Experiments with growth of the eel trypanosome, Trypanosoma granulosum Laveran & Mesnil, 1902, in semi-defined and defined media

Abstract. Growth of the eel trypanosome, Trypanosoma granulosum , was attempted in five semi-defined and three defined media. Growth was poor in four semi-defined and two defined media, but one semi-defined medium, a modified version of SDM-79 without MEM F-14, supported good growth of the trypanosome. In this medium, doubling time was between 1 and 2 days, over 90% of organisms seen in culture after 6 days were trypomastigotes, and 1.8 × 0.1 × 10⁷ trypanosomes ml⁻¹ was achieved in 7 days. When foetal calf serum from the modified SDM-79 was substituted with insulin to create a fully defined medium, a modest but significant increase in cell numbers occurred compared with controls. In vitro experiments with D, L-alpha-difluoromethylomithine (DFMO) showed morphological changes leading to destruction of the trypanosome with increasing concentrations of the drug. A 50-mM concentration of DFMO inhibited growth by more than 90%, and the IC₅₀ was found to be 16 × 2 mM.     

Structural abnormalities of common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> spermatozoa

Spermatozoa of common carp Cyprinus carpio are typically consist of a primitive head without acrosome, a midpiece with several mitochondria, a centriolar complex (proximal and distal centriole), and one flagellum. During an evaluation of the motility of common carp spermatozoa, we found spermatozoa with more than one flagellum and/or “double head” in three different individuals. This may be related to abnormal spermatogenesis. Ultrastructure and physiological parameters of spermatozoa were examined using light microscopy (dark field with stroboscopic illumination), transmission and scanning electron microscopy, and flow cytometry. The recorded pictures and videos were evaluated using Olympus MicroImage software. All spermatozoa with more than one flagellum had a larger head and shorter flagella. They occasionally demonstrated several cytoplasmic channels separating the flagella from the midpiece. Each flagellum was based upon its own centriolar complex, with the connection of the flagellum to the head always at a constant angle. The flagella always consisted of nine peripheral pairs and one central doublet of microtubules. Sperm exhibited a relative DNA content similar to that found in sperm from normal males, with higher coefficients of variation. Although similar abnormalities have been found in livestock, where they were described as a defect in spermiogenesis, no comparable results have been reported in fish. The frequency at which these abnormalities occurs, the fertilization ability of males with defects in spermiogenesis, the influence of these abnormalities on progeny in terms of ploidy level, and the occurrence of deformities warrant further investigation.     

虾夷扇贝精子形态结构和超低温冷冻损伤的电镜观察

应用透射电镜和扫描电镜技术,研究了超低温冷冻保存前后虾夷扇贝精子超微结构和形态的变化。结果显示,虾夷扇贝精子由头部、中段和尾部3部分组成,外被光滑质膜;顶体位于头部最前端,呈倒"v"形,细胞核近似圆柱状,电子密度较高;4个线粒体和两个相互垂直的中心粒构成了精子的中段;鞭毛细长,轴丝为典型的"9 2"结构。经超低温冷冻保存后,冷冻损伤的精子表现为:精子被膜肿胀、被膜与核膜分离、丢失;顶体破裂、内容物流出;线粒体解体、线粒体嵴变形;鞭毛被膜肿胀、部分精子鞭毛脱落。可以推测,超低温冷冻保存对精子膜、顶体、线粒体和鞭毛的损伤可导致冻精活力和受精能力的下降。     

Surplus spermine does not protect salmon cells against lipopolysaccharide (LPS)‐induced stress,but excess spermine is eliminated through spermidine/spermine N1‐acetyltransferase (SSAT)

The present trials tested the efficiency of surplus spermine to reduce inflammation and oxidative stress following LPS‐induced stress using an in vitro model of head kidney and liver cells isolated from Atlantic salmon. Spermine did not protect cells from LPS‐induced inflammatory response at either 0.3, 0.6 or 0.9 mM. However, as the gene expression of spermidine/spermine N1‐acetyltransferase (SSAT) increased with increasing spermine concentration, we addressed possible oxidative effects of the increased SSAT using its activator DENSPM or inhibitor of polyamine oxidation of the acetylated polyamines using MDL72527 at a spermine concentration of 0.6 mM. There was no significant effect of DENSPM, but MDL72527 decreased gene expression of GPX‐3 (p = .04), while gene expression of catalase and MnSOD was unaffected by treatment (p = .30 and p = .48, respectively). In conclusion, spermine did not protect cells from LPS‐provoked inflammation. The higher the spermine concentration, the more SSAT producing acetylated spermine occurred. Inhibiting the acetylated polyamine oxidases by MDL72527 improved oxidation status as expected due to a lower endogenous production of H₂O₂ by polyamine and acetylated polyamine oxidases. Probably care should be taken using polyamines or arginine as functional ingredients to avoid any increased oxidation within cells.     

