. swinhoei has a close relationship with Pelodiscus sinensis, and they shared a common ancestor ~54.4 Ma. Population history analyses indicated that R. swinhoei has the lowest heterozygosity among all turtles examined, and its population size has declined dramatically over the past 10 million years. Many core genes involved in autophagy and DNA damage response, such as Rab5a and Parp1, were under positive selection and/or evolving rapidly, and these genes may make important contributions to the long lifespan and evolutionary adaptation of R. swinhoei. The loss of several tooth-related genes in turtle genomes explains the genetic basis of their toothless phenotype. This study not only provides a genomic resource for scientists to study the species using the strategy of comparative genomics, but also provides a data basis for us to find more potential R. swinhoei individuals in the wild.

Turtle blood flukes ("spirorchiids") comprise a paraphyletic assemblage including the monophyletic Schistosomatidae Stiles and Hassall, 1898 as a crown group. We herein morphologically diagnose the natural groups of turtle blood flukes and propose family names for them. Spirorchiidae Stunkard, 1921 (Spirorchis MacCallum, 1919 [type]; Spirhapalum Ejsmont, 1927; Plasmiorchis Mehra, 1934; MonticelliusMehra, 1939; VasotremaStunkard, 1928; provisionally UterotremaPlatt and Pichelin, 1994) has a ventral sucker (lost in Spirorchis), an esophageal gland surrounding the entire esophagus, lateral esophageal diverticula (plicate organ and medial esophageal diverticulum present or absent), a glandular mass at the esophagus base, paired ceca surrounded by vitelline follicles for their entire length, a non-filamented, ovoid egg, and typically a Manter's organ. Baracktrematidae n. fam. (Baracktrema Roberts, Platt, and Bullard, 2016 [type]; Unicaecum Stunkard, 1925; NeospirorchisPrice, 1934) has no ventral sucker, a single cecum or cyclocoel, a coiled or looped testis extending into the anterior body half, a convoluted post-cecal and post-gonadal uterus, a uterine pouch, no metraterm, no Manter's organ, and non-filamented eggs. Plattidae n. fam. (Platt Roberts and Bullard, 2018 [type]; Hapalorhynchus Stunkard, 1922; Coeuritrema Mehra, 1933; Enterohaematotrema Mehra, 1940; CardiotremaDwivedi, 1967; Ruavermis Dutton and Bullard, 2020) has the anatomical sequence of a ventral sucker, external seminal vesicle, cirrus sac, anterior testis, ovary, transverse vitelline duct, and posterior testis (except Enterohaematotrema) plus a pars prostatica, dorsal genital pore, globular excretory vesicle, and no Manter's organ. Carettacolidae Yamaguti, 1958 (CarettacolaManter and Larson, 1950 [type]) has a spinose oral sucker, a spinose and contractile ventral sucker (transverse cavity present), numerous testes distributing in a post-ovarian, inter-cecal column, and an ovary and transverse vitelline duct that are pre-testicular. Hapalotrematidae (Stunkard, 1921) Poche, 1926 (Hapalotrema Loos, 1899 [type]; AmphiorchisPrice, 1934; LearediusPrice, 1934; Cheloneotrema Simha and Chattopadhyaya, 1980; NeocaballerotremaSimha, 1977; Satyanarayanotrema Simha and Chattopadhyaya, 1980; Shobanotrema Simha and Chattopadhyaya, 1980) has the anatomical sequence of a ventral sucker, anterior testis(es), external seminal vesicle and cirrus sac, ovary, and posterior testis(es). Atamatamidae n. fam. (Atamatam Bullard and Roberts, 2019 [type]; Paratamatam Bullard and Roberts, 2019; Pitiutrema Dutton and Bullard, 2019) has an oral sucker with anteroventral spines, vasa efferentia connecting to a pre-ovarian external seminal vesicle, a cirrus sac directed postero-laterad and having an internal seminal vesicle and glandular luminal wall, and a dendritic ovary flanked by the external seminal vesicle and oviducal seminal receptacle. A 28S phylogenetic analysis failed to reject the monophyly of these families.

