Blood cells and hematological parameters of Chiala Mountain Salamander, Batrachuperus karlschmidti (Urodela, Hynobiidae).

Hematological parameters are essential indices for assessing the function of blood and reflecting not only the health status of animal but also their physiological adaptation to the environment. Herein, the composition of blood cells and the hematological parameters of wild Batrachuperus karlschmidti were examined for the first time, and the effects of sex, body size, body mass, and age on the hematological parameters were explored. The morphology and morphometric data of the blood cells, as well as the hematological parameters, of B. karlschmidti were slightly differ from those of its congener. However, hematological differences between sexes were only found in erythrocyte and leukocyte count, and mean cell volume (MCV), which possibly reflecting the need for better oxygen distribution and stronger immune protection for reproduction. Hematocrit (Hct) and mean cell hemoglobin (MCH) were strongly dependent on body mass. These also might have been attributed to higher oxygen requirements with larger body masses. This is a pilot project exploring the hematology of this species that may help establish hematological parameters in future for supporting species protection and monitoring studies, as well as help understanding the physiological adaptation of this species.

Seasonal variations in the composition and diversity of gut microbiota in white-lipped deer (Cervus albirostris).

The gut microbiota has key physiological functions in host adaptation, although little is known about the seasonal changes in the composition and diversity of the gut microbiota in deer. In this study, seasonal variations (grassy and withering season) in the gut microbiota of white-lipped deer (Cervus albirostris), which lives in alpine environments, were explored through 16S rRNA high-throughput sequencing based on sixteen fecal samples collected from Gansu Qilian Mountain National Nature Reserve in China. At the phylum level, Firmicutes, Bacteroidota, and Actinobacteriota dominated the grassy season, while Firmicutes, Proteobacteria, and Actinobacteriota dominated the withering season. At the genus level, Carnobacterium dominated the grassy season, while Arthrobacter and Acinetobacter dominated the withering season. Alpha diversity results (Shannon: P = 0.01, ACE: P = 0.00, Chao1: P = 0.00) indicated that there was a difference in the diversity and richness of the gut microbiota between the two seasons, with higher diversity in the grassy season than in the withering season. Beta diversity results further indicated that there was a significant difference in the community structure between the two seasons (P = 0.001). In summary, the composition, diversity, and community structure of the gut microbiota showed significant seasonal variations, which could be explained by variations in the seasonal food availability, composition, diversity, and nutrition due to phenological alternations. The results of this study indicate that the gut microbiota can adapt to changes in the environment and provide the scientific basis for health assessment of white-lipped deer.

Blood cells and hematological parameters of the mountain dragon, Diploderma micangshanensis (Squamata: Lacertilia: Agamidae).

Hematological characteristics reflect the health status of animals and their physiological adaptation to the environment. However, few studies focused on the species of Diploderma. In this study, the blood cells and the hematological parameters of Diploderma micangshanensis, a species endemic to China, were examined based on 48 healthy adult (32 males and 16 females). The blood cells and hematological parameters of D. micangshanensis were similar to those of other lizard species. Although the values of erythrocyte morphometric characters and hematological parameters varied between males and females, the differences were only significant in the case of the hematocrit and erythrocyte size, which may allow for higher oxygen availability in males. Hemoglobin, mean corpuscular hemoglobin concentration, and mean corpuscle volume were strongly affected by the snout-vent length and/or body mass, which reflect the physiological adaptation to the oxygen requirement of different individuals. This is the first report of hematological data from a species of Diploderma, and the results will provide data for research on the adaptive evolution and health assessment in this species and other congeners.

Toxicity of amine-functionalized single-carbon nanotube (NH2 f-SWCNT) to Channel Catfish (Ietalurus Punetaus): Organ pathologies, oxidative stress, inflammation, and apoptosis.

