Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei)

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.     

Gyrodactyloides bychowskii (Monogenea: Gyrodactylidae) from sea-caged Atlantic salmon Salmo salar in Scotland: occurrence and ribosomal RNA sequence analysis

Gyrodactyloides bychowskii has been recorded in Scottish waters for the first time. The parasite was found on the gills of Atlantic salmon reared in seawater. An integrated morphological and molecular examination of the parasite was carried out. Prevalence of the parasite was greatest in February and declined to 0 by June in 1999. During 2000, parasites were located in March and November. An overdispersion of parasites was recorded, with intensity of infection reaching over 200 parasites per gill arch in some fish. Parasitised gill tissue showed hyperplasia and hypertrophy but not always at the site of parasite attachment. The internal transcribed spacer of the ribosomal RNA gene array was amplified by PCR and sequenced. This sequence shared greatest similarity with the internal transcribed spacer of Gyrdicotylus gallieni, followed by Gyrodactylus species. This is the first molecular analysis of this parasite and provides sequence data that may be used in comparison of G. bychowskii from other locations or in phylogenetic analysis of this group of Monogenea.     

The nuclear rDNA region of Gyrodactylus arcuatus and G. branchicus (Monogenea: Gyrodactylidae)

The primary structure of the ribosomal DNA internal transcribed spacers (ITS-1 and ITS-2) and 5.8S rRNA gene were used to characterize and identify 2 monogenean species of Gyrodacrylus living externally on the threespine stickleback (Gasterosteus aculeatus). The ITS region was amplified by PCR from freshwater, brackish, and marine isolates of Gyrodactylus arcuatus and G. branchicus, and the ends of the coding regions were identified by comparative alignment. No intraspecific and very low interspecific variation were observed in the 5.8S rRNA gene; high inter- and low intraspecific variation were revealed in the ITS-1 and ITS-2 regions. The morphological species identification was in all cases confirmed by the molecular identification. Intraspecifically, samples from 2 locations in the North Sea could be differentiated, but the Baltic sample resembled North Sea genotypes. Our approach offers perspectives for a multimetric genetical, morphometrical, and ecological taxonomy of the genus Gyrodactylus.     

Characterisation of a low pathogenic form of Gyrodactylus salaris from rainbow trout

Gyrodactylus salaris was isolated from rainbow trout in a Danish freshwater trout farm, and a laboratory population of this particular parasite form was established on rainbow trout. Challenge infections were performed using different salmonid strains and species, including East Atlantic salmon Salmo salar (from the Danish River Skjern?), Baltic salmon S. salar (from the Swedish River Ume Alv) and rainbow trout Oncorhynchus mykiss (from the Danish rainbow trout farm Fousing). These were compared to infection studies on the Norwegian Laerdalselva parasite form kept under exactly the same conditions in the laboratory. The Danish G. salaris form had low virulence towards both Atlantic and Baltic salmon, whereas rainbow trout proved susceptible to the parasite. The Danish G. salaris form was able to maintain a very low infection on East Atlantic salmon, but not on the Baltic salmon, which eliminated the infection within 2 wk. Rainbow trout developed infection intensities ranging up to several hundred parasites per host. The host colonization patterns of the parasite differed clearly from those of previous studies on microhabitats of the Norwegian form of G. salaris. A comparative study on morphological characters (opisthaptoral hard parts) from the Danish parasite form and Norwegian G. salaris showed no significant differences. Selected genes comprising internal transcribed spacers 1 and 2 (ITS), ribosomal RNA intergenic spacer (IGS) and cytochrome c oxidase subunit I (COI) regions were cloned and sequenced. Five sequenced ITS clones from 5 individuals of the Danish strain consistently revealed a single base substitution compared to ITS sequences from all other known species and strains of Gyrodactylus. Mitochondrial COI gene sequences demonstrated that the Danish G. salaris form is closely similar to the Laerdalselva parasite form found in Norway. The IGS sequences were highly variable, but very similar to those obtained from German isolates of G. salaris.     

Infestations by Gyrodactylus sp. of Atlantic salmon, Salmo salar L., in Norwegian rivers

Young Atlantic salmon, Salmo salar , brown trout, Salmo trutta , and Arctic char, Salvelinus alpinus , from eight rivers in North and Mid Norway were examined for Gyrodactylus. The fish were collected from 1975 to 1980. A Gyrodactylus salaris type was observed infecting salmon from six of these rivers. No trout or char were infected. A high frequency and intensity of infection of salmon were observed in all but one of the rivers surveyed. In the Saltdalselva, only one specimen of Gyrodactylus infecting one fish was observed. No subsequent mortality of salmon was observed in this river while there were signs of a high mortality of salmon in the other rivers. The salmon parr were more frequently attacked than the fry, the mortality of salmon seemed to have happened in most of the rivers one year after the first observations of Gyrodactylus were made. The mortality of salmon and the high infection rate of Gyrodactylus in these rivers appears unique, and as far as known to the authors there are no other described cases of mortality due to Gyodactylus in Atlantic salmon in natural waters. The reasons for the outbreak of Gyrodactylus in these rivers are not known. Two theories are discussed: one that the fish were weakened by environmental factors and the other that Gyrodactylus was introduced from some of the infected salmon hatcheries in Scandinavia.     

