车轮虫齿体特征的量化及其种内种间系统发育的研究

齿体特征是车轮虫物种鉴定的一个重要依据。本文首次提出一种齿体结构面积比例量化法,即通过对齿钩、齿锥、齿棘、齿体环各部分反口面观面积的测量,分别计算出前三者与所在齿体的面积比以及所有齿体总面积与齿体环的面积比,从而将齿体特征转换为量化指标依据所测量化值,采用相似性聚类法,我们首先探讨了显著车轮虫(Trichodina nobbillis chen,1963)的三个种群的亲缘关系,然后以劳牧小车轮虫(Trichodinella lomi Xu,Song ＆ Warren,1999)为外类群,选择显著车轮虫(Trichodina nobillis chen,1963)、异齿车轮虫(Trichodina heterodentata Duncan,1977)、杜氏车轮虫(Trichodina dormergui Wallengren,1897)和Trchodina fahaka(Khaled A．S．Al-Rasheid,2000)为研究对象,对车轮虫种间亲缘关系进行了分析。结果表明,Pdr值可以有效地表现种群间的差异;同时还发现齿棘面积比例较高的杜氏车轮虫和异齿车轮虫进化较晚,而齿棘面积比例较低的显著车轮虫进化较早,并得出了齿棘越发达,车轮虫越进化这一结论,支持了徐奎栋等所提出的游走亚目六种基本齿型的进化顺序。这种量化研究方法不但可对车轮虫齿体的各部分结构以及它们之间的相互关系进行定量描述,而且还可以这些数据为基础对车轮虫种群间及物种间亲缘关系进行研究。     

鲫寄生车轮虫一新种的描述

本文记述了寄生在鲫鱼鳃上的车轮虫属一新种——Trichodina paranigra sp．nov．。文中采用Lom(1960)所倡导的统一特定方法及由Van As ＆ Basson(1989)提出的齿体各鉴定特征的定位描述,对本新种的各形态学特征(包括虫体外形、附着盘、齿体、辐线、中央颗粒、核器、口围绕体等)进行了较为详尽的描述,并对本种与相似种进行了比较研究。文中还为齿体定位描述模式图新增了两个参数。     

广西地区罗非鱼外寄生车轮虫的形态分类学及多样性研究

采用活体观察、吉姆萨与干银法染色等技术,运用国际间统一特定方法及齿体定位描述法对采自广西地区的罗非鱼(单性鱼吉富、奥尼系列)的外寄生车轮虫进行详细的形态分类学与多样性研究,研究结果共计获得车轮虫属2种,拟车轮虫属与三分虫属各1种,分别为:刺纹车轮虫Trichodina centrostrigata Basson et al.,1983、异齿车轮虫Trichodina heterodentata Duncan,1977、非洲拟车轮虫Paratrichodina africana Kazubski&El-Tantawy,1986与直齿三分虫Tripartiella orthodens Basson&Van As,1987。其中,非洲拟车轮虫为中国新纪录,刺纹车轮虫和直齿三分虫为中国广西地区新纪录。非洲拟车轮虫表现出寄主和寄生部位的专一性;进一步应用SPSS统计软件对非洲拟车轮虫进行了种群差异性分析。     

抗菌肽HBβ-C在全雄和杂交黄颡鱼对多子小瓜虫抗性差异中的作用

为研究全雄黄颡鱼(Pelteobagrus fulvidraco)、瓦式黄颡鱼(Pelteobagrus vachelli)和杂交黄颡鱼(黄颡鱼P. fulvidraco♀×瓦氏黄颡鱼P. vachelli♂)对多子小瓜虫(Ichthyophthirius multifiliis)的抗性差异, 通过生物信息学分析黄颡鱼皮肤黏液蛋白质组, 发现其血红蛋白源抗菌肽(HBβ-C)位于血红蛋白β链HBβ的碳端, 共33个氨基酸。利用化学合成的不同浓度的HBβ-C肽段进行体外抗虫实验, 研究发现其能有效杀死滋养体、包囊体和掠食体阶段的多子小瓜虫, 其中15 μg/mL的HBβ-C能在3min内杀死所有滋养体。基因表达量分析显示, 在杂交黄颡鱼的鳃和皮肤组织中, HBβ的mRNA表达量高于全雄黄颡鱼; 但在应对小瓜虫感染的过程中, 全雄黄颡鱼的HBβ mRNA转录水平快速提升, 其表达水平和上升倍率显著高于杂交黄颡鱼。蛋白表达量分析显示, HBβ在全雄黄颡鱼鳃组织中的蛋白表达量明显高于杂交黄颡鱼。免疫荧光定位结果显示, 抗菌肽HBβ-C特异地在红细胞中表达, 可以分泌并附着在滋养体上。综上所述, 相对于杂交黄颡鱼, 全雄黄颡鱼中HBβ具有更高的翻译效率, 可以更高效地应对多子小瓜虫的感染。     

