MORPHOLOGY,SYSTEMATICS,EVOLUTION Malaria Vector Anopheles (Nyssorhynchus) nuneztovari Comprises One Genetic Species in Colombia Based on Homogeneity of Nuclear ITS2 rDNA 1 1 2 DIANA M. SIERRA, IVAN DARIO VELEZ, AND YVONNE-MARIE LINTON J. Med. Entomol. 41(3): 302Ð307 (2004) ABSTRACT Previous studies indicated that two distinct chromosomal forms of Anopheles nunez- tovari Gabaldo´n, cytotypes B and C, occurred on the west and east of the Latin American Andes Mountains, respectively. To determine the taxonomic status of Anopheles (Nyssorhynchus) nuneztovari in Colombia, link-reared specimens were collected from four sites: in the departments of Choco´ (La Pacurita)and Valle (Sitronella)in the west, and Norte de Santander (Guaramito and Tibu ´ )in the east. Nuclear ITS2 sequences were generated for 46 individuals. Only two specimens (4.4%) showed divergent haplotypes, varying from the consensus by a single-base polymorphism (0.18%). These results suggest that populations of An. nuneztovari corresponding to cytotypes (B and C)are conspeciÞc. KEY WORDS Anopheles nuneztovari, cytotype B and C, ITS2, Colombia Anopheles (Nyssorhynchus) nuneztovari Gabaldo´nis Despite being found infected with both P. vivax and widely distributed in eastern Panama and northern P. falciparum in the Brazilian Amazon (Arruda et al. South America, including Amazonia. SigniÞcant dif- 1986), An. nuneztovari populations (cytotype A)are ferences in host preference and biting behavior be- predominantly zoophilic, and not implicated as im- tween populations of An. nuneztovari in Colombia and portant malaria vectors in this region. Conversely, Venezuela compared with those in the Brazilian Am- An. nuneztovari populations in Colombia and Vene- azon led Elliott (1972)to propose that these geo- zuela are highly anthropophilic and act as primary graphical distinct populations of An. nuneztovari com- malaria vectors (Rey and Renijifo 1950, Gabaldo´n and prised two allopatric races. Differences in autosomal Guerrero 1959). Because of the vector signiÞcance of inversion polymorphisms and a Þxed XR chromosome cytotype B and C populations in Colombia, the current inversion between Venezuelan and Brazilian An. nun- study endeavored to accumulate molecular evidence eztovari (Kitzmiller et al. 1973)corroborated this the- to assess the taxonomic status of the two cytotypes. ory. After additional chromosomal analyses, Conn The Colombian An. nuneztovari were collected (1990)designated them cytotype A (Amazonia)and from four localities in three geographically distinct B (Venezuela/Colombia), respectively. Further sup- regions in Colombia, including sites from west and east port for a two-species separation based on cytotype of the Andes. Nuclear ITS2 sequences were generated was provided by a taxonomic comparison of the egg to compare the haplotypes with those generated by structures (Linley et al. 1996), male genitalia (Hribar Fritz et al. (1994). Sequence data tested the null hy- 1994), isoenzymes (Scarpassa et al. 1996, 1999), and pothesis that An. nuneztovari exists as two separate various DNA studies (Fritz et al. 1994, Conn et al. 1998, genetic forms in Colombia, conforming to the cyto- Scarpassa et al. 2000). Later, a comparative study of types B and C of Conn (1990)and Conn et al. (1993). chromosomes from western Colombia and Venezuela revealed a third cytotype, differing from cytotypes A Materials and Methods and B by the presence of a unique chromocenter and differences in the frequency of a large, complex in- Mosquito Samples. Specimens of An. nuneztovari version. This chromosomal form, found in An. nunez- were collected from three departments in Colombia: tovari populations west of the Andes, was designated La Pacurita, Choco´ (5Њ 42Ј N, 76Њ 39Ј W), Valle, cytotype C (Conn et al. 1993). Sitronella (3Њ 53Ј N, 77Њ 3Ј W), and two sites in Norte de Santander, Tibu´ (8Њ 38Ј N, 72Њ 44Ј W)and 1 Programa de Estudio y Control de Enfermedades Tropicales Guaramito (9Њ 41Ј N, 68Њ 39Ј W)(Fig. 1).Wild-caught (PECET), Facultad de Medicina, Universidad de Antioquia, AA.1226 larvae were individually link-reared. The adults were Medellin, Colombia. 