Eukaryotic food sources analysis in situ of tropical common sea cucumber Holothuria leucospilota based on 18S rRNA gene high-throughput sequencing

Sea cucumber Holothuria leucospilota is one of the most widespread tropical holothurian species.In this study,eukaryotic organism composition in foregut and hindgut contents of H.leucospilota and surrounding sediments was assessed by 18S rRNA gene high-throughput sequencing.Eukaryon richness and diversity in the habitat sediments were significantly higher than those in foregut and hindgut contents of the sea cucumbers(P<0.05).The foregut content group,hindgut content group,and marine sediment group sequences were respectively assigned to 18.20±1.32,19.40±1.03,and 21.80±0.37 phyla.In the foregut contents,Nematoda(20.18%±9.59%),Mollusca(16.12%±10.49%),Chlorophyta(10.04%±4.85%),Annelida(8.72%±10.93%),Streptophyta(8.46%±4.65%),and Diatomea(5.99%±2.01%)were the predominant phyla,which showed the eukaryotic food sources of H.leucospilota were primarily belong to the above phyla.The predominant phyla in the hindgut contents were Streptophyta(45.55%±17.32%),Mollusca(4.93%±4.82%),Arthropoda(5.37%±3.08%),Diatomea(3.88%±2.34%),and Chlorophyta(3.79%±1.59%);and Annelida(37.80%±17.00%),Arthropoda(24.49%±12.53%),Platyhelminthes(7.14%±3.02%),Nematoda(4.14%±0.91%),and Diatomea(5.11%±1.35%)had large contents in the sediments.The comparatively high content of Paris genus in phylum Streptophyta in foregut contents indicated that land plants were one of the primary food sources of H.leucospilota,however the significantly higher contents of Streptophyta in hindgut contents than that in foregut contents might suggest a large part of the terrigenous detritus ingested might not be digested by H.leucospilota.UPGMA and PCoA analysis revealed that eukaryotic organism composition differed significantly between foregut contents of H.leucospilota and ambient sediments,indicating selective feeding feature of H.leucospilota.This study provided useful references for artificial feed of tropical sea cucumbers and enhanced understanding of the ecological roles of detritus-feeding macrobenthos.

陕西省榆林地区鸡蛔虫核糖体18S rRNA与ITS-2序列分析及种系发育关系研究

为了探究陕西省榆林地区鸡蛔虫核糖体18S rRNA、ITS-2基因序列特征及其种系发育关系,试验以采集于该地区的28条鸡蛔虫为样品,PCR扩增其18S rRNA、ITS-2基因的部分序列,测序后分别对鸡蛔虫18S rRNA、ITS-2基因序列的碱基组成进行分析,计算A、T、G和C的含量,并对这两个基因的遗传多样性进行分析,比较其种内、种间差异,构建种系发育树。结果表明:测序所获得的鸡蛔虫18S rRNA、ITS-2基因序列的长度分别为637,350 bp;其中18S rRNA基因序列的A、T、G和C的含量分别为24.5%~25.2%、26.1%~26.4%、28.3%~28.7%和20.3%~20.9%,A+T含量为50.6%~51.6%,G+C含量为48.6%~49.6%,种内差异为0~1.6%;与猪蛔虫(MF358962)、人蛔虫(LC422643)、犬弓首蛔虫(FJ418788)及狮弓蛔虫(JF837179)种间差异为5.18%~61.38%;ITS-2基因序列中的A、T、G和C的含量分别为28.9%~29.1%、38.0%~38.6%、19.1%~19.7%与12.9%~13.7%,A+T含量为66.9%~67.7%,G+C含量为32.0%~33.4%,种内差异为0~2.3%,与猪蛔虫(MF358962)、人蛔虫(LC422643)、犬弓首蛔虫(FJ418788)及狮弓蛔虫(JF837179)种间差异为55.95%~58.42%。此外,所构建的两个种系发育树具有类似的拓扑结构,均显示来自于陕西省榆林地区的28条鸡蛔虫共同聚为一个分支。说明来自于陕西省榆林地区鸡蛔虫的ITS-2序列呈AT偏好,其18S rRNA、ITS-2基因序列均具有种内差异小而种间差异大的特点,且个体之间存在一定程度的遗传差异,但它们之间的亲缘关系较近。

