卵形鲳鲹胚后发育阶段鳃的分化和发育

采用组织学和扫描电镜等方法,鲹研究了卵形鲳(Trachinotus ovatus)胚后发育阶段鳃的分化和发育及其结构和功能的关系。观察发现,仔稚鱼鳃的早期发育可分为3个阶段:第1阶段(0~3日龄)为原基期,鳃原基形成但未分化,鳃耙未出现,仔鱼主要依靠鳍褶、皮肤和卵黄囊上的微血管进行呼吸;第2阶段(4~17日龄)为鳃丝分化、发育期,鳃弓、鳃丝、鳃小片、鳃耙逐渐形成,具备鳃的基本结构和形态特点;第3阶段(18日龄之后)为鳃器官生长发育完善期,鳃弓、鳃丝、鳃小片、鳃耙发育完善,鳃的形态和功能与成鱼相似。进一步研究发现,鳃丝总数随仔稚鱼全长和体质量的增加而增加,单个鳃小片面积和总呼吸面积随仔稚鱼体质量的增加而增大。结果表明,卵形鲳鲳鲹的分化和发育是与仔、稚鱼的生长、形态发育和功能的完善相一致的。     

基于不同纯化策略的4种对虾血蓝蛋白的凝集活性及分子基础对比分析

既往研究表明,对虾血蓝蛋白(hemocyanin, HMC)是一种具有抗病毒、抗菌等多种免疫学活性的免疫球蛋白超家族(immunoglobulin superfamily, IgSF)分子,但迄今为止,其功能多样性的分子基础尚不是很清楚。本研究以凡纳滨对虾HMC为研究对象,采用亲和层析、凝集素层析技术获得4种HMC成分:A-HMCs、A-HMCl、AL-HMCs和AL-HMCl,发现其对不同细菌的凝集活性存在较大差异。其中,AL-HMCs和AL-HMCl对大肠杆菌和副溶血弧菌的凝集活性明显强于A-HMCs和A-HMCl,前者约为后者的2～32倍。继而,通过双向凝胶电泳(two-dimensional gel electrophoresis, 2-DE)和凝集素印迹技术对不同HMC的蛋白质组成和糖基化修饰水平进行对比分析。结果显示,4种HMC在2-DE图谱上表现为6～7个差异明显的蛋白质点,且与伴刀豆凝集素(concanavalin A, ConA)、花生凝集素(peanut agglutinin, PNA)、荆豆凝集素(ulex europaeus agglutinin, UEA)和双花扁豆凝集素(dolichos bifows agglutinin, DBA)等4种凝集素的识别存在显著性差异。其中,A-HMCl 6个蛋白点均可识别4种凝集素,而A-HMCs 6个蛋白点中仅有4、3个点分别与UEA、DBA反应呈阳性,AL-HMCs和AL-HMCl可以与UEA、PNA特异性显色的点分别为其总蛋白点的3/7、2/6。由此推测,对虾血蓝蛋白功能多样性的分子基础可能与其蛋白质组成和糖基化修饰水平的多样性密切相关。     

翘嘴鳜皮肤、鳞片、鳍色素细胞显微观察

采用冰冻切片和常规苏木精—伊红染色切片,在光学显微镜下观察了体质量500 g 1龄翘嘴鳜皮肤、鳞片和鳍色素细胞的组成、分布及形态特征。皮肤纵切显微观察显示,眼后头部、体侧黑斑、体侧下部皮肤主要含黑色素细胞,喉部及腹部皮肤主要含虹彩细胞。皮肤横切显微观察显示,在皮肤表面,黑色素细胞聚集呈团状或块状,黄色素细胞呈颗粒状,虹彩细胞呈长梭形。苏木精—伊红染色下,皮肤黑色素细胞呈棕黑色,虹彩细胞呈浅灰色。皮肤色素层主要分布在表皮层与真皮疏松层之间、真皮致密层与肌肉相连处,少量分布在真皮疏松层纤维组织上。鳞片显微观察显示,眼后头部、体侧黑斑、体侧下部鳞片主要含黑色素细胞、黄色素细胞;喉部及腹部为透明状,不含或含少量黑色素细胞及黄色素细胞。鳞片黑色素细胞胞体大,胞体周边分枝多且细,黄色素细胞呈颗粒状。鳍显微观察显示,鳍主要含黑色素细胞及黄色素细胞,不同部位各色素细胞占比、大小及着色存在较大的区别。翘嘴鳜在长期的进化过程中,通过黑色素细胞、黄色素细胞、虹彩细胞不同的调配比及着色深浅,从而形成了具有独特特征的条纹及图案,利于隐藏及捕食,同时具有一定的观赏性。     

