洪湖碘泡虫的再描述及其近缘种的鉴别性研究

研究基于形态特征和18S rDNA序列相似度、遗传距离、变异位点、GC含量和系统发育比较分析,对采自河南龙湖的寄生于异育银鲫鳃部的一种黏孢子虫以及相似性极高且易混淆的黏孢子虫种类(洪湖碘泡 Myxobolus honghuensis Liu,et al. 2012、瓶囊碘泡虫Myxobolus ampullicapsulatus Zhao,et al. 2008、咽碘泡虫Myxobolus pharynae Lu,et al. 2012和吴李碘泡虫Myxobolus wulii (Wu Li,1986) 进行了系统的鉴别研究。研究结果显示: 河南龙湖异育银鲫鳃部所检获的黏孢子虫为洪湖碘泡虫,该种群对所寄生的异育银鲫未造成疾病症状; 咽碘泡虫与洪湖碘泡虫各种群在形态上极相似,两者间18S rDNA序列相似度为99%-100%,遗传距离为0-0.0013,GC含量均为44.31%,变异位点为2个,表明咽碘泡虫与洪湖碘泡虫应为同一物种。     

鲤肠道寄生岳阳碘泡虫新种的形态特征及系统发育分析

在洞庭湖岳阳地区开展鱼类寄生虫调查中,发现一种寄生于鲤Cyprinus carpio L.肠道的黏孢子虫。该黏孢子虫的孢囊呈白色,椭圆形,大小为(1.0±0.2) mm (0.8—1.2 mm)。成熟孢子具有壳瓣,壳面观近似圆形,后端有4—6个“V”形褶皱;缝面观呈纺锤形,缝脊直而粗;孢质均匀,含有一个嗜碘泡;孢子长(9.8±0.6) μm (9.6—10.0 μm),孢子宽(8.2±0.3) μm (8.0—8.5μm),孢子厚(7.3±0.1) μm (7.0—7.5 μm);2个极囊梨形,位于孢子顶端,大小相等,呈“八”字形;极囊长(4.4±0.4) μm (3.8—5.1 μm),宽(2.7±0.2) μm (2.2—3.2 μm),极丝4—5圈。该黏孢子虫与肠膜碘泡虫、丑陋圆形碘泡形态特征非常相似,但其极囊/孢子小于1/2;与文献已报道的鲤肠道寄生北京碘泡虫和鲤肠碘泡虫相比较,其在孢子形态、孢子和极囊大小方面分别存在明显差异。基于该黏孢子虫18S rDNA基因序列(GenBank登录号KY203795)比对分析,该黏孢子虫与山东碘泡虫相似率最高,仅为96%。系统发育分析发现,该黏孢子虫与山东碘泡虫、倪李碘泡虫、住心碘泡虫、Myxobolus encephalicus、Sphaerospora molnari、多涅茨尾孢虫和Henneguya zikaweiensis聚为独立分支,和其他已报道的黏孢子虫亲缘关系较远。综合形态学和18S rDNA基因序列数据,文章报道的鲤肠道寄生黏孢子虫为碘泡虫属一新物种,将其命名为岳阳碘泡虫。     

洪湖碘泡虫SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及其应用

洪湖碘泡虫(Myxobolus honghuensis)引起的鲫“喉孢子虫病”严重危害我国异育银鲫养殖。病原丰度是决定病害发生的最重要因素之一, 因此建立洪湖碘泡虫的定量检测方法, 不仅可用于异育银鲫“喉孢子虫病”的早期诊断, 也可应用于养殖系统中洪湖碘泡虫的定量监测, 为该病的暴发风险预警及防控措施的效果评价提供技术手段。研究根据洪湖碘泡虫的ITS基因序列, 设计合成一对特异性引物HHF/R, 建立了洪湖碘泡虫的SYBR Green Ⅰ实时荧光定量PCR方法, 并对该方法的特异性、灵敏性、重复性及应用性进行了验证。结果显示, 该方法能特异性检测出洪湖碘泡虫, 而与多涅茨尾孢虫、倪李碘泡虫、普洛宁碘泡虫、吴李碘泡虫之间无交叉反应; 最低检测限为3.02×101copies/μL, 灵敏性较常规PCR高出1000倍; 组内和组间重复性试验的变异系数均小于2%。应用该方法可定量检出洪湖碘泡虫全生活史阶段, 包括鱼体内移行发育的前孢子阶段及养殖系统环境, 如池塘水样及底泥样品中分布的洪湖碘泡虫。因此, 所建立的洪湖碘泡虫SYBR Green Ⅰ实时荧光定量PCR方法特异性好、灵敏度高、重复性稳定, 可应用于异育银鲫全养殖阶段洪湖碘泡虫的定性、定量监测。     