A reduction in adult survival in long-living species may compromise population growth rates. The spur-thighed tortoise (Testudo graeca) is a long-lived reptile that is threatened by habitat loss and fragmentation. Golden eagles (Aquila chrysaetos), whose breeding habitats overlap that of tortoises, may predate them by dropping them onto rocks and breaking their carapaces. In SE Spain, the number of golden eagles has increased in the last decades and the abundance of their main prey (i.e., rabbits Oryctolagus cuniculus) has decreased. Our aims were to 1) describe the role of tortoises in golden eagles’ diet, and 2) estimate the predation impact of golden eagles on tortoises in eagles’ territories and in the regional tortoise population. We collected regurgitated pellets and prey remains under eagle nests and roosts, and obtained information on tortoise abundance and population structure and rabbit abundance. We found that tortoises were an alternative prey to rabbits, so that eagles shifted to the former where the latter were scarce. The average predation rate on tortoises was very low at the two studied scales. However, eagles showed a marked selection for adult female tortoises, which led the tortoise sex ratio to be biased towards males in those eagle territories with higher tortoise predation. Whether this may compromise the spur-thighed tortoise long-term population viability locally deserves further attention., Ministry of Science and Innovation, Spain (MICINN), Spanish Government CGL2012-33536 CGL2015-64144 FPU1700633, European Commission CGL2012-33536 CGL2015-64144, MCIN/AEI PID2019-105682RA-I00, Regional Valencian Government AICO/2021/145 APOSTD/2020/090, Ramon y Cajal research contract from MINECO RYC-2015-19231, European Union-Next Generation EU in the Maria Zambrano Program ZAMBRANO 21-26

Sea turtles are among several hundred species whose sex is determined by incubation conditions during critical developmental periods. Consequently, these marine reptiles may be vulnerable to global climate change, and under the assumption of continued climate warming, numerous studies pose dire predictions for future populations based primarily on hatchling sex ratio data. Alternatively, as long-lived species that take decades to reach maturity, without inherent coping mechanisms for such change, sea turtles could not have persisted across geological epochs. Globally, loggerhead Caretta caretta [Linnaeus, 1758] sea turtles occupy temperate zones, with ontogenetic development that spans the entirety of gyres associated with respective ocean basins. The largest rookery for this species occurs in the Northwest Atlantic Ocean (NWA) population, where a 30-year cycle in annual nest counts is reported through 2018. Complementary studies document a lagged association between these annual nest counts and the Atlantic Multidecadal Oscillation (AMO); however, the underlying mechanism for this association remains elusive. Therefore, objective 1 evaluated the effect of AMO-mediated cohort resonance on the demographic structure of a theoretical neritic assemblage under variable cohort abundance and female proportion but stable annual survival during 165-year runs (i.e., extent of AMO data). For objective 2, blood samples were used to assign sex to 2217 loggerhead sea turtles captured by research trawling (2000 to 2019) on the inner continental shelf from St. Augustine, FL (29.9°N) to Winyah Bay, SC (33.1°N). Shorter oceanic duration of less female-biased cohorts from the AMO cold phase synchronized peak adult male and adult female co-occurrence during subsequent warm phases three decades later. Grand sex ratio predicted from testosterone was 67% female (n = 1484), with a slight temporal female decline. Our findings suggest greater population sex ratio plasticity than predicted solely from terrestrial nesting data.

All sea turtles exhibit temperature-dependent sex-determination, where warmer temperatures produce mostly females and cooler temperatures produce mostly males. As global temperatures continue to rise, sea turtle sex-ratios are expected to become increasingly female-biased, threatening the long-term viability of many populations. Nest temperatures are dependent on sand temperature, and heavy rainfall events reduce sand temperatures for a brief period. However, it is unknown whether these short-term temperature drops are large and long enough to produce male hatchlings. To discover if short-term temperature drops within the sex-determining period can lead to male hatchling production, we exposed green and loggerhead turtle eggs to short-term temperature drops conducted in constant temperature rooms. We dropped incubation temperature at four different times during the sex-determining period for a duration of either 3 or 7 days to mimic short-term drops in temperature caused by heavy rainfall in nature. Some male hatchlings were produced when exposed to temperature drops for as little as 3 days, but the majority of male production occurred when eggs were exposed to 7 days of lowered temperature. More male hatchlings were produced when the temperature drop occurred during the middle of the sex-determining period in green turtles, and the beginning and end of the sex-determining period in loggerhead turtles. Inter-clutch variation was evident in the proportion of male hatchlings produced, indicating that maternal and or genetic factors influence male hatchling production. Our findings have management implications for the long-term preservation of sea turtles on beaches that exhibit strongly female-biased hatchling sex-ratios.

