Microsatellite markers for population genetic studies of the blowfly Chrysomya putoria (Diptera: Calliphoridae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The investigation of the genetic variation and population structure of Chrysomya species is of great interest for both basic and applied research. However, very limited genetic information is available for this genus across its geographical distribution. Here, we describe 12 polymorphic microsatellite loci isolated from Chrysomya putoria with expected heterozygosities ranging from 0.1402-0.8312. These markers are of potential applied interest for forensic entomologists and for the characterisation of the genetic structure of C. putoria from recently colonised regions, with great promise for understanding the colonisation dynamics and spread of the genus Chrysomya in the New World.104710471050Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [03/01458-9]CNPq [471132/01-2

Evolution and structural organisation of mitochondrial DNA control region of myiasis-causing flies

This study reports the molecular characterization of the mtDNA control region (called the A + T-rich region in insects) of five dipteran species which cause myiasis: Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, Chrysomya megacephala Fabricius, Lucilia eximia Wiedemann (Diptera: Calliphoridae) and Dermatobia hominis Linnaeus Jr (Diptera: Oestridae). The control region in these species varies in length from 1000 to 1600 bp. Two structural domains with specific evolutionary patterns were identified. These were (1) conserved sequence blocks containing primary sequence motifs, including dinucleotide pyrimidine-purine series and long T-stretches, located at the 5' end adjacent to the tRNA(Ile) gene and (2) a hypervariable domain at the 3' end characterized by increased nucleotide divergence and size variation. A high frequency of A <----> T transversions at nucleotide substitution level indicated directional mutation pressure. The phylogenetic usefulness of the insect control region is discussed.141718

Characterization of polymorphic microsatellite markers for the blowfly Chrysomya albiceps (Diptera : Calliphoridae)

Chrysomya albiceps is a blowfly of great medical, sanitary and forensic importance widely distributed in the Afrotropical, southern Palaearctic, northern Oriental regions and, recently, in Central and South Americas. Here, we report the characterization of 13 polymorphic microsatellite markers for C. albiceps. The number of alleles ranged from three to 13 alleles with expected heterozygosities ranging from 0.4668 to 0.8408. These markers will be extremely useful for investigating many important aspects of this species such as population structure, dispersal and colonization dynamics.8120821

Evaluation of the internal transcribed spacer 2 (ITS2) as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies (Diptera: Calliphoridae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The internal transcribed spacer 2 (ITS2) is a small non-coding region located inside the nuclear ribosomal DNA cluster. ITS2 sequence variability is thought to be appropriate to differentiate species and for phylogenetic reconstructions analyses, which can be further improved if structural information is considered. We evaluated the potential of ITS2 as a molecular marker for phylogenetic inference in Calliphoridae (Diptera: Brachycera) using a broad range of inference methods and different substitution models, accounting or not for structural information. Sequence analyses revealed a hierarchically organized pattern of sequence variation and a small level of nucleotide substitution saturation. Intragenomic variation due to small sequence repeats was found mainly in the most variable domain (IV), but it has no significant impact on the phylogenetic signal at the species level. Inferred secondary structures revealed that GC pairs are more frequently found flanking bulges and loops regions in more conserved domains, thus ensuring structure stability. In the phylogenetic analyses, the use of substitution models accounting for structural information significantly improves phylogenetic inference in both neighbour-joining and Bayesian analyses, although the former provides limited resolution for dealing with highly divergent sequences. For Bayesian analyses, a significant improvement in likelihood was observed when considering structure information, although with small changes in topology and overall support, probably reflecting better evolutionary rates estimates. Based on these findings, ITS2 is a suitable molecular marker for phylogenetic analyses in Calliphoridae, at both species and generic level.139911891207Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [09/51723, 05/50100-5, 06/61217-3, 08/56769-2

