Preliminary results of random amplification of polymorphic DNA among Triatominae of the phyllosoma complex (Hemiptera, Reduviidae)

In Mexico, Triatoma longipennis (Usinger), Triatoma picturata (Usinger), and Triatoma pallidipennis (Stal), primary Chagas disease vector species of the phyllosoma complex, were analyzed by randomly amplified polymorphic DNA (RAPD). Sixteen decametric primers resolved individual profiles not identical, but partially discriminative between species. Analysis based on pairwise presence/absence comparisons between the three species was performed using three primers and two outgroup species Triatoma infestans (Klug) and Triatoma barberi (Usinger). Fifty-three bands in total were scored, although only two bands were constant among the three phyllosoma complex species. Two other bands were constant only for T. longipennis and T. picturata together, and not present in T. pallidipennis. Neighbor Joining tree and the multiple correspondence analysis discriminated T. pallidipennis clearly from the other two species, although there was overlap between T. longipennis and T. picturata. The results indicate a close relationship between the studied species and support the hypothesis of their recent evolution. The suitability of RAPD to discern populations within the species is discussed.

Triatoma bassolsae sp. n. from Mexico with a key to species of "phyllosoma" complex

According to the descriptions of five closely related species of the genus Triatoma Laporte, 1832: T. phyllosoma (Burmeister, 1835), T. pallidipennis (Stal, 1872), T. picturata Usinger, 1939, T. longipennis Usinger, 1939 and T. mazzottii Usinger, 1941 and further published studies, these species could be included in a "specific complex" named as the species formerly described. All these species are typical from Mexico and another species was found in the same country, in the State of Puebla: Triatoma bassolsae sp. n. This species was morphologically compared with the other five of the "phyllosoma" complex, including the external male genitalia. The most important characters used to separate T. bassolsae from T. phyllosoma (which is the most similar to the other species) are the morphometric relationships on the head, with a longer anteocular region and a significant longer second rostral segment, a long and conspicuous pilosity in different areas of the body and specially on the head, and the characters of the anterolateral, lateral and discal tubercles of the pronotum, very long and sharp in the new species. The male genitalia has several differences between T. bassolsae and T. phyllosoma specially significant on the surface of the endosome process and on the branches of the phallosome support, separated at the apex in the new species. Types and paratypes are incorporated in the respective institutions in Mexico DF and Rio de Janeiro.

Biological and genetic aspects of experimental hybrids from species of the Phyllosoma complex (Hemiptera: Reduviidae: Triatominae)

The present work is a thorough investigation of the degree of reproductive isolation between Meccus mazzottii and Meccus longipennis, Meccus picturatus, Meccus pallidipennis and Meccus bassolsae, as well as between M. longipennis and M. picturatus. We examined fertility and segregation of morphological characteristics in two generations of hybrids derived from crosses between these species. The percentage of pairs with (fertile) offspring was highest in the set of crosses between M. longipennis and M. picturatus, and lowest between M. mazzottii and M. picturatus. Most first-generation (F1) individuals from crosses involving M. mazzottii were morphologically similar to this species, while only F1 x F1 progeny of parental crosses between M. mazzottii and M. longipennis had offspring second generation that looked like M. mazzottii. The results indicate that different degrees of reproductive isolation apparently exist among the species of the Phyllosoma complex examined in this study. The biological evidence obtained in this study does not support the proposal that M. longipennis and M. picturatus are full species. It could indicate on the contrary, that both could be considered as subspecies of a single polytypic species. On the other hand, biological evidence supports the proposal that M. mazzottii is a full species.

Occurrence of hybrids and laboratory evidence of fertility among three species of the Phyllosoma complex (Hemiptera: Reduviidae) in Mexico

In seven studied communities of Western Mexico, triatomine specimens were sympatrically collected, some with atypical morphological characteristics in contrast to pure specimens, which were presumed to be hybrids. More than 200 specimens of Meccus pallidipennis and Meccus longipennis with brown-yellow markings on dorsal connexival segments were collected in Ahuacapán and Quitupan. In La Mesa, more than 60 specimens similar to Meccus picturatus in most morphological characteristics (including size) were collected, although they presented a largely yellowish corium like M. pallidipennis. Interfertility was proven between all of the studied wild hybrid specimens, as well as between all the experimental laboratory hybrids. Two different phenotypes (M. picturatus and M. longipennis) were obtained from crosses between M. picturatus x M. picturatus and M. longipennis x M. longipennis from the three studied localities in state of Nayarit as from La Mesita. Results support the hypothesis that the subspecific ranking of those triatomines may, therefore, be more appropriate because reproductive isolation has not been developed and complete interbreeding was recorded.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

AbstractINTRODUCTION:Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.METHODS:Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.RESULTS:Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.CONCLUSIONS:We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Identification of Anopheles (Nyssorhynchus) albitarsis complex species (Diptera: Culicidae) using rDNA internal transcribed spacer 2-based polymerase chain reaction primes