Trace elements in aquatic environments pose a risk to biological communities; this study investigates the total concentrations of arsenic (As), silver (Ag), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), selenium (Se), lead (Pb), and zinc (Zn) within muscle, carapace, liver, and adipose tissues of diamondback terrapins and common snapping turtles in New Jersey. The effects of tissue type, sex, size, and location upon trace element accumulation were studied. The data obtained indicates that within diamondback terrapins and common snapping turtles, trace element accumulations displayed a significant difference among tissue types and sex (p

Variation in developmental conditions can affect a variety of embryonic processes and shape a number of phenotypic characteristics that can affect offspring throughout their lives. This is particularly true of oviparous species where development typically occurs outside of the female, and studies have shown that traits such as survival and behavior can be altered by both temperature and exposure to steroid hormones during development. In species with temperature-dependent sex determination (TSD), the fate of gonadal development can be affected by temperature and by maternal estrogens present in the egg at oviposition, and there is evidence that these factors can affect gene expression patterns. Here, we explored how thermal fluctuations and exposure to an estrogen metabolite, estrone sulfate, affect the expression of several genes known to be involved in sexual differentiation: Kdm6b, Dmrt1, Sox9, FoxL2 and Cyp19A1. We found that most of the genes responded to both temperature and estrone sulfate exposure, but that the responses to these factors were not identical, in that estrone sulfate effects occur downstream of temperature effects. Our findings demonstrate that conjugated hormones such as estrone sulfate are capable of influencing temperature-dependent pathways to potentially alter how embryos respond to temperature, and highlight the importance of studying the interaction of maternal hormone and temperature effects.

The implications of logger accuracy and precision are rarely considered prior to their application in many ecological studies. We assessed the accuracy and precision of three temperature data loggers widely used in ecological studies (Hobo®, iButton® and TinyTag®). Accuracy was highest in TinyTags (95% of readings were within 0.23°C of the true temperature) and lowest in HOBOs and iButtons (95% of were readings within 0.43°C and 0.49°C of the true temperature, respectively). The precision (standard deviation of the repeat measurements) was greatest in TinyTags (0.04°C), followed by iButtons (0.17°C) and then HOBOs (0.22°C). As a case study, we then considered how modelled estimates of sea turtle hatchling sex ratios (derived from temperature), could vary as a function of logger accuracy. For example, at 29°C when the mean sex ratio derived was 0.47 female, the sex ratio estimate from a single logger could vary between 0.40 and 0.50 for TinyTags and 0.29 and 0.56 for both HOBOs and iButtons. Our results suggest that these temperature loggers can provide reliable descriptions of sand temperature if they are not over-interpreted. Logger accuracy must be considered in future ecological studies in which temperature thresholds are important.

Sea turtles are considered as bio-indicators for monitoring the efficiency of restoration measures to reduce marine litter impacts on health. However, the lack of extended and standardised empirical data has prevented the accurate analysis of the factors influencing litter ingestion and the relationships with individual health. Historic data collected from 1988 and standard data collected from 2016 were harmonised to enable such analyses on necropsied loggerhead turtles (Caretta caretta) in eight Mediterranean and North-East Atlantic countries. Litter was found in 69.24 % of the 1121 individuals, mostly single-use and fishing-related plastics. Spatial location, sex and life history stage explained a minor part of litter ingestion. While no relationships with health could be detected, indicating that all individuals can be integrated as bio-indicators, the mechanistic models published in literature suggest that the high proportion of plastics in the digestive contents (38.77 % per individual) could have long-term repercussions on population dynamics. © 2022

Temperature-dependent sex determination (TSD) is a mechanism in which environmental temperature, rather than innate zygotic genotype, determines the fate of sexual differentiation during embryonic development. Reeves’ turtle (also known as the Chinese three-keeled pond turtle, Mauremys reevesii) exhibits TSD and is the only species whose genome has been determined in Geoemydidae to date. Thus, M. reevesii occupy phylogenetically important position for the study of TSD and can be compared to other TSD species to elucidate the underlying molecular mechanism of this process. Nevertheless, neither embryogenesis nor gonadogenesis has been described in this species. Therefore, herein, we investigated the chronology of normal embryonic development and gonadal structures in M. reevesii under both female- and male-producing incubation temperatures (FPT 31 °C or MPT 26 °C, respectively). External morphology remains indistinct between the two temperature regimes throughout the studied embryonic stages. However, the gonadal ridges present on the mesonephros at stage 16 develop and sexually differentiate at FPT and MPT. Ovarian and testicular structures begin to develop at stages 18–19 at FPT and stages 20–21 at MPT, respectively, and thus, the sexual differentiation of gonadal structures began earlier in the embryos at FPT than at MPT. Our results suggest that temperature sensitive period, at which the gonadal structures remain sexually undifferentiated, spans from stage 16 (or earlier) to stages 18–19 at FPT and to stages 20–21 at MPT. Understanding the temperature-dependent differentiation in gonadal structures during embryonic development is a prerequisite for investigating molecular basis underlying TSD. Thus, the result of the present study will facilitate further developmental studies on TSD in M. reevesii.

