Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene

Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene. To analyze the genetic variability of populations of Aedes aegypti, 156 samples were collected from 10 municipalities in the state of Paraná, Brazil. A 311 base pairs (bp) region of the NADH dehydrogenase subunit 4 (ND4) mitochondrial gene was examined. An analysis of this fragment identified eight distinct haplotypes. The mean genetic diversity was high (h = 0.702; p = 0.01556). AMOVA analysis indicated that most of the variation (67%) occurred within populations and the F ST value (0.32996) was highly significant. F ST values were significant in most comparisons among cities. The isolation by distance was not significant (r = -0.1216 and p = 0, 7550), indicating that genetic distance is not related to geographic distance. Neighbor-joining analysis showed two genetically distinct groups within Paraná. The DNA polymorphism and AMOVA data indicate a decreased gene flow in populations from Paraná, which can result in increased vectorial competence.

Gene flow networks among American Aedes aegypti populations

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Population structure of the malaria vector Anopheles darlingi in Rondônia, Brazilian Amazon, based on mitochondrial DNA

Anopheles darlingi is the most important Brazilian malaria vector, with a widespread distribution in the Amazon forest. Effective strategies for vector control could be better developed through knowledge of its genetic structure and gene flow among populations, to assess the vector diversity and competence in transmitting Plasmodium. The aim of this study was to assess the genetic diversity of An. darlingi collected at four locations in Porto Velho, by sequencing a fragment of the ND4 mitochondrial gene. From 218 individual mosquitoes, we obtained 20 different haplotypes with a diversity index of 0.756, equivalent to that found in other neotropical anophelines. The analysis did not demonstrate significant population structure. However, haplotype diversity within some populations seems to be over-represented, suggesting the presence of sub-populations, but the presence of highly represented haplotypes complicates this analysis. There was no clear correlation among genetic and geographical distance and there were differences in relation to seasonality, which is important for malarial epidemiology.

Population structure of the malaria vector Anopheles darlingi in Rondônia, Brazilian Amazon, based on mitochondrial DNA

Anopheles darlingi is the most important Brazilian malaria vector, with a widespread distribution in the Amazon forest. Effective strategies for vector control could be better developed through knowledge of its genetic structure and gene flow among populations, to assess the vector diversity and competence in transmitting Plasmodium. The aim of this study was to assess the genetic diversity of An. darlingi collected at four locations in Porto Velho, by sequencing a fragment of the ND4 mitochondrial gene. From 218 individual mosquitoes, we obtained 20 different haplotypes with a diversity index of 0.756, equivalent to that found in other neotropical anophelines. The analysis did not demonstrate significant population structure. However, haplotype diversity within some populations seems to be over-represented, suggesting the presence of sub-populations, but the presence of highly represented haplotypes complicates this analysis. There was no clear correlation among genetic and geographical distance and there were differences in relation to seasonality, which is important for malarial epidemiology.

A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis

Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species-specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real-time high-resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species-specific genetic mutations that result in PCR products with unique melt profiles. A real-time HRM PCR species-diagnostic assay (RT-HRM-PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.

Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) rDNA and partial sequences of the cytochrome coxidase subunit I (COI) mtDNA and white gene nDNA were obtained from specimens of Anopheles nuneztovari A collected in Macapá (state of Amapá), Óbidos, Prainha and Almeirim (state of Pará), Itacoatiara and Parintins (state of Amazonas), Brazil, and compared with previously published sequences of A. nuneztovari s.l. Results of the Bayesian phylogenetic analyses performed using either COI or combined ITS2, COI and white gene sequences suggest that An. nuneztovari B/C is distinct from specimens obtained in the Amazonas/Solimões River basin. Anopheles goeldii, currently in synonymy with An. nuneztovari, was described from individuals collected in Belterra (= Fordlândia) in the Tapajós River, state of Pará, Southern Amazonas River. Morphological comparisons of the characteristics of the male genitalia indicated that An. nuneztovari A and An. goeldii are similar but distinct from An. nuneztovariB/C by the apex of the aedeagus. In considering the results of the phylogenetic analyses and morphological comparisons, An. goeldii is resurrected from synonymy with An. nuneztovari. Additionally, Anopheles dunhamiis reported for the first time in Parintins. This species can be distinguished from An. goeldiiby characters of the male genitalia and molecular data

