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Faculty Publications: Department of Entomology Entomology, Department of

1997

Mitochondrial DNA Variation among Muscidifurax spp. (: ), Pupal Parasitoids of Filth Flies (Diptera)

David B. Taylor University of Nebraska-Lincoln, [email protected]

Richard D. Peterson II University of Nebraska-Lincoln, [email protected]

Allen L. Szalanski University of Nebraska-Lincoln

James J. Petersen University of Nebraska-Lincoln

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Taylor, David B.; Peterson, Richard D. II; Szalanski, Allen L.; and Petersen, James J., "Mitochondrial DNA Variation among Muscidifurax spp. (Hymenoptera: Pteromalidae), Pupal Parasitoids of Filth Flies (Diptera)" (1997). Faculty Publications: Department of Entomology. 206. https://digitalcommons.unl.edu/entomologyfacpub/206

This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Mitochondrial DNA Variation Among Muscidifurax spp. (Hymenoptera: Pteromalidae) ,Pupal Parasitoids of Filth Flies (Diptera)

DAVID B. TAYLOR, RICHARD D, PETERSON 11, ALLEN L. SZALANSKI,' A~DJAMES J. PETERSEN Midwest Livestock Research Laboratory. USDA-ARS, Department of Entomology, University of Nebraska, Lincoln. NE 68583

Ann. Entomol. Soc. Am. W(6): 814-824 (1997) ABSTRACT Polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) and sequencing analyses were used to characterize an amplicon of -625 bp in 4 of the 5 nominate species of Muscidifim Girault & Sanders, pupal parasitoids of muscoid flies. A single polymorphic nucleotide site was observed among 2 samples of M. mptor Girault Br Sanders. No sequence variation was observed among 3 samples of M. rqh-ellur Kogan & Legner. The sequence of M. uniraptor Kogan & Legner was identical to that of M. raptwellw. Nucleotide divergence among the Muscidijmnx spp. ranged from 0.14 to 0.18 substitutions per nucleotide. Muscidifitm urraptor Kogan & Legner exhibited multiple haplotypes, 2 of which were char- acterized by sequencing and 4 others by PCR-RFLP. The sequenced haplotypes differed by 0.08 nucleotide substitutions per site. Restriction site analysis indicated that nucleotide divergence ranged from 0.03 to 0.10 among all 6 haplotypes. Analysis of progeny from individual females indicated that the observed variation in M. mraptor was caused by multiple haplotypes within individuals rather than differentiation among individuals. These results bring to question the specific status of M.unimptorand indicate that the genus is native to the Western Hemisphere, and not introduced with their primary host, Murcn domestics L,as previously proposed. Heteroplasmy and translocation of aportion of the mitochondrial genome to the nuclear genome are discussed as possible causes for the variation observed in M. zamptw.

KEY WORDS Muscidifimx, polymerase chain reaction-restriction fragment length polymor- phism, mitochondrial DNA, phylogeny

WASEm TAF, genus Musddifirax are pupal parasi- species known only from the island of Puerto Rico toids of muscoid flies, especially the house fly, Mu~cn (Kogan and Legner 1970). domtica L., and the stable fly, Stmnoxys colcitrm The geographic origins and phylogenetic rela- (L.),and are among the most promising biological tionships of Muscidifirrax are unclear. Kogan and control agents for these flies in the confined live- Leper (1970) proposed 2 alternatives for the ori- stock environment (Miller and Rutz 1990, Petersen gins of the genus. First, they originated in the Ethi- et al. 1990). Murcidifirax includes 5 species. Mus- opian region and wereintroduced to the New World cidifirax mptor Girault & Sanders, the most wide- along with house flies, or 2nd, they are native to the spread, is found throughout the temperate and semi- New World and have secondarily adapted to house tropical regions of the world (Kogan and Legner flies. Kogan and Legner conclude that the "remark- 1970). The remaining 4 species are limited to the able preference of Musctdifirax spp. for house flies New World. Muscidifurax zaraptor Kogan & Legner as compared to native Nearctic flies" indicates an is found sympatrically with M. raptor in western Old World origin for the genus. Legner (1983)in- North America (Kogan and Legner 1970, Lysyk dicates that the dependence of Musctdifirax upon 1995), M. raptoroides Kogan & Legner and M.rup- the "barnyard" environment outside of Africa is torellus Kogan & Legner are found allopatrically in further evidence that the genus was not native to the Central and South America, respectively (Kogan New World. However, to accept an Old World or- and Legner 1970). Two forms of M. raptorellus have igin of the genus and account for the 4 species been reported, one solitary and the other gregarious endemic to the New World, one must accept a very (Kogan and Legner 1970, Legner 1988). MuPcidi- rapid rate of speciation following their introduction. &rax mtraptw Kogan & Legner is a parthenogenic Despite the interest in these species for biological control of filth flies and the questions concerning their geographic origin, little work has been done on "~spartrnmt of Plant Pathology, University of Nebmkq Lin- the population genetics and genetic structure of coln, NE 68583. Muscidifirax. Ropp (1986) used allozymes to ex- November I997 TAYLOREX AL.: mtDNA VARIATIONLY M~cidifimxSPP. 815

