Contemporary Asymmetric Genetic Introgression Between Two Pelophylax Species in Shanghai

Human disturbances are considered to break reproduction barriers among species.Significant increases in hybridization events have been reported among a large number of taxonomic groups in anthropogenic environments,providing novel insights into species evolution mechanisms and conservation management in the Anthropocene.The Eastern Golden Frog(Pelophylax plancyi)and BlackSpotted Frog(P.nigromaculatus)are two sympatric anuran species with a long history of mitochondrial genome introgression in highly urbanized continental East Asia.However,there is only limited understanding of the pattern of their contemporary hybridization and factors influencing their interspecific relationship under anthropogenic disturbances.Here,interspecific hybridization between P.plancyi and P.nigromaculatus at the population level was investigated in Shanghai.All except two haplotypes obtained from both species in Shanghai were mixed together,and located in the introgression clade,implying multiple ancient mitochondrial introgression events occurred in the populations of our study area.Asymmetric genetic introgression was detected by microsatellite markers,with 0.7%of P.plancyi and 14.6%of P.nigromaculatus identified as contemporary admixed individuals.Consistent with the trend of population density,higher genetic diversity of neutral microsatellite loci was found in the more abundant P.plancyi;however,variation in mitochondrial(Cyt-b)and nuclear(POMC)genes was higher in relatively rare P.nigromaculatus.The population density of P.plancyi and number of water patches within local habitats were significantly positively correlated with both occurrences and proportions of admixed individuals in the populations of P.plancyi and P.nigromaculatus.Considering the prevalent transformation of habitats in urbanized areas,these results imply that a high population density in isolated artificially altered habitats is likely to increase interspecific hybridization.Thus,population monitoring and improvement of landscape connectivity between habitats would be needed to control the intensity of interspecific hybridization between P.plancyi and P.nigromaculatus in anthropogenic-disturbed environments.

Tracing the Origin of the Black-spotted Frog,Pelophylax nigromaculatus,in the Xinjiang Uyghur Autonomous Region

t Identifying the origin of a biological invasion is critical for controlling the invaders. To explore the genetic diversity and identify the source region of introductions of Pelophylax nigromaculatus to the Xinjiang Uyghur Autonomous Region, we sequenced 695 bp of the mitochondrial Cyt b gene in 140 individuals of P. nigromaculatus and identified 42 haplotypes in Heilongjiang, Beijing, Jiangsu, Shaanxi, Zhejiang provinces, Chongqing and the Xinjiang Uyghur Autonomous Region. We detected only four mitochondrial haplotypes in 20 specimens from Yining city in Ili Kazak Autonomous Prefecture. We traced the origin ofYining P. nigromaculatus to the Beijing and Chongqing area. Our results extend the known distribution range of this species in Asia.

Altitudinal Variation in Digestive Tract Length in Yunnan Pond Frog (Pelophylax pleuraden)

The digestive tract plays an important role in digestion and the acquisition of food energy. Understanding the impact of abiotic environments on digestive tract morphology is especially important for evolution of digestive tract across different environments. Here, we investigated altitudinal variation in digestive tract length in the Yunnan Pond Frog （Pelophylaxpleuraden） across five populations ranging from 1413 m to 1935 m a.s.1, in Ningnan County, Sichuan province in western China. Frogs were collected during the breeding season, from 1-5 June 2012. Our results revealed that females had longer digestive tract and relative digestive tract （i.e. digestive tract length / body size） lengths in com- parison to males, on average, but the differences between them decreased with increasing altitude. Digestive tract and relative digestive tract lengths increased with increasing altitude suggesting that a higher proportion of indigestible ma- terials may be consumed at high-altitude sites and result in a relative increase in digestive tract dimensions.

Haematology of <i>Pelophylax ridibundus</i>(Amphibia: <i>Ranidae</i>) of <i>Striata</i>and <i>Maculata</i>Morphs in Populations Living in Conditions of Anthropogenic Pollution in Southern Bulgaria

Basic haematological parameters were studied in adult, sexually mature individuals of colour morphs (striata and maculata) in the populations of Pelophylax ridibundus living in biotopes with various degrees of anthropogenic pollution (control, domestic sewage pollution and heavy metal pollution) in Southern Bulgaria. We found that in the polluted biotopes in individuals of both morphs of P. ridibundus the blood parameters: erythrocyte (RBC) and leukocyte (WBS) count, haemoglobin concentration (Hb), haematocrit (PCV) were statistically reliably higher, the number of lymphocytes (Ly) decreased, and the parameters: mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV) and differential blood formula changed considerably in comparison with the control group. In terms of anthropogenic pollution, the average values of RBC and Hb were higher in P. ridibundus of striata morph.

