SMALL SUBUNIT rDNA SEQUENCE ANALYSIS OF SYMBIOTIC DINOFLAGELLATES FROM SEVEN SCLERACTINIAN CORALS IN A TAHITIAN LAGOON

The diversity of symbiotic dinoflagellates from reef-building corals collected in the lagoon of Tahiti (South Pacific ocean) was investigated by using a molecular approach. Populations of symbionts (strains or species) of 7 coral species ( Fungia scutaria , F. paumotensis Stutchbury, Pavona cactus Forskål, Leptastrea transversa Kluzinger, Pocillopora verrucosa Ellis and Solender, Montastrea curta Dana, and Acropora formosa Dana) were delimited by phylogenetic analysis of small subunit rDNA sequences. Coral P. verrucosa harbored 2 populations of symbiont SSU rDNA sequences that may correspond to two different Symbiodinium species. Corals F. scutaria and M. curta also seemed to contain two different Symbiodinium species. SSU rDNA dinoflagellate sequences from P. cactus , L. transversa , F. scutaria , F. paumotensis , and P. verrucosa were in the same phylogenetic cluster and showed low variability. For these distantly related coral species, dinoflagellate strains from the same species, rDNA paralogues from the same strain, or closely related Symbiodinium species could not be distinguished because monophyletic subgroups were not observed. SSU rDNA dinoflagellate sequences from A. formosa and M. curta were clearly different from the other Symbiodinium sequences and may represent specific species. This molecular approach highlighted a greater diversity of symbiotic dinoflagellates from corals in South Pacific ( Symbiodinium groups A, B, and C) than that observed in the rest of the Pacific ocean ( Symbiodinium group C). The diversity of symbiotic associations in a restricted area of the lagoon of Tahiti may reflect the complexity of interactions between species of Symbiodinium and corals.     

The comparison of rDNA spacer regions of Nosema ceranae isolates from different hosts and locations

Nosema ceranae is a common microsporidian pathogen, one of two Nosema species that cause "nosema disease" in honeybees, Apis cerana and Apis mellifera. Samples of N. ceranae rDNA from isolates collected in different locations were sequenced and one 5S rRNA was found to be upstream of SSUrRNA. The rDNA arrangement, 5'-5S rRNA-IGS-SSUrRNA-ITS-LSUrRNA-3', was found in all isolates. In order to better understand the distribution relationship between N. ceranae isolates from A. cerana and A. mellifera, their rRNA spacer regions were also sequenced for analysis. Results showed that there are no significant differences between the IGS sequences of the isolates and no difference in the ITS sequence with the exception of one transition found in an isolate from Martinique. These isolates showed consistency in the IGS phylogenic analysis suggesting that no transmission barrier exists between A. mellifera and A. cerana and there is no difference between isolates from geography separated areas.     

Molecular divergence of the ssu rRNA gene and internal transcribed spacer 1 in Porphyra yezoensis (Rhodophyta)

Nucleotide sequences from the downstream of ssu rDNA to ITS1 region of the individual thalli of both wild-collected Porphyra yezoensis from three different sites and culture strains were determined to obtain the molecular features of strains in the P. yezoensis lineage. Wild-collected thalli identified by morphological systematics, included the individuals that were separate from the P. yezoensis lineage based on ssu rDNA and ITS1 sequence homologies and phylogenetic relationships constructed using ITS1 sequences. Ssu rDNA exon region nucleotide sequences were identical among the wild-collected and clture strains of P. yezoensis. However, all individual wild-collected P. yezoensis thalli had different ITS1 sequences, even among individuals from the same sites. Furthermore, two different ssu rDNA structures with and without an intron were found in individuals from the same site. These results indicated the possibility that the presence and sequence of introns and ITS1 sequences can be used as a characteristic to determine the origin of culture strains. Four of six culture strains examined had an identical sequence from the ssu rDNA to ITS1, while the sequences of another two strains differed. In this study, wild-collected and culture strain thalli sequences were not identical, although similar pairs were identified. This revised version was published online in July 2006 with corrections to the Cover Date.     

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N =9 isolates gave N. apis RFLPs and sequences, N =20 isolates gave N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze N =115 isolates from across the world. Our data, combined with N =36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping.     

