DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda)

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.

Molecular characterisation of dengue virus type 1 reveals lineage replacement during circulation in Brazilian territory

Dengue fever is the most important arbovirus infection found in tropical regions around the world. Dispersal of the vector and an increase in migratory flow between countries have led to large epidemics and severe clinical outcomes, such as dengue haemorrhagic fever and dengue shock syndrome. This study analysed the genetic variability of the dengue virus serotype 1 (DENV-1) in Brazil with regard to the full-length structural genes C/prM/M/E among 34 strains isolated during epidemics that occurred in the country between 1994-2011. Virus phylogeny and time of divergence were also evaluated with only the E gene of the strains isolated from 1994-2008. An analysis of amino acid differences between these strains and the French Guiana strain (FGA/89) revealed the presence of important nonsynonymous substitutions in the amino acid sequences, including residues E297 (Met→Thr) and E338 (Ser→Leu). A phylogenetic analysis of E proteins comparing the studied isolates and other strains selected from the GenBank database showed that the Brazilian DENV-1 strains since 1982 belonged to genotype V. This analysis also showed that different introductions of strains from the 1990s represented lineage replacement, with the identification of three lineages that cluster all isolates from the Americas. An analysis of the divergence time of DENV-1 indicated that the lineage circulating in Brazil emerged from an ancestral lineage that originated approximately 44.35 years ago.

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

Mesocoelium lanfrediae sp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.

Virulence profiles of bacteremic extended-spectrum β-lactamase-producing Escherichia coli: association with epidemiological and clinical features.

There is scarce data about the importance of phylogroups and virulence factors (VF) in bloodstream infections (BSI) caused by extended-spectrum β-lactamase-producing Escherichia coli (ESBLEC). A prospective multicenter Spanish cohort including 191 cases of BSI due to ESBLEC was studied. Phylogroups and 25 VF genes were investigated by PCR. ESBLEC were classified into clusters according to their virulence profiles. The association of phylogropus, VF, and clusters with epidemiological features were studied using multivariate analysis. Overall, 57.6%, 26.7%, and 15.7% of isolates belonged to A/B1, D and B2 phylogroups, respectively. By multivariate analysis (adjusted OR [95% CI]), virulence cluster C2 was independently associated with urinary tract source (5.05 [0.96-25.48]); cluster C4 with sources other than urinary of biliary tract (2.89 [1.05-7.93]), and cluster C5 with BSI in non-predisposed patients (2.80 [0.99-7.93]). Isolates producing CTX-M-9 group ESBLs and from phylogroup D predominated among cluster C2 and C5, while CTX-M-1 group of ESBL and phylogroup B2 predominantes among C4 isolates. These results suggest that host factors and previous antimicrobial use were more important than phylogroup or specific VF in the occurrence of BSI due to ESBLEC. However, some associations between virulence clusters and some specific epidemiological features were found.

Emergence of young human genes after a burst of retroposition in primates.

The origin of new genes through gene duplication is fundamental to the evolution of lineage- or species-specific phenotypic traits. In this report, we estimate the number of functional retrogenes on the lineage leading to humans generated by the high rate of retroposition (retroduplication) in primates. Extensive comparative sequencing and expression studies coupled with evolutionary analyses and simulations suggest that a significant proportion of recent retrocopies represent bona fide human genes. We estimate that at least one new retrogene per million years emerged on the human lineage during the past approximately 63 million years of primate evolution. Detailed analysis of a subset of the data shows that the majority of retrogenes are specifically expressed in testis, whereas their parental genes show broad expression patterns. Consistently, most retrogenes evolved functional roles in spermatogenesis. Proteins encoded by X chromosome-derived retrogenes were strongly preserved by purifying selection following the duplication event, supporting the view that they may act as functional autosomal substitutes during X-inactivation of late spermatogenesis genes. Also, some retrogenes acquired a new or more adapted function driven by positive selection. We conclude that retroduplication significantly contributed to the formation of recent human genes and that most new retrogenes were progressively recruited during primate evolution by natural and/or sexual selection to enhance male germline function.

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation.

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

Novel Chlamydiales strains isolated from a water treatment plant.

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers (Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae; two closely related strains belonged to the Criblamydiaceae; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.

Evolutionary simulations to detect functional lineage-specific genes.

