Molecular approaches for eco-epidemiological studies of Paracoccidioides brasiliensis

Medical mycology has greatly benefited from the introduction of molecular techniques. New knowledge on molecular genetics has provided both theoretical and practical frameworks, permitting important advances in our understanding of several aspects of pathogenic fungi. Considering Paracoccidioides brasiliensis in particular, important eco-epidemiological aspects, such as environmental distribution and new hosts were clarified through molecular approaches. These methodologies also contributed to a better understanding about the genetic variability of this pathogen; thus, P. brasiliensis is now assumed to represent a species complex. The present review focuses on some recent findings about the current taxonomic status of P. brasiliensis, its phylogenetic and speciation processes, as well as on some practical applications for the molecular detection of this pathogen in environmental and clinical materials.

A morphologically distinct Phlebotomus argentipes population from active cutaneous leishmaniasis foci in central Sri Lanka

Although the reported aetiological agent of cutaneous leishmaniasis (CL) in Sri Lanka is Leishmania donovani, the sandfly vector remains unknown. Ninety-five sandflies, 60 females and 35 males, collected in six localities in the district of Matale, central Sri Lanka, close to current active transmission foci of CL were examined for taxonomically relevant characteristics. Eleven diagnostic morphological characters for female sandflies were compared with measurements described for Indian and Sri Lankan sandflies, including the now recognised Phlebotomus argentipes sensu lato species complex. The mean morphometric measurements of collected female sandflies differed significantly from published values for P. argentipes morphospecies B, now re-identified as Phlebotomus annandalei from Delft Island and northern Sri Lanka, from recently re-identified P. argentipes s.s. sibling species and from Phlebotomus glaucus. Furthermore, analysis of underlying variation in the morphometric data through principal component analysis also illustrated differences between the population described herein and previously recognised members of the P. argentipes species complex. Collectively, these results suggest that a morphologically distinct population, perhaps most closely related to P. glaucus of the P. argentipess. I. species complex, exists in areas of active CL transmission. Thus, research is required to determine the ability of this population of flies to transmit cutaneous leishmaniasis.

Biogeographical aspects of the occurrence of Nyssomyia neivai and Nyssomyia intermedia (Diptera: Psychodidae) in a sympatric area of the Brazilian savannah

Nyssomyia intermedia and Nyssomyia neivai constitute a species complex associated with Leishmania transmission. The aim of this study was to analyse the ecological profiles of the Ny. intermedia and Ny. neivai populations in a sympatric area in the Brazilian savannah along the banks of the Velhas River. Captures were performed from July 2003-June 2005 in two distinct environments: a gallery forest with various degrees of anthropogenic modification and animal shelters. A total of 20,508 Ny. neivai (86%) and Ny. intermedia (14%) sandflies were collected. The difference between the proportions of the sandflies that were collected (Ny. neivai/Ny. intermedia) per bank was significant. The right bank presented a greater number of sandflies (65%) and more preserved vegetation. The abundance of Ny. neivai was higher than that of Ny. intermedia on both banks. The results demonstrate that anthropic activities can affect the sandfly populations in this area, thereby leading to a reduction in species abundance. Nevertheless, the environments with higher levels of antropogenic modification displayed sandfly population numbers that favour the Leishmania transmission cycle.

Rickettsia amblyommii infecting Amblyomma sculptum in endemic spotted fever area from southeastern Brazil

The Rickettsia bacteria include the aetiological agents for the human spotted fever (SF) disease. In the present study, a SF groupRickettsia amblyommii related bacterium was detected in a field collected Amblyomma sculptum (Amblyomma cajennense species complex) tick from a Brazilian SF endemic site in southeastern Brazil, in the municipality of Juiz de Fora, state of Minas Gerais. Genetic analysis based on genes ompA,ompB and htrA showed that the detected strain, named R. amblyommii str. JF, is related to the speciesR. amblyommii.

Is the Gibraltar strait a barrier to gene flow for the bat Myotis myotis (Chiroptera: Vespertilionidae)?

Because of their role in limiting gene flow, geographical barriers like mountains or seas often coincide with intraspecific genetic discontinuities. Although the Strait of Gibraltar represents such a potential barrier for both plants and animals, few studies have been conducted on its impact on gene flow. Here we test this effect on a bat species (Myotis myotis) which is apparently distributed on both sides of the strait. Six colonies of 20 Myotis myotis each were sampled in southern Spain and northern Morocco along a linear transect of 1350 km. Results based on six nuclear microsatellite loci reveal no significant population structure within regions, but a complete isolation between bats sampled on each side of the strait. Variability at 600 bp of a mitochondrial gene (cytochrome b) confirms the existence of two genetically distinct and perfectly segregating clades, which diverged several million years ago. Despite the narrowness of the Gibraltar Strait (14 km), these molecular data suggest that neither males, nor females from either region have ever reproduced on the opposite side of the strait. Comparisons of molecular divergence with bats from a closely related species (M. blythii) suggest that the North African clade is possibly a distinct taxon warranting full species rank. We provisionally refer to it as Myotis cf punicus Felten 1977, but a definitive systematic understanding of the whole Mouse-eared bat species complex awaits further genetic sampling, especially in the Eastern Mediterranean areas.

The effect of recombination in Aeromonas

Although several approaches have been attempted, the estimation of recombination frequencies in natural populations ofbacteria remains challenging. Previous studies have demonstrated awide variety of situations among bacterial species, ranging from theclonal diversification of Salmonella or Escherichia coli, which aremainly due to mutation, to the frequent recombination found inNeisseria gonorrhoeae or Helicobacter pylori. Most of the populationstudies done with bacterial species suggest that recombination occursin nature but that it is infrequent compared to mutation. Consequently,bacterial populations consist largely of independent clonal lineages.Our research suggests little or null influence of recombination in thegenetic structure of "Aeromonas hydrophila Species Complex", despite the presence of some strains with recombinant gene fragments.

