Dependence on body size of respiratory function in Hoplias malabaricus (Teleostei,Erythrinidae) during graded hypoxia

Oxygen uptake and ventilatory responses to environmental hypoxia were assessed for two groups of Hoplias malabaricus of nearly tenfold different body weights (mean weights, group I = 39 g; group II = 365 g). The main purpose of the study was to evaluate the relationships between body weight and the critical O₂ tension (P_CO₂) for maintenance of normal resting O₂ uptake. The smaller specimens had a threefold larger weight-specific O₂ uptake compared to that of the larger specimens. Moreover, smaller size coincided with a higher P_CO₂, i.e., a more limited range for maintenance of O₂ uptake during hypoxia. Likewise, at any inspired PO₂ the weight-specific gill ventilation was largest for group I. This was most pronounced during severe hypoxia where both tidal volume and respiratory frequency increased markedly. In addition, the oxygen cost of breathing tended to be highest in the smaller individuals. The results open questions as to correlations between development, weight-dependent O₂ demands, and tolerance to hypoxia on an intraspecific level.     

Gill morphometry of the facultative air-breathing loricariid fish,Hypostomus plecostomus (Walbaum) with,special emphasis on aquatic respiration

Gill respiratory surface area and oxygen consumption during aquatic respiration were measured in the facultative air-breathing loricariid fish,Hypostomus plecostomus. The fish did not surface to breathe atmospheric air in normoxic water; air-breathing was evoked by environmental hypoxia (water oxygen tension=35±2, mmHg) and did not show size-related threshold differences for air breathing.During gradual hypoxia, without access to atmospheric, air,H. plecostomus was found to be an oxyregulator and showed a reduced range of water oxygen tension in which the oxygen consumption remained constant in smaller fish. The critical oxygen tensions were 55 and 33 mmHg at 25°C for fish of 14–30 g and 31–80g body weight, respectively.The gill respiratory surface area (total lamellae area) is reduced, however, the lamellar frequency per mm of gill filament is high which facilitates the gas exchange. Moreover, the increase of gill respiratory surface area (b=0.666) is higher than the increase in routine VO₂ (b=0.338) showing a positive relationship between the gill respiratory surface area /VO₂ ratio and body mass (b=0.328); this indicates that the fish have greater gill respiratory surface area per unit of routine VO₂ as they grow.     

急性氨氮胁迫对黄颡鱼组织抗氧化酶活性及HSP70和HSP90基因mRNA表达水平的影响

为了探讨急性氨氮胁迫对黄颡鱼组织中抗氧化酶活性及HSP70和HSP90基因mRNA表达水平的影响,实验随机挑选了360尾黄颡鱼[初体质量(17.25±0.05)g],分别暴露于含有0(对照)、5.70(低浓度组)、28.50(中浓度组)和57.00(高浓度组)mg/L总氨氮浓度的水体中,进行96 h的急性胁迫实验。实验开始后,分别于0、12、24、48和96 h取样。结果显示,氨氮胁迫发生后,低、中浓度组实验鱼肝脏中超氧化物歧化酶(SOD)活性呈先升高后降低趋势,而高浓度组则持续降低;低、中、高浓度组实验鱼肝脏中丙二醛(MDA)含量在胁迫开始后显著升高;3 h时,高浓度组实验鱼肝脏中SOD活性达到最低,而MDA含量最高;24 h后,高浓度组实验鱼肝脏中过氧化氢酶(CAT)活性显著升高;低、中、高浓度组实验鱼肝脏中HSP70基因的mRNA表达量呈先降低后升高趋势,而鳃中HSP70基因表达量持续升高,但脑中HSP70基因在0 h后显著降低;氨氮胁迫3 h时,低、中、高浓度组实验鱼肝脏和脑中HSP70基因表达量显著低于对照组,而在鳃中正好相反;相比HSP70基因,高氨氮浓度组实验鱼肝脏和鳃中HSP90基因的mRNA表达量在24 h时达到最高。研究表明,不同浓度的氨氮胁迫会对黄颡鱼抗氧化酶活性造成不同程度的抑制,原因与丙二醛的积累量有关;相比HSP90基因,黄颡鱼HSP70基因的表达量在氨氮胁迫发生后迅速上调,这种生理调控机制提示HSP70在应对急性氨氮胁迫时发挥着更重要的作用。     