苏丹鱼甘露糖受体的基因克隆表达和免疫特性

为了解苏丹鱼MR(Lh MR)的结构特点及其在抗感染免疫反应中的作用,本研究克隆并分析了LhMR基因,采用实时荧光定量PCR(qRT-PCR)技术检测了LhMR在正常及柱状黄杆菌感染苏丹鱼组织中的表达。结果显示,LhMR开放阅读框4296 bp,编码1431个氨基酸(aa)。LhMR的氨基酸序列和分子结构与其他物种高度相似,胞外1个富含蓖麻类β型三叶草的结构域(RICIN)、1个纤连蛋白Ⅱ型结构域(FNⅡ)、8个串连的C型凝集素样结构域(CLECTs)、1个跨膜区和1个胞内区。通过qRT-PCR检测显示,LhMR在脾脏、体肾、心脏、脑、皮肤、肌肉、鳃、肝脏、后肠、前肠和中肠11种组织中均有表达,其中脾脏中表达量最高;经柱状黄杆菌感染苏丹鱼后,脾脏、肠、体肾和鳃中LhMR基因的相对表达量显著升高。通过免疫组织化学(IHC)检测发现,在脾脏、肾脏和鳃中的LhMR蛋白表达水平提高。通过苏木精-伊红染色病理组织切片表明,在体肾、肠、肝脏和鳃中均有不同程度的病变。体肾的肾小管有大量的血细胞浸润,上皮细胞发生坏死;肠壁变薄,组织大量弥散坏死;肝脏组织中有多个空泡;鳃小片肿胀、混乱、坏死和脱落。研究表明,LhMR在苏丹鱼感染柱状黄杆菌免疫反应中发挥重要作用,为苏丹鱼柱形病的防控提供新的参考。     

鳜肤孢虫新种的分离和鉴定

为准确鉴定鳜肤孢虫病的病原,实验基于形态学比较,结合寄生特征分析和分子系统学方法开展研究,并观察超微结构。形态学观察结果显示,纺锤形或哑铃形白色孢囊,长约1.9～6.8 mm,寄生于鳍条、皮肤、口腔、鳃盖内外表面和鳃丝;圆形内生孢子,直径约(9.8±1.8)(7.3～13.8)μm;孢子内环状细胞质和直径约(6.5±1.2)(3.9～8.3)μm的圆形折光体,鉴定其为肤孢虫。该物种测量值区别于其他肤孢虫,与寄生于鳜鳃的未定种(HB)测量值基本一致;与广东省和河南省的鳜源鳗鲡肤孢虫(GD和HN)以及HB寄生于相似部位。分子序列比对和系统发育结果显示,该物种与GD、HN、芬兰肤孢虫、鲑肤孢虫和寄生美洲鳗鲡的鳗鲡肤孢虫序列的一致性大于99%;与GD和HN的亲缘关系最近,与鲑肤孢虫次之,与芬兰肤孢虫和鳗鲡肤孢虫的亲缘关系较远。综上,该物种与已报道的寄生与鳜的肤孢虫为同种,并修订寄生鳜的鳗鲡肤孢虫的GD和HN分离株记录;将本研究肤孢虫和HB、GD和HN分离株一同命名为鳜肤孢虫(Dermocystidium sinipercae sp. n.),新种。鳜肤孢虫种内形态差异可能与采样时间和发育阶段有...     

