Population Genetics Of New World Screwworm From The Caribbean: Insights From Microsatellite Data

Diseases affecting livestock can have a significant impact on animal productivity and on trade of live animals, meat and other animal products, which, consequently, affects the overall process of economic development. The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is an important parasitic insect pest in Neotropical regions. This species has been successfully eradicated from North and most of Central America by the sterile insect technique, but continues to affect the development of the livestock sector in most Caribbean economies. Here, we provide some insight into the patterns of genetic variation and structure and gene flow of C. hominivorax populations from the Caribbean. Analysis of populations from 10 geographical sites in four islands revealed a moderate genetic variability within the populations. Surprisingly, a high population differentiation was found even in intra-island comparisons between populations. This observation can reflect either highly structured populations resulting from a lack of gene flow or a source-sink dynamic. Our study also suggests that New World screwworm populations can recover very rapidly from population contractions. 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LondonInfante-Malachias, M.E., Yotoko, K.S.C., Azeredo-Espin, M.L., Random amplified polymorphic DNA of screwworm fly populations (Diptera: Calliphoridae) from Southeastern Brazil and Northern Argentina (1999) Genome, 42, pp. 772-779Infante-Vargas, M.E., Azeredo-Espin, M.L., Genetic variability in mitochondrial DNA of the screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae) from Brazil (1995) Biochemical Genetics, 33, pp. 237-256Krafsur, E.S., Whitten, C.J., Breeding structure of screwworm fly populations (Diptera: Calliphoridae) in Colima, Mexico (1993) Journal of Medical Entomology, 30, pp. 477-480Lyra, M.L., Fresia, P., Gama, S., Cristina, J., Klaczko, L.B., Azeredo-Espin, M.L., Analysis of mitochondrial DNA variability and genetic structure in populations of New World screwworm flies (Diptera: Calliphoridae) from Uruguay (2005) Journal of Medical Entomology, 42, pp. 589-595Méndez, L., García, R., Serrano, E., Myiasis for Cochliomyia hominivorax: Development and evaluation of an adult suppression system (2005) Book of Extended Synopses, pp. 288-289. , pp. Food & Agriculture Organization/ International Atomic Energy Agency International Conference on Area-Wide Control of Insect Pests: Integrating the Sterile Insect and Related Nuclear and Other Techniques, 9-13 May 2005, Vienna. IAEA, ViennaMichel, A.P., Ingrasci, M.J., Schemerhorn, B.J., Al, E., Rangewide population genetic structure of the African malaria vector Anopheles funestus (2005) Molecular Ecology, 14, pp. 4235-4248Piry, S., Luikart, G., Cornuet, J.M., Bottleneck: A computer program for detecting recent 536 reductions in the effective population size using allele frequency data (1999) Journal of Heredity, 90, pp. 502-503Pritchard, J.K., Stephens, M., Donnelly, P., Inference of population structure using multilocus genotype data (2000) Genetics, 155, pp. 945-959Raymond, M., Rousset, F., Genepop (Version 1.2): Population genetics software for exact tests and ecumenicism (1995) Journal of Heredity, 86, pp. 248-249Rice, W.R., Analysing table of statistical tests (1989) Evolution, 43, pp. 223-225Robertson, B.C., Gemmell, N.J., Defining eradication units to control invasive pests (2004) Journal of Applied Ecology, 41, pp. 1042-1048Taylor, D.B., Peterson Ii, R.D., Population genetics and gene variation in primary and secondary screwworm (Diptera: Calliphoridae) (1994) Annals of the Entomological Society of America, 87, pp. 626-633Taylor, D.B., Peterson Ii, R.D., Moya-Borja, G., Population genetics and gene variation in screwworms (Diptera: Calliphoridae) from Brazil (1996) Biochemical Genetics, 34, pp. 67-76Torres, T.T., Azeredo-Espin, M.L., Development of new polymorphic microsatellite markers for the New World screwworm Cochliomyia hominivorax (Diptera: Calliphoridae) (2005) Molecular Ecology Notes, 5, pp. 815-817Torres, T.T., Brondani, R.P.V., Garcia, J.E., Azeredo-Espin, M.L., Isolation and characterization of microsatellite markers in the New World screwworm Cochliomyia hominivorax (Diptera: Calliphoridae) (2004) Molecular Ecology Notes, 4, pp. 182-184Torres, T.T., Lyra, M.L., Fresia, P., Azeredo-Espin, M.L., Assessing genetic variation in the New World screwworm Cochliomyia hominivorax populations from Uruguay (2007) Area-Wide Control of Insect Pests: From Research to Field Implementation, pp. 183-192. , ed. by. 