Complete mitochondrial genome of a forensically important muscid, Hydrotaea chalcogaster (Diptera: Muscidae), with notes on its phylogenetic position

Muscidae are a dipteran family which is important for forensic investigations. However, it has received limited attention in forensic entomological experiments as a reason of identification issues. It is hard to identify specimens by morphological methods, especially in developmental stages. Therefore, complete mitochondrial genome sequences can be important tool in forensic entomology for identifying species. In this study we sequenced and analyzed the first complete mitochondrial genome from a forensically important Muscidae species Hydrotaea (=Ophyra) chalcogaster by next-generation sequencing. The mitochondrial genome of the sequenced species is circular molecules of 17,076?bp which have the typical mitochondrial genome complement of 13 protein-coding genes, 22 tRNAs, two ribosomal RNA genes and a control region. Rearrangements of gene positions are identical with the ancestral insect genome. Furthermore, phylogenetic relationships of the family Muscidae were evaluated in regard to mitochondrial protein coding genes. The inferred trees indicate that the Muscidae is a paraphyletic family. These data provide additional information for molecular identification of muscid species.     

Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 2.4% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannamyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.     

金鱼寄生三代虫的形态学及基于rDNAits序列的分子鉴定

为查明我国金鱼三代虫的种类, 研究采用形态学与分子生物学相结合的方法, 对我国6个金鱼主要养殖区的三代虫进行了调查。通过形态测量分析, 发现了4种三代虫, 分别为: 小林三代虫（Gyrodactylus kobayashii）、G. gurleyi、G. longoacuminatus和三代虫未定种（Gyrodactylus sp.）。而通过核糖体rDNA its序列比对分析, 发现有2种三代虫分别与小林三代虫和G. longoacuminatus相似性在99.1%以上, 有1种与G. gurleyi的相似性在98.2%以上, 另外, 还有1种三代虫与现有序列的相似性都较低, 虽然它与小林三代虫的相似性最高, 也仅为91.0%, 暂为未定种。系统发育分析也显示, 金鱼寄生的3种三代虫分别与小林三代虫、G. gurleyi和G. longoacuminatus聚集在一起, 另外1种单独聚在一起, 这4种三代虫均隶属于G. （Limnonephrotus）亚属, 与G. （Gyrodactylus）亚属的秀丽三代虫（G. elegans）的亲缘关系很远。通过对三代虫样品的抽样调查发现, 小林三代虫存在于所有采样点, 在广州、武汉、郑州3个采样点为优势种, 所占的比率都在60%以上; G. gurleyi在海口、南京和上海为优势种, 所占的比率都在50%以上; G. longoacuminatus和三代虫未定种所占比率均较小。由此可见, 小林三代虫和G. gurleyi是我国金鱼常见的三代虫, G. gurleyi 和G. longoacuminatus是中国三代虫新记录种。       

Analysis of ribosomal RNA intergenic spacer (IGS) sequences in species and populations of Gyrodactylus (Platyhelminthes: Monogenea) from salmonid fish in Northern Europe

The intergenic spacer (IGS) region of ribosomal RNA genes was amplified and sequenced from a variety of Gyrodactylus specimens collected from wild and farmed Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, and grayling Thymallus thymallus, from various locations in Northern Europe. Phylogenetic analysis of the sequences confirmed the distinction between G. salaris Malmberg, 1957 and G. thymalli Zitnan, 1960, supporting their validity as separate species. G. salaris adapted to rainbow trout are also distinct from the parasites found on Atlantic salmon, supporting the existence of a rainbow-trout form that was initially identified on the basis of morphological differences. Analysis of the IGS did not provide good resolution of different populations of G. salaris sensu stricto, but was consistent with epidemiological evidence which indicates that introduction of the parasite to Norway was recent and limited. The IGS may be helpful in distinguishing forms of G. salaris that are pathogenic to Atlantic salmon from those that are not.     