Evolución de la agregación y separación de sexos: ¿Qué hemos aprendido de las poblaciones ibéricas de Mercurialis annua?

Most plant species are hermaphrodites, with both male and female functions performed by the same individuals. However, separate sexes (dioecy) have evolved on numerous independent occasions, probably either in response to selection for inbreeding avoidance, or because it pays individuals to specialize in one gender or the other. Although the evolution of dioecy from hermaphroditism tends to be thought of as a one-way path, dioecy has broken down to yield hermaphroditic populations on several occasions. One such case is found in the mainly dioecious genus Mercurialis (Euphorbiaceae). In the species complex M. annua, diploids are dioecious, but polyploid populations are variously monoecious or androdioecious (where males co-exist with functional hermaphrodites). This species complex offers rich material for addressing questions concerning the evolution and ecology of combined versus separate sexes, the evolution of secondary sexual dimorphism, which likely contributes to the stability of dioecy in the genus, and the evolution and genetics of sex determination and sex chromosomes. The species also offers itself as a valuable teaching tool for addressing topics ranging from sex-ratio selection to inter-sexual competition.

The effect of recombination in Aeromonas

Although several approaches have been attempted, the estimation of recombination frequencies in natural populations ofbacteria remains challenging. Previous studies have demonstrated awide variety of situations among bacterial species, ranging from theclonal diversification of Salmonella or Escherichia coli, which aremainly due to mutation, to the frequent recombination found inNeisseria gonorrhoeae or Helicobacter pylori. Most of the populationstudies done with bacterial species suggest that recombination occursin nature but that it is infrequent compared to mutation. Consequently,bacterial populations consist largely of independent clonal lineages.Our research suggests little or null influence of recombination in thegenetic structure of "Aeromonas hydrophila Species Complex", despite the presence of some strains with recombinant gene fragments.

The phylogeography of an alpine leaf beetle: divergence within Oreina elongata spans several ice ages.

The genetic landscape of the European flora and fauna was shaped by the ebb and flow of populations with the shifting ice during Quaternary climate cycles. While this has been well demonstrated for lowland species, less is known about high altitude taxa. Here we analyze the phylogeography of the leaf beetle Oreina elongata from 20 populations across the Alps and Apennines. Three mitochondrial and one nuclear region were sequenced in 64 individuals. Within an mtDNA phylogeny, three of seven subspecies are monophyletic. The species is chemically defended and aposematic, with green and blue forms showing geographic variation and unexpected within-population polymorphism. These warning colors show pronounced east-west geographical structure in distribution, but the phylogeography suggests repeated origin and loss. Basal clades come from the central Alps. Ancestors of other clades probably survived across northern Italy and the northern Adriatic, before separation of eastern, southern and western populations and rapid spread through the western Alps. After reviewing calibrated gene-specific substitution rates in the literature, we use partitioned Bayesian coalescent analysis to date our phylogeography. The major clades diverged long before the last glacial maximum, suggesting that O. elongata persisted many glacial cycles within or at the edges of the Alps and Apennines. When analyzing additional barcoding pairwise distances, we find strong evidence to consider O. elongata as a species complex rather than a single species.

Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi.

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Molecular genetic differentiation in earthworms inhabiting a heterogeneous Pb-polluted landscape

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages ('A' and 'B'), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central 'ecological island' site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in ESTPb libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies.

Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens

Background: Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. Results: Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. Conclusions: P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

The impact of genome defense on mobile elements in Microbotryum

Repeat induced point mutation (RIP), a mechanism causing hypermutation of repetitive DNA sequences in fungi, has been described as a ‘genome defense’ which functions to inactivate mobile elements and inhibit their deleterious effects on genome stability. Here we address the interactions between RIP and transposable elements in the Microbotryum violaceum species complex. Ten strains of M. violaceum, most of which belong to different species of the fungus, were all found to contain intragenomic populations of copia-like retrotransposons. Intragenomic DNA sequence variation among the copia-like elements was analyzed for evidence of RIP. Among species with RIP, there was no significant correlation between the frequency of RIP-induced mutations and inferred transposition rate based on diversity. Two strains of M. violaceum, from two different plant species but belonging to the same fungal lineage, contained copia-like elements with very low diversity, as would result from a high transposition rate, and these were also unique in showing no evidence of the hypermutation patterns indicative of the RIP genome defense. In this species, evidence of RIP was also absent from a Class II helitron-like transposable element. However, unexpectedly the absolute repetitive element load was lower than in other strains.

Diversity in a honey bee pathogen: first report of a third master variant of the Deformed Wing Virus quasispecies

Treatment of emerging RNA viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies. Declining honey bee populations have been attributed to the ectoparasitic mite Varroa destructor and its affiliation with Deformed Wing Virus (DWV). In the current study we use next-generation sequencing to investigate the DWV quasispecies in an apiary known to suffer from overwintering colony losses. We show that the DWV species complex is made up of three master variants. Our results indicate that a new DWV Type C variant is distinct from the previously described types A and B, but together they form a distinct clade compared with other members of the Iflaviridae. The molecular clock estimation predicts that Type C diverged from the other variants ~319 years ago. The discovery of a new master variant of DWV has important implications for the positive identification of the true pathogen within global honey bee populations.