rRNA Molecules Research Articles

Measurement of rRNA Synthesis and Degradation Rates by 3H-Uracil Labeling in Yeast.

In order to measure the actual synthesis and degradation rates (SR, DR) for rRNA in yeast, we developed a method based on the pulse labeling and quantification of newly synthesized large rRNA molecules by a known mass of cells. The SR is calculated as the ratio of new rRNA molecules (synthesized after a short [5,6-3H]-uracil pulse) to total rRNA (a proxy of cell mass), calculated by northern blotting after hybridization with a 32P-labeled rRNA probe. Then to measure the DR we perform a chase of the existing 3H-labeled rRNA for several hours during yeast culture growth. We have used this method in control experiments where the yeast cell volume varies as a way to check if the SR and DR are constant with the cell volume.

Uniparental silencing of 5S rRNA genes in plant allopolyploids - insights from Cardamine (Brassicaceae).

While the phenomenon of uniparental silencing of 35S rDNA in interspecific hybrids and allopolyploids is well documented, there is a notable absence of information regarding whether such silencing extends to the 5S RNA component of ribosomes. To address this gap in knowledge, we analyzed the 5S and 35S rDNA expression in Cardamine (Brassicaceae) allopolyploids, namely C. × insueta (2n = 3x = 24, genome composition RRA), C. flexuosa (2n = 4x = 32, AAHH), and C. scutata (2n = 4x = 32, PPAA) which share a common diploid ancestor (AA). We employed high-throughput sequencing of transcriptomes and genomes and phylogenetic analyses of 5S rRNA variants. The genomic organization of rDNA was further scrutinized through clustering and fluorescence in situ hybridization. In the C. × insueta allotriploid, we observed uniparental dominant expression of 5S and 35S rDNA loci. In the C. flexuosa and C. scutata allotetraploids, the expression pattern differed, with the 35S rDNA being expressed from the A subgenome, whereas the 5S rDNA was expressed from the partner subgenome. Both C. flexuosa and C. scutata but not C. × insueta showed copy and locus number changes. We conclude that in stabilized allopolyploids, transcription of ribosomal RNA components occurs from different subgenomes. This phenomenon appears to result in the formation of chimeric ribosomes comprising rRNA molecules derived from distinct parental origins. We speculate that the interplay of epigenetic silencing and rDNA rearrangements introduces an additional layer of variation in multimolecule ribosomal complexes, potentially contributing to the evolutionary success of allopolyploids.

Active Microbiota of Penaeus stylirostris Larvae: Partially Shaped via Vertical and Horizontal Transmissions and Larval Ontogeny.

During their entire lifecycle, mariculture animals are farmed in water that contains various microorganisms with which they are in close associations. Microbial exchanges between the animals and their surrounding water can occur. However, little is known about the interactions between shrimp larvae and water, and more especially, about larval bacterial selection and microbiota modulation across ontogeny. To address this gap, using HiSeq sequencing targeting the V4 region of the 16S rRNA molecule, we investigated the active prokaryotic diversity and structure of healthy Penaeus stylirostris larvae and seawater. Comparisons between different larval stages revealed evidence of stage-specific microbiotas and biomarkers, a core microbiota common to all stages, and shared taxa between successive stages, suggesting vertical transmission of bacterial taxa. Comparisons between stage-specific microbiotas and core microbiotas with water storages highlighted that many taxa associated with the larvae were originally present in the natural seawater, underlining horizontal transmission of bacteria from water to larvae. As some of these lineages became active at specific larval stages, we suggest that larvae were able to modulate their microbiota. This study provides insight into larvae-microbiota interactions at the larval stage scale.

A scalable and cost-efficient rRNA depletion approach to enrich RNAs for molecular biology investigations.