Date Source* Species Orldn inllected . raptor Nebraska I995 MLIRL New York Unknown Cornell University . mp~IlwChile Unknown Legner via MLIRL Nebraska 1991 MLIRL Peru Unknown Leper ria MLlRL M.miaptor Puerto Rico Unknown Rochester University M. zamptor Nebraska 1991-1993 MLIRL Nebraska 1995 hu.lRL

'MLEU+ Midwest Livestock Inrect Research Irpboratory. USDA-ARS, Lincoln. NE; Legner via MLIRL,originally collected by E. F. Leper and transferred to MLlRL in 1989. amine several populations of M. raptor and M.zarap- tor. He found relatively low levels of differentiation among geographic isolates of the 2 species, but a high level of differentiation was observed between species. Antolin et al. (1996) used RAPD-PCR to examine 3 Muscidifiraz spp. They were able to dif- ferentiate the species and associate the gregarious North American Muscidifurox sp. with M.ruptorel- Ius, but indicated they were unable to explore phy- logenetic relationships because of the nature of the RAPD-PCR data. The purpose of this study was to develop molec- Fig. 1. Intact amplicon from 4 Muscidifirrax spp., A. ular diagnostic characters and examine genetic dif- mellifera, and C. macelloria on 2.5% Metaphor agarosc gel. ferentiation among Muscidifumx spp. A region of Primers werc 5' ATACCTCGACG'ITA?TCACA 3' and 5' the mitochondria1genome, =625 bp, including parts TCAATATCA'ITCATCACCMT 3'. of the cytochrome oxidase I (COI) and I1 (COII) genes as well as the entire tRNA leucine (~RNA''~) gene was examined by polymerase chain reaction- extracted with thc chloroform-phenol tcchniquc as restriction fragment length polymorphism (PCR- outlined in Taylor ct al. (1996). RFLP) and sequencing. Representative samples of A region of mtDNA, -625 bp long, was amplified 4 of the 5 species in the genus (samples of M. using thc primcrs 5' ATACCTCGACGTTATC- raptomides were not available) were included in ACA 3' (= S2792; Bogdanowicz ct al. 1993) and 5' this study. TCAATATCATTGATGACCAAT 3' (K. Prucss, personal communication). Thc 5' ends of thcric primcrs wcrc located at bp 2773 and 3400 of the Materials and Methods Drosghila yakuba mtDNA map (Clary and Wol- SampIes. Mumidifirax samples were obtained stenholme 1985), respcctivcly. from established colonies (Table 1).The Chile and For amplification, 1 p1 of sample DNA was added Nebraska samples of M. raptorellus represented gre- to a reaction mixture containing 2.5 p1 of reaction garious populations and the Peru sample was soli- buffer (Perkin-Elmer, Norwalk, CT), 2 p1 of dNTP tary. The Apis mellifera lingustica L control sample mix (10 mM each-dATP, $ITP, dCTP and dCTP), was collected from an apiary in Lincoln, NE, and the 1pl of each primer (20 mM). 1.0 unit of Taq poly- Cochliomyia naacellaria (F.) samples were from a merase (Perkin-Elmer) and deionized water to a laboratory colony originating in Fargo, ND. Mus- volume of 25 pl. Amplifications were donc in a cidifcnvlx colonies were maintained on freeze-killed Perkin Elmer Cetus Model 9600 therrnocycler pro- M. rlomestica pupae (Petersen and Matthews 1984). grammed for 35 cycles of 92°C for 1 min, 55°C for 1 Isofemale lines were initiated by isolating indi- min, and 72OC for 1or 2 min. Amplification products vidual females (presumably mated) from the main were stored at 4OC. colony and placing them in small plastic cups (2 cm Restriction Endonuclease Digests. Twenty-seven diameter, 2 cm high) with -150 freeze-killed fly restriction enzymes-Alu I, Apo I, Ase I, Ava I. Ban pupae. Progeny emerged in 3 wk. Specimens were Zl, Bfa I, Bsr I, Dde I, Dpn 11, Dra I, EcoR I, EcoR V, stored at -80%. Hae Ill, Hinc 11, Hind Ill. Hinf 1, Hpa I, Mse I, Msp I, Voucher specimens from each of the Murcidifimx Puu 11, Rsa I, Sac I, Sau96 I, ScrF I, SJ~I, Taq I, and samples have been placed in the collection of the Xba I-were screened. Digests for polyacrylamide University of Nebraska State Museum, Lincoln. gel electrophoresis (PAGE) were done using 1.5 p1 DNA Extraction and Amplification. Pools of 5-25 of PCR product, 0.2 p1 enzyme (New England Bio- wasps were used for each DNA extraction. DNA was labs, Beverly, MA), 1X buffer (New England Bio- ANNALS OF THE E@STOMOUX:ICALSOCIEN OF AMERICA Vol. 90, no. 6

