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Faculty Publications Department of Entomology Entomology Department of

2002

Random amplified polymorphic DNA markers fordiscriminating Cochliomyia hominivorax from Cmacellaria (Diptera Calliphoridae)S R SkodaUSDA-ARS SteveSkodaarsusdagov

Saowaluck PornkulwatUniversity of Nebraska-Lincoln

John E FosterUniversity of Nebraska-Lincoln johnfosterunledu

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Part of the Entomology Commons

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Skoda S R Pornkulwat Saowaluck and Foster John E Random amplified polymorphic DNA markers for discriminatingCochliomyia hominivorax from C macellaria (Diptera Calliphoridae) (2002) Faculty Publications Department of Entomology 551httpdigitalcommonsunleduentomologyfacpub551

Bulletin of Entomological Research (2002) 92 89ndash96 DOI 101079BER2001135

Random amplified polymorphic DNAmarkers for discriminating Cochliomyia

hominivorax from C macellaria(Diptera Calliphoridae)

SR Skoda1 S Pornkulwat2 and JE Foster2

1USDA-ARS-MLIRU University of Nebraska Lincoln NE 68583-0938USA 2Department of Entomology Insect Genetics Laboratory University

of Nebraska Lincoln NE 68583-0816 USA

Abstract

The screwworm Cochliomyia hominivorax (Coquerel) is one of the mostimportant pests of livestock in the Western Hemisphere During early immaturestages it is morphologically very similar (first instars are virtually indistinguish-able) to the secondary screwworm C macellaria (Fabricius) Here the utility of therandom amplified polymorphic DNAndashpolymerase chain reaction (RAPDndashPCR)was explored as a technique for developing molecular genetic markers for thesetwo species Of the 120 arbitrary primers screened 21 primers produced markersthat were further investigated Seven of the 21 primers produced clear andreproducible markers that were tested with DNA of five individuals from fourpopulations of each species five of these primers showed 12 RAPD markers thatdifferentiated the species in all populations Analyses of data from these sevenprimers also suggested that intraspecific polymorphisms exist that could be usefulin distinguishing populations of screwworms Some population genetic tools suchas genetic distance cluster analysis and bootstrapping were used to statisticallyexplore these polymorphisms The resulting statistics showed 100 support for theability of RAPDndashPCR to discriminate between the two species Bootstrapping withdata from one of the genetic distance calculations produced a tree with allindividual screwworms in the correct populations indicating that RAPDndashPCR haspromise for displaying intraspecific genetic variation that could be used inestablishing the general geographic origin of screwworm samples

Introduction

The screwworm Cochliomyia hominivorax (Coquerel)(Diptera Calliphoridae) also known as the New World(NW) screwworm is unlike other calliphorids in the WesternHemisphere because it causes primary myiasis in livestockand sometimes humans This species once a very importanteconomic pest in the USA and wherever it occurs in theWestern Hemisphere has been eradicated from the USAMexico and much of Central America by application of the

sterile insect technique through formal screwwormeradication programmes (Wyss amp Galvin 1996)

Other non-pest species particularly the secondaryscrewworm C macellaria (Fabricius) (DipteraCalliphoridae) which normally deposits eggs on anddevelops in the carcasses of dead animals may also infestwounds already infested by C hominivorax (Metcalf ampMetcalf 1993) Early larval stages particularly first instars ofC hominivorax and C macellaria are very difficult todistinguish (Hall 1948 Leite amp Guevara 1993) In factCushing amp Patton (1933) recognized that C hominivorax andC macellaria were in fact two species and not one as they hadbeen mistakenly considered Typically eradicationprogramme personnel collect suspected screwworm cases as

Author for correspondenceFax (402) 437ndash5260E-mail sskoda1unledu

third instars which are reasonably easy to identify especiallyby experienced personnel in an eradication programme Butunder various circumstances (ie when animals are preparedfor export or when suspected cases of myiasis are closelymonitored in areas where C hominivorax has recently beeneradicated) samples that consist of first instars aresubmitted for identification and may be misidentifiedReliable rapid and cost effective identification techniquesthat complement identification based on morphologicalcharacters would increase efficiency in the screwwormeradication programme and in exclusion efforts

