Mem Inst Oswaldo Cruz, Rio de Janeiro, 100(2) April 2005
Original Article

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Freddy RuizI, Martha L Quiñones, Holmes F ErazoII, David A Calle, Juan F Alzate, Yvonne-Marie LintonI

Programa de Estudio y Control de Enfermedades Tropicales, Facultad de Medicina, Universidad de Antioquia, Medellín,u00a0Colombia
IMosquitoes Programme, Department of Entomology, Natural History Museum, Cromwell Road, Londonu00a0SW7 5BD, England
IIDivisión Administrativa de Salud, Putumayo, Colombia

Page: 155-160
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Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi,u00a0andu00a0An. (N.) rangeliu00a0are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Althoughu00a0An. rangeliu00a0is easy to identify, the morphological variant ofu00a0An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia.


Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochiu00a0andu00a0An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length inu00a0An. benarrochiu00a0(n = 9) and 531 bp inu00a0An. oswaldoiu00a0(n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzymeu00a0Haeu00a0III as follows:u00a0An. benarrochiu00a0(365, 137, and 38 bp) andu00a0An. oswaldoiu00a0(493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. Thatu00a0An. benarrochiu00a0comprises a complex of at least two species across Latin America is discussed.

Malaria transmission in Colombia is mainly attributed to Anopheles (Nyssorhynchus) albimanus Wiedemann, An. (N.) darlingi Root or An. (N.) nuneztovari Gabaldón (Faran 1980, Herrera et al. 1987, Olanoet al. 2001). Despite the absence of these major vectors, reported cases of malaria in the southern Colombian state of Putumayo averaged 1894 cases per year between 1999-2003. Human malaria in Putumayo is exclusively due to Plasmodium vivax, except in the municipality of Puerto Leguízamo where cases of P. falciparum are also reported (OPAS 2003).

Early studies in Putumayo, including internal reports of the now-defunct Malaria Eradication Service, showed thatAn. evansae (Brèthes) (as An. noroestensis Galvão and Lane) was highly anthropophilic, and thus it was circumstantially incriminated in the transmission of malaria in Southern Colombia (Ferro 1979). However a 1988 survey detected no An. evansae in Putumayo, and, most unusually, reported natural infection of P. vivax in 6.2% of 419 An. rangeli Gabaldón, Cova García and Lópeztested by ELISA (Suárez et al. 1990).

Integrated molecular and morphological studies of progeny material reared from wild-caught females captured on human bait in Putumayo were undertaken in our laboratories. These showed that the highly anthropophilic specimens found in Putumayo, previously believed to be An. evansae, were infact a morphological variant of An. benarrochi Gabaldón, Cova García and López(Quiñones et al. 2000, 2001). An. benarrochi is believed to be predominantly zoophilic and therefore not of importance in malaria transmission (Faran 1980). A susceptibility trial of P. vivax with An. benarrochi from Rondônia, Brazil also proved negative (Klein et al. 1991), and in Venezuela the species is zoophilic, present in low densities, and therefore not believed to be involved in malaria transmission (Rubio-Palis, pers. comm.). Conversely, and more in line with our findings in Southern Colombia,An. benarrochi was reported to be the dominant vector in the west of Loreto Province in Perú, which shares a border with Putumayo (Aramburú et al. 1999, Schloeler et al. 2003). Flores-Mendoza et al. (2004) reported that An. benarrochi was indeed a vector of both P. falciparum and P. vivax in Eastern Perú with 0.14% (9 of 6323 pools containing 1-10 mosquitoes each) ELISA positive for either Plasmodium species.

An. oswaldoi (Peryassú) is regarded as an efficient vector in Brazil (Branquinho et al. 1996), and a secondary vector in Venezuela where it was found ELISA positive for P. vivax (Rubio-Palis et al. 1992). The species has been reported biting man in Putumayo, where it is suspected to be involved in malaria transmission (Quiñones et al. 2000). On the basis of sequence data for the nuclear second internal transcribed spacer (ITS2), Marrelli et al. (1999) suggested that An. oswaldoi comprised a complex of at least four species.

Thus, it appeared that three species, An. rangeliAn. benarrochi, and An. oswaldoi, could prove important in vector incrimination studies in Putumayo. An. rangeli is morphologically distinct and easy to identify, but overlapping characters in the adult females made the correct identification of An. oswaldoi and An. benarrochi nearly impossible (Quiñones et al. 2001, Calle et al. 2002), and hampered our vector incrimination studies. Eggs of An. benarrochi can be differentiated from all other species of the subgenus Nyssorhynchus by the presence of a small, short-walled deck at the anterior end and this finding led to the development of an indirect method of identifying wild-caught females based on the morphology of their induced eggs (Estrada et al. 2003). This method is extremely effective but proved difficult to implement on a large scale under field conditions, hence a more rapid approach to facilitate the identification of adult females of An. oswaldoi and An. benarrochi was needed.