用18S rRNA序列探讨8种绿藻分子系统发育

测定了宽礁膜Monostroma latissimum、浒苔Enteromorpha prolifera、孔石莼Ulva pertusa的18S rRNA基因序列,并与肠浒苔E.intestinalis、穿孔石莼U.fenesrtata、石莼U.lactuca、礁膜M.nitidum和格氏礁膜Monostroma grevillei5种已知的大型海洋绿藻的同一基因序列合并进行比较,分析了核苷酸序列差异和碱基替换特点,并构建系统发生树。结果表明:浒苔与肠浒苔之间的Kimura双参数距离大于浒苔与石莼属3个物种之间的Kimura双参数距离;用UPGMA法和MP法构建的系统树基本一致,在构建的系统发生树中,浒苔与石莼属物种聚为一类,表现出与石莼属物种更近的亲缘关系。

枝睾阔盘吸虫凉山分离株18S rRNA基因部分序列测定与种系发育分析

为探究山羊源枝睾阔盘吸虫的种内遗传变异和系统发育关系,对采集自四川省凉山州9个样品的18S rRNA基因部分序列进行PCR扩增、序列测定及分析,并构建种系发育树。测序结果显示,所测得的9个山羊源枝睾阔盘吸虫18S rRNA基因目标序列长度均为1758 bp,核苷酸同源性为99.9%~100%,共检测到3个单倍型、5个变异位点;系统发育树显示,山羊源枝睾阔盘吸虫凉山州分离株与瘤牛源枝睾阔盘吸虫孟加拉共和国分离株聚在同一小支上,且与胰阔盘吸虫、腔阔盘吸虫亲缘关系最近。本研究结果为枝睾阔盘吸虫病的诊断、分子流行病学调查和枝睾阔盘吸虫种群遗传的进一步研究奠定了基础。

Epiphytic zooplankton community profiles in a typical urban wetland as revealed by DNA metabarcoding

Zooplankton,a crucial component of urban wetland,are one of the effective bioindicators for monitoring the feeding stocks of organisms at higher trophic levels and assessing the ecological quality of ecosystems.However,information about the characteristics of epiphytic zooplankton community structure resulted from traditional methods is limited and hindered by the large amount of detritus and sludge attached to the macrophytes.We investigated the epiphytic zooplankton communities associated with macrophytes(Vallisneria,Nymphaea,and Thalia dealbata)in a subtropical wetland using as DNA markers of the 18 S rRNA gene and the mitochondrial cytochrome c oxidase subunit I(COI)gene.A total of 241 OTUs of zooplankton were obtained from COI amplicons,including 194 OTUs of Rotifera,22 of Cladocera,and 25 of Copepoda,while only 62 OTUs of zooplankton were obtained from 18 S rDNA amplicons including 34 OTUs of Rotifera and 28 of Copepoda.The zooplankton communities associated with the three macrophytes were similar,but they differed significantly from those in the open waters.However,there were no significant temporal differences among the zooplankton communities.Epiphytic zooplankton communities were dominated by littoral zooplankton such as Testudinella,Lecane,and Philodina.Microzooplankton,especially littoral species,utilize macrophytes as food sources and as refuges against predation.This further led to an increase inαandβdiversity of zooplankton communities in urban wetlands.Our result suggests that the joint use of multiple molecular markers could improve the taxonomic resolution and generate a comprehensive biodiversity profile of zooplankton.