Identification of a host-associated species complex using molecular and morphometric analyses,with the description of Gyrodactylus rugiensoides n. sp. (Gyrodactylidae,Monogenea)

Gyrodactylus rugiensis was originally described as a parasite occurring on the marine gobies Pomatoschistus minutus and Pomatoschistus microps. In our preliminary survey this species was also frequently found on Pomatoschistus pictus and Pomatoschistus lozanoi. Subsequent molecular analysis of the internal transcribed spacers rDNA region revealed that this parasite actually represents a complex of two apparently cryptic species, one restricted to P. microps and the other shared by P. minutus, P. lozanoi and P. pictus. Morphometric analyses were conducted on 17 features of the opisthaptoral hard parts of specimens collected from all four host species. Standard discriminant analysis showed a clear separation of both genotypes by significant differences in marginal hook and ventral bar features. Statistical classifiers (linear discriminant analysis and nearest neighbours) resulted in an estimated misclassification rate of 4.7 and 3.1%, respectively. Based on molecular, morphological and statistical analyses a new species, Gyrodactylus rugiensoides is described. This species seems to display a lower host-specificity than generally observed for Gyrodactylus species as it infects three sympatric host species.However, seasonal and host-dependent morphometric variation is shown for G. rugiensoides collected on P. pictus. Host-switching and gene flow might be important factors preventing speciation on closely related and sympatric host species. The presence of host associated species complexes in this Gyrodactylus-Pomatoschistus system is also confirmed by the presence of two host-dependent genotypes within G. micropsi found on P. minutus and P. lozanoi, and P. microps, respectively. By comparing host and parasite phylogeny, phylogenetic and ecological factors influencing host-specificity are discussed.     

Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Phiasterides dicentrarchi (Ciliophora: scuticociliatida)

In the course of identifying scuticociliates recently obtained from systemically infected olive flounder Paralichthys olivaceus in Korea, we found a scuticociliate species whose small subunit ribosomal RNA (SS rRNA) gene was not amplified by species-specific primers previously designed for Uronema marinum and Pseudocohnilembus persalinus. By studying morphological characteristics of wet-mounted and stained specimens, we identified the species as Philasterides dicentrarchi, which has been reported to cause systemic infection in the European sea bass Dicentrarchus labrax and turbot Scophthalmus maximus. In this study, we compared morphological characteristics of our specimens with previously reported Philasterides species, including P. dicentrarchi, and sequenced the SS rRNA gene in order to design P. dicentrarchi specific primers. This is the first report on scuticociliatosis caused by P. dicentrarchi from marine fish in Asia.     

The first complete monogenean ribosomal RNA gene operon: sequence and secondary structure of the Gyrodactylus salaris Malmberg, 1957, large subunit ribosomal RNA gene

The sequence of the Gyrodactylus salaris Malmberg, 1957, large subunit, or 28S, ribosomal RNA (rRNA) gene has been determined. This gene is the final portion of the Gyrodactylus rRNA gene operon to be sequenced and results in the first complete sequence of all rRNA genes and spacers from a monogenean. The nucleotide sequence was used to predict the secondary structure of the large subunit rRNA, and regions of conserved and variable sequence and structure were identified. The site where the 5' terminus of the 5.8S rRNA binds to a region within the large subunit rRNA was predicted and complements the anticipated interaction of the 3' terminus of the 5.8S with the 5' terminus of the large subunit rRNA. The large subunit gene may be useful in phylogenetic analysis of the Monogenea or Platyhelminthes and comparisons with other eukaryotes. The variable domains C and H may be most suitable for this purpose.     

A molecular phylogenetic investigation of zoothamnium (ciliophora, peritrichia, sessilida)

The gene coding for 18S small subunit ribosomal RNA (ssu rRNA) was sequenced in seven free-living, marine species of the sessiline peritrich genus Zoothamnium. These were Zoothamnium niveum, Zoothamnium alternans, Zoothamnium pelagicum, and four unidentified species. The ssu rRNA gene also was sequenced in Vorticella convallaria, Vorticella microstoma, and in an unidentified, freshwater species of Vorticella. Phylogenetic trees were constructed using these new sequences to test a previously published phylogenetic association between Zoothamnium arbuscula, currently in the family Zoothamniidae, and peritrichs in the family Vorticellidae. Trees constructed by means of neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods all had similar topologies. The seven new sequences of Zoothamnium species grouped into three well-supported clades, each of which contained a diversity of morphological types. The three clades formed a poorly supported, larger clade that was deeply divergent from Z. arbuscula, which remained more closely associated with vorticellid peritrichs. It is apparent that Zoothamnium is a richly diverse genus and that a much more intensive investigation, involving both morphological and molecular data and a wider selection of species, will be necessary to resolve its phylogeny. A greater amount of molecular diversity than is predicted by morphological data exists within all major clades of sessiline peritrichs that have been included in molecular phylogenies, indicating that characteristics of stalk and peristomial structure traditionally used to differentiate taxa at the generic level and above may not be uniformly reliable.     