First detection of Nosema ceranae, a microsporidian parasite of European honey bees (Apis mellifera), in Canada and central USA

Nosema ceranae is an emerging microsporidian parasite of European honey bees, Apis mellifera, but its distribution is not well known. Six Nosema-positive samples (determined from light microscopy of spores) of adult worker bees from Canada (two each from Nova Scotia, New Brunswick, and Prince Edward Island) and two from USA (Minnesota) were tested to determine Nosema species using previously-developed PCR primers of the 16S rRNA gene. We detected for the first time N. ceranae in Canada and central USA. One haplotype of N. ceranae was identified; its virulence may differ from that of other haplotypes.     

A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences

Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.     

The major histocompatability complex (MHC) contains conserved polymorphic genomic sequences that are shuffled by recombination to form ethnic-specific haplotypes

The major histocompatibility complex (MHC) consists of polymorphic frozen blocks (PFBs) that are linked to form megabase haplotypes. These blocks consist of polymorphic sequences and define regions where recombination appears to be inhibited. We have been able to show, using a highly polymorphic sequence centromeric of HLA-B (within the beta block), that PFBs are conserved and contain specific insertions/deletions and substitutions that are the same for individuals with the same MHC haplotype but that differ between at least most different haplotypes. A sequence comparison between ethnic-specific haplotypes shows that these sequences have remained stable and predate the formation of these haplotypes. To determine whether the same conserved block has been involved in the generation of multiple haplotypes, we compared the block typing profiles of different ethnic specific haplotypes. Block typing profiles have previously been shown to be identical in individuals with the same MHC haplotype but, generally, to differ between different haplotypes. It was found that some PFBs are common to more than one haplotype, implying a common ancestry. Subsequently, haplotypes have been generated by the shuffling and exchange of these PFBs. The regions between these PFBs appear to permit the recombination sites and therefore could be expected to exhibit either low polymorphism or a localized ``hotspot.' Received: 20 January 1997 / Accepted: 11 March 1997     

Comparison of within hive sampling and seasonal activity of Nosema ceranae in honey bee colonies

Nosema ceranae is a microsporidian parasite of the European honey bee, Apis mellifera, that is found worldwide and in multiple Apis spp.; however, little is known about the effects of N. ceranae on A. mellifera. Previous studies using spore counts suggest that there is no longer a seasonal cycle for N. ceranae and that it is found year round with little variation in infection intensity among months. Our goal was to determine whether infection levels differ in bees collected from different areas of the hive and if there may be seasonal differences in N. ceranae infections. A multiplex species-specific real-time PCR assay was used for the detection and quantification of N. ceranae. Colonies were sampled monthly from September 2009-2010 by collecting workers from honey supers, the fringe of the brood nest, and the brood nest. We found that all bees sampled were infected with N. ceranae and that there was no significant difference in infection levels among the different groups of bees sampled (P=0.74). However, significant differences in colony infection levels were found at different times of the year (P<0.01) with the highest levels in April-June and lower levels in the fall and winter. While our study was only performed for one year, it sheds light on the fact that there may be a seasonality to N. ceranae infections. Being able to predict future N. ceranae infections can be used to better advise beekeepers on N. ceranae management.     

Species Diversity of Uncultured and Cultured Populations of Soil and Marine Ammonia Oxidizing Bacteria

Although molecular techniques are considered to provide a more comprehensive view of species diversity of natural microbial populations, few studies have compared diversity assessed by molecular and cultivation-based approaches using the same samples. To achieve this, the diversity of natural populations of ammonia oxidising bacteria in arable soil and marine sediments was determined by analysis of 16S rDNA sequences from enrichment cultures, prepared using standard methods for this group, and from 16S rDNA cloned from DNA extracted directly from the same environmental samples. Soil and marine samples yielded 31 and 18 enrichment cultures, respectively, which were compared with 50 and 40 environmental clones. There was no evidence for selection for particular ammonia oxidizer clusters by different procedures employed for enrichment from soil samples, although no culture was obtained in medium at acid pH. In soil enrichment cultures, Nitrosospira cluster 3 sequences were most abundant, whereas clones were distributed more evenly between Nitrosospira clusters 2, 3, and 4. In marine samples, the majority of enrichment cultures contained Nitrosomonas, whereas Nitrosospira sequences were most abundant among environmental clones. Soil enrichments contained a higher proportion of identical sequences than clones, suggesting laboratory selection for particular strains, but the converse was found in marine samples. In addition, 16% of soil enrichment culture sequences were identical to those in environmental clones, but only 1 of 40 marine enrichments was found among clones, indicating poorer culturability of marine strains represented in the clone library, under the conditions employed. The study demonstrates significant differences in species composition assessed by molecular and culture-based approaches but indicates also that, employing only a limited range of cultivation conditions, 7% of the observed sequence diversity in clones of ammonia oxidizers from these environments could be obtained in laboratory enrichment culture. Further studies and experimental approaches are required to determine which approach provides better representation of the natural community.     

