<Emphasis Type="Italic">Sarcocystis</Emphasis> in moose (<Emphasis Type="Italic">Alces alces</Emphasis>): molecular identification and phylogeny of six <Emphasis Type="Italic">Sarcocystis</Emphasis> species in moose,and a morphological description of three new species

Muscle tissues from 34 moose from Southeastern Norway and two moose from Canada were examined. Sarcocysts were excised and morphologically classified by light microscopy, and some cysts were further examined by scanning electron microscopy or DNA amplification and sequencing at the small subunit (ssu) rRNA gene. In Norwegian moose, three sarcocyst types were recognized, yet five Sarcocystis species were found by sequence analysis. New names were proposed for three species which could be characterised by both morphological and molecular methods, i.e., Sarcocystis alces, Sarcocystis ovalis, and Sarcocystis scandinavica. S. alces was the most prevalent species, whereas S. scandinavica and the two unnamed species were rare and might either use another principal intermediate host or a rare definitive host. The five species in Norwegian moose were different from Sarcocystis alceslatrans isolated from a Canadian moose. Phylogenetic analyses based on complete ssu rRNA gene sequences revealed a close relationship between the six Sarcocystis species from moose and species from reindeer and Sika deer. We conclude that molecular methods are necessary for unequivocal species identification, as different cervid hosts harbour morphologically indistinguishable sarcocysts.     

Morphometric and molecular characteristics of <Emphasis Type="Italic">Labeonema synodontisi</Emphasis> n. comb. (Nematoda: Atractidae) from the West African fishes

Numerous nematodes were found in the rectum of three fish species Synodontis ocellifer, S. nigrita, and S. schall (Mochokidae, Siluriformes) from the Gambia River and Mare Simenti, National Park Niokolo Koba, Senegal. A nematode species Raillietnema synodontisi Vassiliadès, 1973 (host S. ocellifer), is redescribed using morphometric (including scanning electron microscopy) and molecular characteristics and transferred into the genus Labeonema Puylaert, 1970. It is morphologically and metrically similar to Labeonema intermedium Puylaert, 1970, the other congeneric species (L. bainae Baker, 1982; L. bakeri Van Waerebeke, Chabaud, Bain et Georges, 1988; and L. africanum Moravec et Van As, 2004) differ from them either by the spicule and gubernaculum lengths, distribution and number of pre-cloacal papillae, position of the vulva, as well as hosts and geographical distribution. The partial sequences of small ribosomal subunit rDNA of L. synodontisi were analyzed and compared with other nematode sequences. Molecular analyses seem to support the position of this nematode species based on the morphological observation.     

Transmission experiments on the host specificity ofHeterophyes species in 16 potential definitive hosts

Sixteen different species of piscivorous mammals and birds were tried as experimental definitive hosts forHeterophyes heterophyes, H. aequalis andH. dispar. The hosts were classified in four categories, by fluke longevity, recovery and size, and the number of uterine eggs (embryonated/unembryonated): (1) Canidae and the cat were highly susceptible hosts for all three species ofHeterophyes; (2) several mammals and herons showed a reduced susceptibility to infection (H. aequalis, 6 species;H. dispar, 1 species;H. heterophyes, 0 species); (3) In a group of hosts specific to each trematode, flukes were recovered up to 14 days post infection, but their uterine eggs did not become embryonated; (4) In a fourth category of hosts, chiefly Mustelidae, flukes could not be recovered. Taking also the literature into account it is concluded that man is a highly susceptible host forH. heterophyes, and that probablyH. aequalis andH. dispar may reach reproductive maturity in humans also. The described wide host range ofH. aequalis appears to be more typical for Heterophyidae than the comparably narrow host range ofH. heterophyes.     