Detection of invasive and cryptic species in marine mussels (Bivalvia,Mytilidae): A chromosomal perspective

Marine mussels illustrate a stunning variability in shape and color. Such variability, added to the scarcity of reliable morphological characters for their identification, can mislead recognition prompting the assignation of specimens of a single species to different ones or incorporate specimens belonging to different taxa into a single one. DNA barcoding is widely used for species identification; however, as this method relies on the previous morphological identification of the specimens, some of the DNA sequences stored in DNA databases are incorrectly assigned to a given species. In view of this uncertainty, further criteria beyond morphological characters and DNA sequences in databases are required to more reliably and accurately identify marine mussels. In this work we mapped ribosomal RNA and histone gene clusters to chromosomes of four species of marine mussels and compared them with those from another eight marine mussel taxa. Specimens of these twelve taxa were also DNA barcoded. Our results clearly demonstrated that the chromosomal analysis of marine mussels could shed light on their identification and, therefore, solve contradictions posed by morphological and molecular data.     

中国甘肃省狭义丝盖伞属二新种

依据形态学和分子系统学研究结果,描述了产自中国甘肃省的狭义丝盖伞属2个新种,即拟黄囊丝盖伞Inocybe muricellatoides和甘肃丝盖伞I. gansuensis。对新种的ITS、LSU和rpb2片段进行了测序和分析,并提供了详细描述、线条图、生态照片及与相似种的区别。拟黄囊丝盖伞以菌盖翘起的鳞片、菌柄纤维状、孢子光滑和厚壁的侧生囊状体为主要识别特征。甘肃丝盖伞具有粗壮的子实体、较大的孢子和厚壁侧生囊状体。基于LSU和rpb2联合数据的分子系统发育分析显示这两个新种隶属于狭义丝盖伞属且分别占据独特的分支。     

Gene trees, species trees, and morphology converge on a similar phylogeny of living gars (Actinopterygii: Holostei: Lepisosteidae), an ancient clade of ray-finned fishes

Extant gars represent the remaining members of a formerly diverse assemblage of ancient ray-finned fishes and have been the subject of multiple phylogenetic analyses using morphological data. Here, we present the first hypothesis of phylogenetic relationships among living gar species based on molecular data, through the examination of gene tree heterogeneity and coalescent species tree analyses of a portion of one mitochondrial (COI) and seven nuclear (ENC1, myh6, plagl2, S7 ribosomal protein intron 1, sreb2, tbr1, and zic1) genes. Individual gene trees displayed varying degrees of resolution with regards to species-level relationships, and the gene trees inferred from COI and the S7 intron were the only two that were completely resolved. Coalescent species tree analyses of nuclear genes resulted in a well-resolved and strongly supported phylogenetic tree of living gar species, for which Bayesian posterior node support was further improved by the inclusion of the mitochondrial gene. Species-level relationships among gars inferred from our molecular data set were highly congruent with previously published morphological phylogenies, with the exception of the placement of two species, Lepisosteus osseus and L. platostomus. Re-examination of the character coding used by previous authors provided partial resolution of this topological discordance, resulting in broad concordance in the phylogenies inferred from individual genes, the coalescent species tree analysis, and morphology. The completely resolved phylogeny inferred from the molecular data set with strong Bayesian posterior support at all nodes provided insights into the potential for introgressive hybridization and patterns of allopatric speciation in the evolutionary history of living gars, as well as a solid foundation for future examinations of functional diversification and evolutionary stasis in a "living fossil" lineage.     

Identification and phylogenetic analysis of morphologically similar naked amoebae using small subunit ribosomal RNA

Fan-shaped, naked amoebae are commonly encountered in samples from freshwater and marine habitats suggesting that they are an important component of the microbial food web. However, there are considerable problems in both detecting these amoebae and identifying them, given their morphological similarity. In this study we used restriction analysis and partial sequence analysis of the small-subunit 18S ribosomal RNA gene to examine the phylogenetic relationships between nine "fan-shaped" Vannella and Platyamoeba species. The molecular phylogeny showed that the marine Vannella and Platyamoeba isolates are closely related, whereas the freshwater isolates are disparate. Thus, the current reliance on the fine structure of the cell coat (glycocalyx) used to separate these genera is not justified. The study also highlights sequence elements that might be targeted by fluorescent probes for the direct detection of these amoebae in field samples. The molecular data were also used to aid the identification of three unknown fan-shaped isolates. All three unknowns resembled Vannella or Platyamoeba. However, one of the strains (a small < 10 microm, benthic, fan-shaped amoeba) probably represents a new genus.