Journal of the American Mosquito Contol Association, 12(3):477-482, 1996Copyright O 1996 by the American Mosquito Control Association, Inc.

RESTRICTION ANALYSIS OF THE RIBOSOMAL DNAINTERNAL TRANSCRIBED SPACER REGION OFCULEX RESTUANS AND MOSQUITOES IN THE

CULEX PIPIENS COMPLEX

BETTINA A. DEBRUNNER-VOSSBRINCK.' CHARLES R. VOSSBRINCK,'MICHAEL H. VODKINT AND ROBERT J. NOVAICa

ABSTRACT, Members of the Culex pipiens Linn. complex in the eastern, southern, and central UnitedStates are the primary vectors of St. Louis encephalitis virus. Although species and subspecies in thecomplex can be identified as 4th-instar larvae and by characters on the male genitalia, adult femalescannot be identified accurately. In this study a ribosomal DNA (rDNA) segment that includes the internaltranscribed spacer region (ITS) was amplified from Culex pipiens pipiens Linn., Culex quinquefasciatusSay, and Culex restuans Theobald. The DNA was amplified from single abdomens or single legs. Theamplified rDNA segment from C.r. restuans is 90 base pairs smaller than those from members of the Cx.pipiens complex. Ribosomal DNA was amplified separately from 3 individuals for each population ofCx. pipiens and analyzed by restriction digestion. Intrapopulation variation is seen, because for eachpopulation, bands are present that are common to all 3 individuals within the population, but are alsounique to that population. These results indicate that this method may provide a means for distinguishingamong the mosquitoes in the Cx. pipiens complex.

INTRODUCTION

The primary epidemic vectors associated withthe transmission cycle of St. Louis encephalitisvirus (SLEV) are all members of the subgenusCulex. ln the United States these mosquitoes in-clude Culex tarsalis Coq. in the west, the Culexpipiens complex in the eastern, southern, andcentral States, and Culex nigripalpus Theobaldin Florida (Hammon and Reeves 1942, Reeveset al. 1942, Chamberlain et al. 1964, Dow er al.1964). The Cx. pipiens complex is composed of2 species, Culex pipiens pipiens Linn. and Culexquinquefasciatas Say, and a single subspeciesCulex pipiens pallens Coquillett (Knight andStone 1977; Harbach et al. 1984, 1985). Harbachet al. (1984, 1985) synonymized both Culex pi-piens molestus Forskal and Culex pipiens callotiRioux and Pech as behavioral/physiological var-iants of Cx. p. pipiens. Two other members ofthe subgenus Culex have also been implicated asplaying a role in the enzootic transmissionamong vertebrate animals as well as epidemictransmission of SLEV to humans. These twospecies are Culex salinarius Coq. and Culex res-tuans Theobald (Monath 1980).

Based on morphologic characteristics of adultfemales, only C-r. tarsalis and Cx. nigripalpuscan be easily identified to species. The only re-liable diagnostic characters that can be used to

rlllinois Natural History Survey, Center for Eco-nomic Entomology, Medical Entomology Program,6O7 E. Peabody Drive, Champaign, IL 61820.

'�University of Illinois, Department of Entomology,Urbana, IL 6l8Ol.

3 To whom reprint requests should be addressed.

separate Cx. pipiens from Cx. quinquefasciatusare the phallosome of the male genitalia (Belkin1962), and the shape of the larval siphon (Car-penter and LaCasse 1955). Altholgh Cx- sali-narius and Cx. restuans have diagnostic char-acteristics in the adult female (Carpenter andLaCasse 1955), these characters are often lostduring sampling, transport, or aging. The prob-lem of female identification is further compli-cated in areas where Cx. pipiens and, Cx. quin-quefasciatus are sympatric. Jacob et al. (1979)used DV/D ratios to determine that 39-5OVo ofthe Culex males collected in Memphis, TN, wereCx. pipienslCx. quinquefosciarus hybrids. Sun-dararaman (1949) deflned DV/D as the exten-sion of the ventral arm of the phallosome be-yond the intersection with the dorsal arm (DV)relative to the distance between the dorsal arms(D). Collections of the Cx. pipiens complex fromdifferent sites in North America were analyzedusing the DV/D ratio, and a north-south clinewas found between the 2 subspecies (Barr1957). Culex pipiens was found north of latitude39T.{, and Cx. quinquefasciatus occurred southof latitude 36'N. Both species and hybrids oc-curred between the latitudes 36'N and 39'N.

