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2008

Insight into Anopheles (Nyssorhynchus)

(Diptera: Culicidae) species from Brazil full

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Insight into Anopheles (Nyssorhynchus) (Diptera: Culicidae)Species from BrazilAuthor(s) :M. A M. Sallum, M. T. Marrelli, S. S. Nagaki, G. Z. Laporta, and C. LS. Dos SantosSource: Journal of Medical Entomology, 45(6):970-981. 2008.Published By: Entomological Society of AmericaDOI: 10.1603/0022-2585(2008)45[970:IIANDC]2.0.CO;2URL: http://www.bioone.org/doi/full/10.1603/0022-2585%282008%2945%5B970%3AIIANDC%5D2.0.CO%3B2

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MORPHOLOGY, SYSTEMATICS, EVOLUTION

Insight into Anopheles (Nyssorhynchus) (Diptera: Culicidae) Speciesfrom Brazil

M.A.M. SALLUM,1 M. T. MARRELLI, S. S. NAGAKI, G. Z. LAPORTA, AND C.L.S. DOS SANTOS

Departamento de Epidemiologia, Faculdade de Saude Publica, Universidade de Sao Paulo, Avenida Doutor Arnaldo 715,CEP 01246-904, Sao Paulo, Brazil

J. Med. Entomol. 45(6): 970Ð981 (2008)

ABSTRACT Anopheles (Nyssorhynchus) benarrochi s.l.,Anopheles (Nyssorhynchus) oswaldoi s.l., andAnopheles (Nyssorhynchus) konderi s.l. collected in Acrelandia, state of Acre, Brazil, were identiÞedbased on morphological characters of the male genitalia, fourth-instar larvae, and pupae. Morpho-logical variation was observed in the male genitalia of these species in comparison with specimens fromother localities in Brazil. DNA sequence from the nuclear ribosomal second internal transcribed spacerof individuals identiÞed as An. benarrochi s.l. by using male genitalia characteristics showed that thevarious morphological forms are conspeciÞc but are distinct from An. benarrochi B from Colombia.Anopheles konderi s.l. andAn.oswaldoi s.l. both misidentiÞed asAn.oswaldoi s.s. (Peryassu) throughoutBrazil, may actually comprise at least two undescribed species. Diagnostic morphological character-istics of the male genitalia are provided to distinguishAnopheles benarrochi s.l.,Anopheles oswaldoi s.l.,and Anopheles konderi s.l. from morphologically similar species. Incrimination of An. oswaldoi s.s. inmalaria transmission inBrazil needs further investigationbecauseotherundescribedspecies fromAcremay have been confounded with this taxon.

KEY WORDS Anopheles, Nyssorhynchus, male genitalia, identiÞcation, internal transcribed spacer 2

In the second half of the 20th century, the control ofhuman malaria transmission was the object of a world-wide campaign to eliminate the vector mosquitoes byusing DDT. As a result, malaria transmission was elim-inated from developed countries and from developedareas of developing countries. In areas where livingconditions were poor and the climate and ecologicalconditions were ideal for the proliferation of vectormosquitoes, malaria transmission continued to be in-tense. This is the case in areas in the Brazilian Amazon(Tauil 2006). Because of a continuous migration ofsusceptible humans from nonendemic areas to theAmazon region and among distinct localities in theAmazon, the annual incidence of malaria has in-creased. In 1999, the Brazilian federal government andthe Amazonian states government adopted an aggres-sive strategy for malaria control, targeting 32% of themunicipalities in the Amazon that accounted for 93.6%of cases (de Castro et al. 2007). Consequently, activemonthly searches and immediate treatment of bothsymptomatic and asymptomatic infections were car-ried out in communities throughout the Brazilian Am-azon (Macauley 2005). As observed by Macauley, thestrategy adopted would be effective for the control ofmalaria if carried out with the following criteria: 1)effective treatment; 2) monitoring of human migra-tion for detection of infected individuals; and, 3) col-

laboration of the local community. The control mea-sures provided a quick and dramatic reduction in theannual incidence of malaria. However, the program,which was almost exclusively based on the search andtreatment of infected individuals, was not continuedand the incidence of malaria again began to increasein almost all locations. Unfortunately, other controlmeasures were not adopted, e.g., vector control, hu-man migration control, and improvement of livingconditions. Tauil (2006) took into consideration thatthe rational use of insecticides and larvicides for mos-quito control, reduction, elimination, and cleaning oflarval habitats, as well as participation of the localcommunity, would be required for the success of anyvector control strategy.

The Brazilian National Program for Malaria Preven-tion and Control is divided into nine components(Coura et al. 2006). One of these is the selectivecontrol of vectors. Thus, any measures adopted shouldbe dependent on which mosquito species are impor-tant in malaria transmission in a given locality. There-fore, entomological studies are necessary for bothknowing the species composition in a certain locationand for understanding ecological and biological as-pects of those species that have vector potential.

