How incubation temperature affects hatchling performance in reptiles: an integrative insight based on plasticity in metabolic enzyme.

Evaluating the effects of temperature variations on animals plays an important role in understanding the threat of climate warming. The effects of developmental temperature on offspring performance are critical in evaluating the effects of warming temperatures on the fitness of oviparous species, but the physiological and biochemical basis of this developmental plasticity is largely unknown. In this study, we incubated eggs of the turtle Pelodiscus sinensis at low (24 °C), medium (28 °C), and high (32 °C) temperatures, and evaluated the effects of developmental temperature on offspring fitness, and metabolic enzymes in the neck and limb muscles of hatchlings. The hatchlings from eggs incubated at the medium temperature showed better fitness-related performance (righting response and swimming capacity) and higher activities of metabolic enzymes (hexokinase, HK; lactate dehydrogenase, LDH) than hatchlings from the eggs incubated at high or low temperatures. In addition, the swimming speed and righting response were significantly correlated with the HK activities in limb (swimming speed) and neck (righting response) muscles, suggesting that the developmental plasticity of energy metabolic pathway might play a role in determining the way incubation temperature affects offspring phenotypes. Integrating the fitness-related performance and the activities of metabolic enzymes, we predict that the P. sinensis from high latitude would not face the detrimental effects of climate warming until the average nest temperatures reach 32 °C.

Collective effects of rising average temperatures and heat events on oviparous embryos.

Survival of the immobile embryo in response to rising temperature is important to determine a species' vulnerability to climate change. However, the collective effects of 2 key thermal characteristics associated with climate change (i.e., rising average temperature and acute heat events) on embryonic survival remain largely unexplored. We used empirical measurements and niche modeling to investigate how chronic and acute heat stress independently and collectively influence the embryonic survival of lizards across latitudes. We collected and bred lizards from 5 latitudes and incubated their eggs across a range of temperatures to quantify population-specific responses to chronic and acute heat stress. Using an embryonic development model parameterized with measured embryonic heat tolerances, we further identified a collective impact of embryonic chronic and acute heat tolerances on embryonic survival. We also incorporated embryonic chronic and acute heat tolerance in hybrid species distribution models to determine species' range shifts under climate change. Embryos' tolerance of chronic heat (T-chronic) remained consistent across latitudes, whereas their tolerance of acute heat (T-acute) was higher at high latitudes than at low latitudes. Tolerance of acute heat exerted a more pronounced influence than tolerance of chronic heat. In species distribution models, climate change led to the most significant habitat loss for each population and species in its low-latitude distribution. Consequently, habitat for populations across all latitudes will shift toward high latitudes. Our study also highlights the importance of considering embryonic survival under chronic and acute heat stresses to predict species' vulnerability to climate change.

Efectos colectivos del aumento de las temperaturas promedio y los eventos de calor en embriones ovíparos Resumen La supervivencia de los embriones inmóviles en respuesta al incremento de temperatura es importante para determinar la vulnerabilidad de las especies al cambio climático. Sin embargo, los efectos colectivos de dos características térmicas claves asociadas con el cambio climático (i. e., aumento de temperatura promedio y eventos de calor agudo) sobre la supervivencia embrionaria permanecen en gran parte inexplorados. Utilizamos mediciones empíricas y modelos de nicho para investigar cómo el estrés térmico crónico y agudo influye de forma independiente y colectiva en la supervivencia embrionaria de los lagartos en todas las latitudes. Recolectamos y criamos lagartos de cinco latitudes e incubamos sus huevos en un rango de temperaturas para cuantificar las respuestas específicas de la población al estrés por calor crónico y agudo. Posteriormente, mediante un modelo de desarrollo embrionario parametrizado con mediciones de tolerancia embrionaria al calor, identificamos un impacto colectivo de las tolerancias embrionarias al calor agudo y crónico en la supervivencia embrionaria. También incorporamos la tolerancia embrionaria crónica y aguda al calor en modelos de distribución de especies híbridas para determinar los cambios de distribución de las especies bajo el cambio climático. La tolerancia embrionaria al calor crónico (T­crónico) permaneció constante, mientras que la tolerancia al calor agudo (T­agudo) fue mayor en latitudes altas que en latitudes bajas. La tolerancia al calor agudo ejerció una influencia más pronunciada que la tolerancia al calor crónico. En los modelos de distribución de especies, el cambio climático provocó la pérdida de hábitat más significativa para cada población y especie en su distribución de latitudes bajas. En consecuencia, el hábitat para poblaciones en todas las latitudes se desplazará a latitudes altas. Nuestro estudio también resalta la importancia de considerar la supervivencia embrionaria bajo estrés térmico crónico y agudo para predecir la vulnerabilidad de las especies al cambio climático.

