Analysis of Mitochondrial DNA Variability and Genetic Structure in Populations of New World Screwworm Flies (Diptera: Calliphoridae) from Uruguay

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Analysis of Mitochondrial DNA Variability and GeneticStructure in Populations of New World Screwworm Flies(Diptera Calliphoridae) from UruguayAuthor(s) Mariana Luacutecio Lyra Pablo Fresia Santos Gama Juan Cristina LouisBernard Klaczko and Ana Maria Lima de Azeredo-EspinSource Journal of Medical Entomology 42(4)589-595 2005Published By Entomological Society of AmericaDOI httpdxdoiorg1016030022-2585(2005)042[0589AOMDVA]20CO2URL httpwwwbiooneorgdoifull1016030022-2585282005290425B05893AAOMDVA5D20CO3B2

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POPULATION BIOLOGYGENETICS

Analysis of Mitochondrial DNA Variability and Genetic Structure inPopulations of New World Screwworm Flies (Diptera Calliphoridae)

from Uruguay

MARIANA LUCIO LYRA1 PABLO FRESIA2 SANTOS GAMA3 JUAN CRISTINA4

LOUIS BERNARD KLACZKO5 AND ANA MARIA LIMA DE AZEREDO-ESPIN5

Laboratorio de Genetica Animal Centro de Biologia Molecular e Engenharia Genetica Instituto de BiologiaUniversidade Estadual de Campinas (UNICAMP) PO Box 6010 Campinas Sao Paulo 13083-875 Brazil

J Med Entomol 42(4) 589ETH595 (2005)

ABSTRACT The New Word screwworm Cochliomyia hominivorax (Coquerel 1858) (DipteraCalliphoridae) is one of the most important insect pests of livestock in the Neotropical region In thiswork polymerase chain reaction-restriction fragment length polymorphism of mitochondrial DNA(mtDNA) was used to study the diversity and population structure of seven geographically distinctpopulations of C hominivorax from most of the important livestock areas in Uruguay The controlregion (AT12S) and subunits 1 and 2 of cytochrome oxidase (cox1cox2) were ampliTHORNed anddigested with restriction endonucleases Nine haplotypes were observed among the populationssampled The mean nucleotide diversity and the haplotype diversity indicated high mtDNA variabilityin this species The similarity index average nucleotide divergence and analysis of molecular varianceresults showed no evidence of subpopulation differentiation indicating that the C hominivoraxpopulations of Uruguay form a single panmitic population The distribution pattern of the geneticvariation in natural populations ofC hominivorax and the implications of these results for establishingcontrol program are discussed

KEY WORDS screwworm mitochondrial DNA polymerase chain reaction-restriction fragmentlength polymorphism population structure Uruguay

THE NEW WORLD SCREWWORM Cochliomyia homini-vorax (Coquerel 1858) (Diptera Calliphoridae) is anobligate ectoparasite that causes myiasis in warm-blooded vertebrates throughout the Neotropical re-gion (Guimaraes et al 1983) Adult females lay eggs inopen wounds where the emerging larvae feed(Guimaraes et al 1983) The infestation by these lar-vae generates an exudate that stimulates more femalesto lay their eggs in the wound resulting in injuriescontaining hundreds to thousands of larvae (Thomasand Mangan 1989) This mode of infestation makes Chominivorax one of the most important pests of live-stock and one of the most expensive to control andtreat (Hall and Wall 1995)

Historically the distribution of C hominivorax ex-tended from the southern United States to ArgentinaHowever this species has been successfully eradi-cated from North and most of Central America byusing the sterile insect technique (SIT) but it stilloccurs in the Caribbean islands and South Americaexcept for Chile (Wyss and Galvin 1996 IAEAFAO2000) In 1988C hominivoraxwas recorded outside ofthe Americas for the THORNrst time (in Libya) but it wasefTHORNciently combated using SIT (Vargas-Teran et al1994)

In Uruguay as in other South American countriesC hominivorax is one of the most important insectpests and represents a signiTHORNcant health problem forthe livestock industry often causing great economiclosses (Carballo et al 1990 IAEAFAO 2000)