The rapid development of carbon nanotubes (CNTs) in the field of fish disease control and prevention raises concerns about the toxicity and safe use in fish. This study was performed to assess the effect on histological changes, oxidative stress related markers in response to various concentrations of amine-functionalized single carbon nanotubes (NH2 f-SWCNT) (1, 10 and 100 mg kg-1 fish) in Channel Catfish (Ietalurus Punetaus) for up to 10 days. Moreover, pro-inflammatory cytokine genes and apoptotic genes were analyzed to obtain a better understanding of molecular mechanism of NH2 f-SWCNT induced toxicity. As a result, intraperitoneal (i.p.) administration of NH2 f-SWCNT caused dose-dependent and time-dependent injuries in the sampled tissues. In comparison with the control groups, decrease of catalase (CAT) activity and superoxide dismutase (SOD), and increase of malondialdehyde (MDA) and lactin dehydrogenase (LDH) were observed in all treatments. Real-time PCR assay showed inflammatory response with dose-dependent increase of tumor necrosis factor alpha (TNFα) and transient increase of interleukin 1ß (IL-1ß) in the liver. NH2 f-SWCNT administration induced increase of p38 as well as caspase-3 in all treatments compared to the control groups, indicating the involvement of p38-MAPK cascade and caspase-3 cascade in liver cell apoptosis. Overall, we conclude that NH2 f-SWCNT exert effects by direct injury and indirectly oxidative stress, resulting in inflammation and apoptosis, which provides data for understanding of the biological mechanisms underlying the toxicity of CNTs in fish.

Emerging important roles of circRNAs in human cancer and other diseases.

CircRNAs are a large class of endogenous single-stranded RNA that is different from other linear RNA, which are produced by back-splicing and fusion of either exons, introns, or both exon-intron into covalently closed loops. CircRNAs are found in almost all living organisms and have emerged as potentially important players effecting on all life activities. It was characterized by stable structure, resistant to RNA degradation, highly abundance and conservation and tissue-specific expression. Early circRNAs were ignored as a by-product of meaningless abnormally cut RNA and had little biological function. Currently, circRNAs have become a research hotspot due to its special characteristics. CircRNAs could function as miRNA sponges, interfere with splicing and bind to protein to regulate the expression of parental genes and so on. In recent years, an increasing number of studies have revealed that circRNAs are closely related to a series of physiological and pathological processes. Additionally, circRNAs play an important role in the occurrence and development of a variety of diseases, suggesting circRNAs may be as novel indicators or biomarkers for cancer and other diseases with which they are associated. In this article, we review the biogenesis, biological functions of circRNAs and recent advances in circRNAs research in human diseases. Results will provide new insights on the roles and new ideas of circRNAs for the diagnosis and treatment of diseases and possible directions and approach for future circRNA applications.

Mitochondrial genome of Diploderma micangshanense and its implications for phylogeny of the genus Diploderma.

The lizard Diploderma micangshanense, which belongs to the family Agamidae is endemic to China. Here, we determined the complete mitogenome of D. micangshanense using an Illumina Hiseq X Ten sequencer. This mitogenome's structure is a typical circular molecule of 16,467 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region. The overall base composition of D. micangshanensis is 34.1% A, 23.64% T, 13.62% C, and 28.64% G with a slight AT bias of 57.74%. Most mitochondrial genes except ND6 and seven tRNAs were encoded on the heavy strand. Notably, the trnP gene was encoded on the heavy strand instead of its typical light strand position, providing an example of gene inversion in vertebrate mitogenomes. Phylogenetic analysis indicated that D. micangshanensis had a close relationship with D. zhaoermii.

A new species of the genus Batrachuperus (Urodela: Hynobiidae) from Southwestern China.

A new stream salamander species, Batrachuperus daochengensis sp. nov., from southwestern China, is described herein based on morphological and molecular evidence. Molecular phylogeny derived from the mitochondrial gene together with previous nuclear data revealed that B. daochengensis sp. nov. is sister to B. yenyuanensis. The new species differs from all other species of the genus by the following combination of characters: brown horny epidermis on tips of fingers and toes absent; tubercles on palms and soles absent; costal grooves 12; dorsal brown, mottled with blackish spots; fingers 2-3-4-1 in order of decreasing length; tips of longest digits of fore- and hindlimbs largely separated by one to two costal spaces when adpressed towards each other along sides of body. The new species is currently known in the central and southern Shaluli Mountains in southwestern China.

Molecular Insights of Copper Sulfate Exposure-Induced Nephrotoxicity: Involvement of Oxidative and Endoplasmic Reticulum Stress Pathways.