花鲈ncc、nkcc基因在海、淡水适应中鳃组织的表达与定位分析

为探究ncc、nkcc基因在花鲈渗透调节中发挥的作用,实验通过全基因组鉴定、多重序列比对、系统进化树构建以及蛋白结构预测对花鲈ncc进行了鉴定及序列分析,利用实时荧光定量PCR (qRT-PCR)检测ncc和nkcc在海水、淡水花鲈鳃组织中的表达水平,利用原位杂交技术确定ncc2和nkcc1a在海水及淡水花鲈鳃中的表达位置。结果显示,从花鲈中鉴定出2个ncc基因,即ncc1和ncc2,其编码序列(CDS)长度分别为2 691和3 120bp,编码896和1 039个氨基酸,在进化上具有保守性。ncc2在淡水花鲈鳃组织中的表达量显著高于海水,而nkcc1a在海水花鲈鳃组织中的表达量显著高于淡水,ncc1、nkcc1b、nkcc2在海淡水中的表达量则无显著差异。淡水适应过程中花鲈鳃组织中的ncc2的表达量逐渐上调,而nkcc1a的表达量逐渐下调;海水适应过程则呈现相反的表达趋势。此外,原位杂交结果显示,ncc2和nkcc1a基因分别位于淡水与海水中鳃组织的相邻鳃小片间的鳃丝上皮。以上结果表明,ncc2和nkcc1a基因分别编码淡水及海水花鲈鳃中重要的Na⁺及Cl     

草鱼呼肠孤病毒Ⅱ型自然发病与人工注射感染草鱼的病理症状和病毒分布研究

为了探究自然发病和人工注射感染草鱼呼肠孤病毒Ⅱ型(GCRV-Ⅱ型)草鱼的临床症状、病理特征和病毒分布的区别，实验采用临床剖检、组织病理学观察、分子生物学检测、实时荧光定量PCR(qRT-PCR)和蛋白质印迹法(Western blot)等检测方法开展实验，通过对自然发病和人工注射感染草鱼的临床症状进行比较，结果显示，患病草鱼的眼眶、鳃盖、口腔、腹部、鳍条基部、肠道、肌肉出现明显的点状出血，且后者的出血情况比前者更为严重；比较组织病理切片，发现草鱼感染组织出现不同程度的充血和红细胞充盈现象，其中，人工注射感染草鱼的肠道、肌肉和肝胰脏的病变程度更为严重，呈现出较为严重的组织内出血和病变，而自然发病草鱼的鳃和脾脏的病变程度更为严重，鳃呈现出较为严重的充血和炎症增生物，脾脏出现大面积含铁血黄素沉积块病灶。qRT-PCR和Western blot检测结果显示，GCRV在不同的组织中均有分布，头肾在两种感染方式的患病草鱼中的病毒量都比较高，人工注射感染草鱼的肝胰脏、肠道和肌肉的病毒量较高，自然发病草鱼的中肾、鳃和脾脏的病毒量较高。因此，在人工注射感染时，肝胰脏、肠道和肌肉可能是GCRV入侵的主要靶...     

Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single‐sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.     

Arachidonic acid metabolism in gill homogenate and isolated gill cells from rainbow trout,Oncorhynchus mykiss: the effect of osmolality,electrolytes and prolactin

An assay method based on thin layer chromatography to study the arachidonic acid (AA) metabolism in gill tissues was optimized and the effect of osmotically different incubation mediums on AA metabolism was evaluated. Rainbow trout gill tissues metabolize AA into PGE₂ in highest concentration followed by PGD₂, PGF₂ and 6-keto-PGF₁ (the stable metabolite of PGI₂) among the prostanoids tested. Approximately 40% of PGE₂ is synthesized within the first minute of incubation and is directly dependent on the substrate concentration (AA). As in mammalian tissues, PGE₂ synthesis in fish gills is inhibited by the cyclooxygenase inhibitor indomethacin. PGE₂ synthesis in gill homogenate and isolated gill cells incubated in trout Ringer was 0.45 and 1.9 ng/mg protein, respectively, and increased to 8.9 and 4.3 ng/mg protein, respectively, when incubated in KPO₄ buffer, due to a ten-fold increase in the free AA. The hydroxy acid synthesis of the gill homogenate was higher (13%), and that of the isolated gill cells incubated in KPO₄ buffer was lower (44%) compared to gill homogenate and cells incubated in trout Ringer. Gill homogenate incubated in 50 mM phosphate buffer with increasing sodium or potassium concentrations (up to 250 mM) exhibited a concentration-dependent increase in PGE₂ synthesis (220% and 72%, respectively). Prolactin stimulated the PGE₂ synthesis up to 30% while PGD₂, PGF₂ and 6-keto-PGF₁ synthesis was not affected. This effect of prolactin was maximal when PGE₂ synthesis was estimated 30 minutes after prolactin addition and diminished after two hours. These results suggest that rainbow trout gills possess the ability to metabolize AA through the cyclooxygenase and lipoxygenase pathways. PGE₂ synthesis may be under the influence of ion balance and prolactin availability, indicating the probable involvement of AA metabolites in the regulation of ion balances across the gill membrane.     