Structural Characterization of the Internal Transcribed Spacer 2 (ITS2) of the Ribosomal DNA (rDNA) Cluster in Calyptratae (Diptera: Schizophora) and its Implications for Molecular Phylogenetic Analyses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The internal transcribed spacer 2 (ITS2) of the eukaryotic ribosomal DNA (rDNA) cluster plays an essential role in processing of the ribosomal RNA, which is primarily accomplished by the secondary structures acquired by the molecule after transcription. Two possible structural conformation models have been proposed for the ITS2 region, the 'ring model' and the 'hairpin model,' and the former has been widely used in many molecular phylogenetic analyses incorporating structural information available to date. To evaluate the validity of this model, in vitro transcribed ITS2 molecules from species representing the three superfamilies of the Calyptratae clade (Diptera: Schizophora), namely Cochliomyia hominivorax, Musca domestica, and Glossina morsitans, were submitted to enzymatic digestion with single- and double-stranded specific nucleases (RNases I, A, T1, and V1). The resulting fragments were analyzed by capillary electrophoresis and digestion sites were mapped in the secondary structure models which were obtained by in silico prediction with further refinement by homology comparisons. The pattern of RNA fragments generated by these RNases show a high degree of correlation to most of the predicted helix-loop regions and structural motifs. Discrepancies to the models can be explained by alternative structural conformation dynamics (in M. domestica and G. morsitans) and by higher-order factors (such as tertiary interactions) that may stabilize thermodynamically unfavored structures (in C. hominivorax).763158171Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [06/61217-3

Methods for the recovery of mitochondrial DNA sequences from museum specimens of myiasis-causing flies

Mitochondrial DNA (mtDNA) sequences from eight species of myiasis-causing flies, stored for up to 50 years, were amplified successfully. Universal primers were used to amplify six specific regions from total genomic DNA, including five mtDNA genes. The comparison of phenol/chloroform, DNAzol(R) and Chelex techniques for DNA extraction showed that the DNAzol(R) reagent was the most efficient in retrieving DNA from museum specimens, although the Chelex extraction procedure is currently the most frequently reported method. Comparison of the universal primer sequences with the homologous sequences of Cochliomyia hominivorax Coquerel and Chrysomya putoria Wiedemann (Diptera: Calliphoridae) revealed mismatches that could contribute to the low recovery of a short sequence from subunit II of cytochrome oxidase. The ability to characterize mtDNA markers from museum specimens should be useful in comparative studies of contemporary samples and should help in elucidating species introduction, colonization and dispersal.161394

The Phylogeographic History of the New World Screwworm Fly, Inferred by Approximate Bayesian Computation Analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Insect pest phylogeography might be shaped both by biogeographic events and by human influence. Here, we conducted an approximate Bayesian computation (ABC) analysis to investigate the phylogeography of the New World screwworm fly, Cochliomyia hominivorax, with the aim of understanding its population history and its order and time of divergence. Our ABC analysis supports that populations spread from North to South in the Americas, in at least two different moments. The first split occurred between the North/Central American and South American populations in the end of the Last Glacial Maximum (15,300-19,000 YBP). The second split occurred between the North and South Amazonian populations in the transition between the Pleistocene and the Holocene eras (9,100-11,000 YBP). The species also experienced population expansion. Phylogenetic analysis likewise suggests this north to south colonization and Maxent models suggest an increase in the number of suitable areas in South America from the past to present. We found that the phylogeographic patterns observed in C. hominivorax cannot be explained only by climatic oscillations and can be connected to host population histories. Interestingly we found these patterns are very coincident with general patterns of ancient human movements in the Americas, suggesting that humans might have played a crucial role in shaping the distribution and population structure of this insect pest. This work presents the first hypothesis test regarding the processes that shaped the current phylogeographic structure of C. hominivorax and represents an alternate perspective on investigating the problem of insect pests.810Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAO/IAEA [11822/RO]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [03/13598-0]FAO/IAEA [11822/RO

The mitochondrial DNA control region of muscidae flies: Evolution and structural conservation in a dipteran context

