Ana Cláudia Lessinger

The mitochondrial genome of the primary screwworm fly Cochliomyia hominivorax (Diptera: Calliphoridae)

The complete sequence of the mitochondrial genome of the screwworm Cochliomyia hominivorax was determined. This genome is 16 022 bp in size and corresponds to a typical Brachycera mtDNA. A Serine start codon for COI and incomplete termination codons for COII, NADH 5 and NADH 4 genes were described. The nucleotide composition of C. hominivorax mtDNA is 77% AT‐rich, reflected in the predominance of AT‐rich codons in protein‐coding genes. Non‐optimal codon usage was commonly observed in C. hominivorax mitochondrial genes. Phylogenetic analysis distributed the Acalypterate species as a monophyletic group and assembled the C. hominivorax (Calyptratae) and the Acalyptratae in a typical Brachycera cluster. The identification of diagnostic restriction sites on the sequenced mitochondrial genome and the correlation with previous RFLP analysis are discussed.

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 tRNAIle 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.

AMiGA: the arthropodan mitochondrial genomes accessible database

UNLABELLED The Arthropodan Mitochondrial Genomes Accessible database (AMiGA) is a relational database developed to help in managing access to the increasing amount of data arising from developments in arthropodan mitochondrial genomics (136 mitochondrial genomes as of September 2005). The strengths of AMiGA include (1) a more accessible and up-to-date database containing a more comprehensive set of mitochondrial genomes for this phylum, (2) the provision of flexible search options for retrieving detailed information such as bibliographical data, genomic graphics, FASTA sequences and taxonomical status, (3) the possibility of enhanced comparative analyses by multiple alignment of single or concatenated sets of genes, (4) more accurate and updated information resulting from a specific curation process called AMiGA Notes and (5) the possibility of including unpublished sequences in a password-restricted area for comparative analysis with the other sequences stored in the database. AVAILABILITY http://amiga.cbmeg.unicamp.br CONTACT lessinger@amiga.cbmeg.unicamp.br SUPPLEMENTARY INFORMATION Detailed information, including an illustrated tutorial, is available from the above URL.

Structure and evolution of the mitochondrial genomes of Haematobia irritans and Stomoxys calcitrans : The Muscidae (Diptera: Calyptratae) perspective

We present the first two mitochondrial genomes of Muscidae dipterans for the species Haematobia irritans (the horn fly) and Stomoxys calcitrans (the stable fly). Typical insect mtDNA features are described, such as a high A+T content (79.1% and 78.9%, respectively), the preference for A+T-rich codons, and the evidence of a non-optimal codon usage. The strong A+T enrichment partially masks another nucleotide content bias maintained by A+C mutation pressure in these Muscidae mtDNAs. The analysis of this data provides a model of metazoans tRNA anticodon evolution, based on the selection hypothesis of anticodon versatility. H. irritans mitochondrial genome (16078 bp) is structurally similar to the hypothetical ancestral mitochondrial genome of arthropods and its control region (A+ T-rich region in insects) organization is consistent with the structure described for Brachycera dipterans. On the other hand, the mitochondrial genome of S. calcitrans is approximately 2kb longer (18 kb), characterized by the presence of approximately 550 bp tandem repeats in the control region, and an extra copy of trnI remarkably similar to a duplicated element of blowflies mtDNA. Putative sequence elements, involved in the regulation of transcription and replication of the mtDNA, were reliably identified in S. calcitrans control region despite the 0.8-1.5 kb gap uncovered from this genome. The use of amino acid and nucleotide sequences of concatenated mitochondrial protein-coding genes (PCGs) in phylogenetic reconstructions of Diptera does not support the monophyly of Muscomorpha, as well as the monophyly of Acalyptratae. Within the Calyptratae group, the inclusion of Muscidae (Muscoidea) as a sister group of Calliphoridae (Oestroidea) implies in a potential conflict concerning the monophyly of the superfamily Oestroidea.

Genetic Approaches for Studying Myiasis-causing Flies: Molecular Markers and Mitochondrial Genomics

Abstract“Myiasis-causing flies” is a generic term that includes species from numerous dipteran families, mainly Calliphoridae and Oestridae, of which blowflies, screwworm flies and botflies are among the most important. This group of flies is characterized by the ability of their larvae to develop in animal flesh. When the host is a live vertebrate, such parasitism by dipterous larvae is known as primary myiasis. Myiasis-causing flies can be classified as saprophagous (free-living species), facultative or obligate parasites. Many of these flies are of great medical and veterinary importance in Brazil because of their role as key livestock insect-pests and vectors of pathogens, in addition to being considered important legal evidence in forensic entomology. The characterization of myiasis-causing flies using molecular markers to study mtDNA (by RFLP) and nuclear DNA (by RAPD and microsatellite) has been used to identify the evolutionary mechanisms responsible for specific patterns of genetic variability. These approaches have been successfully used to analyze the population structures of the New World screwworm fly Cochliomyia hominivorax and the botfly Dermatobia hominis. In this review, various aspects of the organization, evolution and potential applications of the mitochondrial genome of myiasis-causing flies in Brazil, and the analysis of nuclear markers in genetic studies of populations, are discussed.

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

Abstract 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® and Chelex techniques for DNA extraction showed that the DNAzol® 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.

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 ∼550-bp element tandemly repeated and a duplicated tRNAIle 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.

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: 870 bp from Cytochrome oxidase subunit I and 2100 bp 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.

Evolutionary and structural analysis of the cytochrome c oxidase subunit I (COI) gene from Haematobia irritans, Stomoxys calcitrans and Musca domestica (Diptera: Muscidae) mitochondrial DNA

This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A+T-rich sequence is linked to three features: a high A+T content throughout the entire gene, a high A+T content in the third codon position, and a predominance of A+T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.

The value of PCR-RFLP molecular markers for the differentiation of immature stages of two necrophagous flies (Diptera: Calliphoridae) of potential forensic importance

The identification of insect species involved in corpses decomposition is of particular importance in estimating the post-mortem interval (PMI) in forensic science, since the PMI is based on information about the life cycle of necrophagous insects. However, the identification of some insect species, especially in their immature stages, may be complicated by many factors, even for experienced taxonomists. Species of the same genus such as Hemilucilia segmentaria (Fabricius) and H. semidiaphana (Rondani) (Diptera: Calliphoridae) are morphologically and behaviorally very similar, but differ in their growth and maturation rates. These species are abundant in forests, exclusively necrophagous and, therefore, are of potential medicolegal importance for estimating the PMI in criminal events that would take place in those areas. In this study, we assessed the usefulness of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in the differentiation of these two species. Two specific regions of mitochondrial DNA, the Cytochrome oxidase subunit I (COI) and the control region (CR), were amplified by PCR and digested using restriction endonucleases. The cleavage patterns generated by the endonucleases DraI and SspI were suitable for differentiating the two Hemilucilia species. This method can be helpful for the forensic entomologist in estimating the PMI because it provides a fast identification, also making possible the use of the insect at any life stage, including immature specimens, regardless of the conditions of preservation (dead or live specimens).