Morphological and molecular identification of the metacestode parasitizing the liver of rodent hosts in bamboo growing areas of mizoram, northeast India

In Mizoram (Northeast India), rodent outbreaks are known to occur periodically with the onset of bamboo flowering causing a tremendous destruction to food grains and as per the folk belief, often resulting in famine. In an exploratory survey of rodent pests in bamboo growing atreas for their helminth parasite spectrum, metacestodes of tapeworms were frequently encountered infecting the liver lobes and body cavity of the host. The morphological criteria were found to be closely consistent with the metacestode of Taenia species. In molecular characterization of the parasite, the ribosomal DNA (ITS1, ITS2) and mitochondrial COI were amplified and sequenced. Based upon both morphological data and molecular analysis using bioinformatic tools, the metacestode is identified as confirmed to be representing Cysticercus fasciolaris. The adult form of which (Taenia taeniaeformis) commonly occurs in felid and canid mammalian hosts.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

RNA reveals a succession of active fungi during the decay of Norway spruce logs

Current knowledge of the succession of fungi in decaying wood is mostly based on fruit bodies and in vitro culture. Here, we investigated the changing community of metabolically active fungi during the decomposition of fallen Picea abies logs by directly extracting and barcode sequencing precursor rRNA. We also compared rRNA-derived amplicons of the 18S and ITS regions in 21 isolates and discuss the use of RNA as a marker of metabolically active fungi. The richness of active fungi, revealed as separated bands in DGGE, peaked in logs at an advanced stage of decay. Soft-rot fungi were common in the early stages but white- and brown-rot fungi became dominant as decay progressed. Ectomycorrhizal fungi were detected at an early stage, and they became the most abundant group in the late stages of succession. A comparison of rRNA-derived amplicons revealed that although ITS was detected in the form of precursor rRNA, introns within 18S rDNA were already spliced. As such, rRNA- and rDNA-derived amplicons would yield different profiles of active and total communities if profiling method is affected by amplicon length.     

Evolution of the Euphrasia transmorrisonensis complex (Orobanchaceae) in alpine areas of Taiwan

Aims To unravel isolation and differentiation of the genetic structure of the Euphrasia transmorrisonensis complex, a showy herb, among alpine regions of mountain peaks in subtropical Taiwan and to infer its evolutionary history. Location Alpine ecosystems of high‐montane regions of Taiwan. Methods Phylogenetic analyses of the trnL intron and the trnL–trnF intergenic spacer of chloroplast (cp) DNA, and the intertranscribed spacer (ITS) of nuclear ribosomal (nr) DNA between 18S and 26S were carried out on 18 populations of the E. transmorrisonensis complex in Taiwan. Results In total, 10 haplotypes for cpDNA and 14 haplotypes for nrDNA were detected. Three population groups located in the northern, north‐eastern, and south‐central regions of the Central Mountain Range (CMR) were revealed according to the frequencies of haplotypes and haplotype lineages of nrDNA. Balancing selection might have played a role in the evolution of Euphrasia in Taiwan. Main conclusions By integrating the spatial‐genetic patterns of cpDNA and nrDNA, two possible evolutionary histories of Euphrasia in Taiwan were inferred. The favourable hypotheses for interpreting the data suggest at least three origins of the E. transmorrisonensis complex in Taiwan, corresponding to each nuclear lineage in the northern (II), northern/north‐eastern (I), and central/southern regions (III) with subsequent hybridization between lineages I and II and lineages II and III. These lineage boundaries are strengthened by the finding that haplotypes of C derived from cpDNA were found in the geographical region of lineage II of nrDNA, while haplotypes of A derived from cpDNA were found in the region of lineage III of nrDNA. Thus, the origin of chloroplasts exclusive to lineages II and III supports their long‐term isolation from one another.     

Multiple Independent Losses of Photosynthesis and Differing EvolutionaryRates in the Genus Cryptomonas (Cryptophyceae): Combined Phylogenetic Analyses of DNA Sequences of the Nuclear and the Nucleomorph Ribosomal Operons

In this study, evidence for at least three independent losses of photosynthesis in the freshwater cryptophyte genus Cryptomonas is presented. The phylogeny of the genus was inferred by molecular phylogenetic analyses of the nuclear internal transcribed spacer 2 (nuclear ITS2), partial nuclear large subunit ribosomal DNA (LSU rDNA), and nucleomorph small subunit ribosomal DNA (SSU rDNA, NM). Both concatenated and single data sets were used. In all data sets, the colorless Cryptomonas strains formed three different lineages, always supported by high bootstrap values (maximum parsimony, neighbor joining and maximum likelihood) and posterior probabilities (Bayesian analyses). The three leukoplast-bearing lineages displayed differing degrees of accelerated evolutionary rates in nuclear and nucleomorph rDNA. Also an increase in A+T-content in highly variable regions of the nucleomorph SSU rDNA was observed in one of the leukoplast-bearing lineages.This article contains three online-only supplementary tables.Reviewing Editor: Dr. Yves Van de Peer     

Identification of an EcoRI restriction site for a rapid and precise determination of beta-asarone-free Acorus calamus cytotypes

Calamus (Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of beta-asarone [(Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid beta-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography-mass spectrometry (GC-MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of beta-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-beta-ocimene (3.28%), camphor (1.54%), calarene (1.42%), alpha-selinene (5.02%) and tau-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), beta-sesquiphellandrene (3.28%), preiso calamendiol (22.81%) and acorone (26.33%), and completely lacked of beta-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid beta-asarone-free A. calamus was also discussed.     

Molecular criteria for determining new hybrid species--an application to the Sonneratia hybrids

The possible hybrid origin of new species can usually be corroborated by molecular means. Here, we suggest that the segregation patterns of the molecular markers be further analyzed. A true hybrid species should show the patterns under continuous breeding among its members, at least beyond the F2 generation. We applied the guidelines to the putative hybrid species of Sonneratia, a widespread mangrove genus, and concluded that all the observed hybrids in this genus are simple F1's. Thus, S. x gulngai and S. x hainanensis are not true hybrid species. The segregation patterns of molecular markers should be heeded in interpreting the existence of hybrid species.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

Ribosomal DNA ITS-1 Intergenic Spacer Polymorphism in Triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.