Abstract:The Saker Falcon (Falco cherrug) has been rapidly becoming endangered in the world. We analyzed the gender, signal duration, and missing month of 67 Saker Falcons tracked by satellites in the past 10 years, and their causes of death, threat factors and loss rates. We performed a more detailed analysis of the causes of death of 10 sakers from Russia and Mongolia during 2016 and 2017. Backpack method was used to fit the transmitters with the weight (17 g to 22 g) less than 3% of sakers′ body weight (Fig. 1). When the transmitters no longer returned information, we went to the last locations of the GPS to search for the bodies of died birds in a certain area. We also investigated the animal traces and surrounding environment, and performed an autopsy to analyze the cause of death. As to the 67 sakers, 37 were females (55.2%), 29 were males and one was unknown. The binomial test showed that there was no difference in sex ratio (P > 0.05). The average signal duration was 201 ± 129.9 days (n = 64); with 39 sakers less than 200 days (accounted for 60.9%), and 59 sakers less than 400 days (accounted for 92.2%) (Fig. 2). The annual return rate was 31.4%; and 45 sakers (accounted for 67.2%) were lost during August to December (Fig. 3). Of the 10 sakers being tracked in 2016, we obtained information on four lost ones, one was hunted in Mongolia, one was unclear, one died of electric shock (Fig. 4) in the west of China, and one died due to collision and hunger (Fig. 5) in China (Table 1). The significant low annual return rate of satellite tracking sakers indicated that the situation of Saker Falcon was not optimistic, causing concerns among the raptor research experts.

Abstract:We made population census on the Mute Swan (Cygnus olor) during the breeding season and pre-migration period in the Wuliangsuhai Lake from 2014 to 2017 by line transects and point counts methods (Fig. 1), compared with data and records in past decades, and analyzed the causes of the changes of the populations. The breeding adults in springs of 2015 to 2017 remained basically stable, with the number of 84, 92, and 80 individuals, however, the autumn population of 2014 to 2017 declined year by year, with the number of 411, 302, 281, and 153 individuals respectively (Table 1), possibly because that the reduction of suitable breeding and feeding habitat, predation, egg collecting by local people and poisoning factors have affected the survival of sub-adults and juveniles in recent years. From 1996 to 2004, the number of Mute Swan populations increased as the establishment of the nature reserves, with hunting and egg-collecting being forbidden. From 2005 to 2013, the number of populations declined due to drought and water shortage, development and utilization of waters, expansion of reed (Phragmites australis) and broad leaf cattail (Typha latifolia), deterioration of water quality, and increased eutrophication. The research showed that the Wuliangsuhai Lake has been overexploited and utilized, and frequent human interference affecting the breeding of Mute Swan in recent years. Scientific management on the habitats and more strictly control of egg collecting and poisoning are essential to the survival and population growth of Mute Swan.

Abstract:The Siberian Crane (Grus leucogeranus), an IUCN Critically Endangered species, comprises 3 500﹣4 000 individuals, with about 98% of its global population wintering in the Poyang Lake, China. Originally, Siberian cranes fed tubers of submerged plants, especially the Eel Grasses (Vallisneria spp.) in shallow water habitat of the lake. However, in the past few years, as the agricultural fields became their important foraging places, the diet of the Siberian Crane should have changed. To study the food composition of Siberian cranes in agricultural fields, we used microscopy method to analyze 70 fecal samples collected from 5 rice paddies and 2 lotus ponds during November 2017 and April 2018. We also collected plants at the foraging grounds to construct a referencing library of plant cell morphology, which was used to identify plants in fecal samples. Frequency conversion method was used to calculate density of each food resource. The food of Siberian cranes composed of 15 plant species belonging to 10 families (Table 1), with Rice (Oryza sativa, relative density 34.34%), Lotus Roots (Nelumbo nucifera, 22.99%), and Chinese Milk Vetch (Astragalus sinicus, 10.61%) being their main food items. However, the tuber of eel grasses, which formerly was their typical food, became extremely low proportion (2.05%). Therefore, the diet of Siberian cranes has changed very much, with the crops becoming their important food, as the degradation of submerged plants in the Poyang Lake. Foraging in agricultural fields, the Siberian cranes are facing a series of problems, especially high intensity of human disturbance. To provide a safe and stable foraging environment for cranes, we propose some protective measures, including enhancing environment education, promoting ecological compensation, restoring submerged plants, and constructing mini protected areas.