Because of the economic importance of C homini-vorax and its inszliguence on the trade of live animalsamong infested and noninfested countries interna-tional efforts have been aimed at designing a programto control and eventually eradicate this species fromendemic areas and to prevent invasions into screw-worm-free areas A fundamental component of a suc-cessful pest management strategy such as SIT is agood understanding of the genetic diversity and struc-

1 E-mail marilyraunicampbr2 Seccion de Genetica Evolutiva Dpto de Biologotildea Animal Fac-

ultad de Ciencias Universidad de la Republica Avda Mataojo 2055Montevideo 11400 Uruguay

3 Laboratorio de Veterinaria Centro de Investigaciones NuclearesUniversidad de la Republica Avda Mataojo sn Montevideo 11400Uruguay

4 Dpto Tecnologotildea Nuclear Aplicada Centro de InvestigacionesNucleares Universidad de la Republica Avda Mataojo sn Montev-ideo 11400 Uruguay

5 Departamento de Genetica e Evolucao Instituto de BiologiaUniversidade Estadual de Campinas (UNICAMP) PO Box 6010Campinas SP 13083-875 Brazil

0022-2585050589ETH0595$04000 2005 Entomological Society of America

ture of the target populations (Roehrdanz 1989 In-fante-Vargas and Azeredo-Espin 1995)

Mitochondrial DNA (mtDNA) is a suitable markerfor studying microevolutionary processes in animalpopulations and can be a suitable marker for estimat-ing the genetic variability within populations (Avise etal 1987 Avise 1994 Rokas et al 2003) The restrictionfragment length polymorphism (RFLP) analysis ofmtDNA has been successfully used to study the ge-netic variability inC hominivorax populations and hasrevealed high variation in this molecule (Roehrdanzand Johnson 1988 Roehrdanz 1989 Infante-Vargasand Azeredo-Espin 1995 Taylor et al 1996) Anotherapproach for studying mtDNA variation is polymerasechain reaction (PCR)-RFLP analysis This powerfulmethod has been used in several population analyses(Ross et al 1997 Duenas et al 2002)

The aim of this study was to examine the geneticvariability among geographically distinct populationsof C hominivorax from Uruguay at the southern limitof this speciesOtilde distribution by using mtDNA PCR-RFLP analysis Inferences regarding the degree ofisolation of the populations analyzed were made bytesting the subdivided population hypothesis by usinganalysis of molecular variance (AMOVA)

Materials and Methods

Samples Larvae of C hominivorax from seven Uru-guayan localities including important livestock areaswere obtained from wounded sheep dogs or cattle inJanuary 2003 Collected larvae were reared in thelaboratory for species identiTHORNcation (Guimaraes et al1983) and were allowed to pupate or THORNxed in 100ethanol in the third instar The adults that emergedand the THORNxed larvae were used for DNA extractionsTotal nucleic acids were isolated using the phenolchloroform method as reported in Infante-Vargas andAzeredo-Espin (1995) and the DNA extracted wasstored at 20C In total 175 larvae were obtainedfrom 48 wounds

The geographic locations of the seven areas sam-pled (all low hills and plains in the transition from theArgentinean pampas to the hilly uplands of southernBrazil) are Banados de Medina-Cerro Largo (32 2300 S 54 21 00 W) Cerro Colorado-Florida (33 52 00S 55 33 00 W) Colonia-Colonia (34 20 00 S 57 8667 W) Dayman-Paysandu (31 33 00 S 57 57 00 W)Joaquotilden Suarez-Canelones (34 44 01 S 56 02 12 W)Paso Munoz-Salto (31 27 00 S 56 23 00 W) and SanAntonio-Salto (31 24 00 S 57 58 00 W) (Fig 1)

For haplotype frequency estimations and geneticcomparisons each haplotype found in a wound wasconsidered only once This approach avoids a bias inthe analysis caused by sampling the same mitochon-dria but it could bias the results toward higher esti-mates of diversity because common haplotypes wouldbe counted only once in multiple infections Howeverthis conservative approach that reduces sample sizewas preferred to an artiTHORNcial overestimation of theprecision of this study Moreover because sibling lar-

vae have a gregarious behavior this putative bias tendsto be actually minimized

Using this approach the number of C hominivoraxconsidered in the analysis of genetic variation was 65and it was sufTHORNcient to provide information at thehierarchical level of the populationAmplifications Two speciTHORNc mtDNA regions were