Molecular techniques that can differentiate closely related species on the basis of their ITS2 are well documented in Anopheles, and the low level of intra-specific sequence variation has proven useful as the basis for species-specific PCR assays, including the African An. (Cellia) gambiae Giles complex (Scott et al. 1993) and the Holarctic An. (Anopheles) maculipennis Meigen group (Cornel et al. 1996, Proft et al. 1999). ITS2 sequences have also been used in taxonomic studies of closely related species in the subgenus Nyssoryhnchus, including An. nuneztovari Gabaldón (Fritz et al. 1994, Sierra et al. 2004) and An. oswaldoi (Marrelli et al. 1999), for species characterisation (Lounibos et al. 1998), and in phylogenetic reconstruction (Manguin et al. 1999, Sallum et al. 2002).

In this study we obtained ITS2 sequence data for specimens of An. benarrochi and An. oswaldoi from Colombia's southern state of Putumayo.These sequences were used as a basis for designing a PCR-RFLP assay that would allow the rapid identification of An. benarrochi and An. oswaldoi adult females to facilitate vector incrimination studies in the region. Similar PCR-RFLP assays have proven useful in the differentiation of closely related members of species complexes, for example, in the An. (N.) albitarsis Lynch-Arribalzaga species complex (Narang et al. 1993).



Mosquito specimens - Mosquitoes were collected using human landing catches (both indoors and out) between 18-21 h. Specimens were collected from three localities in two towns (Puerto Asís and Puerto Leguízamo) in the Department of Putumayo, Southern Colombia (Fig. 1). Efforts to collect from wider localities were thwarted by civil unrest in the area. Progeny of wild-caught females were individually reared to obtain adults with associated fourth-instar larval and pupal exuviae (see procedures of Belkin et al. 1965 and Reinert et al. 1997). Only those isofamilies positively identified as An. benarrochi and An. oswaldoi in all life stages, using the existing morphological keys (Faran 1980, Faran & Linthicum 1981, Suárez et al. 1988, Rubio-Palis 2000) and the original descriptions (Peryassú 1922, Gabaldón et al. 1941), were used to generate the DNA sequences. Their siblings, housed at the PECET laboratory (Programa de Estudio y Control de Enfermedades Tropicales, Universidad de Antioquia, Medellín), serve as morphological voucher specimens for this work.

Associated wild-caught adults were killed using ethyl acetate, and individually transferred to clean, labelled 1.5 ml Eppendorf® tubes. Tubes were capped, pierced with a mounted needle to allow the escape of moisture, and placed in plastic bags containing silica gel to ensure best DNA preservation. Those specimens morphologically grouped as either An. benarrochi or An. oswaldoi were identified to species using the PCR-RFLP assay developed herein.

Molecular methods - Template DNA was acquired using either the phenol-chloroform extraction protocol listed in Linton et al. (2001), or from a single leg placed directly into the PCR reaction (after Scott et al. 1993). Amplification of the nuclear ribosomal spacer (ITS2) was achieved using 5.8SF and 28SR primers listed in Collins and Paskewitz (1996). PCR products were amplified using the reaction and thermocycler parameters described by Linton et al. (2001), and cleaned using a commercially available PCR purification kit (QIAgen Ltd, Sussex, England), prior to direct sequencing. Sequencing reactions were carried out in both directions using the Big Dye Terminator Kit (PE Applied Biosystems, Warrington, England) and chromatograms were read on an ABI 377 automated sequencer (PE Applied Biosystems).

Sequences were edited using SequencherÔ version 3.1.1 (Genes Codes Corporation, Ann Arbor, MI) and aligned in CLUSTAL X (Thompson et al. 1997) software packages. Sequence similarity of the An. benarrochi and An. oswaldoi ITS2 genotypes generated in this study were compared with those available in GenBank using FASTA ( Intra-specific variation was calculated using MEGA2 (Kumar et al. 2001). Most of the template DNA was dried and retained at _70oC in the Molecular Systematics Laboratory, Department of Entomology (BMNH) for future reference. Sequences generated in this study are available in GenBank, under the following accession numbers: An. benarrochi (AY684976-AY684984) and An. oswaldoi (AY679149-AY679155). All sequenced individuals were collected in La Manuela, Puerto Asís, except two An. benarrochispecimens (AY684979 and AY684980) that were collected in Lisberia, Puerto Asís (Table).