Several investigators have used biochemicaltechniques to characterize and distinguish mem-bers of the Cx. pipiens complex. Cupp and Ib-rahim (1973) used immunoelectrophoresis to de-tect differences among Cx. pipiens, Cx. quin-quefasciatus, and their hybrids. Cupp and lbra-him (1973) were also able to distinguish C.r.pipiens (molestus) from Cx. p. pipiens and Cx.quinquefasciafzs. Saul et al. (1977) used poly-acrylamide gel electrophoresis and a doublestaining technique to distinguish among Cx. res-

478 JourulaL oF THE AMERICIN Mosqurro Covrnol Assocrerron VoL. 12, No. 3

tuans, Cx. p. pipiens, and Culex territans Walk-er. Preliminary work indicated that Cx. quinque-fasciatus and Cx. salinarius could also be iden-tified using this method. Cheng et al. (1982) de-termined the biochemical genetics of the Cx.pipiens complex through investigations of sev-eral isoenzyme loci. All of these biochemicaltechniques require that the specimen be sacri-ficed.

Several investigations have utilized the ribo-somal DNA (rDNA) locus to distinguish amonganopheline species. Two of the studies (Mclainand Collins 1989, Collins et al. 1990) demon-strated variability in this locus by Southern blot-ting of DNA digested with a restriction enzyme.An alternative strategy was developed by Porterand Collins (1991). They sequenced the rDNAfrom 2 sibling species and designed 2 species-specific primers that yielded products of differ-ent size.

In a recent report, Crabtree et al. (1995) usedthe rDNA internal transcribed spacer (ITS) re-gion to differentiate among Culex species. TheITS region was amplified from 2 individuals ofeach population studied in order to design spe-cies-specific or population-specific primers. Theamplified DNA was cloned and 2 independentclones from each individual were sequenced.Based on this sequence information the authorswere able to design primers to differentiateamong Cx. salinarius, Cx. restuans, and Cx. p.pipiens, but were not able to resolve Cx. p. pi-piens from Cx. quinquefasciatus.

The objective of this study was to distinguishamong populations of the subgenus Culexby re-striction analysis of the amplifled rDNA ITS re-gion. We wished to develop a rapid assay thatcould be done without destruction of the mos-quito, leaving it available for genetic crossing,morphologic analysis, or viral detection studies.

MATERIALS AND METHODS

Mosquitoes: Culex restuans was collected inIllinois. Egg rafts were collected in the field andreared to adults in the laboratory. Fourth-instarlarvae reared from each egg raft were used forspecies identification. Laboratory colonies wereestablished from Cx. p. pipiens collected in Il-linois, Cx. quinquefasciarus collected in Florida,Cx. quinquefasciatus collected in Louisiana, andCx. p. pipiens (variant molestus) collected inRome, Italy. Mosquitoes were maintained at 18-30oC, a photoperiod of 16:8 (L:D), and an RHof 6OVo. Adults were provided with a lO-ZOVohoney solution and were fed on quail l-2 timesa week. Eggs were collected and hatched asneeded. Larvae were reared on Tetramin(TetraWerke, Melle, Germany) and rabbit chow.

Specimen preparation: Homogenates wereprepared from the abdomen or a leg of individ-ual female mosquitoes. Single abdomens andlegs were homogenized with a Teflon pestle ina 1.5-ml microcentrifuge tube containing 150 plor 50 pl, respectively, of STE buffer (l0O mMNaCl, 10 mM Tris-HCl, pH 8.0, and I mMEDTA). Homogenates were incubated for 5 minat 95"C and then centrifuged in a Microfuge-E(Beckman, Fullerton, CA) for 5 min at 16.000x g, 4oC. Supernatants were transferred to newtubes and held at -20'C until frozen. The su-pernatants were then thawed, centrifuged for 5min at 16,000 X g, 4'C, and transferred to newtubes.