Transcriptomics analyses play pivotal roles in understanding the complex regulatory networks that govern cellular processes. The abundance of rRNAs, which account for 80%-90% of total RNA in eukaryotes, limits the detection and investigation of other transcripts. While mRNAs and long noncoding RNAs have poly(A) tails that are often used for positive selection, investigations of poly(A)- RNAs, such as circular RNAs, histone mRNAs, and small RNAs, typically require the removal of the abundant rRNAs for enrichment. Current approaches to deplete rRNAs for downstream molecular biology investigations are hampered by restrictive RNA input masses and high costs. To address these challenges, we developed rRNA Removal by RNaseH (rRRR), a method to efficiently deplete rRNAs from a wide range of human, mouse, and rat RNA inputs and of varying qualities at a cost 10- to 20-fold cheaper than other approaches. We used probe-based hybridization and enzymatic digestion to selectively target and remove rRNA molecules while preserving the integrity of non-rRNA transcripts. Comparison of rRRR to two commercially available approaches showed similar rRNA depletion efficiencies and comparable off-target effects. Our developed method provides researchers with a valuable tool for investigating gene expression and regulatory mechanisms across a wide range of biological systems at an affordable price that increases the accessibility for researchers to enter the field, ultimately advancing our understanding of cellular processes.

Epicoccum sp. as the causative agent of a reddish-brown spot defect on the surface of a hard cheese made of raw ewe milk

Colour defects can affect the appearance of cheese, its flavour, the safety of its consumption, and the price it can demand. This work reports the identification of five fungal isolates from a dairy plant where the surface of most cheeses was affected by patent, reddish-to-brown stains. One of these isolates was obtained from cheese, two from brine, and two from a bulk tank containing ewe milk. Molecular identification by partial amplification, sequencing, and database comparison of the concatenated sequence of the genes coding for the largest subunit of RNA polymerase II (RPB2), β-tubulin (β-TUB), and the large subunit of the rRNA molecule (LSU), plus the internal transcribed sequence (ITS) regions, assigned the isolates to Epicoccum layuense, Epicoccum italicum, and Epicoccum mezzettii. Features of the growth of these different species on different agar-based media, and of the morphology of their conidia following sporulation, are also reported. The strain isolated from cheese, E. layuense IPLA 35011, was able to recreate the reddish-brown stains on slices of Gouda-like cheese, which linked the fungus with the colour defect. In addition, two other strains, E. italicum IPLA 35013 from brine and E. italicum IPLA 35014 from milk, also produced stains on cheese slices. Epicoccum species are widely recognized as plant pathogens but have seldom been reported in the dairy setting, and never as human or animal pathogens.

Morphological and Molecular Characterization of Cyclopoid Copepod in and around Coimbatore Lakes Using 18s rRNA Analysis

Published ecological research on the variety and distribution of freshwater planktonic cyclopoid copepods rely on DNA techniques for precise taxonomic identification. Large-scale investigations of plankton identification using molecular techniques are now more practical due to the increased coverage of reference species in the genetic database GenBank and the falling prices for DNA sequencing. Here, we offer a useful morphological and molecular method for identifying Mesocyclops aspericornis, M. dissimilis, M. pehpiensis, Thermocyclopsdecipiens, and T. hyalinus, which were found in Coimbatore, India, at the Sulur, Singanallur, Ukkadam, and Muthanna lakes. A microscope was used to observe morphological identification. Amplification of 18S rRNA was one technique used for cyclopoids' molecular identification. The evolutionary relationship between 5 species of freshwater cyclopoid copepods is the focus of the current study on molecular phylogenetic analysis. The findings revealed that there was no interpopulation variation in the 18S rRNA molecules of M. dissimilis, M. pehpiensis, T. decipiens, and T. hyalinus. It is intriguing to look at genetic distances within and across taxa to see if a particular group of cyclopoids has diverged, on average, more or less than others. The greatest genetic divergence among cyclopoid taxa was caused by the extreme variance in M. aspericornis, which was more than twice as high as the other cyclopoids. Finally, during the investigation, T. hyalinus was discovered for the first time in the lakes of Coimbatore, India.

2D approach to reconstruct the evolutionary history of clevelandellids (Ciliophora, Armophorea) inhabiting the hindgut of the Panesthiinae cockroaches