ampliTHORNed one region with 2100 bp included the com-plete control region and partial rRNA 12S sequences(AT-rich12S) and another region with 2360 bp in-cluded the entire sequences of cytochrome oxidasesubunits 1 and 2 (cox1cox2) PCR assays were doneas described by Lessinger and Azeredo-Espin (2000)and Litjens et al (2001) by using a PTC-200 (MJResearch Watertown MA) thermal cycler The UBC-insect mtDNA oligonucleotide set described by Simonet al (1994) was used for AT rich12S ampliTHORNcationwith the primers TM-N-193 and SR-J-14233 the prim-ers TY-J-1460 and TK-N-3785 were used for cox1cox2ampliTHORNcation The PCR products were evaluated byelectrophoresis in 10 agarose gels stained withethidium bromide (EtBr) in 1 TAE (40 mM Tris-acetate 1 mM EDTA) bufferRFLP Procedures For a preliminary survey to de-

tect mtDNA polymorphisms three individuals wererandomly chosen from different Uruguayan and Bra-zilian localities The Brazilian samples were from pre-vious DNA extractions used in RFLP analysis ofmtDNA (Infante-Vargas and Azeredo-Espin 1995)The samples were ampliTHORNed and digested to provideinitial information on the restriction sites present inthe mtDNA regions analyzed and their usefulness forpopulation analysis

Aliquots of PCR products of the AT-rich12S andcox1cox2 regions were single digested for 4 h at 37Caccording to the enzyme supplierOtildes protocols (Invitro-gen Carlsbad CA and PTHORNzer Inc New York NY)The digested fragments were separated by electro-phoresis in 2 agarose gels stained with EtBr andphotographed using the Kodak EDAS 290 software inan UV trans-illuminator The size of the fragments wasestimated by comparison with the molecular size stan-dard DNA Ladder Plus 1Kb (12 kb-100 pb Invitrogen)by using regression analysis carried out manuallyDigestions with enzymes that produced different re-

Fig 1 Geographic locations of the screwworm popula-tions sampled in this study

590 JOURNAL OF MEDICAL ENTOMOLOGY Vol 42 no 4

striction patterns were repeated to conTHORNrm that theobserved patterns do not result from partial digestsFragments 200 bp were not included in the analysis

The PCR products of the AT-rich12S sequenceswere digested with nine restriction endonucleasesCla IDra I EcoRVHae IIIHind IIIMsp IMun I SspI andSau96 I three of the enzymes (Hae IIIMsp I andMun I) did not cut the PCR product THORNve of the en-zymes (Cla I EcoR V Hind III Ssp I and Sau96 I)produced only a single cleavage pattern for the sam-ples analyzed and onlyDra I detected polymorphismin this region (Table 1) The PCR products of thecox1cox2 region were digested with 13 restrictionendonucleasesAse IDde IDra IEcoR IEcoRVHindIIIHpa IMsp IRsa I Sma I Ssp I Sst I andXho I fourof the enzymes (EcoR IHind IIIHpa I and Sma I) didnot cut the PCR product seven of the enzymes (DdeIDra IEcoRVRsa I Ssp I Sst I andXho I) producedonly a single cleavage pattern for the samples analyzedand the restriction endonucleases Ase I and Msp Ishowed diagnostic restriction patterns (Table 1) Theenzymes were selected based on a previous RFLPanalysis and PCR-RFLP results for C hominivorax(Infante-Vargas and Azeredo-Espin 1995 Litjens et al2001) and on an analysis of the mtDNA AT rich12Sand cox1cox2 sequences available for mtDNA C

hominivorax species (Lessinger et al 2000) by usingthe WebCutter software (Heiman 1997) The Uru-guayan samples were analyzed after the diagnosticenzymes had been selected for both regionsData Analysis Haplotype Analyses The different

restriction patterns for both regions obtained witheach enzyme were designated with capital lettersaccording to the order in which they were detectedFor each individual these letters were compiled intoa composite haplotype designated by numbers (Table2) Each haplotype was scored by a vector of 0s and 1s(absence and presence of a fragment respectively)that represented the components of their PCR-RFLPphenotypes and a matrix was constructed to be usedas an input THORNle in the analyses of genetic variation Theevolutionary distance (d) between all pairwise com-parisons of haplotypes was estimated according to Neiand Li (1979) and Nei (1987) (equation 555) by usingthe REAP software (McElroy et al 1992) consideringthe different size of restriction endonuclease recog-nitions sequences (r-value 4 or 6) (Nei 1987)Diversity The genetic diversity within each popu-