黑斑侧褶蛙抗菌肽Pelophylaxin 2PG的cDNA筛选与性质研究

通过cDNA文库筛选在黑斑侧褶蛙中发现一种抗菌肽Pelophylaxin 2PG.Pelophylaxin 2PG前体由74个氨基酸残基组成,与蛙科抗菌肽具有相同的组织形式.成熟的Pelophylaxin 2PG包括30个氨基酸残基,含有一对二硫键,理论分子量为3 012.7 Da,等电点为9.6.Pelophylaxin 2PG对大肠杆菌具有抑菌作用,最小抑菌浓度为6.25μg/mL,经血清孵育6 h后抗菌活性未受明显影响.Pelophylaxin 2PG可作为药物开发的候选分子.

Karyological and morphometric variation of the North African green frog Pelophylax saharicus (Anura) in northeastern Africa

Morphometric variation of Pelophylax saharicus was analysed using univariate and multivariate statistics, with bothtraditional and geometric morphometries, based on 148 specimens from four different geographical localities in Tunisia and Algeria.The results show the existence of three morphotypes in Tunisia and one in Algeria, and indicate a significant degree of variationin morphometric characters between regions. Specimens from the southernmost region have the smallest body size and thegreatest morphometric divergence from other populations. This pattern of morphometric variation probably results from phenotypicplasticity in response to local environmental factors. The results of our chromosomal study (C-, Ag-NOR-, endonucleasedigestion, DAPI, CMA_3 and Q-banding) reveal this species to exhibit the plesiomorphic Pelophylax karyotype of 2n = 26 biarmedchromosomes and NORs on the eleventh pair. Similarities and differences of the North African green frog are discussed in relationto the different forms of data collected (chromosomal, morphometric, ecological)

增温对黑斑侧褶蛙蝌蚪形态表型和生理特征的影响

探究增温对蝌蚪形态表型和生理特征的影响,可为深入理解气候变暖背景下蝌蚪生存适应策略提供基础数据。2021年4月,设置了26℃、28℃和30℃3个温度梯度组,每组设平行处理3次,测定了黑斑侧褶蛙(Pelophylax nigromaculatus)从37期蝌蚪发育至46期幼蛙的时长、46期幼蛙身体和内脏器官大小、血液中各型白细胞的百分比及嗜中性粒细胞与淋巴细胞的比值(N/L)、植物血凝素(phytohemagglutinin,PHA-P)反应的变化情况。结果显示:(1)随着温度升高,变态时长缩短,但组间差异不显著(P>0.05);变态完成期幼蛙的体型随温度升高显著变小(P<0.05),肾脏湿重系数下降明显(P<0.05),其他内脏器官湿重系数和消化道各段长度系数的组间差异均不显著(P>0.05);(2)增温使幼蛙血液中嗜中性粒细胞的百分比和N/L下降(P<0.05),但嗜碱性粒细胞的百分比明显增加(P<0.05);(3)26℃、28℃和30℃幼蛙PHA-P反应最高值分别出现于注射后1、2和2 h,组间差异均不显著(P>0.05)。增温使幼蛙的体型趋于变小、肾脏湿重下降,免疫功能和应激反应能力均趋弱,嗜中性粒细胞与嗜碱性粒细胞的数量存在权衡关系,气候变暖将不利于幼蛙的存活。

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone（TH）signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A（TBBPA）, a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3,whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.

Effects of triclosan on gonadal differentiation and development in the frog Pelophylax nigromaculatus

