Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations.

Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research.

Trait Loss in Evolution: What Cavefish Have Taught Us about Mechanisms Underlying Eye Regression.

Reduction or complete loss of traits is a common occurrence throughout evolutionary history. In spite of this, numerous questions remain about why and how trait loss has occurred. Cave animals are an excellent system in which these questions can be answered, as multiple traits, including eyes and pigmentation, have been repeatedly reduced or lost across populations of cave species. This review focuses on how the blind Mexican cavefish, Astyanax mexicanus, has been used as a model system for examining the developmental, genetic, and evolutionary mechanisms that underlie eye regression in cave animals. We focus on multiple aspects of how eye regression evolved in A. mexicanus, including the developmental and genetic pathways that contribute to eye regression, the effects of the evolution of eye regression on other traits that have also evolved in A. mexicanus, and the evolutionary forces contributing to eye regression. We also discuss what is known about the repeated evolution of eye regression, both across populations of A. mexicanus cavefish and across cave animals more generally. Finally, we offer perspectives on how cavefish can be used in the future to further elucidate mechanisms underlying trait loss using tools and resources that have recently become available.

Pleiotropic function of the oca2 gene underlies the evolution of sleep loss and albinism in cavefish.

Adaptation to novel environments often involves the evolution of multiple morphological, physiological, and behavioral traits. One striking example of multi-trait evolution is the suite of traits that has evolved repeatedly in cave animals, including regression of eyes, loss of pigmentation, and enhancement of non-visual sensory systems.1,2 The Mexican tetra, Astyanax mexicanus, consists of fish that inhabit at least 30 caves in Mexico and ancestral-like surface fish that inhabit the rivers of Mexico and southern Texas.3 Cave A. mexicanus are interfertile with surface fish and have evolved a number of traits, including reduced pigmentation, eye loss, and alterations to behavior.4-6 To define relationships between different cave-evolved traits, we phenotyped 208 surface-cave F2 hybrid fish for numerous morphological and behavioral traits. We found differences in sleep between pigmented and albino hybrid fish, raising the possibility that these traits share a genetic basis. In cavefish and other species, mutations in oculocutaneous albinism 2 (oca2) cause albinism.7-12 Surface fish with mutations in oca2 displayed both albinism and reduced sleep. Further, this mutation in oca2 fails to complement sleep loss when surface fish harboring this engineered mutation are crossed to independently evolved populations of albino cavefish with naturally occurring mutations in oca2. Analysis of the oca2 locus in wild-caught cave and surface fish suggests that oca2 is under positive selection in 3 cave populations. Taken together, these findings identify oca2 as a novel regulator of sleep and suggest that a pleiotropic function of oca2 underlies the adaptive evolution of albinism and sleep loss.

The history, taxonomy, and geographic origins of an introduced African monkey in the southeastern United States.

The origins and taxonomy of the introduced vervet monkey population in Dania Beach, Florida has been unconfirmed due to a lack of documentation and genetic research. Our goal was to determine the introduction history, species identification, and geographic origins of the monkeys. Through interviews, historical archives, and popular media, we traced the monkeys to an escape from the Dania Chimpanzee Farm in 1948. The facility imported primates from Africa for medical research purposes. Historical archives suggest the monkeys were caught in Sierra Leone. We tested the hypothesis of West African origins using three genetic markers: one mitochondrial DNA gene (cytochrome b) and two fragments from the Y-chromosome, the sex-determining gene and the zinc-finger gene. We ran Bayesian and maximum-likelihood analyses to reconstruct phylogenetic trees. Results from all loci confirmed the species identification is Chlorocebus sabaeus. We found no variation among the sampled individuals and found the cytochrome b haplotype to be a complete match to a C. sabaeus sample from Senegal. Phylogenetic analyses showed strong support for the Dania Beach mitochondrial and Y-chromosome lineages to group within a monophyletic C. sabaeus clade endemic to West Africa. Our study provides critical baseline information to the scientific community about a little-known population of Chlorocebus monkeys that have adapted to a novel environment in the southeastern United States.