Life-history theory predicts that investment in reproduction should increase as future reproductive potential (i.e., residual reproductive value [RRV]) decreases. Researchers have thus intuitively used age as a proxy for RRV and assume that RRV decreases with age when interpreting age-specific investment. Yet age is an imperfect proxy for RRV and may even be a poor correlate in some systems. We used a 31-year study of the nesting ecology of painted turtles (

A new glossiphoniid leech species, Placobdelloides tridens sp. n., is discovered on the Malayan Giant Turtle (Orlitia borneensis) at the Nakhon Ratchasima Zoo in Thailand. The morphological study of this new species revealed that it is distinguished from P. siamensis, a turtle leech species that can be found commonly in Thailand. Placobdelloides tridens presented the following diagnostic morphological characteristics: a pear-shaped and triannulate body, well-developed rod-like papillae on the dorsal surface, smooth posterior and anterior suckers with nominal pits inside, a single pair of dark contiguous eyes, light yellow-brown to greenish dorsal color, absence of median line, male and female gonopore separated by a single annulus and a unique trident shape at the tip of the crop ceca. The phylogenetic relationships of P. tridens sp. n., was clarified, and shown to be a sister clade to the P. siamensis and P. sirikanchanae clade. Furthermore, this is a new host record for P. siamensis, which was found on O. borneensis, Batagur affinis and B. borneoensis in the Khao Kheow Open Zoo, Chonburi, Thailand.

Ontogenetic development of hearing sensitivity has been verified in many groups of vertebrates, but not turtles. Turtles exhibit sexual dimorphism in hearing. To examine the development of hearing in female turtles, auditory brainstem responses (ABR) were compared by assessing the hearing-sensitivity bandwidth, ABR threshold, and latency of female Trachemys scripta elegans aged 1 week, 1 month, 1 yr, and 5 yr. The hearing-sensitivity bandwidths were 0.2-1.1, 0.2-1.1, 0.2-1.3, and 0.2-1.4 kHz in each age group, respectively. Below 0.6 kHz, the ABR threshold decreased from the 1-week to 1-yr age group, with a significant difference between age groups. No significant difference was detected between the 1- and 5-yr age groups (within a stimulus frequency of 0.2-0.6 kHz). Above 0.6 kHz, ABR thresholds decreased significantly from the 1-yr to 5-yr age group (within a stimulus frequency of 0.7-1.0 kHz). There was no significant difference between the 1-month and 1-yr age groups (within a stimulus frequency of 0.7-1.0 kHz), or between the 1-week and 1-month age groups (within a stimulus frequency of 0.7-1.0 kHz, except 0.9 kHz). Thus, female turtle hearing shows frequency-segmented development.

Temperature-dependent sex determination (TSD) decides the sex fate of an individual based on incubation temperature. However, other environmental factors, such as pollutants, could derail TSD sexual development. Cadmium is one such contaminant of soils and water bodies known to affect DNA methylation, an epigenetic DNA modification with a key role in sexual development of TSD vertebrate embryos. Yet, whether cadmium alters DNA methylation of genes underlying gonadal formation in turtles remains unknown. Here, we investigated the effects of cadmium on the expression of two gene regulators of TSD in the painted turtle

In oviparous reptiles, parental care is often limited to the energy allocated to embryos before oviposition. Reproducing females can allocate energy toward vitellogenesis, determining the number and size of eggs, fertilization, eggshell calcification, retention of eggs within the oviduct after fertilization (oviductal egg retention), and nesting activities. Oviductal egg retention in turtles ranges from 2 wk to half a year, permitting flexibility in the timing of oviposition. The energetic cost of oviductal egg retention in eastern musk turtles (

