Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

Background  

Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.     

Freshwater fishes of thailand as experimental intermediate hosts for Capillaria philippinensis

Bhaibulaya M., Indra-Ngarm S. and Ananthapruti M. 1979. Freshwater fishes of Thailand as experimental intermediate hosts for Capillaria philippinensis. The International Journal for Parasitology9: 105–108. Nine species of freshwater fishes from Thailand have been exposed to infection with embryonated eggs of Capillaria philippinensis. The eggs were fed to fishes with food or through a polyethylene tube. After 10–30 days larvae capable of establishing intestinal capillariasis in Mongolian gerbils were recovered from six fish species: Cyprinus carpio, Puntius gonionotus, Aplocheilus panchax, Gambusia holbrookii, Rasbora borapelensis, and Trichopsis vittatus. Larvae were not recovered from Tilapia nilotica, Osteochilus hasseltii and Trichogaster trichopterus. Five human cases of intestinal capillariasis have been reported from widely scattered areas of Thailand and the natural intermediate and definitive hosts remain to be determined.     

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

Mitochondrial genetic diversity and gene flow of common carp from main river drainages in China

1. The common carp (Cyprinus carpio L.) is one of the most widely distributed and important freshwater fishes in the world. In China, the common carp has been recognised as three subspecies: C. carpio haematopterus, C. carpio rubrofuscus and C. carpio carpio. The Nanling Mountains have been suggested as providing the dividing line between C. carpio haematopterus and C. carpio rubrofuscus. However, the demographic history and gene flow of the common carp in China is not clear. 2. We collected mitochondrial COII and D‐loop sequences (1494 bp) from 241 individuals distributed in eight major river drainages across China. The objective was to provide the first investigation into population genetic structure, demographic history and migration patterns of the common carp from these river drainages, and to assess the validity of the three subspecies. 3. Phylogenetic analysis did not result in three major monophyletic lineages corresponding to the three subspecies. The Nanling Mountains do not form a border separating C. carpio haematopterus and C. carpio rubrofuscus. amova showed low population differentiation, with 11.60% of the molecular variance found among river drainages. Pairwise F_ST values between river drainages were moderate (0.0331–0.2617). Substantial gene flow detected in coalescent analysis between drainages showed that the Yangtze drainage was the centre from which migrants moved northward, southward and north‐westward. Human‐mediated translocation has confounded our ability to identify subspecies of common carp in China.     

Ecological fish production in Long Pond,a lakeshore lagoon on Long Point,Lake Erie

Synopsis Long Pond, a recently formed 2.4 ha lagoon on Long Point, Lake Erie (Canada), was treated with piscicide and found to contain 47,768 fishes of 22 species. Nine dominant species (Notropis heterolepis, Pimephales notatus, Notemigonus crysoleucas, Lepomis gibbosus, L. macrochirus, Perca flavescens, Cyprinus carpio, Micropeterus salmoides and Carassius auratus) comprised 91% and 97% of total numbers and standing crop respectively. Age, growth and production were determined giving a total fish production estimate (corrected for all species) of 87.5 kg ha^–1 y^–1 for this lagoon. Dominant species were deemed either invaders (Cyprinus carpio, Carassius auratus), which had entered the lagoon to spawn and been trapped, or residents. The invaders comprised 82% of the 277.8 kg ha^–1 total standing crop. Their successful spawning and subsequent escape of the young from the lagoon would have resulted in some 615 kg y^–1 of fish production outside the lagoon. Resident stocks were dominated by young fishes, a characteristic of exploitation or early colonization. This may have been due to the recent formation and subsequent expansion of the lagoon or high annual mortality due to extreme physical conditions but was most likely the result of excessive predation by Micropterus salmoides. Relationship between biomass and production show that the chance inclusion of the invaders produces an immediate shift from a characteristically early to a characteristically late phase of ecological development.     

New species of myxosporidians in the genus Myxobolus (Myxosporidia, Myxobolidae) from cyprinid fishes of the Amur River

Five new species are described as follows: M. haematopterus from gills, fins and skin of Cyprinus carpio haematopterus; M. gibelio from connective tissue of gill filaments, fin rays and kidneys of Carassius auratus gibelio; Myxobolus waleckii from gills of Leuciscus waleckii; Myxobolus junchisi from gills, muscles, kidneys and spleen of Cyprinus carpio haematopterus; M. alacaudatus of Cyprinus and Carassius Myxobolus divergens carassii from gills, muscles, fins, and cartilaginous tissue of operculim of Carassius auratus gibelio and Cyprinus carpio haematopterus.     