Random amplified polymorphic DNA (RAPD) (Welsh ampMcClelland 1990 Williams et al 1990) is a polymerase chainreaction (PCR) technique that allows detection of manypolymorphisms within the genomic DNA in a short timeRAPD markers are generated by the amplification ofrandom DNA segments with single primers of arbitrarynucleotide sequence Polymorphisms most of which areinherited as dominant traits are detected as the presence orabsence of amplification products from a single locus(Williams et al 1990) This technique has been used formany systematic and population genetic studiesRAPDndashPCR requires very small amounts of DNA and can beused with very small insects (Black et al 1992) It is rapidand relatively inexpensive compared with restrictionfragment length polymorphism analysis or DNA sequencing(Hoy 1994) Furthermore this technique allows linkagemaps to be developed (Hunt amp Page 1995)

Here RAPDndashPCR was used to examine DNA primingand banding patterns among wild and laboratorypopulations of C hominivorax and C macellaria Fourpopulations of both species representative of NorthAmerica Central America South America and theCaribbean were used to determine which primers produceddiagnostic markers for these two species over theirgeographic range Also examined was the potential ofRAPDndashPCR for detecting genetic polymorphisms in samplesof C hominivorax that could be useful for determining theirgeographic origin

Materials and methods

Insect specimens

Fifteen third instars from the C hominivorax straindesignated CR-91 and the C macellaria strain designatedCMN both reared in the Biosecure Facility (BSL-II) of theMidwest Livestock Insect Research Unit (MLIRU) in LincolnNebraska were used for initial screening Then fiveindividuals from each of four populations of C hominivorax(CR-91 Costa Rica RJS southern Brazil PA-34 southernMexico and CHJ Jamaica) and C macellaria (CMNNebraska RJM southern Brazil CRMB Costa Rica andCMJ Jamaica) were used for confirming species-specificmarkers and detecting potential C hominivorax populationpolymorphisms Specimens were maintained at 80degC untilDNA isolation

DNA isolation

Total genomic DNA was isolated from frozenindividuals The gut of each insect was removed beforeindividual insects were ground with a pestle within an15 ml microcentrifuge tube Then 50 l of 2-mercaptoethanol

and 50 l of stock buffer (100 mM of NaCl 100 mM of TrisHCl and 100 mM EDTA) were added to the minced tissue ofindividual flies The DNA solution was incubated afterproteinase K and lysis buffer (stock buffer and 25 SDS)were added for 2 h at 55degC and then overnight at 37degC Thiswas followed by chloroformphenol extraction DNA wasprecipitated using 95 ice-cold ethanol DNA pellets werecollected and dissolved in 50 l of TE buffer and diluted 20times before use in RAPDndashPCR

Polymerase chain reaction

All PCRs were performed using the Perkin-ElmerGeneAmpreg PCR System 9600 and reagents used for PCRwere obtained from Applied Biosystems (Foster CityCalifornia USA) The diluted DNA solution(approximately 25 ng l1) was used for the PCRs Threeto five DNA samples from the C hominivorax strain CR-91and the C macellaria strain CMN were used for initialscreening with 120 primers (sets OPA OPB OPC OPDOPE and OPF consisting of 20 unique 10-mer primers ineach set Operon Technologies Inc Alameda CaliforniaUSA) Optimization of the PCR followed Pornkulwat et al(1998) Primers that generated many bands with complexbanding patterns were eliminated Those primers withpotentially discriminating patterns were further testedusing DNA from 10ndash15 insects After the PCR was shownto be reproducible using DNA from the same individualsthen primers with consistent banding patterns were usedwith five individuals from four populations of both Chominivorax and C macellaria

Polymerase chain reactions were carried out in a totalvolume of 25 l for each primer A master mix consisted ofeach of the following components (multiplied by the totalnumber of tubes) 122 l of sterile distilled water 1 l of 1Nonidet P-40 25 l of 10 Stoffel buffer (AppliedBiosystems Foster City California) 3 l of dNTPs (10 mM ofeach dATP dCTP dGTP dTTP) 4 l of 25 mM MgCl2 1 l ofeach diluted DNA sample (120) and 03 l of AmpliTaqregDNA Polymerase Stoffel fragment (Applied BiosystemsFoster City California) Twenty-four microlitres of themaster mix were aliquoted to individual tubes One hundredpicomoles (1 l) of each 10-mer primer were then added tothe appropriate tube A negative control (PCR mix withoutDNA template) was prepared to check whether there wascarry-over (or contamination)

