多子小瓜虫滋养体糖蛋白残基的种类和分布研究

应用伴刀豆凝集素A、麦胚凝集素、大豆凝集素和荆豆凝集素Ⅰ,检测寄生于金鱼皮肤、鳃和鳍上的多子小瓜虫滋养体糖蛋白残基的种类和分布。研究结果发现,伴刀豆凝集素A和麦胚凝集素免疫阳性染色在金鱼皮肤、鳃和鳍寄生的滋养体上均有分布,麦胚凝集素免疫阳性染色强于伴刀豆凝集素A,未见有大豆凝集素和荆豆凝集素Ⅰ免疫阳性染色。鳃上寄生的滋养体伴刀豆凝集素A免疫阳性染色最强,皮肤次之,鳍最弱。鳍上寄生的滋养体麦胚凝集素免疫阳性染色最强,皮肤次之,鳃最弱。研究结果表明,滋养体有单糖D-甘露糖和D-葡萄糖,以及氨基衍生物乙酰氨基葡萄糖。寄生于鳃上的滋养体D-甘露糖和D-葡萄糖可能较多,寄生在鳍上的滋养体乙酰氨基葡萄糖可能较多。     

Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of the structural and functional relationships with respect to cryoprotectant penetration

The structure of the chorion and plasma membranes of gastrula‐stage zebrafish Brachydanio rerio embryos were studied using field emission scanning electron microscopy (FE‐SEM) and transmission electron microscopy (TEM). These studies confirm the outer chorion membrane complex to be 1.5–2.5 μm in thickness and to consist of three layers, electron‐dense outer and innermost layers (0.2–0.3 and 1.0–1.6 μm in thickness respectively) separated by an electron‐lucent middle layer (0.3–0.6 μm in thickness). The middle and inner layers are pierced by pore canals. A granular to farinaceous nature of the thin outer surface of the outer layer of the chorion has been revealed for the first time. The study provides original TEM images of the plasma membrane structures of gastrula‐stage embryos, and FE‐SEM and TEM images showing the plasma membrane to have three morpohologically distinct regions, being prominently ridged and folded at the surface of the blastoderm, smooth over the syncytial layer at the vegetal pole and with an intermediate region between the animal and vegetal pole where folding develops in advance of the expanding blastodermal disc of cells. FE‐SEM and TEM studies reveal details of the syncytial layer (1–4 μm thick) beneath the smooth plasma membrane at the vegetal pole, containing cytoplasmic organelles and small yolk globules. The significance of the structural detail shown in these studies is considered in the light of the difficulties experienced in cryopreservation of the embryo resulting from the inability of achieving cryoprotectant penetration of the yolk mass.     

中华绒螯蟹卵黄发生期卵母细胞和卵泡细胞超微结构观

通过透射电镜技术观察了中华绒螯蟹第二次卵巢发育过程中卵巢的超微结构变化。结果表明：（1）中华绒螯蟹第二次卵巢发育过程中卵黄发生期可分为初期和后期;（2）卵黄发生初期（雌蟹第一次排卵后的16 d内）,卵黄生成以卵母细胞内源性合成为主,此时卵母细胞胞质中存在大量内质网囊泡、高尔基体和线粒体,这些细胞器参与胞内卵黄物质的合成。内源性合成后期,卵母细胞膜形态多样,呈现触手状、波浪状和断裂状,为外源合成期做准备。此期卵泡细胞还未向卵母细胞靠近,两类细胞间存在着由淋巴细胞吐出的絮状物;（3）卵黄发生后期,首先为卵泡细胞与卵母细胞的结合阶段（排卵后16~21 d）,此后,卵泡细胞胞质中含有大量内质网囊泡、卵黄颗粒和脂滴,卵母细胞与卵泡细胞膜变为链珠状便于物质交换,卵母细胞的卵黄合成能力减少,转由卵泡细胞进行外源性物质吸收和卵黄物质合成（21~36 d）;（4）卵黄发生结束后,双层卵膜形成,卵黄体和脂肪滴均匀分布在卵母细胞胞质中。     