Abstract:Two bats (sample number are HUNNU16SF16 male and HUNNU16SF38 female) have been captured by mist net in Xianfei Cave (26°58′25″N, 113°3′23″E, altitude 463 m), Sifang Mountain, Hengdong County, Hunan Province. The bats were identified as Myotis petax based on its external morphology, skull characteristics and molecular data. The measurement of forearm length was 40.73 mm and 42.40 mm for male and female which is slightly longer than that of M. petax captured from other locations (Table 2). The baculum Length in M. petax is 1.088 mm, nearly twice as large as in M. daubentonii. Maximum Likelihood tree based on Cyt b sequences was constructed (Fig. 4). The cladogram indicated the sequence of the samples from Hengdong County form a clade with M. petax that collected from Jilin Province China, Russia and Japan. The bat is a new record of Chiroptera in Hunan province. The samples were preserved in Hunan Normal University Vertebrate Specimens Museum.

Abstract:We measured totally 9 types of body characteristic parameters of 23 female frogs (Odorrana tormota) including snout-vent length (SVL), head length (HL), head width (HW), width of lower arm (WLA), length of lower arm and hand (LAHL), forelimb length (FLL), hindlimb length (HLL), tibia length (TL), body mass (BM) in Huangshan Fuxi area after they ovulating to explore whether there is a correlation between fertility and the body characteristic parameters. We found out that the 9 types of body characteristic parameters frogs had significant positive correlation with clutch size (P < 0.05, Table 1). We also found out that SVL was also positively correlated with the rest 8 types of body characteristic parameters (P < 0.05, Table 2). By using the SVL as the control variable, we conducted a partial correlation analysis and got a positively correlation of the clutch size with BM (P < 0.05, Table 3). These results indicated those females who with longer SVL and heavier BM produced a larger clutch size and had a stronger fertility. The clutch size between female individuals was differences with average around 646.5 ± 37.6 (590﹣706). The larger individual females have stronger fertility and larger reproductive output, which may be played an important role for the high degree sexual dimorphism of the concave-eared torrent female frogs. In order to explore what kind of mating patterns do male and female frogs choose, we measured and compared the 9 body characteristic parameters mentioned above from 21 mating paires and found out that the body characteristic parameters did not affect the female frogs selecting their matings (P > 0.05, Table 4). The fact means that the female frogs may prefer males with other quality characteristics, such as croaks.

Abstract:Botiinae was the second largest group of Cobitidae fish. In order to investigate the morphological differences of the four species (Sinibotia superciliaris, S. reevesae, Leptobotia taeniops, L. microphthalrna) of Botiinae fishes, anatomical methods were used in this study. Standard length, body weight and other biological indices of each specimen were measured. Statistical analysis of all data was performed using Microsoft Excel 2007 and SPSS 19.0. The results showed that the morphological structures of the brain in four Botiinae fishes were similar to other teleosts, consisting of five parts: telencephalon, diencephalon, mesencephalon, cerebellum and myelecephalon (Fig. 1 and 2). Among them, there were significant differences in the relative volumes of cerebrum and corpus cerebelli, while there was no significant difference in other parts (Table 3). The more closely related the fish, the more similar the morphological structure of the brain. The L. taeniops and L. microphthalrna prefer to live in the environment with flowing water, suggesting that possession of the larger relative volume of cerebrum and corpus cerebelli might be related to a higher motor coordination ability.

Abstract:Melanocortin receptor 1 gene (mc1r) is a crucial regulatory factor for body color formation in animals. In order to explore the role of mc1r gene during body color variation in Amphilophus citrinellus, mc1r full-length cDNA was obtained for the first time by rapid-amplification of cDNA ends (RACE) technique. The total length of mc1r was 1 699 bp containing 497 bp 5′ untranslated region (UTR), 224 bp 3′-UTR, and 978 bp open reading frame (ORF) encoding 325 amino acids (Fig. 1). Sequence analysis of amino acids and phylogenetic analysis indicated that the similarity of mc1r encoded protein sequence of A. citrinellus showed 55.1%, 77.1%, 90.15% and 96.92% similarity with that of Homo sapiens, Danion rerio, Larimichthys crocea and Oreochromis niloticus, respectively (Fig. 4). Analysis revealed that mc1r was expressed at different levels in all 9 embryonic periods. With the development of embryo, the expression level of mc1r decreased gradually (Fig. 7). The results suggested that the basic expression level of mc1r initiated adenylate cycle in early stage of embryonic development, then involved in the formation of pigment cells. In three periods of body color changes (black to hoar to yellow), the expression of mc1r was decreased first and then increased slightly in caudal fin, scales and skin and its expression was the lowest in the gray white transition stage (Fig. 8). It was presumed that there might be a competitive combination between mc1r and Agouti, and that the color might be related to appropriate balance among three genes, mc1r, tyr, and tyr1 in the process of body color changes of A. citrinellus. When the fish grew to near maturity, individuals with incomplete black-fading showed higher mc1r expression compared to those with complete black-fading (P < 0.05, Fig. 9), and surface melatonin was negatively correlated with the mc1r expression. mc1r gene was expressed in all 11 tissues, and its expression in scale was significantly higher than in other tissues (P < 0.05, Fig. 10). The present study revelaed expression features of genes related to color variation, and the data are important for further studying fish body color heredity and body color improvement.