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Development Of New Polymorphic Microsatellite Markers For The New World Screw-worm Cochliomyia Hominivorax (diptera: Calliphoridae)

In this report, we describe the development of seven new polymorphic microsatellite markers for Cochliomyia hominivorax, a parasitic insect pest of primary agricultural and veterinary importance throughout the Neotropics. The number of alleles found ranged from 3 to 13 per locus, with the expected heterozygosity ranging from 0.4220 to 0.9045. The across-taxa amplification of some of these new microsatellite loci was successful in four additional Calliphoridae species. In combination with the 10 polymorphic microsatellite markers previously described, the markers developed here should provide a high resolution for assessing the fine-scale genetic structure of New World screw-worms. © 2005 Blackwell Publishing Ltd.54815817Billotte, N., Lagoda, P.J.L., Risterucci, A.M., Baurens, F.C., Microsatellite-enriched libraries: Applied methodology for the development of SSR markers in tropical crops (1999) Fruits, 54, pp. 277-288Edwards, K.J., Barker, J.H.A., Daly, A., Jones, C., Karp, A., Microsatellite libraries enriched for several microsatellite sequences in plants (1996) BioTechniques, 20, pp. 758-759(2000) Genetic Sexing and Population Genetics of Screw Worms, , August 7-11, 2000, Vienna, Austria. 19ppRaymond, M., Rousset, F., GENEPOP (version 1.2): Population genetics software for exact tests and ecumenicism (1995) Journal of Heredity, 86, pp. 248-249Rice, W.R., Analyzing tables of statistical tests (1989) Evolution, 43, pp. 223-225Rozen, S., Skaletsky, H.J., (1998) PRIMER 3, , http://www-genome.wi.mit.edu/genome_software/other/primer3.html, Code availableTorres, T.T., Brondani, R.P.V., Garcia, J.E., Azeredo-Espin, A.M.L., Isolation and characterization of microsatellite markers in the New World screw-worm Cochliomyia hominivorax (Diptera: Calliphoridae) (2004) Molecular Ecology Notes, 4, pp. 182-184Wyss, J.H., Galvin, T.J., Central America regional screw-worm eradication program (benefit/cost study) (1996) Annals of the New York Academy of Sciences, 791, pp. 241-24

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. © Springer 2006.12601/02/15111131Avancini, R.M.P., Linhares, A.X., Selective attractiveness of rodent-baited traps for female blowflies (1988) Med. Vet. Entomol., 2, pp. 73-76Avancini, R.M.P., Prado, A.P., Oogenesis in Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) (1986) Int. J. Insect Morphol. 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Complex Patterns Of Genetic Variability In Populations Of The New World Screwworm Fly Revealed By Mitochondrial Dna Markers

Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), the New World screwworm fly, is an important agent of traumatic myiasis, which is endemic in the Neotropical region and which has great economic impact on the development of the livestock industry. International efforts have been aimed at designing programmes to control and eradicate this species from endemic areas. Thorough knowledge of the population genetics of an insect pest is a fundamental component to ensuring the success of a pest management strategy because it enables the determination of an appropriate geographic scale for carrying out effective treatments. This study undertook an analysis of mtDNA polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) in 34 populations of C. hominivorax from 10 countries, encompassing almost all the current distribution of the species. Results showed high levels of mitochondrial DNA variability (π = 2.9%) and a complex pattern of population genetic structure for this species. Significant population structure (>st = 0.5234) and low variability were found in Caribbean populations, suggesting that, in general, islands constitute independent evolutionary entities connected by restricted gene flow. By contrast, high variability and low, but significant, differentiation was found among mainland populations (>st = 0.0483), which could not be attributed to geographic distance. 