Evidence for mating between islates of Colletotrichum gloeosporioides with different host specificities

Individual isolates of the ibiquitous plant pathogen Colletotrichum gloeosporioides (teleomorph Glomerella cingulata) can have very restricted host ranges. Isolates that share the same host range are considered to be genetically discrete units, and sexual compatibility has been reported to be limited to individuals that share the same host range. However, we have recently observed that some isolates of C. gloeosporioides that are specifically pathogenic to different, distantly-related hosts are sexually compatible. Ascospore progeny from one such cross were randomly isolated and outcrossing was verified by the reassortment of several RFLP markers among the progeny. In addition, the progeny were analyzed for pathogenicity to parental hosts. The implications of sexual compatibility between C. gloeosporioides isolates with different host specificities on the evolution of Colletotrichum species are discussed.     

Identification of new isolates of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> that cluster with less common viral strains

Summary Turnip mosaic virus (TuMV) was found infecting cultivated brassicas and wild and cultivated ornamental Brassicaceae plants in different regions of Spain. Five new TuMV isolates, originating from different host plant species (Brassica cretica, Brassica juncea, Brassica napus, Eruca vesicaria subsp. sativa and Sisymbrium orientale), have been identified. The nucleotide sequences of the coat protein (CP) genes of the five isolates were determined. Phylogenetic analysis of the CP sequences showed that the five isolates grouped into two different clusters. The three isolates from the central region of Spain clustered with a previously reported Pisum sativum isolate from southeastern Spain, whereas the other two isolates from the eastern region clustered with two Italian and two Greek isolates. Both clusters were genetically distinct and belonged to the multi-lineage group OBR. The OBR group contains mainly TuMV isolates from hosts other than Brassica spp. and Raphanus sativus and mostly originating from Mediterranean countries. These new sequences provide further phylogenetic resolution of the OBR group. Although new TuMV isolates have been found in Spain, they were not associated with any serious disease outbreaks.     

Multiple polyadenylated RNA viruses detected in pooled cultivated and wild plant samples

RNA extracted from 120 leaf specimens from 17 plant species was pooled, and polyadenylated RNA species were sequenced together without barcoding in one lane using massively parallel sequencing technology. After analysis, complete or partial genome sequences representing 20 virus isolates of 16 polyadenylated RNA species were identified. In three cases, 2-3 distinct isolates of a virus species co-infected the same plant. Twelve of the viruses identified were described previously and belonged to the genera Potyvirus, Nepovirus, Allexivirus, and Carlavirus. Four were unknown and are proposed as members of the genera Potyvirus, Sadwavirus, and Trichovirus. Virus sequences were subsequently matched to original host plants using RT-PCR assays.     

Phylogenetic studies explain the discrepant host distribution of <Emphasis Type="Italic">Allopodocotyle heronensis</Emphasis> sp. nov. (Digenea,Opecoelidae) in Great Barrier Reef serranids

We report a new species of Allopodocotyle Pritchard, 1966 from the intestine of two species of Serranidae, Cromileptes altivelis and Epinephelus fuscoguttatus, from the southern Great Barrier Reef. Despite the examination of eight other species of Epinephelus from the same region this species appears anomalous in its distribution in one species of Epinephelus and the single species of Cromileptes. Molecular phylogenetic studies of the Epinephelinae suggest, however, that these two species are closely related so that the host specificity demonstrated by this species is actually stenoxenic (phylogenetically related hosts) rather than euryxenic.     

The mallard duck (Anas platyrhynchos) as intermediate host for Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis)

Morphometric and DNA investigation results of Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis) and Sarcocystis sp. (cyst type IV) from the mallard duck (Anas platyrhynchos) are presented. No significant morphometric differences between the investigated Sarcocystis species were found. ITS-1, 18S rRNA, and 28S rRNA gene sequences of these species showed 100% identity. The conclusion is drawn that it is one and the same Sarcocystis species in different intermediate hosts.     