Ultrastructure of the microgamont and microgamete of Eimeria funduli, a coccidium parasitizing killifishes

Abstract. The ultrastructure of the microgamont and microgamete of the coccidium Eimerici funduli was studied in hepatocytes of the killifish Fundulus grandis and F. similis . During microgametogenesis, the nuclear chromatin condensed, marginated, then differentiated into a peripheral dense portion that became the nucleus of the microgamete and a central clear portion that remained in the gamont. Near each nucleus was a mitochondrion and four microtubules which were incorporated into the microgamete. In microgamonts, dense polysaccharide granules (50–60 nm diameter) were demonstrated with the periodic acid-thiocarbohydrazide-silver proteinate method. Flagella extended into the parasitophorous vacuole from basal bodies situated near nuclei. Microgametes budded from the surface of the microgamonts. The microgamete consisted of a nucleus, two flagella and basal bodies, a mitochondrion, a rudimentary basal body, four microlubules, a perforatorium, and a dense rod-like structure. The elongate nucleus was narrow anteriorly and bulb-like posteriorly. One of the flagella was attached to the body of the microgamete. A mitochondrion that was swollen anteriorly and constricted posteriorly ran alongside the nucleus. The four microtubules appeared to arise from a rudimentary basal body and were closely related to the mitochondrion. Two of them extended nearly the length of the nucleus. The rod-like structure appeared to develop from a thickening of the limiting membrane of the microgamont. Fertilization was not directly observed, but microgametes were found in the parasitophorous vacuoles of several macrogamonts.     

Studies on the ultrastructure of spermiogenesis and spermatozoon of tongue fish,Cynoglossus semilaevis günther

Ultrastructures of spermiogenesis and spermatozoon of commercially valuable tongue fish Cynoglossus semilaevis Günther were investigated by means of transmission electron microscopy. The study shows that the spermiogenesis of C. semilaevis can be divided into four stages, and the course of spermatid differentiation included flagellum development, chromatin compaction, nuclear fossa formation, migration of diplosome and mitochondria with structural change and disappearance of residual cytoplasm. No acrosome was observed in spermatozoon of C. semilaevis. The nucleus of sperm was U‐shaped; the nuclear fossa was deep with a centriolar complex at the bottom composed by proximal centriole and basal body. Five to six mitochondria were located behind the nucleus in a circle, forming the midpiece of sperm. The sperm tail, 43±2.4 μm (n=5) long, had lateral fins and sacciform tissues in some parts of the tail, with axoneme shown of a typical ‘9+2’ pattern. The ultrastructural observation revealed that the sperm of C. semilaevis is of a primitive type among teleostean fish species.     

Sperm motility and seminal plasma characteristics in Barbus sharpeyi (Günther, 1874)

Spermatozoa concentration, ionic composition, osmolality, glucose and total protein contents of seminal plasma and sperm motility were determined in Barbus sharpeyi (Cyprinidae, Teleosotei). Spermatozoa concentration ranged from 9.77 to 20.20 × 10⁹ spermatozoa mL^?1. Osmolality (mOsmol kg^?1) and ionic contents (mM L^?1) of the seminal plasma were 274.5±9.0, 70.0±3.4 Na⁺, 28.8±0.9 K⁺, 101.7±3.1 Cl^?, 0.9±0.1 Mg²⁺ and 2.1±0.1 Ca²⁺ respectively. Total protein and glucose were 5.3±0.2 g L^?1 and 76.7±4.3 mM L^?1 respectively. Sperm motility was initiated in a hypo‐osmotic condition, composed of either an ionic (KCl or NaCl) or a non‐ionic (sucrose) activation medium. Duration of sperm motility was very short: <2 min after activation in distilled water. Percentage of motile spermatozoa was significantly higher in an activation medium containing NaCl compared with that of distilled water. An activating medium containing NaCl or KCl higher than 150 mM or sucrose higher than 275 mM totally inhibited the activation of sperm motility. Immediately after sperm activation, wave(s) propagated along the flagellum, but waves were restricted to the proximal part of the flagellum (close to the head) at 1 min post activation. Studied characteristics in the present study were compared with those of other cyprinids for understanding inter‐species differences.     