Understanding the evolution and regulation of nucleolar organizing regions (NORs) is important to elucidate genome structure and function. This is because ribosomal gene (rDNA) copy number and activity mediate protein biosynthesis, stress response, ageing, disease, dosage compensation and genome stability. Here, we found contrasting dosage compensation of sex-linked NORs in turtles with male and female heterogamety. Most taxa examined exhibit homomorphic rRNA gene clusters in a single autosome pair (determined by 28S rDNA fluorescence in situ hybridization), whereas NORs are sex-linked in Apalone spinifera, Pelodiscus sinensis and Staurotypus triporcatus. Full-dosage compensation upregulates the male X-NOR (determined via silver staining-AgNOR) in Staurotypus (who lacks Y-NOR) compared with female X-AgNORs. In softshell Apalone and Pelodiscus, who share homologous ZZ/ZW micro-chromosomes, their enlarged W-NOR is partially active (due to 28S rDNA invasion by R2 retroelements), whereas their smaller Z-NOR is silent in females but active in both male-Zs (presumably because the W-NOR meets cellular demands and excessive NOR activity is costly). We hypothesize that R2 disruption favoured W enlargement to add intact 28S-units, perhaps facilitated by reduced recombination during sex chromosome evolution. The molecular basis of the potentially adaptive female Z-silencing is likely intricate and perhaps epigenetic, as non-ribosomal Z genes are active in Apalone females. Yet, Emydura maquarii exhibit identical heteromorphism in their autosomal NOR (R2 invaded 28S-units and the small-autosome NOR is silent), suggesting that the softshell turtle pattern can evolve independent of sex chromosome evolution. Our study illuminates the complex sex chromosome evolution and dosage compensation of non-model systems that challenges classic paradigms.

Between 2011 and 2020, the Ontario Turtle Conservation Centre's veterinary hospital admitted 6,335 turtles of eight different species, with 3,246 being midland painted turtles (Chrysemys picta marginata). At the time of admission from the wild, aural abscesses were present in 26 of the painted turtles (0.8%). Aural abscesses were never observed in juvenile turtles and were not found in any other species of turtle admitted to the hospital. An equal number of males and females were affected. All cases showed signs of chronicity. Abscesses alone did not appear to affect the overall health of turtles regardless of clinical signs. All turtles with abscesses as the sole cause for admission were released after successful treatment.

Mesenchymal stem cell (MSC) secretome refers to a variety of bioactive compounds that represents the more important pathway by which MSCs participate in tissue regeneration. Many of these compounds have shown variable functional activity even across nonmammalian vertebrate species, although MSCs in turtles have not yet been described. Canine adipose MSC secretome has been successfully used experimentally in skin healing. Our aim was to conduct a blinded controlled study to evaluate the effect of canine adipose MSC secretome (cS-MSC) as an alternative for the healing of soft skin, second intention wounds of red-eared slider turtles (Trachemys scripta). Under general anesthesia, one circular, 6-mm full thickness wound was made in each rear leg of 14 females. After randomization, cS-MSC was injected subcutaneously around one wound at days 1, 7, and 14, whereas the other wound acted as control. Biopsies from three animals' wounds were obtained at days 21, 28, 42, and 63. Differences in mean wound retraction at days 21 (n=14) and 28 (n=11) were statistically nonsignificant. The clinical and histopathologic scores performed blind by two different investigators were similar for treated and control wounds. In conclusion, we could not detect a significant functional activity of cS-MSC on wound healing of Trachemys scripta.

KDM6B-mediated epigenetic modification of the testicular regulator Dmrt1 has previously been identified as the primary switch of the male pathway in a temperature-dependent sex-determination (TSD) system; however, the molecular network of the female pathway has not yet been established. Here, we have functionally characterized for the first time an upstream regulator of the female pathway, the forkhead transcription factor FOXL2, in Trachemys scripta, a turtle species with a TSD system. FOXL2 exhibited temperature-dependent female-specific expression patterns before the onset of gonadal differentiation and was preferentially localized in ovarian somatic cells. Foxl2 responded rapidly to temperature shifts and estrogen. Importantly, forced expression of Foxl2 at the male-producing temperature led to male-to-female sex reversal, as evidenced by the formation of an ovary-like structure, and upregulation of the ovarian regulators Cyp19a1 and R-spondin1. Additionally, knockdown of Foxl2 caused masculinization at the female-producing temperature, which was confirmed by loss of the female phenotype, development of seminiferous tubules, and elevated expression of Dmrt1 and Sox9. Collectively, we demonstrate that Foxl2 expression is necessary and sufficient to drive ovarian determination in T. scripta, suggesting a crucial role of Foxl2 in female sex determination in the TSD system.

