Investigation of co‐infections with pathogens associated with gill disease in Atlantic salmon during an amoebic gill disease outbreak

Gill diseases are a complex and multifactorial challenge for marine farmed Atlantic salmon. Co‐infections with putative pathogens are common on farms; however, there is a lack of knowledge in relation to the potential effect co‐infections may have on pathology. The objective of this study was to determine the prevalence and potential effects of Neoparamoeba perurans, Desmozoon lepeophtherii, Candidatus Branchiomonas cysticola, Tenacibaculum maritimum and salmon gill poxvirus (SGPV) during a longitudinal study on a marine Atlantic salmon farm. Real‐time PCR was used to determine the presence and sequential infection patterns of these pathogens on gill samples collected from stocking until harvest. A number of multilevel models were used to determine the effect of these putative pathogens on gill health (measured as gill histopathology score), while adjusting for the effect of water temperature and time since the last freshwater treatment. Results indicate that between 12 and 16 weeks post‐seawater transfer (wpst), colonization of the gills by all pathogens had commenced and by week 16 of marine production each of the pathogens had been detected. D. lepeophtherii and Candidatus B. cysticola were by far the most prevalent of the potential pathogens detected during this study. Detections of T. maritimum were found to be significantly correlated with temperature showing distinct seasonality. Salmon gill poxvirus was found to be highly sporadic and detected in the first sampling point, suggesting a carryover from the freshwater stage of production. Finally, the model results indicated no clear effect between any of the pathogens. Additionally, the models showed that the only variable which had a consistent effect on the histology score was N. perurans.     

Methacarn preserves mucus integrity and improves visualization of amoebae in gills of Atlantic salmon (Salmo salar L.)

Two aqueous fixation methods (modified Davidson's solution and modified Davidson's solution with 2% (w/v) Alcian blue) were compared against two non‐aqueous fixation methods (methacarn solution and methacarn solution with 2% (w/v) Alcian blue) along with the standard buffered formalin fixation method to (a) improve preservation of the mucous coat on Atlantic salmon, Salmo salar L., gills and (b) to examine the interaction between the amoebae and mucus on the gill during an infection with amoebic gill disease. Aqueous fixatives demonstrated excellent cytological preservation but failed to deliver the preservation of the mucus when compared to the non‐aqueous‐based fixatives; qualitative and semi‐quantitative analysis revealed a greater preservation of the gill mucus using the non‐aqueous methacarn solution. A combination of this fixation method and an Alcian blue/Periodic acid–Schiff staining was tested in gills of Atlantic salmon infected with amoebic gill disease; lectin labelling was also used to confirm the mucus preservation in the methacarn‐fixed tissue. Amoebae were observed closely associated with the mucus demonstrating that the techniques employed for preservation of the mucous coat can indeed avoid the loss of potential mucus‐embedded parasites, thus providing a better understanding of the relationship between the mucus and parasite.     

Site of acid-base relevant ion transfer in the gills of rainbow trout (Oncorhynchus mykiss) exposed to environmental hypercapnia

In order to delineate the contribution of primary vs. secondary circulatory circuits in the gill for acid-base and ionic regulation, the flow and composition of the fluids in the central venous sinus (CVS) and the systemic circuit of rainbow trout were studied by application of a previously developed microcannulation technique during normocapnic and hypercapnic conditions. The average haematocrit (Hct) of blood from dorsal aorta (DA) and sinus venosus (SV) ranged from 20.1 to 26.7%, whereas average Hct in the fluid from the branchial vein (BV), representing drainage from the central venous sinus (CVS), was in the range of 4.2 to 7.0%. Under normocapnic conditions, the largest fraction of cardiac output, 92.9%, was directed through the systemic vascular circuit, whereas the CVS circuit was perfused with 7.1 % of cardiac output. Hypercapnia did not significantly affect the blood flow distribution between the two circuits. The pattern of acid-base regulation in dorsal aortic blood reflected the characteristic response of fish exposed to environmental hypercapnia. Upon initiation of environmental hypercapnia (2% CO₂), plasma P_{CO 2} was elevated in all three flow compartments (CVS, DA, SV), inducing an extracellular respiratory acidosis of about 0.4 pH units. pH and [HCO₃ ^-] values in the DA were consistently lower, than those for both CVS and SV sites throughout the hypercapnic period. During the 8h of exposure plasma bicarbonate concentration was elevated by about 12 mM, complemented by a fall in extracellular [Cl^-] of about 10 mM in all three compartments. The amount of HCO₃ ^- gained at the CVS site during 8h of hypercapnia (3.3 mmol · kg^-1) exceeds the amount accumulated in the extracellular space (2.1 mmol·kg^-1), suggesting the CVS as the main site of ionic acid-base regulation in trout.     