嘉庚蛸精子发生的超微结构

应用透射电镜技术研究嘉庚蛸精子发生过程中各阶段细胞的形态、细胞器及细胞核的动态结构变化。精原细胞呈椭圆形,细胞质少、核大,有线粒体、高尔基体等细胞器;初级精母细胞呈圆形或卵圆形,线粒体多,索条状内质网发达,高尔基体常集中分布,中心粒形成;次级精母细胞呈圆形、椭圆形或不规则形,线粒体嵴发达、基质丰富,高尔基囊泡积累电子致密物,中心粒发出的“9＋2”轴丝结构形成。精细胞分化过程分为6个时期,主要特点为：①核内染色质凝集经颗粒化、纤维化及片层化,最后,形成高电子密度的均质结构;与此相应,核形态由椭圆形、变成长椭圆形、再变成细长柱状,核后端最后向前凹入形成核内沟;精子核的形态建成为染色质的凝集与核周微管加压协同作用的结果。②高尔基囊泡积累电子致密物、融合发育成顶体囊,逐渐延长演变成钻头形的顶体。③线粒体向核后端迁移,形成精子尾部的线粒体鞘。     

银鲳精子的超微结构

运用扫描与透射电镜对银鲳精子的超微结构进行了观察。银鲳精子由头部、中段和尾部(鞭毛)3部分组成。精子全长约39.51±1.64μm,头部长约1.98±0.30μm,表面粗糙。头部的主要结构包括细胞核和中心粒复合体。细胞核位于头部腹侧,卵圆形。细胞核中染色质致密,存在着不规则的网络状间隙。近端中心粒为9组三联微管结构,与远端中心粒相互垂直。中段的主要结构为袖套和线粒体。袖套与细胞核后端相连,含有4~5个线粒体及少量囊泡。尾部细长,长约37.52±1.68μm,至末端逐渐变细。尾部的主要结构为轴丝,为典型的"9+2"微管结构。尾部具有波纹状侧鳍,排列不对称。     

Changes in intracellular polyamine concentration during growth of Heterosigma akashiwo (Raphidophyceae)

ABSTRACT:   Of the free, conjugated and bound forms of polyamines, the free form of spermidine was the most abundant polyamine in Heterosigma akashiwo throughout the growth period except for the lag phase. Free spermidine content increased remarkably during the exponential growth phase and increased as the growth rate increased. The maximum growth yield of H. akashiwo was reduced by the addition of methylglyoxal bis-guanylhydrazone (MGBG) and the reduced growth yield could be counteracted by the addition of spermidine to the medium. It is concluded that spermidine plays a significant role in the growth of H. akashiwo . These results are similar to those obtained in Chattonella antiqua that belongs to same taxonomic Class as H. akashiwo .     

褐牙鲆精子超微结构观察

利用扫描电镜和透射电镜对自然成熟的褐牙鲆精子超微结构进行了观察。褐牙鲆精子的头部近似圆形,直径1.49±0.17μm,尾部鞭毛较长,达40.17±0.65μm。褐牙鲆精子由头部、中段和尾部(鞭毛)构成。头部的顶端无顶体,细胞核为圆形,染色质致密。核膜分由内核膜和外核膜构成,其间有核周腔。核膜与质膜间有较大间隙,其间分布有许多细胞质、囊泡和溶酶体,在细胞核后端,核膜与质膜之间除细胞质外,还分布有5~6个呈环形单层排列的线粒体。植入窝位于细胞核后端的凹陷处,中心粒复合体位于植入窝内。基体的头端由电子致密物质构成,基体的末端与鞭毛起始端相连,鞭毛从袖套腔中伸出。鞭毛中心结构是轴丝,轴丝为"9+2"微管结构。轴丝外侧有侧鳍。     

超低温冷冻对西伯利亚鲟精子形态结构损伤的观察

为改进西伯利亚鲟精子超低温冷冻保存技术,探讨精子损伤机制,应用扫描和透射电子显微镜,观察了西伯利亚鲟鲜精与冷冻后精子的形态结构.结果显示,经过冷冻保存后精子的形态发生了很大变化.精子顶体长、精子头中部宽、头中部宽与前部宽比值及中段宽与鲜精相比显著增加(P<0.05);中段长度、后外侧延伸物长度比鲜精显著变短(P<0.05).精子经过冷冻后有30.5%的精子在形态、结构上受到不同程度损伤,受损精子显微结构表现为顶体后外侧延伸物与核糅合,顶体内容物丢失;核膜囊泡化、核膜断裂,核内出现空泡;线粒体内嵴弥散,线粒体脱落;鞭毛外膜松弛与鞭毛脱离等.部分受损伤精子出现顶体丢失,中段脱落,鞭毛自中段基部断裂的现象.精子损伤主要集中在膜系统,中心粒等微管系统基本完好.     