AbstractThe ciliate order Clevelandellida unites endosymbionts of the digestive tract of a variety of invertebrates and vertebrates. In the present study, the primary and secondary structures of nuclear and hydrogenosomal rRNA molecules were employed to reconstruct the phylogenetic relationships and to estimate the divergence times of clevelandellids inhabiting the hindgut of the Panesthiinae cockroaches. The secondary structure information was incorporated in phylogenetic analyses using two different strategies, viz., indirectly through 2D‐guided alignments and directly through so‐called pseudo‐protein data. Nuclear and hydrogenosomal markers carried a consistent phylogenetic signal and robustly supported the monophyletic origin of the family Clevelandellidae as well as of its four genera. According to Bayesian relaxed molecular clock analyses, the last common ancestor (LCA) of the family Clevelandellidae very likely emerged during the Late Cretaceous in the Oriental region. Its descendants most likely expanded to Australia in concert with the Neogene colonization and radiation of their host Panesthiinae cockroaches. Taking into account the time‐calibrated phylogenies and the fact that early branching members of the order Clevelandellida inhabit the digestive tracts of amphibians, it is tempting to speculate that the LCA of the Clevelandellida evolved in ectothermic vertebrates. Amphibians could have brought clevelandellids to the land, where they may have been transmitted to the cockroach digestive tract upon feeding on amphibian faeces.

Canis MitoSNP database: a functional tool useful for comparative analyses of human and canine mitochondrial genomes.

Canis MitoSNP is a tool allowing assignment of each mitochondrial genomic position a corresponding position in the mitochondrial gene and in the structure of tRNA, rRNA, and protein. The main aim of this bioinformatic tool was to use data from other bioinformatic tools (TMHMM, SOPMA, tRNA-SCAN, RNAfold, ConSurf) for dog and human mitochondrial genes in order to shorten the time necessary for the analysis of the whole genome single nucleotide polymorphism (SNP) as well as amino acid and protein analyses. Each position in the canine mitochondrial genome is assigned a position in genes, in codons, an amino acid position in proteins, or a position in tRNA or rRNA molecules. Therefore, a user analysing changes in the canine and human mitochondrial genome does not need to extract the sequences of individual genes from the mitochondrial genome for analysis and there is no need to rewrite them into amino acid sequences to assess whether the change is synonymous or nonsynonymous. Canis mitoSNP allows the comparison between the human and canine mitochondrial genomes as well. The Clustal W alignment of the dog and human mitochondrial DNA reference sequences for each gene obtained from GenBank (NC_002008.4 dog, NC_012920.1 human) was performed in order to determine which position in the canine mitochondrial genome corresponds to the position in the human mitochondrial genome. This function may be useful for the comparative analyses. The tool is available at: https://canismitosnp.pl .

Small Nucleolar (Sno)RNA: Therapy Lays in Translation

The ribosome is one of the largest complexes in the cell. Adding to its complexity are more than 200 RNA modification sites present on ribosomal RNAs (rRNAs) in a single human ribosome. These modifications occur in functionally important regions of the rRNA molecule, and they are vital for ribosome function and proper gene expression. Until recent technological advancements, the study of rRNA modifications and their profiles has been extremely laborious, leaving many questions unanswered. Small nucleolar RNAs (snoRNAs) are non-coding RNAs that facilitate and dictate the specificity of rRNA modification deposition, making them an attractive target for ribosome modulation. Here, we propose that through the mapping of rRNA modification profiles, we can identify cell-specific modifications with high therapeutic potential. We also describe the challenges of achieving the targeting specificity needed to implement snoRNAs as therapeutic targets in cancers.

Antibacterial Activity and 16S rRNA Gene Sequencing of Lactic Acid Bacteria from Homemade Fermented Milk in Medan, Indonesia

Research indicates that fermented products have nutritional and non-nutritional components that can improve health. Milk is commonly used for fermentation products because its rich nutrients support the growth of Lactic Acid Bacteria (LAB). This paper investigates antibacterial activity and 16S rRNA gene sequencing of LAB from homemade fermented milk in Medan City, Indonesia. This paper was an experimental study by In Vitro Models conducted in August-October 2019. This study used three different homemade fermented milk (SF2-4) and positive control of manufactured fermented milk (SF1). All isolated LABs underwent an antibacterial assay by the Disc diffusion method against two pathogens, including Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). In addition, there was molecular identification based on 16S rRNA gene sequencing. The zone of inhibition from samples against Escherichia coli ranged from 5.600-12.23 mm. The most potent antibacterial activity was found in SF4 (12.23 mm) and the least in SF2 (5.60 mm). Some samples (SF1 and SF2) showed no antibacterial effect against Staphylococcus aureus bacteria. The antibacterial activity against Staphylococcus aureus bacteria was found only in SF2 and SF3, which were 6.60 mm and 7.14 mm, respectively. Based on the characteristics, enumeration, and antibacterial activity, the authors chose isolated LAB from SF4 for molecular identification based on 16S rRNA gene sequencing. SF4 isolates had a similar 16S rRNA molecule to Lactobacillus fermentum strain NBRC 15885 with a homology level of 99.78%. In conclusion, some homemade fermented milk in Medan City, Indonesia, are potential probiotics.

