Journal of the American Contol Association,9(3\:294-301, 1993

A NEW CYTOTYPE OF ANOPHELES NUNEZTOVARI FROM WESTERN AND

JAN CONN,'2 YADIRA RANGEL PUERTAS,,tND JACK A. SEAWRIGHT3

ABSTRACT. Cytogeneticanalysis of the larval polytene chromosomesof Anophelesnuneztovari from 5 collection sites in T6chira and Zulia statesnorthwest of the Andean Cordillera in westem Venezuela and from 2 sitesin the Department of Valle, westernColombia, revealedwhat appearsto be a distinctive cytotype informally designatedas An. nuneztovariC.lts chromosomesare homosequentialwith those of An. nuneztovariB from westernVenezuela southeast ofthe Cordillera but differ in the presenceofa well- defined chromocenter and unique inversion polymorphisms. The large complex inversion in western Venezuela, 2Lb, is present at a frequency of 0.263 and deviates significantly from Hardy-Weinbery equilibrium in 3 of the 5 sites.Two smaller inversions (2Ir and 2Id) that are included in 2Lb arepresent in the Colombian samplesat a frequency of 0.300.

INTRODUCTION Anophelesnuneztovari has been incriminated as the main vector of the malaria parusite,Plas- The distribution of the malaria vector Anoph- modium vlvax (Grassi and Feletti), in northern eles nuneztovari Gabald,6n includes an extensive Colombia and westernVenezuela (Gabald6n and portion of northern (Fig. 1) as Guerrero 1959,Gabald6n et al. 1963).More re- well as eastern Panama (Faran 1980). Early be- cently, results of monoclonal antibody havioral and ecological observations (Elliott surveys ofCS proteins and/or salivary gland 1972) as well as cytological differences (Kitz- dissections for sporozoiteshave implicated An. nuneztovari miller et al. 1973) suggested that An. nuneztovari in the transmission of P. vivax in Par6 State, consisted of 2 geographically distinctive popu- Brasil (de Armda et al. 1986),eastern (Hayes lations, one a nonvector in the Amazonian drain- et al. 1987) and Amap6 Brasil (Tadei age basin and the other a vector in western Ven- State, et al. I 99 I as well in ezuela and northern Colombia. These populations ), as the transmission of Plas- modiumfalciparumin Amap6 State(Tadei et al. have been designated informally as A (Amazo- 199l). These findings nian) and B (Colombian/Venezuelan) (Conn confound the earlier, sim- pler hypothesis of vector and nonvector 1990) and can be distinguished cytologically by status of An. nuneztovaribased,primarily on geograph- a fixed inversion in the XR arm (Kitzmiller et ical distribution (Kitzmiller et al. 1973). al. 1973). Unfortunately, no voucher specimens In this report, we present evidence for a dis- from the collection sites of Kitzmiller et al. (1973) tinctive cytotype, C, of An. nuneztovari from were saved to determine whether concomitant western Colombia and Venezuelanorthwest morphological differentiation existed. The re- of the Andean Cordillera, and we document the sults of an electrophoretic study (Steiner et al. existence of deviation from Hardy-Weinberg 1980) comparing samples from western Vene- equilibrium for the complex inversion 2Lb in zuela with showed allele frequency dif- Venezuela. ferences at the Est-5 locus that might be consid- ered diagnostic. The authors suggested, however, that additional sites and larger samples should MATERIALS AND METHODS be analyzed. Within Venezuela a comparative In Venezuela, larvae and adult females (the study of male genitalia of An. nuneztovari from latter for progeny rearings) were collected at 5 2 sites, one to the north and the other to the sites:Caffo Macho (8'22'0'N, 7221'0"W), Gua- south of the Andean Cordillera, suggested that ramito (8"13'48'N,72"19'59'W) and Moravia these represent 2 distinct populations (Avila Nu- (8"11'45'N, 72"21'14'W) in T6chira State;and fiez 1989a). Casigua(8'50'43'N, 72o30'10'W)and Rio So- cuav6(8'54'0'N, 72"38'0'W)in Zulia State(Figs. rPresent addressand address for reprint requests: 2 and 3). This area is just northwest of the An- % USDA/ARS, Medical and Veterinary Entomology dean Cordillera and southwest of Lake Mara- ResearchI-aboratory, P. O. Box 14565, Gainesville, caibo. The averagerainfall in the Maracaibo Ba- FL32604. sin variesfrom I,800 to 3,800mm annually,and 2 Instituto de Zoologia Tropical, Universidad Cen- the averagetemperature is 27"C. The mean al- tral de Venezuela, Caracas, Venezuela. 3 titude is 90 m above sealevel (Ewel et al. 1976). USDA/ARS, Medical and Veterinary Entomology The Colombian collection sites of Sitronela and Research Laboratory, Gainesville, FL 32604. a Avila Nufrez, J. L. 1989. Estudio bionomico de dos poblacionesde Anophelesnufteztovari Gabald6n, en el Occidente de Venezuela. Lic. Thesis. Univ. de 1940 (Diptera: Culicidae) separadas geograficamente los Andes. M6rida. Venezuela. 294 SBsrBlaseR1993 Anopnztz s wuNz zrovARI Cvrorvpr