Family and genetic group effects for resistance to proliferative gill disease in channel catfish, blue catfish and channel catfish×blue catfish backcross hybrids

The severity of gill damage (percentage of gill lamellae with lytic lesions) was determined in juveniles from 10 USDA 103 line channel catfish Ictalurus punctatus full‐sib families, 10 channel catfish×blue catfish I. furcatus backcross hybrid (7/8 channel catfish, 1/8 blue catfish) full‐sib families and a mixed‐family group of blue catfish placed in a commercial catfish pond experiencing proliferative gill disease (PGD)‐related fish mortalities. An initial challenge was conducted with all families, and a second challenge was conducted using the two most susceptible (most gill damage) channel catfish and backcross hybrid families and the two most resistant (least gill damage) channel catfish and backcross hybrid families. In the initial challenge, percentage gill damage was not different between channel catfish (12.3%) and backcross hybrids (11.6%), but was lower in blue catfish (0.2%). Mean percentage gill damage in the second challenge was not different among resistant backcross hybrid families (6.9%), resistant channel catfish families (7.6%) and blue catfish (4.8%), but was higher in susceptible backcross hybrid and channel catfish families (19.0% and 11.9% respectively). The correlation among family means for gill damage from challenge 1 and challenge 2 was r=0.87. Consistent differences between channel catfish and blue catfish and between resistant and susceptible families within genetic groups for gill damage after PGD challenge suggest that there is a genetic component for resistance to PGD and that improving PGD resistance through selection may be possible.     

Morphological diversity of Paramoeba perurans trophozoites and their interaction with Atlantic salmon,Salmo salar L., gills

Amoebic gill disease (AGD) caused by the ectoparasite Paramoeba perurans affects several cultured marine fish species worldwide. In this study, the morphology and ultrastructure of P. perurans in vitro and in vivo was investigated using scanning and transmission electron microscopy (SEM and TEM, respectively). Amoebae cultures contained several different morphologies ranging from a distinct rounded cell structure and polymorphic cells with pseudopodia of different lengths and shapes. SEM studies of the gills of AGD‐affected Atlantic salmon, Salmo salar L., revealed the presence of enlarged swellings in affected gill filaments and fusion of adjacent lamellae. Spherical amoebae appeared to embed within the epithelium, and subsequently leave hemispherical indentations with visible fenestrations in the basolateral surface following their departure. These fenestrated structures corresponded to the presence of pseudopodia which could be seen by TEM to penetrate into the epithelium. The membrane–membrane interface contained an amorphous and slightly fibrous matrix. This suggests the existence of cellular glycocalyces and a role for extracellular products in mediating pathological changes in amoebic gill disease.     

Coping with climate change: Phenotypic plasticity in an imperilled freshwater fish in response to elevated water temperature

1. Climate change has emerged as an increasingly important threat to freshwater systems. To cope with rapidly changing thermal regimes, freshwater fishes must either relocate or adjust through genetic adaptation and/or phenotypic plasticity. Short-term responses to elevated water temperature have been well studied in freshwater fishes; however, far less is understood about change induced by long-term exposure. Furthermore, few studies have investigated the effects of temperature on already imperilled species, which may be more sensitive to environmental change.
2. This study investigated the effects of rearing temperature on critical thermal maximum (CT_max), agitation temperature (T_ag, temperature at which fish show behavioural signs of thermal stress) and gill size in pugnose shiner, Notropis anogenus, a threatened species in Canada. Juvenile pugnose shiner were reared for 4 months across five different ecologically relevant temperatures. CT_max and T_ag were measured under normoxia and acute exposure to hypoxia to test for oxygen sensitivity of the upper thermal limits in this species.
3. CT_max increased with elevated water temperature. T_ag also increased with rearing temperature and occurred, on average, 4.3°C above acclimation temperatures. The CT_max and T_ag were lower when fish were exposed acutely to hypoxia. Interestingly, gill size (e.g. total gill filament length) increased with rearing temperature, which may increase oxygen uptake capacity and support increased metabolic demands of warmer waters.
4. Overall, pugnose shiner show plasticity in several traits in response to long-term exposure to elevated water temperature that may facilitate persistence in warmer waters. However, acute hypoxia exposure reduced thermal tolerance, stressing the importance of evaluating interactive effects of multiple stressors.
5. Identifying source populations of pugnose shiner with greater thermal tolerance or implementing captive breeding under higher temperature regimes may improve the success of re-introduction efforts in the face of climate change, but the consequences to fitness of increased thermal tolerance should be examined.