拟穴青蟹一种含CTLD结构域基因的克隆及表达分析

从拟穴青蟹(Scylla Paramamosain)中克隆得到一个含有CTLD(C-type lectin-like domain)结构域的C型凝集素基因,经分析未发现同源基因,将其命名为SpCTLD。该基因cDNA序列全长为1 237 bp,包含一个468 bp的开放阅读框,编码155个氨基酸,预测分子量为18.32 kDa,等电点为7.56。经一代测序,该基因在第390位(A390G)和第1 020位(T1020C)含有两个SNP位点。对SpCTLD基因在拟穴青蟹幼体不同时期和组织中的表达模式分析发现,SpCTLD在幼体的各个发育时期均有表达,其中在溞状幼体Ⅴ期表达量最高,仔蟹Ⅰ期次之,仔蟹Ⅱ期最低。对SpCTLD的组织分布分析表明,SpCTLD在鳃中表达量最高,其次为射精管,在血淋巴、肝胰腺等其他免疫组织中也有表达,其中在鳃和射精管的表达量远高于在其他组织中的表达量。副溶血弧菌胁迫实验表明,活菌组的肝胰腺、血淋巴、鳃中的SpCTLD表达量发生显著上调,且都在12 h达到最高。根据研究结果,推测SpCTLD是一种含CTLD结构域的新基因,它可能参与了拟穴青蟹抗细菌反应。该基因的发现丰富了拟穴青蟹C型凝集素相关基因的研究,有利于丰富甲壳动物先天免疫机制的研究。     

缢蛏2-CRD型半乳糖凝集素(Galectin)基因的克隆和表达分析

利用cDNA末端快速扩增(RACE)技术获得了缢蛏半乳糖凝集素基因(ScGL)。ScGL全长为1 282 bp,5′非编码区35 bp,3′非编码区329 bp,开放阅读框(ORF) 918 bp,编码305个氨基酸。氨基酸序列分析显示,ScGL无跨膜结构域,与已报道的缢蛏半乳糖凝集素含1个糖识别结构域(CRD)不同,ScGL具有2个CRD。相似性分析显示,ScGL与其他软体动物的半乳糖凝集素具有较高的相似性,与菲律宾蛤仔相似性最高,达65%;与已报道的2个缢蛏半乳糖凝集素相似性分别为39.74%和44.76%。系统进化上ScGL与菲律宾蛤仔半乳糖凝集素聚为一支。重组表达发现ScGL在包涵体表达,分子量约34.4 ku。荧光定量PCR分析显示,ScGL在缢蛏鳃、肠、唇瓣、外套膜、出水管、入水管、足和内脏团中均表达;其中肠、内脏团、唇瓣和足中表达量较高,出水管和入水管中表达量最低。消化腺ScGL表达量分别在金黄色葡萄球菌感染后3 h和48 h显著高于对照组;鳃ScGL表达量分别在鳗弧菌和金黄色葡萄球菌感染后6 h和48 h显著高于对照组,说明ScGL参与了病原体诱导的缢蛏免疫应答。本研究为深入探索ScGL在缢蛏免疫中的作用奠定基础。     

