Primary Structure of 28S rRNA Gene Confirms Monophyly of Free-Living Heterotrophic and Phototrophic Apicomplexans (Alveolata).

Phylogenetic analysis of large subunit ribosomal RNA (LSU rRNA or 28S rRNA) gene sequences from free-living predatory flagellates Colpodella angusta, Voromonas pontica, and Alphamonas edax (Apicomplexa) confirms their close relationship with chromerids Chromera velia and Vitrella brassicaformis, which possess a functional photosynthetic plastid. Together these organisms form a sister group to parasitic apicomplexans (coccidians and gregarines, or sporozoans sensu lato). This result agrees with the previous conclusion on monophyly of colpodellids and chromerids (chrompodellids) based on phylogenomic data. The revealed relationships demonstrate a complex pattern of acquisition, loss, or modification of plastids and transition to parasitism during alveolate evolution.

Local damage detection by nonlinear coda wave interferometry combined with time reversal.

Coda wave interferometry (CWI) is a sensitive ultrasound method for the detection of weak and local changes in complex inhomogeneous media. In a nonlinear modification of the method discussed here, a high-frequency probe coda is compared to its replica obtained in the presence of low-frequency pumping. If, after the filtering-out of low frequencies, the coda signals are different, this is attributed to nonlinear pump-probe interaction induced by contact acoustical nonlinearity in the damaged zone. Actually, the CWI methods are used for global inspection of complex media, such as for example, concrete structures. In this work, a step forward is made; it consists in combining the CWI with the time-reversal (TR) technique in order to allow one to focus the pump wave on a selected area in the structure and to detect and localize a flaw. Time-reverse pump is possible only in pulsed mode due to the spatio-temporal wave compression. By this reason, the particularities of coda wave mixing in conventionally used continuous and pulsed pump mode are considered. It has been experimentally observed that an aftereffect of a pulsed pump provides a nonlinear interaction between pump and probe waves of a sufficient overall level for defect detection with TR. Finally, it was shown that a TR focusing even with the minimal available quality i.e., with only one transducer produces a sufficient contrast allowing to distinguish intact and damaged zones with nonlinear CWI.

[On the phylogenetic position of Hexapoda within the Pancrustacea].

Contemporary views on the phylogeny of arthropods are at odds with the traditional system, which recognizes four independent arthropod classes: Chelicerata, Crustacea, Myriapoda and Insecta. There is compelling evidence that insects in fact comprise a monophyletic lineage with Crustacea within a larger clade of Pancrustacea (=Tetraconata). Which crustacean group is the closest living relative of insects remains an open question. Recent phylogenetic analyses based on multiple genes suggest their sistership with "lower" crustaceans, the Branchiopoda. This relationship was often impeached to be caused by the long branch attraction artifact. We analyzed concatenated data on 77 ribosomal proteins, elongation factor 1 alpha (EF1A), initiation factor 5 alpha (alF5A) and other selected nuclear and mitochondrial proteins. Nuclear protein data supports the monophyly of Hexapoda, the clade uniting entognath and ectognath insects. Hexapoda and Branchiopoda comprise a monophyletic lineage in most analyses. Maxillopoda occupies the sister position to the Hexapoda + Branchiopoda. "Higher" crustaceans, the Malacostraca, in most reconstructions comprise a more basal lineage withinthe Pancrustacea. Molecular synapomorphies in low homoplastic regions are found for the clades Hexapoda Branchiopoda + Maxillopoda and the monophyletic Malacostraca containing the Phyllocarida. Therefore, the sistership of Hexapoda and Branchiopoda and their position within Entomostraca may in fact represent bona fide phylogenetic relationships.

[Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes].

Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.

The unusually long small subunit ribosomal RNA gene found in amitochondriate amoeboflagellate Pelomyxa palustris: its rRNA predicted secondary structure and phylogenetic implication.