Singer and de Castro (2006) pointed out that ma-laria transmission in the Amazon over the past 100�years has been favored by ecosystem transformationscaused by human migration, expanding of frontier1 Corresponding author, e-mail: masallum@usp.br.

0022-2585/08/0970Ð0981$04.00/0 � 2008 Entomological Society of America

lands for agricultural, settlement, cattle ranching, andnatural resource extraction resulting in extensive de-forestation. Furthermore, Singer and de Castro (2006)considered that the phenomenon deÞned as frontiermalaria operates in three spatial scales in conjunctionwith a temporal scale, and they discussed some eco-logical characteristics ofAn.darlingiRoot, the primarymalaria vector species in the Amazon. A fundamentalpoint is that human behavior can promote the creationof ecological conditions for the proliferation of vectorspecies and human exposure to mosquito bites. More-over, Singer and de Castro (2006) observed that thebimodal biting pattern of An. darlingi, at dawn anddusk, may be related to the phase of the deforestation/human incursion process. Vittor et al. (2006) exam-ined the impact caused by deforestation in the pop-ulation of An. darlingi in an area situated in thePeruvian Amazon. They found that the An. darlingibiting rate was 278 times higher in deforested areasthan in predominantly forested areas. It is noteworthythat in highly deforested area in Brazil (Dourado mu-nicipality, in Sao Paulo state), An. darlingi showedbimodal dusk and dawn biting peaks (Forattini 1987),whereas in the Peruvian Amazon, Vittor et al. (2006)observed a unimodal biting peak, from 2100 to 2300hours. An. darlingiwas rare in areas of primary forest,suggesting that the immatures were not ovipositing inthe forest habitats. According to Singer and de Castro(2006), malaria transmission rapidly increases in areasof colonization projects where substantial govern-ment-sponsored and informal human migration is ac-companied by deforestation. After 6 or 7 yr of landoccupation, when a more organized urbanization pro-cess is established, malaria transmission becomes morestable and malaria rates decrease. Along with the pro-cess of land occupation and urbanization, ecologicalconditions may become inhospitable for An. darlingi;however, they may be adequate for other AnophelesMeigen vector species. Vasconcelos et al. (2006) sug-gested that a reduction in relative humidity in defor-ested Amazon areas, the presence of larval habitatsand the increase in temperature may be favorable forthe proliferation of thoseAnophelesvector species thathave capacity to adapt to those environmental con-ditions. Boete and Paul (2006) pointed out the possi-bility that changes in anopheline species or genotypecomposition within a population of mosquito vectors,as a result of, for example, vector-control measures,could have a considerable impact on transmission ofsympatric parasite species. For example, Povoa et al.(2003), in a study conducted in the city of Belem, Para,observed a change in the species composition ofAnopheles,with the reappearance of An. darlingi. Thiswas followed by an increase in malaria cases caused byPlasmodium vivax Grassi & Feletti and a decrease ofthose caused by Plasmodium falciparumWelch.

Using individuals collected in three deforested ar-eas in the state of Acre, Brazil, Branquinho et al.(1993) tested 2,610 individuals of An. oswaldoi s.l. byenzyme-linked immunosorbent assay (ELISA) by us-ing speciÞc monoclonal antibodies for detecting P.falciparum, P. vivax, P. vivax V247, and Plasmodium

malariae Feletti & Grassi. The infection rates of An.oswaldoi s.l. for all Plasmodium tested were higherthan those observed for An. deaneorum Rosa-Freitas.No samples ofAn. darlingi andAn. triannulatus (Neiva& Pinto) were found positive. Because of these results,An. oswaldoi s.l. was considered to be the main malariavector in those localities. Later, Branquinho et al.(1996) dissected the midguts and the salivary glands todetermine oocyst and sporozoite rates in Anophelesspecies collected in Senador Guiomard and Placido deCastro, state of Acre. As a result, only oneAn. oswaldois.l. collected from a Shannon trap was found to bepositive for both sporozoites and oocysts. In the sameareas, Marrelli et al. (1998) recorded An. oswaldoi s.l.and humans infected with P. vivax-like/P. simiovaleStephens parasites corroborating the importance ofAn. oswaldoi s.l. as a vector in recently settled areas inthe state of Acre. Subsequently, Marrelli et al. (1999a)compared the susceptibility ofAn. oswaldoi s.l. andAn.konderi s.l. to infection withP. vivaxby using mosquitopopulations obtained in Sena Madureira, in the stateof Acre, and in Sao Miguel, in the state of Rondonia,respectively. They demonstrated that An. oswaldoi s.l.was involved in the transmission of P. vivax, whereasAn. konderi s.l. developed P. vivax oocysts in the mid-gut, sporozoites failed to develop in the salivaryglands. Incrimination of An. oswaldoi s.l. as a vector ofP. vivax in Putumayo, southern Colombia, was shownby Quinones et al. (2006).

