Vector Biology JournalISSN: 2473-4810

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Research Article, Vector Biol J Vol: 1 Issue: 3

Cytochrome Oxidase I as Tool to Evaluate the Lutzomyia longipalpis Complex: Useful Molecular Marker or Not?

Moises Thiago de Souza Freitas1*, César Raimundo Lima Costa Jr1, Lidiane Gomes da Silva1, Tereza Cristina Leal-Balbino2 and Valdir de Queiroz Balbino1
1Department of Genetics, Federal University of Pernambuco, Cidade Universitária, 50740-600 Recife, Pernambuco, Brazil
2Department of Microbiology, Research Center Aggeu Magalhaes, Avenida, Cidade Universitária, 50740-965 Recife, Pernambuco, Brazil
Corresponding author : Moises Thiago de Souza Freitas
Departament of Genetic, Federal University of Pernambuco, Avenida Professor Moraes Rego S/N, Recife, Pernambuco 50740-600, Brazil
E-mail: [email protected]
Received: September 13, 2016 Accepted: September 30, 2016 Published: October 06, 2016
Citation: Freitas MTS, Costa Jr CRL, Silva LG, Leal-Balbino TC, Balbino VQ (2016) Cytochrome Oxidase I as Tool to Evaluate the Lutzomyia longipalpis Complex: Useful Molecular Marker or Not?. Vector Biol J 1:3. doi: 10.4172/2473-4810.1000112


In Latin America, Lutzomyia longipalpis is the principal vector of Leishmania infantum, and is associated with the majority of cases of visceral leishmaniasis. This species has distribution from southern Mexico to northern Argentina. Although widespread, its geographical distribution is discontinuous due to its low flight capacity associated with the large number of geographical barriers. Its geographic range includes climatic and environmental discontinuities that are associated with patterns of genetic divergence. In this study, a 487 bp fragment of the Cytochrome Oxidase I (COI ) mitochondrial gene was used to evaluate two sympatric populations of L. longipalpis in the municipalities from Sobral (CE-1S and 2S) and Caririaçu (CE- 1S and 2S) from Northeast, Brazil. A combination of probabilistic methods such as maximum likelihood and Bayesian Inference were applied to populations studied. Phylogenetic analysis revealed the presence of a single monophyletic clades composed of L. longipalpis morph types 1S and 2S. These results suggest that mitochondrial gene COI is not able to evaluate the genetic structure of the complex L. longipalpis.

Keywords: Lutzomyia longipalpis; Cytochrome Oxidase I; Phylogenetic analysis


Lutzomyia longipalpis; Cytochrome Oxidase I; Phylogenetic analysis


Lutzomyia longipalpis sensu lato, the principal vector of Leishmania infantum, the etiologic agent of American visceral leishmaniasis, has a low flight capacity and discontinued distribution throughout the Neotropical region exhibiting bionomics and genetics features compatible with a species complex [1-3]. In Brazil, L. longipalpis complex was first proposed by Manga Beira [4] and was based on the number of abdominal male spots (1 spot and 2 spot) on tergites. Subsequently, analysis of sex pheromones and male “lovesongs” that are produced during copulation supported the existence of a species complex in L. longipalpis [5-9]. These two features can have an important role in the reproductive isolation among closely related species.
The presence of cryptic species in Lutzomyia longipalpis was supported initially by studies using morphological, isozyme and Cytochrome oxidase I (COI ) molecular marker in populations from Central and South America leading to the identification of L. pseudolongipalpis [10], formally recognized as the first taxon of the L. longipalpis complex [11]. Genetic analyses were also realized using microsatellite markers and speciation genes (period gene), have provided further evidence of a species complex [3]. Thereafter, Vigoder [12] suggested that L. cruzi should be regarded as a cryptic species within the L. longipalpis complex based in copulation songs and period gene analysis.
Mitochondrial DNA (mt DNA) has proven useful in molecular phylogenetics of several subfamilies of vectors insects (Table 1) due to its maternal inheritance, rapid rate of divergence and lack of recombination [13-18]. Phylogenetics analysis realized on the gene COI of L. longipalpis revealed the existence of four clades in Latin America for this species [19]. However, little is known about the ability of this molecular marker evaluating the L. longipalpis complex. In light of this possibility, this study aimed to assess the phylogenetic structure of populations L. longipalpis from the state of Ceará using the COI mitochondrial gene. This mitochondrial gene (COI) has been implemented in many evolutionary studies of species of the subfamily Phlebotominae.