Genetically Encoded Lizard Color Divergence for Camouflage and Thermoregulation.

Local adaptation is critical in speciation and evolution, yet comprehensive studies on proximate and ultimate causes of local adaptation are generally scarce. Here, we integrated field ecological experiments, genome sequencing, and genetic verification to demonstrate both driving forces and molecular mechanisms governing local adaptation of body coloration in a lizard from the Qinghai-Tibet Plateau. We found dark lizards from the cold meadow population had lower spectrum reflectance but higher melanin contents than light counterparts from the warm dune population. Additionally, the colorations of both dark and light lizards facilitated the camouflage and thermoregulation in their respective microhabitat simultaneously. More importantly, by genome resequencing analysis, we detected a novel mutation in Tyrp1 that underpinned this color adaptation. The allele frequencies at the site of SNP 459# in the gene of Tyrp1 are 22.22% G/C and 77.78% C/C in dark lizards and 100% G/G in light lizards. Model-predicted structure and catalytic activity showed that this mutation increased structure flexibility and catalytic activity in enzyme TYRP1, and thereby facilitated the generation of eumelanin in dark lizards. The function of the mutation in Tyrp1 was further verified by more melanin contents and darker coloration detected in the zebrafish injected with the genotype of Tyrp1 from dark lizards. Therefore, our study demonstrates that a novel mutation of a major melanin-generating gene underpins skin color variation co-selected by camouflage and thermoregulation in a lizard. The resulting strong selection may reinforce adaptive genetic divergence and enable the persistence of adjacent populations with distinct body coloration.

Can nesting behaviour allow reptiles to adapt to climate change?

A range of abiotic parameters within a reptile nest influence the viability and attributes (including sex, behaviour and body size) of hatchlings that emerge from that nest. As a result of that sensitivity, a reproducing female can manipulate the phenotypic attributes of her offspring by laying her eggs at times and in places that provide specific conditions. Nesting reptiles shift their behaviour in terms of timing of oviposition, nest location and depth of eggs beneath the soil surface across spatial and temporal gradients. Those maternal manipulations affect mean values and variances of both temperature and soil moisture, and may modify the vulnerability of embryos to threats such as predation and parasitism. By altering thermal and hydric conditions in reptile nests, climate change has the potential to dramatically modify the developmental trajectories and survival rates of embryos, and the phenotypes of hatchlings. Reproducing females buffer such effects by modifying the timing, location and structure of nests in ways that enhance offspring viability. Nonetheless, our understanding of nesting behaviours in response to climate change remains limited in reptiles. Priority topics for future studies include documenting climate-induced changes in the nest environment, the degree to which maternal behavioural shifts can mitigate climate-related deleterious impacts on offspring development, and ecological and evolutionary consequences of maternal nesting responses to climate change. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

A reciprocal egg-swap experiment reveals sources of variation in developmental success among populations of a desert lizard.

Identifying intrinsic and extrinsic sources of variation in life history traits among populations has been well-studied at the post-embryonic stage but rarely at the embryonic stage. To reveal these sources of variation in the developmental success of embryos, we measured the physical characteristics of nest environments and conducted reciprocal egg-swap experiments in two populations of the toad-headed agamid lizard (Phrynocephalus przewalskii), isolated from each other by a mountain range. We determined the effects of population origin and nest environment on embryonic and offspring traits related to developmental success, including incubation period, hatching success, and offspring growth and survival. Females from the northern population constructed deeper nests that were colder and wetter than those from the southern population. Northern embryos had higher hatching success than the southern embryos when incubated at the northern nest environment, but not when they were incubated at the southern nest environment. The southern hatchlings grew faster than the northern hatchlings when incubated at the southern nest environment, but not after incubation at the northern nest environment. These phenomena likely reflect local adaptation of embryonic development to their nest environments among populations in lizards. In addition, the southern hatchlings had higher survivorship than the northern hatchlings regardless of nest environment, suggesting the southern population has evolved a superior phenotype at the hatchling stage to maximize its fitness.