The epidemiological importance ofAn. oswaldoi s.l.may be over or underestimated in the state of Acre asa consequence of species misidentiÞcation. Natal et al.(1992) collected 4,588 culicid specimens in the set-tlement Pedro Peixoto, located in the Purus RiverBasin. Pedro Peixoto settlement is situated in the mu-nicipalities of Rio Branco, Senador Guiomard andPlacido de Castro, Acre. Among 53 species or speciesgroups identiÞed, An. oswaldoi s.l. (as An. oswaldoi)was the most numerous species collected in theregion and made up 86% of the anophelines col-lected (3,156 specimens). However, two males iden-tiÞed as An. oswaldoi s.l. by Natal et al. (1992) thatare deposited in Faculdade de Saude Publica (FSP/USP) collection, it was possible to identify them asAn. rangeliGabaldon, Cova Garcia, and Lopez. Con-sequently, some individuals ofAn. rangeliwere misi-dentiÞed as An. oswaldoi s.l.

The Oswaldoi Group (Faran 1980) of the subgenusNyssorhynchusBlanchard comprises 18 species, Þve ofwhich have been incriminated as vectors of humanPlasmodium and at least four, An. aquasalisCurry, An.benarrochi Gabaldon, Cova Garcia & Lopez, An. Os-waldoi, and An. nuneztovari Gabaldon are complexesof sibling species. Marrelli et al. (1999b) using nucle-otide sequences of the ITS2 of the rDNA showed thatAn. oswaldoi s.l. consists of a complex of at least fourspecies, one of which may correspond to An. konderiGalvao & Damasceno, recently elevated from synon-ymy with An. oswaldoi by Flores-Mendoza et al.(2004). There are various internal transcribed spacer(ITS)2 sequences of An. oswaldoi s.l. available inGenBank, i.e., Brazil: Acre AF055068, Amapa AF056318,

November 2008 SALLUM ET AL.: Anopheles SPECIES IN THE STATE OF ACRE 971

Amazonas AF056317, Rondonia AF055069 (Marrelli etal. 1999b); Colombia: Putumayo, AY679149-55 (Ruizet al. 2005); Peru: Yurimaguas, AF055071 (Marrelli etal. 1999b); Venezuela: Ocama, AF055070 (Marrelli etal. 1999b); and from unnamed localities: U92344,U92352-3 (Danoff-Burg & Conn direct submission). AFASTA search revealed that the ITS2 sequences ofAn.oswaldoi s.s. (n� 12) generated from individuals col-lected in the type locality in Espõrito Santo state andalso in southern Sao Paulo state (Espõrito SantoEF457228-37; Sao Paulo EF457238-9) were uniquewith regard to those deposited in GenBank (Motoki etal. 2007). Consequently, the identiÞcation of the spec-imens mentioned above remains unresolved. Addi-tionally, when using nucleotide sequences of thecytochrome oxidase I gene (COI) from the mitochon-drial DNA to evaluate genetic variability among fourpopulations ofAn. oswaldoi s.l. from the states of Acre,Rondonia, Amazonas, and Para, Brazil, Scarpassa andConn (2006) detected no gene ßow among those pop-ulations, and the COI haplotypes grouped into fourclusters that may correspond to distinct species.

Morphological and behavioral differences suggestthatAn. benarrochi s.l. comprise a species complex.An.benarrochi is zoophilic in Rondonia, Brazil (Klein et al.1991), whereas it is anthropophilic in Colombia (Qui-nones et al. 2001), and in the west of Loreto, Peru(Aramburu et al. 1999). Ruiz et al. (2005) suggestedthat An. benarrochi from southern Colombia and

Western Peru may comprise a distinct species in theAn. benarrochi complex, designated An. benarrochi B,whereas An. benarrochi is present in Venezuela andBrazil. However, because the type specimen of An.benarrochi is missing, the identity of the species needsto be established using topotypic specimens from LaCeiba, Venezuela (Ruiz et al. 2005).

Characters of the male genitalia have proven effec-tive for separating species of Culicidae, includingthose of the genus Anopheles. Anopheles oswaldoi andAn. konderi can be easily separated based on morphol-ogy of the aedeagus (Flores-Mendoza et al. 2004).Similarly, Bergo et al. (2007) hypothesized that An.goeldii Rozeboom & Gabaldon may be a valid speciesand that there is an unnamed morphological form thatcan be misidentiÞed as An. nuneztovari when usingadult female characters. In this study, with the primaryobjective of collecting An. oswaldoi s.l. to verify spe-cies identiÞcation, we obtained reared-associatedspecimens of all Anopheles species encountered inAcrelandia municipality, state of Acre, Brazil.

Materials and Methods

Mosquito Collection. Immature collections wereconducted in Linha 14 (9�41�03.5� S, 67�08�05.3� W),Ramal do Granada, Acrelandia municipality, state ofAcre, Brazil (Fig. 1), in the area of the Acre Project(Silva-Nunes et al. 2006). Acrelandia borders the

Fig. 1. Map of the state of Acre, showing Acrelandia municipality and the mosquito immature collection localities.