Details of larval rearing, salivary gland dis- section and polytene chromosome slide prepa- ration can be found in Conn (1990). From the venezuelan sites, chromosomes of 177 larvae were examined. Of these, 59 larvae were field collectedand l18 were F, progeny(l-2 larvae per female fro m a total of 72 mothers).The large, complex inversion polymorphism initially de- scribedby Kitzmilleret al. (1973)in the 2L chro- mosome arm has been named 2Lb following the convention ofColuzzi et al- (1973\. The break- points of the new inversion polymorphisms de- scribed here are based on the standard An. nu- neztovari chromosome map (Kitzmiller et al. 1973)and on the photomapofConn (1990).In the Department of Valle, Colombia, both larvae (n : 5) and Fr progeny (n : 8) from 5 adult females were analyzed from Zabaletas,and Ft progeny (n: 2) from one adult female from Si- tronela. Link-reared voucher specimens have depositedat the National Museum of Nat- l. The presumed distribution of the 3 cyto- been Fig. Washing- types(A, B and C) ofthe Anophelesnuneztovari ural History, Smithsonian Institution, complex. ton, DC.

Zabaletas are located at sea level near Buena- RESULTS ventura in the Cauca Valley (Fig. a). This valley is part ofthe marshy coastal lowland with a mean All larvae analyzed (Table l) had identical temperature of 27"C and an average annual rain- polytene chromosome banding pattems for all fall of 7,400 mm (Box 1990). chromosomes. The chromosomal banding pat-

VENEZUELA

Elevqtion lmeid I = ovet 975 ffi=500-9zs B = 200'500 I = Under200

Fig.2. Venezuela.The rectangleis the study area that is enlargedin Fig, 3. JounNal or rrrE ArusRlclN Moseutro Coxrrol Assocrlrror Vor-. 9, No. 3

o CASIGUA (0.l9tll

rlA FRIA I o GUARAMITO (0.2651 o MORAVTA(0.0831 \

ffi oxm60

Fig. 3. The study area in westernVenezuela. The 5 collection sitesare Rio Socuav6,Casigua, Cafro Macho, Guaramito and Moravia. The frequencyof the inverted constituent (b) in the complex inversion 2Lb is found in brackets after each site.

cotoMBlA

Elevotion lmeled- = Ovet 2000 Buenoventuro | E = 975'2@0 & = 500'9zs I = Under500