The following temperature profile was used an initialdenaturation at 95degC for 5 min 10 cycles of 94degC for 1 min36degC for 30 s and 72degC for 1 min followed by 30 cycles of94degC for 10 s 35degC for 30 s and 72degC for 30 s An additionalextension step of 72degC for 5 min was done About 10 l ofthe PCR products were loaded on 15 Ultrapure AgaroseGel (Gibco-BRL Gaithersburg Maryland USA) and elec-trophoresed at 90 V for 3 h Each primerrsquos RAPD productsfrom C hominivorax (top 20 wells) and C macellaria (bottom20 wells) samples were run on the same gel (Horizon 20ndash25electrophoresis chamber Gibco-BRL GaithersburgMaryland) DNA molecular weight standards were addedto the outside two wells of all C hominivorax and Cmacellaria sample sets negative controls were added to thebottom set of wells on each gel The gel was then stained inethidium bromide solution for 15ndash20 min andphotographed

90 SR Skoda et al

Data analysis

Photographs of the gels were scanned (600 dpi Hewlett-Packard Greeley Colorado USA) and analysed with imageanalysis software (Advanced QuantifierTM GenomicSolutions Inc Ann Arbor Michigan USA) this standardizedband detection and the interpretation of all bandsrsquo molecularweights Only the most visible and reproducible bands wereselected as species-specific markers (Roderick 1996)

For statistical analyses of inter- and intra-specific geneticvariation the first step was to acknowledge severalnecessary assumptions (such as genotype frequencies atRAPD loci are in Hardy-Weinberg proportions) andprecautions (such as variability of the RAPDndashPCRtechnique) (Black 1993 Haymer 1994 Lynch amp Milligan1994) RAPD banding information was coded as a matrix of1rsquos (band present) and 0rsquos (band absent) and used incomputer programs (written in FORTRAN programminglanguage and available by anonymous file transfer protocol(FTP) from lamarcolostateedupubwbc4) writtenspecifically for use with data generated by RAPDndashPCR(Apostol et al 1996) The program RAPDDIST was used tocalculate Neirsquos genetic distances (Nei 1972) applying Lynchamp Milliganrsquos (1994) correction between the populations ofthe two species RAPDPLOT was used to calculate geneticdistances between individuals from populations of bothspecies using the formula of Nei amp Li (1985) RAPDPLOTwas also used separately to calculate genetic distances ofthe individuals in the screwworm populations values fromboth Nei amp Li (1985) and Apostol et al (1993) were compared(designated 1-S and 1-M respectively in the RAPDPLOTprogram) Bootstrapping (100 replicates) available in bothRAPDDIST and RAPDPLOT was used to test theconsistency of the estimated relationships amongpopulations and individuals Genetic distance data fromresultant bootstrapping with either the RAPDPLOT orRADPDIST programs were then used in the PHPYLIPprograms (Felsenstein 2000) of NEIGHBOR to develop treesand then CONSENSE to develop the consensus tree Thecluster analysis technique of unweighted pair-group methodof arithmetic averages (UPGMA) was used to develop thetrees that represented the calculated relationships Also thevariance in allele frequencies among subpopulations (ieWrightrsquos FST ) and estimates of effective migration rates (Nm)were investigated using the program RAPDFST (Apostol etal 1996)

Results

The initial screening of 120 random primers with Chominivorax (CR-91) and C macellaria (CMN) revealed 21primers that gave clear consistent and discrete bandingpatterns that were further screened (table 1) From these 21primers seven were selected (OPA-12 OPB-08 OPD-02OPE-04 OPE-09 OPE-16 and OPF-01) that were easilyamplified and produced consistent results for furthertesting with DNA from five individuals representing fourgeographically distant populations of both C hominivoraxand C macellaria Five primers produced 12 RAPD markersthat distinguished the two species (table 2 fig 1)

