Glutathione content and GSH S-transferase activity in midgut gland of Procambarus clarkii. Sex differences, the effect of fasting, and their implications in cadmium toxicity

1. Glutathione content and GSH S-transferase activity in the midgut gland of Procambarus clarkii (P. c.) of different sex and body weight are presented. 2. Procambarus clarkii females' GSH concentration in the midgut gland decreases to a higher extent upon fasting, compared with males. 3. Procambarus clarkii females, both in control and fasting conditions, have a slightly higher GSH S-transferase activity than males. 4. Cadmium present in water only affects GSH content and GSH S-transferase activity (after 96 hr) in midgut gland, with cadmium chloride concentrations higher than 100 micrograms/l.     

镉对克氏原螯虾肝胰腺抗氧化系统的影响

采用毒性试验方法,研究了不同浓度Cd2+(1.72、3.44、6.89、13.77和27.55mg·L-1)对克氏原螯虾肝胰腺抗氧化酶(SOD、CAT、GSH-PX)和抗氧化物质(GSH、Vc)的影响.结果表明:超氧化物歧化酶(SOD)活性受低浓度Cd2+诱导和高浓度Cd2+抑制,且受抑制程度与Cd2+浓度呈正相关.过氧化氢酶(CAT)活性总体表现为先激活后下降,且在第3天达最大值,CAT活性对低浓度Cd2+(≤6.89 mg·L-1)暴露敏感,而高浓度Cd2+对其影响较小.谷胱甘肽过氧化物酶(GSH-PX)活性对Cd2+浓度敏感,当Cd2+≤6.89 mg · L-1时,GSH-PX活性先升高后下降,更高浓度下GSH-PX活性在暴露后第1天即表现出抑制作用.还原型谷胱甘肽(GSH)含量在Cd2+≤6.89 mg·L-1时始终被诱导,且均在第1天达最大值,而高浓度Cd2+(≥13.77 mg·L-1)对GSH含量影响不明显.维生素C(Vc)对Cd2+胁迫敏感,各处理组Vc含量在第1天均显著下降,且下降程度与Cd2+浓度呈正相关,之后具有一定的合成恢复能力.抗氧化酶和非酶抗氧化物质在抵御Cd2+胁迫反应中共同发挥作用,且大多表现出明显的时间和剂量效应性.GSH-PX和Vc可以作为Cd2+污染的潜在生物指示物.     

镉和十二烷基硫酸钠对克氏原螯虾的单一和联合毒性

采用静水生物测试法,研究了十二烷基硫酸钠(SDS)和Cd2+对克氏原螯虾的单一和联合毒性效应。结果表明,SDS对克氏原螯虾24、48、72和96h的半致死浓度(LC50)分别为1365.60、1171.72、1128.43和1120.43mg·L-1,安全浓度(SC)为258.79mg·L-1;Cd2+对克氏原螯虾24、48、72和96h的LC50分别为1197.09、142.06、90.85和82.64mg·L-1,SC值为0.6mg·L-1。SDS和Cd2+对克氏原螯虾的毒性分别为低毒和高毒,毒性大小为Cd2+>SDS。联合毒性试验表明,二者毒性比为1∶1,暴露时间为24、48、72和96h时,相加指数(AI)分别为0.335、0.017、0.030和0.032,联合作用结果均为毒性增强的协同作用。     

