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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 approximately 550-bp element tandemly repeated and a duplicated tRNA(Ile) 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.

In view of the medical, sanitary and forensic importance of Chrysomya species, a knowledge of their nucleotide sequences would be useful for the molecular characterization of this genus, and would help in designing primers and in improving the molecular identification of Calliphoridae species. In this work, the mitochondrial genome of the blowfly Chrysomya chloropyga (Diptera: Calliphoridae) was completely sequenced. The entire mitochondrial DNA (mtDNA) molecule was 15,837 bp long and was sequenced using the shotgun approach. The overall nucleotide composition was heavily biased towards As and Ts, which accounted for 76.7% of the whole genome. The cox1 gene had a serine as the start codon, while incomplete termination codons mediated by tRNA signals were found for cox2 , nd4 and nd5 . The C. chloropyga genes were in the same order and orientation as the mitochondrial genome of other dipteran species, except for the occurrence of a 123 bp region that included a complete duplication of tRNA Ile and a partial duplication of tRNA Gln genes. C. chloropyga is the first species of Diptera with 23 tRNA genes instead of the usual 22 already described. A phylogenetic analysis showed a split of Brachycera into Calyptratae and Acalyptratae subdivisions. The complete sequence of C. chloropyga mtDNA described here will be a useful source of sequence information for general molecular and evolutionary studies in Diptera.

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.

The amplification of complete mitochondrial genomes by long PCR (polymerase chain reaction) has been a major contribution to the large-scale sequencing of arthropodan mitochondrial genomes. In this work, we designed six conserved long-PCR primers to successfully recover the entire mitochondrial genome of the horn fly Haematobia irritans (Diptera: Muscidae) in two overlapping fragments. The conservation and versatility of these primers were tested for 17 other species from four major insect orders: Diptera (14), Coleoptera (1), Lepidoptera (1) and Hymenoptera (1). The amplification of complete mitochondrial genomes in orders other than Diptera suggested an even broader application of these primers, especially within the Hexapoda.

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.

Summary: 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: Contact: lessinger@amiga.cbmeg.unicamp.br Supplementary information: Detailed information, including an illustrated tutorial, is available from the above URL.