A chromosome-level genome of Astyanax mexicanus surface fish for comparing population-specific genetic differences contributing to trait evolution.

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species.

Repeated evolution of eye loss in Mexican cavefish: Evidence of similar developmental mechanisms in independently evolved populations.

Evolution in similar environments often leads to convergence of behavioral and anatomical traits. A classic example of convergent trait evolution is the reduced traits that characterize many cave animals: reduction or loss of pigmentation and eyes. While these traits have evolved many times, relatively little is known about whether these traits repeatedly evolve through the same or different molecular and developmental mechanisms. The small freshwater fish, Astyanax mexicanus, provides an opportunity to investigate the repeated evolution of cave traits. A. mexicanus exists as two forms, a sighted, surface-dwelling form and at least 29 populations of a blind, cave-dwelling form that initially develops eyes that subsequently degenerate. We compared eye morphology and the expression of eye regulatory genes in developing surface fish and two independently evolved cavefish populations, Pachón and Molino. We found that many of the previously described molecular and morphological alterations that occur during eye development in Pachón cavefish are also found in Molino cavefish. However, for many of these traits, the Molino cavefish have a less severe phenotype than Pachón cavefish. Further, cave-cave hybrid fish have larger eyes and lenses during early development compared with fish from either parental population, suggesting that some different changes underlie eye loss in these two populations. Together, these data support the hypothesis that these two cavefish populations evolved eye loss independently, yet through some of the same developmental and molecular mechanisms.

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.

Identifying Sex of Neonate Turtles with Temperature-dependent Sex Determination via Small Blood Samples.

Temperature-dependent sex determination, present in most turtle species, is a mechanism that uses temperature to direct the sex of the embryo. The rapid increase of global temperatures highlights the need for a clear assessment of how sex ratios of organisms with TSD are affected. In turtles with TSD, quantifying primary sex ratios is challenging because they lack external dimorphism and heteromorphic sex chromosomes. Here we describe a new technique used to identify sex in neonate turtles of two TSD species, a freshwater turtle (Trachemys scripta) and a marine turtle (Caretta caretta) via analysis of small blood samples. We used an immunoassay approach to test samples for the presence of several proteins known to play an important role in sex differentiation. Our results show that Anti-Mullerian Hormone (AMH) can be reliably detected in blood samples from neonate male turtles but not females and can be used as a sex-specific marker. Verification of sex via histology or laparoscopy revealed that this method was 100% reliable for identifying sex in both T. scripta and C. caretta 1-2 day-old hatchlings and 90% reliable for identifying sex in 83-177 day-old (120-160 g) loggerhead juveniles. The method described here is minimally invasive, and for the first time, greatly enhances our ability to measure neonate turtle sex ratios at population levels across nesting sites worldwide, a crucial step in assessing the impact of climate change on imperiled turtle species.

The Genome of the Endangered Dryas Monkey Provides New Insights into the Evolutionary History of the Vervets.

Genomic data can be a powerful tool for inferring ecology, behavior, and conservation needs of highly elusive species, particularly, when other sources of information are hard to come by. Here, we focus on the Dryas monkey (Cercopithecus dryas), an endangered primate endemic to the Congo Basin with cryptic behavior and possibly <250 remaining adult individuals. Using whole-genome sequencing data, we show that the Dryas monkey represents a sister lineage to the vervets (Chlorocebus sp.) and has diverged from them ∼1.4 Ma with additional bidirectional gene flow ∼750,000-∼500,000 years ago that has likely involved the crossing of the Congo River. Together with evidence of gene flow across the Congo River in bonobos and okapis, our results suggest that the fluvial topology of the Congo River might have been more dynamic than previously recognized. Despite the presence of several homozygous loss-of-function mutations in genes associated with sperm mobility and immunity, we find high genetic diversity and low levels of inbreeding and genetic load in the studied Dryas monkey individual. This suggests that the current population carries sufficient genetic variability for long-term survival and might be larger than currently recognized. We thus provide an example of how genomic data can directly improve our understanding of highly elusive species.

Hox Genes in Reptile Development, Epigenetic Regulation, and Teratogenesis.