虾肝肠胞虫感染病理特征、18S rRNA基因序列及系统进化树分析

为调查浙江省部分地区虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)暴发来源及与其他宿主来源的微孢子虫进化关系,本研究利用形态学特征和分子鉴定,尝试对引起浙江多地凡纳对虾生长缓慢综合征的病原进行探究.同时基于18S rRNA基因序列分析该病原与其他地区及其他宿主来源微孢子虫的进化关系.结果显示,所采病虾均为EHP阳性,采样地区EHP 18S rRNA基因具有高度同源性,为98.5%~100%.对国内外EHP基因序列进行同源性分析,其同源性为86.2%~100%,所采EHP与印度EHP聚为一个分支. EHP 18S rRNA基因序列与其他水生生物微孢子虫18S rRNA均处于不同分支,表明18S rRNA可作为EHP的主要鉴别序列.将本研究所采EHP与NCBI中同源性较高的比氏肠胞虫和金头鲷微孢子虫比较分析,结果表明微孢子虫基因序列同源性与宿主进化发育程度有关,宿主之间进化距离越近,则微孢子虫基因序列同源性相聚越近.

16S rRNA及18S rRNA同时检测脑脊液中的病原菌

目的 提高脑脊液中病原菌的快速、准确鉴定率。方法 以细菌的保守序列 16SrRNA和真菌的 18SrRNA为通用引物 ,用多重PCR技术对标准菌株及 4 6例中枢神经系统感染患者的脑脊液进行扩增 ,同时与脑脊液的培养结果进行比较。结果 标准菌株金黄色葡萄球菌、大肠埃希菌、脑膜炎奈瑟菌、结核分枝杆菌、化脓性链球菌及白色念球菌均出现特异条带 ,人白细胞DNA为阴性。 4 6份脑脊液标本培养阳性 39例 ,另有 2例PCR阳性而培养阴性 ,后经诱导传代培养证实为L型金黄色葡萄球菌。PCR阳性 4 1例 ,与培养分离鉴定结果符合率为 10 0 %。结论 多重PCR可快速鉴定脑脊液中病原菌 。

湖南省怀化地区白纹伊蚊核糖体18S rRNA和线粒体cox 1基因的序列测定及分析

为探究湖南省怀化地区白纹伊蚊的基因变异、遗传多样性及其遗传进化关系,本试验以收集于怀化地区的30只白纹伊蚊为研究对象,利用PCR技术扩增其核糖体18S rRNA及细胞色素c氧化酶亚基Ⅰ(cox1)基因序列,并对其基因变异、遗传多样性及遗传进化关系进行分析。结果显示,所获全部样品的18S rRNA和cox 1基因序列长度均为489和1004 bp;基于18S rRNA基因序列,怀化地区白纹伊蚊的种内差异为0~2.9%,种间差异为28.74%~61.46%;基于cox 1基因序列,怀化地区白纹伊蚊的种内差异为0~0.2%,种间差异为6.08%~12.65%。基于18S rRNA、cox 1基因序列所构建的进化树发现,来自于怀化市的白纹伊蚊全部位于同一分支上。结果表明,怀化地区白纹伊蚊之间存在一定程度的基因变异和遗传多样性,但它们之间的亲缘关系较近,且其18S rRNA、cox 1基因序列的种内差异小、种间差异大,可作为白纹伊蚊鉴定的理想遗传标记。

Comparative evaluation of microscopy,OptiMAL® and 18S rRNA gene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner,India

Objective:To evaluate microscopy,OptiMAL? and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL? and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL? was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL?.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL?,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.

福建省蛇舌形虫18S rRNA基因的扩增及虫种鉴定

以我国福建省三明市某蛇场的病蛇为研究对象,对其进行剖检,观察其器官组织病变,并从体腔中分离得到1对寄生虫成虫,经过剖检观察初步怀疑为舌形虫病。提取虫体和肠道结节DNA,利用18S rRNA基因特异性的引物对提取的DNA进行PCR扩增,经琼脂糖凝胶电泳得到目的条带,切胶回收纯化,连接转化培养得到单菌落。对菌落进行PCR扩增测序。结果显示:病蛇肺脏、肠道病变严重有肿大、淤血和大量结节。经测序得到序列总长均为770 bp,与NCBI公布的相关舌形虫18S rRNA基因序列进行比对分析,三明蛇场得到的舌形虫成虫和肠道结节的DNA序列与EU370434.1(Raillietiella sp.1 BMR-2008 strain C129);AY744887.1(Raillietiella sp.MCZ DNA101107)的相似性为99.74%;与KC904945.1(Raillietiella orientalis)的相似性为97.40%;与JX088397.2(Linguatula serrate isolate NoDog1)的相似性为93.51%。表明此次分离的舟山眼镜蛇舌形虫属于Raillietiella属舌形虫。