lation analyzed was interpreted using the estimate ofhaplotype diversity (Hs) and nucleotide diversity ()Haplotype frequency distributions for each popula-tion and the associated d values among haplotypeswere used to estimate these diversity indices Haplo-type diversity was estimated according to Nei (1987)(equations 84 85 and 812) and nucleotide diversitywas estimated according to Nei and Tajima (1981) byusing the REAP software (McElroy et al 1992)PopulationDifferentiation The variation among the

populations analyzed and the population differentia-tion were interpreted using different indices The sim-ilarity index (F) or the proportion of shared fragmentsbetween populations was calculated for each possiblepairwise comparison of populations This index wasestimated according to Nei and Li (1979) as F 2Nxy(Nx Ny) where Nx and Ny are the numbers offragments in populations x and y respectively and Nxy

is the number of fragments shared by the two popu-lations The nucleotide divergence () was estimatedaccording to Nei and Tajima (1981) by using theREAP software (McElroy et al 1992)

Table 1 Diagnostic restriction patterns obtained by PCR-RFLP of C hominivorax from Uruguay

Region Enzyme mtDNA Pattern (bp)

AT12S Dra I A 800 320 200 200B 1000 320 200C 1900 320 200 200

cox1cox2 Ase I A 1100 460 350 220B 1100 680 350

Msp I A 1500 480B 1400 480C 1500 300D 870 680 300E 1400 300

Capital letters indicate polymorphic restriction patterns Frag-ments 200 bp were not considered The existence of an unknown noof small fragments accounts for the sum of the fragments being lessthan that of the intact ampliTHORNed segment C represents the fragmentsize polymorphism

Table 2 Distribution of mtDNA haplotypes in Uruguayan populations

Haplotype JoS BaM Col CeC PaM Day SaA Total Freq

1 AAC 2 (7) 5 (14) 3 (12) 6 (15) 3 (12) 7 (17) 7 (10) 33 (87) 0512 AAD 1 (3) 1 (3) 0023 ABA 3 (17) 4 (11) 3 (5) 3 (8) 1 (1) 14 (42) 0224 ABB 2 (8) 2 (10) 6 (10) 1 (2) 1 (4) 12 (34) 0185 BAC 1 (1) 1 (1) 0026 ABE 1 (2) 1 (2) 0027 CAC 1 (3) 1 (3) 0028 AAA 1 (2) 1 (2) 0029 ABC 1 (1) 1 (1) 002Total 5 (24) 12 (36) 5 (22) 16 (32) 7 (22) 10 (21) 10 (18) 65 (175) 100No wounds 5 8 5 10 4 9 7 48

The haplotypes are designated by a number and a combination of the three restriction enzymes patterns (DraIAseI andMspI) The numbersin parentheses indicate the total number of individuals found with a haplotype and those without parentheses indicate the number of individualsused for population analyses BaM Banados de Medina CeC Cerro Colorado Col Colonia Day Dayman JoS Joaquotilden Suarez PaM PasoMunoz SaA San Antonio Freq frequency

July 2005 LYRA ET AL C hominivorax POPULATIONS FROM URUGUAY 591

To study the distribution of genetic variation withinand among populations an AMOVA was done usingARLEQUIN version 20 (Schneider et al 2001) Thedegree of isolation of the populations was interpretedusing the -statistic (ST) parameter This analysiswas done by considering the number of pairwise dif-ferences and the evolutionary distance (d) betweenhaplotypes The signiTHORNcance of the variance compo-nents and ST was computed using a nonparametricpermutation test (ExcofTHORNer et al 1992)

Results

Genetic Variation Two fragments were observedafter ampliTHORNcation of the AT-rich12S sequencesThe most frequent fragment in the Uruguayan popu-lations contained 2100 bp and occurred in 98 of thesamples this fragment was previously described byLitjens et al (2001) The second fragment contained3200 bp and was identiTHORNed in three individuals fromthe same wound in the locality of San Antonio Thenature of this variation is still under investigation TheampliTHORNed cox1cox2 sequences showed no size poly-morphism