The thermal ecology and physiology of reptiles and amphibians: A user's guide.

Research on the thermal ecology and physiology of free-living organisms is accelerating as scientists and managers recognize the urgency of the global biodiversity crisis brought on by climate change. As ectotherms, temperature fundamentally affects most aspects of the lives of amphibians and reptiles, making them excellent models for studying how animals are impacted by changing temperatures. As research on this group of organisms accelerates, it is essential to maintain consistent and optimal methodology so that results can be compared across groups and over time. This review addresses the utility of reptiles and amphibians as model organisms for thermal studies by reviewing the best practices for research on their thermal ecology and physiology, and by highlighting key studies that have advanced the field with new and improved methods. We end by presenting several areas where reptiles and amphibians show great promise for further advancing our understanding of how temperature relations between organisms and their environments are impacted by global climate change.

Heat tolerance of reptile embryos: Current knowledge, methodological considerations, and future directions.

Aspects of global change result in warming temperatures that threaten biodiversity across the planet. Eggs of non-avian, oviparous reptiles (henceforth "reptiles") are particularly vulnerable to warming due to a lack of parental care during incubation and limited ability to behaviorally thermoregulate. Because warming temperatures will cause increases in both mean and variance of nest temperatures, it is crucial to consider embryo responses to both chronic and acute heat stress. Although many studies have considered embryo survival across constant incubation temperatures (i.e., chronic stress) and in response to brief exposure to extreme temperatures (i.e., acute stress), there are no standard metrics or terminology for determining heat stress of embryos. This impedes comparisons across studies and species and hinders our ability to predict how species will respond to global change. In this review, we compare various methods that have been used to assess embryonic heat tolerance in reptiles and provide new terminology and metrics for quantifying embryo responses to both chronic and acute heat stress. We apply these recommendations to data from the literature to assess chronic heat tolerance in 16 squamates, 16 turtles, five crocodilians, and the tuatara and acute heat tolerance for nine squamates and one turtle. Our results indicate that there is relatively large variation in chronic and acute heat tolerance across species, and we outline directions for future research, calling for more studies that assess embryo responses to acute thermal stress, integrate embryo responses to chronic and acute temperatures in predictive models, and identify mechanisms that determine heat tolerance.

Captivity Influences Gut Microbiota in Crocodile Lizards (Shinisaurus crocodilurus).

Captivity is an important measure for conservation of an endangered species, and it is becoming a hot topic in conservation biology, which integrates gut microbiota and endangered species management in captivity. As an ancient reptile, the crocodile lizard (Shinisaurus crocodilurus) is facing extreme danger of extinction, resulting in great significance to species conservation in the reserve. Thus, it is critical to understand the differences in gut microbiota composition between captive and wild populations, as it could provide fundamental information for conservative management of crocodile lizards. Here, fecal samples of crocodile lizards were collected from two wild and one captive populations with different ages (i.e., juveniles and adults) and were analyzed for microbiota composition by 16S ribosomal RNA (rRNA) gene amplicon sequencing. This study showed that the lizard gut microbiota was mainly composed of Firmicutes and Proteobacteria. The gut microbiota composition of crocodile lizard did not differ between juveniles and adults, as well as between two wild populations. Interestingly, captivity increased community richness and influenced community structures of gut microbiota in crocodile lizards, compared with wild congeners. This was indicated by higher abundances of the genera Epulopiscium and Glutamicibacter. These increases might be induced by complex integration of simple food resources or human contact in captivity. The gut microbiota functions of crocodile lizards are primarily enriched in metabolism, environmental information processing, genetic information processing, and cellular processes based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. This study provides fundamental information about the gut microbiota of crocodile lizards in wild and captive populations. In the future, exploring the relationship among diet, gut microbiota, and host health is necessary for providing animal conservation strategies.

Adaptive responses of the embryos of birds and reptiles to spatial and temporal variations in nest temperatures.