50 bp SSP ladder frag

Apo I ssp I

Fis 2. Apo I and Ssp I digests of Mutddifumx spp. on 2.5%Metaphor agarose gels. labs) and water to bring the volume to 5 pl in 200-pl onto a 10% acrylamide gel (1X Tris:borate:EDTA tubes. For agarose gel electrophoresis, all quantities (TBE).0.5% Photoflo-20 [Kodak, Rochester, NY]. were increased 2.5 times. Samples were incubated at 0.15% TJ3MED, and 0.05% ammonium persulfate). A 37°C for 3-16 h and stored at 4°C until further molecular size standard, pGEM (Promega. Madison, analysis. WI), was included on each gel. Hoefer (Hoefer Electrophoresis. For PAGE, 1.5 pI of loading Scientific Instruments, San Francisco, CA) SE 600 buffer (10%Ficoll400 [Sigma,St. Louis,MO], 0.25% electrophoresis units with gels (16 by 20 cm by 0.75 Bromophenol Blue [Sigma], 50 mM EDTA, 10 mM mm) and 28 well combs were used for electrophore- Tris-HCI pH 7.5) was added to the 5-p1 digest prod- sis. Gels were run in 1X TBE buffer at a constant 300 uct. The entire digest product (6.5 pl) was loaded V (15 rnAlgel) for 1.5 h at 20°C. Gels were stained

Tabla 2. Rmtrietion +at leu& mtinuted on 2.540 Metaphor qmmm for Mmc- @m

Restriction M. raptor NE M. nrptorsllut M,unimptor M. zarapror enzymes

-1 247.219.52.42,28 Asel 327,297 mn 373,239 IkaI 321,243,65 Hue III . 614 Hinf 1 600-34 Mse I 149.98, 94, 71, 53. 33, 14 Ssp1 292,239,98

* Poaaibly double bands. November 1997

H. raptor YPD SYL CWNM ISS M. raptorel l us M. zaraptor I M. zaraptar I1 ...... V.. M. raptor 1 atacctcgac gttattcaga TTATCCGGAT TCATATTTAT GTTGAAATAT AATTTCTTCC M. raptorellus 1 ...... A...... G. ...C..A..A M. zaraptar I 1 ...... A...... A M. zaraptor I1 1 ...... T...... G......

...... co I------raptor LGS FISM MST MLF FFII WES raptorell us M.. I.. . LF...... zaraptor I . I... V IF. LL FEKE zaraptor I1 . I..V.IF.Y ..... rapt or 61 CTGGGAAGAT TTATTTCTAT AATAAGAACA ATATTATTTT TCTTCATTAT TTGAGAAAGA raptorellus 61 A.A...... A ....C..A...... T T....T...... T...... zaraptor I 61 T.A...... A ...... G...... T..T..-- -- ..T...... zaraptor I1 61 T.A..G...A ...... A.. .G...... T..T..C. .T.A...... G

...... CO I------H. raptor MIS NRTV IFN KNM NNSI EWI M. raptorellus I.. ..I1 ... SIv ... M. zaraptor I LFL IEL M. zaraptor 11 I.. ..I. ... S....v M. raptor 12 1 ATAATTTCTA ATCGTACAGT AATTTTTAAT AAARATATAA ATAATTCAAT TGAATGAATT M. raptorellus 121 ..T ...... C..A.TTA. C...... GA..C...... TG. A ...... C M. zaraptor 1 117 ..T...... A.TTA...... G...... T...... M. zaraptor I1 121 ..T ...... TT...... G...... T...... G..

...... CO I------"-- M. raptor MSF PPSF HSF SEI PKIY K' M. raptorellus A...... FN* M. zaraptor I M. zaraptor I1 N.M ... N.L. I" M. raptor 181 ATATCTTTTC CACCTTCATT TCATTCATTT AGAGAAATTC CA?JAWTTTA TAWTMTTT M. raptorellus 181 ...G.A.... .C..A...... T...... T ...... T C..T ...A.. M. zaraptor I 177 ....GC ...... A. A.. ...T... .AT...... , .....C.... .T.T...... M. zaraptor I1 181 ...AAC...... TA. G. ....T. .. .AT...... T...C.... .T.T......