矩形车轮虫的再描述

目的:我们对泸州市鲇类寄生车轮虫进行调查,对于已经发现,但由于原来的技术水限制需要重新描述的,或缺少资料的车轮虫,给予重新描述或补充新的资料。方法:用国际间统一的干银法染色以显示车轮虫的附着盘结构和口围绕度,用Lom(1958)倡导的"统一的特定方法"(Uniform specifi characteristic system)进行测量,用Van&Bassion(1989)提供的方法描述车轮虫的齿体。结果:对寄生在泸州地区养殖鱼类斑点叉尾鮰(Ietalurus punetaus)的鳃上的车轮虫进行了描述,该车轮虫是矩形车轮虫(Trichodina rectangli)。结论:矩形车轮虫是陈启鎏和谢杏人1964年在青鱼、草鱼等鱼身体上发现的,但是没有显微照片。国际上有许多它的同种异名的车轮虫的描述,这些文献也只有附着盘的显微照片。斑点叉尾鮰是矩形车轮虫的新寄主记录。本研究用国际统一的方法对矩形车轮虫进行描述,并绘出了矩形车轮虫的齿体定位图,提供了完整的统计数据,以及完整的显微照片,即附着盘、口围绕度和细胞核显微照片。     

重寄生车轮虫的新分布和再描述

于乌鳢（Chanaa argas）鳃上获得一外寄生原动物,经鉴定为一缘毛类目纤毛虫——重寄生车轮虫（Trchodina hypervatositis Chen ＆ Hsish,1984）。按照国际上对车轮虫形态学特征鉴定的标准,本工作用齿体定位描述对该虫进行了研究,补充并完善了重寄生车轮虫的形态描述,如缘膜宽、大核内外径、齿长及齿棘长等数据,同时提供了显微照片（图1）及齿体定位描述图（图2）。     

重庆地区泥鳅体外寄生车轮虫的形态分类学研究

在重庆地区鱼类寄生虫的调查过程中,从淡水鱼泥鳅的鳃上获得四种外寄生车轮虫,经鉴定隶属车轮虫科（Trichodinidae）、车轮虫属（Trichodina）,含1车轮虫未定种Trichodina．sp．和3已知种,即：适度车轮虫（TrichodinamodestaLom,1970）,斜齿车轮虫（Trichodina lechriodentata Zhao＆Tang,2007）及拟黑色车轮虫（TrichodinaparanigraTangetal,2005）。车轮虫的形态学特征统计采用Lom（1958）倡导的统一特定方法,齿体的定位描述则采用Van As＆Basson（1989）和Tang,Zhao＆Chen（2005）改进的方法进行     