Abstract:Black Carp (Mylopharyngodon piceus) is an important aquaculture species in China. Developing microsatellite DNA markers from genome of M. piceus will provide a tool for its genetic diversity analysis. In this study, two biotin probes (CA)10 and (GACA)6 were used to construct two microsatellite enrichment libraries of M. piceus by magnetic beads enrichment. A series of microsatellite primers was designed and their polymorphisms were tested in a population of M. piceus. Softwares GeneMapperv4.0, Cervus3.0 and PopGen32 were used to analyze their genetic diversity information. Microsatellite markers with high polymorphism and rich genetic diversity were screene. Through analysis, 849 sequences containing microsatellite loci were obtained from 993 positive clones. The repeat number of the repeating unit was then counted (Fig. 1). The statistics showed that most numbers of the repeats were between 5 and 21. The repeating units were classified according to the number of nucleotides in each repeating unit. The results showed that the number of dinucleotide microsatellite loci was the most, as high as 1 076. It accounted for 82.45% of the total. There were 217 trinucleotide type microsatellite loci, accounting for 16.63% of the total. In trinucleotide type, only 0.77% of all 39 microsatellite markers were designed and assessed in 36 individuals of black carp from Wujiang population. Results showed that 25 microsatellite loci showed polymorphism (Table 1). The mean number of alleles (Na) and effective number of alleles (Ne) were 7.08 and 3.526, respectively. The mean value of observed heterozygosity (Ho) and expected heterozygosity (He) were 0.602 and 0.619, respectively. The mean value of polymorphic information content was 0.568. Mp23, Mp27 and Mp35 were extremely significant deviating from Hardy-Weinberg equilibrium (P < 0.01) (Table 2). The microsatellite primers developed in this study will provide a tool and theoretical basis for evaluation and protection of germplasm resources, and genetic diversity analysis in M piceus.

Abstract:Boule gene, one of the DAZ (Deleted in Azoospermia) gene family members, is specifically expressed in animal germ cells. In mammals, the boule null caused spermatogenic failure, leading to male infertility, while the loss of boule in invertebrate, such as Caenorhabditis elegans resulted in female sterility due to a failure of oogenesis. Turtles are considered as a link between the high and low vertebrates, however, the molecular mechanisms behind germ cell development in reptile including turtles remain largely unclear. In this study, we used the Chinese Soft-shell Turtle (Pelodiscus sinensis) as a model to reveal the mechanisms under germ cell differentiation in turtles. Firstly, we cloned a turtle boule cDNA fragment of 1 005 bp, containing a 3′ untranslated region (UTR) of 57 bp and an open reading frame (ORF) of 948 bp, encoding 315 amino acid residues (Fig. 1). The predicted turtle Boule is maximally 92% identical to that of Chelonia mydas, 83% to that of mouse, 53% to that of Drosophila melanogaster and 42% to that of Oryzias latipes (Fig. 2). The reverse transcription quantitative real-time PCR (RT-qPCR) showed that boule transcript of P. sinensis was abundantly expressed in adult ovary and testis but barely in all the somatic tissues examined in this study (Fig. 4). Chemical in situ hybridization revealed that the boule mRNA of P. sinensis was exclusively expressed in germ cells but absent in somatic cells, and displayed dynamic expression patterns in germ cells during gametogenesis (Fig. 5 & 6). In testis, the boule mRNA signals were strongly dispersed in cytoplasm of primary spermatocytes and weakly in spermatogonia and secondary spermatocyte, hardly detected in spermatids and spermatozoa (Fig. 5). In ovary, the boule mRNA signals were strongly dispersed in cytoplasm of oocytes at early stages. Subsequently, the signals were concentrated in the perinuclear area of growing oocytes and weakened gradually with the growth of oocytes (Fig. 6). Therefore, our findings indicated that boule gene might play an important role in germ cell meiosis in Pelodiscus sinensis, which shed new insight into understanding the functions of boule gene during germ cell differentiation in vertebrates.