Morphological studies of peripheral blood cells of the Chinese sturgeon, <Emphasis Type="Italic">Acipenser sinensis</Emphasis>

The peripheral blood cells of one-year-old Chinese sturgeon (Acipenser sinensis) have been studied by light microscopy and transmission electron microscopy. The erythrocyte count was 84.86 × 10⁴ cell mm⁻³ in the peripheral blood of the fish and that of leukocytes was 2.24 × 10⁴ cell mm⁻³. The erythrocytes and four main types of leucocyte—thrombocytes, lymphocytes, granulocytes (including neutrophils and eosinophils), and monocytes, were identified in the peripheral blood. In addition to normal erythrocytes, reticulocytes and division of erythrocytes were observed. Thrombocytes were the most numerous among the leukocytes, and the number of neutrophils with lobated nuclei was larger than for other fish. The structures of the erythrocytes, lymphocytes, monocytes, granulocytes, and thrombocytes of the fish were studied. The erythrocytes were almost completely devoid of organelles, except for some mitochondria and granules. A large number of vacuoles and a few organelles were observed in cytoplasm of the monocytes. There were many microvilli on the membrane and pseudopodia-like cytoplasm bulge in the lymphocytes. The neutrophils were round or oval in shape with bilobed, trilobed, or multilobed nuclei whereas the eosinophils had big special granules, dark stained. There were many vesicles in some thrombocytes, which were related to its phagocytosis; some thrombocytes had almost no cytoplasm or organelles.     

中华绒螯蟹肝胰腺白化症组织病理变化

采用常规石蜡切片技术和透射电镜技术,对患有肝胰腺白化症的中华绒螯蟹病变组织和细胞进行观察。发现病蟹肝胰腺、鳃、肌肉等组织细胞发生了不同程度的病变,其病理特征为肝细胞水肿、变性、坏死,细胞中脂肪颗粒锐减,线粒体水肿、嵴消失、形成空泡,内质网、溶酶体解体成小泡。鳃组织增厚,上皮细胞微绒毛排列疏松、不规则,部分线粒体嵴消失,出现膜性退化。坏死的肝细胞和鳃上皮细胞中出现细菌颗粒。肌细胞的病变主要表现为细胞核固缩,肌丝松驰、水肿、断裂、肌质网溶解形成小泡。中华绒螯蟹具有重要生理功能的肝胰腺、鳃组织发生了严重病变,使其功能损伤,不耐运输;而肌肉的松驰、肝胰腺脂肪的锐减影响了中华绒螯蟹的品质。同时,病变组织和细胞中未见病毒等生物病原,表明该病为非生物性疾病。图2表0参12     

Optimization of UV treatment to induce haploid androgenesis in the stinging catfish, Heteropneustes fossilis

We studied the microdistribution of the artificial radionuclide ²⁴¹Am in the components of Elodea canadensis - a submerged macrophyte of the Yenisei River. The alpha-track analysis showed that the microdistribution of ²⁴¹Am within different components of the submerged plant E. canadensis was not uniform. ²⁴¹Am distribution was found to be affected by the age of the leaf blades, state of the cells, and morphological features of the plant stem. The radionuclide ²⁴¹Am penetrated into the plant cells through the cell wall of E. canadensis, but it was accumulated in the vacuoles rather than in the cell wall or cytoplasm. In this case, the integrity of the cell membranes was not damaged.     