半滑舌鳎甘露糖结合凝集素相关丝氨酸蛋白酶1基因的克隆及表达分析

甘露糖结合凝集素相关丝氨酸蛋白酶1(Mannan-binding lectin associated serine protease 1,MASP1)是补体凝集素途径中起重要作用的激活蛋白。本研究以半滑舌鳎(Cynoglossus semilaevis)为研究对象,应用RACE技术和实时荧光定量qRT-PCR技术对半滑舌鳎MASP1基因(Cynoglossus semilaevis MASP1,CsMASP1)进行了克隆和表达模式分析。结果显示,CsMASP1基因c DNA序列全长为2507 bp,5′非编码区长82 bp,3′非编码区长142 bp,开放阅读框长2283 bp,共编码760个氨基酸;CsMASP1基因包含13个外显子和12个内含子,与多数已知鱼类的MASP1基因结构一致;SMART分析显示,CsMASP1包含6个结构域,与哺乳动物、鸟类、其他鱼类的结构域一致;同源比对发现,CsMASP1和鱼类的相似度较高,与金目鲈(Latescalcarifer)的相似性最高,为76%。CsMASP1基因在11种健康组织(血液、脑、鳃、性腺、心脏、头肾、肠、肝、皮肤、脾和后肾)中均有表达,其中,在肝、脾和头肾中表达量较高;鳗弧菌(Vibro anguillarum)感染后,CsMASP1基因在6种免疫组织(血液、鳃、头肾、肠、肝和脾)中呈现不同的表达模式,在6种免疫组织中呈现明显的上调表达,肝的表达峰值出现在感染后24 h;脾和鳃的表达峰值出现在感染后6 h;肠、头肾和血液的表达峰值均出现在感染后12h;随着病原菌感染时间增加,基因表达量逐渐降低并恢复至正常水平。研究表明,CsMASP1基因参与了半滑舌鳎的免疫应答过程,本结果为半滑舌鳎的免疫机理研究奠定了基础。     

淋巴囊肿病毒27.8 ku受体蛋白在牙鲆组织中的分布

应用抗牙鲆淋巴囊肿病毒(lymphocystis disease virus,LCDV)受体蛋白(27.8 ku)的单克隆抗体(2G11和3D9)定位LCDV受体蛋白在牙鲆组织中的分布。通过对牙鲆外周血、白细胞、鳃、胃、肠、表皮、肝脏、头肾、体肾、脾、性腺、脑、心脏等进行LCDV受体蛋白的间接免疫荧光与免疫组织化学定位观察,发现在牙鲆外周血白细胞的细胞膜、鳃上皮细胞、表皮、胃黏膜上皮细胞顶端、肠上皮细胞、肝细胞、脾表层结缔组织细胞及头肾后端的肾小管上皮细胞内均有较强的阳性信号,表明这些部位分布有LCDV的27.8 ku受体蛋白,但在体肾、性腺、脑、心脏及外周血红细胞中未观察到阳性信号。推测LCDV通过与鳃、表皮及消化道上皮的受体结合进入牙鲆体内,通过与外周血白细胞上的受体结合侵染白细胞而进入血液循环,进而感染肝脏、脾脏、头肾等器官。     

一例乌苏里拟鲿体表溃疡症的病理学诊断

为了确定乌苏里拟鲿(Pseudobagrus ussuriensis)体表溃疡症的病因,在无菌操作下从发病鱼的溃疡灶边缘、脾、肾中分离病原菌;利用湿片法对鳃组织、体表黏液和肠道内容物压片,观察寄生虫寄生;利用病理学技术观察病鱼的病理损伤特点,分析病因。结果发现,采用BHI培养基作为该病例的首次分离培养基效果不佳,未见细菌生长。该病损伤的主要靶器官为皮肤肌肉、脾脏、头肾和体肾,所有鱼均主要表现为溃疡性坏死性皮炎和脂膜炎,脾炎和间质性肾炎。还可见轻微的灶性坏死性鳃炎。眼、脑、肝脏、心脏、消化道、鳔等未见明显病理改变。将病变组织的石蜡切片进行吉姆萨染色,在肌肉组织内可发现大量细长、可弯曲,犹如"乱发样"细菌。此外,在病灶内还可见少量真菌菌丝。综上结果,判定该病为细菌和真菌共同感染引起。证明了病理学技术在该病诊断中的重要作用。     

Antiparasitic activity of the antimicrobial peptide HbβP‐1, a member of the β‐haemoglobin peptide family