M. raptor M. raptorel 1 us M. zaraptor I M. zaraptor 11 M. raptor 241 TTAAAATGGC AGATTAGTGC AATAGACTTA AATTCTATAT ATATAATAAT TAGTATTATT M. raptorellus 241 ...... T...... T. A.AC...... M. zaraptor I 237 ...... T...... - - ..-A...... M. zaraptor I1 24i ...... T... ..C...... A ...... - - ..AA......

Fig. 3. Sequence comparison for MwddifiKm spp., represents the same amino acid or nucleotide is present as that in M,ruptor; -, indicates a deletion. The M. ruptmellw sequence represents both that species and M. uniraptor. Lower case letters in the M. raptor sequence represent primer sequences. 818 ANNU OF THE ENTOMOLOGICAL SOCEIY OF A~~~ERICA Vol. 90. no. 6

M. raptor M SMW SQLM LQD SNS PIME M. raptorellus . AL. N.M. F...... M. zaraptor I .... N.I.F.. *.. *..a M. zaraptor I1 . L.NIF...... H. raptor 301 TTTAAAAATT TCAATATGAA GTCAATTAAT ATTACAAGAT AGTAATTCTC CTATTATAGA M. raptorellus 301 ...... G..T...... A....A...... T...... A.....A. .C ...... M. zaraptor I 294 A....A.T.. ...C...... A.....A...... M. zaraptor I1 299 ...... T...... A....A.T .....T.....C ..G..C.... .A......

...... co II------M. raptor MMI YFH DHSM MVI MVI ISLI M. raptorellus S.. M.G. LI. IM. M. M. zaraptor I S.M...G.L..II ..... M. zaraptor I1 S.M.GLII. .... M. raptor 361 AATAATAATT TATTTTCATG ATCATAGAAT AATAGTTATT ATAGTTATTA TTAGTTTRAT M. raptorellus 361 ..G...... ATA...... C...G.... .T..A...... TA.A...... A. ... M. zaraptor 1 354 ..G...... ATA ...... G.... .T....A... ..CA...... C..A..... M. zaraptor I1 359 ..G...... ATA..C,...... G.... .T....A... ..TA...... A.....

------+------co I------*-- M. raptor MY1 ILF MFFN NLM NRF MLEG M. raptorellus L.. M.IT1 ...... M. zaraptor I L.M. VTI Y .... M. zaraptor I1 L.. M.LMI ...... M. raptor 421 TATATATATT ATTTTATTTA TATTTTTTAA TAATTTAATA AATCGATTTA TATTAGAAGG M. raptorellus 421 .C.T...... A....C...... A...... CA.....T .....G...... M. zaraptor I 414 .T...... A...... G.C.. ..CA .....T ...... A...... M. zaraptor I1 419 .T....C...... A....C...... A.. ..TA..G..C ......

...... ------co II------M. raptor QMI EII WTII PIF FLI ILAI H. raptorellus . . v.. VF.-. M. zaraptor I . . v.R... . F... H. zaraptor I1 . . v.. F... M. raptor 481 TCAAATAATT GAAATTATTT GAACAATTAT TCCAATTTTT TTTTTAATCA TTTTAGCAAT M. raptorellus 481 C...... G.A.. ...T...... G.AT ...... M. zaraptor I 474 ...G...... G.A.. ..GT...... TT ...... M. zaraptor I1 479 ...G...... TG.A.. ...T...... TT ......

...... co II------M. raptor PSL KIL YMTD EMN LPN LSIK M. raptorellus ...... L. I.. TH. M. zaraptor I . L.s* ...... M. zaraptor I1 . L... .T.* M. raptor 541 TCCTTCATTA AAAATTCTTT ATATAACTGA TGAAATAAAT TTACCTAATT TATCTATTAA M. raptorellus 541 ...... T...... T.A. .C..G...... T.... . A.T..A...... A....A. . M. zaraptor I 534 ...G...... T...... GTC...... A*.... M. zaraptor I1 539 ...A..C...... T...... A.A. ....