First molecular identification of Cryptosporidium by 18S rRNA in goats and association with farm management in Terengganu

Objective:To identify the prevalence of Cryptosporidium from goats in three types of farm management systems in Terengganu,Malaysia and to determine the Cryptosporidium species infecting goats by using 18 S r RNA.Methods:A total of 478 fecal samples were randomly collected from goats in three farms;199 samples were collected from intensive farm,179 samples from semi-intensive farm and 100 samples from extensive farm.The samples were processed by using formolether concentration technique and stained by using modified Ziehl–Neelsen.Positive samples were performed by using nested PCR analysis by using 18 S r RNA.Results:Out of 478 goats,207(43.3%) were found to be infected with Cryptosporidium.Goats reared under the intensive farm management system reported the highest prevalence of infection(49.7%),followed by intensive farm management system(41%) and the lowest prevalence was reported in the goats reared under semi-intensive management system(37.4%).Conclusions:The identified species found in goat was Cryptosporidium parvum.Future study on the zoonotic transmission of Cryptosporidium parvum in goats needs to be done in order to find the source of transmission of this parasite.

基于18S rRNA基因的芜湖市疟疾病例溯源研究

目的研究芜湖市疟疾病例疟原虫种系发育和地理聚类。方法采集2018年8月至2019年9月皖南医学院附属弋矶山医院收治的4例疟疾患者血样,提取血样疟原虫DNA,采用巢式PCR技术对其18S rRNA基因进行扩增、克隆测序分析,并与GenBank中疟原虫相应序列进行比较分析,采用MEGA X软件建树,运用分子进化树分析芜湖市疟疾的地理起源。结果 4例患者PCR电泳结果获得片段均为206 bp,经BLAST比对证实均为恶性疟原虫感染。系统进化树显示,4例病例检测基因片段与GenBank中非洲分离株聚集在相同亚支,其余由GenBank上获取的恶性疟原虫序列也都分别按照地理来源聚集在相同的进化亚支内。结论 18S rRNA基因是能够确定疟疾感染来源地的准确、特异性的溯源分子标记物。

云南省蛋鸡源隐孢子虫鉴定及18S rRNA生物信息学分析

为了了解云南省规模化蛋鸡场腹泻病鸡隐孢子虫的感染情况,试验采用卢戈氏碘液和改良抗酸染色镜检、巢式PCR扩增隐孢子虫18S rRNA基因、BLAST程序测序及遗传进化分析等方法对2020年12月份从云南省多个规模化蛋鸡场采集的71份腹泻蛋鸡的粪便样品进行研究。结果表明:经卢戈氏碘液染色后可见呈棕色近似香蕉形的子孢子,经改良抗酸染色后可见呈红色近似球形的卵囊和近似香蕉形的子孢子。巢氏PCR扩增得到大小为821 bp的目的片段,将获得的虫株命名为YX202106虫株。YX202106虫株与GenBank中已公布的火鸡隐孢子虫的同源性最高,为99.14%~99.51%,且在进化树上处于同一个分支,该虫株还与能引起人畜共患的五种主要隐孢子虫——人隐孢子虫、微小隐孢子虫、火鸡隐孢子虫、猫隐孢子虫、犬隐孢子虫在进化树上构成一个大分支,具有重要的公共卫生学意义。说明云南地区蛋鸡群中存在火鸡隐孢子虫感染,应加强蛋鸡隐孢子虫病的防治。

Morphogenesis and Molecular Phylogeny of a Marine Urostylid Ciliate Apokeronopsis wrighti(Protista, Ciliophora, Hypotrichia)

