红螯螯虾酚氧化酶原及其特性研究

采用同源克隆策略和RACE技术, 从红螯螯虾Cherax quadricarinatus血细胞中克隆得到酚氧化酶原基因的全长cDNA序列, 共2951 bp, 开放读码框为1995 bp, 编码665个氨基酸. 预测的分子量和等电点分别为75.7 kD和6.23. 酚氧化酶原含有两个推测的tyrosinase copper-binding motifs (带有六个组氨酸残基)和一个thiol-ester-like motif, 这些特征和其他甲壳动物的酚氧化酶原特征相同. 红螯螯虾酚氧化酶原氨基酸序列与通讯螯虾Pacifastacus leniusculus、欧洲龙虾Homarus gammarus、美洲龙虾Homarus americanus 和克氏原螯虾Procambarus clarkii 酚氧化酶原的相似率分别为68%、63%、63%和59%. 酚氧化酶原基因双酶切后连接入pET-28a原核表达载体, 转化到大肠杆菌BL21后重组表达酚氧化酶原蛋白. 在重组蛋白纯化后, 免疫新西兰大耳兔制备得到的酚氧化酶原多克隆抗体, 其效价大于1:12800. 红螯螯虾血淋巴、肝和鳃组织中的酚氧化酶原mRNA表达和酚氧化酶活性较高, 而神经、心、肠和肌肉中较低. 中华绒螯蟹螺原体和嗜水气单胞菌免疫红螯螯虾后, 血淋巴细胞、肝和鳃组织中的酚氧化酶原和酚氧化酶活性在免疫后的不同时间均出现了显著性的增加, 此结果表明酚氧化酶原和酚氧化酶在红螯螯虾对抗细菌感染的过程中起到重要的免疫作用. 此结果为进一步深入研究酚氧化酶原基因和酚氧化酶的功能及其调控机理奠定基础.       

Gonad maturation,morphological and physiological changes during the first reproductive cycle of the crayfish Cherax quadricarinatus female

Summary

Concurrent morphological, anatomical and physiological changes took place during the first reproductive cycle in the Australian red-claw crayfish Cherax quadricarinatus, which prepared the female for spawning and holding of the newly deposited eggs. The endopod became longer and wider than the exopod and developed a mixture of plumose and long thin simple (ovigerous) setae. Small oocytes (0.24±0.05 mm) were present in the immature ovary. The growing ovary contained two distinct oocyte populations: one consisted of small (0.55±0.07 mm), barely growing oocytes, while the other consisted of large oocytes, which increased in size continuously (0.73 to 2.55 mm) until egg laying took place. A gradual change in the relative abundance of ovarian polypeptides occurred until the late vitellogenic stage (large oocytes < 1.8 mm). Three predominant female-specific, SDS-PAGE separated, polypeptides were observed (103, 78 and 73 kDa) that may represent vitellin subunits. The most abundant carotenoid in the ovary was astaxanthin, while β-carotene was present at a lower concentration. The strong correlation between the increasing diameter of the oocyte and the concentration of astaxanthin in the ovary and in the hemolymph suggested an association of astaxanthin with vitellin and vitellogenin.     

Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus

Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56 kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1,4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012 bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194 bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.     

Effect of long‐term injection of dopamine on the ovarian growth of Cherax quadricarinatus juvenile females (Parastacidae,Decapoda)

The neurotransmitter dopamine (DA) modulates many physiological processes in decapod crustaceans, including reproduction. This study was aimed at evaluating whether the long‐term injection of DA affects ovarian growth in the freshwater crayfish Cherax quadricarinatus. Three experiments were performed with females of different mean initial size: 4.15 g (Group A); 9.65 g (Group B); and 13.98 g (Group C). Treated females were injected with DA and control females with physiological saline twice a week for 90 (Group B), 105 (Group A) and 120 (Group C) days. At the end of the experiments, the animals were killed, and the stage of ovarian development, gonadosomatic index, and mean oocyte diameter were determined. DA had a differential effect according to female size: it negatively affected ovarian growth of females in a weight range of 4–14 g and had no effect on ovarian maturation when injected to females with an initial weight of 14 g. The results are compared with previous reports in freshwater decapod crustaceans.     

Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l(-1)). On the other hand, pH varied greatly, from 8.7+/-0.15 in the gastrolith cavity through 7.6+/-0.2 in muscle to 6.9+/-0.5 in the stomach.     

红螯光壳螯虾卵黄磷蛋白的分离纯化和鉴定

采用凝胶层析、聚丙烯酰胺凝胶电泳及SDS-聚丙烯酰胺凝胶电泳法对红螯光壳螯虾(Cherax quadricarinatus)卵巢中的卵黄磷蛋白进行了分离、纯化和鉴定。结果显示该虾的卵黄磷蛋白是一个分子量为369ku的多聚体,由分子量为85．4、80．6、76．6、73．7ku的四个亚基组成。染色分析表明卵黄磷蛋白为糖-磷-类胡萝卜素结合的复合蛋白。氨基酸组成分析显示天冬氨酸(Asp)和谷氨酸(Glu)为主要氨基酸。     

Circadian oscillations of RPCH gene expression in the eyestalk of the crayfish Cherax quadricarinatus

The RPCH and β-actin cDNAs from the crayfish Cherax quadricarinatus were amplified, cloned and sequenced. The primary structure sequences of these cDNAs were compared to other members of the AKH/RPCH family. Fluctuations in the amount of the C. quadricarinatus RPCH and β-actin mRNAs, as cDNAs, were quantified every 3 h by RT-PCR. Single cosinor analysis supports the notion of β-actin and RPCH mRNA circadian behavior in animals subjected to 12 h:12 h light/dark regimes. In constant darkness RPCH mRNA concentration changes to ultradian cycles.     