大亚湾养殖鱼类寄生车轮虫的种类组成与季节动态

2017年8月至2018年7月对大亚湾8种养殖鱼类的车轮虫感染情况进行了调查, 采用Lom倡导的“统一特定方法”及Van As & Basson提出的“齿体定位描述法”对采获的车轮虫标本进行了形态鉴定与描述。结果在卵形鲳鲹(Trachinotus ovatus)、星点笛鲷(Lutjanus stellatus)、鞍带石斑鱼(Epinephelus lanceolatus)、眼斑拟石首鱼(Sciaenops ocellatus)和虎龙杂交斑[E. fuscoguttatus (♀) × E. lanceolatus (♂)]等5种鱼上共检获5种车轮虫: 亚卓车轮虫(Trichodina jadranica)、莱普斯车轮虫(T. lepsii)、长须鳠车轮虫(T. guliae)、劳牧小车轮虫(Trichodinella lomi)和斜拟车轮虫(Paratrichodina oblique); 其中卵形鲳鲹5种虫均有寄生, 虎龙杂交斑感染了4种虫, 星点笛鲷、眼斑拟石首鱼和鞍带石斑鱼分别感染了3种、2种和1种虫。周年感染数据表明: 5种虫的感染率均以卵形鲳鲹最高, 眼斑拟石首鱼上2种虫的感染率最低; 莱普斯车轮虫平均感染强度最高的宿主是虎龙杂交斑, 其他4种虫的均是卵形鲳鲹。寄生车轮虫的季节动态表明: 冬季的感染率和平均感染强度较低; 不同虫种感染高峰出现的季节不一, 其中斜拟车轮虫的感染高峰出现在秋季, 其春夏秋季的平均感染强度均大于其他4种虫。研究填补了大亚湾海区鱼类寄生车轮虫研究的空缺, 为该海区车轮虫病的诊断与防控提供了参考。     

洞庭湖四种黄颡鱼基因组DNA遗传多样性的RAPD分析

以洞庭湖瓦氏黄颡鱼、光泽黄颡鱼、长须黄颡鱼、普通黄颡鱼的基因组DNA为材料对 4种黄颡鱼进行了遗传多样性随机扩增多态性DNA(RAPD)分析。通过筛选的 90个引物对 4种黄颡鱼基因组DNA的扩增 ,获得了 36个有效引物 ,并计算出了 4种黄颡鱼群体间的遗传相似系数和遗传距离 ,遗传距离最大的为普通黄颡鱼和瓦氏黄颡鱼 (D =0 9895 ) ,遗传距离最小的为普通黄颡鱼和光泽黄颡鱼 (D =0 672 0 )。同时运用聚类分析 (UPGMA)的方法建立了 4种黄颡鱼的聚类图。     

云南地区水生经济动物外寄生车轮虫的研究

在对云南地区淡水车轮虫的调查研究过程中,在几大重要水生经济动物,即泥鳅、鲫、鲤、鳙、斑点叉尾鮰及牛蛙蝌蚪上获得8种外寄生车轮虫。其中车轮虫属4种,即虱性车轮虫Trichodina pediculus、杜氏车轮虫T.domerguei、拟黑色车轮虫T.paranigra和适度车轮虫T.modesta;拟车轮虫属1种,即圆形拟车轮虫Paratrichodina rotundiformis;小车轮虫属3种,即周丛小车轮虫Trichodinella epizootica、卡普小车轮虫T.carpi和纤细小车轮虫T.subtilis。采用活体观察、甲基绿-派洛宁染色及干银法,并运用国际统一的特定方法及齿体定位描述法对该8种车轮虫进行了详细的形态学研究。     

Interaction and ovicidal activity of nematophagous fungus Pochonia chlamydosporia on Taenia saginata eggs

The ovicidal activity of the nematophagous fungi Pochonia chlamydosporia (isolates VC1 and VC4), Duddingtonia flagrans (isolate AC001) and Monacrosporium thaumasium (isolate NF34) on Taenia saginata eggs was evaluated under laboratory conditions. T. saginata eggs were plated on 2% water-agar with fungal isolates and controls without fungus and examined after 5, 10 and 15 days. At the end of the experiment P. chlamydosporia showed ovicidal activity against T. saginata eggs (p < 0.05), mainly for internal egg colonization with results of 12.8% (VC1) and 2.2% (VC4); 18.1% (VC1) and 7.0% (VC4); 9.76% (VC1) and 8.0% (VC4) at 5, 10 and 15 days, respectively. The other fungi showed only lytic effect without morphological damage to the eggshell. Results demonstrated that P. chlamydosporia was effective in vitro against T. saginata eggs unlike the other fungi.     