Natural nests of egg-laying birds and reptiles exhibit substantial thermal variation, at a range of spatial and temporal scales. Rates and trajectories of embryonic development are highly sensitive to temperature, favouring an ability of embryos to respond adaptively (i.e. match their developmental biology to local thermal regimes). Spatially, thermal variation can be significant within a single nest (top to bottom), among adjacent nests (as a function of shading, nest depth etc.), across populations that inhabit areas with different weather conditions, and across species that differ in climates occupied and/or nest characteristics. Thermal regimes also vary temporally, in ways that generate differences among nests within a single population (e.g. due to seasonal timing of laying), among populations and across species. Anthropogenic activities (e.g. habitat clearing, climate change) add to this spatial and temporal diversity in thermal regimes. We review published literature on embryonic adaptations to spatio-temporal heterogeneity in nest temperatures. Although relatively few taxa have been studied in detail, and proximate mechanisms remain unclear, our review identifies many cases in which natural selection appears to have fine-tuned embryogenesis to match local thermal regimes. Developmental rates have been reported to differ between uppermost versus lower eggs within a single nest, between eggs laid early versus late in the season, and between populations from cooler versus warmer climates. We identify gaps in our understanding of thermal adaptations of early (embryonic) phases of the life history, and suggest fruitful opportunities for future research.

The Embryos of Turtles Can Influence Their Own Sexual Destinies.

Sessile organisms with thermally sensitive developmental trajectories are at high risk from climate change. For example, oviparous reptiles with temperature-dependent sex determination (TSD) may experience strong (potentially disastrous) shifts in offspring sex ratio if reproducing females are unable to predict incubation conditions at the time of oviposition. How then have TSD reptile taxa persisted over previous periods of extreme climatic conditions? An ability of embryos to move within the egg to select optimal thermal regimes could buffer ambient extremes, but the feasibility of behavioral thermoregulation by embryos has come under strong challenge. To test this idea, we measured thermal gradients within eggs in semi-natural nests of a freshwater turtle species with TSD, manipulated embryonic thermoregulatory ability, and modeled the effects of embryonic thermoregulation on offspring sex ratios. Behavioral thermoregulation by embryos accelerated development and influenced offspring sex ratio, expanding the range of ambient conditions under which nests produce equal numbers of male and female offspring. Model projections suggest that sex ratio shifts induced by global warming will be buffered by the ability of embryos to influence their sexual destiny via behavioral thermoregulation.

Anticipatory parental effects in a subtropical lizard in response to experimental warming.

Parental effects may produce adaptive or maladaptive plasticity that either facilitates persistence or increases the extinction risk of species and populations in a changing climate. However, empirical evidence of transgenerational adaptive plastic responses to climate change is still scarce. Here we conducted thermal manipulation experiments with a factorial design in a Chinese lacertid lizard (Takydromus septentrionalis) to identify the fitness consequences of parental effects in response to climate warming. Compared to present climate conditions, a simulated warming climate significantly advanced the timing of oviposition, depressed the immune capability of post-partum females, and decreased the hatching success of embryos, but did not affect female reproductive output (clutch size and egg mass). These results indicate that maternal warming negatively affects female health, and embryonic hatchability. More interestingly, we found that offspring from parents exposed to warming environments survived well under a simulated warming climate, but not under a present climate scenario. Accordingly, our study demonstrates anticipatory parental effects in response to a warming climate in an ectothermic vertebrate. However, the fitness consequences of this parental effect will depend on future climate change scenarios.

The vulnerability of developing embryos to simulated climate warming differs between sympatric desert lizards.

The vulnerability of species to climate warming varies along latitudinal and elevational clines, but how sympatric species vary in vulnerability to climate warming remains largely unknown. We experimentally simulated nest temperatures of two sympatric lizards with divergent microhabitat preferences (Phrynocephalus przewalskii and Eremias argus), under climate warming senarios, to determine the response of embryos to increased mean temperatures and heat waves. Our study demonstrated that simulated climate warming reduced hatching success and hatchling size and growth in E. argus (that prefers closed microhabitats), but had less effect in P. przewalskii (that occupies open microhabitats). The reduced growth rate of E. argus hatchlings was associated with a decrease in metabolic rate, which was more evident in hatchling E. argus than in P. przewalskii. Our results suggest lizards that prefer closed microhabitats may be more vulnerable to climate warming than those that prefer open microhabitats; further studies are needed to test this hypothesis. More generally, the divergent responses of sympatric species to climate warming highlights the importance of distinguishing the thermal sensitivity of behavior and physiology for each species of a community, in order to make predictions about the impacts of climate warming at regional scales.