------CO II------M. raptor I M. raptorellus v M. zaraptor I v M. zaraptor I1 v M. raptor 601 AATTattggt catcaatgat attga M. raptorellus 601 .G.A M. zaraptor I 694 .G.A I'P. zaraptor I1 699 .G.A

Fig 3. Continued November 1997 TAYLOR AL.: mtDNA VARIAT~ONM Murcidifimx SPP. 819

Table 3. Nnclcotidc mbrittrtna (d) rmtu(Id- the diyoarl) mdtrnmitioa to truuvadioa don(he he dimpod) mow Mwcid#ka* rpp., A. nullifera, d D. pkuba

Species Species 123456 1 A. nclliJ%ra - 0.41 0.33 0.33 0.36 0.37 2 D. yakuba 0.53 - 0.27 0.31 0.28 0.32 3 M. mptor 0.46 0.53 - 0.50 0.52 0.56 4 M. mptonlku 0.53 0.61 0.19 - 0.66 0.82 5 M.zomptor 1 0.47 0.62 0.14 0.15 - 1.22 6M.zamptor2 0.54 0.60 0.17 0.18 0.08 -

Statistical Analyses. Sequence divergence (d)was calculated from restriction site data using The re- Fig. 4. Cladogram based upon sequence data of Mw- striction enzyme analysis package (McEI- cfdifirax spp., A. ntellffem, and D. ynkuba. All branches (REAP) were supported by 297 of 100 bootstrap replicates. roy et al. 1992) following the procedures of Nei and Tajima (1981) and Nei and Miller (1990). The DNA- DIST procedure of PHYLIP 3.5 (Felsenstein 1993) was used to calculate d values from the sequence for 5 min with ethidium bromide (1 pglml). Meta- data using the Kimura %parameter model (Kimura Phor agarose (FMC Bioproducts, Rockland, ME ) 1980). The consensus dendrogram of the mtDNA gel electrophoresis was used to estimate fragment haplotypes was derived using the Wagner parsi- lengths. Gels (2.5%) were run for 4 h in Cibco-BRL mony method (DNAPARS and CONSENS proce- (Gaithersburg, MD) Horizon 11-14gel boxes at 80 V dures of PIRLIP). Bootstrap replicates (100) of the with lx T5E buffer. A 50-bp ladder (Gibco-BRL, scquence data were generated with the SEQBOOT Grand Island, NY) was used as a size standard. procedure of PHYLIP. Ethidium bromide was added to the gel, 0.06 pglml final concentration, and 8 pg of ethidium bromide Results was added to the buffer tray at the anodal end. Gels were interpreted on an ultraviolet (312 nm) tran- The primer pair produced an amplicon estimated silluminator. Fragment sizes were calculated with to be 632 bp for all of thc Muscidifirax spp. and C. the computer program GELJML (LaCroix 1994). macellatia with a 2.5% metaphor agarosc gel (Fig. DNA Sequencing. Amplified DNA was purified 1). Thc A, mellifera amplicon was cstimatcd to bc using Geneclean I1 (Bio 101, Vista, CA) following 856 bp. manufacturer protocols and resuspended in 30 p1 of Restriction Fragment Length Patterns. Thc rc- TE: (pH 7.5). DNA was blunt-ended using New striction enzymes Alu 1,Aua I, Ran 11. Bfa I, Bsr I, D& England Biolabs (Beverly, MA) reagents and ligated I, EcoR I, EcoR V,Hinc 11, Hind 111, Hpa I, Msp I, Pou into pBluescript sk+ plasmid using Stratagene (La 11, Rsa 1, Sau96 I, Sac I, SctfI, and Xha I did not cut Jolla, CA) reagents (Sarnbrook et al. 1989). Com- the amplicon from any of the Mtlscidifirux samplcs. petent E. coli were transformed and positive colo- Twenty-four restriction sites were identified among nies were verified by picking colonies with a sterile the 4 Mwcidifirar spp. for thc remaining 9 restric- toothpick, replating and dipping thc toothpick into tion enzymes. The NY and NE samples of M. raptor a 50-4 Pm reaction mixture (Sambrook et al. gave identical restriction fragment patterns for all 1989).PCR reaction was performed as before but for REs except Apo I. One of 2 wasps from the NY only 20 cycles. Two pl of PCR product was run on colony had an 81-bp band and lacked the 28-bp an agarosegel to determine if the insert was present. band. These bands were absent and present. respec- Positive clones were sequenced by the University of tively, in both of the NE and the other NY M. raptor Nebraska-Lincoln Center for Biotechnology DNA (Fig. 2). The 3 samples of M. raptorellus and M. Sequencing Laboratory (Lincoln, NE) using a LI- uniraptor gave identical restriction fragment pat- COR Model 4000 DNA Sequencer (LI-COR, Lin- terns (Fig. 2; Table 2). Several of the restriction coln, NE). Two primers, T3 and T7 promoters digests for M.zaraptor had faint bands in addition to (Gibco-BRL, Gaithersbug, MD),were used for se- the stronger bands, and the sum of the lengths of the quencing. Two clones were sequenced in both di- bands consistently exceeded the length of the intact rections for each sample. Published sequences for A. amplicon. Although these bands appeared to be mellifera (Crozier and Crozier 1993) and D. yakuba caused by incomplete digests, they were not ob- (Clary and Wolstenholme 1985) were used for com- served for the other species under similar digest parisons with those species. conditions and could not be eliminated by increas- Nucleotide sequences for M. raptor, M. reptorel- ing enzyme concentration or the duration of the [us, and M. zaruptor (sequences 1 and 2) have been digest. Because of these difficulties in interpreting deposited in Genbank with accession numbers the M. zarnptur restriction digests. this species was U97506 -U97515. not included in the restriction site analysis. The 820 ANNU OF THE ENTOMOLOGICALSO(~IE~ OF AMERICA Vol. 90, no. 6