圆形碘泡虫和关桥碘泡虫孢子的光镜及扫描电镜观察

对寄生于鲫的圆形碘泡虫(Myxobolus rotundus Nemeczek,1911)及异育银鲫的关桥碘泡虫(M．guanqiaoensis Wu ＆Wang,1997)的成熟孢子进行光镜及扫描电镜观察。圆形碘泡虫主要寄生于鲫体表、头部、鳃、吻部及鳍条,形成许多大小不一的乳白色胞囊。关桥碘泡虫主要寄生于异育银鲫的肝脏,肝脏基本上被虫体所充满,肝脏组织被破坏。扫描电镜观察表明圆形碘泡虫成熟孢子表面光滑,缝脊直而明显,宽0．2μm,缝脊上下两侧对称。关桥碘泡虫成熟孢子表面皱褶,其程度不等,有的虫体一侧向内凹陷形成盆状,缝脊呈“S”形,宽0．5μm,缝脊上下两侧不对称,其中间有一不很明显凹陷的沟。     

长江中游长吻鮠、蛇鮈寄生粘孢子虫感染率的季节动态

1.寄生长吻鮠上的粘孢子虫有3种:巨囊两极虫和四极虫一种,寄生在胆囊中,碘泡虫一种寄生在肾脏中;寄生蛇鮈上的粘孢子虫有2种:湖北碘泡虫,寄生在脑、鳃、肾、体腔、肠,主要寄生在脑,楚克拉虫一种,寄生在胆囊中。2.粘孢子虫对长吻鮠、蛇鮈的感染率存在明显的季节变化;对长吻鮠的感染率最高值出现在6月(95%),最低值出现在2月(43%),年平均值为65%;湖北碘泡虫对蛇鮈的总感染率最高值出现在12—2月(63—67%),其次在8月(58%),春秋两季感染率较低(36—44%),年平均值为51%。3.总感染率(IR)与鱼体全长(TL)的关系:长吻鮠IR=0.956-0.009TL;蛇鮈IR=-2.0678+0.3838(TL)-0.0136(TL)2,8厘米以下的蛇鮈未发现感染。4.粘孢子虫的感染率与长吻鮠、蛇鮈的性别无关。5.感染粘孢子虫的长吻鮠,肥满度下降不大(0.04),感染粘孢子虫的蛇鮈,肥满度下降0.12,体重平均下降12.4%。6.长吻鮠中寄生粘孢子虫的总感染率(IR)与长江中游水温(T℃)呈显著正相关,二者关系可用下式描述:IR=0.3129+0.0192(T℃)。蛇鮈寄生粘孢子虫的总感染率(IR)与水温(T℃)之间的关系呈一反抛物线型、两者关系可用下式描述 IR=1.1228-0.073777T+0.001795T2       