Histochemical and immunohistochemical characterization of rodlet cells in the intestine of two teleosts,Anguilla anguilla and Cyprinus carpio

Rodlet cells (RC) are characterized by a distinctive cell cortex and conspicuous inclusions named “rodlets.” These cells are particularly abundant and large in size in intestine of eels. Histochemical, immunohistochemical and ultrastructural investigations were carried out on European eel Anguilla anguilla and Common carp Cyprinus carpio from Northern Italy. Eight biotinylated lectins were used to probe for specific carbohydrate residues in deparaffinized, hydrated intestinal sections of eel and carp. Five antibodies were tested on intestinal sections of both fish species: inducible nitric oxide synthase (i‐NOS), leu‐enkephalin, lysozyme, serotonin and tumour necrosis factor‐α. Lectin histochemistry revealed rodlet cells (RCs) of the eel intestine to react with two of the eight lectins tested, specifically Concanavalin A (ConA) and Sambucus Nigra Agglutinin (SNA). This contrasted to lectin staining of RCs in the intestine of common carp, where four of the eight lectins showed a positive reaction; Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), SNA and ConA. RCs in eel and carp intestine were immunoreactive with antibodies to lysozyme and i‐NOS. The occurrence of the inflammatory peptides lysozyme and i‐NOS in RCs of the eel and common carp poses in favour that these cells are involved in the mechanism of defence against pathogens.     

Cell membrane glycoconjugates on virulent and avirulent strains of the haemoflagellate Cryptobia salmositica (Kinetoplastida)

The glycoconjugates on the cell membranes of Cryptobia salmositica (virulent and avirulent strains) were analysed using 13 highly purified lectins. The virulent strain of C. salmositica was agglutinated by two of these lectins (Concanavalin A (Con A) Canavalia ensiformis , specific for α-mannose and α- D -glucosyl residues; Pisum sativau agglutinin (PSA), specific for N-acetyl α- D -galactosaminyl) but the avirulent strain of C. salmositica was agglutinated by 11 lectins. No agglutination of C. salmositica (virulent and avirulent strains) was observed with lectin Tetragonolobus purpureas agglutinin (TPA, specific for α- L -fucose). Glycoprotein analysis with digoxigenin or biotin labelled lectins showed strain specific staining patterns for C. salmositica; with the avirulent strain showing stronger reactions than the virulent strain. These results indicate that the surface carbohydrate residues changed with attenuation of the pathogen and this change may be related to its loss of virulence.     

Biochemical characterization and physiological role of cortical rods in black tiger shrimp, Penaeus monodon

Cortical rods (CRs), precursors of egg jelly investment in many penaeoid shrimp, are composed of different proportions of proteins and carbohydrates, the physiological role of which still requires extensive investigation. In this study, we demonstrated the biochemical properties of the CRs and their role in the induction of the acrosome reaction (AR). Profiles of the isolated CRs revealed a number of major protein bands ranging from 35 to 230 kDa. These CR proteins were extensively glycosylated and sulfated. Lectin-based carbohydrate analysis further revealed the highest reactivity of concanavalin A (Con A) among other lectins used. In addition, the selective interference of Con A binding with mannose but not glucose indicated that CR glycoproteins were of high-mannose type. Using immunoblotting with anti-CR antibody, we further demonstrated that part of egg water (EW, a natural AR inducer) was derived from miscible components of the CRs. Physiological tests of water-soluble CR (wsCR) revealed its high AR inducing competency comparable to that of EW, which was far superior to that of acid-urea treated CR (auCR). Furthermore, the wsCR-induced AR was selectively inhibited by Con A, suggesting the significance of the exposing mannose residues in regulating P. monodon sperm AR response.     