Tubla 4, Remt.iction ribiu Mw&&tkt.* spp.

Restriction hi. zampfw clones M.mptor M. roptoreUw M. zoraptorI M. zamptor Il enzyme site 1 2 3 456 Apo I 183 - - - - + - - + - + 215 + + + + + + + +++ 224 + + ------236 - + ------270 k + + - - -+- - - 306 + - + + ++++++ 343 - + + - - - + - - - 552 + + + + - + + ++- 577 + - + + ++++++ 586 - + ------A381 234 - - + + -++- + + 326 + ------392 - + ------415 - - + + - + + - - f 455 - + + - 7-??-? 458 - - + - ?-??-? Ddef €4 - - - + -+- -++ mn 379 + - + + ++++++ 455 - - - + - + - - + - Dra I 242 + + + + ++++++ 269 - + + + +++ + + + 303 + + + + ++++++ 446 - - - + ++- +++ 549 - + - - + - - - - + Hoe llI 480 - + ------Hinf 1 28 + + + + ++++++ 155 - + ------176 - + ------552 - - - - + - - - - + 571 - - + - - - + - - - Mse 1 I46 + + + + eon - - + + 234 - + + + 211 + + + + 268 t + + + 289 - + - - 3032 + + -+ + 326 + - - - 392 - - + - 416 + - + + 447 + - - + 455 + + + - 458 - + + - 524 + - + + 548 + - + + 562 - - + + 575 - + - - 590 - + - + 597 + - + + ssp) 92 + ------287 - + - - + - - + - - 330 + + ------390 - + + + -+++++ 580 - + ------Tw 1 6 + + + + ++++++ I29 - - + - - - + - - - 170 - - - - + - - - - - 463 + - + + ++++++

S4te posittons are bwed on M. mpfmmap (Fig 3). M.uniropior wu identfcd to M.raptoreUw for all restriction dtcs. Site presence (+) and absence (-1 were based on the sequences and vdedby FWLPanalysisfor M. raptar, M.mptowCh, M. unimptor, and M.zmoptor types I and 11. DPta for clones 1-6 of M. zamptw are based on RFLP data only. November 1997 TAYLORw N..: mtDNA VARIATIONIN Muscidifurex SIT. 821

Tdio 5. Fbt.iation frqmem~lcn& ufimud on 2.5% MeuPhor agarom for 6 M. sumpfor clona - Restrlc tion Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 enzymes

- " Poksibty double bands. estimated number of nucleotide substitutions per M.ruptorfrom New York. Sequences for Chile, Peru, nucleotide, d, calculated from restriction site data and Nebraska M. raptorellus and M. uniraptor were (Nei and Tajima 1981) was 0.23 between M. raptor identical. Two distinct sequences were obtained and M. raptorellus-M. uniraptor. All 9 restriction from 2 clones of M. zaraptor: clone 1 was 618 bp long enzymes gave restriction patterns that were diag- and clone 2 was 623 bp. The 4-bp deletion observed nostic between M.mptor and M.raptorellus-M. un- in clone 1 (bps 99-102 in Fig. 4) imparted a frame imptor. shift in the COI gene. This frame shift changed 12 Sequence Analysis. Sequencing the amplicon re- amino acids bcfore reaching a stop codon and trun- vealed that it was 625 bp long for M. raptor, M. cating the final 31 amino acids of the subunit. A. mptorellus, and M. uniraptor (Fig. 3). Sequences for mellifma differed from D. yakuha and the Muscidi- Nebraska and New York M. raptor samples were firax spp. by a 30-bp insert at the 3' end of the COI identical except for nucleotide bp 273 (Fig. 3), gene (after base 234 of the M. raptor sequcncc) and which exhibited a T to G transvenion in 1 of the 2 a 192-bp A + T-rich insert between ~HNA~""and

clones 4'8 # 4'B clones wea@ $123456$ $1234569 8 50 bp SSP ladder frag

Dpn II ssp I

Fig. 5. Dpn I1 and S~II digests of ampIicon from 6 M. mmptm clones on 2.5%Metaphor agarose gels, 822 ANNALSOF THE ENTOMOL~~CALSocm OF AMERICA Vol. 90, no. 6