The precise pathogenic mechanism in Cu exposure-cause nephrotoxicity remains unclear. This study investigated the underlying molecular mechanism of copper sulfate (CuSO4)-induced nephrotoxicity. Mice were treated with CuSO4 at 50, 100, 200 mg/kg/day or co-treated with CuSO4 (200 mg/kg/day) and 4-phenylbutyric acid (4-PBA, 100 mg/kg/day) for 28 consecutive days. HEK293 cells were treated with CuSO4 (400 µM) with or without superoxide dismutase, catalase or 4-PBA for 24 h. Results showed that CuSO4 exposure can cause renal dysfunction and tubular necrosis in the kidney tissues of mice. CuSO4 exposure up-regulated the activities and mRNA expression of caspases-9 and -3 as well as the expression of glucose-regulated protein 78 (GRP78), GRP94, DNA damage-inducible gene 153 (GADD153/CHOP), caspase-12 mRNAs in the kidney tissues. Furthermore, superoxide dismutase and catalase pre-treatments partly inhibited CuSO4-induced cytotoxicity by decreasing reactive oxygen species (ROS) production, activities of caspases-9 and -3 and DNA fragmentations in HEK293 cells. 4-PBA co-treatment significantly improved CuSO4-induced cytotoxicity in HEK293 cells and inhibited CuSO4 exposure-induced renal dysfunction and pathology damage in the kidney tissues. In conclusion, our results reveal that oxidative stress and endoplasmic reticulum stress contribute to CuSO4-induced nephrotoxicity. Our study highlights that targeting endoplasmic reticulum and oxidative stress may offer an approach for Cu overload-caused nephrotoxicity.

Nerve Growth Factor Confers Neuroprotection against Colistin-Induced Peripheral Neurotoxicity.

Neurotoxicity is an unwanted side effect for patients when receiving parenteral colistin therapy. The development of effective neuroprotective agents that can be coadministered during colistin therapy remains a priority area in antimicrobial chemotherapy. The present study aimed to investigate the protective effect of nerve growth factor (NGF) against colistin-induced peripheral neurotoxicity using a murine model. C57BL/6 mice were randomly divided into the following 6 groups: (i) untreated control, (ii) NGF alone (36 µg/kg/day administered intraperitoneally), (iii) colistin alone (18 mg/kg/day administered intraperitoneally), and (iv-vi) colistin (18 mg/kg/day) plus NGF (9, 18, and 36 µg/kg/day). After treatment for 7 days, neurobehavioral and electrophysiology changes, histopathological assessments of sciatic nerve damage, and oxidative stress biomarkers were examined. The mRNA expression levels of Nrf2, HO-1, Akt, Bax, and caspase-3 and -9 were assessed using quantitative RT-PCR. The results showed that, across all the groups wherein NGF was coadministered with colistin, a marked attenuation of colistin-induced sciatic nerve damage and improved sensory and motor function were observed. In comparison to the colistin only treatment group, animals that received NGF displayed upregulated Nrf2 and HO-1 mRNA expression levels and downregulated Bax and caspase-3 and -9 mRNA expression levels. In summary, our study reveals that NGF coadministration protects against colistin-induced peripheral neurotoxicity via the activation of Akt and Nrf2/HO-1 pathways and inhibition of oxidative stress. This study highlights the potential clinical application of NGF as a neuroprotective agent for coadministration during colistin therapy.

The First Transcriptome Assembly of Yenyuan Stream Salamander (Batrachuperus yenyuanensis) Provides Novel Insights into Its Molecular Evolution.

The Yenyuan stream salamander (Batrachuperus yenyuanensis) has been previously evaluated with regards to phylogeny, population genetics, and hematology, but genomic information is sparse due to the giant genome size of salamanders which contain highly repetitive sequences, thus resulting in the lack of a complete reference genome. This study evaluates the encoding genetic sequences and provides the first transcriptome assembly of Yenyuan stream salamander based on mixed samples from the liver, spermary, muscle and spleen tissues. Using this transcriptome assembly and available encoding sequences from other vertebrates, the gene families, phylogenetic status, and species divergence time were compared or estimated. A total of 13,750 encoding sequences were successfully obtained from the transcriptome assembly of Yenyuan stream salamander, estimated to contain 40.1% of the unigenes represented in tetrapod databases. A total of 88.79% of these genes could be annotated to a biological function by current databases. Through gene family clustering, we found multiple possible isoforms of the Scribble gene-whose function is related to regeneration-based on sequence similarity. Meanwhile, we constructed a robust phylogenetic tree based on 56 single-copy orthologues, which indicates that based on phylogenetic position, the Yenyuan stream salamander presents the closest relationship with the Chinese giant salamander (Andrias davidianus) of the investigated vertebrates. Based on the fossil-calibrated phylogeny, we estimated that the lineage divergence between the ancestral Yenyuan stream salamander and the Chinese giant salamander may have occurred during the Cretaceous period (~78.4 million years ago). In conclusion, this study not only provides a candidate gene that is valuable for exploring the remarkable capacity of regeneration in the future, but also gives an interesting insight into the understanding of Yenyuan stream salamander by this first transcriptome assembly.