972 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 45, no. 6

counties of Senador Guiomard and Placido de Castroin Acre, and the states of Amazonas and Rondonia andthe country of Bolivia. The county is located betweenthe Abuna and Iquiri Rivers in the Acre River Valley.Ramal do Granada is part of the Pedro Peixoto Orga-nized Agricultural Settlement, which was imple-mented by the National Institute for Colonization andAgrarian Reform in the mid-1970s. Details about thearea of the Acre Project are in Silva-Nunes et al.(2006).

In this study, rDNA ITS2 nucleotide sequenceswere derived from seven individually reared adultmale specimens, with associated fourth-instar larvaland pupal exuviae, and adult male genitalia kept asvouchers. Species identiÞcation was based on the malegenitalia characteristics.DNA Extraction, ITS2 Amplification, Cloning, andSequencing. DNA was extracted from the specimensfollowing the tissue DNA extraction protocol pro-vided by the QIAgen DNeasy blood and tissue kit(QIAGEN, Crawley, United Kingdom). All bufferswere supplied in the kit. Because DNA often remainsbound to the membrane after the Þrst elution, theelution step was repeated and stored in a separatetube.

One microliter of the Þrst elution was used as DNAtemplate in the polymerase chain reactions (PCR).AmpliÞcation of the ITS2 region was carried out using5.8SF, 5�-ATC ACT CGG CTC GTG GAT CG-3�, and28SR, 5�-ATG CTT AAA TTT AGG GGG TAG TC-3�primers. PCR was carried out in a 25-�l reaction mixcontaining 1 �l of DNA, 10 mM Tris-HCl, pH 8.3, 50mM KCl, 1.5 mM MgCl2, 2.5 �l of dimethyl sulfoxide,5 pmol of each primer, 200 �M each dNTPs, and 2.5 Uof Taq polymerase (New England Biolabs, Ispwich,MA). PCR ampliÞcation protocol consisted of a 2-mindenaturation at 94�C, 34 cycles at 94�C, 57�C and 72�Cfor 30 s each, followed by a 10 min extension at 72�C.PCR products were electrophoresed in 1% TAE aga-rose gels stained with ethidium bromide. ITS2 PCRamplicons obtained from two individuals of An. os-waldoi s.l. were puriÞed directly from bands excisedfrom agarose gel using the QIAQuik gel extraction kitand cloned into pGem-T Easy Vector (Promega, Mad-ison, WI). Four positive clones were sequenced.

Sequencing reactions were carried out in both di-rections using the above PCR primers and the Big DyeTerminator kit, version 1.3 (PE Applied Biosystems,Warrington, England). Sequences were analyzed ei-ther on an ABI Prism 3100-Avant genetic analyzer(Applied Biosystems, Foster City, CA) or on a 377-ABIsequencer (Applied Biosystems) (An. oswaldoi s.l.clones).Sequence Analysis. Sequences were edited using

Sequence Navigator, version 1.0.1 (PE Applied Bio-systems), aligned in CLUSTAL X (Thompson et al.1997) and optimized manually in MacClade, version4.3 (Maddison and Maddison 2000). Sequence simi-larity of the ITS2 sequences generated in this studywith those previously available in GenBank was as-sessed using FASTA search (http://www.ncbi.nlm.nih.gov/BLAST/). IntraspeciÞc sequence differenti-

ation was assessed using mean uncorrected P distancein PAUP (Swofford 2003).Vouchers. Template DNA from this study is re-

tained dry at �70�C in the FSP-USP for future refer-ence (DNA reference nos. are AC18-16, An. konderis.l.; AC18-102; AC18-107, An. oswaldoi s.l.; AC18-104;AC15-109; AC18-115; AC18-117, An. benarrochi s.l.).Immatures and male genitalia slides of the same spec-imens used for DNA extraction are deposited in theFSP-USP entomological collection as vouchers, acces-sion numbers from E-12928 until E-12934.

Results and Discussion

In a larval habitat in Linha 14 (9�41�03.5� S,67�08�05.3� W), Ramal do Granada, Acrelandia, Acre,we obtained 32 specimens that were preliminarilyidentiÞed as either An. oswaldoi s.l. (n � 12) or An.benarrochi s.l. (n � 20) by adult female and fourth-instar larvae. When examining characters of the dis-sected male genitalia, we identiÞed distinct morpho-logical forms: two similar to An. benarrochi s.l. (Fig.2AÐD), one similar to An. oswaldoi s.l. (Fig. 3AÐD),and one similar to An. konderi s.l. (Fig. 4AÐD).Molecular Characterization. The ITS2 was se-

quenced for seven individuals, four from An. benar-rochi s.l. (GenBank accession nos. EU636797ÐEU636800), two (�eight clones) fromAn. oswaldoi s.l.(EU636802-EU636809), and one from An. konderi s.l.(EU636801). The ITS2 base composition was 20.6% T,26.8% A, 26.7% C, and 25.7% G for An. benarrochi s.l.;20.9% T, 27.7% A, 27.0% C, and 24.2% G for An. os-waldoi s.l.; and 20% T, 27% A, 28% C, and 25% G forAn.konderi s.l.