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Extreme Mitochondrial Dna Variability And Lack Of Genetic Structure In Populations Of Dermatobia Hominis (diptera: Cuterebridae) From Brazil

Restriction fragment-length polymorphism analysis of mitochondrial DNA (mtDNA) was used to characterize the genetic variation and population structure of the human bot fly, Dermatobia hominis (Diptera: Cuterebridae), in parasite populations from cattle in southeastern, southern and central regions of Brazil. Forty-eight haplotypes with a nucleotide sequence divergence of 2.75% were found among 227 individuals. Haplotypes could be divided into three clades, with interclade variation ranging from 0.94% to 2.00%. 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Evol., 9, pp. 552-569Rohlf, F.J., (1988) NTSYS: Numerical Taxonomy and Multivariate Analysis System, Version 1.4, , Exeter Software, Setauket, NYSambrook, J., Fritsch, E.F., Maniatis, T., (1989) Molecular Cloning. A Laboratory Manual, , Cold Spring Harbor Laboratory, Cold Spring Harbor, NYSancho, E., Dermatobia, the neotropical warble fly (1988) Parasitol. Today, 4, pp. 242-246Schneider, S., Kueffer, J.M., Roessli, D., Excoffier, L., (1997) ARLEQUIN Version 1.0. A Software for Population Genetic Analysis, , Genetics and Biometry Laboratory, University of Geneva, SwitzerlandSneath, P.H.A., Sokal, R.R., (1973) Numerical Taxonomy, , Freeman, San Francisco, CAStevens, J., Wall, R., Species, subspecies and hybrid populations of the blowflies Lucilia cuprina and Lucilia sericata (Diptera: Calliphoridae) (1996) Proc. R. Soc. Lond. B, 263, pp. 1335-1341Taylor, D.B., Szalanski, A.L., Peterson, R.D., Mitochondrial DNA variation in screwworm (1996) Med. Vet. 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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. © 2005 Taylor & Francis Ltd.162156160Beard, C.B., Hamm, D.M., Collins, F.H., The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects (1993) Insect Mol Biol, 2, pp. 103-124Bernasconi, M.V., Valsangiacomo, C., Piffaretti, J.C., Ward, P.I., Phylogenetic relationships among Muscoidea (Diptera: Calyptratae) based on mitochondrial DNA sequences (2000) Insect Mol Biol, 9, pp. 67-74Caterino, M.S., Cho, S., Sperling, F.A.H., The current state of insect molecular systematics: A thriving Tower of Babel (2000) Annu Rev Entomol, 45, pp. 1-54Crozier, R.H., Crozier, Y.C., The mitochondrial genome of the honeybee Apis mellifera: Complete sequence and genome organization (1993) Genetics, 133, pp. 97-117Foster, P.G., Jermiin, L.S., Hickey, D.A., Nucleotide composition bias affects amino acid content in proteins coded by animal mitochondria (1997) J Mol E, 44, pp. 282-288Greenberg, B., Flies and disease (1973) Biology and Disease Transmission, 2. , New Jersey: University Press PrincetownInfante, M.E., Azeredo-Espin, A.M.L., Genetic variability in mitochondrial DNA of screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae), from Brazil (1995) Biochem Genet, 33, pp. 737-756Kumar, S., Tamura, K., Nei, M., (1993) MEGA: Molecular Evolutionary Genetics Analysis, Version 1.01, , University Park, Pennsylvania: The Pennsylvania State UniversityLessinger, A.C., Azeredo-Espin, A.M.L., Evolution and structural organisation of mitochondrial DNA control region of myiasis-causing flies (2000) Med Vet Entomol, 14, pp. 71-80Litjens, P., Lessinger, A.C., Azeredo-Espin, A.M.L., Characterization of the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria by PCR-RFLP of mitochondrial DNA (2001) Med Vet Entomol, 15, pp. 183-188Lunt, D.H., Zhang, D.