Digestion of the AT-rich12S sequences with theenzymeDraI produced three diagnostic patterns Pat-terns A and B were digestion products of the 2100-bpfragment (restriction site polymorphism) and patternC was identiTHORNed when the 3200-bp fragment was di-gested (fragment size polymorphism) (Table 1) Pat-tern A occurred in 96 of the samples analyzed Forcox1cox2 sequences the enzyme MspI yielded THORNvediagnostic patterns for the populations whereas theenzyme Ase I presented two different patterns (Table1) The existence of an unknown number of smallfragments meant that the total size for the sum of thefragments was less than that of the intact ampliTHORNedfragment

We examined 25 (42 kb) of the C homini-vorax mitochondrial genome (Lessinger et al 2000)and nine haplotypes were identiTHORNed on the basis of therestriction patterns Table 2 shows the distribution andfrequency of the haplotypes found at the differentUruguayan localities sampled Haplotype 1 was themost frequent (51) in the sample as a whole and atmost of the localities and it was present in all popu-lations Haplotypes 3 and 4 represented 22 and 18 ofthe total sample respectively and they were widelydistributed among the populations The distance es-timates between pairs of common haplotypes wasd(1vs3) 00227 d(1vs4) 00719) and d(3vs4) 00110 The others six haplotypes had a local distribu-

tion and represented a very low proportion of the totalsample (2 each)

Only one of these six rare haplotypes was found inthe three southern locations (Colonia Joaquotilden Suarezand Cerro Colorado) near the Uruguayan coast andTHORNve of these haplotypes were found near the borderwith Brazil in northwestern and northeastern loca-tions Although this distribution of haplotype wouldsuggest a decrease in diversity at the edge of thespeciesOtilde range in Uruguay an AMOVA was conductedto compare the two groups and the results (data notshown) suggest that they are not different

On average the gene diversity within populationsbased on halotypes (Hs) was 06355 and the nucleotidediversity () was 00229 (Table 3) The levels of di-versity did not differ greatly among the populationsBoth indices indicated that the Uruguayan C homi-nivorax populations were highly polymorphicPopulationDifferentiationThe overall estimates of

nucleotide divergence () similarity (F) and ST

parameters are shown in Table 3 The degree of ge-netic divergence of DNA sequences between twopopulations is expected to be correlated with the pro-portion of DNA fragments that they share (Nei and Li1979) The high similarity (967) and the low nucle-otide divergence ( 000055) estimated for the pop-ulations agreed with this correlation and indicate thatthe sampled populations were very similar

Two hierarchical AMOVA were used to investigatepopulation differentiation one considered the pair-wise differences among populations and the otherconsidered NeiOtildes genetic distance The THORNrst revealedthat 924 of the genetic variation was attributable tothe variance within populations and the second that855 of this variation can be ascribed to the samehierarchical level Although both estimates of the ST

values among populations indicated some subpopula-tion differentiation they were not statistically signif-icant (Table 3)

Discussion

PCR-RFLP analysis of the two ampliTHORNed regions ofthe C hominivorax mitochondrial genome by usingthree restriction endonucleases revealed high geneticvariability with nine haplotypes in the seven Uru-guayan screwworm populations sampled (Fig 1 Table2)

The PCR-RFLP analysis revealed marked polymor-phism in the cox1cox2 region at the intra- and inter-populational levels and was useful for deTHORNning differ-ent haplotypes However the AT12S region

Table 3 Estimates for the indices of genetic variation within and among populations

Genetic variation within populations Genetic variation among populations

Hs F ST (pd) ST (nd)

0022975( 209 105)

06355( 117 103)

0967 0000555( 12 106)

00762(df 6 58 P 00865)

01449(df 6 58 P 08768)

nucleotide diversity Hs haplotype diversity F similarity nucleotide divergence ST (pd) F-statistic based on pairwise differences(W) ST (nd) F-statistic based on NeiOtildes evolutionary distance (d)


revealed lower PCR-RFLP than expected For exam-ple only one pattern was found for 96 of the samplesanalyzed indicating that the PCR-RFLP approach wasnot efTHORNcient in detecting variation in the AT12Sregion This result could be attributed to the lowresolution of the 2 agarose gel used to screen re-striction patterns containing fragments 200 bp insize In any case the size variation in the AT12Sregion is an indication of length polymorphism as amolecular marker for screen intraspeciTHORNc variation