克氏原螯虾Rab5、Rab6蛋白及其抗体对血淋巴细胞吞噬活性的影响

为研究克氏原螯虾(Procambarus clarkii)Rab5(PcRab5)和Rab6(PcRab6)的生物学功能, 利用同源重组技术构建了克氏原螯虾pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体, 并进行了诱导表达和多克隆抗体的制备, 采用ELISA和Western Blot技术检测了抗体效价和特异性。将PcRab5和PcRab6的原核表达蛋白和多克隆抗体注射到健康克氏原螯虾体内, 研究了其对血淋巴细胞吞噬活性的影响。实验结果表明构建的pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体经诱导后可表达目的蛋白, 分子量均为67 kD, 纯化后蛋白条带单一, 纯度较高。重组表达纯化后的PcRab5和PcRab6蛋白免疫日本大耳兔分别获得效价为1:2048 K和1:512 K的兔抗血清, 制备的抗体可分别特异性识别PcRab5和PcRab6蛋白。将PcRab5和PcRab6蛋白注射健康克氏原螯虾后, 其血淋巴细胞中可吞噬荧光微球的细胞比例分别显著上升至38%和30%(P＜0.01)。而将纯化的PcRab5和PcRab6多克隆抗体分别注射至螯虾体内后, 其血淋巴细胞的吞噬细胞比例均出现显著下降(P＜0.05), PcRab5和PcRab6蛋白参与了血淋巴细胞吞噬功能。实验为进一步研究克氏原螯虾PcRab5和PcRab6分子功能奠定基础, 也可为理解甲壳动物Rab5和Rab6在血淋巴细胞吞噬功能中的作用提供帮助。     

Population genetic structure and post-establishment dispersal patterns of the red swamp crayfish Procambarus clarkii in China

The red swamp crayfish (Procambarus clarkii) was introduced to China in the early 20(th) century. It has been spread to almost all forms of fresh water bodies including lakes, rivers and even paddyfields in most provinces of China. To clarify issues such as the initial entry point(s), dispersal pattern, genetic diversity and genetic structure of Procambarus clarkii in China, the genetic structure and diversity of P. clarkii populations at 37 sampling sites (35 from China, one from the USA and one from Japan) were analyzed using both mitochondrial gene sequences (COI and 16S rRNA) and 12 nuclear microsatellites. Multiple tests including phylogenetic analyses, Bayesian assignment and analysis of isolation by distance showed that (i) the population from Japan and those collected from China, particularly from NanJing (BGt and XG) and its some neighboring sites (CJr, NT and NB), have similar genetic composition, (ii) relatively high genetic diversity was detected in Chinese populations, (iii) the P. clarkii populations in China did not experience significant population expansions. Taken together, Nanjing, Jiangsu province is the presumed initial entry point, and human-mediated dispersal and adaptive variation are likely responsible for the observed genetic pattern of P. clarkii in China.     

Regulation of ion concentration by Drosophila hydei Na+, K+, Ca2+, and Cl-

The ionic composition of the haemolymph of osmotically unencumbered larvae of Drosophila hydei shows a pattern that is typical (Bone, 1944) for highly developed phytophagous insect larvae: 36 mval/l. Cl^?; 56 mval/l. Na⁺; 31 mval/l. K⁺; approximately 18 mval/l. Ca²⁺ at an osmolality of 299 mOsmol/l.The larvae are able to maintain their most favourable ionic concentrations in the haemolymph after experimental osmotic stress in hypertonic as well as in hypotonic media. The reactions are most distinct with an increase or decrease of Cl^? concentration of the external medium. Characteristic regulating processes begin, and the Cl^? concentration of the haemolymph adjusts to the ‘standard’ again. The principal lapse shows the functional representation of an intensely suppressed oscillation. There seems to be a two-point regulation which requires the existence of a Cl^? ion depot and the existence of Cl^?-sensitive receptors.     

Effect of variation in photoperiod and light intensity on oxygen consumption, lactate concentration and behavior in crayfish Procambarus clarkii and Procambarus digueti

The effects of light intensity and duration on metabolic and behavioral parameters of two species of crayfish, Procambarus clarkii and Procambarus digueti, were studied. Sixty animals of each species were submitted to high irradiance conditions of two different photoperiod lengths, one normal light/dark (LD) 12:12 and one extreme LD 20:4 for 2 weeks. Hemolymph, lactate and oxygen consumption were determined throughout the experimental period. Simultaneously in 18 additional animals of each species, motor activity was individually recorded under the same control and experimental conditions. Both species showed a decrease in oxygen uptake and an increase in hemolymph lactate concentration. The statistical significance of this finding was higher for LD 20:4. This extreme condition evoked a significant decrease of motor activity in P. clarkii and a high mortality rate in P. digueti. P. digueti did not survive after the experiment, whereas P. clarkii survived and adapted to the laboratory conditions. Changes in metabolic and behavioral parameters could indicate different adaptation abilities in these species.     