     

稀有鮈鲫鳃的组织结构及水体镉暴露对其影响

运用石蜡切片技术和扫描电镜技术对稀有鮈鲫(Gobiocypris rarus)鳃结构进行观察,并采用浓度为2 mg/L的Cd Cl_2水溶液对稀有鮈鲫进行急性毒理实验。结果显示:稀有鮈鲫具4对鳃,由鳃耙、鳃弓、鳃丝及鳃小片组成。鳃的各个部位表面均有上皮细胞覆盖;鳃耙和鳃弓具味蕾,鳃弓上还具不同类型的黏液细胞;相邻鳃小片的上皮细胞间也分布有黏液细胞,在鳃小片基部有氯细胞着生。鳃丝表面有不规则凹陷和微嵴,具沟、坑、孔等结构;鳃小片两面均呈凹凸状。在2 mg/L的Cd Cl_2水溶液暴露下,鳃受到损伤,发生鳃小片充血呈球状,鳃上皮水肿、脱落,黏液细胞增多等现象;并随时间延长受损程度加重。     

Production of salmonid amoebic gill disease by exposure to Paramoeba sp. harvested from the gills of infected fish

Atlantic salmon, S almo salar L., were exposed to different concentrations of Paramoeba sp. The lowest concentration which induced amoebic gill disease (AGD) was 230 Paramoeba sp. L^–1 and the severity of infection increased with increasing concentration. The concentration of Paramoeba sp. positively correlated with the number of gill lesions ( R ² > 0.7). This study provides evidence that Paramoeba sp. is the causative agent of AGD and describes an experimental model that enables the severity of the induced disease to be controlled.     

壬基酚对1月龄泥鳅的急性毒性及鳃、肝脏组织学损伤研究

为了研究壬基酚(Nonylphenol,NP)对淡水鱼类幼鱼的急性毒性及相关组织的损伤效应,选取平均体质量6.0 g、平均体长9.2 cm的1月龄泥鳅(Misgurnus anguillicaudatus)为受试材料,检测壬基酚对幼鱼的急性毒性效应,按等对数间距法,将NP设置为1.69、1.95、2.25、2.60、3.00 mg/L共计5个浓度处理组进行急性毒性实验,同时设置空白对照组,每组设3个平行样,每个样投放10条泥鳅,观察NP对泥鳅鳃、肝脏组织结构的损伤变化,并统计半致死浓度和安全浓度。结果表明,壬基酚对1月龄泥鳅24、48、72、96 h的半致死质量浓度分别为3.722、2.939、2.466、2.175 mg/L,安全浓度为0.550 mg/L。组织学切片观察显示,壬基酚高浓度处理使泥鳅鳃小片细胞脱落严重,鳃小片变细;泥鳅肝脏细胞出现空泡、肿大、核脱落等现象,细胞间隙变大,边界模糊。实验表明,壬基酚属于高毒性酚类污染物,对1月龄泥鳅的毒性作用随着暴露浓度增加、作用时间的延长而增强,在高剂量暴露下可造成泥鳅鳃和肝脏组织的严重损伤。     

Identification of a novel gill-specific calpain from rainbow trout (Oncorhynchus mykiss)

Calpains are calcium-dependent neutral proteases responsible for many cellular functions. The two forms of calpain ubiquitously expressed in mammalian tissues are known as μ-calpain and m-calpain. We report here the identification of a novel calpain that is similar to but distinct from the μ- and m-calpains in rainbow trout. The cDNA of the novel gene is 2623 bp in length with a single open reading frame. The predicted protein (676 amino acids) contains the conserved calpain characteristic domains that include: domain I (pro peptide), II (cysteine catalytic site), III (electrostatic switch), and IV (calmodulin-like) with five Ca²⁺-binding EF hands. Northern blot and RT-PCR analyses demonstrated that the novel calpain gene is predominantly expressed in rainbow trout gills. Comparison of the novel protein with the ubiquitously expressed calpains and several mammalian tissue-specific calpains revealed that the novel calpain is an orthologue of the mammalian digestive tract specific calpain (calpain 9).     

Acute lion's mane jellyfish,Cyanea capillata (Cnideria: Scyphozoa), exposure to Atlantic salmon (Salmo salar L.)

Jellyfish‐induced gill pathology relies upon occasional diagnostic observations yet the extent and impact of jellyfish blooms on aquaculture may be significant. Idiopathic gill lesions are often observed in apparently healthy fish. This study exposed Atlantic salmon (Salmo salar L.) smolts to macerated Cyanea capillata at 2.5 and 5 g/L for 2 hr under controlled laboratory conditions. Blood chemistry and gill histopathology were examined over a subsequent 4‐week period. Fish showed an acute response to the presence of jellyfish, including characteristic external “whiplash” discoloration of the skin and acute increases in blood electrolytes and CO₂ concentration; however, these were resolved within 4 days after exposure. Histopathologically, gills showed first an acute oedema with epithelial separation followed by focal haemorrhage and thrombus formation, and then progressive inflammatory epithelial hyperplasia that progressively resolved over the 4 weeks post‐exposure. Results were consistent with the envenomation of gills with cytotoxic neurotoxins and haemolysins known to be produced by C. capillata. This study suggests that many focal hyperplastic lesions on gills, especially those involving focal thrombi, may be the result of jellyfish stings. Thus, the presence of jellyfish and their impact may be severe and understated in terms of marine fish aquaculture and fish welfare.     