-X., Szymura, J.M., Hewitt, G.M., The insect cytochrome oxidase I gene: Evolutionary patterns and conserved primers for phylogenetic studies (1996) Insect Mol Biol, 5, pp. 153-165Morlais, I., Severson, D.W., Complete mitochondrial DNA sequence and amino acid analysis of the cytochrome c oxidase subunit I (COI) from Aedes aegypti (2002) DNA Seq, 13, pp. 123-127Nirmala, X., Hypsa, V., Zurovec, M., Molecular phylogeny of Calyptratae (Diptera: Brachycera): The evolution of 18S and 16S ribosomal rDNAs in higher dipterans and their use in phylogenetic inference (2001) Insect Mol Biol, 10, pp. 475-485Saccone, C., De Giorgi, C., Gissi, C., Pesole, G., Reyes, A., Evolutionary genomics in Metazoa: The mitochondrial DNA as a model system (1999) Gene, 238, pp. 195-209Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers (1994) Ann Entomol Soc Am, 87, pp. 651-701Szalanski, A.L., Owens, C.B., Sequence change and phylogenetic signal in muscoid COII DNA sequences (2003) DNA Seq, 14, pp. 331-334Tajima, F., Nei, M., Estimation of evolutionary distance between nucleotide sequences (1984) Mol Biol Evol, 1, pp. 269-285Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools (1997) Nucleic Acids Research, 25, pp. 4876-488

Assessing Genetic Variation In New World Screwworm Cochliomyia Hominivorax Populations From Uruguay

The New World screwworm Cochliomyia hominivorax (Coquerel) is an important parasitic insect pest in Neotropical regions. NewWorld screwworm myiasis is caused by the larval stage of the fly infesting tissues of warm-blooded vertebrates. This species represents a serious threat to the livestock sector across its current distribution, which includes part of the Caribbean and all of South America (except for Chile). Knowledge of the extent and distribution of genetic variability of C. hominivorax is of great interest for the description of populations and for contributing to future strategies of control. This paper describes the analysis of genetic variability and structure of New World screwworm populations in Uruguay using two different molecular markers, mitochondrial DNA and microsatellites.183191Agis, M., Schlötterer, C., Microsatellite variation in natural Drosophila melanogaster populations from New South Wales (Australia) and Tasmania (2001) Molecular Ecology, 10, pp. 1197-1205Avise, J.C., (1994) Molecular Markers, Natural History and Evolution, , Chapman and Hall, New York, USACallen, D.F., Thompson, A.D., Shen, Y., Phillips, H.A., Richards, R.I., Mulley, J.C., Sutherland, G.R., Incidence and origin of "null" alleles in the (AC) n microsatel-lite marker (1993) American Journal of Human Genetics, 52, pp. 922-927Dueñas, J.C.R., Panzetta-Dutari, G.M., Blanco, A., Gardenal, C.N., Restriction fragment length polymorphism of the mtDNAAT-rich region as a genetic marker in Aedes aegypti (Diptera: Culicidae) (2002) Annals of the Entomological Society of America, 95, pp. 352-358Gaggiotti, O.E., Lange, O., Rassmann, K., Gliddon, C., A comparison of two indirect methods for estimating average levels of gene flow using microsatellite data (1999) Molecular Ecology, 8, pp. 1513-1520Goldstein, D., Schlötterer, C., (1999) Microsatellites: Evolution and Applications, , Oxford University Press, Oxford, UKGoudet, J., Fstat version 1.2: A computer program to calculate F-statistics (1995) Journal of Heredity, 86, pp. 485-486Goudet, J., Raymond, M., De Meeüs, T., Rousset, F., Testing differentiation in diploid populations (1996) Genetics, 144, pp. 1933-1940Guo, S.W., Thompson, E.A., Performing the exact test of Hardy-Weinberg proportion for multiple alleles (1992) Biometrics, 48, pp. 361-372Infante-Malaquias, M.E., (1999) Estrutura Genética de Populações de Cochliomyia Hominivorax (Diptera: Calliphoridae) da Região Sudeste do Brasil: Análise Através de Três Tipos de Marcadores Genéticos, , Ph. D. Dissertation. State University of Campinas Unicamp, Campinas, SP, BrazilInfante-Vargas, M.E., Azeredo-Espin, A.M.L., Genetic variability in mito-chondrial DNA of the screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae) from Brazil (1995) Biochemical Genetics, 33, pp. 237-256Infante-Malaquias, M.E., Yotoko, K.S.C., Azeredo-Espin, A.M.L., Random amplified polymorphic DNA of screwworm fly populations (Diptera: Calliphoridae) from southeastern Brazil