Phenotypic plasticity may help lizards cope with increasingly variable temperatures.

Temperature variability is predicted to increase in the coming century due to climate change. However, the biological impact of increased temperature variability on animals remains largely unexplored. Here, we experimentally exposed gravid viviparous lizards (Eremias multiocellata) to two thermal environments [constant daily maximum (CDM) versus variable daily maximum (VDM) treatment with the same average temperature] to address maternal and offspring responses to increased variability in ambient temperature. Females from the VDM treatment delayed parturition, but produced similar litter sizes and litter masses as did CDM females. Offspring from the VDM treatment selected higher body temperatures, had higher metabolic rates and higher growth rates, and grew to a similar size as those from the CDM treatment despite having a shorter growth period prior to hibernation. Therefore, phenotypic plasticity may be critical for lizards to respond effectively to climate change, and its role in responding to increasingly variable temperatures warrants further attention.

[Clinical characteristics and risk factors affecting outcomes of elderly patients with non-small cell lung cancer complicated by chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the clinical features, treatment strategy and risk factors affecting the prognosis of elderly patients with non-small cell lung cancer (NSCLC) complicated by chronic obstructive pulmonary disease (COPD). METHODS: We retrospectively analyzed the data of elderly patietns (>60 years) with newly diagnosed NSCLC complicated by COPD at the Geriatric Institution of General Hospital of PLA between January, 2000 and June, 2015. The clinical data collected included history of smoking, pulmonary function test results, initial treatments, TNM stage, chief complaints, comorbidities and laboratory tests. The Cox proportional hazards regression model was used to explore the prognostic factors in these patients. RESULTS: A total of 200 NSCLC patients were reviewed, of which 107 (53.5%) patients had the co-morbidity of COPD as confirmed by spirometry using bronchodilator test. The median survival of the patients with NSCLC complicated by COPD was 45.8 months with 1-, 3-, 5-, and 10-year survival rates of 80.4%, 55.4%, 41.0% and 20.0%, respectively. Stratification analysis showed that patients with COPD Gold grades 1 and 2 had a significant longer median overall survival (51.7 and 43.1 months, respectively) than those with grade 3/4 (16.9 months; P=0.020 and 0.043, respectively). Univariate and multivariate analyses using Cox proportional hazards regression model showed that an older age, a higher Gold grade, advanced disease stage (stages III and IV), squamous cell carcinoma, nonsurgical initial treatment, coughing and an elevated serum CEA level were independent risk factors for shorter survival of the patients. CONCLUSION: Multiple prognostic factors can affect the outcomes of elderly patients with NSCLC complicated by COPD, and a higher COPD Gold grade that fails to respond to treatment within 3 months is the independent risk factor for survival of the patients.

A propensity-matched analysis of surgery and stereotactic body radiotherapy for early stage non-small cell lung cancer in the elderly.