红螯螯虾输精管的结构及精荚的形成

运用组织学、组织化学和蛋白电泳等多种方法,对红螯螯虾输精管结构和精荚形成进行研究,结果发现,红螯螯虾精荚为一个连续的管状结构,由精子群、精荚基质和精荚壁组成; 精荚壁完全包裹精子,分初级、次级精荚壁2层,初级精荚壁由致密的纤丝组成,包裹精子和精荚基质;次级精荚壁由大小不等的小泡和疏松的纤丝组成,包裹在初级精荚壁之外。红螯螯虾输精管可分为前、中、后输精管。其上皮细胞均具有合成、分泌精荚形成物质的功能。前输精管卷曲段的高柱状上皮,分泌弱嗜酸性丝状分泌物,主要成分为中性粘多糖和蛋白质,形成均匀的初级精荚壁;中输精管的高柱状上皮,成丛状向内腔突起,具有分泌泡,其分泌物为嗜酸性、颗粒状物质,主要成分为酸性粘多糖和蛋白质,形成不均匀,黏性极好的次级精荚壁。后输精管结构较简单,肌层较前两者厚,同时管腔更大,其内精荚结构完整。后输精管与射精管相连处有一狭部,内有瓣膜相隔。射精管肌层厚,上皮明显突起,内无精荚。     

Viral-like particles associated with cuticular epithelium necrosis in cultured Litopenaeus vannamei (Decapoda: Crustacea) in Ecuador

A new viral agent was found associated with the endoplasmic reticulum of epithelial cells of the Pacific white shrimp Litopenaeus vannamei (Boone) sampled during mass mortalities. A 40% mortality rate affected nursery and grow‐out ponds during the first 50–60 days of culture, and peak mortality in ponds occurred when shrimp reached 2–4 g. Histopathological changes of affected shrimp showed different grades of necrosis in epithelial cells and, in some cases, other tissues were affected. Transmission electron microscopy (TEM) of columnar cells of the cuticular epithelium showed the accumulation of viral particles, either dispersed in the cytoplasm or in a string‐like or paracrystalline array. These arrays of virions were within membrane‐bound vesicles formed from the endoplasmic reticulum (ER), in orderly arrays on the outer nuclear membrane or along the ER. The virus particles had apparently proliferated in the ER. The virions had an opaque area with an approximate diameter of 20 nm and an electron‐lucent surface layer. The approximate diameter of the non‐enveloped virions was 25 nm. The cytological changes observed are similar to those associated with the Picornaviridae and Nodaviridae families. The histopathology and ultrastructure of a new disease in L. vannamei is associated with the presence of a putative new virus. Until further isolation and characterization is performed, it is recommended to refer to the agent as Litopenaeus vannamei viral‐like particles (LvVLPs).     

Genus Pleistophora (Phylum Microspora): redescription of the type species, Pleistophora typicalis Gurley, 1893 and ultrastructural characterization of the genus

Abstract. Pleistophora typicalis Gurley, 1893, a parasite of the striated muscle of Myoxocephalus scorpius has been re-examined at light- and electron-microscope levels. Foci of infection were almost exclusively found in the ventral body wall where a mixture of schizonts, sporonts and masses of pansporoblasts lay in direct contact with unaltered host myofibrils and mitochondria. All stages were surrounded by a thick (0-5 μm) amorphous coat, external to the plasmalemma. In schizonts this was traversed by channels passing from the plasma-lemma to a layer of vesicles in contact with the muscle. This coat became modified as a pansporoblast envelope surrounding the mature spores: at first a layer of fine granules was laid down about mid-way across the previously amorphous coat, while the channels disappeared. At the time when the sporogonial plasmodium retracted away from the coat to produce the pansporoblast cavity, the layer of the coat within the granules disintegrated into spherical vesicles. The pansporoblast envelope around the mature spores was composed of two or three layers of different electron density, including one which was strongly electron dense. The pansporoblast envelope as defined here corresponds in function, but not necessarily in origin, to the sporophorous vesicle that encloses the spores in other pansporoblastic microsporidia. Schizonts had an extensive system of smooth endoplasmic reticulum, composed of expanded vesicles and divided by plasmotomy into smaller multinucleate segments. The endoplasmic reticulum of sporogonial stages was comprised of a network of fine channels, parallel arrays of flat cysternae and close-packed stacks of membranes. The sporogonial plasmodium divided stepwise through smaller multinucleate segments into uninucleate sporoblasts. Nuclei were isolated throughout development. Two types of sporonts were recognized: large sporonts gave rise to pansporoblasts containing up to 200 microspores. Macrospore pansporoblasts always contained eight macrospores. Microspores measured 4–4 × 2·3 μm (fresh) and were probably uninucleate. Macrospores measured 7·5 × 3·0 μm (fresh) and were probably binucleate. Another microsporidium, found in the muscles of Blennius pholis, which had been attributed to the same species by Thélohan (1895), was distinguished on the basis of spore size (microspores 3·9 × 2·3 μm and macrospores 7·7 × 3·8 μm fresh) and by the fact that electron dense components were laid down in the pansporoblast wall early in sporogony. The species was named Pleistophora littoralis n.sp.     