Glyphosate treatment mediates the accumulation of small discrete 5'- and 3'-terminal fragments of 18S rRNA in plant cells.

Under many kinds of stress, eukaryotic cells rapidly decrease the overall translation level of the majority of mRNAs. However, some molecular mechanisms of protein synthesis inhibition like phosphorylation of eukaryotic elongation factor 2 (eEF2), which are known to be functional in animals and yeast, are not implemented in plants. We suggest that there is an alternative mechanism for the inhibition of protein synthesis in plant cells and possibly, in other eukaryotes, which is based on the discrete fragmentation of 18S rRNA molecules within small ribosomal subunits. We identified four stress-induced small RNAs, which are 5'- and 3'-terminal fragments of 18S rRNA. In the present work, we studied the induction of 18S rRNA discrete fragmentation and phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) in germinated wheat embryos in the presence of glyphosate, which imitates the condition of amino acid starvation. Using northern and western blotting, we have shown that stress-induced 18S rRNA fragments started to accumulate in wheat embryos at glyphosate concentrations that did not evoke eIF2α phosphorylation. It was also found that cleavage of 18S rRNA near the 5'-terminus began much earlier than eIF2α phosphorylation, which became noticeable only at higher concentration (500 μM) of glyphosate. This result suggests that discrete fragmentation of 18S rRNA may constitute a regulatory mechanism of mRNA translation in response to stress and may occur in plant cells in parallel with and independently of eIF2α phosphorylation. The identified small 5'- and 3'-terminal fragments of 18S rRNA that accumulate during various stresses may serve as stress resistance markers in the breeding of economically important plant crops.

The layered costs and benefits of translational redundancy.

The rate and accuracy of translation hinges upon multiple components - including transfer RNA (tRNA) pools, tRNA modifying enzymes, and rRNA molecules - many of which are redundant in terms of gene copy number or function. It has been hypothesized that the redundancy evolves under selection, driven by its impacts on growth rate. However, we lack empirical measurements of the fitness costs and benefits of redundancy, and we have poor a understanding of how this redundancy is organized across components. We manipulated redundancy in multiple translation components of Escherichia coli by deleting 28 tRNA genes, 3 tRNA modifying systems, and 4 rRNA operons in various combinations. We find that redundancy in tRNA pools is beneficial when nutrients are plentiful and costly under nutrient limitation. This nutrient-dependent cost of redundant tRNA genes stems from upper limits to translation capacity and growth rate, and therefore varies as a function of the maximum growth rate attainable in a given nutrient niche. The loss of redundancy in rRNA genes and tRNA modifying enzymes had similar nutrient-dependent fitness consequences. Importantly, these effects are also contingent upon interactions across translation components, indicating a layered hierarchy from copy number of tRNA and rRNA genes to their expression and downstream processing. Overall, our results indicate both positive and negative selection on redundancy in translation components, depending on a species' evolutionary history with feasts and famines.

FUS regulates a subset of snoRNA expression and modulates the level of rRNA modifications

FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2’-O methylation (2’-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2’-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2’-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression.

Isolating Mitochondria, Mitoplasts, and mtDNA from Cultured Mammalian Cells.

Mitochondria are double membrane-bound eukaryotic organelles with roles in a range of cellular activities including energy conversion, apoptosis, cell signalling, and the biosynthesis of enzyme cofactors. Mitochondria contain their own genome, called mtDNA, which encodes subunits of the oxidative phosphorylation machinery as well as the rRNA and tRNA molecules required for their translation within mitochondria. The ability to isolate highly purified mitochondria from cells has been instrumental in a number of studies of mitochondrial function. Differential centrifugation is a long-established method for the isolation of mitochondria. Cells are subjected to osmotic swelling and disruption, followed by centrifugation in isotonic sucrose solutions to separate mitochondria from other cellular components. We present a method using this principle for the isolation of mitochondria from cultured mammalian cell lines. Mitochondria purified by this method can be further fractionated to investigate protein localization, or act as a starting point to purify mtDNA.