Amplification of the rDNA ITS region: Theprimers used for the polymerase chain reaction(PCR), rS52156f (5' �-CTGGGCTGCACGCG-CGCT-3') and ILS228r (5'-GTTAGTTTCTTT"I-CCTCC-3'), were selected to amplify a segmentof the rDNA with about 370 bases of the 3' endof the l8S rDNA, the ITSI region, the 5.8SrDNA, the ITS2 region, and 75 bases at the 5'end of the 28S rDNA. Each 100-pl reaction mixcontained 4 pl of homogenate, 4.2 pmoles ofeach primer,0. I mM each of dATB dGTP, dTIP,and dCTP, 1.5 mM MgCl, lx buffer containing50 mM KCl, l0 mM Tris-HCl, pH 8.3, andO.lVoTriton X-100, and 2.5 units of Taq DNA poly-merase (Promega Corporation, Madison, WI).Reactions were incubated at94"C for 3 min, fol-lowed by 40 cycles of 94'C for 30 sec, 4O'C forI min, and 72"C for 1.5 min. For DNA extractedfrom single abdomens, 3 100-pl PCR reactionswere pooled for each sample, and the DNA wasprecipitated with 0.5 volume of 7.5 M ammo-nium acetate and 2.5 volumes of ethanol at-2O"C. The precipitated DNA was resuspendedin 50 pl of sterile water. For single legs, theproduct of a l0o-pl PCR reaction was precipi-tated with ethanol as above and resuspended in1O pl of sterile water. Four microliters of eachsample was analyzed on a lqo agarose gel in I XTAE buffer (40 mM Tfis-HCl, pH 8.0, 5 mMsodium acetate, and 2 mM NaTEDTA).

Restriction analysis of PCR products: Am-plified DNA (0.35 pg per reaction, as deter-mined by ethidium bromide-stained gels) wasdigested in separate reactions with Hae III, HpaII, Alu I, and Rsa I restriction enzymes (BRL,Gaithersburg, MD). Several other 4-base recog-nition restriction enzymes were tried, but onlythe above 4 proved informative. Digested DNAwas analyzed on3qo or 5Vo 3:l NuSieve agarosegels (FMC BioProducts, Rockland, ME) in I xTAE buffer.

RESULTS

The region of the rDNA that was amplified isshown in Fig. l. As seen in Fig. 2, the amplified

482 JounNaL oF THE AMERTCAN Moseurro CoNrnol AssocrnuoN Vor . 12 , No.3

tus; J. Day, Florida Medical Entomology Lab, forproviding Cx. quinquefasciatus; Karen McClellanand Hilary Lee for providing field-collected C.r.restuans: and Nina Krasavin for providing lab_reared Cx. pipiens. This work was supported inpart by funds from the former Illinois Departmentof Energy and Natural Resources, currently theIllinois Department of Natural Resources, S'ENRTM-5 (R.J.N.). Computer resources were provid-ed by the Pittsburgh Supercomputer Center grantPSCB DMB89O07 7 p (M.H.V.).

REFERENCES CITEDBarr, A. R. 1957. The distributionof Culex p. pipiens

and C. p. quinquefasciatus in North America. Am.J. Tlop. Med. Hyg. 6:153-165.

Barr, A. R. 1982. T\e Culex pipiens complex, pp.551-572. 1n.' W. W. M. Steiner, W. J. Tabachnick,K. S. Rai and S. Narang (eds.). Recent developmentsin the genetics of insect disease vectors. Stipes pub-lishing Co., Champaign, IL.

Belkin, J. N. 1962. The mosquitoes of the South Pa-cific (Diptera: Culicidae), Volumes l-2. Universityof California Press, Berkeley, CA.

Carpenter, S. J. and W. J. LaCasse. 1955. Mosquitoesof North America. University of California Press,Berkeley, CA.

Chamberlain, R. W., W. D. Sudia, P H. Coleman andL. D. Beadle. 1964. Vector studies in the St. Louisencephalitis epidemic, Tampa Bay area, Florida.Am. J. Trop. Med. Hyg. 13:456-461.

Cheng, M. L., C. S. Hacker, S. C. Pryor, R. E. Ferrelland G. B. Kitto. 1982. The ecological genetics ofthe Culex pipiens complex in North America, pp.581-627. 1n; W. W. M. Steiner. W. J. Thbachnick.K. S. Rai and S. Narang (eds.). Recent developmentsin the genetics of insect disease vectors. Stipes Pub-lishing Co., Champaign, IL.