Revisiting the rDNA sequence diversity of a natural population of the arbuscular mycorrhizal fungus<Emphasis Type="Italic"> Acaulospora colossica</Emphasis>

In 1999, the diversity of a field population of the arbuscular mycorrhizal (AM) fungus Acaulospora colossica was characterized using DNA sequence data. Since 1999, AM fungal sequences have accumulated rapidly within public databases. Moreover, novel phylogenetic tools have been developed and can be used to interpret the data. A second analysis of those sequences collected in 1999 demonstrates that while the majority of the sequences are, in fact, sequences of A. colossica; a minority of the sequences still cannot be identified with confidence. Those sequences identified as A. colossica can be used to show that (1) the nuclear rDNA ITS regions are remarkably diverse, and (2) sequences isolated from different spores of the same site may be more closely related to each other than to sequences of other sites, so that the genetic diversity of an AM fungal field population may be spatially structured; however, identical sequences can also be recovered from different sites.     

Linkage disequilibrium between incompatibility locus region genes in the plant Arabidopsis lyrata

We have studied diversity in Arabidopsis lyrata of sequences orthologous to the ARK3 gene of A. thaliana. Our main goal was to test for recombination in the S-locus region. In A. thaliana, the single-copy ARK3 gene is closely linked to the non-functional copies of the self-incompatibility loci, and the ortholog in A. lyrata (a self-incompatible species) is in the homologous genome region and is known as Aly8. It is thus of interest to test whether Aly8 sequence diversity is elevated due to close linkage to the highly polymorphic incompatibility locus, as is theoretically predicted. However, Aly8 is not a single-copy gene, and the presence of paralogs could also lead to the appearance of elevated diversity. We established a typing approach based on different lengths of Aly8 PCR products and show that most A. lyrata haplotypes have a single copy, but some have two gene copies, both closely linked to the incompatibility locus, one being a pseudogene. We determined the phase of multiple haplotypes in families of plants from Icelandic and other populations. Different Aly8 sequence types are associated with different SRK alleles, while haplotypes with the same SRK sequences tend to have the same Aly8 sequence. There is evidence of some exchange of sequences between different Aly8 sequences, making it difficult to determine which ones are allelic or to estimate the diversity. However, the homogeneity of the Aly8 sequences of each S-haplotype suggests that recombination between the loci has been very infrequent over the evolutionary history of these populations. Overall, the results suggest that recombination rarely occurs in the interval between the S-loci and Aly8 and that linkage to the S-loci can probably account for the observed high Aly8 diversity.     

Genetic Diversity and Mosaicism at the por Locus of Neisseria gonorrhoeae

The por genes of the predominant serovars of Neisseria gonorrhoeae circulating in a high-frequency transmitter core group located in Nairobi, Kenya, were examined for nucleotide sequence polymorphism. The level of por gene diversity did not differ significantly between core group-derived gonococcal strains and gonococcal strains originating elsewhere. However, por mosaicism appeared to be more frequent among core group-derived strains, suggesting that recombination of different por sequences may be a important strategy by which N. gonorrhoeae generates por gene diversity within core group populations. Despite extensive sequence variability, por expressed by gonococcal isolates of different geographic origin exhibited conserved patterns of nucleotide change, suggesting that diversity among por alleles may also be finite.     