史氏鲟精子超微结构

利用扫描和透射电镜观察了史氏鲟精子形态和超微结构。精子具有顶体、头部、中段和单鞭毛等部分。顶体长0.99±0.06μm,宽0.87±0.09μm。细胞核从后往前逐渐变细,前端宽度为0.88±0.04μm,后端宽度为1.26±0.06μm,细胞核长7.29±0.32μm。核内含有3条核管(E),核管上行至顶体下行至植入窝而止。中段紧接头后部,长为0.51±0.12μm,宽0.91±0.05μm。中段含有线粒体、中心粒复合体和鞭毛的起始部分。线粒体分2~3层排列,线粒体中可见髓样嵴结构。在细胞核与中段接合部细胞核向内凹陷形成植入窝,纤维体位于植入窝内,其后是中心粒复合体。中段后缘延长为袖套,袖套腔中含有线粒体、脂质空泡。鞭毛从袖套中伸出,由轴丝组成,为典型的"9+2"微管结构。     

An amoeba associated with gill disease in turbot,Scophthalmus maximus (L.)

An amoeba associated with a severe gill disease affecting farmed turbot, characterized by pronounced hyperplasia of the branchial epithelia and accumulation of mucus between the secondary lamellae, is described for the first time. The amoeba was isolated, cloned and cultured on Bactoagar. Light microscopy showed that trophozoites had a mean length of 12 μm (range 8–20 μm), with a nucleus 3–5 μm in diameter containing a large central nucleolus 1–3 μm in diameter. Transmission electron microscopy showed that the mitochondria, Golgi bodies, rough endoplasmic reticulum, microfilaments, cytoplasmic microtubules and glycocalyx were similar to those previously reported for the genus Platyamoeba. These and other findings strongly suggest that the amoeba isolated is of the genus Platyamoeba, while its morphometric characteristics suggest that it may represent a new species. This is the first report of a species of the genus Platyamoeba in association with gill disease in turbot.     

Semen biology and stimulation of milt production in the European smelt (Osmerus eperlanus L.)

Basic characteristics of the European smelt (Osmerus eperlanus) sperm are reported here for the first time. Smelt spermatozoa had a bullet-shaped head (1.42 μm length), a short midpiece and a long flagellum (27.72 μm). Two mitochondria were located along the flagella. The volume of smelt sperm was small (30-60 μl) and the duration of sperm motility was short (22 s in distilled water and 41 s in 20 mM sodium bicarbonate solution). Sodium chloride at concentrations ranging from 0-120 mM did not influence the percentage of motile spermatozoa but caused a steady increase in the duration of sperm movement. Potassium ions clearly reduced the percentage of motile sperm at a concentration of 10 mM. Spermatozoa were motile through a broad range of pH with an optimum from 7.5 to 8.5. Testicular spermatozoa had a different motility pattern compared to stripped spermatozoa (the latter exhibiting a reduction of motile spermatozoa by 30%, lower ALH and VCL and higher LIN and VSL). These results indicate that maturation of smelt spermatozoa occurring in sperm ducts is related not only to an increase of the percentage of motile spermatozoa but also to changes in the sperm motility pattern. Maintaining males with females resulted in stimulation of milt production. Our results indicate that European smelt sperm characteristics are similar to those of ayu (Osmeridae).     

Ultrastructural morphogenesis of salmonid alphavirus 1

Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE‐214). Different stages of viral replication were observed under electron microscopy. Virus‐like particles were observed inside membrane‐bound vesicles as early as 1 h following contact of the virus with the cells. Membrane‐dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome‐like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive‐sense single‐stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by ‘fuzzy’‐coated membranes was greater in virus‐infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post‐infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.     

美洲鳗鲡致病性鲍曼不动杆菌的分离、鉴定及致病性分析

为确定海南某鳗鲡养殖场患病美洲鳗鲡大量死亡的原因,从患病美洲鳗鲡肝脏和脾脏中分离获得1株优势菌株AB01,回归感染证实该菌株为导致此次美洲鳗鲡患病的病原菌。经生理生化鉴定、16S rDNA基因序列比对及系统发育分析,判定菌株AB01为鲍曼不动杆菌。致病性分析结果显示,菌株AB01对美洲鳗鲡的半致死浓度为4.63×106CFU/mL。组织病理学观察显示,患病美洲鳗鲡的肝脏、脾脏均发生不同程度的病变,其中,肝脏中部分肝索排列紊乱,细胞肿胀,胞核溶解,胞质疏松,结构模糊,有大量大小不等的空泡;脾脏中胞质多处稀疏,伴有大面积损伤。药敏检测显示,鲍曼不动杆菌AB01对头孢噻吩、头孢唑啉、阿奇霉素、丁胺卡那霉素、利福平、依诺沙星、环丙沙星、氧氟沙星敏感,对氟苯尼考、氨苄西林等9种抗生素中度敏感,对氨曲南和四环素等20种抗生素有多重耐药性。