Comparative phylogeography of two open forest frogs from eastern Australia

We investigated the phylogeography of two closely related Australian frog species from open forest habitats, Limnodynastes tasmaniensis and L. peronii, using mitochondrial ND4 sequence data. Comparison of our results with previous work on Litoria fallax allowed us to test the generality of phylogeographic patterns among non-rainforest anurans along the east coast of Australia. In general, there was no strong evidence for congruence between overall patterns of genetic structure in the three species. However, phylogenetic breaks congruent with the position of the Burdekin Gap were detected at some level in all species. As previously noted for closed forest taxa, this area of dry habitat appears to have been an important influence on the evolution of several open forest taxa. There were broad geographic similarities in the phylogenetic structuring of southern populations of L. peronii and L. tasmaniensis. Contrarily, although the McPherson Range has previously been noted to coincide geographically with a major mtDNA phylogenetic break in Litoria fallax this pattern is not apparent in L. peronii or L. tasmaniensis. It appears that major phylogeographic splits within L. peronii and L. tasmaniensis may predate the Quaternary. We conclude that phylogeographies of open forest frogs are complex and more difficult to predict than for rainforest taxa, mainly due to an absence of palaeomodels for historical distributions of non-rainforest habitats. (C) 2001 The Linnean Society of London.

Moluscos hospedeiros intermediários de tremátodes: Estudo molecular de helisoma sp. (Gastropoda: planorbidae) de diferentes áreas geográficas

Ao longo dos tempos a caracterização das diferentes espécies da classe Gastropoda baseava-se apenas em características fenotípicas (morfologia da concha e partes moles), as quais eram insuficientes para distinguir espécies e subespécies. Assim, a caracterização genética desenvolvida nos últimos anos, tem-se mostrado uma boa ferramenta aplicada á diferenciação molecular de espécies, permitindo uma melhor compreensão sobre moluscos com um importante papel como hospedeiros intermediários de tremátodes e qual a sua posição dentro da família Planorbidae. Os objectivos deste trabalho foram, por um lado fazer um estudo comparativo de populações de Helisoma sp., de Portugal e Cabo Verde, baseado num estudo molecular utilizando marcadores moleculares, nomeadamente o gene COI do DNA mitocondrial (mtDNA) e o gene 16S do RNA ribossomal (rRNA) e a região interna transcrita (ITS) do DNA ribossomal, e recorrendo à técnica PCR-RFLP, direccionada para a região ITS para a identificação de possíveis polimorfismos e, por outro lado estabelecer uma relação filogenética entre as populações portuguesas e cabo verdianas de Helisoma e outros planorbideos, hospedeiros intermediários de tremátodes. Os resultados obtidos, para os genes em análise permitiram a identificação de três regiões distintas: Cabo Verde, Madeira e Portugal Continental, esta última formada pelas amostras de Algarve e Coimbra, apesar da distante geográfica que separa cada umas destas duas áreas. Os resultados obtidos para os genes COI e 16S e para a região ITS, mostraram uma elevada homologia com as espécies Helisoma trivolvis e H. duryi.

Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) rDNA and partial sequences of the cytochrome coxidase subunit I (COI) mtDNA and white gene nDNA were obtained from specimens of Anopheles nuneztovari A collected in Macapá (state of Amapá), Óbidos, Prainha and Almeirim (state of Pará), Itacoatiara and Parintins (state of Amazonas), Brazil, and compared with previously published sequences of A. nuneztovari s.l. Results of the Bayesian phylogenetic analyses performed using either COI or combined ITS2, COI and white gene sequences suggest that An. nuneztovari B/C is distinct from specimens obtained in the Amazonas/Solimões River basin. Anopheles goeldii, currently in synonymy with An. nuneztovari, was described from individuals collected in Belterra (= Fordlândia) in the Tapajós River, state of Pará, Southern Amazonas River. Morphological comparisons of the characteristics of the male genitalia indicated that An. nuneztovari A and An. goeldii are similar but distinct from An. nuneztovariB/C by the apex of the aedeagus. In considering the results of the phylogenetic analyses and morphological comparisons, An. goeldii is resurrected from synonymy with An. nuneztovari. Additionally, Anopheles dunhamiis reported for the first time in Parintins. This species can be distinguished from An. goeldiiby characters of the male genitalia and molecular data.

Mitochondrial DNA mutations induce mitochondrial dysfunction, apoptosis and sarcopenia in skeletal muscle of mitochondrial DNA mutator mice

Background: Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings: We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase c, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Dym). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance: These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

Population dynamics, structure and behavior of Anopheles darlingi in a rural settlement in the Amazon rainforest of Acre, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differentiation and species status of the Neotropical yellow-eared bats Vampyressa pusilla and V. thyone (Phyllostomidae) with a molecular phylogeny and review of the genus

A systematic re-evaluation of Vampyressa pusilla warrants the elevation of V. p. thyone from subspecies to species rank based on its distinction from the allopatric V. p. pusilla. Morphological, mensural, chromosomal, and mitochondrial differences define each of these two taxa as divergent lineages. Vampyressa pusilla is endemic to the Atlantic Forest of southeastern South America and V. thyone is found allopatrically in northwestern South America, Central America, and southern Mexico. A molecular phylogenetic analysis of the mtDNA ND3-4 gene region using restriction endonuclease cut sites resulted in a monophyletic, although weakly supported Vampyressa ingroup with Chiroderma, and a clade of Mesophylla and Ectophylla as successive basal outgroup lineages. The phylogeny within Vampyressa, with the exception of V. melissa which is most similar to V. thyone based on karyotypes and morphology, had a topology of ((pusilla + thyone) + ((brocki + nymphaea) + bidens))).