Previous studies have reported that triclosan（TCS） could possess an androgenic activity.We aimed to investigate the effects of TCS on gonadal differentiation and development in the frog Pelophylax nigromaculatus, a sensitive amphibian species to androgenic chemicals.P. nigromaculatus tadpoles at stage 24 were exposed to TCS（3, 30, and 300 nmol/L） to stage46 in a semi-static exposure system. At the end of exposure, gonadal morphology and histology, sex ratio and gonadal expression of sex-biased genes were examined in P. nigromaculatus. In each TCS treatment group, we found several individuals whose gonads exhibited morphological and/or histological abnormalities. Gonadal histological abnormalities were characterized by few oocytes and many somatic cells. Although the percentage of the individuals with abnormal gonads was low（7.8%） among all animals treated with TCS,statistical test revealed the sex ratios in the 3 and 300 nmol/L TCS treatment groups were significantly different from the solvent control. In the 30 nmol/L TCS treatment group,abnormal gonads were also observed, although the sex ratio was not changed compared with the solvent control, which was possibly due to the smaller sample size in this group. In all the TCS treatment groups, the sex ratios were not obviously male-biased, but the expression levels of some sex-biased genes were significantly altered by TCS. Altogether,our results suggest that TCS, even at environmentally relevant concentrations, could disrupt gonadal differentiation and development in P. nigromaculatus, but we are not sure whether the disrupting effects were associated with masculinization or feminization.

稻田套养不同密度黑斑蛙对水稻产量和土壤肥力的影响初探

为优化稻蛙共作模式中水稻施肥技术,大田条件下设置4个黑斑蛙(Pelophylax nigromaculatus)投放密度处理,分析稻田套养不同密度黑斑蛙对水稻(Oryza sativa L.)产量和土壤肥力的影响。结果表明,随着稻田套养黑斑蛙密度的增加,水稻单位面积有效穗数显著增加(P<0.05),产量增加,稻田土壤pH、有机质和养分含量也呈上升趋势。与试验前相比,种养一季结束后,稻田土壤pH、有机质、总氮、总磷、碱解氮和速效磷含量均呈增加趋势,增幅为2.3%~89.0%,而总钾和速效钾含量降低,降幅为6.9%~24.4%。因此,稻蛙共作模式中水稻生产可不施氮肥和磷肥,只需施用适量的钾肥。

六种农药对黑斑侧褶蛙的毒理效应

稻-蛙种养是一种绿色农业的发展模式,为了筛选高效低毒农药,优化稻-蛙种养体系,为农药的合理使用与两栖动物资源保护提供基础资料,测定了6种农药对农田常见的黑斑侧褶蛙的毒理效应.结果表明:在水温(25±1)℃条件下施毒96 h后,对黑斑侧褶蛙的毒害作用由强到弱依次为阿维菌素、戊唑醇、毒死蜱、氰氟草酯、吡虫啉和噻虫嗪;以稻田常用农药吡虫啉和戊唑醇为实验试剂进行生长毒性实验后发现,黑斑侧褶蛙生长发育随着这2种农药质量浓度和作用时间的增加而减缓,表明这2种农药在黑斑侧褶蛙体内有一定的蓄积作用,短时间就可以对蛙的生长发育造成较大的影响;不同天数及不同浓度的染毒组都可以激活黑斑侧褶蛙体内的氧化应激机制,低浓度吡虫啉和戊唑醇处理后,黑斑侧褶蛙的过氧化氢酶(CAT)酶活降低、谷胱甘肽(GSH)含量下降;升高2种农药浓度,该抑制现象更为显著,机体自由基清除和抗氧化防御系统被破坏,同时丙二醛(MDA)含量显著上升,引起生物体内蛋白质和核酸等交联聚合,产生毒害作用.研究结果可为稻-蛙综合种养体系的完善提供参考依据.

黑斑侧褶蛙脑微血管内皮细胞的原代培养及鉴定

为了建立蛙脑微血管内皮细胞原代培养方法,实验以黑斑侧褶蛙脑组织为实验材料,在无菌条件下分离出大脑灰质,利用0.1%Ⅱ型胶原酶和胶原酶-分散酶进行消化,经过Percoll密度梯度离心后,将获取的脑微血管段接种于鼠尾胶包被的培养瓶中,于28℃、5%CO_(2)条件下原代培养蛙脑微血管内皮细胞,并通过细胞形态学观察和Ⅷ因子相关抗原免疫荧光对细胞进行鉴定,最后用四唑蓝比色法(MTT)测定细胞的生长状态。结果显示,微血管段在体外培养1 d后开始贴壁生长,并逐渐扩大成团簇状,培养至7 d时,细胞呈“铺石路样”镶嵌式排列,表现为区域性单层生长;Ⅷ因子免疫荧光检测发现胞质呈红色,表达为阳性,阳性细胞率达97%以上;MTT实验显示,细胞在第3~5天时生长速率最快。本研究首次建立了蛙脑微血管内皮细胞的原代培养与鉴定方法,为体外研究蛙类神经性疾病的发病机理奠定基础,也为相关药物的研发和筛选提供了细胞模型。