Structural changes in the spermatophore of the freshwater 'red claw' crayfish Cherax quadricarinatus (Von Martens, 1898) (Decapoda, Parastacidae)

López Greco, L.S. and Lo Nostro, F.L. 2007. Structural changes in the spermatophore of the freshwater ‘red claw’ crayfish Cherax quadricarinatus (Von Martens, 1898) (Decapoda, Parastacidae). —Acta Zoologica (Stockholm) 88 : 000–000 The structure of the spermatophore was studied in Cherax quadricarinatus. Pieces of the distal vas deferens and transferred spermatophore from the females were fixed, cut and stained. Within the distal vas deferens, the primary layer and the secondary layer of the spermatophore were distinguishable. In the latter, two components were detected: cytoplasmic droplets and a homogeneous matrix. During the first 10 minutes post‐extrusion the cytoplasmic droplets drastically changed from looking like ‘empty droplets’; at this time the spermatophore changed from a liquid stage to a sticky one. One hour after extrusion the spermatophore began to harden and within the first 24–48 h post‐mating it was a solid and intense white structure tightly attached to the female; after 72 h it acquired a softer aspect, completely dehiscing between 96 and 120 h post‐mating. Histologically, the primary layer maintained its integrity surrounding the spermatozoa while the secondary layer lost the cytoplasmic droplets. The spermatophore began to hydrate between 24 and 48 h and by 72–96 h many sections of the sperm cord began to coalesce. From 48 h post‐mating some fissures appeared within the matrix that enlarged between 72 and 120 h. We propose that both manipulation by the female and hydration are the mechanisms involved in the release of the spermatozoa from the spermatophore.     

Effect of different diets on digestive enzyme activities,in vitro digestibility,and midgut gland structure in juvenile crayfish,Cherax quadricarinatus

This study investigated the effects of food quality on digestive enzyme activities, in vitro protein digestibility and histological traits of the midgut gland in juvenile crayfish Cherax quadricarinatus. Animals of a wide weight range were fed different diets: two commercial diets with high or low lipid content (high lipid and low lipid, respectively) and were compared with a reference diet (RF) previously formulated for this species. Proteinase, lipase and amylase activities were significantly influenced by diet and weight. Specific trypsin activity was significantly higher for crayfish fed with the HL diet. Trypsin activity depended on diet and weight. Protein digestibility showed that HL was the most digestible diet and RF the least. The weight of the animals did not affect protein digestibility. Structural disorganization, hypertrophy of B‐cells and presence of large vacuoles in R‐cells were mainly observed in juveniles fed with HL, indicative of malnutrition. Thus, our data suggest that the HL diet would not be the most appropriate for C. quadricarinatus, while RF diet would be more convenient for culture of this species.     

In vitro effects of both dopaminergic and enkephalinergic antagonists on the ovarian growth of Cherax quadricarinatus (Decapoda, Parastacidae), at different periods of the reproductive cycle

The in vitro effect of both spiperone (dopaminergic antagonist) and naloxone (enkephalinergic antagonist), was assayed on small pieces of ovary dissected from C. quadricarinatus females, with the eventual addition of some neuroendocrine organs, such as thoracic ganglion or eyestalk tissue. The incorporation of tritiated leucine by the ovary was measured in order to estimate the ovarian growth. During the post-reproductive period, both mentioned antagonists were able to significantly stimulate the ovary in the presence of thoracic ganglion, but did not produce any significant effect in the preparation containing ovary and eyestalk tissue, or only ovary. No significant effects of the assayed antagonists were noted during the pre-reproductive period. These results were in accordance with previous models describing the neuroendocrine control of crustacean reproduction, and represent new findings about the hormonal context in different periods of the reproductive cycle of crayfish. Besides, by means of the experimental combination of the tested antagonists with dopamine or met-enkephalin, a new model dealing with the interaction of these two neurotransmitters on the hormonal secretion of thoracic ganglion has been proposed.     