A family of antimicrobial peptides (AMPs) derived from the β‐subunit of haemoglobin was recently isolated from channel catfish, Ictalurus punctatus, infected with Ichthyophthirius multifiliis (ich), an important freshwater fish parasite that causes ichthyophthiriosis. We previously discovered that one of these AMPs, HbβP‐1, had strong cidal activity against ich as well as another ectoparasite, Tetrahymena pyriformis. HbβP‐1 toxicity was specific, primarily affecting the trophozoite (trophont) stage of ich. Here, we show that HbβP‐1 acts more rapidly to kill smaller (presumably less mature) trophonts of ich, taking almost twice as long to kill larger trophonts (P < 0.0001). It acts more rapidly than an unrelated AMP, piscidin 1, which is haemolytic and also lethal to ich trophonts. HbβP‐1 is potently and selectively lethal to the trophont stage of the dinoflagellate ectoparasite, Amyloodinium ocellatum, one of the most important pathogens of warmwater marine fish. HbβP‐1 has no effect on the fish gill cell line feeder layer (G1B cells) used to propagate Amyloodinium, further suggesting a highly selective action. These findings suggest that HbβP‐1 or related AMPs might function in protecting marine as well as freshwater fish and that HbβP‐1 has highly selective activity against specific life stages of important fish ectoparasites.     

Protective Immunity of Goldfish Against Ichthyophthirius multifiliis Infection Induced by Different Trophont Vaccine Preparations

Ichthyophthirius multifiliis, commonly called “Ich,” is a protozoan parasite that infects the epidermis and gills of freshwater fish. Here, we used goldfish (Carassius auratus) to determine whether the four vaccine preparations (live trophonts, formalin‐fixed trophonts, freeze‐thawed trophont lysates, and trophont cilial and cell cortical fractions) of I. multifiliis (Fouquet) can elicit resistance of immunized fish against subsequent theront challenge, and whether a relationship exists between in vitro immobilization of theronts in sera or mucus from immunized fish and host protective immunity against the parasite. Experimental goldfish were randomly divided into five groups with five parallel controls, with each group or subgroup consisting of 20 individuals. Each test goldfish was immunized with one of the four Ich vaccine preparations administered by one of the three routes: live trophonts by gavage (Group 1), formalin‐fixed trophonts by injection (Group 2) or by immersion (Group 3), freeze‐thawed trophont lysates by injection (Group 4), and trophont cilial and cell cortical fractions by injection (Group 5). The fish were then challenged by a standard Ich theront challenge on Day 21 postimmunization. For every 10 d following immunization, we tested the in vitro immobilization of Ich theronts by sera or mucus from immunized and control fish. We found that although all control (nonimmunized) and Group 3‐immunized goldfish died following theront challenge, more than half (55–65%) of the immunized fish from Groups 1, 2, 4, and 5 survived. Furthermore, except for the fish from Groups 3 and 4, the number of mean days to death in each test group was significantly higher than that in the respective control. These results indicate that goldfish immunized by injection or by gavage with any of the four vaccine preparations gained resistance to Ich infection. Further analysis indicated that this protective immunity was closely associated with the in vitro immobilization of Ich theronts by fish sera or mucus.     

Hatching envelope formation in the egg of the black tiger shrimp,Penaeus monodon (Decapoda,Penaeidae)

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)‐labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti‐FITC‐conjugated gold‐lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar‐associated components of the HE. This revealed that both Con A and WGA‐binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold‐labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N‐acetyl‐β‐d ‐glucosamine (sugar affinity to WGA).     

Induced cross-protection in channel catfish, Ictalurus punctatus (Rafinesque), against different immobilization serotypes of Ichthyophthirius multifiliis