MOTIVATION: Supporting the functionality of recent duplicate gene copies is usually difficult, owing to high sequence similarity between duplicate counterparts and shallow phylogenies, which hamper both the statistical and experimental inference. RESULTS: We developed an integrated evolutionary approach to identify functional duplicate gene copies and other lineage-specific genes. By repeatedly simulating neutral evolution, our method estimates the probability that an ORF was selectively conserved and is therefore likely to represent a bona fide coding region. In parallel, our method tests whether the accumulation of non-synonymous substitutions reveals signatures of selective constraint. We show that our approach has high power to identify functional lineage-specific genes using simulated and real data. For example, a coding region of average length (approximately 1400 bp), restricted to hominoids, can be predicted to be functional in approximately 94-100% of cases. Notably, the method may support functionality for instances where classical selection tests based on the ratio of non-synonymous to synonymous substitutions fail to reveal signatures of selection. Our method is available as an automated tool, ReEVOLVER, which will also be useful to systematically detect functional lineage-specific genes of closely related species on a large scale. AVAILABILITY: ReEVOLVER is available at http://www.unil.ch/cig/page7858.html.

False phylogenies on wood mice due to cryptic cytochrome-b pseudogene.

The phylogeny and phylogeography of the Old World wood mice (subgenus Sylvaemus, genus Apodemus, Muridae) are well-documented. Nevertheless, the distributions of species, such as A. fulvipectus and A. ponticus remain dubious, as well as their phylogenetic relationships with A. sylvaticus. We analysed samples of Apodemus spp. across Europe using the mitochondrial cytochrome-b gene (cyt-b) and compared the DNA and amino-acid compositions of previously published sequences. The main result stemming from this study is the presence of a well-differentiated lineage of Sylvaemus including samples of various species (A. sylvaticus, A. fulvipectus, A. ponticus) from distant locations, which were revealed to be nuclear copies of the mitochondrial cyt-b. The presence of this cryptic pseudogene in published sequences is supported by different pathways. This has led to important errors in previous molecular trees and hence to partial misinterpretations in the phylogeny of Apodemus.

Discovery of catalases in members of the Chlamydiales order.

Catalase is an important virulence factor for survival in macrophages and other phagocytic cells. In Chlamydiaceae, no catalase had been described so far. With the sequencing and annotation of the full genomes of Chlamydia-related bacteria, the presence of different catalase-encoding genes has been documented. However, their distribution in the Chlamydiales order and the functionality of these catalases remain unknown. Phylogeny of chlamydial catalases was inferred using MrBayes, maximum likelihood, and maximum parsimony algorithms, allowing the description of three clade 3 and two clade 2 catalases. Only monofunctional catalases were found (no catalase-peroxidase or Mn-catalase). All presented a conserved catalytic domain and tertiary structure. Enzymatic activity of cloned chlamydial catalases was assessed by measuring hydrogen peroxide degradation. The catalases are enzymatically active with different efficiencies. The catalase of Parachlamydia acanthamoebae is the least efficient of all (its catalytic activity was 2 logs lower than that of Pseudomonas aeruginosa). Based on the phylogenetic analysis, we hypothesize that an ancestral class 2 catalase probably was present in the common ancestor of all current Chlamydiales but was retained only in Criblamydia sequanensis and Neochlamydia hartmannellae. The catalases of class 3, present in Estrella lausannensis and Parachlamydia acanthamoebae, probably were acquired by lateral gene transfer from Rhizobiales, whereas for Waddlia chondrophila they likely originated from Legionellales or Actinomycetales. The acquisition of catalases on several occasions in the Chlamydiales suggests the importance of this enzyme for the bacteria in their host environment.

Arbuscular mycorrhizal fungi (Glomeromycota) harbour ancient fungal tubulin genes that resemble those of the chytrids (Chytridiomycota).

The genes encoding alpha- and beta-tubulins have been widely sampled in most major fungal phyla and they are useful tools for fungal phylogeny. Here, we report the first isolation of alpha-tubulin sequences from arbuscular mycorrhizal fungi (AMF). In parallel, AMF beta-tubulins were sampled and analysed to identify the presence of paralogs of this gene. The AMF alpha-tubulin amino acid phylogeny was congruent with the results previously reported for AMF beta-tubulins and showed that AMF tubulins group together at a basal position in the fungal clade and showed high sequence similarities with members of the Chytridiomycota. This is in contrast with phylogenies for other regions of the AMF genome. The amount and nature of substitutions are consistent with an ancient divergence of both orthologs and paralogs of AMF tubulins. At the amino acid level, however, AMF tubulins have hardly evolved from those of the chytrids. This is remarkable given that these two groups are ancient and the monophyletic Glomeromycota probably diverged from basal fungal ancestors at least 500 million years ago. The specific primers we designed for the AMF tubulins, together with the high molecular variation we found among the AMF species we analysed, make AMF tubulin sequences potentially useful for AMF identification purposes.

Genetic consequences of the ice ages on nurseries of the bat Myotis myotis: a mitochondrial and nuclear survey.