Glycoproteins isolated from scallop shells inhibit differentiation of 3T3-L1 preadipocyte cells

We previously demonstrated that the organic components isolated from scallop shells (scallop shell extract) inhibit the differentiation of 3T3-L1 preadipocyte cells. In this study, we show that a pronase-treated scallop shell extract inhibited differentiation, but degradation of sugar chains in the scallop shell extract with trifluoromethane sulfonic acid resulted in the loss of the differentiation-inhibiting activity, suggesting that the sugar chain modifications were responsible for the inhibitory activity. To identify the bioactive substance in the scallop shell extract, we isolated glycoproteins from the scallop shell extract via affinity chromatography using concanavalin A (Con A), wheat germ agglutinin, lens culinaris agglutinin (LCA), and ricinus communis agglutinin. LCA and Con A binding fractions inhibited the differentiation of 3T3-L1 preadipocyte cells. In addition, a glycoprotein with a molecular weight of 16 kDa that was purified from the LCA binding fraction reduced lipid accumulation in 3T3-L1 cells during differentiation. The glycoprotein inhibited differentiation-associated mitotic clonal expansion and suppressed the expression of an adipocyte-specific protein, fatty acid binding protein. These results suggest that the sugar chains of glycoproteins in the scallop shell extract inhibit the differentiation of 3T3-L1 preadipocyte cells.     

Lectin histochemical studies on Sphaerospora sp. (Myxosporea) from Italian brown trout, Salmo trutta L.

A Sphaerospora sp. (Myxosporea) infection (presumably S. truttae ) was identified on a trout farm in northeastern Italy. Parasites were detected in kidneys from infected brown trout, Salmo trutta L., over a 2-year period. Extrasporogonic, sporogonic stages and mature spores were simultaneously detected in the same fish. Traditional diagnostic methods for Sphaerospora spp. rely on the detection of the myxosporean developmental stages in Giemsa-stained kidney smears or haematoxylin-eosin stained tissue sections. A histochemical method was employed where 10 biotinylated lectins (Con-A, DBA, SBA, GS-I, PHA-P, LEA, PWM, RCA₁, WGA and UEA-I) and the avidin-biotin-peroxidase complex (ABC) were used on Sphaerospora -infected brown trout renal tissues and kidney imprints. Five monoclonal antibodies against PKX (Mab12, MabA3, MabC5, MabD4 and MabB4) were also tested. A lectin glycoconjugate binding pattern for Sphaerospora spp. is presented. This staining method shows that SBA lectin ( Glycine max agglutinin) is a useful tool for the detection of the Sphaerospora spp. Only MabB4 bound some of the most mature sporogonic stages. In contrast Mabs12, A3, C5 and D4, and GS-I lectin ( Griffonia simplicifolia agglutinin) did not stain any of the Sphaerospora spp. stages, but did bind very specifically to the sporogonic and extrasporogonic stages of PKX, the causative agent of proliferative kidney disease (PKD).     