The high genetic variability was consistent withconclusions reached using independent data setsbased on mtDNA RFLP for populations from NorthAmerica Central America and Brazil which de-scribedChominivorax as a polymorphic species (Roe-hrdanz 1989 Azeredo-Espin 1993 Infante-Vargas andAzeredo-Espin 1995 Taylor et al 1996)

The nucleotide diversity (23) estimated for Chominivorax in this study is the highest value obtainedfor this species and is much higher than the samediversity previously described for Brazilian popula-tions (092) (Infante-Vargas and Azeredo-Espin1995) and for North and Central American popula-tions (13) (Roehrdanz 1989) This high value prob-ably reszligects the inclusion of only polymorphic frag-ments However gene diversity is not affected by thechoice of fragments It can be interpreted as the prob-ability that two randomly chosen szligies in a populationhave different haplotypes The average value of genediversity estimated here (Hs 0633) indicates a highlevel of genetic variation for C hominivorax and re-inforces the results previously obtained

Comparisons among the Uruguayan screwwormpopulations clearly indicated that there was no evi-dence of subpopulation differentiation The presenceof the common haplotype 1 at a high frequency in allpopulations and the wide distribution of haplotypes 3and 4 suggested that the populations were very similarThe high value of the similarity index (967) con-THORNrmed this observation There were six local haplo-types but the divergence between each of them andthe common haplotypes was very low (just one mu-tational step d data not shown) For this reason theestimates of nucleotide divergence between popula-tions was very low ( 000055) indicating that thepopulations analyzed were very similar

The AMOVA showed that the genetic variabilitywas distributed mainly within populations This THORNnd-ing and the ST estimates provide evidence that therewas no genetic differentiation by natural forces suchas drift and selection thus reinforcing the evidencethat the screwworm populations of Uruguay are aunique panmictic population

The population structure of C hominivoraxthroughout its distribution has been a controversialtopic Allozyme studies have found no differentiationamong North and Central American screwworm pop-ulations (Krafsur and Whitten 1993 Taylor and Peter-son 1994) and only moderate differentiation in Bra-zilian populations (Infante-Malachias 1999) Infante-Malachias et al (1999) studied C hominivoraxpopulations from southeastern Brazil and northern

Argentina by using random ampliTHORNcation of polymor-phic DNA (RAPD) and found moderate populationdifferentiation despite the low genetic distance ThemtDNA RFLP analysis of four populations from thestate of Sao Paulo in Brazil (Infante-Vargas and Az-eredo-Espin 1995) corroborated the results obtainedwith RAPD and allozymes for South American pop-ulations and suggested that the populations were con-nected by reduced gene szligow Roehrdanz and Johnson(1988) and Roehrdanz (1989) used RFLP of mtDNAto study populations from Texas Mexico JamaicaCosta Rica and Guatemala both studies analyzed se-quence diversity among populations and concludedthat there was some differentiation among the ldquomain-landrdquo versus Jamaican samples and less variationamong samples from northern Mexico and Texas

The distribution of mtDNA polymorphism at a geo-graphic level among the Uruguayan screwworm pop-ulations suggested a panmictic population in the ex-treme south of this speciesOtilde occurrence Based on thedistribution ofC hominivoraxmtDNA polymorphismsin the Neotropical region we concluded that the pop-ulations of this szligy cannot be differentiated at the limitsof the speciesOtilde distribution in contrast to the variationfound in southeastern Brazil based on RFLP ofmtDNA (Infante-Vargas and Azeredo-Espin 1995)Further analyses using populations from other SouthAmerica countries with emphasis in BrazilianC homi-nivorax populations are being done to elucidate theintraspeciTHORNc genetic variability in this species at ageographic level

The lack of genetic structure among the screwwormpopulations in Uruguay may reszligect the effect thatthere are no geographical barriers or important cli-matic differences among the regions studied This ab-sence of barriers would facilitate the natural disper-sion of szligies within Uruguay Second according to theUruguayan Ministry of Agriculture (MGAP 2002) allregions of the country have a large livestock popula-tion such that the trading of animals possibly infectedwith C hominivorax could contribute to the dispersalof this species and results in more homogeneous pop-ulations