患白内障病黑斑蛙米尔伊丽莎白菌的分离鉴定与PNGase基因克隆

为给安徽舒城地区患白内障病黑斑蛙(Pelophylax nigromaculatus)提供治疗方案,对病蛙进行了细菌分离、鉴定和药敏检测。从典型患病黑斑蛙的肝脏、眼睛、脑和脊柱中分离出4株优势菌株,经16S rRNA和gyrB基因序列分析,结合生理生化鉴定,结果表明,4株菌株均为米尔伊丽莎白菌(Elizabethkingia miricola)。对分离菌的N-糖苷酶(PNGase)基因进行了克隆和分析,结果显示,其包含PNGase F和PNGase F-Ⅱ两种类型,进化分析结果同样显示,其与米尔伊丽莎白菌聚为一支,表明PNGase基因序列可作为伊丽莎白菌属内部菌分类的参考依据。药敏试验结果显示,4株分离株仅对酰胺醇类中的氟苯尼考敏感,对香豆素类中的新生霉素、大环内酯类中的红霉素和利福霉素类中的利福平等3种抗生素中介,而对测试的其他14种抗生素耐药。实验结果为伊丽莎白菌属细菌的分类鉴定提供了参考方法,且为黑斑蛙细菌病防治提供参考依据。

异噻唑啉酮类杀菌剂对黑斑蛙胚胎和蝌蚪的急性毒性

异噻唑啉酮类杀菌剂1,2-苯并异噻唑-3-酮(BIT)和甲基异噻唑啉酮(MIT)虽已在多种行业中广泛使用,但目前有关其毒性尤其对水体中生物毒性的数据还较少。鉴于BIT和MIT在水体中普遍存在,本文研究了这两种污染物对两栖动物黑斑蛙胚胎和蝌蚪的急性毒性。黑斑蛙胚胎和蝌蚪分别暴露系列浓度的BIT和MIT,观察化学品对其生长、发育和运动的影响,计算96小时半数致死浓度(96 h-LC50)和96小时半数致畸浓度(96 h-TC50),确定最小生长抑制浓度(MCIG)。结果发现,BIT对黑斑蛙胚胎的96 h-LC50和96 h-TC50分别为2.99 mg·L-1和0.60 mg·L-1,MCIG小于0.40 mg·L-1,对蝌蚪的96 h-LC50为6.44 mg·L-1。MIT对黑斑蛙胚胎的96 h-LC50和96 h-TC50分别为5.30 mg·L-1和2.36 mg·L-1,MCIG为2.59 mg·L-1,对蝌蚪的96 h-LC50为7.58 mg·L-1。根据《化学农药环境安全评价准则报批稿》中两栖动物蝌蚪急性毒性的分级标准,判定BIT和MIT的毒性等级为中等。该毒性数据可为异噻唑啉酮类杀菌剂的环境管理提供参考。

双酚A与其替代品对黑斑蛙急性毒性的比较

双酚F(BPF)和双酚S(BPS)作为双酚A(BPA)替代品广泛使用,然而有关BPF和BPS的毒性数据非常有限。采用系列浓度的BPA、BPF、BPS溶液,暴露黑斑蛙胚胎和蝌蚪96 h,通过半致死浓度(LC50)、最小生长抑制浓度(MCIG)和致畸率等指标比较3种化合物的急性毒性。结果显示:100 mg·L-1BPS未导致黑斑蛙胚胎及蝌蚪畸形和死亡。BPA和BPF对黑斑蛙胚胎的96h-LC50分别为7.68 mg·L-1和7.99 mg·L-1,MCIG分别为4.47 mg·L-1和4.77 mg·L-1,最大致畸率为33.33%;对蝌蚪的96 h-LC50分别为9.00 mg·L-1和9.52 mg·L-1。依据《化学农药环境安全评价准则》的毒性分级标准,判定BPA和BPF的毒性等级为中毒,BPS的毒性等级为低毒。表明BPF急性毒性与BPA相当,BPS急性毒性低于BPA。本研究数据可为BPF、BPS作为BPA替代品的生产和使用以及相应的环境管理提供毒理学参考。

金线侧褶蛙和黑斑侧褶蛙皮肤显微结构

采用常规石蜡切片及H.E染色法,对金线侧褶蛙(Pelophylax plancyi)和黑斑侧褶蛙(P.nigro-maculatus)体表9个部位皮肤的显微结构进行观察和比较.结果显示:2者皮肤基本结构与其他无尾两栖类相似,由表皮和真皮组成,但是各部位皮肤薄厚存在差异.分布于真皮疏松层的皮肤腺包括粘液腺和颗粒腺,2者粘液腺的分布范围没有差别,只在腺体数量和大小上存在不同;颗粒腺的分布范围存在种间差异.结果表明不同生态机制下物种间的差别和对环境的适应性.