Distribution of X-cell disease in common dab, Limanda limanda L., in the North Sea, and ultrastructural observations of previously undescribed developmental stages

Abstract. The distribution and prevalence of common dab, Limanda limanda L., affected with X-cell lesions was investigated at various localities around the British Isles. Extreme variability in the prevalence of affected gills was recorded in the Moray Firth (NE Scotland), where samples obtained from hauls taken in close proximity ranged from nil to 60% of the collected dab. The condition was not detected in dab from the Irish Sea, and was found only sporadically in samples in the southern North Sea and the northwest coast of Scotland, Areas of high prevalence are typically coastal or relatively shallow habitats. The patchy distribution of the disease throughout dab populations suggests that an infectious agent may be involved in its aetiology. A method is described for rapid, accurate screening of fish samples for X-cell disease, which utilizes the autofluorescence of the X-cell. Ultrastructural studies of lesions from external and internal organs in dab disclosed some previously undescribed features of X-cells. Stages of the X-cell development which occurred in'nests', and possibly preceded the commonly found X-cells, were found in gill lesions. Electron dense inclusions found in X-cells in the spleen and kidney were rod- or dumbbell-shaped structures and possibly represented secretory granules.     

Simulation of the effects of oxygen on food consumption and growth of Nile tilapia, Oreochromis niloticus (L.)

Fish need oxygen for aerobic generation of energy for body maintenance, locomotion, feeding and biosynthesis. The rate of oxygen uptake of most fish is limited by diffusion through the gills, and gill surface area grows at a slower rate than body mass. We hypothesize, therefore: (1) that the maximum rate of feed intake is related to the capacity to take in oxygen for processing of this feed; and (2) that the maximum rate of feed intake relative to body size decreases with increasing body size to a point where growth is zero. An oxygen limitation module based on this theory was incorporated into an existing dynamic simulation model for the Nile tilapia, Oreochromis niloticus (L.). The module calculates the maximum potential oxygen supply to the fish on the basis of Fick's law of diffusion and the allometric relationship between body weight and gill surface area. Total oxygen demand of the fish is computed as the sum of routine metabolism, feeding metabolism and energy needed for biosynthesis. In the module, the feeding rate is limited to a level where total oxygen demand does not exceed the potential supply. The model is used to simulate feeding and growth of O. niloticus. Simulation results provide strong support for the oxygen limitation theory. Hence, the model can be used for the analysis of fish growth as affected by feed amount, feed composition, as well as environmental conditions (temperature, dissolved oxygen concentration). It also explains differences in final size between fish species, and within species under different conditions.     

Fate of plasma glucose in tissues of brown trout in vivo: effects of fasting and glucose loading

We report the fate of glucose, both as a source of energy and as a temporary store, in the tissues of brown trout (Salmo trutta) in control, fasted and glucose-loaded fish. Tissue glucose utilization (³H-2-deoxyglucose phosphorylation) and storage (conversion of ¹⁴C-glucose into glycogen, protein, and lipid) were measured in immature brown trout, and the oxidation rate was calculated as glucose utilization minus storage and ¹⁴C-ionic metabolites remaining in the tissue. The glucose utilization rate is tissue-specific, the highest values being found in spleen, kidney, hindgut, brain, and gill. All these tissues also showed a highly active glycolytic pathway. The lowest utilization indices were observed in white and red muscles, skin, stomach and caeca, which also presented the largest proportion of glucose converted into stores (mainly protein and glycogen). Fasting reduced the glucose disappearance rate by 24%, although there were no significant variations in glucose utilization indices or distribution profile. After a glucose load, plasma glucose and insulin levels rose and the rates of glucose utilization, storage, and oxidation also increased in all tissues (from 1.5- to 4-fold). The relative importance of each tissue in glucose disposal was similar to that in normoglycaemia. In liver, only glucose storage was measured reliably; the conversion of glucose to glycogen was higher than in other tissues, and rose markedly (35-fold) in glucose-loaded fish. In most tissues glucose flux into lipids, glycogen and protein increased. The distribution of glucose may not be a merely substrate-mediated process because fasting in glucose-loaded fish caused lower tissue glucose utilization, particularly in gut, red muscle and gills. Conversion of glucose to tissue stores was reduced, lipids being the most affected.