For the statistical analyses 52 bands (including thebands representing the 12 species diagnostic markers) fromall seven primers were used Table 2 and fig 1 illustrate thebands used from the primer OPA-12 The average geneticdistance between populations (RAPDDIST calculations)was 00845 (range = 00581ndash0105) for C macellariapopulations 0216 (range = 0151ndash0304) for C hominivoraxpopulations 0799 (range = 0654~1000) betweenpopulations of C hominivorax and C macellaria (table 3) Theaverage genetic distances between individuals withinpopulations of C macellaria were higher than those ofindividuals within populations of C hominivorax (table 4)The consensus trees resulting from bootstrapping usingboth population and individual data showed 100 supportfor the branch separating the two species The clusteranalysis based on genetic distances for individuals did notdisplay relationships for C macellaria while it did for Chominivorax (data not presented) One consensus treeresulting from individual data and using the Nei amp Li (1985)designated 1-S genetic distances correctly clustered most Chominivorax samples with the exception of two from Mexico(they formed a separate cluster branching from aMexicondashCosta Rica branch) but bootstrapping support wasvariable (data not presented) The other consensus treeresulting from individual data but using the 1-M geneticdistances of Apostol et al (1993) correctly clustered allscrewworms but again bootstrapping support wasvariable for the various branches (fig 2)

The estimated variance in allele frequencies amongsubpopulations (FST

) and estimated migration rates (Nm) forpopulations of C hominivorax and C macellaria were 0538and 020 and 0189 and 11 respectively

RAPD markers for screwworms 91

Table 1 The 21 arbitrary 10-mer primers used to determine if genetic markers could beestablished for distinguishing Cochliomyia hominivorax from C macellaria

Primer Sequence Primer Sequence

OPA-07 GAAACGGGTG OPA-12 TCGGCGATAGOPB-08 GTCCACACGG OPB-10 CTGCTGGGACOPB-12 CCTTGACGCA OPC-03 GGGGGTCTTTOPC-15 GACGGATCAG OPD-02 GGACCCAACCOPD-11 AGCGCCATTG OPD-13 GGGGTGACGAOPD-16 AGGGCGTAAG OPD-18 GAGAGCCAACOPD-20 ACCCGGTCAC OPE-04 GTGACATGCCOPE-09 CTTCACCCGA OPE-16 GGTGACTGTGOPE-17 CTACTGCCGT OPF-01 ACGGATCCTGOPF-10 GGAAGCTTGG OPF-13 GGCTGCAGAAOPF-15 CCAGTACTCC

Discussion

Fingerprinting genomes with arbitrary primers is aversatile method for detecting genetic polymorphismsuseful for genetic mapping phylogenetics and populationbiology (McClelland amp Welsh 1995) Most RAPD bands aredominant traits (Rafalski amp Tingey 1993) and their presencereflects priming sites flanking a segment of DNA suitable foramplification (Williams et al 1990 Black 1993) RAPDndashPCRgenerates a fingerprint using arbitrarily selected primersand conditions of reduced stringency so the primer willinitiate synthesis on DNA even when the match with thetemplate is imperfect The most efficient of these primingevents compete with each other during amplification toproduce a fingerprint of a few to over 100 prominent PCRproducts (McClelland amp Welsh 1995)

The RAPDndashPCR method has proved to be valuable inidentifying large numbers of genetic polymorphisms inseveral insect species refractory to or little used for classicalgenetic analysis (Haymer 1994) Results presented hereshow that RAPDndashPCR products from the primers OPA-12OPD-02 OPE-04 OPE-16 and OPF-01 exhibited species-specific markers capable of discriminating C hominivoraxfrom C macellaria Although RAPDndashPCR can be veryversatile it must be approached with care because of extremesensitivity to changes in buffers the condition andconcentration of template DNA the source of Taqpolymerase and amplification parameters (Black 1993) The

RAPDndashPCR procedures used in this study were optimizedThus adherence to our conditions including the use ofAmpliTaqreg DNA Polymerase Stoffel fragment should allowothers to use RAPDndashPCR for identifying screwworms withconfidence But it would be prudent for each laboratory tooptimize the technique based on existing conditions

When a New World screwworm eradication programmeis initiated in a country myiasis cases are common and mostsamples submitted for identification consist of third instarsthat are fairly easy to distinguish But as a programme issuccessful and cases are reduced or after the programmehas been completed and surveillanceexclusion activitiesproceed it is not uncommon to receive suspected casesconsisting of samples that are first instars One requirementin a New World screwworm eradication programme isefficient correct identification of unknown insect samplessent from various countries where suspected re-infestationsby C hominivorax occur Currently identification depends onmorphological traits that are not reliable in earlyparticularly first instars (Knipling 1939 Leite amp Guevara1993) It is important that rapid and efficient techniques areavailable that correctly identify C hominivorax and avoid theunnecessary and costly lsquoeradicationrsquo of areas erroneouslyidentified as being infested or perhaps more importantlythat appropriate eradication efforts be implemented as earlyas possible after confirming new infestations Nowcomparing morphological and molecular data can enhancethe accuracy of identifications Any of the 12 markers could