In order to ascertain a phylogenetic position of the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be 3502 bp long. The predicted secondary structure of its rRNA includes at least 16 separate expansion zones located in all the variable regions (V1-V9), as well as in some conservative gene regions. Most insertions are represented by sequences of low complexity that have presumably arisen by a slippage mechanism. Relatively conservative, uniformly positioned motifs contained in regions V4 and V7, as well as in some others, made it possible to perform folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P. palustris tends to cluster with amitochondriate and secondary lost mitochondria amoebae and amoeboflagellates Entamoeba, Endolimax nana, and Phreatamoeba balamuthi, comprising together with them and aerobic lobose amoebae Vannella, Acanthamoeba, Balamuthia, and Hartmannella a monophyletic cluster. Another pelobiont, Mastigamoeba invertens, does not belong to this cluster. No specific similarity was discovered between the SSU rRNA of P. palustris and amitochondriate taxa of 'Archezoa': Diplomonada, Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA does not occupy a basal position in the phylogenetic trees and could be ascribed to the so-called eukaryotic 'crown' group if the composition of the latter were not so sensitive to the methods of tree building. Thus, molecular and morphological data suggest that P. palustris represents a secondarily modified eukaryotic lineage.

The novel transmembrane Escherichia coli proteins involved in the amino acid efflux.

A novel gene of Escherichia coli, rhtB, has been characterized. Amplification of this gene provides resistance to homoserine and homoserine lactone. Another E. coli gene, rhtC, provides resistance to threonine. The homologues of RhtB are widely distributed among various eubacteria and archaea, from one to 12 copies of family members that differ in their primary structure were found in the genomes. Most of them are genes that encode hypothetical transmembrane proteins. Experimental data that indicate participation of the rhtB product in the excretion of homoserine have been obtained.

Secondary structure of some elements of 18S rRNA suggests that strongylid and a part of rhabditid nematodes are monophyletic.

Analysis of the secondary structure of 18S rRNA molecules in nematodes revealed some new traits in the secondary structure peculiar to their hairpin 17. Some of them are characteristic of all the nematodes, whereas others are characteristic exclusively of the order Rhabditida. The loss of a nucleotide pair in the highly conservative region of hairpin 17 distinguishes 18S rRNA of the Strongylida and some species of the Rhabditida from other nematodes and, moreover, from all other organisms. Hence, it is possible to regard the Strongylida and a part of the Rhabditida including Caenorhabditis elegans as a new monophyletic taxon.

The broad host range plasmid pLF1311 from Lactobacillus fermentum VKM1311.

The complete nucleotide sequence (2389 bp) of the cryptic plasmid pLF1311 from Lactobacillus fermentum VKM1311 was determined. DNA sequence analysis revealed the putative coding regions for a replicative protein (RepB), its repressor (RepA) and double-stranded (dso) and single-stranded (sso) origins. pLF1311 belongs to the pE194 family of rolling circle-replicating plasmids. A derivative of pLF1311 that contains the cat gene of plasmid pC194 of Staphylococcus aureus and the oriT of RP4 was constructed and transferred by conjugative mobilization from Escherichia coli to various Gram-positive bacteria. The stable maintenance of this derivative was shown in some strains of Lactobacillus, Lactococcus, Enterococcus and Bacillus under non-selective conditions.

[Heterogeneity and homologies of the repeating and unique DNA of dragonflies (Odonata, Insecta)]. / Geterogennost' i gomologii povtoriaiushcheisia i unikal'noi DNK strekoz (Odonata, Insecta).

A relative content of unique and reiterated nucleotide sequences in DNA of eleven dragonfly species was estimated. The degree of intra- and intergenomic divergence of these DNA sequences was determined by means of DNA-DNA hybridization. Species from different genera share 40-45% of the repetitive sequences and those from different families--from 11 to 20% only. Data on the thermostability of homo- and heteroduplexes suggest that new families of the repetitive sequences have arisen repeatedly during dragonflies evolution. The quality of homologous unique sequences in the DNA compared (20-97%) correlates with the taxonomic relationships of species. Phylogenesis of some dragonfly families is discussed in view of the results obtained.

[Primary structure of hairpin 35 and 48 small rRNA is proof that Trefusiidae is a subtaxon of marine Enoplida (Nemotoda)]. / Pervichnaia struktura petel' 35 i 48 maloi rRNK svidetel'stvuet, chto Trefusiidae iavliaiutsia subtaksonom morskikh Enoplida (Nematoda).