COJI (after base 307 of the M. raptor sequence) of the bands observed in the other 9, but no new (Fig. 3). These inserts were ignored in the compar- bands were observed. isons with A. mellijera. Muscidifurax spp. differed from A, mlltfera by 0.50 nucleotide substitutions Discussion per site and from D. yakubaby 0.59 substitutions per site. Divergence among the Muscidifirax spp. The estimated length of the amplicons was close ranged between 0.14 and 0.19 substitutions per site to that predicted by the D. yakuha sequence (Clary (Table 3). The 2 M. zaruptor sequences differed by and Wolstenholme 1985). The 193-bp A + T-rich, 0.08 substitutions per site. Transition to transversion noncoding insert found between the ~RNA'"~and ratios ranged between 0.50 and 1.22 among the Mus- COII genes by Crozier and Crozier (1993) in A. cidijkax spp., with the lower values being associ- mellijka was not present in Murcidifirax sp. As in ated with higher d values (Table 3). The A A T other non-Apis hymenopterans (Cornuet et al. content of the amplicon was 81-84% in the Mus- 1991), no nucleotides are present between the cidifirrax sp. compared with 75 and 84%in D. yakuba and COII genes. and A. mllifm, respectively. Four restriction enzymes, Apo 1, Ase 1, Mse I, and Parsimony analysis resulted in a single most par- Ssp I, resulted in digest patterns which were diag- simonious cladogram with 467 state changes (Fig. nostic among M. -tor, M. raptorellw-M, uniraptor, 4). The 2 M.zaraptor sequences clustered together. and M. zaraptor. An additional 5 enzymes, Dpn 11, All branches of the cladogram were supported by Dra I, Hae 111, Hinf I, and Taq I, differentiated M. 297 of 100 bootstrap replicates of the dataset. raptorellus-M. uniraptor from thc other species. Sequences were scanned for restriction enzyme RFLP analysis could not differentiate the geo- sites with the computer program DIGEST (written graphic isolates of M. raptor or M. raptorellus nor by Ramin Nakisa). All of the restriction patterns could they separate M. raptoellus from M. uniraptw. observed in the RFLP analysis were supported by Use of PCR-RFLP patterns for species diagnostics restriction enzyme sites in the sequences (Table 4). of Muscidifirax spp. must be used with caution until The M. zaraptor colony used for the study de- the questions regarding the multiple, divergent se- quences in M, zuraptor is resolved. scribed study was discarded before realizing that it contained distinct mitochondrial haplotypes. In an The sequencing analysis supported the variation detected by PCR-RFLP analysis. Predicted restric- effort to see if both haplotypes were present in the M, tion sites were observed in all sequences to account new zaraptor colony, amplification products for the observed RFLP patterns. Although a single from the new colony were cloned as for sequencing. polymorphic nucleotide sight was observed in the DNA from individual clones was amplified by PCR, New York sample of M. raptor, no fixed differences and arnplicons from the 6 positive clones were di- were observed among the sequences from the geo- gested with 9 restriction enzymes (Table 5). These graphic isolates of M,raptor and M. raptorellus. The enzymes were chosen because the sequence data M. uniraptor sequence was identical to that of M. indicated they were diagnostic between the 2 M. raptorellus. These data support the characterization zamptor sequences. Each of the 6 clones resulted in of the Nebraska isolate of M. raptorellus as being a unique composite digest pattern (Fig. 5).Two of true to that species, with the probable origin being the patterns, clones 3 and 2, matched the digest an introduction from South America (Antolin et al. patterns predicted for the M. zaraptor I and I1 se- 1996). quences. Restriction site analysis indicated that d The status of M. uniraptor is not resolved. The values ranged from 0.03 to 0.10 among the clones. mtDNA amplicon sequence for this species was To isolate M.zaraptor lines with each of the ob- identical to that of M. raptwellus. Kogan and Legner served haplotypes for morphological comparisons (1970) indicated that M. uniraptor was "extremely and further genetic studies, we initiated 10 isofe- difficult" to distinguish from M. raptor and "almost male lines from the M. zaraptor colony. DNA was impossible" to differentiate from M. raptoroides. isolated from F, wasps (5 wasps pooled and indi- However, a fringe of setae on the margins of M. vidual wasps) fiom each line, amplified, and di- uniraptw wings "easily" distinguish it from M rap- gested with the diagnostic restriction enzymes Apo torellus and M. zaraptor. Fringe setae were apparent I, Ase I, Dde I, Dpn II, Hinf I, Mse I, Ssp I, and Taq on the wings of M. uniraptor from our