Variance analysis of Cd2+, Zn2+, and Mn2+ effect on membrane Na+,K(+)-ATPase activity of loach embryos

The evaluation of influences Cd2+, Zn2+, Mn2+ in concentrations 10(-6), 10(-5), 10(-4) M (factor of dose) on the Na+, K(+)-ATP-ase activity in the early period of development (60-330 min.) of loach embryos (time factor) using one- and two-factor analysis of variance has been performed. It has been detected, that the changes of enzyme activity are mainly caused by action of the explored cations and do not depend on time of embryos development. The most influence on activity in the indicated period of embryos development of loach renders Cd2+ in concentration 10(-4) M, relative value of its influence being 95.7% (p = 0.01). Substantial concentration dependence of the Na+, K(+)-ATP-ase activity is exposed to the action of each of cations. The values of the influence of their concentration changes during the studied period of development differ insignificantly for all cations and make 76.2-77.5% (p < 0.01).     

Primary structure and relative potency of an analog of beta-PDH (pigment-dispersing hormone) from the crayfish Procambarus clarkii.

A pigment-dispersing hormone (PDH) from eyestalks of the crayfish Procambarus clarkii was purified by gel filtration, cation-exchange chromatography, partition chromatography, and reversed-phase HPLC. Based on automated sequencing and by the identical chromatographic behavior of the native PDH and the synthetic amidated form of the deduced sequence, the primary structure of Procambarus PDH has been established as: Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Glu-Ala- NH2. This peptide differs from beta-PDH of the fiddler crab Uca pugilator at a single position, Glu17 in place of Asp17. Because of this substitution, Procambarus PDH was 4 to 7-fold less potent than beta-PDH in causing pigment dispersion in the erythrophores, leucophores, and melanophores of Uca. In contrast, Procambarus PDH was 4-fold more potent than beta-PDH in eliciting pigment dispersion in the erythrophores of Procambarus. These peptides displayed less marked differences in potency in triggering leucophore pigment dispersion and light-adaptational distal eye pigment movement in Procambarus. These findings indicate that the structural requirements for PDH-receptor interactions vary with the species and with the target cell type within a given species.     

克氏原螯虾嗜水气单胞菌噬菌体的分离鉴定和应用

为分离获得致病性嗜水气单胞菌烈性噬菌体,鉴定和研究其生物学特性,为克氏原螯虾细菌性疾病的防治和治疗提供依据,研究以患病克氏原螯虾分离到的致病性嗜水气单胞菌为宿主菌,从湖泊水体中分离到多株噬菌体,对其中一株烈性噬菌体Ph-0进行了生物学特性的分析,包括噬菌斑形态的观察,最佳感染复数的测定,一步生长曲线的绘制,热稳定性的测...     

克氏原螯虾的入侵生态学研究进展

克氏原螯虾(Procambarus clarkii)原产于美国南部和墨西哥北部,是一个著名入侵物种,作为一种水产经济资源物种在世界各地扩散。克氏原螯虾抗逆性强,所具有的广泛生境适应性、生长迅速、高生殖率等特点使它们迅速建立野生种群。近10余年的研究认为,克氏原螯虾通过捕食和资源竞争等机制严重威胁引入地的水生植物、无脊椎动物、两栖类等的生存,显著降低引入地的生物多样性。当前,由于克氏原螯虾的经济价值高,它会借助于人力的作用而继续扩散。为认清和减少克氏原螯虾对引入地的生态影响,应加强以下方面的研究:1)在中国开展克氏原螯虾的生态危害的调查和研究;2)克氏原螯虾种群调节和控制对策研究;3)被入侵地的生态恢复工作。     

One century away from home: how the red swamp crayfish took over the world

Reviews in Fish Biology and Fisheries - The red swamp crayfish (Procambarus clarkii) (hereafter RSC), native to the southern United States and north-eastern Mexico, is currently the most widely...     