The intermediate hosts of Wardium cirrosa (Krabbe, 1869) Spassky, 1961 (Cestoda,Cyclophyllidea, Aploparaksidae) in Ukraine

The metacestodes of aploparaksid cestode Wardium cirrosa Krabbe, 1869 parasitic in gulls were found in polychaetes of the family Nereidae collected off the Black Sea coast, Ukraine. Two species of polychaetes, Hediste diversicolor (prevalence 5.3%; intensity 1–3 specimens) and Neanthes succinea (prevalence 9.9%; intensity 1–39 specimens), were infected with cysticercoids that were observed either individually or in accumulations. The preliminary identification of the material based on morphological characteristics was later confirmed by experimental infection of the definitive host, Larus cachinnans (Charadriiformes: Laridae) with metacestodes, and by the identity of the partial 28S rDNA sequences of cysticercoids and experimentally obtained adults. Although previous studies suggested freshwater leeches as the intermediate host for W. cirrosa, our study provides the evidence for marine polychaetes to serve as intermediate hosts. This study is the first to present the morphological characteristics of metacestodes of W. cirrosa in addition to molecular data for this species, as well as reporting the possibility of several cysticercoids developing from a single oncosphere. Morphology of the adult specimens obtained in the experiment was compared with adults of W. cirrosa previously collected from L. cachinnans in Ukraine. The results of our study suggest that further research focused on the elucidation of the life cycles of cestodes within the genus Wardium should consider marine invertebrates as potential intermediate hosts.

     

Molecular characterization and phylogeny of whipworm nematodes inferred from DNA sequences of cox1 mtDNA and 18S rDNA

A molecular phylogenetic hypothesis is presented for the genus Trichuris based on sequence data from the mitochondrial cytochrome c oxidase 1 (cox1) and ribosomal 18S genes. The taxa consisted of different described species and several host-associated isolates (undescribed taxa) of Trichuris collected from hosts from Spain. Sequence data from mitochondrial cox1 (partial gene) and nuclear 18S near-complete gene were analyzed by maximum likelihood and Bayesian inference methods, as separate and combined datasets, to evaluate phylogenetic relationships among taxa. Phylogenetic results based on 18S ribosomal DNA (rDNA) were robust for relationships among species; cox1 sequences delimited species and revealed phylogeographic variation, but most relationships among Trichuris species were poorly resolved by mitochondrial sequences. The phylogenetic hypotheses for both genes strongly supported monophyly of Trichuris, and distinct genetic lineages corresponding to described species or nematodes associated with certain hosts were recognized based on cox1 sequences. Phylogenetic reconstructions based on concatenated sequences of the two loci, cox1 (mitochondrial DNA (mtDNA)) and 18S rDNA, were congruent with the overall topology inferred from 18S and previously published results based on internal transcribed spacer sequences. Our results demonstrate that the 18S rDNA and cox1 mtDNA genes provide resolution at different levels, but together resolve relationships among geographic populations and species in the genus Trichuris.     

PCR-RFLP analysis: a promising technique for host species identification of blood meals from tsetse flies (Diptera: Glossinidae)

A polymerase chain reaction with the restriction fragment length polymorphism (PCR-RFLP) method using universal primers complementary to the conserved region of the cytochrome b gene (cyt b) of the mitochondrion DNA (mtDNA) of vertebrates was applied to the identification of the origin of blood meals in tsetse flies. Blood samples from ten potential tsetse hosts of the family bovidae (cattle, water buffalo, red buffalo, waterbuck, springbok, goat, sheep, sable antelope, oryx and dik-dik) were included in this study. Sites for appropriate restriction endonucleases cuts were chosen by pairwise alignment of the amplified 359 bp fragments. A flow chart of endonucleases digestion using three restriction enzymes (e.g. TaqI, AluI and HindII) for the unequivocal identification of the respective bovid species was developed. A number of additional non-specific DNA fragments attributed to the co-amplification of cytochrome b pseudogenes were observed in some species (e.g. in red buffalo and dik-dik after digestion with AluI) but did not hamper assignment of bovid species. The detection rate of host DNA in tsetse by PCR-RFLP was 100, 80, 60 and 40% at 24, 48, 72 and 96 h after in vitro feeding, respectively. Identification of the last blood meal was possible even when tsetse had previously fed on different hosts.     