Hypotrichs are one of the highly differentiated ciliated lineages which play important roles in ecological, environmental,evolutionary and basic biological studies. In the present study, we investigated the living characteristics, infraciliature, nuclear apparatus, ontogenesis and phylogenetic position of a marine hypotrichous ciliate, Apokeronopsis wrighti Long et al., 2008, which was isolated from coastal waters in Shenzhen, China. The new isolate resembles the type population in terms of morphological characteristics, morphometrics, and SSU rRNA gene sequence that is with a 99.7% similarity. Ontogenesis of A. wrighti is characterized by oral primordium for the proter as well as marginal and dorsal kineties anlagen in both filial products formed de novo, and the cirral row arranged along the paroral and endoral arises from several anterior frontoventral-transverse cirral streaks. Phylogenetic analyses based on SSU and concatenated gene data suggest that five species of Apokeronopsis form a monophyletic clade, and the genus Apokeronopsis is closely related to Thigmokeronopsis and Metaurostylopsis.

鱼类肌肉组织18S rRNA降解变化与死亡时间的关联性

自我国实施了海洋伏季休渔制度以来,确定市售鱼类的死亡时间成了渔业执法领域的难题。本论文借鉴了法医学研究,探索死亡鱼类肌肉组织中18S核糖体RNA(18s)降解程度来推断死亡时间的可行性。先通过分析在(0~1℃)低温贮存条件下斑马鱼肌肉组织18s的降解动力学,结果表明肌肉组织内18s呈现区域偏好性的时序降解。在此基础上,建立了一种基于逆转录PCR检测多个特定片长18s降解物的技术,推测鱼类死亡时间的方法原型。虽然该方法仍存在局限性,但在1~7 d的死亡后冷藏时间范围内具有可行性。

台湾小孔蛸寄生丛集球虫新种的形态特征及系统发育分析

为探究感染台湾小孔蛸(Cistopus taiwanicus)消化道中常见的寄生虫-从集球虫属(Aggregata)种类,本研究在浙江南麂岛、福建东山、广东阳江和海南三亚周边海域采捕台湾小孔蛸,在其盲囊和肠组织内发现了丛集球虫新种,新种命名为疣丛集球虫(Aggregata verrucosa sp.nov.)。采用光镜和电镜观察发现:疣丛集球虫的成熟卵囊为球形,有时不规则,长径范围936.96~1563.31μm,短径范围860.13~1227.85μm;孢子囊为球形或近球形,长径范围12.87~14.24μm,短径范围12.39~13.70μm。孢子囊壁为双层,外层光滑,内层具疣状突起。孢子囊内子孢子通常呈螺旋状,数目范围4~10个,子孢子长径范围12.81~16.01μm,短径范围2.02~2.62μm。利用18S rRNA基因构建的系统发育树显示该物种隶属于丛集球虫属,并高度支持了属内(中华丛集球虫A.sinensis+疣丛集球虫A.verrucosa)+A.octopiana+A.eberthi+多刺丛集球虫A.octaculeata的系统发生关系。本研究鉴定出的丛集球虫新种补充了丛集球虫属的生物学资料,同时本研究可为头足类疾病诊断和组织学病理研究提供参考。