Differential diagnosis of Taenia asiatica using multiplex PCR

Taenia asiatica and T. saginata are frequently confused tapeworms due to their morphological similarities and sympatric distribution in Asian regions. To resolve this problem, a high-resolution multiplex PCR assay was developed to distinguish T. asiatica infections from infection with other human Taenia tapeworms. For molecular characterization, the species specificity of all materials used was confirmed by sequencing of the cox1 gene. Fifty-two samples were analyzed in this study, comprising 20 samples of T. asiatica genomic DNA from China, Korea, and the Philippines; 24 samples of T. saginata from Belgium, Chile, China, Ethiopia, France, Indonesia, Korea, Laos, the Philippines, Poland, Taiwan, Thailand, and Switzerland; and 10 samples of T. solium from Cape Verde, China, Honduras, and Korea. The diagnostic quality of the results obtained using PCR and species-specific primers designed from valine tRNA and NADH genes was equal to that based on the nucleotide sequencing of the cox1 gene. Using oligonucleotide primers Ta4978F, Ts5058F, Tso7421F, and Rev7915, the multiplex PCR assay was useful for the differentially diagnosing T. asiatica, T. saginata, and T. solium based on 706-, 629-, and 474-bp bands.     

Hc-daf-2 encodes an insulin-like receptor kinase in the barber’s pole worm, Haemonchus contortus, and restores partial dauer regulation

Infective L3s (iL3s) of parasitic nematodes share common behavioural, morphological and developmental characteristics with the developmentally arrested (dauer) larvae of the free-living nematode Caenorhabditis elegans. It is proposed that similar molecular mechanisms regulate entry into or exit from the dauer stage in C. elegans, and the transition from free-living to parasitic forms of parasitic nematodes. In C. elegans, one of the key factors regulating the dauer transition is the insulin-like receptor (designated Ce-DAF-2) encoded by the gene Ce-daf-2. However, nothing is known about DAF-2 homologues in most parasitic nematodes. Here, using a PCR-based approach, we identified and characterised a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2 displays significant sequence homology to insulin receptors (IR) in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2 is consistent with that of the human IR, suggesting that it is involved in the formation of the IR complex. The Hc-daf-2 gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the iL3 compared with other stages. To compare patterns of expression between Hc-daf-2 and Ce-daf-2, reporter constructs fusing the Ce-daf-2 or Hc-daf-2 promoter to sequence encoding GFP were microinjected into the N2 strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2. Taken together, these findings provide a first insight into the roles of Hc-daf-2/Hc-DAF-2 in the biology and development of H. contortus, particularly in the transition to parasitism.     

Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses

Hyaloscyphaceae is the largest family in Helotiales, Leotiomycetes. It is mainly characterized by minute apothecia with well-differentiated hairs, but its taxonomic delimitation and infrafamilial classification remain ambiguous. This study performed molecular phylogenetic analyses using multiple genes including the ITS-5.8S rDNA, the D1–D2 region of large subunit of rDNA, RNA polymerase II subunit 2, and the mitochondrial small subunit. The primary objective was to evaluate the phylogenetic utility of morphological characters traditionally used in the taxonomy of Hyaloscyphaceae through reassessment of the monophyly of this family and its genera. The phylogenetic analyses inferred Hyaloscyphaceae as being a heterogeneous assemblage of a diverse group of fungi and not supported as monophyletic. Among the three tribes of Hyaloscyphaceae only Lachneae formed a monophyletic lineage. The presence of hairs is rejected as a synapomorphy, since morphologically diversified hairs have originated independently during the evolution of Helotiales. The true- and false-subiculum in Arachnopezizeae are hypothesized to have evolved through different evolutionary processes; the true-subiculum is likely the product of a single evolutionary origin, while the false-subiculum is hypothesized to have originated multiple times. Since Hyaloscyphaceae sensu lato was not resolved as monophyletic, Hyaloscyphaceae sensu stricto is redefined and only applied to the genus Hyaloscypha.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum. Manila clams, with variable levels of infection with Perkinsus olseni, were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum. After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions.Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.     