Abstract Channel catfish, Ictalurus punctatus (Rafinesque), were immunized with Ichthyophthirius multifiliis (Ich) theronts and trophonts, and the immune response and host protection against both homologous and heterologous serotypes of Ich were evaluated. Immunizations were done with two immobilization serotypes (ARS4 and ARS6) of live theronts by bath immersion (trial I) and with sonicated trophonts by intraperitoneal (i.p.) injection (trial II). Cutaneous and serum antibody titres against Ich following immunization were measured and survival of catfish was determined after theront challenge. Theronts were immobilized by the antiserum from fish immunized with homologous theronts or trophonts, but not by the serum of fish immunized with the heterologous serotype. Serum from fish immunized by immersion with live theronts showed higher enzyme-linked immunosorbent assay titres against both homologous and heterologous serotypes than fish immunized by i.p. injection of trophonts. Channel catfish immunized by immersion with live theronts or by i.p. injection with sonicated trophonts developed an immune response against Ich and provided cross-protection against challenge from both serotypes (ARS4 and ARS6) of the parasite. Sonicated trophont antigens in aqueous solution by i.p. injection could stimulate an immune response in fish, but the immunity was of short duration.     

Cryptocaryon irritans (Ciliophora): primary infection in thick-lipped mullet, Chelon labrosus (Risso)

Abstract. Cryptocaryon irritans (Ciliophora), isolated from the reef fish, Grammistes sexlineatus Thumberg, was established as primary infections in mullet, Chelon labrosus (Risso). Only a small percentage (<22%) of exposed theronts established infection under controlled conditions. This is discussed in relation to the probability of host parasite contact and susceptibility. There was no evidence of density-dependent or intra-specific competition in trophont establishment, a strong linear relationship being found between the levels of exposure and infection. Infection intensity was assessed in relation to host survival, indicating a 90% probability of host death following levels of >200 trophonts per gram of host. A close temporal association between host death and trophont exit was found, possibly as a result of epidermal disruption leading to dysfunction of osmoregulatory and respiratory systems.     

Ichthyophthiriasis in carp, Cyprinus carpio L.: fate of parasites in immunized fish

Abstract. The host-parasite relationship between O-group carp and the ciliatc Ichthyophthirius multifiliis Fouquet was investigated, with the specific aim of characterizing the fate of parasites encountering immunized fish. Carp were immunized by repeated controlled infections; immunized fish and control fish naive to I. multifiliis were then infected in the caudal fin epidermis by a single controlled exposure to theronts, which were applied in a droplet suspension to the tail surface. The number of parasites present within the caudal fin was monitored over a subsequent 5-day period by means of in situ parasite mapping. Results indicated that, contrary to previous reports, theronts penetrated the skin of immunized fish in numbers comparable to those of fish receiving a primary infection. However, the majority of parasites which penetrated immune skin did not complete normal development; 79% of the parasites which had initially penetrated the immune skin were not relocated within 2h of exposure, and since no parasite material was detected at penetration sites, it was concluded that these parasites had prematurely exited the skin rather than been killed in situ. Subsequently, these sites became populated by leucocytes, predominantly macrophages, and the infiltrations continued for up to 5 days after the initial exposure. In contrast, at sites where mature trophonts had exited the skin of fish following a primary infection, more diffuse leucocytic infiltrations were recorded, and these were predominated by neutrophils. Differences in the response to parasite exit from immunized and previously unexposed control fish skin are discussed, with particular reference to the mode of protection and the fate of parasites encountering immune fish.     

人工感染的刺激隐核虫各期虫体的超微结构

从天然感染的卵圆鲳鲹（Trachiruotus blochii）分离到1株刺激隐核虫（Cryptocaryon irritans），再经人工感染的方法收集各期虫体，制成电镜样品，对虫体进行超微结构研究。刺激隐核虫质膜的基本结构与淡水的多子小瓜虫的质膜相似，分为3层；外限制膜、外胞膜和内胞膜。刺激隐核虫的质膜小泡内充满大量电子致密物质（EDM）。幼虫、幼体和滋养体均具有复杂的口器，由胞咽、口肋、双动基体口纤毛和线带等组成，但不具备膜口类纤毛虫所具有的独特的细胞器：Lieberkuhn氏器。单动基体的体纤毛存在于幼虫、幼体和滋养体，但在包囊期，体纤毛和口器消失。各期虫体的胞质中具有线粒体、高尔基体和脂肪体等细胞器，有的阶段具有粘液囊、伸缩泡、食物泡、共生细菌等。粘液囊在大小、形态和分布上与多子小瓜虫的不同。文中分析了刺激隐核虫在形态上与多子小瓜虫的诸多差异，认为刺激隐核虫的分类更适合归于原口类（Promotome），而不是膜口类（Ophryoglenina）。     