Fig. 4. Colombia. Buenaventura is the nearest town to the collection sites of Sitronela and Zabaletas. SePTpr{spn1993 ANoPHE LE s vuugzrovtat CvtoTvpr 297 tern of An. nuneztovari C is also identical (i.e., Table l. Collections of An. nuneztovari C homosequential) to that of An. nuneztovari B from Venezuelaand Colombia used for from Barinas, Venezuela(Kitzmiller et al. 1973, cytogeneticanalysis. Conn 1990).However, one striking differenceis Collection Number the presenceof a marked chromocenter in each Location datet analyzed polytenecell of theAn. nuneztovariC mosquitoes (Fig. 5). l. Larval collections Severalpreviously unreported inversion poly- Venezuela morphisms were observed.Inversion 2Ra (l0A/ Caflo Macho July 18, 1989 2 B-l2AlB) was encountered in one larva from Nov. 8, 1989 4 Casigua. A single larva from Guaramito dis- June6, 1990 29 played inversion 3Ra (3lB/C-348). Inversion Guaramiro July 19, 1989 6 3Rb (28AlB-338) was observedin one larva from Casigua June 7, 1990 l8 Cafio Macho. A very small inversion loop in- volving 2 sets of double bands in the 3L arm Colombia "knot" 1992 s (3Lb, 38A/B-38C), was seenas a small Zabaletas Apm25, and found in 2 larvae from Cafio Macho. Asyn- 2. F, progeny from adult female collections apsis for the band in section l5,A',just proximal Venezuela to the sectionlimit l4l15 in the 2R arm (Fig. 5), Caffo Macho July 17, 1989 8 was detectedin 3 larvae from Casigua,in 4 from Nov. 7. 1989 9 Cafio Macho and in 2 from Guaramito. June5, 1990 4 The complex inversion (2Lb) involving much Guaramito Aug. 19, 1989 9 of the 2L arm was first described by lGtzmiller Nov. 8, 1989 19 et al. (1973, Fig. 8) basedon the analysisof 4 Moravia June6, 1990 l8 larvae collected from Casigua.This same inver- Rio Socuav6 July 8, l99l 14 sion was found from all 5 collection sitesin Ven- July 9, l99l 9 ezuela in the present study. A test for homoge- July 10, l99l 12 neity of genotype frequencies between the 5 Sept.22,1992 16 localities showed no significant differences for 2Lb (x2 -- 5-9O,4 df, P < 0.30). For the most Colombia 2 part we agreewith Kitzmiller et al. ( I 973) on the Sitronela Apil24,1992 designation of the major breakpoints (from the 7-,abaletas Apil26,1992 8 centromereto the limit of 22/23; Fig. 6), but the Total 192 larger synaptic region within this complex in- ' Datefor Fr progenyis thedate adults were caught. versionappears to extend from 19/20 to 2lA/B rather than regions2lB,2lC and 20A. We also determined that the included region from 16,4 had 2 inversions, 2I-c and 2l-d (Fig. 6), that ap- to l8C actually appearsto extend farther, from pear to be included within the complex 2Lb in- l6,4.to lgB/C. We agreethat the small inversion version from Venezuela. Inversion 2k com- 2La(15/16to l64lB) inAn. nuneztovariB(Kitz- mencesat the centromere(breakpoint I 5/ I 6) and miller et al- 1973,Conn 1990)is involved in the endsat l8B/C. This inversion polymorphism has complex 2Lb inversion, as suggestedby Kitz- the proximal breakpoint coincident both with miller et al. ( I 973).Simple inspection of the data the small inversion 2La (found in An. nunezto- on frequencyofthe 2Lb inversion indicated that variB from Venezuelasoutheast of the Cordille- several ofthe populations analyzed were not in ra) and the large complex inversion 2Lb from Hardy-Weinberg equilibrium, primarily because the Maracaibo Basin, Venezuela.Inversion 2Ld there were no inversion homozygotes. Because (I9B/C-21B' between the 2nd and 3rd heavy the frequencies of the 2Lb heterokaryotype in dark bands; Fig. 6) is also included within in- field-collected Iawae (26 of 59) and F, progeny version 2Lb, and is presumably linked to 2Lc, (66 of I 18) were not significantly different (1' : as they were always found together. The re- 1.77, I df, P < O.2O),these 2 types of samples maining 6 larvae from Colombia all displayed were combined for each collection site for cal- the standard 2L sequence.Inversion frequencies culation of 12 for the fit to a Hardy-Weinberg of 2Lc * 2Lddid not deviate from Hardy-Wein- equilibrium. When analyzed by geographic lo- berg equilibrium (x' : 2.76, I df, P < 0.10). cation, 3 sites,Cafro Macho, Guaramito and Rio Socuav6,deviated significantly from the Hardy- DISCUSSION Weinberg equilibrium for inversion 2Lb (Table 2). Although homosequential speciesas first de- In the Colombian samples,9 of the I 5 larvae scribed for Drosophila showed significant mor-

SsF-rEr{snn1993 Atto pnz tn s nul r zro vARI CYrorypE 299

Table 2. Frequency ofinversion 2Lb by site. Genotype Freq. inv. Site +/+ +/b b/b const.r x2 P Cafio Macho Obs. 28 28 0 (n: 56) Exp. 31.500 21.000 3.500 0.250 6.22 0.01 Guaramito Obs. t6 l8 0 @:3a) Exp. 18.368 r3.245 2.387 0.265 4.40 0.01 Moravia Obs. l5 J 0 (tz: 18) Exp. 15.1 36 2.740 o.t24 0.083 0.l5 0.70 Casigua Obs. 1t 7 0 (n: 18) Exp. 1t.693 s.629 0.677 o.t94 1.05 0.31 R. Socuav6 Obs. t4 JI 0 (n:51) Exp. 20.694 23.s86 6.720 0.363 16.51 <0.001 I Freq. inv. const.: frequencyinverted constituent (b).