海城碘泡虫(黏体动物门，黏孢子纲)补充描述及基于18S rDNA系统发育分析

海城碘泡虫原始描述中形态数据较为简单,且存在多个宿主及寄生部位,其有效性有待确定。利用现行主流的黏孢子虫形态特征和基因标记系统分析相结合的分类学方法,对采自太湖棒花鱼鳃丝的海城碘泡虫进行了补充描述。该碘泡虫孢囊呈白色,圆形,大小为(0.6—1.1) mm。成熟孢子正面观近似椭圆形,上端稍尖,侧面观呈纺锤型,孢子长(10.8±0.7) μm (10.1—11.5 μm),孢子宽:(8.1±0.5) μm (7.5—9.0 μm),孢子厚:(5.7±0.4) μm (5.2—9.0 μm);两极囊呈梨形,大小存在细微差别,极囊顶端存在突起,大极囊长:(4.7±0.5) μm (4.8—6.7 μm),宽:(2.5±0.2) μm (3.2—4.3 μm),小极囊长:(4.4±0.2) μm (4.1—4.8 μm),宽:(2.2±0.1) μm (2.0—2.5 μm);极丝盘绕4—5圈。基于18S rDNA序列(GenBank登录号:KY965936)比对分析,该碘泡虫与放射孢子虫Hexactinomyxon type 2相似率最高,为97%。系统发育分析表明,该碘泡虫与Hexactinomyxon type 2、Hexactinomyxon type 1、Hexactinomyxon type SH-2006、Myxobolus pfeifferi、Myxobolus caudatus和Myxobolus squamae聚为独立分支,和其他已报道的黏孢子虫亲缘关系较远。研究在补充了海城碘泡虫形态学、基因标记序列信息基础上,推断了该虫生活史。     

重庆淡水鱼寄生粘孢子虫二新种(粘孢子纲:双壳目)

报道了中华倒刺鲃鳃上的粘体虫一新种,即倒刺鲃粘体Myxosoma spinibarbi sp.nov.;寄生于白鲢肾中的碘泡虫一新种,即马氏碘泡虫Myxobolus mai sp.nov.,将2种与近似种进行了比较。     

中国西部淡水鱼类寄生粘孢子虫的记述：粘孢子纲：双壳目

本文报道新疆与四川淡水鱼类寄生粘孢子虫７种,即阿勒泰两极虫,白甲两极虫,宁南两极虫,Ｊｕ楚克拉虫,杜氏粘体虫,宜宾碘泡虫,鲈尾孢虫,除杜氏粘体虫和鲈尾孢虫外,其余５种为新种。本文是首次对新疆地区粘孢子虫研究的报道。     

吴李碘泡虫的重描述及其长江流域不同江段株系的比较研究

研究在对吴李碘泡虫Myxobolus wulii (Wu & Li) Landsberg & Lom, 1991重描述的基础上, 基于形态和分子数据对长江流域不同江段的吴李碘泡虫(重庆株系、湖北株系及江苏株系)进行了比较研究。结果表明: 吴李碘泡虫重庆株系孢子及极囊量度比湖北株系略小, 重庆株系两极囊等大而湖北株系两极囊大小不等。重庆株系、湖北株系及江苏株系18S rDNA序列相似度为99.2%—99.9%, 遗传距离为0.002—0.007。系统发育分析显示: 吴李碘泡虫并未形成地理种群特有的进化枝, 也并未依宿主种类而聚支, 而是依据寄生部位不同分为鳃寄生和肝胰脏寄生2大支系。这表明, 相同寄生部位的吴李碘泡虫具有更近的亲缘关系。吴李碘泡虫的2大支系中, 鳃寄生种群先分化出来, 这可能与体表寄生和体内寄生的演化有关, 而鳃寄生的吴李碘泡虫可能是较早定居的群体。     

浙江省淡水鱼类寄生粘孢子虫一新种和属的新纪录：粘孢子目：碘泡科

本文报道寄生于淡水鱼斑尾复鰕虎鳃弓骨骨膜上一种罕见的粘孢子虫,形态结构特殊,孢质中含有明显的嗜碘泡,分类地位应属碘泡科的三角孢子虫属 Trigono-sporous。这次发现,是该属在我国的新纪录,并定名浙江三角孢子虫新种。文内叙述该种的形态结构和孢囊内发育各阶段的母孢子及泛成孢子母细胞等。     