ITS2 sequences ofAn. benarrochi s.l. are available inGenBank from Brazil (Rondonia AF462384 andAF462383; Marrelli et al. 2005), unlisted localities(U92325; Danoff-Burg & Conn direct submission),and Colombia (Puerto Asis AY684976-84; La Manuela,Liberia). A FASTA search revealed that the ITS2 se-quences of An. benarrochi s.l. from Acrelandia, Acre(n� 4, reported here) are identical, but distinct fromthose already in GenBank, sharing highest sequencesimilarity at 97% withAn. benarrochiB from Colombia,AY684984ÐAY684976. Along an ITS2 456 bp align-ment, 12 bases varied, including one 1-bp (base 316),one 4-bp (bases 334, 335, 336 and 337), and two 2-bp(bases 370, 371 and 381, 382) indels, and three single-ton polymorphic sites (bases 282, 347, and 348) (Fig.5). Mean uncorrected P distance among sequences ofAn. benarrochi B (AY684984) and An. benarrochi s.l.ranged from 0.00665 to 0.00671 (AC18-104). In com-paring two An. benarrochi s.l. ITS2 sequences fromRondonia state deposited in GenBank (AF462383 andAF462384) by Marrelli et al. (2005), we observed thatthese sequences are 97% similar to each other. Inaddition, ITS2 sequence identity among AF462383-4(Rondonia) and AY684976-84 (Colombia) is only 86%.Similarly, sequence identity among AF462383,AF462384, and thosegenerated fromAn.benarrochi s.l.collected in Acrelandia, Acre, is 93 and 91%, respec-tively.

November 2008 SALLUM ET AL.: Anopheles SPECIES IN THE STATE OF ACRE 973

We cloned and sequenced four copies of the entireITS2 per individual for two An. oswaldoi s.l. mosqui-toes from Acrelandia, Acre. The fragment ampliÞedincluded 60 bp of 5.8S and 42 bp of the 28S. The clonedsequences were aligned with reference sequences ofAn. oswaldoi s.s. from Espõrito Santo and Sao Paulo(EF457237 and EF457239; Motoki et al. 2007) andother ITS2 sequences of An. oswaldoi s.l. available inGenBank from Brazil (Acre AF055068, AmazonasAF056317, Rondonia AF055069; Marrelli et al. 1999b).The length of the ampliÞed sequences varied from 488to 490 bp, with length variation due to a 2-bp indel(Fig. 6). A few base substitutions were observed; how-ever, they were not consistent among the clones. Twocloned sequences of four of each individual were 100%identical; however, there were no identical sequencesamong the clones generated from samples AC18-107and AC18-102. We estimated mean sequence diver-gence of ITS2 variants within individuals by usinguncorrected P distance in PAUP (Swofford 2003).Sequence divergence among the clones of AC18-107ranged from 0.00000 to 0.01435, whereas those of

AC18-102 ranged from 0.00000 to 0.00615. By compar-ing cloned sequences of AC18-107 to AC18-102, weobserved that uncorrected P distance ranged from0.00205 to 0.01435. No diagnostic loci were detectedfor individuals AC18-107 or AC18-102. All of the dis-tinctive clones sequenced for An. oswaldoi s.l. weredeposited in GenBank (accession nos. EU636802ÐEU636809).

ITS2 sequences of An. oswaldoi s.l. are available inGenBank from Brazil (Acre AF055068, AmapaAF056318, Amazonas AF056317, Rondonia AF055069;Marrelli et al. 1999b), Colombia (Putumayo,AY679149-55; Ruiz et al. 2005), Peru (Yurimaguas,AF055071; Marrelli et al. 1999b), Venezuela (Ocama,AF055070; Marrelli et al. 1999b), unlisted localities(U92352-3, U92344; Danoff-Burg & Conn direct sub-mission), and of An. oswaldoi s.s. (Espõrito SantoEF457228-37, Sao Paulo EF457238-9; Motoki et al.2007). A FASTA search revealed that all cloned se-quences of An. oswaldoi s.l. from Acrelandia are dis-tinct from those already in GenBank. In comparing thetwo identical cloned sequences from AC18-102 with

Fig. 2. Male genitalia of An. benarrochi s.l. from Acrelandia, state of Acre and from Dourado, state of Sao Paulo, Brazil.Form 1: ventral claspette (ventral aspect) (A) and detail of ventral claspette (ventral aspect) (from Acrelandia, AC15-109)(B). Form 2: ventral claspette (ventral aspect) (C), detail of ventral claspette (ventral aspect) (from Acrelandia, AC18-115)(D), dissected ventral claspette (ventral aspect) (E), and detail of dissected ventral claspette (ventral aspect) (fromDourado) (F).