An. aquasalisCurry (Frizzi and Ricciardi 1955, from the Maracaibo Basin in westernVenezuela Kitzmiller and Chow l97l). All polytenechro- include environmental heterogeneities, subdi- mosome preparationsexarnined for thesespecies vided population structure, clinal variation or show distinct chromocenters.In the An. nunez- small sample sizes. In both An. gambiae Giles tovari complex,none ofthe 602 individuals from and An. arabiensisPatton significant diflerences population B in western Venezuelahad a chro- in heterokaryotype frequencies in villages less mocenter (Conn 1990) nor, surprisingly, did the than l0 km apart were attributed to environ- 4 individuals examined from Casigua by IGtz- mental heterogeneities(Coluzzi et al. 1979). In miller et aI. (1973\. We consider the chromocen- particular, in An. gambiae,inversion 2Rd varied ter to be a prominent cytological marker. It has from 0.430 to 0.590; in An. arabiensls,the 2Ra been usedto identify 2 out of I 2 cytotypesin the arrangement ranged from 0.400 to 0.667. One Eusimulium vernum(Macquart) black species hypothesissuggested by theseauthors to account complex (Brockhouse1985). for these different frequenciesinvolved the oc- The presenceof the chromocenter,in addition currenceofpopulation bottlenecksduring the dry to the complex inversion (2Lb), as well as the seasonaccompanied by genetic drift. The abun- other inversions suggeststl:rat An. nuneztovariC dance of An. nuneztovariat El Vigia (8"37'N, is genetically differentiated from B. Lack ofthe 71'39'W), M6rida State,western Venezuela (pre- 2Lb homozygote indicates either complete le- sumably also C) is extremely low (i.e., less than thality or a very low fitness. We cannot deter- 25 adult femalescollected per month [Scorzaet mine which of these explanations is more likely al. I 98 I ]) during the dry season(August-October becauseour Venezuelan sample size (n : 177) and January-March). Becausett.e 5 An. nunez- might fail to detect the homozygote if its fitness tovari C sites in T6chira and Zulia states have is very low. The persistenceof this inversion at similar precipitation patterns (Atlas de Vene- a frequencyof O.263is probably due to some zuela 1979) comparedto those found in El Vigia, form of hybrid superiority. A similar caseof hy- each may be subject to annual bottleneck effects brid superiority hasbeen documented for species and subsequentgenetic drift. However, the ef- A and B of the An. quadrimaculatus complex fects may differ from site to site if inversion 2Lb (Seawright et al. l99l). The left arm of chro- is strongly influenced by microhabitat differen- mosome 3 occursin 2 forms, 3L, and 3L, (Kaiser tiation. This may explain, in part, the deviation et al. 1988),and high frequencies(0.723 and 0.733 from Hardy-Weinberg in samples from Cafro in speciesA and B, respectively) of the hetero- Macho, Guaramito and Rio Socuav6 and not karyotype (3Lt/3L) were observedfor field col- from the other 2 sites (Table 2). lections (Seawrightet al. l99l). A subdivided population structure may result Normally, with a complex inversion such as in part from poor adult dispersalwith a concom- 2Lb, there are severaladditional genotypicclass- itant reduction in geneflow, as was suggestedto es present that result in simple inversion loops explain the distribution ofintergenic spacerfrag- (suchas 2I*and 2Ld in the Colombian samples) ments in An. gambiae from western Kenya orinverted homozygotes.Presumably in the Ma- (Mclain et al. 1989). Their study demonstrated racaibo Basin these other genotypes are being restricted geneflow in 2 populations only l0 km selectedagainst at some level. apart based on significant differencesin the fre- Possibleexplanations for the discrepanciesin quencies of 4 ribosomal DNA array types. Al- Hardy-Weinberg equilibrium in the 5 samples though adult dispersaland flight range have not 300 JounNlr or rHe Al,mRrcAN Mosqurro Coxrnol AssocllrroN Vol.9, No. 3 been systematically measured,in An. nunezto- may be due in part to the genetic differentiation vari, inAn. albimanus (also in the subgenusNys- we have documented. sorhynchus),reports rangefrom 1,800m for wild In order to clarify the genetic structure ofthe populations in Panama (Zetek l9 I 5) to 3 km for An. nuneztovarl complex, we and several col- laboratory-rearedmosquitoes released in El Sal- leaguesare currently involved in analyzing the vador (Hobbs et al. 1974). isozymes,mitochondrial DNA and morpholog- There is no strong evidence suggestingclinal ical characteristics of field populations from variation in the frequency of inversion 2Lb in acrossthe range