Environment and host-related factors modulate gut and carapace bacterial diversity of the invasive red swamp crayfish (Procambarus clarkii)

Hydrobiologia - The invasive red swamp crayfish Procambarus clarkii is present in most of the Portuguese rivers with well-known economic and environmental impacts, being also an important reservoir...     

一种新的螯虾丝氨酸蛋白酶抑制物基因的克隆表达及其活性分析

根据本课题组从克氏原螯虾中新发现的丝氨酸蛋白酶抑制物的基因序列(GenBank登录号CD644775)设计一对引物,应用逆转录-聚合酶链式反应(RT-PCR)技术,从螯虾血淋巴细胞中扩增出丝氨酸蛋白酶抑制物基因PCI188,将其连入原核表达载体pET-32a,转化至大肠杆菌Rosetta株和BL21株中进行蛋白表达,结果该蛋白只在前者表达。表达产物用免疫转印检测,出现50kD的特异性条带,与螯虾PCI188基因编码的蛋白大小相符。将融合蛋白纯化后免疫新西兰兔,用免疫血清与螯虾血淋巴作用后测定酚氧化酶活力,结果显示,酚氧化酶活力有所升高,从而首次证实螯虾PCI188编码的蛋白对丝氨酸蛋白酶有抑制作用。     

Physiological biomarkers of hypoxic stress in red swamp crayfish Procambarus clarkii from field and laboratory experiments

The crayfish industry in Louisiana is the largest in the United States, with crayfish frequently harvested from waters that experience episodic or chronic hypoxia (dissolved oxygen [DO]≤ 2 mg/l). We examined physiological biomarkers (hemolymph lactate, glucose, and protein concentrations) of hypoxic stress in the red swamp crayfish Procambarus clarkii from chronically hypoxic natural habitats and laboratory hypoxia experiments. P. clarkii from normoxic and hypoxic areas in the Atchafalaya River Basin were sampled monthly from April to July 2010. Laboratory experiments subjected P. clarkii to severe hypoxia (1 mg/l DO), moderate hypoxia (2 mg/l DO), or normoxic conditions (control: DO>7.5 mg/l) for 12, 24, and 48 h. P. clarkii from normoxic and hypoxic natural habitats did not display significantly different hemolymph lactate or glucose concentrations; however, mean hemolymph protein concentration was significantly lower in crayfish from hypoxic areas. P. clarkii exposed to severe hypoxia in laboratory experiments had significantly higher hemolymph lactate and glucose concentrations for all three exposure times, whereas large differences in protein concentrations were not observed. These results suggest that elevated hemolymph lactate and glucose concentrations are responses to acute hypoxia in P. clarkii, while differences in protein concentrations are the result of chronic hypoxic exposure.     

Behavioral effects of serotonin and serotonin agonists in two crayfish species, Procambarus clarkii and Orconectes rusticus

Exogenous serotonin elicits several behaviors in Procambarus clarkii, including a flexed, elevated posture, reduced locomotion, and changes in aggressive behavior. We conducted experiments to determine if several serotonin agonists mimicked the behavioral effects of serotonin in two crayfish species, P. clarkii and Orconectes rusticus. Drugs tested were 1-(3-Chlorophenyl)-piperazine dihydrochloride (mCPP), Oxymetazoline, 2,5-dimethoxy-4-iodoamphetamine (DOI), CGS-12066A, and (+/-)-8-hydroxy-2-(di-n-dipropylamino) tetralin (8-OH-DPAT). In P. clarkii, mCPP most closely mimicked the effects of serotonin, significantly increasing the performance of the flexed, elevated posture and reducing locomotion; 8-OH-DPAT significantly reduced locomotion as well. Both of these drugs produced significant increases in elevated posture and decreases in locomotion in O. rusticus, and in this species, the drugs at test concentrations were more effective in eliciting these effects than serotonin. The effects of the drugs on behaviors performed during fighting bouts were variable. In both species, only 8-OH-DPAT significantly reduced several agonistic behaviors, and no agonist or 5-HT itself produced significant increases in agonistic behavior.     