金线侧褶蛙和泽陆蛙的两性异形与生育力

比较金线侧褶蛙和泽陆蛙的两性异形和生育力,首次通过种间比较来验证生育力选择理论是否能解释两栖动物两性异形的进化。金线侧褶蛙和泽陆蛙均为雌体大于雄体的两性异形类型,其两性异形程度存在显著差异,金线侧褶蛙两性异形程度指数为0 .30 ,而泽陆蛙仅为0 .0 8。然而,两者的怀卵量无显著差异。因此,结果与生育力选择理论预测不一致。此种不一致性可能由下述原因导致:选择压力作用于雄体而非雌体、能量分配策略和死亡率的种间差异、以及系统发育历史的种间差异。金线侧褶蛙和泽陆蛙雄体的身体状态指数显著高于雌体。金线侧褶蛙的头,眼和四肢都显著大于泽陆蛙;两者雌体的前后肢长度显著大于雄体。两性金线侧褶蛙的前后肢随体长呈同速生长。

黑斑侧褶蛙的两性异形和雌性繁殖特征

测定了黑斑侧褶蛙成体的体长、体重、头长、头宽、眼径、鼓膜径、前肢长、后肢长等形态指标以及雌体的怀卵数量。黑斑侧褶蛙雌体的体长和体重显著大于雄体。其它局部形态特征指标与体长呈正相关 ,协方差分析表明 ,雄体的鼓膜径大于雌体 ,其余形态指标不存在明显的两性差异。黑斑侧褶蛙雌体怀卵数量与体长和体重皆成正相关关系 ,表明黑斑侧褶蛙通过增加个体大小增加繁殖输出。

广义金线侧褶蛙河南3个地理种群的形态分析及分类探讨

运用典型判别分析和聚类分析,对河南省内3个广义金线侧褶蛙(Pelophylax plancyi)种群16个特征进行形态测量和比较分析.结果显示:广义金线侧褶蛙在河南应分为两个物种,分布于黄淮平原及以北的种群属于金线侧褶蛙,分布于大别山丘陵地区的种群属于湖北侧褶蛙(Pelophylax hubeiensis);金线侧褶蛙伏牛山种群的分类地位尚不能确定,湖北侧褶蛙和金线侧褶蛙是否在伏牛山区交叉分布,需要更多的形态学比较和分子生物学证据.

黑斑侧褶蛙肥满度及脏器重量的性别和季节差异

内脏器官的大小能反映动物的生长发育情况和生理生态状况,可作为动物对环境适应的近似指标。以黑斑侧褶蛙Pelophylax nigromaculata为研究对象,测定了2012年夏季、秋季及2013年春季其体重、体长、含水量、肥满度及脏器重量的性别和季节差异。结果显示:1)雌蛙的体重和体长均高于雄蛙,两性都在秋季最高;胴体湿重和干重无性别差异,胴体湿重夏季最高,胴体干重无季节差异。2)含水量和肥满度无性别差异,前者夏季和春季高于秋季,后者秋季高于春季;躯体肥满度和干体肥满度雄蛙均高于雌蛙,雄蛙无季节差异,雌蛙夏季最高,春季最低。体重与体长的比值(K WL)雌蛙高于雄蛙,两性都在秋季最高,春季最低。3)除肺干重雄蛙大于雌蛙,肾脏干重雌蛙大于雄蛙外,其他内脏器官的湿重和干重均无性别差异。肺湿重春季高于夏季和秋季,肾脏湿重无季节性差异;雄蛙的肺干重春季高于夏季,肾干重秋季高于夏季和春季,雌蛙均无季节差异。心脏和脾脏湿重都在夏季最高,秋季或春季最低,但干重均无季节差异。脂肪体和肝脏及卵巢和输卵管的湿重和干重均在秋季最高,春季或夏季最低。精巢的湿重及干重均无季节差异。结果表明,K WL可较好地反映成年黑斑侧褶蛙整体水平的营养和生理状况,多种器官重量有季节性的可塑性,这可能与雌、雄蛙的繁殖状态有关。