Reptiles are ancestral organisms presenting a variety of shapes, from the elongated vertebral column of the snake to the turtle dorsalized ribs or retractile neck. Body plans are specified by a conserved group of homeobox-containing genes (Hox genes), which encode transcription factors important in cell fate and vertebral architecture along the anteroposterior axis during embryonic development; thus, dysregulation of these genes may cause congenital malformations, from mild-sublethal to embryonic-lethal. The genetic pool, maternal transfer, and environmental conditions during egg incubation affect development; environmental factors such as temperature, moisture, oxygen, and pollution may alter gene expression by epigenetic mechanisms. Thus, in this review, we present information regarding Hox genes and development in reptiles, including sex determination and teratogenesis. We also present some evidence of epigenetic regulation of Hox genes and the role of the environment in epigenetic modulation of gene expression. So far, the evidence suggests that the molecular instructions encoded by Hox genes to build a snake, a lizard, or a turtle represent the interplay between genome and epigenome after years of evolution, with occasional environmentally induced molecular mistakes leading to abnormal body shapes.

Methylation status of the putative Pax6 promoter in olive ridley sea turtle embryos with eye defects: An initial approach.

Normal development involves the interplay of genetic and epigenetic regulatory mechanisms. Pax6 is an eye-selector factor responsible for initiating the regulatory cascade for the development of the eyes. For the olive ridley sea turtle (Lepidochelys olivacea), a threatened species, eye malformations have been reported. In order to study the DNA methylation status of the putative promoter of the Pax6 gene in embryos with ocular malformations, an exploratory study was carried out in which DNA was isolated from embryos with anophthalmia, microphthalmia, and cyclopia, as well as from their normal counterparts. The 5'-flanking region from the Pax6 gene was isolated, showing two CpG islands (CGIs). The methylation status of CGIs in malformed embryos was compared with that of normal embryos by bisulfite sequencing. Putative transcription factor binding sites and regulatory features were identified. Methylation patterns were observed in both CpG and non-CpG contexts, and were unique for each malformed embryo; in the CpG context, an embryo with cyclopia showed a methylated cytosine upstream the CGI-1 not present in other embryos, an embryo with left anophthalmia presented two methylated cytosines in the CGI-1, whereas an embryo with left anophthalmia and right microphthalmia showed two methylated cytosines in the CGI-2. Normal embryos did not show methylated cytosines in the CGI-1, but one of them showed one methylcytosine in the CGI-2. Methylated transcription factor-binding sites may affect Pax6 expression associated to the cellular response to environmental compounds and hypoxia, signal transduction, cell cycle, lens physiology and development, as well as the transcription rate. Although preliminary, these results suggest that embryos with ocular malformations present unique DNA methylation patterns in the putative promoter of the Pax6 gene in L. olivacea, and probably those subtle, random changes in the methylation status can cause (at least in part) the aberrant phenotypes observed in these embryos.

A new approach for measuring temperature inside turtle eggs.

For turtles, the thermal environment experienced during development plays critical roles in many biological processes. While the temperature inside an egg is assumed to match the substrate temperature, many factors such as evaporative cooling, metabolic heating and the insulating properties of extra-embryonic components can lead to thermal differences. However, no method developed to date has allowed for measurement of the embryonic temperature in live chelonian eggs. We designed a thermocouple-based technique to measure embryonic temperature, achieving 94% survival in Trachemys scripta This methodology may be applicable to other reptile species. We found that, while the temperature in the substrate adjacent to the eggshell accurately reflects the internal egg temperature, it differs from air temperature (∼2°C) in a moisture-dependent manner. Our results demonstrate that external egg temperature, but not air temperature, is suitable for assessing the effects of temperature on biological processes, which could be critical when considering that processes such as temperature-dependent sex determination in turtles occurs within a 4°C window.

Hydric environmental effects on turtle development and sex ratio.