ofthis species.For 8 restriction An. nuneztovari C from western Venezuela, al- enzymes surveyed, estimates of mtDNA se- though this phenomenon is common in well- quence divergence between 5 populations of B studied anophelines(e.g-, An. gambiae fColuzzi and C for 8 I individual mosquitoes(0.00 l{.0 l0 et al. 19 79, l 9 8 5l, A n. quadri mac u l a/as Say[Kai- [Conn et al., unpublished data]), is in the range ser et al. 19881).The collectionsites in western of intraspecific distancescalculated for members Venezuela (Fig. 3) are within I degree of each of other anophelinespecies complexes (,4 n. quad- other, both latitudinally and longitudinally. The rimaculatus [Mitchell et al. 19921,An. aquasalis mean altitude is 90 m (Ewel et al. 1976),and [Conn et al. 1993]), suggestingthat although B they are all in the same ecological zone (humid and C may be separablecytologically, they are tropical forest [Ewel et al. 1976])in the Mara- very closely related. In concordancewith these caibo Basin,formed during the Pleistocene(Atlas data, an electrophoretic analysis of 28 enzyme de Venezuela1979) and surroundedby the An- loci of samplesfrom either side of the Andes did dean Cordillera to the southeastand the Serrania not show any discriminatory or diagnostic locus de Perij6 to the west. differences (Nei's unbiased genetic distance < A power test for contingency tables (Glantz 0.001 [G. Fritz, unpublisheddata]). The mor- 1992) revealed that the statistical confidence in phological data have not been completely ana- detecting a difference (P < 0.05) for inversion lyzed.No crosseshave beenmade between mem- 2Lb from Hardy-Weinberg equilibrium is less bers of the complex, becauseattempts at forced than 800/obased on the sample sizes at each of copulation have failed. There are no laboratory the 5 sites in the Maracaibo Basin. This strongly colonies of members of this complex, and there suggeststhat additional collections should be is a generaldearth of formal geneticinformation. made in this area to further evaluate the signif- Therefore,most ofthe insights intogenetic struc- icanceofthe frequenciesofthe 3 genotypic class- ture will necessarilycome from biochemical and es of 2Lb in cytotypeC. cytogeneticanalysis. Becausewe have collected cytotype C from Buenaventura, western Colombia, we hypothe- ACKNOWLEDGMENTS sizethat its distribution includesColombia north and west of the Andean Cordillera (Fig. 4) aswell This study was supported in part by the Di- as the Maracaibo Basin in western Venezuela recci6n de Malariologia y SaneamientoAmbien- (Fig. 2). However, this hypothesis can only be tal (Venezuela),the Instituto Venezolanode In- tested with additional samplesfrom this region. vestigacionesCientificas, National Institutes of Cytotype B occurs in the lower montane areas Health Grant AI 31034(USA), Natural Sciences of western Venezuela (IGtzmiller et al. 1973, and EngineeringResearch Council (Canada),the Conn 1990) but it is also likely to be found in Organizaci6n Panamericanade la Salud (Vene- the Venezuelansavannahs (between the Andean ztela), the Servicio de la Erradicacionde Malaria Cordillera and the Orinoco River basin) and in (Colombia), the United Nations Industrial De- contiguous easternColombia (Fig. l). The range velopment Organization (Contract No. 91047) of cytotype A is believed to include the whole and the U.S. National Academy of Sciences Amazon Basin(Kitzmiller et al. 1973).The Ori- BOSTID grant MVR-72. We thank A. Anselmi, noco River in Venezuela(Fig. l) marks a well- R. Barrera,C. Brancho, E. Brown, M. de la Rosa, defined geologicalboundary between the savan- N. Delgado, R. Dussan, G. Fritz, L. Hribar, C. nah region and the Guiana Shield (consideredto Iveros, P. Kaiser, L. P. Lounibos, X. M6rquez, be part of the Amazon Basin) and appears to H. Montaffez, C. Moreno, L. Munstermann, D. serve as a speciesboundary between cytotypes Ortiz Garcia, B. Pattok, C. Pecefio,H. P6rez,E. A and B. Irritability to DDT of An. nuneztovari L. Peyton, S. Ramos, E. Rojas, J. Scorza,A. Ve- from [Jraba (in the Atlantic region of Colombia l6squez, L. Villarreal, R. Wilkerson, R. Zim- near Panama,presumably cytotype C) and from mernann, and the Instituto de Zoologia Tropical Oriente (in the Colombian Llanos east of the of the Universidad Central de Venezuela for Andean Cordillera along the Venezuelanborder, technical and logistical assistance.This is Uni- presumably'cytotype B) was significantly differ- versity of Florida IFAS Journal Series No. ent (P < 0.01 [Quiflonesand Suarez1989]), and R-03004. SnrrsMsnn 1993 ANo PHE LE s w ut tz zro vtru CxrorYPE 301

