Invasion origin, rapid population expansion, and the lack of genetic structure of cotton bollworm (Helicoverpa armigera) in the Americas.

In 2013, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was officially declared as present in Brazil and, after two years, the species was detected in the Caribbean and North America. Information on genetic features and accurate distribution of pests is the basis for agricultural protection policies. Furthermore, such knowledge is imperative to develop control strategies, understand the geographical range, and genetic patterns of this species in the Americas. Here, we carried out the widest sampling of H. armigera in the South American continent and Puerto Rico, after we estimated the diversity, demographic parameters, and genetic structure. The Internal Transcribed Spacer 1 (ITS1) nuclear marker was used to investigate the presence of putative hybrids between H. armigera and H. zea, and they were observed at a frequency of 1.5%. An ABC analysis, based in COI gene fragment, suggested Europe as the origin of South America specimens of H. armigeraand following a movement northward through the Caribbean. Three mtDNA genes and three nDNA markers revealed high genetic diversity distributed without the defined population structure of H. armigera in South America. Most of the genetic variation is within populations with a multidirectional expansion of H. armigera among morphoclimatic regions. High genetic diversity, rapid population expansion, and hybridization have implications for pest management since they suggest that adaptive alleles are spread through wide areas in South America that favor rapid local adaptation of H. armigera to new and disturbed environments (e.g., in agricultural areas).

The first phylogenetic study of Mesembrinellidae (Diptera: Oestroidea) based on molecular data: clades and congruence with morphological characters.

The Mesembrinellidae (Diptera: Oestroidea) comprise a small group of strictly Neotropical calyptrate flies, with 36 described species. The group has often been treated as a subfamily of Calliphoridae, but there is growing evidence that it corresponds to a distinct Oestroidea lineage. Internal relationships have so far been addressed based only on morphology, with results lacking resolution and support. This is the first molecular phylogeny for the group, which is based on the analyses of 80 terminal taxa (22 mesembrinellid and 28 outgroup species) and 5 molecular markers (ITS2, 28S, COI, COII and 16S). Maximum-parsimony, maximum-likelihood and Bayesian inference methods were used, the latter two with partitioning strategies considering codon position and secondary structure information. Results corroborate the Mesembrinellidae as a monophyletic lineage inside Oestroidea. Three clades were consistently recovered: (1) (Laneella + Mesembrinella patriciae); (2) (Mesembrinella (excluding M. patriciae)  + Eumesembrinella); and (3) (Huascaromusca + Giovanella). Re-examination of the female reproductive tract of M. patriciae revealed a Laneela-type spermatheca, which corroborates the position of the species recovered in the molecular phylogenetic analyses. Mesembrinella and Huascaromusca are in all cases paraphyletic with regards to Eumesembrinella and Giovanella, respectively. These latter two genera should, thus, be seen as subjective junior synonyms.

Selection and validation of reference genes for functional studies in the Calliphoridae family.

The genera Cochliomyia and Chrysomya contain both obligate and saprophagous flies, which allows the comparison of different feeding habits between closely related species. Among the different strategies for comparing these habits is the use of qPCR to investigate the expression levels of candidate genes involved in feeding behavior. To ensure an accurate measure of the levels of gene expression, it is necessary to normalize the amount of the target gene with the amount of a reference gene having a stable expression across the compared species. Since there is no universal gene that can be used as a reference in functional studies, candidate genes for qPCR data normalization were selected and validated in three Calliphoridae (Diptera) species, Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, and Chrysomya albiceps Wiedemann . The expression stability of six genes ( Actin, Gapdh, Rp49, Rps17, α -tubulin, and GstD1) was evaluated among species within the same life stage and between life stages within each species. The expression levels of Actin, Gapdh, and Rp49 were the most stable among the selected genes. These genes can be used as reliable reference genes for functional studies in Calliphoridae using similar experimental settings.

Glutamate-gated chloride channel subunit cDNA sequencing of Cochliomyia hominivorax (Diptera: Calliphoridae): cDNA variants and polymorphisms.

The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel) is one of the major myiasis-causing flies that injures livestock and leads to losses of ~US$ 2.7 billions/year in the Neotropics. Ivermectin (IVM), a macrocyclic lactone (ML), is the most used preventive insecticide for this parasite and targets the glutamate-gated chloride (GLUCLα) channels. Several authors have associated altered GluClα homologues to MLs resistance in invertebrates, although studies about resistance in NWS are limited to other genes. Here, we aimed to characterise the NWS GluClα (ChGluClα) cDNA and to search for alterations associated with IVM resistance in NWS larvae from a bioassay. The open reading frame of the ChGluClα comprised 1,359 bp and encoded a sequence of 452 amino acids. The ChGluClα cDNAs of the bioassay larvae showed different sequences that could be splice variants, which agree with the occurrence of alternative splicing in GluClα homologues. In addition, we found cDNAs with premature stop codons and the K242R SNP, which occurred more frequently in the surviving larvae and was located close to mutation (L256F) involved in ML resistance. Although these alterations were in low frequency, the ChGluClα sequencing will allow further studies to find alterations in the gene of resistant natural populations.