Length–weight relationships of nine fish species from Xingkai (Khanka) Lake,Heilongjiang, China

In this study the length–weight relationships of nine fish species from Xingkai (Khanka) Lake in China belonging to three families and eight genera were analyzed. A total of 273 specimens were used to estimate the relationship parameters. Four of these species (Chanodichthys dabryi, Hemibarbus maculatus, Tachysurus fulvidraco and Cyprinus carpio haematopterus) had no previous estimations and one maximum length is new to science for inclusion in FishBase.     

Goussia carpelli (Protozoa, Apicomplexa) in cyprinid fish of the Duero basin (NW Spain). Aspects of host-parasite relationships

Goussia carpelli was found parasitizing the intestine and gall bladder of Cyprinus carpio, Carassius carassius and Barbus barbus bocagei, and less frequently Leuciscus cephalus cabeda and Chondrostoma polylepis, from the Esla River. The coccidian is described and data on its prevalence, intensity and pathogenicity are given. In histological sections, the oocysts were detected in the lamina propria or submucosa. We did not observe a clear seasonal pattern nor an influence of the host sex on the infections. On the contrary, there was a tendency to a decrease in the prevalence with the age of the host.     

长江野鲤(Cyprinus carpio)及两种养殖鲤群体遗传多样性评估

为了给鲤鱼遗传资源保护及利用提供较为准确的基础数据,利用10对微卫星标记分析了黄河鲤(Huanghe Cyprinus carpio,人工养殖群体)、长江野鲤(Changjiang wild Cyprinus carpio,安徽段)、安徽养殖鲤(Anhui cultured Cyprinus carpio,宿州市)群体遗传多样性。遗传多样性分析实验表明:3个群体均具有较高的遗传水平,且长江野鲤群体遗传多样性高于黄河鲤、安徽养殖鲤;卡方检验表明:3个群体在较大程度上受到人类活动及生态环境变化的影响,30个位点中76.67%的位点显著偏离Hardy-Weinberg平衡。遗传距离、遗传相似度、非加权组平均法(unweighted pair-group method with arithmetic means,UPGMA)聚类树及贝叶斯聚类分析均显示,长江野鲤与安徽养殖鲤亲缘关系较近,研究情况与实际生产中养殖户从长江里获得鲤鱼亲本用于繁殖苗种的现象相符。突变-漂移平衡分析显示,在TPM模式下,Wilcoxon符号秩次检验中黄河鲤群体显著偏离突变-漂移平衡,需进一步扩大选育群体。总体来说,黄河鲤、长江野鲤、安徽养殖鲤群体遗传多样性均较高,具有进一步的选育空间。     

Screening and identification of male‐specific DNA fragments in common carps Cyprinus carpio using suppression subtractive hybridization

In this study, a sex subtractive genomic DNA library was constructed using suppression subtractive hybridization (SSH) between male and female Cyprinus carpio. Twenty‐two clones with distinguishable hybridization signals were selected and sequenced. The specific primers were designed based on the sequence data. Those primers were then used to amplify the sex‐specific fragments from the genomic DNA of male and female carp. The amplified fragments from two clones showed specificity to males but not to females, which were named as Ccmf2 [387 base pairs (bp)] and Ccmf3 (183 bp), respectively. The sex‐specific pattern was analysed in a total of 40 individuals from three other different C. carpio. stocks and grass carp Ctenopharyngodon idella using Ccmf2 and Ccmf3 as dot‐blotting probes. The results revealed that the molecular diversity exists on the Y chromosome of C. carpio. No hybridization signals, however, were detected from individuals of C. idella, suggesting that the two sequences are specific to C. carpio. No significant homologous sequences of Ccmf2 and Ccmf3 were found in GenBank. Therefore, it was interpreted that the results as that Ccmf2 and Ccmf3 are two novel male‐specific sequences; and both fragments could be used as markers to rapidly and accurately identify the genetic sex of part of C. carpio. This may provide a very efficient selective tool for practically breeding monosex female populations in aquacultural production.     