Abstract:To establish a stable culture system of skin fibroblasts from Tree Shrew (Tupaia belangeri), and provide a primary cell model for related experiment on this specific animal species, the inner thigh skin was scraped to isolate primary cells using tissue mass adherence and collagenase Ⅰ digestion, respectively. The obtained cells were purified by using differential digestion with trypsin. MEM (with 10% FBS) and low Serum Growth Supplement added MEM (LSGS) were used for cell culture. The cells were identified by immunofluorescence and Western blot analysis, and the characteristics of cells′ survival and replication were further assessed. The results showed that collagenase digestion was more suitable for isolation of primary skin cells than method of tissue adherence (Fig. 1, 2). According to the survival and growth characteristics of isolated cells (Fig. 5, 6), the LSGS may provide a better culture condition for isolated cells in comparing with that of MEM (Fig. 4). Immunofluorescence observation (Fig. 8 and Fig. 9) and Western blot (Fig. 10) detection revealed that the isolated cells were primary skin fibroblasts. This effective method for isolation of primary skin fibroblasts from Tree Shrews was successfully established and their culture conditions were optimized for further investigation.

Abstract:To explore the optimal injection concentration and the changes of footpad skin histology and hematology of Asiatic toads (Bufo gargarizans) after injection of phytohemagglutinin-P (PHA-P), individuals were injected at footpad with PHA-P at three concentrations (1.0 g/L, 2.5 g/L and 5.0 g/L). The responsiveness to PHA-P in Asiatic toads exposed to different injection concentrations was tested and the optimal injection concentration was selected. Under the condition of the optimal PHA-P injection concentration, variations of different subtypes of leukocytes in the skin tissues of footpad position and circulation blood, as well as serum bactericidal capacity were measured when the PHA-P response reached the peak. All statistical analyses were performed using SPSS software packages (SPSS 17.0 for windows). One or two way ANOVA, or a repeated measurement one way ANOVA, or independent sample t test was used to compare the differences of body mass or percentage of different subtypes of leukocytes from skin tissues and circulation blood, or serum bactericidal capacity, or the responsiveness to PHA-P solutions among six time points. Results were presented as means ± standard error, and P < 0.05 was considered to be statistically significant. The results were as follows: PHA-P response did not display significant sex difference (P > 0.05, Fig. 2); The optimal PHA-P injection concentration was 2.5 g/L with the peak at 12 h after injection (Fig. 2). No significant group differences were observed for different subtypes of leukocytes from skin tissue with injected PHA-P or saline (or without any solution) at footpad positions (P > 0.05, Fig. 5); At peak time, percentage of lymphocytes was increased (P < 0.05, Fig. 7), but percentage of eosinophils was reduced (P < 0.05, Fig. 7) in circulation blood, and no significant difference was found for serum bactericidal capacity (Fig. 8). The optimal 2.5 g/L PHA-P concentration was appropriate for Asiatic toads. The peak of PHA-P response was not correlated with the dynamic changes of any subtype of leukocyte in the footpad skin tissues, but abundant lymphocytes might be activated, and the function of eosinophils might be attenuated in circulation blood, which may be regarded as the main factors causing Asiatic toads′ responsiveness to PHA-P solution.