and northern Argentina (1999) Genome, 42, pp. 772-779Krafsur, E.S., Whitten, C.J., Breeding structure of screwworm fly populations (Diptera: Calliphoridae) in Colima, Mexico (1993) Journal of Medical Entomology, 30, pp. 477-480La Chance, L.E., Bartlett, A.C., Bram, R.A., Gagne, R.J., Graham, O.H., McInnis, D.O., Whitten, C.J., Seawright, J.A., Mating types in screw-worm populations (1982) Science, 218, pp. 1142-1143Lindquist, D.A., Abusowa, M., Hall, M.J.R., The New World screwworm fly in Libya: A review of its introduction and eradication (1992) Medical and Veterinary Entomology, 6, pp. 2-8Lyra, M.L., Fresia, P., Gama, S., Cristina, J., Klaczko, L.B., Azeredo-Espin, A.M.L., Analysis of mitochondrial DNA variability and genetic structure in populations of New World screwworm flies (Diptera: Calliphoridae) from Uruguay (2005) Journal of Medical Entomology, 42, pp. 589-595Mayer, D.G., Atzeni, M.G., Estimation of dispersal distances for Cochliomyia hominivorax (Diptera: Calliphoridae) (1993) Environmental Entomology, 22, pp. 368-374Nei, M., Estimationofaverage heterozy-gosity and genetic distance from a small number of individuals (1978) Genetics, 89, pp. 583-590Raymond, M., Rousset, F., GENEPOP (version 1.2): Population genetics software for exact tests and ecumenicism (1995) Journal of Heredity, 86, pp. 248-249Rice, W.R., Analyzing table of statistical tests (1989) Evolution, 43, pp. 223-225Ross, K.G., Krieger, M.J.B., Shoemaker, D.D., Vargo, E.L., Keller, L., Hierarchical analysis of genetic structure in native fire ant populations: Results from three classes of molecular markers (1997) Genetics, 147, pp. 643-655Rousset, F., Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance (1997) Genetics, 145, pp. 1219-1228Sanetra, M., Crozier, R.H., Patterns of population subdivision and gene flow in the ant Nothomyrmecia macrops reflected in microsatellite and mitochondrial DNA markers (2003) Molecular Ecology, 12, pp. 2281-2295Slatkin, M., Isolation by distance in equilibrium and nonequilibrium populations (1993) Evolution, 47, pp. 264-279Slatkin, M., Ameasureofpopulation subdivision based on microsatellite allele frequencies (1995) Genetics, 139, pp. 457-462Taylor, D.B., Peterson, A.L., II, Population genetics and gene variation in primary and secondary screwworm (Diptera: Calliphoridae) (1994) Annals of the Entomological Society of America, 87, pp. 626-633Taylor, D.B., Peterson, A.L., II, Moya-Borja, G., Population genetics and gene variation in screwworms (Diptera: Calliphoridae) from Brazil (1996) Biochemical Genetics, 34, pp. 67-76Torres, T.T., Azeredo-Espin, A.M.L., Development of new polymorphic microsatellite markers for the New World screw-worm Cochliomyia hominivorax (Diptera: Calliphoridae) (2005) Molecular Ecology Notes, 5, pp. 815-817Torres, T.T., Brondani, R.P.V., Garcia, J.E., Azeredo-Espin, A.M.L., Isolation and characterization of microsatel-lite markers in the new world screw-worm Cochliomyia hominivorax (Diptera: Calliphoridae) (2004) Molecular Ecology Notes, 4, pp. 182-184Van Oosterhout, C., Hutchinson, W.F., Wills, D.P.M., Shipley, P., MICRO-CHECKER: Software for identifying and correcting genotyping errors in microsatellite data (2004) Molecular Ecology Notes, 4, pp. 535-538Vargas-Terán, M., Hursey, B.S., Cunningham, E.P., The eradication of the screwworm from Libya using the sterile insect technique (1994) Parasitology Today, 10, pp. 119-122Vargas-Terán, M., Hofmann, H.C., Tweddle, N.E., Impact of screwworm eradication programmes using the sterile insect technique (2005) Sterile Insect Technique. Principles and Practice in Area-wide Integrated Pest Management, pp. 629-650. , Dyck, V. A., J. Hendrichs, and A. S. Robinson eds., Springer, Dordrecht, The NetherlandsWeir, B.S., Cockerham, C.C., Estimating F-statistics for the analysis of population structure (1984) Evolution, 38, pp. 1358-1370Wright, S., The interpretation of population structure by F-statistics with special regards to systems of mating (1965) Evolution, 19, pp. 395-420Wyss, J.H., Screw-worm eradication in the Americas - Overview (2000) Proceedings: Area-wide Control of Fruit Flies and Other Insect Pests. International Conference on Area-wide Control of Insect Pests, and the 5th International Symposium on Fruit Flies of Economic Importance, pp. 79-86. , Tan, K. H. ed., 28 May-5 June 1998, Penang, Malaysia. Penerbit Universiti Sains Malaysia, Pulau Pinang, Malaysi