92 SR Skoda et al

Table 2 Five of the arbitrary 10-mer primers and the number and size (bp) of RAPD bands found in five individuals from fourpopulations of Cochliomyia hominivorax and C macellaria

C hominivoraxa C macellariab

Primerc CR-91 RJS PA-34 CHJ CMN CRMB RJM CMJ

OPA-12d 860 860 860 860 0 0 0 0630 0 0 0 0 630 (2) 0 630 (1)480 (4) 480 (2) 480 (1) 0 0 0 0 0

0 0 0 0 450 (4) 450 (2) 450 (2) 450 (3)360 0 360 (4) 360 (1) 360 (1) 0 360 (2) 360 (2)331 (2) 331 331 331 0 0 0 0

0 0 0 0 295 295 295 295279 0 0 0 279 (4) 0 279 (2) 0245 (2) 245 245 245 245 245 (4) 245 (4) 245

OPD-02e 485 485 485 485 0 0 485 (1) 485 (2)275 275 275 275 0 275 (1) 275 (1) 275 (1)230 230 230 230 0 230 (1) 0 0

OPE-04 685 685 685 (4) 685 (3) 0 0 0 0360 360 360 360 0 0 0 0

0 0 0 0 345 345 345 345251 251 251 251 0 0 0 0

OPE-16 505 505 505 (4) 505 502 (2) 0 505 (2 ) 00 0 0 0 455 455 455 (3) 455 (4)

OPF-01e 375 375 375 375 375 (2) 375 (2) 375 (1) 0347 (2) 347 0 0 347 347 347 347320 320 320 320 0 0 320 (1) 0

Values in brackets show the number of individuals displaying a band no bracketed value shows that all individuals were alikea Four strains of C hominivorax designated as CR-91 (Costa Rica) PA-34 (southern Mexico) RJS (southern Brazil) and CHJ (Jamica)b Four strains of C macellaria designated as CMN (Nebraska) CRMB (Costa Rica) RJM (southern Brazil) and CMJ (Jamica)c All bands represented here were considered as diagnostic markers unless otherwise notedd An example of the bands used in statistical analyses only the bands of size 850 bp and 295 bp are considered markerse The three band profile is diagnostic for the two species (considered as one marker)

be used to distinguish C hominivorax from C macellaria Itwould be prudent to use at least two primers to verify andstrengthen resultant identifications Also the primers OPA-12 and OPE-04 should be considered for this purpose asthey based on results presented here produced markers thatwere reliable and easy to interpret RAPDndashPCR also takesless time and material with no loss of accuracy thanPCRndashRFLP (restriction fragment length polymorphisms) ofmtDNA (Taylor et al 1996b)

The PA-34 strain (from Mexico) was the oldest strain of Chominivorax examined it was established in 1984 CR-91(from Costa Rica) was established in 1991 and until 1995 itwas the strain mass reared in Mexico for use in the NewWorld screwworm eradication programme RJS represented

wild C hominivorax collected in southern Brazil in 1993 andthe colony of C hominivorax collected from Jamaica(designated CHJ) was started in 1998 The C macellariasamples designated CMN (from Nebraska) and CMJ (fromJamaica) were from colonies established in 1998 while RJM(from southern Brazil) and CRMB (from Costa Rica)represented wild samples Our recommended markers fromprimers OPA-12 and OPE-04 as well as the other markerswere consistently present even though these samplesoriginated from geographically distant populations andpopulations that had been in culture for varying lengths oftime The 100 bootstrap support for the branch in the treeresulting from cluster analysis and separating C hominivoraxfrom C macellaria shows that the technique should be a