Because our data showed that the populations of Chominivorax in Uruguay form a single panmictic andhighly polymorphic population some implications forsterile control programs need to be discussed Roe-hrdanz (1989) showed that the genetic variability inmtDNA in the form of different haplotypes was ex-tensive and that the sequence divergence was notgreat among populations from Texas Mexico CostaRica Guatemala and Jamaica but with greater dif-ferences between Jamaican and ldquomainlandrdquo popula-tions This extensive variability in mtDNA has notadversely affected the eradication programs becauseSIT has effectively eliminated theC hominivorax fromMexico and from some Central American countries(Wyss and Galvin 1996) In Uruguay we obtainedsimilar results to those reported by Roehrdanz (1989)which suggests that this country could be a place ofchoice for testing the efTHORNciency of SIT in South Amer-ica Because sterile insect release programs either as


a holding buffer zone or an eradication campaignrequire knowledge of the composition of the targetspecies to determine optimal strategies we are nowusing other molecular markers such as microsatellite(Torres et al 2004) to obtain a better understandingof the genetic structure of C hominivorax in SouthAmerica

Acknowledgments

We thank R A Rodrigues and M S Couto for valuabletechnical and laboratory assistance M T de Oliveira SLanzzeri E Martines and A Maggi for help in collecting thesamples T T Torres for valuable suggestions on the manu-script and data analyses and R L Roehrdanz and two anon-ymous reviewers for critical reviews of the manuscript Thisresearch was supported by grants to AMLAE from Fun-dacao de Amparo a Pesquisa do Estado de Sao Paulo (grant0301458-9) Conselho Nacional de Desenvolvimento Cien-totildeTHORNco e Tecnologico (grant 47113201-2) and the Interna-tional Atomic Energy Agency (grant 11822RO) MLL wassupported by a fellowship from Fundacao de Amparo a Pes-quisa do Estado de Sao Paulo (grants 0112528-2 and 0313598-0)

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Received 24 January 2005 accepted 23 March 2005


POPULATION BIOLOGYGENETICS

Analysis of Mitochondrial DNA Variability and Genetic Structure inPopulations of New World Screwworm Flies (Diptera Calliphoridae)

from Uruguay

MARIANA LUCIO LYRA1 PABLO FRESIA2 SANTOS GAMA3 JUAN CRISTINA4

LOUIS BERNARD KLACZKO5 AND ANA MARIA LIMA DE AZEREDO-ESPIN5

Laboratorio de Genetica Animal Centro de Biologia Molecular e Engenharia Genetica Instituto de BiologiaUniversidade Estadual de Campinas (UNICAMP) PO Box 6010 Campinas Sao Paulo 13083-875 Brazil

J Med Entomol 42(4) 589ETH595 (2005)

ABSTRACT The New Word screwworm Cochliomyia hominivorax (Coquerel 1858) (DipteraCalliphoridae) is one of the most important insect pests of livestock in the Neotropical region In thiswork polymerase chain reaction-restriction fragment length polymorphism of mitochondrial DNA(mtDNA) was used to study the diversity and population structure of seven geographically distinctpopulations of C hominivorax from most of the important livestock areas in Uruguay The controlregion (AT12S) and subunits 1 and 2 of cytochrome oxidase (cox1cox2) were ampliTHORNed anddigested with restriction endonucleases Nine haplotypes were observed among the populationssampled The mean nucleotide diversity and the haplotype diversity indicated high mtDNA variabilityin this species The similarity index average nucleotide divergence and analysis of molecular varianceresults showed no evidence of subpopulation differentiation indicating that the C hominivoraxpopulations of Uruguay form a single panmitic population The distribution pattern of the geneticvariation in natural populations ofC hominivorax and the implications of these results for establishingcontrol program are discussed

KEY WORDS screwworm mitochondrial DNA polymerase chain reaction-restriction fragmentlength polymorphism population structure Uruguay