Collins, F, H., C. H. Porter and S. E. Cope. 199O.Comparison of rDNA and mtDNA in the siblingspecies Anopheles freeborni and, A. hermsi. Am. LTrop. Med. Hyg. 42:417-423.

Crabtree, M. 8., H. M. Savage and B. R. Miller. 1995.Development of a species-diagnostic polymerasechain reaction assay for the identification of Culexvectors of St. Louis encephalitis virus based on in-terspecies sequence variation in ribosomal DNAspacers. Am. J. Trop. Med. Hyg. 53:lO5-1O9.

Cupp, E. W. and A. N. Ibrahim. 1973. Identificationof members of the Culex pipiens complex by im-munodiffusion and immunoelectrophoresis. J. Med.Entomol. lO''277-281.

Dow, R. P, P H. Coleman, K. E. Meadows and T. H.Work. 1964. Isolation of St. Louis enceohalitis vi-

ruses_from mosquitoes in the Thmpa Bay area ofFl,orida during the epidemic of L962. Am. J. Trop.Med. Hyg. 13:462-468.

Gelfand, D. H. and T, J. White. 1990. ThermostableDNA polymerases. 12..M. A. Innis, D. H. Gelfand.J. J. Sninsky and T J. White (eds.). pCR protocols.A guide to methods and applications. AcademicPress, San Diego, CA.

Hammon, W. McD. and W. C. Reeves. 1942. Cutextarsalis Coquillett, a proved vector of St. Louis en-cephalitis. Proc. Soc. Exp. Biol. Med 5O:142-143.

Harbach, R. 8., C. Dahl and G. B. Graham. 1985.Culex (Culex) pipiens Linnaeus (Diptera: Culici-dae): concepts, type designations and description.Proc. Entomol. Soc. Wash. 87:l-24.

Harbach, R. E., B. A. Harrison and A. M. Gad. 1984.Culex (Culex) molestus Forskal (Diptera: Culicidae):neotype designation, description, variation, and tax-onomic status. Proc. Entomol. Soc. Wash. 86:521-542.

Jacob, W. L., S. A. Daggers, D. B. Francey, J. Mul-lenix and K. Mosely. 1979. T\e Culex pipiens com-plex in Memphis, Tennessee. Mosq. Syst. ll:179-186.

Knight, K. L. and A. Stone. 1977. A catalog of themosquitoes of the world (Diptera: Culicidae), 2nded. Thomas Say Found. 6:1-611.

Kumar, A. and K. S. Rai. 1990. Chromosomal local-ization and copy number of 18S + 28S ribosomalRNA genes in evolutionarily diverse mosquitoes(Diptera, Culicidae). Hereditas 1 13 :277 -289.

Kumar, A. and K. S. Rai. 1993. Molecular organiza-tion and evolution of mosquito genomes. Comp.Biochem. Physiol. 1O6:495-5O4.

Mclain, D. J. K. and E H. Collins. 1989. Structureof rDNA sequence variation within and betweenspecies of the A. gambiae complex. Heredity 62:233-242.

Monath, T. P 1980. St. Louis encephalitis. AmericanPublic Health Association, Washington, DC.

Porter, C. H. and E H. Collins. 1991. Species-diag-nostic differences in a ribosomal DNA internal tran-scribed spacer from sibling species Anopheles free-borni and Anopheles hermsi (Diptera: Culicidae).Am. J. Tiop. Med. Hyg. 45:271-279.

Reeves. W. C.. W. McD. Hammod and E. M. Izumi.1942. Experimental transmission of St. Louis en-cephalitis virus by Culex pipiens Linnaeus. Proc.Soc. Exp. Biol. Med. 5O:125-128.

Saul, S. H., P R. Grimstad and G. B. Craig, Ir. 1977.Identification of Culex species by electrophoresis.Am. J. Trop. Med. Hyg. 26:lOO9-lO12.

Sundararaman. S. 1949. Biometrical studies on inter-gradation in the genitalia of certain populations ofCulex pipiens and, Culex quinquefasciatus in theUnited States. Am. J. Hyg. 6:3O7-314.