Effect of Feeding Regime on Compensatory Growth of Juvenile Abalone,Haliotis discus hannai,Fed on the Dry Sea Tangle,Laminaria japonica

Effect of feeding regime on compensatory growth of juvenile abalone (Haliotis discus hannai) fed on the dry sea tangle (Laminaria japonica) was determined. Thirty juvenile abalone averaging 15.7 g were randomly stocked into 18 50‐L plastic rectangular containers each. Six treatments were prepared in triplicate: Abalone were fed the dry sea tangle once a day at a satiation level with a little leftover for 16 wk as the control (Con) and other abalone were fed the dry sea tangle once a day at a satiation level with a little leftover for 15 wk after 1‐wk starvation (S1 treatment), 14 wk after 2‐wk starvation (S2 treatment), 13 wk after 3‐wk starvation (S3 treatment), 12 wk after 4‐wk starvation (S4 treatment), and 10 wk after 6‐wk starvation (S6 treatment), respectively. A linear relationship between weight change of abalone and wk of starvation was observed: Y (Weight of abalone) = ?0.17X (Wk of starvation) + 15.89 (R ² = 0.9462) (P < 0.0001). The highest survival of abalone was achieved in the S2 treatment, but not different from that of abalone in the Con, S1 and S3 treatments. Weight gain of abalone in the Con treatment was higher than that of abalone in the S4 and S6 treatments. Abalone fed on the dry sea tangle seemed to be able to achieve full compensatory growth up to 3‐wk starvation.     

Effects of Vitamin E with Different Levels or Sources of Dietary Lipid on the Growth and Expression of Inflammatory,Oxidative Stress,and Apoptotic Genes in the Head Kidney of Olive Flounder,Paralichthys olivaceus

This study has assessed the effects of vitamin E (?E, +LE, +HE; 0, 100, 1000 mg/kg, respectively) in fish diets containing high levels (HL; 10%) of fish oil (FO) or mixed vegetable oils (VO) on the growth and inflammatory, oxidative stress, and apoptotic gene expression in the head kidney of olive flounder, Paralichthys olivaceus. Consequently, the highest weight gain was achieved in the FO group and the lowest in the HL‐VO + LE group. The gene expression levels of each group were compared to the 5% FO group. The 5% VO group showed higher expression levels of tumor necrosis factor (TNF) α, interleukin (IL)‐1β, and scinderin‐like (ScinL) genes. Although lysozyme gene expression was higher in the HL‐VO + LE group, the other gene expression levels of the HL‐FO/VO + LE groups were not different from those of the FO group. The HL‐FO/VO?E/+HE groups showed a higher TNFα gene expression, but the cytochrome oxidase subunit III gene expression was higher in the HL‐FO?E and HL‐VO + HE groups. Lysozyme gene expression was higher in the HL‐FO?E and HL‐VO?E/+HE groups. IL‐6 and ScinL gene expression were higher in the HL‐VO‐E and HL‐VO + HE groups, respectively. In conclusion, mixed VO and too high or too low vitamin E levels in fish diets may affect inflammatory, oxidative stress, and apoptotic gene expression in the head kidney of olive flounder.     

