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

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

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

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

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

Wing morphometry as a tool for correct identification of primary and secondary New World screwworm fly

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Cochliomyia hominivorax and Cochliomyia macellaria are endemic Neotropical Calliphoridae species. The former Causes severe myiasis in hosts while the latter is Sarcosaprophagous, but commonly found as a second invader in Wounds. Due to the morphological similarity between them and the potential losses that C. hominivorax represents for cattle breeders, the rapid and correct identification of these two species is very important. In addition to a correct identification of these species, a good knowledge of C. hominivorax biology can be helpful for designing control programs. We applied geometric morphometric methods to assess wing differences between C. hominivorax and C. macellaria and conduct a preliminary analysis of wing morphological variation in C. hominivorax populations. Canonical variate analysis, using wing shape data, correctly classified 100% of the individuals analyzed according to sex and species. This result demonstrates that wing Morphometry is a simple and reliable method for identifying C. hominivorax and C. macellaria samples and can be used to monitor C. hominivorax. Both species show sexual dimorphism, but in C. hominivorax it is magnified. We suggest that this may reflect different histories of selection pressures operating on males and females. Significant differences in wing size and shape were obtained among C. hominivorax populations, witli little correlation with latitude. This result suggests that wing variation is also a good morphological marker for studying Population variation in C. hominivorax.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.10011926Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)International Atomic Energy Agency (IAEA) [11822/RO]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [01/12528-2, 03/13598-0]International Atomic Energy Agency (IAEA) [11822/RO

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

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)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 (1298V. 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. (C) 2010 Elsevier B.V. All rights reserved.17741671190195Conselho 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)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq [578231/2008-5, 141277/2005-0]FAPESP [03/01458-9, 07/54431-1, 04/12532-8

Genetic Structure and Demographic History of New World Screwworm Across Its Current Geographic Range

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The phylogeographical history of the pest fly screwworm, Cochliomyia hominivorax (Coquerel), was studied using partial mitochondrial DNA sequences of the control region, Cytochrome c oxidase (CO) subunit I and CO subunit II from 361 individuals collected across its current geographic range. Analyses showed marked genetic differentiation on a macrogeographic scale. The genetic diversity in the species is structured into four main "regional groups," corresponding to Cuba, the Dominican Republic, and the North and South Amazon region. Results indicated that the distribution of screwworm genetic diversity was mainly shaped by historical events, i.e., colonization of Caribbean islands, vicariance in the Amazon region and population expansion. Demographic history analyses revealed that the population expansion started approximate to 20-25,000 yr ago and recently increased exponentially. We hypothesized that the initial period of expansion was probably associated with environmental amelioration in the late Pleistocene and the exponential increase with resource availability in recent times. The population expansion is probably responsible for the low divergence and the lack of genetic and geographic correlation in the South Amazon region but did not erase the genetic structure pattern on a continental scale. The screwworm is one of the most damaging livestock pests in South and Central America, and the pattern of genetic variability distribution reported here suggests that the Caribbean area and the North and South Amazon regions could be considered as independent units for future pest control programs.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.482280290Coordenação 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

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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, alpha-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.14Fundação 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 [2008/58106-0]CNPq [477335/2009-8]FAPESP [2009/13463-3