Anopheles (Nyssorhynchus) marajoara is a proven primary vector of malaria parasites in Northeast Brazil, and An. deaneorum is a suspected vector in Western Brazil. Both are members of the morphologically similar Albitarsis Complex, which also includes An. albitarsis and an undescribed species, An. albitarsis "B". These four species were recognized and can be identified using random amplified polymorphic DNA (RAPD) markers, but various other methodologies also point to multiple species under the name An. albitarsis. We describe here a technique for identification of these species employing polymerase chain reaction (PCR) primers based on ribosomal DNA internal transcribed spacer 2 (rDNA ITS2) sequence. Since this method is based on known sequence it is simpler than the sometimes problematical RAPD-PCR. Primers were tested on samples previously identified using RAPD markers with complete correlation.

Biological and genetic aspects of crosses between species of the genus Meccus (Hemiptera: Reduviidae Triatominae)

The degree of reproductive isolation between Meccus phyllosomus and the remaining five species of the genus Meccus, as well as between Meccus bassolsae and Meccus pallidipennis, Meccus longipennis and Meccus picturatus, was examined. Fertility and the segregation of morphological characteristics were examined in two generations of hybrids from crosses between these species. The percentage of couples with offspring (fertile) was high in the vast majority of sets of crosses, with the exception of that between ♀M. phyllosomus and ♂Meccus mazzottii. In sets of crosses involving M. bassolsae specimens, no first-generation (F1) individuals were morphologically similar to M. bassolsae, but instead shared the morphology of the other parental species. A similar phenomenon was observed in most sets of crosses involving M. phyllosomus. These results indicated that different degrees of reproductive isolation exist among the species of Meccus involved in this study. The biological evidence obtained in this study does not support the proposal that M. bassolsae is a full species. It could indicate that, on the contrary, it should be considered a subspecies of a single polytypic species. The biological evidence does support the proposal that M. phyllosomus is a full species.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Structure Variation And Dynamics of the Nuclear Ribosomal Intergenic Spacer Region in Key Gibberella Fujikuroi Complex Species

The intergenic spacer (IGS) region of the ribosomal DNA was cloned and sequenced in eight species within the Gibberella fujikuroi species complex with anamorphs in the genus Fusarium , a group that includes the most relevant toxigenic species. DNA sequence analyses revealed two categories of repeated elements: long repeats and short repeats of 125 and 8 bp, respectively. Long repeats were present in two copies and were conserved in all the species analyzed, whereas different numbers of short repeat elements were observed, leading to species-specific IGS sequences with different length. In Fusarium subglutinans and Fusarium nygamai , these differences seemed to be the result of duplication and deletion events. Here, we propose a model based on unequal crossing over that can explain these processes. The partial IGS sequence of 22 Fusarium proliferatum isolates was also obtained to study variation at the intraspecific level. The results revealed no differences in terms of number or pattern of repeated elements and detected frequent gene conversion events. These results suggest that the homogenization observed at the intraspecific level might not be achieved primarily by unequal crossing-over events but rather by processes associated with recombination such as gene conversion events.

Environmental niche variation and evolutionary diversification of the Brachypodium distachyon grass complex species in their native circum-Mediterranean range

PREMISE OF THE STUDY: We conducted environmental niche modeling (ENM) of the Brachypodium distachyon s.l. complex, a model group of two diploid annual grasses ( B. distachyon , B. stacei ) and their derived allotetraploid ( B. hybridum) , native to the circum-Mediterranean region. We (1) investigated the ENMs of the three species in their native range based on present and past climate data; (2) identifi ed potential overlapping niches of the diploids and their hybrid across four Quaternary windows; (3) tested whether speciation was associated with niche divergence/conservatism in the complex species; and (4) tested for the potential of the polyploid outperforming the diploids in the native range. M ETHODS: Geo-referenced data, altitude, and 19 climatic variables were used to construct the ENMs. We used paleoclimate niche models to trace the potential existence of ancestral gene fl ow among the hybridizing species of the complex. KEY RESULTS: Brachypodium distachyon grows in higher, cooler, and wetter places, B. stacei in lower, warmer, and drier places, and B. hybridum in places with intermediate climatic features. Brachypodium hybridum had the largest niche overlap with its parent niches, but a similar distribution range and niche breadth. C ONCLUSIONS: Each species had a unique environmental niche though there were multiple niche overlapping areas for the diploids across time, suggesting the potential existence of several hybrid zones during the Pleistocene and the Holocene. No evidence of niche divergence was found, suggesting that species diversifi cation was not driven by ecological speciation but by evolutionary history, though it could be associated to distinct environmental adaptations.