Recent Advances in RNA m6A Modification in Solid Tumors and Tumor Immunity.

An analogous field to epigenetics is referred to as epitranscriptomics, which focuses on the study of post-transcriptional chemical modifications in RNA. RNA molecules, including mRNA, tRNA, rRNA, and other non-coding RNA molecules, can be edited with numerous modifications. The most prevalent modification in eukaryotic mRNA is N6-methyladenosine (m6A), which is a reversible modification found in over 7000 human genes. Recent technological advances have accelerated the characterization of these modifications, and they have been shown to play important roles in many biological processes, including pathogenic processes such as cancer. In this chapter, we discuss the role of m6A mRNA modification in cancer with a focus on solid tumor biology and immunity. m6A RNA methylation and itsregulatory proteins can play context-dependent roles in solid tumor development and progression by modulating RNA metabolism to drive oncogenic or tumor-suppressive cellular pathways. m6ARNA methylation also plays dynamic roles within both immune cells and tumor cells to mediate the anti-tumor immune response. Finally, an emerging area of research within epitranscriptomics studies the role of m6A RNA methylation in promoting sensitivity or resistance to cancer therapies, including chemotherapy, targeted therapy, and immunotherapy. Overall, our understanding of m6A RNA methylation in solid tumors has advanced significantly, and continued research is needed both to fill gaps in knowledge and to identify potential areas of focus for therapeutic development.

Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.

The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as apowerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don'ts will enable beginners and experienced scientists to study the import and assembly of MRPs.

First molecular evidence of hybridization in endosymbiotic ciliates (Protista, Ciliophora).

Hybridization is an important evolutionary process that can fuel diversification via formation of hybrid species or can lead to fusion of previously separated lineages by forming highly diverse species complexes. We provide here the first molecular evidence of hybridization in wild populations of ciliates, a highly diverse group of free-living and symbiotic eukaryotic microbes. The impact of hybridization was studied on the model of Plagiotoma, an obligate endosymbiont of the digestive tube of earthworms, using split decomposition analyses and species networks, 2D modeling of the nuclear rRNA molecules and compensatory base change analyses as well as multidimensional morphometrics. Gene flow slowed down and eventually hampered the diversification of Lumbricus-dwelling plagiotomids, which collapsed into a single highly variable biological entity, the P. lumbrici complex. Disruption of the species boundaries was suggested also by the continuum of morphological variability in the phenotypic space. On the other hand, hybridization conspicuously increased diversity in the nuclear rDNA cistron and somewhat weakened the host structural specificity of the P. lumbrici complex, whose members colonize a variety of phylogenetically closely related anecic and epigeic earthworms. By contrast, another recorded species, P. aporrectodeae sp. n., showed no signs of introgression, no variability in the rDNA cistron, and very high host specificity. These contrasting eco-evolutionary patterns indicate that hybridization might decrease the alpha-diversity by dissolving species boundaries, weaken the structural host specificity by broadening ecological amplitudes, and increase the nuclear rDNA variability by overcoming concerted evolution within the P. lumbrici species complex.

Microbiota of the Rearing Water of Penaeus stylirostris Larvae Influenced by Lagoon Seawater and Specific Key Microbial Lineages of Larval Stage and Survival.