colony. Leg- I. Restriction digest patterns were identical for the ner (1969) indicated that M. unfraptw males, on pooled and individual DNA isolations, although am- occasion, were able to fertilize M,raptor females plifications were stronger from the pooled samples. successfully, but he did not observe successful hy- Banding patterns were identical for 9 of the 10 lines bridization between M.uniraptor and M. raptorellur. and the same as those observed with the earlier Based upon further hybridization data, Legner pooled samples fmm the discarded colony (Fig. 2). (1987) suggested "the evolution of M. uniraptor The sum of the estimated lengths of the observed from M. rgtor." Isozyme data (Kawooya 1983) ap- bands greatly exceeded the length of the amplicon, pear to support the relationship between M,unirap- and the banding appeared to be composites tor and M. raptor. However, extremely high, within- of the att terns observed in the 6 clones combined. species genetic distances (Nei 119781 unbiased The b&ding pattern of the 10th line lacked several genetic distances of 0.40,0.50, and 0.76 for M. raptor November 1997 TAYLORET AL.: mtDNA VAFUATIONIN Muscidiflrmx SPP. 823 from North Carolina, Utah, and Israel) were ob- the coding regions. Recent translocation and am- served in that study. These distances are far larger plification of mitochondrial sequences in the nu- than expected for within-species variation (Ayala et clear genome has been reported for the desert lo- al. 1974), making Kawooya's results difficult to in- cust (Zhang and Hewitt 1996b). In future studies, terpret. Our mtDNA data clearly indicate that M. the variant digest pattern observed in the excep- miraptw is more closely related to M. raptorellus tional M. zargtw isofemale line will be useful for than to M. mptor. determining the mode of inheritance. Currently, our data are insufficient to evaluate the specific status of M. miraptor. Thelytokous repro- duction was the primary characteristic used by Acknowledgments Kogan and Legner (1970) to distinguish this species. Subsequent studies have implicated Wolbachia, a We thank Wes Watson, J. H. Werren, and M. D. Ellis for samples of M. raptor. M. uniraptw, and A. mellifma, re- cytoplasmically inherited microorganism, as the spectively, and T. 0.Powers for permitting us to do some cause for thelytokous reproduction in M,uniraptor aspects of this work in his laboratory. T. 0. Powers, K. P. (Stouthamer et al. 1993). Wolbachia infections usu- Ruess, R. L. Roehrdanz, and B. 13. Siegfried provided alIy can be eliminated with antibiotics or high tem- helpful suggestions and critical reviews of the manuscript. peratures (Stouthamer and Luck 1988), rendering This work was done in cooperation with the Institute of thelytokous strains arrhenotokous. Crosses between Agriculture and Natural Resources, University of Ne- Wolbachia-free M. unirnptor and M. raptorellus will braska, Lincoln, and is published as Journal Series. Ne- be needed to confirm the specific status of M, un- braska Agricultural Research Division Paper 11709. iraptor (Stoutharner et al. 1990). The levels of sequence divergence observed among M. raptor, M. raptotellus, and M. zaraptur References Cited were higher than expected. Dowton and Austin Antolin, M. F., D. S. Guertin, and J. J. Petersen. 1996. (1995) indicate that a higher rate of mtDNA diver- The origin of gregarious Muscidif;rm (Hymenoptera: gence may be associated with parasitism in Hyrne- Reromalidae) in North America: an analysis using mo- noptera. Nevertheless, the levels of divergence ob- lecular markers. Biol. Control 6: 76-82. served between the species indicate divergence Ayala, F. J.,M. L. Tracey, D. Hedgecock, and R. C.Richmond times of several millions of years (Powell et al. 1974. Genetic differentiation during the speciation pro- 1986); not the 400 yr required if Mrsscidifirax were cess in Drosophilo. Evolution 28: 576-592. Bogdanowin, S. M., W. E. Wallner, J. Bell, T. M. Odell, of Old World origin. These data indicate that the and R. G. Hamson. 1993. Asian gypsy moths (Lep- presence of Murcidiflrrax in the New World pre- idoptera: Lymantriidae) in North America: evidence dates the arrival of house flies and stabIe flies, their from molecular data. Ann. Entomol. Soc. Am. 86: 710- preferred hosts. The phylogeographic structure of 715. the group in the Americas adds further support to Boyce, T. M, M. E. Zwick, and C. F. Aquadro. 1989. this argument. One question which remains to be Mitochondrial DNA in thc bark weevils: sizc, structure resolved is whether M. raptor was a Holarctic spe- and heteroplasmy. Genetics 123: 825- 836. cies, or if its introduction to the Old World was a 1994. 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