美洲鲥(Alosa sapidissima)眼睛早期发育的组织学观察

应用石蜡切片及HE染色的方法对美洲鲥(Alosa sapidissima)早期发育过程中眼睛的发生、分化和形成过程进行了系统的观察.结果显示,受精后21 h 13 min,视泡出现;受精后26h 23 min,视泡发育成视杯;受精后35 h 44 min,原始视网膜和晶状体形成;受精后60h 15 min,角膜在视网膜前缘处形成,由单一的立方上皮构成;2日龄仔鱼,巩膜分化完成,由软骨组织和弹力纤维构成,脉络膜出现;3日龄仔鱼,虹膜出现,视网膜分化完全,由外向内的色素层、视觉细胞层、外界膜、外核层、外网膜层、内核层、内网膜层、视神经节细胞层、视神经纤维层和内界膜10层构成,此时视觉细胞层中视杆细胞出现;14日龄仔鱼,角膜分化完全,由自外向内的复层扁平上皮、前弹性层、基质层、后弹性层和内皮层5层构成;22日龄仔鱼,脉络膜腺出现;26日龄稚鱼脉络膜分化完全,由外向内依次为银膜层、血管层和色素层,虹膜也完全分化,由外向内依次为虹膜内皮层、前缘层、基质层、后缘层和色素层;45日龄幼鱼,视网膜内核层分化成两层水平细胞.此时美洲鲥眼睛的各个结构均发育完善.     

Ultrastructure of Goussia cruciata (Apicomplexa: Coccidia) infecting the liver of horse mackerel, Trachurus trachurus (L.), from Ibero-Atlantic waters

The ultrastructure of developmental stages of Goussia cruciata and the pathology they cause in the liver of Trachurus trachurus (Teleostei: Carangidae) caught off the Galician (North-West Spain) and Portuguese North Atlantic coasts are described. Each oocyst contained four ellipsoidal sporocysts, with two sporozoites. The sporocyst wall consisted of a thick and dense inner layer with transverse striations and a multi-lamellated outer layer formed by parallel dense internal bands alternating with lighter areas. The lamellae formed filamentous extensions of the wall. The sporocyst wall striation period was smaller than that observed in G. clupearum, which has a similar habitat. The dehiscence suture, characteristic of the genus, was present in the sporocysts of G. cruciata. The sporocysts were arranged in a symmetrical and characteristic cross shape. A large number of sporocysts with sporozoites were observed in direct contact with host liver cells. No macroscopic lesions were observed. In heavily infected fish, aggregations of oocysts were often enveloped in a 'yellow body' composed of amylopectin granules derived from the parasite and necrotic or aggregated host cells. Degenerating parasites were frequently observed in liver tissue. Host inflammatory cells were accumulated near some oocysts. The ultrastructure of the parasite, together with its strict host specificity, confirmed G. cruciata as a separate and valid species.     

Dietary supplementation of host‐associated lactic acid bacteria modulates growth,metabolic activities,and immune‐related gene expression in giant freshwater prawn,Macrobrachium rosenbergii

The present study was carried out to evaluate the dietary effects of host‐associated lactic acid bacteria on growth performance, metabolic enzyme activities, and immune response of Macrobrachium rosenbergii juveniles. To formulate the test diets, a control (Con) diet was supplemented with a commercial probiotic and three host‐derived bacteria Enterococcus faecalis (EC), Lactococcus lactis I (LC‐I), and L. lactis II (LC‐II), which were previously isolated from the gastrointestinal tract of adult individuals of M. rosenbergii. Juvenile M. rosenbergii (0.65 ± 0.008 g) were randomly stocked at 20 individuals/100 L of fiberglass tanks with three replications for each test diet. After 50 days, juveniles fed the diets LC‐I and LC‐II showed significantly higher (p < .05) weight gain, specific growth rate, and the lowest feed conversion ratio. The analyses of glutamic oxaloacetate transaminase and glutamic pyruvate transaminase in muscle and hepatopancreas revealed significantly (p < .05) reduced values in LC‐I fed juveniles. The total hemocyte count and phenoloxidase activity were significantly increased (p < .05) in LC‐I and LC‐II fed juveniles. The relative mRNA expression patterns of immune‐related α2‐M, LGBP, ProPO, Cu, Zn‐SOD, TG, PE, AKP, and ACP genes were significantly (p < .05) upregulated in juveniles fed with LC‐I followed by the diet LC‐II. Finally, the study suggests that the growth performance and immune response of M. rosenbergii can be improved through supplementation of host‐associated L. lactis bacteria for its higher production.