红螯光壳螯虾胚胎及仔虾发育过程中卵黄磷蛋白的ELISA检测

建立了间接竞争酶联免疫吸附反应(ELISA)方法,对红螯光壳螯虾(Cherax quadricarinatus)胚胎及仔虾发育过程中的卵黄磷蛋白含量及其亚基组分进行了研究。该方法对卵黄磷蛋白具有良好的特异性,有效检测范围为31.25~250 ng/ml。结果表明,在发育初期胚胎先降解分子量比卵黄磷蛋白大的蛋白;亚基中,分子量较大和较小的亚基都先被消耗;胚胎内卵黄磷蛋白含量总体上呈下降趋势,其中在卵裂囊胚阶段后略有上升(3.19%),至后无节幼体期卵黄磷蛋白含量达最高(4.67%),之后不断下降,到仔虾离开母体独立生活时,含量只剩下卵裂期的1/4。     

Structure and function of a cellulase gene in redclaw crayfish, Cherax quadricarinatus

The most abundant organic compound produced by plants is cellulose; however, it has long been accepted that most animals do not produce endogenous enzymes required for its degradation, but rely instead on symbiotic relationships with microbes that produce the necessary enzymes. Here, we present the genomic organisation of an endogenous glycosyl hydrolase family (GHF) 9 gene in redclaw crayfish (Cherax quadricarinatus), consolidated from a cDNA sequence determined by Byrne et al. [Gene 239 (1999) 317–324.]. Comparison with several other invertebrate GHF9 genes reveals the conservation of both intron position/phase and splice sequence, which adds support to an argument for an ancestral animal cellulase gene. Furthermore, two introns in plant GHF9 genes are also identical in position, implying a more ancient origin for this class of animal cellulase.

Protein purification from redclaw gastric fluid via fast performance liquid chromatography (FPLC) indicated the presence of two endoglucanase enzymes. The molecular weights of these components were determined by matrix-assisted laser desorption/ionisation—time-of-flight (MALDI-TOF) to be 47,887 Da (Cel1) and 50,295 Da (Cel2). Cel1 is possibly the functional product of the described cellulase gene, with N-terminal amino acid residues identical to the translated amino acid sequence from the corresponding gene region. Cel2 was identical to Cel1 for 7 of 11 N-terminal residues and likely to be the product of a paralogous endoglucanase gene. These results suggest that redclaw crayfish possess at least one and possibly two functional, endoglucanase enzymes, although further work is required to confirm their origin and attributes.     

Predation on invasive redclaw crayfish Cherax quadricarinatus by native fishes in the Kafue River,Zambia

The stomach contents of eight species of predatory or omnivorous fish caught in gillnets in the Kafue River in May and June 2010 were examined to determine the relative importance, expressed as ‘prominence value’ (PV), of crayfish Cherax quadricarinatus in their diet. Four species, Clarias gariepinus, Schilbe intermedius, Serranochromis robustus and Serranochromis angusticeps, had fed on C. quadricarinatus. The PV of C. quadricarinatus was highest in C. gariepinus, in which the quantity and size of crayfish eaten was significantly correlated with standard length (SL) of fish >200 mm SL. Predated C. quadricarinatus were significantly smaller (mean carapace length [CL] 30 mm; SE 1.2) than those caught in gillnets (mean CL 76 mm; SE 0.9). No C. quadricarinatus remains were found in the stomachs of Oreochromis andersonii, Synodontis macrostigma, Serranochromis macrocephalus or Mormyrus lacerda.     

The introduction,spread and ecology of redclaw crayfish Cherax quadricarinatus in the Zambezi catchment

Two of the four crayfish species brought to Zambia for aquaculture since 1979 are now naturalised. Procamburus clarkii occurs in the Maramba River at Livingstone, close to a former fish farm, whereas Cherax quadricarinatus, deliberately introduced to a number of sites in the Zambezi and Kafue River catchments since 2001, is now widespread and highly invasive. High rates of dispersal, up to 111 km y?1 downstream, might be the result of passive transport on floating vegetation. Significantly more synodontid Synodontis sp. catfish were caught in Fladen traps in the Kafue River in crayfish-free areas compared with crayfish-infested areas, but the possibility that synodontids became trap averse in the presence of crayfish was not ruled out. No difference was found in the numbers of Single-spined River Crab Potamonautes unispinus caught in crayfish-free and infested areas. Alien temnocephalans, commonly found on crayfish, are now also present on crabs P. unispinus on the Kafue Flats. The artisanal gill net fishery has been harmed by the introduction of C. quadricarinatus and no significant commercial fishery has developed. However, without more information on the adverse impacts, and stronger fisheries regulation, there is a high risk C. quadricarinatus and P. clarkii will be introduced to uninfested catchments with irreversible consequences for artisanal fishermen and the environment.     

Development of the female reproductive system in the freshwater crayfish Cherax quadricarinatus (Decapoda, Parastacidae)

Abstract. The differentiation of the female reproductive system from a macroscopic and microscopic point of view was studied in Cherax quadricarinatus. For this characterization, 184 females were dissected and processed for the histological analysis. From the differentiation of the ovary up to its maturity, three ovarian morphotypes could be distinguished macroscopically: parallel strands without any contact between them, an H-shaped ovary, and a Y-shaped ovary. These morphotypes were compared within the Astacida. Four ovarian developmental stages were recognized based on ovary color, and the histological structure and relative proportion of cellular types. The post-spawning ovary was also characterized. The components of the female reproductive system sheath were described and its modifications in the ovary and oviducts were determined and compared. Theoretical aspects of the study of sexual differentiation in C. quadricarinatus were discussed within a phylogenetic framework.