A rare nucleotide substitution was found in the evolutionarily conserved loop of hairpin 35 of the 18S rRNA gene of marine free-living nematode, Trefusia zostericola (Nematoda: Enoplida). The same substitution was found in all the marine Enoplida studied but not in other nematodes. Such a molecular synapomorphy indicates that marine enoplids are more closely related to T. zostericola than to freshwater Triplonchida. Maximum parsimony, neighbor-joining, and maximum likelihood analyses of complete nucleotide sequences of the gene, with the heterogeneity of nucleotide sites in evolution rates taken into account, support this conclusion. Hence, the hypothesis of particular primitiveness of Trefusiidae among nematodes should be rejected. Phylogenies based on molecular data support the morphological reduction of metanemes in Trefusiidae. Alongside with the unique change in hairpin 35 loop among marine Enoplida (including T. zostericola), hairpin 48 is also modified by a rare transversion which could be found among Mesorhabditoidea nematodes, in related genera Pelodera, Mesorhabditis, Teratorhabditis, Parasitorhabditis, Crustorhabditis, and Distolabrellus, and in 11 orders of Rhodophyta. Rare mutations in hairpins 35 and 48 tend to be fixed correlatively in evolution and could be found in all the Acanthocephala species. X-Ray data show that these regions (H31 and H43, in alternative nomenclature) are spatially brought together in native ribosomes. The nature and distribution of molecular autoapomorphies in phylogenetic trees of high-rank taxa are discussed.

[Conditionally neutral phylogenetic markers of major taxa: a new aspect of the evolution of macromolecules]. / Uslovno-neitral'nye filogeneticheskie priznaki krupnykh taksonov--novyi aspekt évoliutsii makromolekul.

The current phase of molecular phylogenetics can be named the 18S rRNA gene era, which is now approaching the end. To date, almost all phyla of metazoans and many taxa of protists are represented in databases of 18S rRNA gene sequences. The elements of the phylogenetic tree of Metazoa inferred from 18S rRNA genes are characterized by unequal validity: some of them seem to be well grounded; others are not adequately supported, and probably will be revised later. The validity of phylogenetic reconstruction is influenced by two main factors: (1) erroneous grouping of long branches that occur because of abnormally high evolution rate; (2) deficit of phylogenetically informative characters. A method for overcoming these difficulties is suggested in addition to known tools: using phylogenetic markers that are stable within individual taxa and evolve by punctuated equilibrium. These markers are least influenced by the convergence caused by a high evolution rate of the entire gene. The nature of these markers of ancient taxa, paradoxical from the perspective of neutral evolution, is discussed, as well as their importance for establishing monophyly of both new large-scale taxonomic groups of invertebrates (Bilateria + Rhombozoa + Orthonectida + Myxozoa + Cnidaria + Placozoa and Echinodermata + Hemichordata) and some major taxa of Nematoda.

[Cryptic transposable phages of Pseudomonas aeruginosa]. / Skrytye fagi-transpozony Pseudomonas aeruginosa.

Frequencies of nucleotide sequences homologous to phage transposons (PT) of two species, D3112 and B3, were assessed in genomes of natural Pseudomonas aeruginosa strains by the dot-blot hybridization method. These strains were incapable of liberating viable phages on a lawn of the PA01 standard indicator strain of P. aeruginosa. It was shown that the homologies detected belong to two groups, high and intermediate, with respect to homology level. Homology patterns were classified as high when they provided signals comparable to those for hybridization in a positive control; they were classified as intermediate when the hybridization level higher than the background level, but lower than in the positive control. Homologous PT sequences were designated as cryptic PT. Intact cryptic PT prophages were shown to exist in genomes of particular natural strains manifesting a high level of hybridization. However, the growth of these phages was limited by the restriction system of strain PA01. It is possible to isolate strains maintaining the growth of a portion of cryptic PT. These strains differed from P. aeruginosa with respect to the specificity of the restriction and modification system. Nevertheless, in most cases, the attempt to identify a novel host capable of maintaining growth of a cryptic PT failed. Natural strains often carry cryptic PT related to both known PT species, D3112 and B3. The frequency of cryptic PT is extremely high, reaching 30% in only strains with a high level of homology and up to 50% in all strains exhibiting homology. This high PT frequency is assumed to be associated with the considerable variation of P. aeruginosa.

[On the genetic uniformity of the genus Trichoplax (Placozoa)]. / O geneticheskom odnoobrazii v rode Trichoplax (Placozoa).

Fragments of the nuclear and mitochondrial genes for the large-subunit rRNA were compared for Trichoplax sp. and T. adhaerens. High similarity was observed for their sequences, suggesting that different Trichoplax isolates belong to one species.