珠江流域常见鱼类及大型底栖动物DNA条形码空缺分析

条形码数据库是开展基于DNA的生物监测关键先决条件。为在珠江流域有效开展基于DNA的生物监测,迫切需要了解物种DNA条形码的覆盖或空缺状况。整理了珠江流域常见鱼类和大型底栖动物的物种清单,从National Center and Biotechnology Information (NCBI)数据库中检索了物种清单的DNA条形码序列,分析了常见鱼类(包括线粒体组和12s rRNA基因)和大型底栖动物(包括线粒体组、COI和18s rRNA基因)的DNA条形码覆盖范围和空缺程度。数据分析表明:(1)珠江流域共记录了常见鱼类221种,隶属于2纲18目51科和137属;常见大型底栖动物105种/属,隶属于6纲14目53科。(2)共检索到常见鱼类线粒体组序列913条和12s rRNA基因序列962条,分别占总物种的81.45%和57.92%;有12.67%的物种没有线粒体组和12s rRNA基因序列,若将条形码阈值设置为至少包含5个参考序列,则空缺度上升至52.94%;(3)共检索到常见大型底栖动物线粒体组65条序列、COI基因26,988条序列和18s rRNA基因175条序列,分别占总种/属数的29.52%、68.57%和37.14%;有25.71%的种/属在线粒体组、COI和18s rRNA基因区域皆无序列收录,若将条形码阈值设置为至少包含5个参考序列,则空缺度上升至41.90%。总之,本研究将为珠江流域开展基于DNA的鱼类和大型底栖动物监测提供基础数据支撑,为完善珠江本土DNA条形码数据库提供参考建议。

喀什地区某牛场环形泰勒虫的分子生物学鉴定及分析

试验旨在鉴定喀什地区某牛养殖场的西门塔尔牛的病因,对疑似感染泰勒虫的病牛血液进行PCR检测。运用PCR方法对泰勒虫18S rRNA基因进行扩增、测序,通过MEGA 7.0软件构建系统进化树,同时进行遗传进化分析。结果显示,18S rRNA基因序列与环形泰勒虫中国新疆分离株(OM438135.1)同源性为100.0%,表明该场病牛为环形泰勒虫感染;系统进化树分析结果显示,该虫株与中国和巴基斯坦分离株亲缘关系比较近。研究表明,导致喀什某牛场西门塔尔牛患病的病原体是环形泰勒虫。

永州地区犬球虫的感染情况调查、虫种鉴定及种系发育关系研究

为了了解永州地区犬球虫的感染情况,确定优势虫种,并探究不同球虫虫种之间的种系发育关系,试验利用饱和重铬酸钾溶液漂浮法对采自永州地区的362份犬粪便进行虫卵检测,通过宏基因组测序技术鉴定虫种,PCR扩增球虫的18S r RNA、ITS-1基因,经测序、比对确定永州地区流行的优势虫种,并对其种系发育关系进行分析。结果表明:有124份犬粪便为阳性,总体感染率为34.25%,其中宠物犬的感染率为24.31%,流浪犬的感染率为44.20%;在永州地区有犬等孢球虫与俄亥俄等孢球虫流行,其中犬等孢球虫33株(占比26.61%),俄亥俄等孢球虫91株(占比73.39%);犬等孢球虫18S r RNA、ITS-1基因序列的长度分别为738 bp与1000 bp,俄亥俄等孢球虫18S r RNA、ITS-1基因序列的长度分别为1051 bp和398 bp。两种犬球虫18S r RNA、ITS-1基因序列的相似性分别为98.53%~98.76%和99.58%~99.72%。此外,在基于18S r RNA、ITS-1基因序列所组建的2个种系发育树中,犬等孢球虫与俄亥俄等孢球虫均处于每个种系发育树的同一分支上。说明在永州地区犬球虫的感染率较高,俄亥俄等孢球虫为该地区的优势虫种,且犬等孢球虫与俄亥俄等孢球虫之间存在较近的亲缘关系。

林芝地区羊蜱蝇的形态学及分子鉴定

为了鉴定西藏林芝地区绵羊外寄生虫的种属类型,采集林芝地区羊蜱蝇成虫30只、蛹5只,通过形态学作初步鉴定,同时基于18S rRNA序列的PCR方法进行分子鉴定,获得18S rRNA基因序列进行Blast相似性搜索,应用MegAlign和MEGA 7.0软件完成同源性分析及种系发育分析。结果显示:虫体18S rRNA序列与中国新疆2016年上传的羊蜱蝇序列同源性达99.5%~99.9%,差异性在0.1%~0.5%之间;进化树显示与蜱蝇属、羊蜱蝇18S rRNA基因序列聚类。研究表明林芝地区绵羊感染的外寄生虫是羊蜱蝇。