974 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 45, no. 6

AF056317, AF055068, and AF055069 (excluding 6 and7 bp from 5� and 3� end, respectively, because bothends represent nucleotide contamination by insertionof a restriction site that are included in the sequence),we observed that sequence similarity ranged from 98to 99%, the uncorrected P distance between AF056317and AC18-102_F3 is 0.00. The variation consisted of a1-bp indel that may be due to a PCR/cloning error inAF056317. All cloned sequences of AC18-107 are dis-tinct from those already in GenBank with sequencesimilarity ranging from 97 to 99% compared withAF056317, AF055068, and AF055069. In comparing alldistinctive cloned sequences of An. oswadoi s.l. fromAcrelandia, Acre, with sequences of An. oswaldoi s.s.,we detected few diagnostic loci for An. oswaldoi s.s.consisting of two singleton polymorphic sites (bases260 and 314), two 2-bp indel (318-9 and 423-4, respec-tively), and one 4-bp indel (365-8) (Fig. 6).

Three ITS2 sequences ofAn. konderi s.l. from Brazilare available in GenBank (unlisted localities U92342,U92348-9; Danoff-Burg & Conn direct submission). AFASTA search revealed that the ITS2 sequences ofAn.

konderi s.l. from Acrelandia, Acre (n� 1, herein gen-erated) are distinct from those already in GenBank. Incomparing sequences of An. konderi s.l. (U92342,U92348-9) with that from Acrelandia, Acre, we ob-served that the query coverage is 75% only. Along anITS2 356-bp alignment (U92342), sequence similarityis 96%, 11 bases varied, including one 2-bp, one 1-bp,two 2-bp indels (bases 222Ð223, 257, 261Ð262, and272Ð273, respectively), and four singleton polymor-phic sites (bases 300, 313, 336, and 344) (Fig. 7).Furthermore, An. konderi s.l. from Acrelandia, Acre,shares 98% similarity with An. oswaldoi s.s. from SaoPaulo (EF457238-39; Motoki et al. 2007) and EspõritoSanto (EF457228-37; Motoki et al. 2007). Along anITS2 464-bp alignment, nine bases varied, includingone 1-bp, one 2-bp indels (bases 361 and 428Ð429,respectively), and six singleton polymorphic sites(bases 272, 294, 324, 401, 437, and 445, respectively).Morphological Characterization. Anopheles benar-rochi was described by Gabaldon et al. (1941) fromspecimens collected in La Ceiba, Trujillo, Venezuela.Faran (1980) included An. benarrochi in the Strodei

Fig. 3. Male genitalia of Anopheles oswaldoi s.l. from Acrelandia and Placido de Castro, state of Acre and An. oswaldois.s. from Linhares, state of Espõrito Santo, Brazil. (A) Ventral claspette (ventral aspect, AC18-112). (B) Ventral claspette(ventral aspect, AC18-107) (from Acrelandia). (C) Ventral claspette (ventral aspect). (D) Aedeagus, showing detail of theaedeagal apex (ventral aspect) (from Placido de Castro). (E) Ventral claspette (ventral aspect). (F) Aedeagus, showing detailof the aedeagal apex (ventral aspect) [from Linhares, ES8(20)-14].

November 2008 SALLUM ET AL.: Anopheles SPECIES IN THE STATE OF ACRE 975

Complex of the Albimanus Section, and reported thedistribution of the species to the Orinoco basin andeastern versant of the Andes, including the llanosplateau of Colombia, parts of upper Amazonas in Bra-zil and Loreto in Peru. Later, Sallum et al. (1997)reported the species in the state of Sao Paulo, Brazil.Preliminarily, two morphological forms of An. benar-rochiwere found in Acrelandia, designated An. benar-rochiForm 1 (Fig. 2A and B) andAn. benarrochiForm2 (Fig. 2C and D). Anopheles benarrochi Form 1(AC15-109 and AC18-117) is morphologically similarto An. benarrochi s.s. of the original description (Ga-baldon et al. 1941) of Faran (1980) and that reportedin Dourado, state of Sao Paulo, by Sallum et al. (1997)(Fig. 2E and F). Anopheles benarrochi Form 2 (AC18-115 and AC18-104) can be distinguished from Form 1by the apex of the ventral claspette of the male gen-italia, which is moderately expanded laterally, withapicolateral margin sharply angled and pointed inForm 1, whereas it is somewhat rounded and truncatein Form 2. In addition, the apical margin of ventralclaspette has a deep median sulcus and the preapicalplate of ventral claspette is small, circular, homoge-neous and heavily sclerotized in Form 1, whereas theapical margin is somewhat straight and the preapicalplate is small, circular, and not homogeneously scle-rotized in Form 2.