2 Department of Entomology and Biomedical Sciences Theme, The used for DNA extraction, and the associated larval and Natural History Museum, Cromwell Rd., London SW7 5BD, England, pupal exuviae were preserved as voucher specimens. UK (e-mail: [email protected]). Progeny of blood-fed females captured in human bait 0022-2585/04/0302Ð0307$04.00/0 ᭧ 2004 Entomological Society of America May 2004 SIERRA ET AL.: A. nuneztovari IN COLOMBIA 303 Fig. 1. Outline map of Colombia, showing collection sites of An. nuneztovari used in this study, in relation to the position of the Andes mountain range. The species is considered to comprise two cytotypes: B on the east and C on the west of the Andes (Conn 1990, Conn et al. 1993). collections were individually reared to obtain voucher Linton et al. (2001). Polymerase chain reaction (PCR) specimens of all life stages and sexes. IdentiÞcations ampliÞcation and sequencing in both directions were for the progeny broods and larval collections were carried out according to the protocol listed in Linton conÞrmed independently at The Natural History Mu- et al. (2001). Sequences were edited and aligned using seum (BMNH), London. Voucher specimens com- Sequencher version 3.1.1 (Genes Codes Corporation, prising associated larval and pupal exuviae and eggs Ann Arbor, MI)and CLUSTAL X software packages. were deposited at the Programa de Estudio y Control Similarity with those sequences available in GenBank de Enfermedades Tropicales (PECET)Laboratories, was assessed using FASTA search. After sequencing, Universidad de Antioquia, Medellin, Colombia. template DNA was dried and deposited at Ϫ70ЊCin Molecular Techniques. DNA was extracted from the Molecular Systematics Laboratory, Department of individual mosquitoes stored in 100% isopropanol, fol- Entomology, The Natural History Museum, London lowing the phenolÐchloroform extraction protocol in (BMNH)for future reference. 304 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 41, no. 3 Results lombian haplotype 2 sequences generated in the cur- rent study (Fig. 2). Variability in ITS2 Sequences. Forty-six sequences comprising the 3Ј end of the 5.8S gene, the ITS2, and the 5Ј end of the 28S ribosomal gene, were generated for 18 Discussion individuals from Choco, 10 from Sitronella, 13 from Tibu, ´ ´ Concerted evolution of multigene families within and 5 from Guaramito. Sequences are available in Gen- species has resulted in rapidly evolving spacer regions, Bank under the following accession numbers: Choco´ such as the ITS2 rDNA. Sequencing of these regions (AY028109-AY028126), Tibu´ (AY028096-AY028108), has been used to effectively determine the species Sitronella (AY028087-AY028095, AY028128), and composition of Anopheles mosquito complexes (Col- Guaramito (AY028081-AY028085). lins and Paskewitz 1996). Low levels of intraspeciÞc The alignment was 549 bp in length (including variation in the ITS2 region has proven useful for the primers, 43 bp), 53.0% GC rich, and unambiguous. The design of species-speciÞc primers or PCR-restriction ITS2 region was 363 bp in length and two haplotypes, N fragment-length polymorphism assays to differentiate varying by a single base polymorphism (G C)at base members of species complexes and for phylogeny re- 365, were revealed (Fig. 2). Haplotype 1 accounted for construction in mosquitoes. Previous studies of ITS2 44 (95.65%)of the specimens analyzed (GenBank sequences have shown little or no intraspeciÞc vari- accession nos. AY028081-AY028085, AY028087- ation in sibling species of Anopheles. IntraspeciÞc vari- AY028124, and AY028128). The two remaining indi- ation in Þve colony strains of species of the An. gam- viduals, both from Choco´ (GenBank accession nos. biae complex was reported to range between 0 and AY028125 and AY028126), shared haplotype 2. 0.43% (Paskewitz et al. 1993). In contrast, Beebe et al. Comparison with Other Published ITS2 Sequences. (2001)reported population-speciÞc ITS2 sequences Previous studies that generated ITS2 sequences for An. in the An. bancroftii group comparing populations nuneztovari deposited four sequences in GenBank. from Queensland, Australia, and the western province Danoff-Burg and Conn submitted three An. nunezto- of Papua New Guinea vari sequences (U92350, U92351, and U92343 of un- The ITS2 data presented in the current study indi- speciÞed origin)to GenBank in 1997. One consensus cate An. nuneztovari to be a single genetic species on sequence (L22462)of 28 resolved sequences was sub- both sides of the Andes mountain range, despite dif- mitted to GenBank by Fritz et al. (1994). All GenBank ferences in karyotype morphology described by Conn and published An. nuneztovari ITS2 sequences, were et al. (1993). Homosequential chromosomes have aligned with the Colombian haplotypes generated in been documented in other Anopheles species com- the current study (Fig. 2). This alignment comprised plexes, for example, An. labranchiae Falleroni and An. samples (including ours)from Colombia (54),Suri- atroparvus van Thiel of the Maculipennis Complex name (3), Brazil (6), Bolivia (1), and of unspeciÞed (Kitzmiller et al. 1967). However, examples such as origin (3). The alignment was 373 bp in length, re- cytotypes B and C of An. nuneztovari, where distinct ßecting the 363Ð373-bp ITS2 region ampliÞed in other chromosomal forms comprise one genetic species, are studies; only the variable region between bases 264Ð less common.