[Whether variable cleavage of Enoplida (Nematoda) is primitive? Notes to D.A. Voronov article "Comparative embryology of Nematoda and the law of embryologic similarity]. / Primitivno li variabel'noe drobleniie Enoplida (Nematoda)? Zamechanie k stat'e D.A.Voronova "Sravnitel'naia émbriologiia nematod i zakon zarodyshevogo skhodstva".

The early embryonic development of Nematoda proceeds by three ways, which strictly correspond to three phylogenetic lineages. Under the first way the endodermal precursor is localized in the posterior blastomere at the two-cells stage (such a determination is the peculiarity of all the Chromadoria, including Secernentea and Caenorhabditis elegans). Under the second way the endodermal precursor is localized in the anterior blastomere of the egg. This feature is very unusual for Metazoa, but it is the only way of entoderm determination in all the Dorylaimia orders (Mononchida, Mermithida, Trichinellida, Dioctophymida, Dorylaimida). The third way described for the sea Enoplida is characterized with variable location of blastomers and changeable localization of endodermal precursor before eight-cells stage. It is still unknown of these three variants was typical the most recent common ancestor of present Nematoda. D.A. Voronov (2001) produced argument in favour of variable cleavage as primitive one for Nematoda. This opinion is rejected because of the similarity in development between sea Enoplida and C. elegans. Both of them share such features as low-cell gastrula and neurula, identical phylotypic lima bean stage of embryogenesis, identity of some geometrical figures 4 or 8 blastomers, isolating of the endodermal precursor at the eight-cells stage, the lack in development of any plesiomorphous features, which are widely distributed outside Nematoda (under the variable cleavage of Enoplida there are no such locations of blastomers, which are typical for spiral or radial cleavage, there are no embryonic leaves as well). One can see the homology of separate cells at adult Enoplida and Rhabditia. Cell lineage of Triplonchida as far as it is described at Tobrilus gracilis doesn't exclude the hypothesis on their origin from the cleavage similar to one of present Dorylaimia with localization of the endodermal precursor in the anterior blastomere. In view of all the considerations mentioned above one should interpret variable cleavage of Enoplida as derivation from invariant cleavage.

[Molecular evidence of regression in evolution of metazoa]. / Molekuliarnye svidetel'stva regressa v évoliutsii mnogokletochnykh zhivotnykh.

Molecular data permit to construct phylogenetic trees independently of morphological characters. It allows to consider their evolution without the frames of a priori hypothesis of regularities of morphological evolution and independently of palaeontological data. Cladistic analysis of elements of secondary structure of varible areas V7 and V2 in 18S rRNA with different Protozoa as "external" groups shows that Bilateria + Cnidaria are monophyletic, Ctenophora and Porifera are early derivatives of Metazoa, Trichoplax (Placozoa) is a form related to Cnidaria, while Rhombozoa, Orthonectida and Myxozoa were branched within Bilateria. Morphological reduction with losses of any organs and tissues took place many times in early evolution of Metazoa and Bilateria not only in parasitic species. It occurred both at early and late stages of embryonic development and differentiation. Two alternative scenario of morphological degeneration in Trichoplax and the way of their testing are suggested. The similarity of Ctenophora and Calcarea is discussed. Meridional or oblique position of the third cleavage furrow of ovule can be considered as an evidence of their origin from common ancestor.

Phylogenetic analysis of entomoparasitic nematodes, potential control agents of flea populations in natural foci of plague.

Entomoparasitic nematodes are natural control agents for many insect pests, including fleas that transmit Yersinia pestis, a causative agent of plague, in the natural foci of this extremely dangerous zoonosis. We examined the flea samples from the Volga-Ural natural focus of plague for their infestation with nematodes. Among the six flea species feeding on different rodent hosts (Citellus pygmaeus, Microtus socialis, and Allactaga major), the rate of infestation varied from 0 to 21%. The propagation rate of parasitic nematodes in the haemocoel of infected fleas was very high; in some cases, we observed up to 1,000 juveniles per flea specimen. Our study of morphology, life cycle, and rDNA sequences of these parasites revealed that they belong to three distinct species differing in the host specificity. On SSU and LSU rRNA phylogenies, these species representing three genera (Rubzovinema, Psyllotylenchus, and Spilotylenchus), constitute a monophyletic group close to Allantonema and Parasitylenchus, the type genera of the families Allantonematidae and Parasitylenchidae (Nematoda: Tylenchida). We discuss the SSU-ITS1-5.8S-LSU rDNA phylogeny of the Tylenchida with a special emphasis on the suborder Hexatylina.