The Value Of Pcr-rflp Molecular Markers For The Differentiation Of Immature Stages Of Two Necrophagous Flies (diptera: Calliphoridae) Of Potential Forensic Importance [o Valor De Marcadores Moleculares Do Tipo Pcr-rflp Para A Diferenciação De Estágios Imaturos De Duas Moscas Necrófagas (diptera: Calliphoridae) De Potencial Importância Forense]

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).345777783Amorim, J.A., Ribeiro, O.B., Distinction among the puparia of three blowfly species (Diptera: Calliphoridae) frequently found on unburied corpses (2001) Mem. Inst. Oswaldo Cruz, 96, pp. 781-784Carvalho, L.M.L., Linhares, A.X., Seasonality of insect succession and pig carcass decomposition in a natural forest area in Southeastern Brazil (2001) J. Forensic Sci, 46, pp. 604-608Carvalho, L.M.L., Thyssen, P.J., Linhares, A.X., Palhares, F.B., A checklist of arthropods associated with carrion and human corpses in southeastern Brazil (2000) Mem. Inst. Oswaldo Cruz, 95, pp. 135-138Caterino, M.S., Cho, S., Sperling, F.A.H., The current state of insect molecular systematics (2000) Ann. Rev. Entomol, 45, pp. 1-54Catts, E.P., Goff, M.L., Forensic entomology in criminal investigations (1992) Ann. Rev. Entomol, 37, pp. 253-272Catts, E.P., Haskell, N.H., (1990) Entomology and death: A procedural guide, p. 182. , USA, Joyce Print ShopDear, J.P., A revision of the New World Chrysomyini (Diptera) (Calliphoridae) (1985) Rev. Bras. Zool, 3, pp. 109-169Duenas, J.C.R., Panzetta-Dutari, G.M., Blanco, A., Gardenal, C.N., Restriction fragment-length polymorphism of the mtDNA A+T-rich region as a genetic marker in Aedes aegypti (Diptera: Culicidae) (2002) Ann. Entomol. Soc. Am, 95, pp. 352-358Erzinçlioglu, Y.Z., The application of entomology to forensic medicine (1983) Med. Sci. Law, 23, pp. 57-63Greenberg, B., Szyska, M., Immature stages and biology of fifteen species of Peruvian Calliphoridae (Diptera) (1984) Ann. Entomol. Soc. Am, 77, pp. 488-517Harvey, M.L., Dadour, I.R., Gaudieri, S., Mitochondrial DNA cytochrome oxidase I gene: Potential for distinction between immature stages of some forensically important fly species (Diptera) in western Australia (2003) Forensic Sci. Int, 131, pp. 134-139Infante, M.E.V., Azeredo-Espin, A.M.L., Genetic variability in mitochondrial DNA of screwworm Cochliomyia hominivorax (Diptera: Calliphoridae), from Brazil (1995) Biochem. Genet, 33, pp. 737-756Lessinger, A.C., Junqueira, A.C.M., Conte, F.F., Azeredo-Espin, A.M.L., Analysis of a conserved duplicate tRNA gene in the mitochondrial genome of blowflies (2004) Gene, 339, pp. 1-6Lessinger, A.C., Azeredo-Espin, A.M.L., Evolution and structural organisation of the mitochondrial DNA control region of myiasis-causing flies (2000) Med. Vet. Entomol, 14, pp. 71-80Litjens, P., Lessinger, A.C., Azeredo-Espin, A.M.L., Characterization of the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria by PCR-RFLP of mitochondrial DNA (2001) Med. Vet. Entomol, 15, pp. 183-188Liu, D., Greenberg, B., Immature stages of some flies of forensic importance (1989) Ann. Entomol. Soc. Am, 82, pp. 80-93Lunt, D.H., Zhang, D.X., Szymura, J.M., Hewitt, G.M., The insect cytochrome oxidase I gene: Evolutionary patterns and conserved primers for phylogenetic studies (1996) Insect Mol. Biol, 5, pp. 153-165Malgorn, Y., Coquoz, R., DNA typing for identification of some species of Calliphoridae of interest in forensic entomology (1999) Forensic Sci. Int, 102, pp. 111-119Marchenko, M.I., Medicolegal relevance of cadaver entomofauna for the determination of the time of death (2001) Forensic Sci. Int, 120, pp. 89-109Mello, R.P., Chave para identificação das formas adultas das espécies da família Calliphoridae (Díptera, Brachycera, Cyclorrhapha) encontradas no Brasil (2003) Entomol. Vect, 10, pp. 255-268Nuorteva, P., Sarcosaprophagous insects as forensic indicators (1977) Forensic medicine: A study in trauma and