Experimental and field studies of different turtle species suggest that moisture influences embryonic development and sex ratios, wetter substrates tend to produce more males, and drier substrates produce more females. In this study, we used Trachemys scripta elegans to test the effect of moisture on embryonic development and sex ratios. T. s. elegans eggs were incubated under different temperature and moisture regimes. We monitored embryonic development until stage 22 (after sex determination) and, for the first time, we estimated sex ratios using a male-specific transcriptional molecular marker, Sox9. Among treatments, we found differences in developmental rates, egg mass, and sex ratio. Embryos developed slowly in cooler and wetter sand substrate while water uptake by the eggs was significantly greater on wetter substrates. Developmental differences were due to moisture interacting with temperature where increased water content of the sand resulted in temperatures that were 2-3°C lower than air temperatures. The coolest and the wettest substrates produced 100% males compared to 42% males from the warmest and driest treatment. Further, we found that embryonic growth appears to be more sensitive to temperature at earlier stages of development and to moisture at later stages. This study shows how moisture may change the incubation conditions inside nests by changing the temperature experienced by eggs, which affects development, growth and sex ratios. The results of this study highlight the importance of including moisture conditions when predicting embryo growth and sex ratios and in developing proxies of embryonic development.

Molecular and Genomic Characterization of Vibrio mimicus Isolated from a Frozen Shrimp Processing Facility in Mexico.

Vibrio mimicus is a gram-negative bacterium responsible for diseases in humans. Three strains of V. mimicus identified as V. mimicus 87, V. mimicus 92 and V. mimicus 93 were isolated from a shrimp processing facility in Guaymas, Sonora, Mexico. The strains were analyzed using several molecular techniques and according to the cluster analysis they were different, their similarities ranged between 51.3% and 71.6%. ERIC-PCR and RAPD (vmh390R) were the most discriminatory molecular techniques for the differentiation of these strains. The complete genomes of two strains (V. mimicus 87, renamed as CAIM 1882, and V. mimicus 92, renamed as CAIM 1883) were sequenced. The sizes of the genomes were 3.9 Mb in both strains, with 2.8 Mb in ChI and 1.1 Mb in ChII. A 12.7% difference was found in the proteome content (BLAST matrix). Several virulence genes were detected (e.g. capsular polysaccharide, an accessory colonization factor and genes involved in quorum-sensing) which were classified in 16 categories. Variations in the gene content between these genomes were observed, mainly in proteins and virulence genes (e.g., hemagglutinin, mobile elements and membrane proteins). According to these results, both strains were different, even when they came from the same source, giving an insight of the diversity of V. mimicus. The identification of various virulence genes, including a not previously reported V. mimicus gene (acfD) in ChI in all sequenced strains, supports the pathogenic potential of this species. Further analysis will help to fully understand their potential virulence, environmental impact and evolution.

Expression of aromatase in the embryonic brain of the olive ridley sea turtle (Lepidochelys olivacea), and the effect of bisphenol-A in sexually differentiated embryos.

Brain aromatase participates in several biological processes, such as regulation of the reproductive-endocrine axis, memory, stress, sexual differentiation of the nervous system, male sexual behavior, and brain repair. Here we report the isolation and expression of brain aromatase in olive ridley sea turtle (Lepidochelys olivacea) embryos incubated at male- and female-promoting temperatures (MPT and FPT, respectively), at the thermosensitive period (TSP) and the sex-differentiated period. Also, aromatase expression was assessed in differentiated embryos exposed to bisphenol-A (BPA) during the TSP. BPA is a monomer of polycarbonate plastics and is considered an endocrine-disrupting compound. Normal aromatase expression was measured in both forebrain and hindbrain, showing higher expression levels in the forebrain of differentiated embryos at both incubation temperatures. Although no significant differences were detected in the hindbrain, expression was slightly higher at MPT. BPA did not affect aromatase expression neither in forebrains or hindbrains from embryos incubated at MPT, whereas at FPT an inverted U-shape curve was observed in forebrains with significant differences at lower concentrations, whereas in hindbrains a non-significant increment was observed at higher concentrations. Our data indicate that both incubation temperature and developmental stage are critical factors affecting aromatase expression in the forebrain. Because of the timing and location of aromatase expression in the brain, we suggest that brain aromatase may participate in the imprinting of sexual trends related to reproduction and sexual behavior at the onset of sex differentiation, and BPA exposure may impair aromatase function in the female forebrain.