The host range of the pKD1-derived plasmids in yeast

Summary pKDI is a 2-like circular plasmid found in the yeast Kluyveromyces drosophilarum that can also stably replicate in Kluyveromyces lactis. We have found a short intergenic region in this genome that appears to be functionally neutral; that is, the introduction of foreign sequences into the single EcoRI restriction site located near one of the inverted repeats did not affect the high stability of the natural plasmid. By introducing a G418 resistance gene at this site, we constructed an autonomous recombinant plasmid. Since this vector did not require cir ⁺ hosts for its stable maintenance, it could be used to examine the transformation host range of pKD1 among all the species belonging to the genus Kluyveromyces. Both species closely related to K. drosophilarum as well as a few other species that are very different in chromosomal GC % could be transformed to yield highly stable transformant clones.     

Cloning and characterization of the European seabass,Dicentrarchus labrax,mitochondrial genome

Mitochondrial DNA (mtDNA) from the European seabass, Dicentrarchus labrax, has been cloned and characterized. Its gene organization was deduced by a comparison of the sequenced termini of different subclones obtained from European seabass mtDNA to the completely-sequenced mtDNAs from carp and freshwater loach. The difference in genome size between the European seabass mtDNA (approximately 18 kb) and most of the other characterized fish mtDNAs (approximately 16.5 kb) is accounted for by the displacement-loop (D-loop). Comparisons have been performed between the derived amino-acid sequences of three sequenced genes, cytochrome c oxidase subunit 2 (COII), NADH dehydrogenase subunit 4L (ND4L) and ATP synthase subunit 8 (ATPase8), from D. labrax, and their counterparts in other fishes and Xenopus laevis.     

Molecular characterization and evolutionary analysis of <Emphasis Type="Italic">soybean mosaic virus</Emphasis> infecting <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Twenty-nine Pinellia ternata specimens were collected from representative areas in China, including the major production provinces of Zhejiang, Henan, Shanxi, Hunan, Shandong and Hubei. Seven isolates related to soybean mosaic virus (SMV), which could be pathogenic on P. ternata and some soybean [Glycine max (L.) Merr.] cultivars, were detected using double antibody sandwich immunosorbent assay (DAS-ELISA) and RT-PCR amplification performed with degenerate primer of potyviruses. It is revealed that the common potyvirus infecting P. ternata is, indeed, only SMVs rather than Dasheen mosaic virus (DsMV) as previously reported. Further molecular phylogenetic analysis of the coat protein (CP) genes of these SMV isolates from P. ternata and G. max, along with some other potyvirus members, such as DsMV and Watermelon mosaic virus (WMV) reconstructed the evolutionary route on both nucleotide and amino acid levels. Similarity and homology of nucleotide sequences for SMV CP genes demonstrated high host correlation and low partial habitat correlation, while those of amino acid sequences also showed that the host correlation was more notable than the habitat correlation. The amino acid sequence of conserved region within CP determines the main function, which shows high homology between species. This study outspreaded from the viruses themselves and their relationship to the infected hosts and revealed the evolutionary strategies, especially the rapid variation or recombination of SMV of P. ternata, in order to adapt itself naturally to the special host. The GenBank Accession numbers of the sequences reported in this article are DQ360817-DQ360823.     