Aquacultured animals are reared in water, where they interact with microorganisms which can be involved in their development, immunity, and disease. It is therefore interesting to study the rearing water microbiota, especially in the hatcheries of the Pacific blue shrimp Penaeus stylirostris, where larval mass mortalities occur. In this study, using HiSeq sequencing of the V4 region of the 16S rRNA molecule coupled with zootechnical and chemical analyses, we investigated whether any microbial lineages could be associated with certain mortality rates at a given larval stage. Our results indicate that the active microbiota of the rearing water was highly dynamic throughout the rearing process, with distinct communities influenced by progressive water eutrophication, larval stage, and survival rate. Our data also highlighted the role of the lagoon seawater on the rearing water microbiome, as many operational taxonomic units (OTUs) specific to a given larval stage and survival rate were detected in the primary reservoir which contained the lagoon water. We also identified biomarkers specific to water eutrophication, with Alteromonadaceae, Vibrionaceae, and Methylophilaceae, respectively, linked to increases in ammonia, nitrogen, and soluble reactive phosphate, or to increases in colored dissolved organic matter in the rearing water; other biomarkers were specific to certain larval stages and survival rates. Indeed, the Marinobacteraceae were specific to the Nauplii, and the Thalassospiraceae and Saprospiraceae to the Zoea Good condition; when mortality occurred, the Litoricolaceae were specific to the Zoea Bad, Microbacteraceae to the Mysis Bad, and Methylophilaceae to the Mysis Worst condition. Thus, these biomarkers might be used as potential early warning sentinels in water storage to infer the evolution of larval rearing to improve shrimp larval rearing. IMPORTANCE In New Caledonia, rearing of P. stylirostris is one of the main economic activities; unfortunately, mass larval mortalities cause important production decreases, involving major economic losses for the farmers and the Territory. This phenomenon, which has occurred at any larval stage over the past decade, is poorly understood. The significance of our research is in the identification of biomarkers specific to larval stage and survival rate, with some of these biomarkers being already present in the lagoon water. This enhances the role of the lagoon on the active microbiota of the rearing water at various larval stages and survival rates. Together, our results help us understand which active microbial communities are present in the rearing water according to larval stage and health. This might lead to broader impacts on hatcheries by helping to develop useful tools for using the water-lagoon, reservoir, or rearing-to test for the presence of these biomarkers as an early monitoring strategy.

A huge undescribed diversity of the subgenus Hystricochaetonotus (Gastrotricha, Chaetonotidae, Chaetonotus) in Central Europe

The subgenus Hystricochaetonotus Schwank, 1990 is one of the most species-rich subgenera of Chaetonotus Ehrenberg, 1830. It has a worldwide distribution and encompasses 37 species predominantly living in the benthos and periphyton of limnetic habitats. We have discovered further nine new species in running and stagnant waters in Slovakia (Central Europe): Ch. (H.) arcanus sp. nov., Ch. (H.) avarus sp. nov., Ch. (H.) gulosus sp. nov., Ch. (H.) iratus sp. nov., Ch. (H.) luxus sp. nov., Ch. (H.) mirabilis sp. nov., Ch. (H.) optabilis sp. nov., Ch. (H.) slavicus sp. nov., and Ch. (H.) superbus sp. nov. Their morphology was studied using differential interference contrast microscopy and subsequent morphometric analyses were carried out. In addition, the primary and secondary structures of their 18S, ITS2, and 28S rRNA molecules as well as their barcoding mitochondrial gene encoding for cytochrome c oxidase (COI) were analyzed. Species boundaries were tested also using the compensatory base change analysis. The new species could be well separated both morphologically and molecularly. The present barcoding analyses revealed that the nuclear ITS2 sequences represent a powerful DNA barcode in addition to the mitochondrial COI gene. According to the multi-gene phylogenetic analyses, the lineage leading to the last common ancestor of the ‘Hystricochaetonotus’ clade is the longest internal branch within the family Chaetonotidae Gosse, 1864. Since members of the subgenus Hystricochaetonotus are morphologically highly heterogeneous, parallel evolution of Chaetonotus-like and/or Hystricochaetonotus-like characters of scales and spines occurred during its radiation.

28S rRNA gap deletion in two species of Girardia (Platyhelminthes, Tricladida, Dugesiidae).

Studies have shown atypical migration of the 28S rRNA molecule when exposed to heat, dissociates into two subunits (28Sα and 28Sβ), this event is due to a ‘gap deletion’ in the molecule. This rRNA degradation has been correlated to a UA-rich conserved segment. We show the atypical 28S rRNA molecule migration in Girardia tigrina and Girardia sp.. The presence of conserved motifs, 28S rRNA, was confirmed. The UA-rich segment was absent. The 5´-CGAAAGGG-3´ motif was present, indicating that the dissociation 28S rRNA is independent of the UA-rich signal. We propose the existence of a putative secondary structure for the D7a segment of the 28S rDNA molecule and suggest that the atypical RNA profile observed may occur across the Girardia genus.