高榕榕果内Eupristina属两种榕小蜂的遗传进化关系

对生长在福州地区的高榕进行长期追踪观察,发现高榕榕果内仅生活着Eupristina altissima和Eupristina sp.榕小蜂,前者为高榕的传粉小蜂,后者无传粉行为,两者雌蜂之间在体色、触角、花粉袋和花粉刷等部位存在细微的差异,而两者雄蜂之间无形态差异。通过克隆福建地区5个样地的高榕榕果内收集到的E. altissima和Eupristina sp.榕小蜂,以及细叶榕的传粉小蜂Eupristina verticillata(外群)的Cytb及COI基因,并进行碱基组成及遗传距离分析,用邻接法构建系统发育树,分析两榕小蜂群体之间的遗传进化关系,结果显示:(1)榕小蜂COI及Cytb序列碱基组成中A+T的含量(Cytb序列中A+T=75.3%,COI序列中A+T=75.5%)显著高于G+C,符合膜翅目昆虫线粒体基因碱基组成特征。(2)对两群体小蜂进行遗传距离分析显示,Cytb序列中E. altissima和Eupristina sp. 群体内各样本之间的平均遗传距离分别为0.0092和0.0030,而E. altissima与Eupristina sp. 群体间的平均距离为0.1588;COI序列中E. altissima 与Eupristina sp. 群体内各样本之间的平均遗传距离分别为0.0065和0.0205,而二者群体间的平均遗传距离为0.1043,表明两者群体间的遗传距离明显大于各自群体内各样本间的遗传距离。统计GenBank中下载的6个属34种榕小蜂Cytb序列的种间遗传距离为0.0811-0.1723,6个属28种榕小蜂COI序列的种间遗传距离为0.0939-0.1986。由此认为E. altissima和Eupristina sp.之间的遗传距离差异已经达到了种间水平,即E. altissima与Eupristina sp.为两个不同的种。(3)在形态上,两种小蜂的雌蜂之间有微小差异,而二者雄蜂之间无差异,但Cytb与COI序列分析结果一致表明:E. altissima与Eupristina sp.雄蜂之间,以及二者雌蜂之间的遗传距离均差异显著,表明形态变异滞后于基因变异。雌蜂在表型上进化快于雄蜂,可能是由于雌蜂羽化后从榕果出飞,受到外界环境因素的影响较大,且两种雌蜂在传粉功能上存在差异,故二者之间的形态差异较大,而雄蜂寿命短,又终生生活在黑暗封闭、环境变化相对恒定的榕果内,两种雄蜂在行为上不存在差异,故二者表型变异较为缓慢。E. altissima和Eupristina sp.小蜂对宿主的专一性不强,在榕-蜂协同进化过程中,可能发生过宿主转移事件。     

空心莲子草响应南方菟丝子寄生的生长-防御权衡

为探讨全寄生植物南方菟丝子(Cuscuta australis)防治入侵植物空心莲子草(Alternanthera philoxeroides)的可行性,以二者野外天然生长的种群为研究对象,分析南方菟丝子寄生对空心莲子草生长及防御的影响,阐明空心莲子草在受到寄生胁迫时如何权衡自身生长与防御的关系,进而发展出一套应对南方菟丝子寄生的生长-防御策略。结果显示:(1)南方菟丝子寄生显著改变空心莲子草茎的形态,茎直径和平均节间长均增加,茎直径变化极显著(P0.01);(2)南方菟丝子寄生显著减少空心莲子草叶片数,但同时显著增加后者茎的分枝数,而茎上的节是潜在的无性繁殖体,故有利于空心莲子草的克隆繁殖;此外,南方菟丝子寄生显著降低了空心莲子草的根、茎、叶生物量和总生物量,抑制空心莲子草的生长;(3)南方菟丝子寄生显著增加空心莲子草茎的单宁、总酚、三萜皂苷含量,增强其防御能力;(4)南方菟丝子寄生的空心莲子草的生物量与茎部木质素、三萜皂苷、单宁和总酚含量均呈现显著负相关性(P0.01),对照组则不存在相关性;且寄生组较对照组相比,生物量的相对百分比显著低于对照组(P0.01),而用于防御的次生代谢产物总含量的相对百分比显著高于对照组(P0.01)。以上结果表明,受到南方菟丝子寄生胁迫后,空心莲子草改变自身的生长-防御策略,减少营养生长投入而将更多的资源投向克隆繁殖,同时增强对"防御"物质的投入,增强其防御能力,以利于后代生存和繁衍。     

河南省发现宁波滑蜥

2011年7月、2012年7月和10月在河南伏牛山国家级自然保护区采集到5号宁波滑蜥(Scincella modesta)标本,系河南首次发现,标本保存于河南大学生命科学学院动物学标本室。