Elderly patients with early stage non-small cell lung cancer (NSCLC) who undergo surgical resection are at a high risk of treatment-related complications. Stereotactic body radiation therapy (SBRT) is considered an alternative treatment option with a favorable safety profile. Given that prospective comparative data on SBRT and surgical treatments are limited, we compared the 2 treatments for early stage NSCLC in the elderly.We retrospectively collected information from the database at our geriatric institution on patients with clinical stage IA/B NSCLC who were treated with surgery or SBRT. The patients were matched using a propensity score based on gender, age, T stage, tumor location, pulmonary function (forced expiratory volume in 1 second [FEV1]% and FEV1), Charlson comorbidity score, and World Health Organization performance score. We compared locoregional control rate, recurrence-free survival (RFS), overall survival (OS), and cancer-specific survival (CSS) between the 2 treatment cohorts before and after propensity score matching.A total of 106 patients underwent surgery, and 74 received SBRT. Surgical patients were significantly younger (72.6 ±â€Š7.9 vs 82.6 ±â€Š4.1 years, P = 0.000), with a significantly higher rate of adenocarcinoma (P = 0.000), better Eastern Cooperative Oncology Group performance scores (P = 0.039), and better pulmonary function test results (P = 0.034 for predicted FEV1 and P = 0.032 for FEV1). In an unmatched comparison, there were significant differences in locoregional control (P = 0.0012) and RFS (P < 0.001). The 5-year OS was 69% in patients who underwent surgery and 44.6% in patients who underwent SBRT (P = 0.0007). The 5-year CSS was 73.9% in the surgery group and 57.5% in the SBRT group (P = 0.0029). Thirty-five inoperable or marginally operable surgical patients and 35 patients who underwent SBRT were matched to their outcomes in a blinded manner (1:1 ratio, caliper distance = 0.25). In this matched comparison, the follow-up period of this subgroup ranged from 4.2 to 138.1 months, with a median of 58.7 months. Surgery was associated with significantly better locoregional control (P = 0.0191) and RFS (P = 0.0178), whereas no significant differences were found in OS (5-year OS, 67.8% for surgery vs 47.4% for SBRT, P = 0.07) or CSS (67.8% for surgery vs 58.2% for SBRT, P = 0.1816).This retrospective analysis found superior locoregional control rates and RFS after surgery compared with SBRT, but there were no differences in OS or CSS. SBRT is an alternative treatment option to surgery in elderly NSCLC patients who cannot tolerate surgical resection because of medical comorbidities. Our findings support the need to compare the 2 treatments in randomized controlled trials.

The effects of light exposure during incubation on embryonic development and hatchling traits in lizards.

Light is an environmental factor that is known to profoundly affect embryonic development in some oviparous vertebrates, but such effects are unstudied in reptiles. We investigated the light sensitivity of lizard embryos by examining the thickness and light transmittance of eggshells as well as the effect of light on embryonic development and hatchling traits in four lizard species, the Chinese skink (Plestiodon chinensis), the northern grass lizard (Takydromus septentrionalis), the oriental leaf-toed gecko (Hemidactylus bowringii) and the Japanese gecko (Gekko japonicus). The eggshells were thinner and thus had higher light transmittance in Chinese skink than the other three species. Light exposure during incubation significantly accelerated the embryonic development in all species, with higher light intensity resulting in faster embryonic development. Interestingly, light stimulation negatively influenced hatchling size and survival in skinks, but had no effect in lacertids and geckos. This interspecific discrepancy not only relates to the differences in thickness and light transmittance of eggshells, but might also reflect the differences in the reproductive habits of these species. Given the diversity of light conditions that reptile embryos face during development, studies on the response of reptile embryos to light may offer a unique opportunity to understand the mechanisms of embryonic light sensitivity in animals.

Thyroid hormone modulates offspring sex ratio in a turtle with temperature-dependent sex determination.

The adaptive significance of temperature-dependent sex determination (TSD) has attracted a great deal of research, but the underlying mechanisms by which temperature determines the sex of a developing embryo remain poorly understood. Here, we manipulated the level of a thyroid hormone (TH), triiodothyronine (T3), during embryonic development (by adding excess T3 to the eggs of the red-eared slider turtle Trachemys scripta, a reptile with TSD), to test two competing hypotheses on the proximate basis for TSD: the developmental rate hypothesis versus the hormone hypothesis Exogenous TH accelerated embryonic heart rate (and hence metabolic rate), developmental rate, and rates of early post-hatching growth. More importantly, hyperthyroid conditions depressed expression of Cyp19a1 (the gene encoding for aromatase) and levels of oestradiol, and induced more male offspring. This result is contrary to the direction of sex-ratio shift predicted by the developmental rate hypothesis, but consistent with that predicted by the hormone hypothesis Our results suggest an important role for THs in regulating sex steroid hormones, and therefore, in affecting gonadal sex differentiation in TSD reptiles. Our study has implications for the conservation of TSD reptiles in the context of global change because environmental contaminants may disrupt the activity of THs, and thereby affect offspring sex in TSD reptiles.