Mass mortality of pond-reared Carassius gibelio caused by Myxobolus ampullicapsulatus in China

From June to August 2009, allogynogenetic silver crucian carp Carassius gibelio (Bloch) pond-cultured at the Nanquan Experimental Station, China, were found to be heavily infected with myxosporeans, which caused mortalities ranging from 33% (13/40) to 90% (36/40) in the cages. The pharynxes of infected fish were swollen, nodular, and severely damaged. Based on morphological characters and 18S small subunit ribosomal DNA sequence similarity, the myxosporean was identified as Myxobolus ampullicapsulatus. This is the first report of M. ampullicapsulatus causing mass mortality of pond-reared C. gibelio.     

Myxozoan pathogens in cultured Malaysian fishes. II. Myxozoan infections of redtail catfish Hemibagrus nemurus in freshwater cage cultures

Cage-cultured Asian redtail catfish Hemibagrus nemurus (Valenciennes, 1840), a popular food fish in Southeast Asia, proved to be infected by 3 myxozoan species. All the 3 species belonged to the genus Henneguya: 2 were identified as H. mystusia Sarkar, 1985 and H. hemibagri Tchang et Ma, 1993, while the other was described as H. basifilamentalis sp. n. All plasmodia were found in the gills and were characterised by a specific site selection. H. mystusia formed plasmodia in the multi-layered epithelium between the gill lamellae and in the non-lamellar edge of the gill filaments, while H. hemibagri developed in the capillary network of the lamellae. H. basifilamentalis sp. n. had large oval plasmodia located deep among the filaments just above the gill arch.     

外源精子基因组整入雌核生殖银鲫卵子创制异源八倍体的有效方法

银鲫(Carassius auratus gibelio Bloth)具有将其他鱼类精子的染色体组、染色体片段并入到卵核中协同发育的能力, 但通过异精雌核生殖自发形成异源八倍体子代的概率极低。研究采用0.25%、0.5%、1%、2%和4%胰蛋白酶溶液处理白鲫精子10min后以及1%胰蛋白溶液处理白鲫精子5min、10min、15min、20min和25min后, 分别与异育银鲫A+系成熟卵子受精; 接着通过比较对照组和不同处理组合中精子结构和活力的变化, 兼顾受精率、孵化率、成活率等繁育指标与较高的子代八倍体率, 建立了一种将外源精子基因组整入雌核生殖银鲫卵子创制异源八倍体的有效方法。即采用1%胰蛋白溶液处理白鲫精子15min后与异育银鲫A+系成熟卵子受精, 子代的平均存活率为(2.4±0.7)%, 平均八倍体率可达(16.3±0.5)%。批量处理并结合流式细胞术筛选6月龄子代, 获得57尾融入有白鲫精子染色体组的异源八倍体成鱼。研究为创制异育银鲫优异异源八倍体种质提供了一个简单易行的方法, 创制的异源八倍体可作为后续培育生长快、抗病能力强的银鲫新品种的核心育种材料。     

Site preference of fish myxosporeans in the gill

In addition to the morphological and size characteristics of the spores, indicating the exact location and tissue specificity is also essential for differentiation of the large number of species belonging to the group of gill-parasitic fish, the myxosporeans. According to the observations of the present author, Myxobolus, Henneguya and Thelohanellus species are characterised by strict tissue specificity, and species showing affinity to the epithelium, connective tissue, cartilage or vascular tissue usually occur in a strictly defined location within the gill apparatus. Some of the species typically form plasmodia in the lamellae of the gill and others in the gill filaments. Yet other species develop their plasmodia at the base of the gill filament or in the gill arch. Instead of the generally accepted but misleading terms 'intra-' and 'interlamellar', the present author distinguishes interlamellar-epithelial and intralamellar-vascular types in the case of plasmodia developing in the gill lamellae, and intrafilamental-epithelial, intrafilamental-vascular and intrafilamental-chondroidal types in the case of plasmodia developing in the gill filaments. Regarding site of development within the gill, the location of basifilamental plasmodia and that of plasmodia developing in the cartilaginous matrix, connective tissue or blood vessels of the gill arch are well distinguishable from the above types. The different types and their variations are shown in histological illustrations.     

