The complete mitochondrial genome of the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

The oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) currently is one of the economically most destructive pest species of stone and pome fruits worldwide. Here we sequenced the complete mitochondrial genome of this pest. This genome is 15,776 bp long, with an A + T content of 81.24%, containing 37 typical animal mitochondrial genes and an A + T-rich region. All gene are arranged as hypothesized ancestral gene order of insects except for trnM, which was shuffled from 3′ downstream of trnQ to 5′ upstream of trnI. cox1 gene uses unusual CGA start codon, as that in all other sequenced lepidopteran mitochondrial genome. The secondary structures for the two rRNA genes were predicted. All helices typically present in insect mitochondrial rRNA genes are generated. A microsatellite sequence was inserted into the region of H2347 in rrnL in G. molesta and two other sequenced tortricid mitochondrial genomes, indicating that the insertion event in this helix might occurred anciently in family Tortricidae. All of the 22 typical animal tRNA genes have a typical cloverleaf structure except for trnS2, in which the D-stem pairings in the DHU arm are absent. An intergenic sequence is present between trnQ and nad2 as well as in other sequenced lepidopteran mitochondrial genomes, which was presumed to be a remnant of trnM gene and its boundary sequences after the duplication of trnM to the upstream of trnI in Lepidoptera. The A + T-rich region is 836 bp, containing six repeat sequences of “TTATTATTATTATTAAATA(G)TTT.

Accelerated Evolution of Mitochondrial but Not Nuclear Genomes of Hymenoptera: New Evidence from Crabronid Wasps

Mitochondrial genes in animals are especially useful as molecular markers for the reconstruction of phylogenies among closely related taxa, due to the generally high substitution rates. Several insect orders, notably Hymenoptera and Phthiraptera, show exceptionally high rates of mitochondrial molecular evolution, which has been attributed to the parasitic lifestyle of current or ancestral members of these taxa. Parasitism has been hypothesized to entail frequent population bottlenecks that increase rates of molecular evolution by reducing the efficiency of purifying selection. This effect should result in elevated substitution rates of both nuclear and mitochondrial genes, but to date no extensive comparative study has tested this hypothesis in insects. Here we report the mitochondrial genome of a crabronid wasp, the European beewolf (Philanthus triangulum, Hymenoptera, Crabronidae), and we use it to compare evolutionary rates among the four largest holometabolous insect orders (Coleoptera, Diptera, Hymenoptera, Lepidoptera) based on phylogenies reconstructed with whole mitochondrial genomes as well as four single-copy nuclear genes (18S rRNA, arginine kinase, wingless, phosphoenolpyruvate carboxykinase). The mt-genome of P. triangulum is 16,029 bp in size with a mean A+T content of 83.6%, and it encodes the 37 genes typically found in arthropod mt genomes (13 protein-coding, 22 tRNA, and two rRNA genes). Five translocations of tRNA genes were discovered relative to the putative ancestral genome arrangement in insects, and the unusual start codon TTG was predicted for cox2. Phylogenetic analyses revealed significantly longer branches leading to the apocritan Hymenoptera as well as the Orussoidea, to a lesser extent the Cephoidea, and, possibly, the Tenthredinoidea than any of the other holometabolous insect orders for all mitochondrial but none of the four nuclear genes tested. Thus, our results suggest that the ancestral parasitic lifestyle of Apocrita is unlikely to be the major cause for the elevated substitution rates observed in hymenopteran mitochondrial genomes

The complete nucleotide sequence of the mitochondrial genome of Bactrocera minax (Diptera: Tephritidae)

The complete 16,043 bp mitochondrial genome (mitogenome) of Bactrocera minax (Diptera: Tephritidae) has been sequenced. The genome encodes 37 genes usually found in insect mitogenomes. The mitogenome information for B. minax was compared to the homologous sequences of Bactrocera oleae, Bactrocera tryoni, Bactrocera philippinensis, Bactrocera carambolae, Bactrocera papayae, Bactrocera dorsalis, Bactrocera correcta, Bactrocera cucurbitae and Ceratitis capitata. The analysis indicated the structure and organization are typical of, and similar to, the nine closely related species mentioned above, although it contains the lowest genome-wide A+T content (67.3%). Four short intergenic spacers with a high degree of conservation among the nine tephritid species mentioned above and B. minax were observed, which also have clear counterparts in the control regions (CRs). Correlation analysis among these ten tephritid species revealed close positive correlation between the A+T content of zero-fold degenerate sites (P0FD), the ratio of nucleotide substitution frequency at P0FD sites to all degenerate sites (zero-fold degenerate sites, two-fold degenerate sites and four-fold degenerate sites) and amino acid sequence distance (ASD) were found. Further, significant positive correlation was observed between the A+T content of four-fold degenerate sites (P 4FD) and the ratio of nucleotide substitution frequency at P 4FD sites to all degenerate sites; however, we found significant negative correlation between ASD and the A+T content of P4FD, and the ratio of nucleotide substitution frequency at P4FD sites to all degenerate sites. A higher nucleotide substitution frequency at non-synonymous sites compared to synonymous sites was observed in nad4, the first time that has been observed in an insect mitogenome. A poly(T) stretch at the 5′ end of the CR followed by a [TA(A)]n-like stretch was also found. In addition, a highly conserved G+A-rich sequence block was observed in front of the poly(T) stretch among the ten tephritid species and two tandem repeats were present in the CR. © 2014 Zhang et al

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

Isolation and characterization of polymorphic microsatellite loci for the horn fly, Haematobia irritans (L.) (Diptera : Muscidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The horn fly, Haematobia irritans (L.) (Diptera: Muscidae), is a cosmopolitan livestock pest that has caused a great negative impact on the animal production sector throughout the world. Here, we describe 10 polymorphic microsatellite loci isolated from H. irritans. The number of alleles found ranged from two to eight per locus and the expected heterozygosity from 0.1421 to 0.7702. These loci are potentially useful for the fine-scale genetic characterization of horn fly populations and provide fundamental information for pest management and planning of control programs.85971973Fundação 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 [03/01456-9, 05/55033-4]CNPq [131093/2005-3

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 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

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 COT protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COT protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.16215616

Conservation and versatility of a new set of primers for long-PCR amplification of complete insect mitochondrial genomes based on Haematobia irritans mtDNA sequences

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.5488588