RAPD markers for screwworms 93

A

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

850

600

279

100

850

600

245

100

600

295

100

600

295

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Fig 1 (A) RAPDndashPCR products from five individuals each of four strains of Cochliomyia hominivorax with primer OPA-12 From left toright lane 1 100 bp ladder lanes 2ndash6 CR-91 lanes 7ndash11 RJS lanes 12ndash16 PA-34 lanes 17ndash21 CHJ lane 22 100 bp ladder (B) RAPDndashPCR products from five individuals each of four strains of C macellaria with primer OPA-12 From left to right lane 1 100 bpladder lanes 2ndash6 CMN lanes 7ndash11 CRMB lanes 12ndash16 RJM lanes 17ndash21 RJM lane 22 control lane 23 100 bp ladder

robust tool for personnel in any future New Worldscrewworm eradication programme

The cluster analyses also showed promise thatRAPDndashPCR could be useful for indicating the generalgeographic origin of C hominivorax samples All of theindividuals were correctly categorized using the 1-Mgenetic distance calculations (Apostol et al 1993) and manyof the branches were well supported (gt 70) by bootstrapanalysis More detailed investigation is warranted reliableinformation could prove useful to eradication personnelwhen they confront screwworm infestations in previouslyuninfested region(s) (FAO 1992)

The main goal of this work was to evaluate RAPDndashPCRas an identification tool but our data allowed some cautiousexploration of the population genetics of C hominivorax andC macellaria The results indicated more populationsubstructure in C hominivorax than in C macellaria Andresults for C hominivorax are similar to previous reportsusing RAPDndashPCR on populations from Brazil and Argentina(Infante-Malachias et al 1999) and restriction fragmentlength polymorphisms of mtDNA on populations fromBrazil (Infante-Vargas amp Azeredo-Espin 1995) and Northand Central America (Roehrdanz amp Johnson 1988Roehrdanz 1989 Taylor et al 1996c) but are different fromstudies using allozymes (Taylor et al 1996a) Infante-Malachias et al (1999) considered several plausibleexplanations for the differences between DNA and enzymedata that will not be restated here It is noted however that

although the main goals of our research were different fromtheirs our genetic distance data are very similar to those ofInfante-Malachias et al (1999) while the estimated variancein allele frequencies among subpopulations (FST) andestimated migration rates (Nm) differed The differencesprobably reflect the extreme geographic distance in thesamples we used as compared to Infante-Malachias et al

94 SR Skoda et al

Table 3 Neirsquos (1972) genetic distance with Lynch amp Milliganrsquos (1994) correction between populations of Cochliomyiahominivoraxa and C macellariab

CR-91 RJS PA-34 CHJ CMN CRMB RJM CMJ

CR-91 0RJS 0304 0PA-34 0177 0274 0CHJ 0210 0178 0151 0CMN ~10 0813 0812 0882 0CRMB 0993 0745 0798 0801 00581 0RJM 0868 0671 0654 0744 0103 00614 0ˇCMJ 0843 0703 0719 0735 00966 00832 0105 0

a C hominivorax populations are represented by CR-91 (Costa Rica) RJS (southern Brazil) PA-34 (southern Mexico) andCHJ (Jamaica)b C macellaria populations are represented by CMN (Nebraska) CRMB (Costa Rica) RJM (southern Brazil) and CMJ(Jamaica)

Table 4 A comparison of the average genetic distance ofindividuals from populations of Cochliomyia hominivorax and Cmacellaria as calculated by formulae of Nei amp Li (1985) andApostol et al (1996) and designated 1-S and 1-M respectively

1-S 1-M

C hominivoraxCosta Rica 0140 0169Brazil 00916 00923Mexico 0131 0134Jamaica 0119 0114

C macellariaNebraska 0253 0202Mexico 0331 0235Brazil 0429 0291Jamaica 0358 0246

Mexico-1

Mexico-5

Mexico-3

Mexico-2

Mexico-4

Costa Rica-4

Costa Rica-2

Costa Rica-3

Costa Rica-1

Costa Rica-5

Brazil -3

Brazil -2

Brazil -1

Brazil -4

Brazil -5

Jamaica-2

Jamaica-4

Jamaica-3

Jamaica-1

Jamaica-5

94

58

40

46

84

70

98

6050

54

42

54

90

68

74

7051

76

Fig 2 The consensus tree for individual specimens ofCochliomyia hominivorax resulting from UPGMA cluster analysisafter bootstrapping with the genetic distance values from 1-M(Apostol et al 1996) Per cent values above the branches indicatebootstrap support for that branch