Species-diagnostic banding patterns following digestion of the ITS2 fragments with different restriction enzymes were assessed using the program Webcutter 2.0 ( The enzymes HaeIII and Avi II both showedclear species diagnostic bands, but Hae III was employed in this assay, as it was significantly cheaper. RFLP digestions of the ITS2 PCR products were carried out in 20 µl reaction volumes. Four µl of ITS2 PCR product were added to clean 200 µl PCR tubes containing 0.5U of Hae III (GG¯CC) (PROMEGA), 2 µl of the manufacturers incubation buffer (PROMEGA- Y+/Tango TM) and 13.5 µl ddH2O. Tubes were incubated at 37oC overnight to ensure full digest of the ITS2 fragments. The digestion products were visualised on 2% agarose gels, containing 1% ethidium bromide.



In order to obtain baseline ITS2 sequences, DNA was extracted and sequenced from morphologically verified progeny brood specimens collected in Puerto Asís (Table). Sequences were obtained for 9 An. benarrochi (2 from Lisberia, Puerto Asís, and 7 from La Manuela, Puerto Asís), and 7 An. oswaldoi from La Manuela, Puerto Asís (Table). Amplified fragment lengths (including primers, 43 bp) were 540 bp in An. benarrochi and 531 bp in An. oswaldoi. No intra-specific differences were noted in the ITS2 sequences of either species (Fig. 2) and the two species shared 93.6% sequence similarity. Inter-specific variation manifested itself in the ITS2 region, not in the flanking sequences of the 5.8S and 28S genes (Fig. 2).

Intra-specific homogeneity in the ITS2 sequences of these species allowed this region to be exploited in an PCR-RFLP assay. The banding pattern after Hae III digestion was as follows: An. oswaldoi (493 and 38 bp) and An.benarrochi (365, 137, and 38 bp) (Fig. 3). Although fragments of less than 50 bp were difficult to observe on the gel, large ones were clearly differentiable for each species and permitted their clear identification (Fig. 3).

In addition to the sixteen sequences obtained from link-reared An. benarrochi (n = 9) and An. oswaldoi (n = 7) specimensa further 440 Nyssorhynchus females captured landing on humans in Putumayo were identified as An. oswaldoi or An. benarrochi using the PCR-RFLP assay designed in this study. The majority of specimens came from La Manuela in Puerto Asís (441), with an additional 2 specimens from Lisberia, Puerto Asís and 13 more from La Concepción, Puerto Leguízamo. Using both sequencing and PCR-RFLP of the ITS2 region, all 456 specimens tested were successfully identified to species, with 418 An. benarrochi (91.7%) and 38 An. oswaldoi(8.3%) (Table).



Difficulties in morphological identification of adult female Nyssorhynchus mosquitoes from the state of Putumayo in southern Colombia prompted this study. Overlapping morphological characters in adult females resulted in some specimens keying out unsatisfactorily to either An. evansae or An. oswaldoi using available morphological keys (Faran 1980, Faran & Linthicum 1981, Rubio-Palis 2000). However, detailed studies in all the life stages noted that these "odd" specimens were actually a morphological variant of An. benarrochi (Quiñones et al. 2001, Calle et al. 2002, Estrada et al. 2003). Specimens identical to the morphological variant of An. benarrochi from Putumayo have also been detected in Perú (R Wilkerson and C Flores-Mendoza, pers. comm.).

According to the morphological keys of Faran (1980), and Faran and Linthicum (1981), An. oswaldoi and An. benarrochi are easily differentiated on the relative lengths of the basal dark band on hindtarsomere 2. The basal dark band usually occupies less than 25% of hindtarsomere 2 in An. oswaldoi, whereas it is significantly longer (40-55%) in An. benarrochi. This was echoed the keys of Rubio-Palis (2000), where the length of the dark band on hindtarsomere 2 in Venezuelan An. benarrochi was reported to be 45%Quinoñes et al. (2001) reported in the specimens of morphologically variant An. benarrochi from Putumayo, the dark band ranged between 17-33%, overlapping with the range of An. oswaldoi, thus accounting for mistaken identification of these two species using the existing morphological keys.Barring one T insertion, the ITS2 sequences of the Colombian An. benarrochigenerated in this study were identical to the GenBank entry AF055071 from Yurimaguas in Perú. This entry, labelled An. oswaldoi, was mistakenly included in Marrelli et al.'s (1999) characterization of the sibling species in the Oswaldoi Complex. This shows that the correct identification of the morphological variant of An. benarrochiis problematic using existing taxonomic keys and implies that the species could be commonly misidentified across Latin America.