Identification potential biomarkers in pulmonary tuberculosis and latent infection based on bioinformatics analysis.

BACKGROUND: The study aimed to identify the potential biomarkers in pulmonary tuberculosis (TB) and TB latent infection based on bioinformatics analysis. METHODS: The microarray data of GSE57736 were downloaded from Gene Expression Omnibus database. A total of 7 pulmonary TB and 8 latent infection samples were used to identify the differentially expressed genes (DEGs). The protein-protein interaction (PPI) network was constructed by Cytoscape software. Then network-based neighborhood scoring analysis was performed to identify the important genes. Furthermore, the functional enrichment analysis, correlation analysis and logistic regression analysis for the identified important genes were performed. RESULTS: A total of 1084 DEGs were identified, including 565 down- and 519 up-regulated genes. The PPI network was constructed with 446 nodes and 768 edges. Down-regulated genes RIC8 guanine nucleotide exchange factor A (RIC8A), basic leucine zipper transcription factor, ATF-like (BATF) and microtubule associated monooxygenase, calponin LIM domain containing 1 (MICAL1) and up-regulated genes ATPase, Na+/K+ transporting, alpha 4 polypeptide (ATP1A4), histone cluster 1, H3c (HIST1H3C), histone cluster 2, H3d (HIST2H3D), histone cluster 1, H3e (HIST1H3E) and tyrosine kinase 2 (TYK2) were selected as important genes in network-based neighborhood scoring analysis. The functional enrichment analysis results showed that these important DEGs were mainly enriched in regulation of osteoblast differentiation and nucleoside triphosphate biosynthetic process. The gene pairs RIC8A-ATP1A4, HIST1H3C-HIST2H3D, HIST1H3E-BATF and MICAL1-TYK2 were identified with high positive correlations. Besides, these genes were selected as significant feature genes in logistic regression analysis. CONCLUSIONS: The genes such as RIC8A, ATP1A4, HIST1H3C, HIST2H3D, HIST1H3E, BATF, MICAL1 and TYK2 may be potential biomarkers in pulmonary TB or TB latent infection.

High incubation temperatures enhance mitochondrial energy metabolism in reptile embryos.

Developmental rate increases exponentially with increasing temperature in ectothermic animals, but the biochemical basis underlying this thermal dependence is largely unexplored. We measured mitochondrial respiration and metabolic enzyme activities of turtle embryos (Pelodiscus sinensis) incubated at different temperatures to identify the metabolic basis of the rapid development occurring at high temperatures in reptile embryos. Developmental rate increased with increasing incubation temperatures in the embryos of P. sinensis. Correspondingly, in addition to the thermal dependence of mitochondrial respiration and metabolic enzyme activities, high-temperature incubation further enhanced mitochondrial respiration and COX activities in the embryos. This suggests that embryos may adjust mitochondrial respiration and metabolic enzyme activities in response to developmental temperature to achieve high developmental rates at high temperatures. Our study highlights the importance of biochemical investigations in understanding the proximate mechanisms by which temperature affects embryonic development.

Temperature-dependent sex determination ruled out in the Chinese soft-shelled turtle (Pelodiscus sinensis) via molecular cytogenetics and incubation experiments across populations.

The sex determination mechanism for the Chinese soft-shelled turtle (Pelodiscus sinensis) is subject to controversy. Some populations have been shown to possess sex chromosomes and thus genotypic sex determination (GSD), while others were reported to exhibit temperature-dependent sex determination (TSD). To test whether TSD and GSD coexist in this species or whether populations differ in their sex-determining system, we conducted egg incubation experiments to investigate how temperature influences hatchling sex in a wide range of populations of this species in China. In parallel, we used comparative genome hybridization (CGH) to study the micro-sex chromosomes of adult P. sinensis in the 2 populations that were previously identified to be TSD. The incubation experiments showed that temperature did not affect hatchling sex in any of the studied populations. CGH indicated that turtles have micro-sex chromosomes of the female heterogametic (ZZ/ZW) system in the 2 disputed populations. These results indicate that P. sinensis is a GSD rather than a TSD species. Thus, the apparent coexistence of TSD and GSD in this species is the result of previous misdiagnosis in purportedly TSD populations.