Polyphasic characterisation of Burkholderia cepacia complex species isolated from children with cystic fibrosis

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recA sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recA sequencing to identify Bcc species. The recA sequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%), Burkholderia vietnamiensis (30.6%), B. cenocepacia IIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis : C. parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis . In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Analysis of inteins in the Candida parapsilosis complex for simple and accurate species identification

Inteins are coding sequences that are transcribed and translated with flanking sequences and then are excised by an autocatalytic process. There are two types of inteins in fungi, mini-inteins and full-length inteins, both of which present a splicing domain containing well-conserved amino acid sequences. Full-length inteins also present a homing endonuclease domain that makes the intein a mobile genetic element. These parasitic genetic elements are located in highly conserved genes and may allow for the differentiation of closely related species of the Candida parapsilosis (psilosis) complex. The correct identification of the three psilosis complex species C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis is very important in the clinical setting for improving antifungal therapy and patient care. In this work, we analyzed inteins that are present in the vacuolar ATPase gene VMA and in the threonyl-tRNA synthetase gene ThrRS in 85 strains of the Candida psilosis complex (46 C. parapsilosis, 17 C. metapsilosis, and 22 C. orthopsilosis). Here, we describe an accessible and accurate technique based on a single PCR that is able to differentiate the psilosis complex based on the VMA intein. Although the ThrRS intein does not distinguish the three species of the psilosis complex by PCR product size, it can differentiate them by sequencing and phylogenetic analysis. Furthermore, this intein is unusually present as both mini- and full-length forms in C. orthopsilosis. Additional population studies should be performed to address whether this represents a common intraspecific variability or the presence of subspecies within C. orthopsilosis. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

FREQUENCY OF Candida SPECIES IN A TERTIARY CARE HOSPITAL IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL

Infections by Candida species are a high-impact problem in public health due to their wide incidence in hospitalized patients. The goal of this study was to evaluate frequency, susceptibility to antifungals, and genetic polymorphism of Candida species isolated from clinical specimens of hospitalized patients. The Candida isolates included in this study were obtained from blood cultures, abdominal fluids, and central venous catheters (CVC) of hospitalized patients at the Clinical Hospital of the Federal University of Uberlândia during the period of July 2010 - June 2011. Susceptibility tests were conducted by the broth microdilution method. The RAPD-PCR tests used employed initiator oligonucleotides OPA09, OPB11, and OPE06. Of the 63 Candida isolates, 18 (28.5%) were C. albicans, 20 (31.7%) were C. parapsilosis complex species, 14 (22.2%) C. tropicalis, four (6.4%) C. glabrata, four (6.4%) C. krusei, two (3.3%) C. kefyr, and one (1.6%) C. lusitaniae. In vitro resistance to amphotericin B was observed in 12.7% of isolates. In vitroresistance to azoles was not detected, except for C. krusei. The two primers, OPA09 and OPB11, were able to distinguish different species. Isolates of C. albicans and C. parapsilosis complex species presented six and five clusters, respectively, with the OPA09 marker by RAPD-PCR, showing the genetic variability of the isolates of those species. It was concluded that members of the C. parapsilosis complex were the most frequent species found, and most isolates were susceptible to the antifungals amphotericin B, flucozanole, and itraconazole. High genetic polymorphisms were observed for isolates of C. albicans and C. parapsilosis complex species, mainly with the OPA09 marker.

Lutzomyia reducta Feliciangeli et al., 1988, a host of Leishmania amazonensis, sympatric with two other members of the Flaviscutellata complex in southern Amazonas and Rondônia, Brazil (Diptera: Psychodidae)

A member of the Lutzomyia flaviscutellata complex from Rondônia and southern Amazonas States, Brazil, is so close to the Venezuelan Lutzomyia olmeca recuta Feliciangeli et al., 1988, that it is regarded as belonging to the same species. Since this phlebotomine co-extis with L. olmeca nociva in Brazil, the subspecific status of the former is untenable and is rased to specific rank, as Lutzomyia reducta. The Brazilian material is described and illustrated, and compared with specimens of L. o. nociva and L. flaviscutellata from the same area. Keys to the known taxa of the flaviscutellata complex are presented. Leishmania amazonensis was isolated from one heavily infected specimen of L. reducta, making this the third species of the flaviscutellata complex to be implicated as a vector of this parasite in Brazil. The relative abundance of the three sympatric flaviscutellata complex species varies locally and appears to be related to soil drainage. L. reducta constituted about 25% if all phlebotomines captured in Disney traps at poorly drained and well drained site, but appears not to coloniza areas subject to periodic flooding. L. olmeca nociva was restricted to poorly drained areas not subject to flooding, whereas L. flaviscutellata was ubiquitous L. reducta has never been detected north of the Amazon river in Brazil, but absence of recosrds from western and northwestern Amazonas State may reflect lack of collecting in these areas.