鱼类的鳃病及其防治方法

引起鱼类鳃病的病原体主要包括以下几种：细菌、病毒、霉菌、原生动物、单殖吸虫和甲壳类。本文对这些疾病的症状作了描述，并提出了综合防治措施。     

苏氏Mang鲶鳃超微结构观察

对苏氏Mang鲶鳃结构进行扫描和透射电镜观察。Mang鲶鳃丝末端膨大呈杓状结构，每一鳃丝两侧具有许多呈褶状的鳃小片，相邻两鳃丝上的鳃小片紧密镶嵌排列。鳃弓和鳃耙表面分布众多味蕾。鳃丝呼吸面上皮细胞薄，高度血管化，形成呼吸面隆起，非呼吸面由微脊细胞彼此相连，间缝具分泌细胞开口。鳃小片由单层或数层外上皮细胞和由基膜相隔的柱状细胞及其围在血管腔的凸缘构成，氯细胞多分布在鳃小片基部，并有开口通外，还探讨了Mang鲶鳃丝和鳃小片特殊的结构与功能。     

苏氏鲢鲶鳃超微结构观察

对苏氏鲢鲶鳃结构进行扫描和透射电镜观察.鲢鲶鳃丝末端膨大呈杓状结构,每一鳃丝两侧具许多呈褶状的鳃小片,相邻两鳃丝上的鳃小片紧密镶嵌排列.鳃弓和鳃耙表面分布众多味蕾.鳃丝呼吸面上皮细胞薄,高度血管化,形成呼吸面隆起;非呼吸面由微脊细胞彼此相连,间缝具分泌细胞开口.鳃小片由单层或数层外上皮细胞和由基膜相隔的柱状细胞及其围在血管腔的凸缘构成,氯细胞多分布在鳃小片基部,并有开口通外.还探讨了鲢鲶鳃丝和鳃小片特殊的结构与功能.     

福瑞鲤对氨氮胁迫的生理响应

以福瑞鲤(Cyprinus carpio)为材料,研究不同浓度氨氮(0、10、20和30 mg/L)处理对幼鱼鳃组织中抗氧化系统和Na~+/K~+-ATP酶活力的影响。结果显示,氨氮胁迫6 h诱导H_2O_2含量、总抗氧化能力(T-AOC)和超氧化物歧化酶(SOD)活性迅速显著升高,并在胁迫24 h时达到最高水平。胁迫96 h后,各处理组H2O2含量、SOD活性和总抗氧化能力均低于对照组水平,MDA含量显著增加。氨氮胁迫后,Na~+/K~+-ATP酶基因转录水平和蛋白活性呈先降低后升高又降低的变化趋势,参与胁迫过程中渗透压的调控。研究结果表明,低浓度氨氮胁迫后,福瑞鲤鳃组织通过调节细胞抗氧化能力,提高Na~+/K~+-ATP酶活性,有效改善鱼体的抗氨氮胁迫能力,但是高浓度氨氮胁迫对鳃组织造成氧化损伤。     

Effect of cadmium on the cytoskeleton and morphology of gill chloride cells in parr and smolt Atlantic salmon (Salmo salar)

This study documented the effect of cadmium on salmon parr and smolt gill morphology. Cadmium-induced changes in chloride cell (CC) cytoskeletal elements were investigated, as well as the modifications of CC surface area and density. In cadmium-treated parr (10 µg Cd l^-1 for 2 days), immunofluorescent light microscopy revealed the appearance of an intense actin staining located in the CC apical part. Transmission electron microscopic observations revealed a change in the organization of the microfilaments at the CC apex, with the appearance of numerous aggregates of filamentous actin. Higher cadmium concentrations (30 and 50 µg l^-1) and prolonged treatment times (7 to 14 days) did not modify such reorganisation. Microtubules were not significantly affected by similar treatments. Further, scanning electron microscopic analysis revealed that cadmium induces a significant increase of parr CC surface area as early as the second day of exposure. After 2 days, mature CC density had also increased. In smolt, a rise in CC surface area was observed, although CC density did not significantly increase.     

胡子鲶鳃扫描电镜的观察

本文应用扫描电镜对胡子鲶鳃表面形态结构进行了详细的观察,对其丝状鳃、片状鳃与枝状鳃的表面形态特征进行了比较研究,并对三种鳃之间的关系进行了探讨。