In spite of morphological differences on the malegenitalia, the ITS2 sequences generated from speci-mens identiÞed either asAn. benarrochi Form 1 orAn.benarrochi Form 2 are 100% identical, suggesting thatthese forms are conspeciÞc. This observation raises aquestion about male genitalia polymorphism and dis-

sections of those anatomical structures commonlyused to distinguish species of the subgenusNyssorhyn-chus. Hribar (1994) demonstrated polymorphism inthe male genitalia of An. nuneztovari cytotypes A, B,and C, and Faran (1980) reported variation in theventral claspetteof themalegenitaliaofAn.benarrochis.l. Morphological variation in the ventral claspette ofAn. benarrochi Form 1 and Form 2 from Acrelandia,Acre, could be caused by distortion of the structuresduring the dissection and mounting on a microscopeslide. Thus, special care should be taken when dis-secting and mounting male genitalia. Conversely, wecan also question into the utility of ITS2 sequences forseparating morphologically similar species. ITS2 hasbeen shown to be a useful tool for separating closelyrelated species of Anopheles (Marrelli et al. 2006).However, some differences (herein observed) werefound within microsatellite regions showing that notall nucleotide variation may be important for distin-guishing among different taxa.

Quinones et al. (2006) by comparing ITS2 sequencewith male genitalia characteristics of vouchers spec-imens found that An. benarrochi s.l. from Peru com-prises two morphological forms; one form matches theoriginal description of the species and a second formcorresponds to An. benarrochi B of Ruiz et al. (2005).Interestingly, the ITS2 sequences of An. benarrochiForm 1 and Form 2 from Acrelandia share the highestsimilarity (97%) withAn. benarrochiB from Colombia.This difference suggests that the individuals of An.benarrochi s.l. from Acrelandia are distinct from An.benarrochi B and may belong to either An. benarrochis.s. or a distinct species. Supplemental studies are

Fig. 4. Photographs depicting the morphological differences between the male genitalia of Anopheles konderi s.l. fromAcrelandia, state of Acre and An. konderi from Macapa, state of Amapa, Brazil. (A) Aedeagus (ventral aspect, AC18-16). (B)Aedeagus (ventral aspect, AC-103) (from Acrelandia). (C) Aedeagus (ventral aspect). (D) Aedeagus, showing detail of theaedeagal apex (ventral aspect) (from Macapa).

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necessary to establish a morphological and moleculardeÞnition for An. benarrochi s.s. and thus solve thetaxonomic status of the populations found in Colom-bia, Peru, and Brazil. Additionally, it will be worth-while to examine populations of An. benarrochi s.l.across Brazil for a better evaluation of which formsmay occur in the country and to ascertain their tax-onomic identity.

In the same larval habitat of An. benarrochi s.l., wecollected immatures of An. oswaldoi s.l. While exam-ining characters of the male genitalia, it became evi-dent that the specimens could be separated into twomorphological forms designatedAn. oswaldoi s.l. (Fig.3A and B), andAn. konderi s.l. (Fig. 4A and B).Anoph-eles oswaldoi s.l. (Fig. 3A and B) is similar to An.oswaldoi s.s. (Fig. 3E and F). A similar morphologicalformwas found inPlacidodeCastro,Acre(Fig. 3CandD). Based on male genitalia characteristics,An. oswal-doi s.l. can be separated from An. oswaldoi s.s. by theapex of the aedeagus (Fig. 3D and F, respectively).Morphological and ITS2 sequence differences suggestthat specimens of An. oswaldoi s.l. from Acrelandia,Acre, belong to a new species. It is noteworthy that theITS2 sequences from two specimens from Acrelandiaare 99% similar to those generated by Marrelli et al.(2005) from individuals identiÞed as An. oswaldoi s.l.collected in Rondonia (AF055069) and Acre (Placidode Castro) (AF055068). Finally, the identity of thosespecimens from Acre and Rondonia used by Marrelliet al. (2005) remains a problem to be solved by furtherstudies and Þeld collections in the same localities toobtain associated specimens, adult males and femalesand immatures.

Flores-Mendoza et al. (2004) elevated An. konderifrom the synonymy ofAn. oswaldoi and designated theneotype. Unfortunately there is no ITS2 identity avail-able in the GenBank of An. konderi from the typelocality of Coari, Amazonas state, generated from thesame set of specimens used by Flores-Mendoza et al.(2004). Morphological comparisons of samples of An.konderi collected in Coari, state of Amazonas, Macapa,state of Amapa, and Acrelandia, state of Acre, stronglysuggest that specimens from Amapa (Fig. 4C) andCoari (Motoki et al. 2007) are identical, whereas thosefrom Acre can be distinguished by the apex of theaedeagus (Fig. 4A and B). In An. konderi s.s. the sub-apical, collarlike, subtriangular sclerotinization of theaedeagus is somewhat U-shaped, whereas it isV-shaped inAn. konderi s.l. from Acre. Additionally, indorsal aspect, the longitudinal dorsomedial cleft ex-tends apical to the subtriangular sclerotinization andthe apex is short in An. konderi s.s. (Fig. 4C and D),whereas in An. konderi s.l. the longitudinal dorsome-dial cleft does not extend beyond the level of thesubtriangular sclerotinization and the apex is some-what longer (Fig. 4A and B).