The levels of six non-dioxin-like polychlorinated biphenyls (PCBs 28, 52, 101, 138, 153, and 180) were determined in the liver of 84 loggerhead turtles (Caretta caretta) stranded along the coasts of the Tyrrhenian Sea in Campania Region (Southern Italy), from 2017 to 2021. The average value of the sum (∑

Turtles are iconic reptiles that inhabit a range of ecosystems from oceans to deserts and climates from the tropics to northern temperate regions. Yet, we have little understanding of the genetic adaptations that allow turtles to survive and reproduce in such diverse environments. Common snapping turtles, Chelydra serpentina, are an ideal model species for studying adaptation to climate because they are widely distributed from tropical to northern temperate zones in North America. They are also easy to maintain and breed in captivity and produce large clutch sizes, which makes them amenable to quantitative genetic and molecular genetic studies of traits like temperature-dependent sex determination. We therefore established a captive breeding colony and sequenced DNA from one female using both short and long reads. After trimming and filtering, we had 209.51Gb of Illumina reads, 25.72Gb of PacBio reads, and 21.72 Gb of Nanopore reads. The assembled genome was 2.258 Gb in size and had 13,224 scaffolds with an N50 of 5.59Mb. The longest scaffold was 27.24Mb. BUSCO analysis revealed 97.4% of core vertebrate genes in the genome. We identified 3.27 million SNPs in the reference turtle, which indicates a relatively high level of individual heterozygosity. We assembled the transcriptome using RNA-Seq data and used gene prediction software to produce 22,812 models of protein coding genes. The quality and contiguity of the snapping turtle genome is similar to or better than most published reptile genomes. The genome and genetic variants identified here provide a foundation for future studies of adaptation to climate.

Cucullanus carettae Baylis, 1923 (Nematoda: Cucullanidae) is found worldwide in loggerhead turtles (Caretta caretta). Regarding the Mediterranean, C. carettae has been identified in the Tyrrhenian and the Ionian Sea and a unique description of a Cucullanus sp. specimen in loggerheads from the Adriatic Sea has been reported in the literature so far. In the framework of a bio-monitoring project of the Abruzzo and Molise coasts, a parasitological survey was performed on stranded and by-caught sea turtles, at the Istituto Zooprofilattico of Abruzzo and Molise “G. Caporale.” During necropsy, the gastrointestinal system of 72 stranded loggerhead turtles was analyzed for the presence of endoparasites and fecal samples were collected for coprological examination. Adult C. carettae (n = 123) was found in the upper intestine of one loggerhead turtle, associated with chronic lymphoplasmocytic enteritis. Additionally, five stool samples (6.9%) were positive for Cucullanus sp. eggs. Molecular characterization of adult nematodes was carried out to study phylogenetic relationships among the Cucullanus species. To our knowledge, this is the first morphological and molecular identification of C. carettae in loggerhead turtles from the Adriatic Sea. Additional studies on the distribution of this parasite in the Mediterranean are encouraged.

Eastern (Terrapene carolina carolina) and ornate (Terrapene ornata ornata) box turtles have robust plasma antibacterial activity, however, the mechanism behind this activity is unknown. We used sheep red blood cell (SRBC) hemolysis assays, mannan-affinity chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to explore the mechanisms of complement activity in box turtles. Plasma from both species demonstrated volume, time, and temperature-dependent SRBC hemolysis, with significantly greater hemolytic activity in ornate box turtle plasma. Hemolytic activity was highly attenuated following treatment with heat, EDTA, and salicylaldoxime in both species, but was unchanged after treatment with methylamine and ammonium hydroxide. Two abundant mannan-binding proteins (presumed C-type lectins) were identified in eastern box turtle plasma using SDS-PAGE and MALDI-TOF, but ornate box turtles did not express either protein. Eastern box turtles appear to rely on the lectin pathway of complement activation while ornate box turtles utilize the alternative pathway. This study provides further evidence that mechanisms underlying immune function are not always conserved between closely related species. This finding may have important implications for explaining species differences in susceptibility to emerging threats such as disease, toxicants, and climate change.

The quantification of repeatability has enabled behavioural and evolutionary ecologists to assess the heritable potential of traits. For behavioural traits that vary across life, age-related variation should be accounted for to prevent biasing the microevolutionary estimate of interest. Moreover, to gain a mechanistic understanding of ontogenetic variation in behaviour, among- and within-individual variance should be quantified across life. We leveraged a 30-year study of painted turtles (Chrysemys picta) to assess how age contributes to variation in the repeatability of nesting behaviours. We found that four components of nesting behaviour were repeatable and that accounting for age increased the repeatability estimate for maternal choice of canopy cover over nests. We detected canalization (diminished within-individual variance with age) of canopy cover choice in a reduced data set despite no shift in repeatability. Additionally, random regression analysis revealed that females became more divergent from each other in their choice of canopy cover with age. Thus, properly modelling age-related variance should more precisely estimate heritable potential, and assessing among- and within-individual variance components in addition to repeatability will offer a more mechanistic understanding of behavioural variation across age.