Molecular survey of aeroplane bacterial contamination

AIMS: To examine bacterial contamination of passenger aircraft and to identify aeroplane environments posing the greatest potential health risk. METHODS AND RESULTS: DNA was extracted from ten environmental samples collected on four different flights (three domestic, one international) from a variety of surfaces frequently touched by passengers. PCR clone libraries were made from the DNA samples using bacterial-specific 16S ribosomal DNA (rDNA) primers. A total of 271 bacterial rDNA sequences were obtained. We used BLAST analysis of the rDNA clone sequences to identify sequences in Genbank with the highest sequence similarity. The majority of the rDNA clones obtained from aeroplane environments were more than 97% identical to rDNA sequences from cultured bacterial species. Samples collected from the cabin surfaces (e.g., tray tables and arm rests) had undetectable levels of DNA and produced no PCR products. Bacterial diversity was highest on lavatory surfaces, including door handles, toilet handles, and sink faucets. Sequence data from these surfaces detected species from 58 different bacterial genera, and many of the best BLAST hits matched rDNA sequences of cultured species known to be opportunistic pathogens. The most frequently observed species came from five genera commonly associated with humans: Streptococcus, Staphylococcus, Cornybacterium, Proprionibacterium and Kocuria. CONCLUSIONS: The results show that there is a large diversity of bacterial contamination on aeroplanes, including organisms known to be opportunistic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that aeroplanes have the potential to spread an enormous diversity of bacterial species among passengers and destinations. Aeroplane lavatories present an especially significant concern to public health.     

Detection of abundant sulphate-reducing bacteria in marine oxic sediment layers by a combined cultivation and molecular approach

The depth distribution and diversity of sulphate-reducing bacteria (SRB) was analysed in the upper intertidal zone of a sandy marine sediment of the Dutch island Schiermonnikoog. The upper centimetre of the sediment included the oxic-anoxic interface and was cut into five slices. With each slice, most probable number (MPN) dilution series were set up in microtitre plates using five different substrates. In the deeper sediment layers, up to 1 x 10(8) cm(-3) lactate-utilizing SRB were counted, corresponding to 23% of the total bacterial count. From the highest positive dilutions of the MPN series, 27 strains of SRB were isolated in pure culture. Sequencing of a 580 bp fragment of the 16S rDNA revealed that 21 isolates had identical sequences, also identical with that of the previously described species Desulfomicrobium apsheronum. However, the diversity of the isolates was higher with respect to their physiological properties: a total of 11 different phenotypes could be distinguished. Genomic fingerprinting by enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) revealed an even higher diversity of 22 different genotypes. A culture-independent analysis by PCR and denaturing-gradient gel electrophoresis (DGGE) revealed that the partial 16S rDNA sequence of the isolated D. apsheronum strains constituted a significant fraction of the Desulfovibrionaceae. The high subspecies diversity suggests that this abundant aggregate-forming species may have evolved adaptations to different ecological niches in the oxic sediment layers.     

Observations on the diversity and ecology of freshwater Nannochloropsis (Eustigmatophyceae), with descriptions of new taxa

The genus Nannochloropsis is well known from the marine environment but has only recently been reported from fresh and brackish waters. A single species, N. limnetica, was first documented from shallow lakes in Germany, where it produced spring blooms. A second unnamed isolate from a river in the United States has been characterized by sequence analysis and light microscopy. All of the Nannochloropsis species that have been described, both marine and freshwater, are small spheres with essentially no distinguishing morphological characteristics. Therefore, they must be characterized using molecular techniques. We have cultured numerous isolates of Nannochloropsis from a series of lakes on the James River in the Arrowwood National Wildlife Refuge, North Dakota, USA, and 1 isolate from a pond in Itasca State Park, Minnesota, USA. The diversity among these isolates was determined by light microscopy and DNA sequence analysis. Seven distinct haplotypes of Nannochloropsis were found, one of which possesses 18S rDNA and rbcL sequences identical to those of N. limnetica from Europe. The 6 new haplotypes vary in rbcL sequences and some are morophologically distinct from each other and from N. limnetica. These types are described as the new taxa N. limnetica var. globosa, N. limnetica var. irregularis, N. limnetica var. dystrophica, and N. limnetica var. gutta. All of the Nannochloropsis isolates from Arrowwood and Itasca were cultured only from samples taken during cold-water periods. These results suggest that Nannochloropsis species may be better adapted to cold water conditions, including temperatures near 0 degrees C and ice cover.     