THE NEW WORLD SCREWWORM Cochliomyia homini-vorax (Coquerel 1858) (Diptera Calliphoridae) is anobligate ectoparasite that causes myiasis in warm-blooded vertebrates throughout the Neotropical re-gion (Guimaraes et al 1983) Adult females lay eggs inopen wounds where the emerging larvae feed(Guimaraes et al 1983) The infestation by these lar-vae generates an exudate that stimulates more femalesto lay their eggs in the wound resulting in injuriescontaining hundreds to thousands of larvae (Thomasand Mangan 1989) This mode of infestation makes Chominivorax one of the most important pests of live-stock and one of the most expensive to control andtreat (Hall and Wall 1995)

Historically the distribution of C hominivorax ex-tended from the southern United States to ArgentinaHowever this species has been successfully eradi-cated from North and most of Central America byusing the sterile insect technique (SIT) but it stilloccurs in the Caribbean islands and South Americaexcept for Chile (Wyss and Galvin 1996 IAEAFAO2000) In 1988C hominivoraxwas recorded outside ofthe Americas for the THORNrst time (in Libya) but it wasefTHORNciently combated using SIT (Vargas-Teran et al1994)

In Uruguay as in other South American countriesC hominivorax is one of the most important insectpests and represents a signiTHORNcant health problem forthe livestock industry often causing great economiclosses (Carballo et al 1990 IAEAFAO 2000)

Because of the economic importance of C homini-vorax and its inszliguence on the trade of live animalsamong infested and noninfested countries interna-tional efforts have been aimed at designing a programto control and eventually eradicate this species fromendemic areas and to prevent invasions into screw-worm-free areas A fundamental component of a suc-cessful pest management strategy such as SIT is agood understanding of the genetic diversity and struc-

1 E-mail marilyraunicampbr2 Seccion de Genetica Evolutiva Dpto de Biologotildea Animal Fac-

ultad de Ciencias Universidad de la Republica Avda Mataojo 2055Montevideo 11400 Uruguay

3 Laboratorio de Veterinaria Centro de Investigaciones NuclearesUniversidad de la Republica Avda Mataojo sn Montevideo 11400Uruguay

4 Dpto Tecnologotildea Nuclear Aplicada Centro de InvestigacionesNucleares Universidad de la Republica Avda Mataojo sn Montev-ideo 11400 Uruguay

5 Departamento de Genetica e Evolucao Instituto de BiologiaUniversidade Estadual de Campinas (UNICAMP) PO Box 6010Campinas SP 13083-875 Brazil

0022-2585050589ETH0595$04000 2005 Entomological Society of America
























AT12S Dra I A 800 320 200 200B 1000 320 200C 1900 320 200 200

cox1cox2 Ase I A 1100 460 350 220B 1100 680 350

Msp I A 1500 480B 1400 480C 1500 300D 870 680 300E 1400 300








Results











Discussion






0022975( 209 105)

06355( 117 103)

0967 0000555( 12 106)

00762(df 6 58 P 00865)

01449(df 6 58 P 08768)















Acknowledgments


References Cited





























































AT12S Dra I A 800 320 200 200B 1000 320 200C 1900 320 200 200

cox1cox2 Ase I A 1100 460 350 220B 1100 680 350

Msp I A 1500 480B 1400 480C 1500 300D 870 680 300E 1400 300








Results











Discussion






0022975( 209 105)

06355( 117 103)

0967 0000555( 12 106)

00762(df 6 58 P 00865)

01449(df 6 58 P 08768)















Acknowledgments


References Cited















































AT12S Dra I A 800 320 200 200B 1000 320 200C 1900 320 200 200

cox1cox2 Ase I A 1100 460 350 220B 1100 680 350

Msp I A 1500 480B 1400 480C 1500 300D 870 680 300E 1400 300








Results











Discussion






0022975( 209 105)

06355( 117 103)

0967 0000555( 12 106)

00762(df 6 58 P 00865)

01449(df 6 58 P 08768)















Acknowledgments


References Cited







































Results











Discussion






0022975( 209 105)

06355( 117 103)

0967 0000555( 12 106)

00762(df 6 58 P 00865)

01449(df 6 58 P 08768)















Acknowledgments


References Cited


















































Acknowledgments


References Cited







































Acknowledgments


References Cited












































Documents

Analysis of Mitochondrial DNA Variability and Genetic Structure in Populations of New World Screwworm Flies (Diptera: Calliphoridae) from Uruguay