environmental hazards, pp. 1072-1095+1680. , In C.G. Tedeshi, W.G. Eckert & L.G. Tedeshi (eds.), Philadelphia, SaundersOtranto, D., Stevens, J.R., Molecular approaches to the study of myiasis-causing larvae (2002) Int. J. Parasitol, 32, pp. 1345-1360Schroeder, H., Klotzbach, H., Elias, S., Augustin, C., Pueschel, K., Use of PCR-RFLP for differentiation of calliphorid larvae (Diptera, Calliphoridae) on human corpses (2003) Forensic Sci. Int, 132, pp. 76-81Séguy, E., Étude sur quelques Calliphorinés testacés rares ou peu connus (1925) Bull. Mus. Natl. Hist. Nat, 31, pp. 439-441Simon, C.F., McIntosh, C., Deniega, J., Standard restriction fragment length analysis of the mitochondrial genome is not sensitive enough for phylogenetic analysis or identification of 17-year periodical cicada broods (Hemiptera: Cicadidae): The potential for a new technique (1993) Ann. Entomol. Soc. Am, 86, pp. 228-238Simon, C.F., Fratti, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers (1994) Ann. Entomol. Soc. Am, 87, pp. 657-701Smith, K.G.V., (1986) A manual of forensic entomology, p. 205. , Trustees of The British Museum National History and Cornell University PressSouza, A.M., Linhares, A.X., Diptera and Coleoptera of potential forensic importance in southeastern Brazil: Relative abundance and seasonality (1997) Med. Vet. Entomol, 11, pp. 8-12Sperling, F.A.H., Anderson, G.S., Hickey, D.A., A DNA-based approach to the identification of insect species used for post-mortem interval estimation (1994) J. Forensic Sci, 39, pp. 418-427Wallman, J.F., Donnellan, S.C., The utility of mitochondrial DNA sequences for the identification of forensically important blowflies (Diptera: Calliphoridae) in southeastern Australia (2001) Forensic Sci. Int, 120, pp. 60-67Wells, J.D., Sperling, F.A.H., DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae) (2001) Forensic Sci. Int, 120, pp. 110-115Zehner, R., Amendt, J., Schutt, S., Sauer, J., Krettek, R., Povolný, D., Genetic identification of forensically important flesh flies (Diptera: Sarcophagidae) (2004) Int. J. Legal Med, 118, pp. 245-247Zehner, R., Zimmermann, S., Mebs, D., Bratzke, H., RFLP and sequence analysis of the cytochrome b gene of selected animals and man: Methodology and forensic application (1998) Int. J. Legal Med, 111, pp. 323-327Zhang, D.X., Hewitt, G.M., Insect mitochondrial control region: A review of its structure, evolution and usefulness in evolutionary studies (1997) Biochem. Syst. Ecol, 25, pp. 99-12

Molecular Characterization Of Esterase E3 Gene Associated With Organophosphorus Insecticide Resistance In The New World Screwworm Fly, Cochliomyia Hominivorax

The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is one of the most important myiasis-causing flies in South America. It is responsible for severe economic losses to livestock producers, mainly because it causes mortality in newborn calves and reductions in the quality of leather and in the production of milk and meat. The economic losses caused by myiasis, along with those caused by other internal and external parasites, are the main factors limiting meat production. In Brazil, C. hominivorax has been controlled by applying insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of OP-resistant strains. This, associated with the fast development of OP resistance in other myiasis-causing flies, shows the importance of investigating resistance in C. hominivorax. Based on the findings of previous studies, the objective of the current work was to isolate and sequence the E3 gene in C. hominivorax. Mutations at the positions (Gly137 and Trp251) responsible for conferring OP resistance in Lucilia cuprina and Musca domestica L. (Muscidae) were identified in C. hominivorax. In addition, the orthologous region in C. hominivorax contained motifs that are highly conserved among carboxyl/cholinesterases and contribute to the catalytic mechanism of the active site. The characterization of this gene in natural populations of New World screwworm can be an important tool for monitoring resistance to insecticides throughout its current geographic distribution. 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