Sexual dimorphism in the Chinese endemic species Pachyhynobius shangchengensis Fei, Qu and Wu, 1983 (Urodela: Hynobiidae).

Sexual dimorphism (SD) is a widespread phenomenon in most vertebrate species and is exhibited in a myriad of ways. In amphibians, sexual size dimorphism, in which females are larger than males, is the most common type, and sexual shape dimorphism varies among species. Different selection forces (sexual selection, fecundity selection, and ecological selection) that act differently upon the sexes form the consequence of SD. Thus, studies of SD provide information about the general intersexual divergence of the same species and allow insights into the impact of selective forces on the sexes. In this study, we analyzed morphometric data of the Shangcheng stout salamander, Pachyhynobius shangchengensis, an endemic and poorly known Chinese salamander, to examine sexual dimorphism in size and shape. The morphometric data included 15 characteristics of 68 females and 55 males which were analyzed using univariate and multivariate methods. A significant difference was found between the sexes in terms of both body size (snout-vent length) and some body shapes (e.g., head length and width, tail length and width, distance between limbs, and limb length and width) in this salamander. The longer snout-vent length in males may be attributed to sexual selection, longer and wider head in males may contribute to male-male competition, longer and wider tail in males may be attributed to energy storage and reproductive success, the larger distance between limbs in females is likely due to a fecundity advantage, and longer and more robust limbs in males may be related to reproductive or competitive behaviors. These results demonstrated that sexual dimorphism of different morphological traits is the consequence of different selection forces that act differently upon the sexes.

Discovery and characterization of conserved and novel microRNAs from blunt snout bream (Megalobrama amblycephala) by deep sequencing.

MicroRNAs (miRNAs) are short, single stranded RNA molecules with approximately 22 nts in length, which regulate the stability and translation of messenger RNAs in several organisms. To increase the repertoire of miRNAs characterized in M. amblycephala, we used the deep sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the 4 different tissues of M. amblycephala. A total of 309 conserved miRNAs that originated from 131 miRNA families were detected. 15 novel candidates miRNA were identified. Randomly selected 6 miRNAs were analyzed by stem-loop qRT-PCR and differential expression patterns were observed in 6 different tissues of M. amblycephala. Furthermore, the potential targets were predicted. GO analysis showed that most of the targets were involved in a broad range of physiological functions including fish growth, development, metabolism, stress responses and so on. Overall, our results significantly increased the number of novel miRNAs in M. amblycephala, which should be useful for further investigation into the role of miRNAs in regulating diverse biological processes.

Identification of the conserved and novel microRNAs by deep sequencing and prediction of their targets in Topmouth culter.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play important roles in regulation of almost all biological processes in organisms. Topmouth culter is an economically important fish species in the Chinese freshwater polyculture system for its delicacy and high economic value. However, to date, no miRNAs have been reported in Topmouth culter species. In this study, the liver, muscle, spleen and kidney of Topmouth culter are sampled and used for construction of a small RNA library for deep sequencing. A total of 273 conserved miRNAs/miRNAs-star and 8 novel miRNAs/miRNAs-star were discovered, these miRNAs belong to 86 different miRNA families. There is a strong base bias on miRNA nucleotide compositions at certain positions. U is the dominant nucleotide, particularly at the beginning, middle, and end of conserved miRNAs. The expression of all 8 novel identified miRNAs in Topmouth culter was validated by using stem-loop qRT-PCR. Furthermore, the potential targets were predicted for these miRNAs, which were found to be involved in diverse biological processes. Our study is the first significantly enriches the Topmouth culter miRNA repertoire and provides a reference for further elucidation of complex miRNA-mediated regulatory networks for gene expression in the growth and developmental progression of Topmouth culter.