An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeniella genera

Summary Sequences hybridzing to mitochondrial DNA probes from Saccharomyces cerevisiae have been mapped in six mitochondrial genomes from the Dekkera/Brettanomyces yeasts and in mtDNA from the closely related Eeniella nana. Sequence order for the 34.5 kbp mtDNA of E. nana is identical to that for mtDNAs from B. custersianus (28.5 kbp) and B. naardenensis (41.7 kbp) thereby suggesting that the former yeast is affiliated with the latter two species. A closer relationship is suggested for D. intermedia and D. bruxellensis as mtDNAs from these yeasts, 73.2 and 85.0 kbp respectively, have the same sequence order and mostly common restriction endonuclease sites. Differences between the two molecules are reminiscent of those found in mtDNA polymorphisms of S. cerevisiae strains thereby suggesting that the two Dekkera yeasts are variants of a single species. An unusual feature of the Dekkera species mtDNA is an inversion of the cytochrome b hybridizable region relative to the LrRNA sequence. Likewise mtDNA from B. anomalus (57.7 kbp) has an inversion of the cytochrome oxidase subunit 1 sequence with respect to the LrRNA sequence. By contrast the largest mtDNA (101.1 kbp) from B. custersd has the cytochrome b and LrRNA sequences in the same orientation. In addition hybridizable regions in this mtDNA are found in three clusters that are separated by several thousand base pairs of sequence deficient in restriction endonuclease sites. This observation together with the low guanine and cytosine content of the mtDNA suggests that the regions separating the sequence clusters are mostly adenine and thymine residues.     

Molecular identification of <Emphasis Type="Italic">Mesocestoides</Emphasis> spp. from intermediate hosts (rodents) in central Europe (Poland)

Genus Mesocestoides is a representative of the small cyclophyllidean family Mesocestoididae that is found parasitizing the small intestine of carnivores. The life cycle of cestodes from this genus is complex and requires two intermediate hosts. Cysticercoids are produced in the first intermediate host (oribatid mites), which when eaten by the second intermediate host (mainly rodents, but also other mammalian species, birds, reptiles, or amphibians) form tetrathyridia in the body cavity. Because of the rich history of nomenclatural evaluation of Mesocestoididae, the taxonomic status within the genus Mesocestoides is still unclear. Additional problem constitute the difficulty or even the impossibility in the determination of tetrathyridia based on morphological features. Thus, the aim of our study was to identify a molecular characteristic of the isolates of Mesocestoides from the second intermediate hosts (rodents) based on nuclear and mitochondrial ribosomal DNA data. We choose to analyze metacestodes isolated from two species of rodents (Apodemus agrarius and Myodes glarolus) from different sites. As a result of amplification of 18S rDNA, we obtained partial sequences from four isolates ranging from 1,116 to 1,162 bp. In relation to mitochondrial sequence, 354 bp product of 12S rDNA was obtained from one isolate. The neighbor joining and maximum parsimony trees were constructed in order to examine the phylogenetic relationship within Mesocestoides spp. occurring in rodents from central Europe. The results of our research on the larval stages from rodents, living in a periphery of urban agglomeration as well as in an area of reserve protection, confirm the data of more frequently occurring Mesocestoides litteratus.     

Effect of host gender on blood digestion in fleas: mediating role of environment

We investigated mechanisms of male-biased parasitism by studying the rate of digestion and survival time after a single blood meal in fleas Xenopsylla ramesis parasitizing males and females of the rodent Meriones crassus. Assuming that male hosts represent better patches for fleas than female hosts, we predicted that fleas (1) will digest blood of a male host faster than blood of a female host and (2) will survive longer after a single blood meal taken from a male host. To understand the possible role of environmental factors in mediation of the relationship between flea performance and host gender, we tested our predictions in male and female fleas under low (75%) and high (95%) relative humidity (RH). Host gender affected duration of digestion only at the middle and late stages of digestion and only interacting with either flea gender or RH or both. At the early stage of digestion, fleas of the same gender at the same RH digested blood at similar rates, independent of host gender. The rate of digestion did not differ between male and female fleas at 75% RH, but the duration of early stage was significantly shorter in female than in male fleas at 95% RH. At the middle stage of digestion, male fleas at both RH digested blood of male and female hosts at similar rates, but at lower RH, female fleas digested blood from male hosts at a significantly faster rate than blood from female hosts. At the late stage of digestion, both male and female fleas digested blood faster from male hosts than from female hosts at 75% RH, but the opposite was true at 95% RH. Survival time of fleas after completion of digestion was affected by RH, being longer at 95%, and flea gender, being longer in females. Fleas fed on female hosts died faster than fleas fed on male hosts, but this was found only at 95% humidity. We concluded that the relationship between flea performance and host gender was mediated by external conditions.