Field Collection

Field collections were done in Sobral (3°41′15″S; 40°21′5″W) and Caririaçu (07°02′31″S; 39°17′02″W), both located in the Ceará State, Northeast Region of Brazil. Sand flies were trapped in the surrounding houses and domestic animal shelters using five CDC-type miniature light traps. Sand flies were identified according to Young and Duncan [20], and L. longipalpis sensu lato males were separated based on the number of abdominal spots into 1S and 2S. Genomic DNA extraction was carried out using Chelex®100 (BioRad, Berkeley, California, USA), according to Costa-Junior [21]. A fragment of 487 bp of the Cytochrome Oxidase I (COI) was amplified by PCR using the universal primers described by Simon [14]. Amplification reactions were done using the Mix Go Taq Colorless kit, according to manufacturer specifications (Promega® Fitchburg, Wisconsin, USA). PCR products were visualized in 1% agarose gel through UV light and posteriorly purified using the Wizard® SV Gel and PCR Clean-Up System kit (Promega® Fitchburg, Wisconsin, USA). Sequencing was carried out in ABI 3500 automatic sequencer (Applied Biosystems, Cleveland, Ohio, USA). Only sequences with a Phred score above 30 were used in the analysis. Contigs assembly was carried out using Codon Code Aligner (Codon Code Corporation). Local alignments were done using BLAST [22]. All new sequences produced in this study have been deposited in GenBank under accession numbers: KT806399 to KT806475.
Nucleotide sequences were aligned using Muscle [23] incorporated in MEGA v. 5.0 [24]. Phylogenetic analysis was carried out with the Maximum Likelihood criterion using PhyML [25]. The consistency of the branches was assessed using 1000 bootstrap replicates. We use also the Bayesian inference (BI) analysis, which was implemented with Mr Bayes [26]. BI analysis included two simultaneous independent runs of the Markov Chain Monte Carlo (MCMC) for 100 million generations, sampling every 1,000 generations with a burn-in of 25%. Phlebotomus sergenti was used as the out group for both phylogenetic methods.


We obtained 77 sequences of the gene COI from L. longipalpis male, being 59 (29 1S and 30 2S) from Caririaçu and 18 (101S and 82S) from Sobral. The Maximum Likelihood analysis (Figure 1) and Bayesian Inference (Figure 2) suggested a single genetic group associated with 1S and 2S morph types, and was unable to indicate the ancestral relationship and geographic origin of populations. In a previous study using the period speciation gene was detected two subgroups (1S and 2S) linked with abdominal spots in L. longipalpis from Sobral, Caririaçu and Bodocó municipalities [27]. According with Costa-Junior [21], the per gene can be used as a barcode to L. longipalpis since it could also indicate the ancestral relationship between individuals with 1S and 2S based on polymorphic sites fixed in the two morpho types [21]. In our analysis were not observed similar results to previous studies when used the speciation gene period suggesting that the mitochondrial gene COI do not have appropriate features to analyze the complex Lutzomyia longipalpis. The lack of sub structuring observed between sympatric populations of L. longipalpis using mitochondrial gene COI is probably related to evolutionary factors e.g. speciation incipient, natural selection, genetic drift and genetic introgression. Despite this molecular marker (COI) has proven effective in the evolutionary approaches of various species of sand flies (Table 1), in our study, the results indicate a lower sensitivity of this COI marker to assess the sympatric populations of the municipalities of Sobral e Caririaçu, Ceará State, Northeast Region of Brazil. Thus, we concluded that the use of other molecular markers (e.g. markers period and paralytic) is necessary to evaluate the sub structuring of sympatric populations of the complex L. longipalpis [21,28].
Figure 1: Maximum likelihood tree obtained of the TVM+ I+G model has shown the results of using 487 bp from Lutzomyia longipalpis COI marker. The localities of Caririaçu (CAR) and Sobral (SOB), State of Ceará, Brazil.
Figure 2: Bayesian Inference (BI) topology tree obtained of the HKY + I model has shown the results of using 487 bp from Lutzomyia longipalpis COI marker. The localities of Caririaçu (CAR) and Sobral (SOB), State of Ceará, Brazil.
Table 1: Evolutionary studies of sandflies using mitochondrial marker Cytochrome Oxidase I.


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