Heterogeneous Rates of Molecular Evolution Among Cryptic Species of the Ciliate Morphospecies Chilodonella uncinata

While molecular analyses have provided insight into the phylogeny of ciliates, the few studies assessing intraspecific variation have largely relied on just a single locus [e.g., nuclear small subunit rDNA (nSSU-rDNA) or mitochondrial cytochrome oxidase I]. In this study, we characterize the diversity of several nuclear protein-coding genes plus both nSSU-rDNA and mitochondrial small subunit rDNA (mtSSU-rDNA) of five isolates of the ciliate morphospecies Chilodonella uncinata. Although these isolates have nearly identical nSSU-rDNA sequences, they differ by up to 8.0% in mtSSU-rDNA. Comparative analyses of all loci, including β-tubulin paralogs, indicate a lack of recombination between strains, demonstrating that the morphospecies C. uncinata consists of multiple cryptic species. Further, there is considerable variation in substitution rates among loci as some protein-coding domains are nearly identical between isolates, while others differ by up to 13.2% at the amino acid level. Combining insights on macronuclear variation among isolates, the focus of this study, with published data from the micronucleus of two of these isolates, indicates that C. uncinata lineages are able to maintain both highly divergent and highly conserved genes within a rapidly evolving germline genome.     

Mitochondrial DNA analysis reveals cryptic large-scale invasion of non-native genotypes of common carp (Cyprinus carpio) in Japan

Wild common carp ( Cyprinus carpio ) are probably suffering from biological invasions of conspecific domesticated strains. However, such invasions may be largely camouflaged by morphological similarities between introduced and native strains. We conducted a large survey of mitochondrial DNA sequences (complete D-loop region) from 11 localities in Japan. From a total of 166 individuals, 28 haplotypes were determined to fit into six divergent clades. One of the six clades included 19 closely related haplotypes with moderate nucleotide differences; however, the remaining five clades each included either a single haplotype or two almost identical haplotypes. Phylogenetic analysis together with the previously published Eurasian haplotypes further demonstrated that the 'monotypic' clades were sisters to various Eurasian lineages, whereas the 19 related haplotypes formed a monophyletic group apart from the whole Eurasian clade. Given their monophyly and genetic diversity, the 19 related haplotypes were thought to originate from the Japanese native strain. Conversely, their phylogenetic affinities to Eurasian lineages and unnaturally low genetic diversities caused the haplotypes of the five monotypic clades to be considered as domesticated strains introduced from Eurasia. These hypotheses were supported by further evidences; i.e. the probable non-native haplotypes were frequently found from Japanese domesticated strains, and the probable native population structure was rescued when the probable non-native haplotypes were excluded from the analyses. This study revealed that almost half or more of the haplotypes in all of the locations studied originated from domesticated strains introduced from Eurasia.     

Limited genetic variability inMegasphaera elsdenii strains

Levels of phenotypic and genotypic diversity among seven Megasphaera elsdenii strains recovered from rumen contents of cattle, sheep and lambs were determined by a combination of antibiotic-resistance analysis and PCR fingerprint techniques targeted both to the ribosomal RNA operon (ARDRA, RISA) and the whole genome (ERIC-PCR, RAPD-PCR). Despite exhibiting different antibiotic resistance profiles, the tested strains represent genetically nearly identical isolates. Close genetic relatedness was found among M. elsdenii isolates that originated from vastly different habitats worldwide, as revealed by the comparison of 16S rDNA sequences.     

Differences between subcultures of the Mesorhizobium loti type strain from different culture collections

Because of differences in the reported 16S rRNA gene sequence of the Mesorhizobium loti type strain available from different culture collections, we collected different subcultures of this strain and compared them by 16S rDNA sequencing, SDS-PAGE of whole-cell protein extracts and RAPD-PCR. Our results indicate that the 16S rDNA sequence differences can be explained by the presence of two different organisms in one of the subcultures. In addition, even for subcultures of the type strain that had identical 16S rDNA sequences, small differences could be observed in the protein profiles and in the RAPD-PCR patterns. These latter observations indicate that maintenance procedures necessary for long-term preservation by freeze-drying can cause subcultures of the same original strain to undergo changes, effectively leading to different fingerprints even though 16S rDNA sequences remain identical.     

Small-subunit rDNA sequencing of the Italian bovine Neospora caninum isolate (NC-PV1 strain).

The small-subunit (SSU) rDNA of the Neospora sp. NC-PV1 strain isolated in Italy from cattle has been sequenced and compared to the other five N. caninum strains SSU rDNA sequences deposited in the data bases. The NC-PV1 strain sequence is identical to three published sequences. Minor differences, respectively four nucleotide bases and one nucleotide base, have been found when comparing the NC-PV1 sequence with two other available sequences of N. caninum. According to these results, the Neospora sp. NC-PV1 strain is assigned to the species N. caninum.