REFERENCES CITED Glantz, S. A. 1992. Primer of biostatistics.McGraw- Hill, Inc., New York. de Amrda, M., M. B. Carvalho, R. S. Nussenzweig, Green, C. A. and R. H. Hunt 1980. Interpretation of M. Maracic, A. W. Ferreira and A. H. Cochrane. variation in ovarian polytene chromosomes of I 986. Potential vectors of malaria and their differ- Anopheles Giles, A. parensis Gillies and,4. ent susceptibility to Plasmodium and funeslas falciparum Genetica 5 I : I 87-l 95. Plasmodium vivax in northern identified by aruni?. Hayes, J., G. Calderon, R. Falc6n and V. Zambrano. immunoassay.Am. J. Trop. Med. Hyg.35:873-881. 1987. Newly incriminated anopheline vectors of Atlas de Venezuela. 1979. Ministerio del Ambiente human malaria parasitesin Junin Department, Peru. y de los RecursosNaturales Renovables.Direcci6n Am. Mosq. Control Assoc.3:418-422. de Cartografia Nacional, segundaedici6n. J. Hobbs, H., R. E. Lowe and C. E. Schreck. 1974. Box, B. 1990. South American handbook, 66th ed. J. flight rangeand survival of Anophelesal- Prentice Hall, New York. Studiesof bimanusWiedemannin El Salvador.I. Dispersaland Brockhouse,C. 1985. Sibling speciesand sex chro- during the dry season.Mosq. News 34:389- mosomes in Eusimulium vernum (Diptera: Simuli- survival 393. idae).Can. J.7noL 63:2145-2161. B. K. Birky. 1988. Carson,H. L., F. E. Claytonand H. D. Stalker. 1967. Kaiser, P. E., J. A. Seawrightand polymorphism in natural populations Karyotype speciationand stability in Hawaiian Dro- Chromosome quadrimaculatzs speciesA and B. sophila. Proc. Nat. Acad. Sci. 57:1280-1285. of Anopheles Say Co/rrzzi, M., M. Di Deco and G. Cancrini. 1973. Genome30:138-146. W. B. Kitzmiller and M. G. Rabbani. Chromosomal inversions in Anopheles stephensi. Keppler, J., Jr., J. gland Anoph- Parassitologia| 5: 129-136. 1973. The salivary chromosomesof News Coluzzi, M., V. Petrarca and M. A. Di Deco. 1985. elesalbimanus. Mosq. 33:42-49. W. 1971. The Chromosomal inversion intergradation and incipi- Kitzmiller, J. B. and G. Chow. salivary gland of Anopheles aquasalis. Rev. ent speciationin Anophelesgambiae. BolL Znol. 52:. chromosomes 4543. Bras. Malariol. DoencasTrop. 23:65-85. Kitzmiller. B., R. D. Kreutzer and E. Tallaferro. Cofuzzi, M., A. Sabatini, V. Petrarca and M. A. Di J. in populations Deco. 1979. Chromosomal differentiation and ad- 1973. Chromosomal differences of Bull. W.H.O. 48:435455. aptation to human environments in the Anopheles Anophelesnuneztovari. D. Brandling-Bennett gambiae complex. Trans. R. Soc. Trop. Med. Hyg. Mcl,ain, D. K., F. H. Collins, A. 73:483497. and J. B. O. Were. 1989. Microgeographic varia- in rDNA intergenic spacersof Anophelesgam- Conn, J. 1990. A geneticstudy ofthe malaria vector tion in western Kenya. Heredity 62:257-264. Anopheles nuneztovari from western Venezuela. J. Diae Am. Mosq. Control Assoc. 