Analyses of mitochondrial DNA (mtDNA) control region polymorphism and of variation at 10 nuclear microsatellite loci were used to investigate the mechanisms and genetic consequences of postglacial expansion of Myotis myotis in Europe. Initial sampling consisted of 480 bats genotyped in 24 nursery colonies arranged along a transect of approximately 3000 km. The phylogeographical survey based on mtDNA sequences revealed the existence of major genetic subdivisions across this area, with several suture zones between haplogroups. Such zones of secondary contact were found in the Alps and Rhodopes, whereas other potential barriers to gene flow, like the Pyrenees, did not coincide with genetic discontinuities. Areas of population admixture increased locally the genetic diversity of colonies, which confounded the northward decrease in nucleotide diversity predicted using classical models of postglacial range expansion. However, when analyses were restricted to a subset of 15 nurseries originating from a single presumed glacial refugium, mtDNA polymorphism did indeed support a northwards decrease in diversity. Populations were also highly structured (PhiST = 0.384). Conversely, the same subset of colonies showed no significant latitudinal decrease in microsatellite diversity and much less population structure (FST = 0.010), but pairwise genetic differentiation at these nuclear markers was strongly correlated with increasing geographical distance. Together, this evidence suggests that alleles carried via male bats have maintained enough nuclear gene flow to counteract the effects of recurrent bottlenecks generally associated with recolonization processes. As females are highly philopatric, we argue that the maternally transmitted mtDNA marker better reflects the situation of past, historical gene flow, whereas current levels of gene flow are better reflected by microsatellite markers.

Domestic, peridomestic and wild hosts in the transmission of Trypanosoma cruzi in the Caatinga area colonised by Triatoma brasiliensis

The role played by different mammal species in the maintenance of Trypanosoma cruzi is not constant and varies in time and place. This study aimed to characterise the importance of domestic, wild and peridomestic hosts in the transmission of T. cruzi in Tauá, state of Ceará, Caatinga area, Brazil, with an emphasis on those environments colonised by Triatoma brasiliensis. Direct parasitological examinations were performed on insects and mammals, serologic tests were performed on household and outdoor mammals and multiplex polymerase chain reaction was used on wild mammals. Cytochrome b was used as a food source for wild insects. The serum prevalence in dogs was 38% (20/53), while in pigs it was 6% (2/34). The percentages of the most abundantly infected wild animals were as follows: Thrichomys laurentius 74% (83/112) and Kerodon rupestris 10% (11/112). Of the 749 triatomines collected in the household research, 49.3% (369/749) were positive for T. brasiliensis, while 6.8% were infected with T. cruzi (25/369). In captured animals, T. brasiliensis shares a natural environment with T. laurentius, K. rupestris, Didelphis albiventris, Monodelphis domestica, Galea spixii, Wiedomys pyrrhorhinos, Conepatus semistriatus and Mus musculus. In animals identified via their food source, T. brasiliensis shares a natural environment with G. spixii, K. rupestris, Capra hircus, Gallus gallus, Tropidurus oreadicus and Tupinambis merianae. The high prevalence of T. cruzi in household and peridomiciliar animals reinforces the narrow relationship between the enzootic cycle and humans in environments with T. brasiliensis and characterises it as ubiquitous.

Low rates of X-Y recombination, not turnovers, account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup).

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X-Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex-linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X-Y recombination in males. Phylogenetic analyses of sex-linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X-Y homomorphy and fine-scale sequence similarity in these species do not stem from recent sex-chromosome turnovers, but from occasional X-Y recombination.

Retrospective distribution of Trypanosoma cruzi I genotypes in Colombia

Trypanosoma cruziis the aetiological agent of Chagas disease, which affects approximately eight million people in the Americas. This parasite exhibits genetic variability, with at least six discrete typing units broadly distributed in the American continent. T. cruziI (TcI) shows remarkable genetic diversity; a genotype linked to human infections and a domestic cycle of transmission have recently been identified, hence, this strain was named TcIDom. The aim of this work was to describe the spatiotemporal distribution of TcI subpopulations across humans, insect vectors and mammalian reservoirs in Colombia by means of molecular typing targeting the spliced leader intergenic region of mini-exon gene. We analysed 101 TcI isolates and observed a distribution of sylvatic TcI in 70% and TcIDom in 30%. In humans, the ratio was sylvatic TcI in 60% and TcIDom in 40%. In mammal reservoirs, the distribution corresponded to sylvatic TcI in 96% and TcIDom in 4%. Among insect vectors, sylvatic TcI was observed in 48% and TcIDom in 52%. In conclusion, the circulation of TcIDom is emerging in Colombia and this genotype is still adapting to the domestic cycle of transmission. The epidemiological and clinical implications of these findings are discussed herein.