Tetrahymena sp. infection in guppies, Poecilia reticulata Peters: parasite characterization and pathology of infected fish

Tetrahymena sp. infection was diagnosed in guppies imported from Singapore. The parasite was isolated (Tet-NI) and optimally cultured in vitro in RM-9 medium. Cytological analyses [silver-staining and scanning electron microscopy (SEM)] revealed a pyriform-shaped, 64 × 41-μm holotrich ciliate without caudal cilium, containing a macro-nucleus (18.25 × 16.83 μm) and micro-nucleus (5.73 × 5.40 μm). Wet-mount examination and histological analyses of fish exposed to the parasite by co-habitation, immersion and infection by i.p. (intra-peritoneal) and i.m. (intra-muscular) injection revealed numerous ciliates on the skin, and in the gill and caudal fin blood vessels. Ciliates surrounded internal organs, the peri-orbital region of the eye, and were observed inside developing guppy embryos. Some muscle necrosis was associated with infection, but little or no inflammatory response. Immersion, co-habitation and i.m. injection caused relatively high infection rates and levels in the skin and tail, and lower infection in the gill blood vessels and internal organs; i.p. injection caused higher infection in the gill blood vessels and internal organs. Co-habited fish had relatively high infection levels in the hind-gut sub-mucosa. This is the first report of controlled systemic infection by Tetrahymena sp.     

The process of infection, migration, growth and development of Sanguinicola inermis Plehn, 1905 (Digenea: Sanguinicolidae) in carp, Cyprinus carpio L.

Abstract. Sanguinicoliasis is a serious disease of cultured carp causing high mortality in early fry. Experimental infections were used to study the pattern of penetration, and subsequent behaviour and development of the parasite. Maximum penetration of cercariae was achieved within 30 min, followed by a period of rapid development and growth marked by a doubling in length within the first 12 h. Subsequent growth was slower, during which major organ development took place, with gonadal maturity being completed between 60 and 90 days p.i. Penetration occurred mainly through the fins, particularly the caudal fin, and large numbers of worms remained in the skin where they continued to develop to maturity. A small proportion of the worms were found in the heart and gill vessels soon after infection. However, migration from the skin began at 60 days p.i. and greater numbers (57%) of the worms were found in heart and gill vessels at 90 days, coinciding with onset of egg production. Decline in worm numbers in the gill/heart region began after 120 days. Gill/heart worms differed in a number of features from skin worms, but egg production occurred simultaneously in both sites.     

Three‐dimensional visualization of green fluorescence protein‐labelled Edwardsiella tarda in whole Medaka larvae

The invasive fish pathogen Edwardsiella tarda is common in aquatic environments and causes the environmentally and economically destructive emphysematous putrefactive disease called edwardsiellosis. In order to understand the organism's infection pathway, medaka larvae (Oryzias latipes) were immersion‐infected with E. tarda labelled with green fluorescence protein (GFP) and then visualized in three dimensions under confocal laser microscopy and light‐sheet fluorescence microscopy. Confocal microscopy revealed GFP‐labelled E. tarda in the mouth, head, gill bridges, gill cover, skin, membrane fin, gastrointestinal tract and air bladder, and in the caudal vein, somite veins, caudal artery and caudal capillaries. Light‐sheet microscopy additionally showed GFP‐labelled E. tarda in the pharyngeal cavity, muscle of the pectoral fin and cardiac atrium and ventricle. These findings suggest that during its infection of fish, E. tarda initially adheres to, and invades, the epithelial cells of the skin, gills and gastrointestinal tract (through the pharyngeal cavity); E. tarda then enters the blood vessels to access organs, including the air bladder and heart.