Abstract:The purpose of this study was to investigate the developmental toxicity of copper sulfate (CuSO4) and the inhibitory effect of rosmarinic acid (RA) on the zebrafish (Danio rerio) embryos. The embryos (1 hour post fertilization, 1 hpf) were exposed to different concentrations of CuSO4 solution and CuSO4 solution containing different concentrations of RA, respectively. Control group was exposed to E3 medium. The mortality, malformation and hatching of embryos were observed under stereo microscope. The lethal rate, malformity rate and hatching rate were calculated and analysed. The embryonic reactive oxygen species (ROS) was detected using the fluorescent probe of DCFH-DA under RA defense. The experimental data were statistically analyzed with variance analysis. The results showed that the exposure to the CuSO4 solution caused serious lethal effect (Fig. 1a) and teratogentic effect (Fig. 1b, 2), decreased the hatching rate of embryos (Fig. 3) in a concentration-dependent manner. The linear regression equation between concentration of CuSO4 and mortality of zebrafish embryos at 96 hpf was Y = 5.59X + 7.1533, Y and X referred to lethal rate (%) and concentration of CuSO4 (μmol/L), respectively. The corresponding half lethal concentration (LC50) of CuSO4 on zebrafish embryos was 7.7 μmol/L. The linear regression equation between concentration of CuSO4 and deformity rate of zebrafish embryos at 96 hpf was Y = 24.213X + 3.5443, Y and X referred to lethal rate (%) and concentration of CuSO4 (μmol/L), respectively, and the half effective concentration (EC50) resulted in the malformation was 1.9 μmol/L. At 96 hpf, RA decreased lethal effect and increased hatching of zebrafish embryos induced by 8 μmol/L CuSO4 (Fig. 4). RA decreased malformation effect of zebrafish embryos induced by 1.6 μmol/L (Fig. 5). CuSO4 and RA inhibited ROS level induced by CuSO4 in zebrafish embryos (Fig. 6). The results indicate that CuSO4 has serious toxic effect on the development of zebrafish embryos, which may be associated with increased ROS level and caused oxidative stress. RA inhibits developmental toxicity of CuSO4 by reducing oxidative stress.

Abstract:In the history, Water Deer (Hydropotes inermis) distributed widely in the Liaodong Peninsula, the North China Plain and the Yangtze River in China. Since 1949, no record any recorde of the water deer has been reported in Jilin province. The discovery of the water deer has been reported for several times in Jilin province in recent years. Then we carried out an infrared cameras survey in Jilin Baishan Musk Deer National Nature Reserveand during December 2017 to March 2018. Totally, 21 photos and 600 seconds video on the water deer were taken (Table 1 and Fig. 1b﹣d). In the subsequent studies, a fawn (Fig. 1e) and a large amount of the traces (Fig. 1f) were photographed in the field. It proved that water deer was existed in Jilin province.

Abstract:Six bird species were firstly recorded in Qinghai Province during our field investigation in 2016 and 2017, including Crested Kingfisher (Megaceryle lugubris), Blue Rock Thrush (Monticola solitaries philippensis), Slaty-bule Flycatcher (Ficedula tricolor), Verditer Flycatcher (Eumyias thalassina thalassina), Chestnut-flanked White-eye (Zosterops erythropleurus) and European Goldfinch (Carduelis carduelis paraponisi).

Abstract:Five warty newt specimens of the genus Paramesotriton were collected on 14th, February, 2017 from Wangdongyang Mountain wetland provincial nature reserve, Jingning, Lishui, Zhejiang (28°11′51.72″N, 119°49′2.28″E，1 100 m). It was preliminary identified as P. aurantius by measurement and morphological characteristics. Then further identification was conducted by mitochondrial COⅠ gene sequence. The specimens were identified as Salamandridae P. aurantius. This species is new to Zhejiang Province.

Abstract:The taxonomic status of the Western Marsh Harrier (Circus aeruginosus) and Eastern Marsh Harrier (C. spilonotus) has been controversial in the past, and those changes might mislead relevant records of these two species. Here, we briefly reviewed the taxonomic history of the two species, and pointed out that the plumage of juvenile of C. spilonotus and its Japanese population are similar to the C. aeruginosus. A comprehensive review of known records of the C. aeruginosus were also made. We found that due to taxonomic changes, misidentification and misinterpretation of reference, there′re some confusion in the historical records of these two species in China indeed. After a comprehensive examination on all known records of C. aeruginosus in China, as checked some relevant specimen, we confirmed that the C. aeruginosus being recorded in Xinjiang, Beijing, Yunnan, Guizhou, Qinghai, Inner Mongolia and Sichuan with plausible proofs, while the known records from other areas of China should be treated with caution.

Abstract:Red-crowned crane (Grus japonensis) is one of the first-class national protected animals in China, which has been listed as an “Endangered” species by IUCN. We collected and sorted out the publications associated with habitat selection and suitability of red-crowned cranes in 2000-2016, analyzed the habitat selection of red-crowned cranes during breeding, migratory, and wintering period, and determined their suitable and avoidant habitat types of red-crowned cranes in different life stages. Moreover, we examined the habitat changes and their causes of red-crowned cranes in breeding, stopover, and wintering sites. This study provides some evidence for scientifically evaluating the impacts of habitat changes on the wild population of red-crowned cranes and carrying out some specific protection and management actions, and would benefit the healthy and sustainable development of wild red-crowned cranes.

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