A comparative study of the hyobranchial apparatus in Hynobiidae (Amphibia: Urodela).

The morphology of the adult hyobranchial apparatus has played an important role in understanding the systematics and evolution of urodeles, but the hyobranchial apparatus of hynobiid salamanders has received little attention so far. In this study, the hyobranchial apparatus of eight hynobiid salamanders (Hynobius leechii, Onychodactylus zhangyapingi, Ranodon sibiricus, Batrachuperus pinchonii, Salamandrella keyserlingii, Liua shihi, Pachyhynobius shangchengensis and Pseudohynobius flavomaculatus) is described and compared based on the clearing and double-staining method. The basic elements of the hyobranchial apparatus of the eight species are similar, including one basibranchial, cornua, one pair of radial loops, one pair of ceratohyals, one pair of hypobranchials II, one pair of ceratobranchials II, one urohyal (absent in O. zhangyapingi), one pair of the complex of hypobranchial I and ceratobranchial I (separated in certain species). Although the hyobranchial apparatus is similar among hynobiid salamanders and shows a unique morphological pattern, there are also certain species-specific distinctions that may be used for specific or generic diagnosis. The results of an ancestral state reconstruction of five traits showed that the ossified basibranchial, the presence of a separated hypobranchial I and ceratobranchial I, the absence of a urohyal, the ossified hypobranchial I and the partially ossified ceratohyal are derived traits. The state shown by the traits of each species is consistent with the phylogenetic position of each species. Compared with other Urodela, the hyobranchial apparatus of this group shows certain distinctive features that may represent the diagnostic characters of the family Hynobiidae. The partially ossified ceratohyal is correlated with the habitat and represents an ecological adaptation.

Tributyltin exposure causes lipotoxicity responses in the ovaries of rockfish, Sebastiscus marmoratus.

Tributyltin (TBT) is a well-studied endocrine disruptor in mollusks and fishes. Recently, TBT is also recognized as a metabolic disruptor. Since abnormal lipids metabolism can induce negative effects on reproduction, the present study was designed to investigate the effects of TBT on ovarian lipid accumulation and testosterone esterification in rockfish (Sebastiscus marmoratus). After exposure for 48 d, there was a decrease of neutral lipid droplets in the ooplasm of ovaries. Exposure has also induced lipotoxicity responses in the ovaries, which shown as an increase of interstitial ectopic lipid accumulation and total lipids. The decrease of serum triiodothyronine and thyroxine concentrations might be responsible for the lipotoxicity responses. In addition, the percentage of testosterone in an esterified form was significantly decreased in the ovaries by TBT exposure, which might be a mechanism by which free testosterone levels increased. The accumulation of ectopic lipids and increase of free testosterone levels in the ovaries might impact ovarian functions and oocyte development in fish. These results strongly indicate that TBT exposure can influence reproductive functions of rockfish through lipotoxic mechanism.

Rediscovery of Protohynobius puxiongensis (Caudata: Hynobiidae) and its phylogenetic position based on complete mitochondrial genomes.

The mysterious Asian hynobiid salamander, Protohynobius puxiongensis, was described based on a single specimen collected in 1965 and never found again since then. Because the specimen had an internasal bone, Pr. puxiongensis was thought to retain a primitive character lost by a common ancestor of all other hynobiid salamanders, and it was thus considered to be not only a new genus, but also a new subfamily. This conclusion bothered herpetologists for decades because it was based on only one specimen and one character without other living specimens being rediscovered. After years of field effort, we rediscovered living individuals of Pr. puxiongensis at its type locality. All characters observed in rediscovered specimens are identical to the original description of the holotype except the internasal bone, implying that the internasal bone observed in the holotype may be just an individual variation. To examine the phylogenetic position of Pr. puxiongensis, we sequenced complete mitochondrial genomes for this species, together with two Pseudohynobius species. By combining 18 published hynobiid mitochondrial genomes and our new sequences, we reconstructed a comprehensive phylogenetic relationship of Hynobiidae at the genus level. Our results indicate that Pr. puxiongensis is deeply nested within the hynobiid phylogeny. It is the sister group of the Pseudohynobius species, and the validity of subfamily Protohynobiinae is not supported.