Myxozoan pathogens in cultured Malaysian fishes. I. Myxozoan infections of the sutchi catfish Pangasius hypophthalmus in freshwater cage cultures

Cage-cultured sutchi catfish Pangasius hypophthalmus (Sauvage, 1878), a favourite food fish in Southeast Asia, proved to be infected by 6 myxozoan species. Three species belonged to the genus Hennegoides (H. berlandi, H. malayensis, and H. pangasii), 1 to Henneguya (H. shariffi) and 2 to Myxobolus (M. baskai, and M. pangasii). Five myxozoans infected the gills and 1 was found on the spleen. Myxozoans infecting the gills were characterised by a specific site selection. H. shariffi sp. n. and H. berlandi sp. n. formed plasmodia in the multi-layered epithelium of the gill filaments. Of the 2 vascular species H. pangasii sp. n. developed in the gill arteries, while M. baskai sp. n. infected the capillary network of the gill lamellae. Plasmodia of H. malayensis sp. n. were found inside the cartilaginous gill rays of the filaments. Large plasmodia of M. pangasii sp. n. were located in a groove of the spleen but they affected only the serosa layer covering the spleen.     

Sublethal copper exposure induces respiratory stress in common and gibel carp but not in rainbow trout

Rainbow trout, common carp, and gibel carp were exposed to sublethal Cu levels (1.0 or 1.7 microM) for 1 week. In rainbow trout, arterial oxygen tension (P(aO(2))) remained normal and there was no indication of anaerobic metabolism. P(aO(2)) was considerably lower in common and gibel carp and Cu exposure decreased this further. The decrease was transient for common carp but persistent in gibel carp and coincided with an elevation in arterial carbon dioxide tension (P(aCO(2))) indicating that all gas exchange was compromised in both cyprinid species. The disturbed gas exchange resulted in acidosis, which was respiratory and metabolic for common carp but mainly respiratory for gibel carp. Gibel carp produced ethanol as end product of their alternative anaerobic pathway. The hypothesis that hypertrophy and hyperplasia, resulting in increased diffusion distances, are reducing P(aO(2)) appeared invalid. Hypoventilation seems a more likely cause. Ionoregulatory parameters responded more uniform among species. Fast and pronounced decreases in plasma sodium and chloride developed for all three species, independent of the observed gill damage. Rainbow trout lost 20% of their plasma Na in the first 3 days, while common and gibel carp had only lost 13 and 16% respectively at that time. This difference might be crucial when challenged with Cu exposure and allow a fish to survive the first shock phase and supports it the hypothesis that sodium turnover is a key factor in predicting Cu toxicity.     

Ultrastructural studies of Henneguya rhamdia n. sp. (Myxozoa) a parasite from the Amazon teleost fish, Rhamdia quelen (Pimelodidae)

Henneguya rhamdia n. sp. is described in the gill filaments of the teleost fish Rhamdia quelen, collected from the Peixe Boi River, State of Pará, Brazil. This myxosporean produced spherical to ellipsoidal plasmodia, up to 300 microm in diameter, which contained developmental stages, including spores. Several dense bodies up to 2 microm in diameter were observed among the spores. The spore body was ellipsoidal (13.1 microm in length, 5.2 microm in width, and 2.5 microm in thickness) and each of the two valves presented a tapering tail (36.9 microm in length). These valves surrounded the binucleated sporoplasm cell and two equal ellipsoidal polar capsules (4.7 x 1.1 microm), which contained 10-11 (rarely 12) polar filament coils. The sporoplasm contained sporoplasmosomes with a laterally eccentric dense structure with a half-crescent section. Based on the data obtained by electron microscopy and on the host specificity, the spores differed from previously described Henneguya species, mainly in their shape and size, number and arrangement of the polar filament coils, and sporoplasmosome morphology.