(1999) where although geographically separated theirsamples were all from the southern range of C hominivoraxInfante-Malachias et al (1999) estimated FST to be 0122 andNm at 18 indicating modest population substructure andwith these and other data suggested that Brazil may havebeen the centre of origin for C hominivorax This point is notdisputed but we note population substructure is littledifferent in data from this current research (collected fromvastly separated geographical origins) than in Infante-Malachias et al (1999) Thus although there is moderatepopulation substructuring in C hominivorax there isappreciable gene flow even between the extreme regions inthe historic range of the New World screwworm

Our results for C macellaria are very similar to those whereallozyme data were used (Taylor amp Peterson 1994)Cochliomyia macellaria are scavengers with high populations ascompared to the obligatory parasite populations of Chominivorax but both species are very mobile (Dear 1985)High-density populations with mobile individuals (such asthose of C macellaria) would be expected to have morediversity within populations and less diversity betweenpopulations therefore results presented here may reflect thetrue state of genetic diversity in populations of C hominivoraxand C macellaria

Mass reared strains of C hominivorax collected fromdifferent geographical locations and cultured for manygenerations may eventually suffer losses of geneticvariability Hence an immediate use of these RAPDndashPCRmarkers could be as a tool to establish the base level ofsimilarity within strains originating from the same localityand then domesticated in mass rearing for use in aneradication programme The baseline information couldserve as a benchmark that signals when straincontamination or strain deterioration occurs

Results from this study confirm the value of the RAPDtechnique for genetic analysis in screwworms Also withfurther development RAPD markers could be used todetermine the geographical origin of an infestation and mayhave utility as a rapid and efficient technique for routine usein ensuring the quality of C hominivorax strains in massproduction for an eradication programme Other uses suchas linkage maps may also be explored

Acknowledgements

The authors wish to thank Dr Dennis Berkebile andJames Lester Figarola for providing insects The assistance ofJulie Ann Champoux Valerie Lacky and Leela Alamalakalaare greatly appreciated This work was inspired by andpartially funded through a Trust Fund CooperativeAgreement No 58ndash5440ndash4-F009 from the Food andAgriculture Organization of the United Nations This isJournal Series paper No 12405 of the Nebraska AgriculturalResearch Division Institute of Agriculture and NaturalResources University of Nebraska-Lincoln LincolnNebraska This article reports the results of research onlyMention of a proprietary product does not constituteendorsement or recommendation for its use by USDA

References

Apostol BL Black WC IV Miller BR Reiter P amp BeatyBJ (1993) Estimation of the number of full sibling familiesat an ovipositon site using RAPDndashPCR markers

applications to the mosquito Aedes aegypti Theoretical andApplied Genetics 86 991ndash1000

Apostol BL Black WC IV Reiter P amp Miller BR (1996)Population genetics with RAPDndashPCR markers thebreeding structure of Aedes aegypti in Puerto Rico Heredity76 325ndash334

Black WC IV (1993) PCR with arbitrary primers approachwith care Insect Molecular Biology 2 1ndash6

Black WC IV Duteau NM Puterka GJ Nicols JR ampPettorini JM (1992) Use of the random amplified polymorphic DNA polymerase chain reaction(RAPDndashPCR) to detect DNA polymorphisms in aphids(Homoptera Aphididae) Bulletin of Entomological Research82 151ndash159

Cushing EE amp Patton WS (1933) Studies on the higherDiptera of medical and veterinary importance Cochliomyiaamericana sp nov the screwworm fly of the New WorldAnnals of Tropical Medicine and Parasitology 27 539ndash551

Dear JP (1985) A revision of the new world Chrysomyini(Diptera Calliphoridae) Revista Brasileira de Zoologia 3109ndash169

FAO (1992) The New World screwworm eradication programmeNorth Africa 1988ndash1992 Food and Agriculture Organizationof the United Nations Rome 1992

Felsenstein J (2000) PHYLIP (Phylogeny Inference Package)version 36 (alpha) Distributed by the author Departmentof Genetics University of Washington Seattle

Hall DG (1948) The blowflies of North America Thomas SayFoundation Entomological Society of America LanhamMaryland

Haymer DS (1994) Random amplified polymorphic DNA andmicrosatellites What are they and can they tell us anythingwe donlsquot already know Annals of the Entomological Societyof America 87 717ndash722