Acetylcholinesterase cDNA sequencing and identification of mutations associated with organophosphate resistance in Cochliomyia hominivorax (Diptera: Calliphoridae).

Altered acetylcholinesterase (AChE) has been identified in numerous arthropod species resistant to organophosphate (OP) and carbamate insecticides. The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel), one of the most important myiasis-causing flies in the Neotropics, has been controlled mainly by the application of OP insecticides in its current geographical distribution. However, few studies have investigated insecticide resistance in this species. Based on previous studies about mutations conferring OP resistance in related dipteran species, AChE cDNA was sequenced allowing a survey for mutations (I298V, G401A, F466Y) in NWS populations. In addition, the G137D mutation in the carboxylesterase E3 gene, also associated with OP resistance, was analyzed in the same NWS populations. Only 2/135 individuals presented an altered AChE gene (F466Y). In contrast, a high frequency of the G137D mutation in the E3 gene was found in some localities of Brazil and Uruguay, while the mutant allele was not found in Cuba, Venezuela or Colombia. These findings suggest that the alteration in the carboxylesterase E3 gene may be one of the main resistance mechanisms selected in this ectoparasite. The knowledge of the frequency of these resistance-associated mutations in the NWS natural populations may contribute to the selection of appropriate chemicals for control as part of pest management strategies.

Changes in the frequency of the G137D and W251S mutations in the carboxylesterase E3 gene of Cochliomyia hominivorax (Diptera: Calliphoridae) populations from Uruguay.

The New World Screwworm (NWS) fly Cochliomyia hominivorax is one of most important myiasis-causing flies in the Neotropics. It is responsible for severe losses to the livestock industry through both mortality and the loss of productivity of infested animals. In Uruguay, NWS represents a significant problem. To date this pest has been controlled by the application of chemical insecticides, mainly the pyrethroid and organophosphate (OP) classes. However, the intensive use of these compounds over many years has led to the evolution of resistance which has the potential to compromise the effectiveness of current control strategies. One mechanism by which resistance has occurred in this and related dipteran species is through two mutations (G137D and W251S) in the carboxylesterase E3 enzyme that have enhanced ability to hydrolyze certain insecticides. In this study changes in the frequency of these mutations in C. hominivorax was investigated in three different Uruguayan regions in 2003 and 2009. All three regions analyzed showed a reduction in the frequency of the G137D mutation and a significant increase in frequency of the W251S mutation, and this may be related to the current intense use of dimethyl-OP and pyrethroid insecticides. The findings of this study provide current information on the frequency of these resistance-associated mutations in NWS in Uruguay and may help select appropriate chemicals for NWS control as part of potential pest management strategies.

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

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.

Development of polymorphic microsatellite markers for the human botfly, Dermatobia hominis (Diptera: Oestridae).

In this report, we describe the development of 17 polymorphic microsatellite markers for the human botfly, Dermatobia hominis, an obligatory parasite of mammals of great veterinary importance in Latin America. The number of alleles ranged from 5 to 21 per locus, with a mean of 12.2 alleles per locus. The expected heterozygosity ranged from 0.2571 to 0.9206 and from 0.2984 to 0.9291 in two populations from Brazil. These markers should provide a high resolution tool for assessment of the fine-scale genetic structure of natural populations of the human botfly.

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.

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

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.

A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles.

Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. In Brazil, C. hominivorax has been controlled mainly with organophosphate (OP) insecticides, although the inappropriate use of these chemicals can result in the selection of resistant flies. Changes in carboxylesterase activity have been associated with OP insecticides in some arthopodan species. In this work, we isolated and characterized part of the E3 gene in C. hominivorax (ChalphaE7), which contained the same substitutions responsible for the acquisition of OP hydrolase activity in Lucilia cuprina (Calliphoridae). Digestion of the polymerase chain reaction products with a restriction enzyme that specifically recognized the mutation site unambiguously differentiated wild and mutated esterase alleles. The PCR-RFLP assay therefore provided a fast, reliable DNA-based method for identifying C. hominivorax individuals with a mutation in the esterase gene. Further bioassays to determine the association of this mutation with OP resistance in C. hominivorax should allow the development of more effective strategies for managing this species.

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.

Genetic approaches for studying myiasis-causing flies: molecular markers and mitochondrial genomics.

"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.

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.