The structure and evolution of the mtDNA control region (CR) and its flanking genes in economically important dipterans from the family Muscidae (Brachycera: Calyptratae), Haematobia irritans, Musca domestica, Atherigona orientalis, and Stomoxys calcitrans are presented in this paper, along with the description of short noncoding intergenic regions possibly related to CR flanking sequences in Stomoxys calcitrans and Ophyra aenescens mtDNAs (ScIR and OaIR, respectively). S. calcitrans showed a large CR with an similar to 550-bp element tandemly repeated and a duplicated tRNA(Ile) stop gene. The characterization of H. irritans, M. domestica, A. orientalis, and S. calcitrans CR sequences led to the identification of seven conserved sequence blocks homologous to the elements previously described for Calliphoridae and Oestridae species (Brachycera: Calyptratae). Comparative analysis with Drosophila species (Brachycera: Acalyptratae) revealed four conserved regions. The putative functional roles of the conserved elements in the regulation of replication and transcription processes are addressed. The characterization of the structural organization of the mitochondrial genome CR demonstrates the plasticity of the mtDNA molecule in family Muscidae.64551952

Characterization of the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria by PCR-RFLP of mitochondrial DNA

The primary screwworm fly Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important insect pests of livestock in neotropical regions, whereas Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), the secondary screwworm, is of medical and sanitary importance because of its role in the dissemination of pathogens. These two species share morphological similarities and both may occur in the same myiasis, but in different developmental stages. In this work, the usefulness of PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) of mitochondrial DNA (mtDNA) for the unambiguous identification of C. hominivorax and C. macellaria was investigated. Two specific regions of mtDNA were amplified: 870bp from Cytochrome oxidase subunit I and 2100bp from the A+T rich/12S region from C. hominivorax and C. macellaria specimens from different areas of Brazil. Reliable species-specific PCR-RFLP results were obtained for the CO I region and the A+T rich/12S region using the restriction enzymes Dra I and Ssp I. These results confirm the conservation of CO I diagnostic restriction sites previously reported and demonstrate the usefulness of the control region sequences as an efficient marker for PCR-RFLP identification of Brazilian screwworm flies. The occurrences of intraspecific polymorphic patterns are discussed based on frequencies and potential conflicts for species identification. PCR-RFLP provides a potentially useful method for identifying samples from the areas where these species are monitored.15218318

Mitochondrial DNA diversity in wild and cultured populations of Brycon opalinus (Cuvier, 1819) (Characiformes, Characidae, Bryconinae) from the Paraiba do Sul Basin, Brazil

Made available in DSpace on 2019-09-12T16:53:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2002RFLP analysis of mitochondrial DNA was carried out to investigate the population genetic structure of cultured and wild populations of Pirapitinga-do-sul-Brycon opalinus (Cuvier, 1819) (Characiformes, Characidae, Bryconinae) from the Paraiba do sul basin, Brazil. mtDNA samples were collected from 257 specimens sampled in seven rivers of the Paraiba basin and in a hatchery used for restocking. An initial screening with 24 restriction enzymes revealed six informative enzymes, which generated 27 haplotypes, Haplotype diversities were high both in the hatchery broodstock (h = 0.75) and in the samples from the wild (h = 0.60). Nucleotide diversity among the 27 B. opalinus haplotypes was 0.825%. Analysis of molecular variance (AMOVA) showed the highest variance within populations (70.48%) while 28.16% of the total diversity was due to interpopulation variance, suggesting the existence of genetic differentiation among B. opalinus populations. (C) 2002 Elsevier Science B.V. All rights reserved.Univ Sao Paulo, Inst Heart INCOR, Lab Genet & Mol Cardiol, BR-05403000 Sao Paulo, Brazil; Universidade de Taubaté (Unitau), Dept Agr Sci, BR-12020270 Sao Paulo, Brazil; Univ Estadual Campinas, Dept Genet & Evolut, BR-13083970 Campinas, SP, Brazil; Univ Estadual Campinas, Dept Physiol, BR-13081970 Campinas, SP, Brazi