6:40O405. Mitchell, S. E., S. K. Narang, A. F. Cockburn, J. A. 1992. Mitochon- Conn, J., A. F. Cockburn and S. E. Mitchell. 1993. Seawright and M. Goldenthal. Population differentiation of the malaria vector drial and ribosomal DNA variation among membeis quadrimaculatzs (Diptera: Anopheles aquasalis using mitochondrial DNA. J. of the Anopheles Culici- Heredity (in press). dae) speciescomplex. Genome 35:939-950. Elliott, R. 1972. The influence of vector behavior on Quifrones,M. L. and M. F. Suarez. 1989. Irritability populations primary malaria transmission. Am. J. Trop. Med. Hyg. 2l: to DDT of natural of the ma- 7 55-763. laria vectors in Colombia. J. Am. Mosq. Control Ewel, J. J., A. Madriz and J. A. Tosi, Jr. 1976. Znnas Assoc.5:56-59. de vida de Venezuela.Memoria explicativa sobre el Seawright,J. A., P. E. Kaiser and S. K. Narang. 1991. in A mapa ecol6gico, segundd edici6n. Editorial Sucre, A unique chromosomal dimorphism species Venezuela. and B of lhe Anophelesquadrimaculatzs complex. Faran, M. E. 1980. Mosquito studies (Diptera, Cu- J. Hered.82:221-227. licidae) XXXN. A revision of the Albimanus sec. Scorza,J. V., E. Rodriguezand G. Moreno. 1981. poblacional tion of the subgenusNyssorhynchus of Anopheles. Ecologia de Anophelesnufieztovari Ga- Contrib. Am. Entomol.Inst. (Ann Arbor) l5:l-215. baldon, I 940 en el occidentede Venezuela.Bol. Dir. Fritz, G. N., S. K. Narang, D. L. Kline, J. A. Seawright, Malariol. San.Amb. 2l:l-27. R. K. Washino,C. H. Porterand F. H. Collins. 1991. Steiner.W. W. M.. J. B. Kitzmillerand D. L. Osterbur. 1980. Gene differentiation in chromosomeraces of Diagnostic characterization of Anophelesfreebornt and An. hermsi by hybrid crosses,frequencies of Anopheles nuneztovari Gabaldon. Mosq. Syst. 12: polytene X chromosomesand rDNA restriction en- 306-3l 9. zyme fragments.J. Am. Mosq. Control Assoc.7: 198- Tadei, W. P., A. W. Ferreira, S. L. M. de Avila, R. S. 206. Nussenzweig,P. A. Xavier and I. E. N. S. Lima. Frizzi, G. and I. Ricciardi. 1955. Introduzione allo 199I . Prevalenceof Plasmodium spp. in Anopheles studio citogeneticadella fauna anofelica del Brasile. spp. in goldmining areas of Amapa State, Brazil, Rev. Bras. Malariol. DoencasTrop.73994O7. detected by immuno-enzymatic assay.Abstract Gabald6n, A. and L. Guerrero. 1959. An attempt to 10.34. IV International Congresson Malaria and eradicate malaria by weekly administration of pyri- Babesiosis,Rio de Janeiro, Brasil, August 14-17, methamine in areasof out-of-doors transmissionin 1991. Venezuela.Am. J. Trop. Med. Hyg. 8;433439. Zntek, J. 1915. Behavior of Anopheles albimanus Gabald6n. A.. G. Guerrero and G. G. Martin. 1963. Wiede. and tarsimaculata Goeldi. Ann. Entomol. Malaria refractaria en el occidente de Venezuela.Rev. Soc.Am. 8:221-271. Venez.Sanid. l4:5 l3-530.