Visual illusions have been widely used as a tool to study animal visual perception. In many cases, identical experimental procedures were adopted to make highly controlled interspecific comparisons. However, reducing methodological variability may prevent reliable comparisons because a certain methodology could be more suitable for some species than others. This study sought to build on previous work that investigated the perception of the Delboeuf illusion in reptiles. Reptiles were presented with trials composed of 2 different-sized food portions on 2 identical plates in which they were expected to maximize their food intake. In contrast to the bearded dragons (Pogona vitticeps), tortoises (Chelonoidis carbonaria) performed poorly in all conditions and therefore no firm conclusion regarding their perception of the illusion could be made. Such results could be due to cognitive challenges or due to the experimental setup, because descending a ramp is demanding for the tortoises. In this study, we adopted the same experimental paradigm but in a flat apparatus. Tortoises significantly discriminated the larger food portions in baseline trials, however, their performance did not differ from chance in illusory trials revealing that, under these conditions, they are not sensitive to the Delboeuf illusion. This nonperception could be ascribed to different factors, such as poorer discrimination ability or a low sensitivity to contrast and assimilation phenomena. Our study highlights the importance of additional investigation to better understand the nature of null results, taking in consideration the ecological needs of the species before drawing any conclusions about its abilities. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Many egg-laying reptiles possess temperature-dependent sex determination (TSD) in which outcome of gonadogenesis is determined by incubation temperature during a temperature-sensitive period of development. Prior studies on Malayemys macrocephala showed that incubation temperatures influence gonadal development and suggested that M. macrocephala exhibits TSD. However, information on the temperature-sensitivity period in this species was unknown until the current study. Turtle eggs were collected from rice fields in central Thailand from December 2016 to February 2017. In the laboratory, eggs were incubated at male-biased temperature (26 °C) and shifted to female-biased temperature (32 °C), or vice versa. Single shift experiments were performed systematically during embryonic stages 13-21. After hatching, sex of individual turtles was determined by histological analysis. We found that the sex determination of M. macrocephala is affected by temperature up to stage 16 of embryonic development.

Emys orbicularis (Linnaeus, 1758) was considered as a specific host of Placobdella costata (Fr. Mûller, 1846). However, since the parasite was recorded from outside the distribution area of its host, some authors suggested a possible relationship with other hosts. Although two accidental associations were found with another turtle, Mauremys leprosa (Schweigger, 1812), the obtained data remain insufficient to better understand this discovered host-parasite ecological system. In this context, the present study was carried out to evaluate the relationship between the Mediterranean pond turtle, M. leprosa, and the freshwater rhynchobdellid leech, P. costata (Hirudinida: Glossiphoniidae), in aquatic ecosystems of Morocco. During the period from April to June 2018, we found leeches attached to turtles in five out of 30 populations sampled with a prevalence of infection significantly higher in adult than that in juvenile turtles. Moreover, the males are the most infested with 51% of the total, followed by females (33.3%) and juveniles (15.7%). The obtained results indicated that 51 turtles were infested by 139 leeches with a mean intensity of infestation of 4.17 ± 0.47 leeches/turtle (up to 10 leeches/turtle). It was higher in males than that in females in almost all sites. The posterior limbs are the most preferred attachment site, and the body condition of turtles was not affected by the intensity of infestation but it is rather a function of altitude. Our findings proved that M. leprosa-P. costata association is more than accidental and that M. leprosa is rather the main host of P. costata in aquatic ecosystems of Morocco.

Synopsis Here we describe examples of studies that have contributed both to a basic understanding of the biology of imperiled marine turtles, and to their management and conservation. Key elements include, first and foremost, correctly identifying species that differ strikingly in their morphology at different life stages because with growth, they change size by several orders of magnitude and have accompanying shape changes. We also review comprehensive field studies documenting the need for management actions to correct abnormal shifts in sex ratios caused by climate change. We highlight the need to describe those perturbations in terms that are clear to regulators and personnel responsible for management and conservation policies. Finally, we review several basic studies that enhance our understanding of how selection has shaped morphological, functional, and performance attributes, and describe how that knowledge can be applied to the tasks required for enhancing species recovery.