Morphological and behavioural differences serve to support the presence of at least two cryptic species within An. benarrochi. It appears zoophilic in Rondônia, Brazil (Klein et al. 1991) and Venezuela (Rubio-Palis 2000), and highly anthrophophilic in Colombia (Quiñones et al. 2000, 2001, herein) and Perú (Aramburú et al. 1999, Marrelli et al. 1999, Schloeler et al. 2003, Flores-Mendoza et al. 2004). Additionally sequences generated from the morphologically and behaviourally distinct An. benarrochi from Putumayo, Colombia (and Yurimaguas, Perú) are genetically distinct (15.4-16.3%, ungapped) from two other An. benarrochi sequences entered in GenBank (AF462383 and AF462384) from Rondônia in Brazil. A FASTA search with a third sequence labelled An. benarrochi (U92325 Danoff-Burg and Conn, direct submission 1997) was found to be An. albimanus (99.47% identity with L78065 [Teco Strain, Colony] and U92323, Danoff-Burg and Conn).

Ecological, morphological and molecular evidence seem to suggest that the specimens conforming to the original description of An. benarrochi are present in Venezuela and Brazil, whereas a second, highly anthro-phophilic species, herein designated An. benarrochi B, is present in Southern Colombia and Western Perú. Given that the type specimen of An. benarrochi is missing (R Wilkerson, pers. comm.), it is now prudent to obtain material from the type locality of La Ceiba, Trujillo State in Venezuela and to designate a neotype to fix the true identity of An. benarrochi. This neotype description should include both a revised morphological study and molecular sequence data, as was done for An. (Cellia) sundaicus (Rodenwaldt) in Southeast Asia (Linton et al. 2001). Fixing the identity of An. benarrochi s.s. will serve as a foundation for further systematic and vector incrimination studies on the species in Latin America, and only then can An. benarrochi B and any other component members in the Benarrochi Complex be clearly defined.

The An. oswaldoi specimens sequenced from Pu-tumayo showed highest similarity with those from the state of Amapá in Brazil (AF056318) and Ocamo in Venezuela (AF055070) (Marrelli et al. 1999). AF056318 and AF055070 are identical (Marrelli et al. 1999) but differ from our sequences by three variable bases and a 2-bp insertion (98.76% gapped; 99.2% ungapped). Following the work of Marrelli et al. (1999), An. oswaldoi was regarded as a species complex of at least four species. One of these is believed to represent An. konderi, then a junior synonym of An. oswaldoi but which has now been re-elevated to separate species status (Flores-Mendoza et al. 2004). It remains unclear which of Marrelli et al.'s four forms this corresponds to, and another has been shown in this study to correspond to Colombia specimens of An. benarrochi B, thus leaving two remaining species in the Oswaldoi Complex. It is now essential to establish the genetic identity of An. konderi and of An. oswaldoi from its type localities of Valle do Rio Doce in the state of Espirito Santo and Baixada Fluminense in the state of Rio de Janeiro, both in Brazil, to facilitate our understanding of the Oswaldoi Complex. Efforts are underway in our laboratories to resolve these issues.

The ITS2 PCR-RFLP assay designed in this study rapidly and reliably identified all mosquito specimens tested, indicating that An. benarrochi B is significantly more prevalent than An. oswaldoi in Putumayo and thus could act as a potential vector in the region, as in Perú (Aramburú et al. 1999). The assay worked equally effectively on PCR products generated from template DNA extracted using the phenol-chloroform method of Linton et al. (2001), or for those products generated by placing a single leg directly into the PCR reaction (Scott et al. 1993). This method is extremely valuable in vector incrimination studies as the remainder of the specimen can be utilised for ELISA or PCR detection of Plasmodium. The assay as it stands has proven invaluable in our efforts to separate and identify female An. benarrochi B and An. oswaldoi in Southern Colombia and has facilitated vector incrimination studies in the region (data presented elsewhere). However, comparison of our sequence data with that in GenBank has brought to light the taxonomic complexity of these two species across their range, and we acknowledge that this assay may have to be modified in other regions, depending on which members of the complexes are present. It is now necessary to obtain specimens of both species across Latin America to facilitate the design of a robust identification tool for all component members of the Benarrochi and Oswaldoi Complexes in all regions in future.



To Dr Ivan Dario Vélez (Director, PECET), Dr William Galarza (Director, DASALUD, Putumayo) and the entomologists at DASALUD for their support, and to Dr Ralph Harbach (BMNH) for morphological confirmation of the progeny broods used in this study. This paper is dedicated to our friends Edison Correa, Carlos Andrés Pérez, and Marcos Pérez, whose devotion to this project cost them their lives.



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+Corresponding author.
Received 3 September 2004
Accepted 21 February 2005
Financial support: The Wellcome Trust (grant 053401), Colciencias (grant 1115-04-460-98)

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