Draft Genome Sequence of Vibrio mimicus Strain CAIM 602T.

Vibrio mimicus is a Gram-negative bacterium associated with gastrointestinal diseases in humans around the world. We report the complete genome sequence of the Vibrio mimicus strain CAIM 602(T) (CDC1721-77, LMG 7896(T), ATCC 33653(T)).

RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle.

The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination.

Post-transcriptional gene silencing by RNA interference in non-mammalian vertebrate systems: where do we stand?

RNA interference (RNAi), the process by which double stranded RNA induces the silencing of endogenous genes through the degradation of its correspondent messenger RNA, has been used for post-transcriptional gene silencing allowing scientists to better understand gene function, becoming a powerful tool in reverse genetics for in vivo and in vitro systems. Successful results in vivo have been obtained from invertebrate animal models, whereas vertebrate systems have been limited primarily to mammalian models and cell lines. Nevertheless, exciting results have also been reported from non-mammalian vertebrate models, such as the knock-down of endogenous genes in Xenopus tadpoles by a construct containing both a Xenopus-specific shRNA sequence and the human Ago2 (which is a key enzyme in the RNAi silencing complex), or the design of a novel vector expressing a miRNA driven by a tissue-specific promoter in zebrafish, and the use of an avian retroviral vector to deliver miRNA and shRNA in chicken embryos proving to be effective in knocking-down endogenous genes with a long lasting effect, to mention some examples. Whether dsRNA is able to initiate a specific RNAi response, or all the factors required for RNAi are present in non-mammalian vertebrates, are still questions which remain to be answered. Further progress in understanding natural RNAi mechanisms in non-mammalian vertebrates will help scientists to overcome difficulties and improve this gene silencing technology. There is no doubt that in few years RNAi silencing approaches will become the tool of choice to knock-down genes in all groups of non-mammalian vertebrates, fulfilling different purposes, from basic research to animal therapeutics and drug discovery.

Hox gene expression in the embryonic genital system of the sea turtle Lepidochelys olivacea (Eschscholt, 1829), a species with temperature-dependent sex determination.

Hox genes are conserved transcription factors which regulate embryonic morphogenesis and differentiation. For the first time, we examined the quantitative and spatial expression of two Hox 5' genes, HoxD11 and HoxA13, in the developing genital system of the olive ridley Lepidochelys olivacea, a species with temperature-dependent sex determination. Quantitative and spatial expression patterns of both genes suggest a role in the female pathway rather than the male pathway. For instance, both genes, especially HoxA13, were expressed in the undifferentiated gonad during the thermosensitive period at a female promoting temperature, and downregulated in the differentiated gonad. By contrast, expression of both genes was low in gonads incubated at a male promoting temperature and did not change significantly in the differentiated gonad. Furthermore, we found high expression levels of HoxA13 in the paramesonephric duct at the male promoting temperature but not at the female promoting temperature, suggesting a role for this Hox gene in the partial regression of the Müllerian duct in males.

Vitellogenin in black turtle (Chelonia mydas agassizii): purification, partial characterization, and validation of an enzyme-linked immunosorbent assay for its detection.

Black turtle plasmatic vitellogenin (VTG) was purified from 17beta-estradiol-induced males using ion-exchange chromatography. The isolated protein was identified as VTG by its glycolipoprotein nature and amino acid sequence homology with other vertebrate VTG. It was characterized as a 500-kDa dimer composed of two identical, 200- to 240-kDa monomers. Polyclonal antibodies raised against black turtle VTG showed high titer and specificity, as demonstrated by enzyme-linked immunosorbent assay and Western blot analysis, respectively. The range of the assay was estimated to be between 15 ng/ml and 2 microg/ml, and the inter- and intra-assay coefficients of variation were 9.4 and 7.3%, respectively. Black turtle antibody cross-reacted with VTG of two other sea turtle species, Caretta caretta (loggerhead) and Eretmochelys imbricata (hawksbill), extending the applicability of the assay as part of a sea turtle health assessment program.