Endoparasite spectrum of wild cats (<Emphasis Type="Italic">Felis silvestris</Emphasis> Schreber, 1777) and domestic cats (<Emphasis Type="Italic">Felis catus</Emphasis> L.) from the Eifel,Pfalz region and Saarland,Germany

Between 1993 and 2002, carcasses from 15 wild cats (Felis silvestris) and 17 domestic cats (Felis catus) from the Eifel region, Pfalz region and the Saarland were collected and examined for endoparasites. Most cats were road casualties (74%), some died from disease (14%), some were shot (3%), or some died of unknown reasons (9%). Three wild cats were too decomposed for parasitological examination. Endoparasites were recovered in 14 wild cats (n = 15) and 11 domestic cats (n = 17). A total of eight endoparasite species were found in wild cats and six in domestic cats. The nematodes Toxocara mystax and Toxascaris leonina and the cestode Taenia taeniaeformis were the most prevalent parasites. Other helminths detected were Capillaria aerophila, Capillaria feliscati, Capillaria plica and Mesocestoides litteratus. The spiruride Petrowospirura petrowi was detected in Germany for the first time. The parasite fauna was more diverse in male than in female cats indicating a male-biased parasitism in the wild cats.     

Analysis of egg protein in five carps

Quantitative and qualitative studies of proteins from the mature eggs of five carps—Cirrhinus mrigala, Labeo rohita, Cyprinus carpio, Hypophthalmichthys molitrix, Ctenopharyngodon idella were made. It is found that there is much difference both in the quantity and quality of proteins in these five species. As egg proteins in a species depend on the genome, it is suggested that the study of these protein types may be considered as an additional parameter for characterisation of the fish types.     

Predator-prey interactions: effects of carp predation on Tubificid dynamics and carp production in experimental fishpond

Effects of predation by carp (Cyprinus carpio L) were analysed in an experimental fishpond where the main components of the benthic fauna were tubificids. Enclosures inaccessible to fishes were compared with the rest of the pond stocked with 30 young carps (1⁺). Fish predation reduced the density, biomass and production of prey; the production of Tubificidae inside the enclosures was 1.7 times higher than that of the part of the pond stocked with fishes. On the other hand tubificid turnover ratios (P/B) inside and outside enclosures were not significantly different (respectively 5.57 and 5.38), and the size distribution of tubificids was not significantly modified. The ratio of the energetic equivalent of net fish production to biomass of tubificids consumed was 5.59%.     

Occurrence of bifidobacteria and lactobacilli in digestive tract of some freshwater fishes

Distal parts of the fish intestine were analyzed for presence of bifidobacteria and lactobacilli using selective agars. Seventy seven samples from Cyprinus carpio, Oncorhynchus mykiss, Carassius auratus, Tinca tinca, Perca fluviatilis, Rutilus rutilus, Scardinius erythrophthalmus, Oreochromis niloticus, and Squalius cephalus were collected randomly throughout years 2008 and 2009. Bifidobacteria were detected in 5 samples from 4 fish species at counts 2.18–4.29 log CFU/g, lactobacilli were present in 6 fish species at counts 1.21–3.65 log CFU/g. Seven bifidobacterial isolates were identified to the species level using biochemical tests and by sequencing of 16S rRNA gene. Three strains belonged to species B. longum, two isolates were identified as B. dentium, one strain as B. asteroides and one isolate was not determined to the species level by employed methods. As identified bifidobacterial species are considered to be of human, animal or honeybee origin, they probably derived as contamination from sewage or other sources. After further more detail testing, the possible use of isolated bifidobacteria as probiotics is promising since they were able to pass through the digestive tract successfully.     

Toxicity of intermittent chlorination to freshwater fish: Influence of temperature and chlorine form

Fingerling size Salmo gairdneri, Oncorhynchus kisutch, Notemigonus crysoleucas, Cyprinus carpio, and Ictalurus punctatus were exposed in the laboratory three times daily for up to seven days to pulses of either free chlorine or monochloramine. This regime simulated conditions often encountered in the outfall of steam electric generating plants which chlorinate intermittently. LC₅₀'s, LT₅₀'s and response isopleths giving various percentage mortalities, were computed from the bioassays. S. gairdneri, O. kisutch, and I. punctatus were the most sensitive to both types of chlorine. C. carpio were most resistant and the N. crysoleucas were intermediate in sensitivity. Temperature had relatively little effect on the toxicity of intermittent chlorine to the species tested. In this type of test regime, free chlorine was three to fourteen-fold more toxic (depending on the species) than monochloramine. Water quality criteria for the protection of fish should, in the future, take this differential toxicity into consideration.     