Hoy MA (1994) Insect molecular genetics an introduction toprinciples and applications San Diego California AcademicPress

Hunt GJ amp Page RE Jr (1995) Linkage map of the honey beeApis mellifera based on RAPD markers Genetics 1391371ndash1382

Infante-Malachias ME Yotoko KSC amp Azeredo-EspinAML (1999) Random amplified polymorphic DNA ofscrewworm fly populations (Diptera Calliphoridae) fromsoutheastern Brazil and northern Argentina Genome 42772ndash779

Infante-Vargas ME amp Azeredo-Espin AML (1995) Geneticvariability in mitochondrial DNA of the screwwormCochliomyia hominivorax (Diptera Calliphoridae) fromBrazil Biochemical Genetics 33 237ndash256

Knipling EF (1939) A key for blowfly larvae concerned inwound and cutaneous myiasis Annals of the EntomologicalSociety of America 32 376ndash383

Leite ACR amp Guevara JDE (1993) Scanning electronmicroscopy of the larval instars of Cochliomyia hominivoraxMedical and Veterinary Entomology 7 263ndash270

Lynch M amp Milligan BG (1994) Analysis of populationgenetic structure with RAPD markers Molecular Ecology 391ndash99

McClelland M amp Welsh J (1995) DNA fingerprinting usingarbitrary primed PCR pp 203ndash212 in Dieffenbach CW ampDveksler GS (Eds) PCR primer a laboratory manual NewYork Cold Spring Harbor Laboratory Press

Metcalf RL amp Metcalf RA (1993) Destructive and usefulinsects New York McGraw Hill

RAPD markers for screwworms 95

Nei M (1972) Genetic distance between populations AmericanNaturalist 106 283ndash292

Nei M amp Li WH (1985) Mathematical model for studyinggenetic variation in terms of restriction endonucleasesProceedings of the National Academy of Sciences USA 765269ndash5273

Pornkulwat S Skoda SR Thomas GD amp Foster JE (1998)Random amplified polymorphic DNA used to identifygenetic variation in ecotypes of the European corn borer(Lepidoptera Pyralidae) Annals of the Entomological Societyof America 91 719ndash725

Rafalski JA amp Tingey SV (1993) Genetic diagnostics in plantbreeding RAPDs microsatellites and machines Trends inGenetics 9 270ndash280

Roderick GK (1996) Geographic structure of insectpopulations gene flow phylogeography and their usesAnnual Review of Entomology 41 325ndash352

Roehrdanz RL (1989) Intraspecific genetic variability inmitochondrial DNA of the screwworm fly (Cochliomyiahominivorax) Biochemical Genetics 27 551ndash569

Roehrdanz RL amp Johnson DA (1988) Mitochondrial DNAvariation among geographical populations of thescrewworm fly Cochliomyia hominivorax Journal of MedicalEntomology 25 136ndash141

Taylor DB amp Peterson RD II (1994) Population genetics andgene variation in primary and secondary screwworm

(Diptera Calliphoridae) Annals of the Entomological Societyof America 87 626ndash633

Taylor DB Peterson RD II amp Moya-Borja GE (1996a)Population genetics and gene variation in screwworms(Diptera Calliphoridae) from Brazil Biochemical Genetics34 67ndash76

Taylor DB Szalanski AL amp Peterson RD II (1996b)Identification of screwworm species by polymerase chainreaction-restriction fragment length polymorphismMedical and Veterinary Entomology 10 63ndash70

Taylor DB Szalanski AL amp Peterson RD II (1996c)Mitochondrial DNA variation in screwworm Medical andVeterinary Entomology 10 161ndash169

Welsh J amp McClelland M (1990) Fingerprinting genomesusing PCR with arbitrary primers Nucleic Acids Research 187213ndash7218

Williams JGK Kubelik AR Livak KJ Rafalski JA ampTingey SV (1990) DNA polymorphisms amplified byarbitrary primers are useful as genetic markers NucleicAcids Research 18 6531ndash6535

Wyss JH amp Galvin TJ (1996) Central America regionalscrewworm eradication program (benefitcost study)Annals of the New York Academy of Sciences 791 241ndash247

(Accepted 18 September 2001)copy USDA 2002

96 SR Skoda et al