Generally, the olfactory organ of vertebrates consists of the olfactory epithelium (OE) and the vomeronasal organ (VNO). The OE contains ciliated olfactory receptor neurons (ORNs), while the VNO contains microvillous ORNs. The ORNs in the OE express odorant receptors (ORs), while those in the VNO express type 1 and type 2 vomeronasal receptors (V1Rs and V2Rs). In turtles, the olfactory organ consists of the upper (UCE) and lower chamber epithelia (LCE). The UCE contains ciliated ORNs, while the LCE contains microvillous ORNs. Here we investigated the distribution of cells expressing vomeronasal receptors in the olfactory organ of turtles. The turtle vomeronasal receptors were encoded by two V1R genes and two V2R genes. Among them, V2R1 and V2R26 were mainly expressed in the LCE, while V1R3 was expressed both in the UCE and LCE. Notably, vomeronasal receptors were expressed by a limited number of ORNs, which was confirmed by the expression of the gene encoding TRPC2, an ion channel involved in the signal transduction of vomeronasal receptors. Furthermore, expression of ORs by the majority of ORNs was suggested by the expression of the gene encoding CNGA2, an ion channel involved in the signal transduction of ORs. Thus, olfaction of turtle seems to be mediated mainly by the ORs rather than the vomeronasal receptors. More importantly, the relationship between the fine structure of ORNs and the expression of olfactory receptors are not conserved among turtles and other vertebrates.

The morphology of the tetrapod nasal cavity has adapted to the environment in terms of olfaction and respiration. Reports indicate that the internal structure of the nasal cavity of green sea turtles is more complex than that of turtles in general, but whether or not it is similar among sea turtle species remains unknown. The present study aimed to define the internal structures of the nasal cavity of green (Chelonian mydas), loggerhead (Caretta caretta) and leatherback (Dermochelys coriacea) sea turtles using computed tomography. The nasal cavity of green and loggerhead sea turtles contained anterodorsal, anteroventral, posterodorsal diverticula and a posteroventral excavation in the middle. In contrast, the nasal cavity of leatherback sea turtles had more complicated dorsal region comprising anterodorsal and posterodorsal diverticula, and two excavations between the nostril and anterodorsal diverticulum, but no distinct structures at the ventral region. The airway in the nasal cavity was shorter and thicker in the leatherback, than in the green and loggerhead turtles. These species differences might reflect ecological variety and different evolutionary strategies.

Substrate type determines nesting success and fitness in turtles and is a critical consideration for nesting area protection and restoration. Here, we evaluated the effect of substrate on nest removal by humans in the eastern Brazilian Amazon. We analyzed substrate composition and fate of 216 P. unifilis nests along 88 km of rivers. River segment and substrate type were the most important predictors of nest removal by humans. We found up to 36% lower removal of nests in fine sand and experimental results support the hypothesis that wind more often obscures tracks of nesting females in substrates with more (>66%) fine sand. Our findings are useful for informing the restoration of artificial nesting areas across the Amazon, as nesting area substrates should be selected not only to maintain hatchling sex ratios, but also to minimize nest removal by humans.

A healthy adult, intact female keeled box turtle (Cuora mouhotii) was found to have a marked heterophilic leukocytosis using normal hematologic parameters established for the eastern box turtle (Terrapene carolina carolina), a related chelonian species. This animal was monitored with serial complete blood counts (CBCs) over the next 15 years despite remaining asymptomatic for an infectious condition. Retrospective CBC data were compiled from 38 presumably healthy keeled box turtles to establish hematologic values for comparison in this species. Using this species-specific data, over the 15-year period, the female keeled box turtle had two times where the white blood cell (WBC) count was greater than 2 standard deviations (SD) above the mean, six times where the WBC count was greater than 1 SD above the mean, six times where the PCV was greater than 2 SD above the mean, and eight times where the PCV was greater than 1 SD above the mean. Infection and inflammation are the most common causes of leukocytosis in reptiles; however, given the clinical presentation of this patient, it was postulated that these clinicopathologic changes could be secondary to a stress response. Establishing reference intervals and understanding how stress impacts CBC parameters are important for evaluating the health status of keeled box turtles kept in captivity and for assessing the effects of environmental changes on the health status of wild populations of this endangered chelonian species.

The microbiota is considered critical for normal vertebrate homeostasis and it may exert its effects at a local level within the gastrointestinal tract, or systemically through the production of bacterial metabolites. To date, investigations into the role that the microbiota plays in reptile physiology are rare. To address this knowledge gap, we explored the relationship between differences in microbial communities to see if they accounted for differences in haematology and biochemistry values, in different populations of nesting flatback turtles (Natator depressus). We found that microbiota composition was not correlated to any of the blood analytes we measured in flatbacks. This study is the first of its kind in reptiles and highlights the need for further investigations to determine mechanisms by which the microbiota influences the physiology and health of reptiles.