Genetic characterization of common carp (Cyprinus carpio) populations from Greece using mitochondrial DNA sequences

Wild common carp from two lakes and two rivers in Greece were genetically characterized with sequencing analysis of two mitochondrial DNA segments: cytochrome b (1119 bp) and D-loop (646 bp). A total of 9 variable singleton sites and 7 unique haplotypes were detected. A common haplotype was found in three out of the four populations examined, which seems to be the ancestral one and represents the European origin of common carp from Greece. This haplotype could be also justified by the introductions reported with individuals belonging to the Central European race, into many natural habitats in Greece. Limited genetic variation — in Evros and Aliakmonas populations — could be due to bottleneck effects and small effective population sizes, whereas the different haplotypes found in Lake Volvi could represent different common carp stocks. Values of sequence divergence among Greek haplotypes ranged from 0.0006 to 0.0023. The Neighbour-Joining (NJ) phylogenetic tree constructed based on the combined sequences, reveals that the populations of common carp from Greece belong to the European group of populations — which is highly divergent from the South East-Asia cluster — and to the subspecies Cyprinus carpio carpio.     

Immunohistochemical localization of urotensin I and other neuropeptides in the caudal neurosecretory system of three species of teleosts and two species of elasmobranchs

Summary In three species of teleosts — carp Cyprinus carpio; grass carp Ctenopharyngodon idella; and crucian carp Carassius auratus — the caudal neurosecretory system displays small, medium-sized and large neurons. Urotensin I (UI)-immunoreactive and UI-nonreactive neurons were found in all three groups; in general, the number of the latter neurons exceeded that of the former. Noteworthy are: (i) UI-immunoreactive fibers in the caudal spinal cord and (ii) dense accumulations of UI-immunoreactive product around the capillaries of the urophysis. In two species of elasmobranchs — cat shark Heterodontus japonicas and swell shark Cephaloscyllium umbratile — neurosecretory neurons decreased in size in rostro-caudal direction. Most of the neurosecretory perikarya, their axons and the corresponding neurohemal areas were UI-immunoreactive, but a small number of secretory neurons was devoid of immunoreaction. Oxytocin, arginine vasopressin, substance P, somatostatin, neurotensin, vasoactive intestinal polypeptide and gastrin-releasing peptide were not detected in the caudal neurosecretory system of the carp.     

Seasonal effects on some serum and muscle enzymes of catfish (Ictalurus melas) and common carp (Cyprinus carpio)1

Serum and muscle activities of enzymes (LDH, ALD, CK and ICDH) in intensively farmed catfish (Ictalurus melas) and common carp (Cyprinus carpio) were determined bimonthly between July 1 and November 2. Catfish serum enzymes showed no significant differences under different environmental conditions (exception = CK; values increased slighfly in the last sample). In carp serum, a remarkable increase of LDH and ALD occurred during the cold season. Muscle enzyme activity decreased in both species with decreasing water temperature and reduced metabolic activity.     

斑马鱼和鲤miR-1-2/133a-1基因间序列活性和调控研究

分别以斑马鱼(Danio rerio)和鲤(Cyprinus carpio L.)为研究材料,通过克隆斑马鱼和鲤miR-1-2和133a-1的基因间增强子序列,利用活体和离体实验探讨其是否具有肌肉特异性;并通过荧光素酶报告系统等研究转录因子MyoD是否调控该序列。研究结果显示:在活体实验中,无论斑马鱼还是鲤, miR-1-2/133a-1基因间序列均有肌肉特异性,且保留有保守区域(cr,含有E-box)的序列,注射72h后GFP表达量明显高于其他突变体。体外细胞实验也显示,转染含有cr序列的实验组,分化后的荧光素酶活性明显高于分化前。进一步探讨MyoD对miR-1-2/133a-1基因间序列的调控作用结果显示:无论是斑马鱼还是鲤, miR-1-2/133a-1基因间序列活性均受MyoD调控。结果为完善鱼类肌肉发育机理,及未来的鱼类分子育种奠定基础。