克氏原螯虾源维氏气单胞菌的分离鉴定及组织病理学观察

为确定克氏原螯虾(Procambarus clarkii)暴发性死亡的病原体, 研究从一例患病克氏原螯虾的肝胰腺分离到了一株优势菌株QD160502, 根据菌株形态、生理生化特性, 16S rDNA及gyrB序列分析, 将其鉴定为维氏气单胞菌(Aeromonas veronii)。回归感染实验证明, 该分离株可使克氏原螯虾出现与自然发病相同症状。组织病理学观察可见克氏原螯虾肝胰腺、肠道与心脏组织病理损伤, 且随感染持续时间而逐渐加重, 在感染12h后肝小管分泌细胞中出现内含棕色颗粒的转运小泡; 感染24h后肝小管间炎性细胞浸润, 肠道结缔组织萎缩, 心脏组织出现空泡样扩张; 感染48h后肝小管储存细胞与分泌细胞大量解体并空泡化, 肠道上皮皱嵴减少, 心肌空泡样扩张增加; 感染72h后肝小管和肠道上皮细胞坏死, 且在心脏组织中发现透明血细胞聚集。药物敏感性检测发现QD160502菌株对恩诺沙星、诺氟沙星、四环素、强力霉素等敏感, 但对青霉素、链霉素、卡那霉素等抗生素耐药。研究为克氏原螯虾的健康养殖和细菌性疾病的防控提供指导依据。     

Asynchronous swimmeret beating during defense turns in the crayfish, Procambarus clarkii

Swimmeret beating was monitored in freely moving specimens of the crayfish Procambarus clarkii as they exhibited defense turn responses to tactile stimuli. Analysis of videotape records revealed alterations in swimmeret beating during turning responses compared to straight, forward walking. During turns, swimmerets beat with shorter periods and smaller amplitude power strokes than during straight walking. Coordination between swimmerets also changed. Swimmerets on the side toward which the animal turned tended to lag behind their contralateral partners, rather than beat in synchrony as in straight walking, and ipsilateral coordination was loosened relative to straight walking. Asynchronous swimmeret beating accompanied asymmetric motions of the uropods in a manner similar to that observed during statocyst-dependent equilibrium reactions in P. clarkii, but removal of the statoliths did not eliminate turn-associated responses of the swimmerets. The coordinated action of the swimmerets and uropods may contribute to the torque that rotates the animal in the yaw plane. Implications of the observed changes in swimmeret coordination for understanding the underlying neuronal control system are discussed.     

Micro-scale fluid and odorant transport to antennules of the crayfish, Procambarus clarkii

A numerical model was developed to determine advective-diffusive transport of odorant molecules to olfactory appendages of the crayfish, Procambarus clarkii. We tested the extent of molecule transport to the surfaces of aesthetasc sensilla during an antennule flick and the degree of odorant exchange during subsequent flicks. During the rapid downstroke of a flick, odorant molecules are advected between adjacent aesthetascs, while during the slower return stroke, these odorants are trapped between the sensilla and molecular diffusion occurs over a sufficient time period to transport odorants to aesthetasc surfaces. During subsequent flicks, up to 97.6?% of these odorants are replaced with new odorant molecules. The concentration of molecules captured along aesthetasc surfaces was found to increase with increased gap spacing between aesthetascs, flick speed, and distance from the proximal end of the aesthetasc, but these changes in morphology and flicking kinematics reduce the animal's ability to take discrete samples of the odorant-laden fluid environment with each flick. Results suggest that antennule flicking allows discrete sampling of the time- and space-varying odorant signal, and high concentration odorant filaments can be distinguished from more diffuse, low concentration filaments through changes in both the timing and the encounter rate of odorant molecules to aesthetasc surfaces.     

Protein and lipid concentration in the haemolymph of Danais chrysippus. Linn

Proteins and lipids in the haemolymph in both the sexes of the Danais chrysippus has been observed during the first gonotropic cycle. The concentration fluctuations in these haemolymph metabolites are correlated with the physiological state of the insect. The total protein and lipid concentration in the haemolymph of unmated females have been found to be low than the concentration found for the normal females although it followed the same type of oscillations in the concentration as have been observed for normal females.