Scarpassa and Conn (2006) used COI sequences toexamine the intra- and interpopulational variability inAn. oswaldoi s.l. Results of the maximum parsimonyanalysis identiÞed four major clusters of the COI hap-lotypes that may correspond to distinct species. Ac-cording to their study, male genitalia characteristics

Fig. 5. A 486-bp ITS2 sequence alignment of An. benar-rochi s.l. (AC15-109, AC18-117, AC18-115, AC18-104) and thenext nearest taxon in GenBank, An. benarrochi B from Co-lombia (AY684984) and An. benarrochi s.l. from Brazil(AF462383-84). (-) indicates an indel, and (?) indicates miss-ing data at 3� end.

November 2008 SALLUM ET AL.: Anopheles SPECIES IN THE STATE OF ACRE 977

Fig. 6. A 490-bp ITS2 sequence alignment ofAn. oswaldoi s.l. (AC18-107, AC18-102),An. oswadoi s.l. in GenBank from statesof Amazonas, Acre, and Rondonia, Brazil (AF056317, AF055068-9) andAn. oswaldoi s.s. from state of Espõrito Santo and Sao Paulo,Brazil (EF457237, EF457239). (-) indicates indel events. The letter_number after the sample code indicates a clone.

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suggest that one group may correspond to An. oswal-doi s.s., a second group toAn. konderi,whereas the twoother clusters could constitute different lineages orspecies within the An. oswaldoi complex. Consideringthe results obtained by Scarpassa and Conn (2006), weobserved that the specimens from the states of Ron-donia and Acre clustered in Groups I and II. Group Icomprises specimens from Rondonia (Sao Miguel)and Acre (Sena Madureira), whereas Group II isformed by individuals from Sao Miguel. Parsimony

bootstrap support for both Groups I and II is strong(100%). This result suggests that there are at least twodistinct species that may be confounded with An.oswaldoi s.s. in those areas. In considering that thesequences in GenBank (AF055068 and AF055069)generated from specimens of An. oswaldoi s.l. (Mar-relli et al. 2005) are dissimilar and were collected inthe same localities in Rondonia and Acre of thosespecimens used by Scarpassa and Conn (2006), wehypothesize that they could belong to the same taxon.

Fig. 7. A 469-bp ITS2 sequence alignment of An. konderi s.l. (AC18-16) and An. konderi s.l. in GenBank from unspeciÞedlocalities from Brazil (U92342, U92348-9). (-) indicates either indel events or missing data at 5� and 3� ends.

November 2008 SALLUM ET AL.: Anopheles SPECIES IN THE STATE OF ACRE 979

If this scenario is correct, the specimens from Acre-landia would be representative of two other distincttaxa that are largely misidentiÞed as An. oswaldoi s.l.It is also plausible to speculate that one of the groupsidentiÞed by Scarpassa and Conn (2006) may be co-speciÞc withAn. owaldoi s.l. from Acrelandia, whereasthe other may be genetically similar to one individualused by Marrelli et al. (2005) from either Acre orRondonia. Moreover, our results strongly suggest thatat least one of the specimens from Acrelandia (AC18-102) belong to the same species of the specimen rep-resentedbyAF056317(fromAmazonas state)becausethe uncorrected P distance is zero and the sequencedifference is a single indel. It is evident that furtherinvestigation will be necessary to establish a corre-spondence among distinct morphological forms andthe phylogenetic groups found by Scarpassa and Conn(2006), the identity of the specimens of Marrelli et al.(2005) and the correspondence with those from Acre-landia. This is especially relevant becauseAn. oswaldois.l. was considered to be involved in malaria transmis-sion in the state of Acre. Finally, variation in malegenitalia characteristics within An. oswaldoi s.l. andAn. benarrochi s.l. seems to have a correspondencewith genetic variation that is worthy of further inves-tigation.

Acknowledgments

We thank Marcelo Urbano Ferreira for providing logisticsupport for the Þeldwork in Acrelandia and Richard C. Wilk-erson (Walter Reed Biosystematics Unit) for comments thatgreatly improved the manuscript. Special thanks to Natal dosSantos and Aquino who made the Þeldwork possible; Darildo,a Ramal do Granada settler, who showed the larval habitat(Igarape) to M.A.M.S. where the immature stages were col-lected. Henry Rupp kindly reviewed the English. This in-vestigation received Þnancial support from Fundacao deAmparo a Pesquisa do Estado de Sao Paulo grant 05/53973-0,Conselho Nacional de Desenvolvimento CientõÞco e Tecno-logico grant 472485/2006-7, and UNICEF/UNDP/WorldBank/WHO Special Programme for Research and Training inTropical Diseases grant A50252.

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Received 19 September 2007; accepted 6 June 2008.

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