Evolution and historical biogeography of the Eastern Atlantic skates

In my thesis, I tested the hypothesis that the diversification of the Eastern Atlantic skate faunas arose through vicariance rather than dispersal, using combined approach of molecular phylogeny reconstruction and zoogeography (namely historical biogeography). This analyses have been carried out independently on four Rajidae genera belonging to two different tribes: Rajini (Raja and Dipturus) and Amblyrajini (Rajella and Leucoraja). These taxa were selected because they displayed high species diversity and richness of endemic species in the Eastern Atlantic and Mediterranean. The verification of this hypothesis was carried out by reconstructing the best phylogenetic relationships among four genera and 26 species (including several endemism) based on mtDNA and nuDNA gene variation and several statistical approaches. Divergence times of taxa have been estimated based on molecular clock and fossil calibration to explain evolutionary patterns in the context of geological framework. Main issues are (i) the evidence that Eastern Atlantic skate evolution and displacement of species diversity occurred from pulsed geographical speciation (i.e. repeated series of parallel and independent speciation events) started in the Late Eocene-Early Miocene and they have occurred prevalently during Miocene; (ii) such relatively ancient origin of diversification has been allowed the sympatric displacement and evolution of several congeneric taxa likely because they have accumulated huge differences in the genomic and physiological/behavioural phenotypic traits; (iii) recently diverged sister species and taxa showed allopatric or parapatric evolution by the presence of oceanographic or hydrogeographical barriers which likely prevent large mixing between parapatric sister species.

Pseudomonas syringae Hrp type III secretion system and effector proteins

Pseudomonas syringae is a member of an important group of Gram-negative bacterial pathogens of plants and animals that depend on a type III secretion system to inject virulence effector proteins into host cells. In P. syringae, hrp/hrc genes encode the Hrp (type III secretion) system, and avirulence (avr) and Hrp-dependent outer protein (hop) genes encode effector proteins. The hrp/hrc genes of P. syringae pv syringae 61, P. syringae pv syringae B728a, and P. syringae pv tomato DC3000 are flanked by an exchangeable effector locus and a conserved effector locus in a tripartite mosaic Hrp pathogenicity island (Pai) that is linked to a tRNALeu gene found also in Pseudomonas aeruginosa but without linkage to Hrp system genes. Cosmid pHIR11 carries a portion of the strain 61 Hrp pathogenicity island that is sufficient to direct Escherichia coli and Pseudomonas fluorescens to inject HopPsyA into tobacco cells, thereby eliciting a hypersensitive response normally triggered only by plant pathogens. Large deletions in strain DC3000 revealed that the conserved effector locus is essential for pathogenicity but the exchangeable effector locus has only a minor role in growth in tomato. P. syringae secretes HopPsyA and AvrPto in culture in a Hrp-dependent manner at pH and temperature conditions associated with pathogenesis. AvrPto is also secreted by Yersinia enterocolitica. The secretion of AvrPto depends on the first 15 codons, which are also sufficient to direct the secretion of an Npt reporter from Y. enterocolitica, indicating that a universal targeting signal is recognized by the type III secretion systems of both plant and animal pathogens.

Gene flow in great bustard populations across the Strait of Gibraltar as elucidated from excremental PCR and mtDNA sequencing

Recent advances in molecular biology have made it possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. Here we use faeces as a source of DNA and mtDNA sequence data to elucidate the relationship among Spanish and Moroccan populations of great bustards. 834 bp of combined control region and cytochrome-b mtDNA fragments revealed four variable sites that defined seven closely related haplotypes in 54 individuals. Morocco was fixed for a single mtDNA haplotype that occurs at moderate frequency (28%) in Spain. We could not differentiate among the sampled Spanish populations of Caceres and Andalucia but these combined populations were differentiated from the Moroccan population. Estimates of gene flow (Nm = 0.82) are consistent with extensive observations on the southern Iberian peninsular indicating that few individuals fly across the Strait of Gibraltar. We demonstrate that both this sea barrier and mountain barriers in Spain limit dispersal among adjacent great bustard populations to a similar extent. The Moroccan population is of high ornithological significance as it holds the only population of great bustards in Africa. This population is critically small and genetic and observational data indicate that it is unlikely to be recolonised via immigration from Spain should it be extirpated. In light of the evidence presented here it deserves the maximum level of protection.