Use and trends of molecular markers in sandflies (Diptera: Psychodidae)

Gabriel Golczer^1* & Jazzmin Arrivillaga²

¹ Department of Biology, Tufts University, Medford, MA, USA 02176 *Autor de correspondencia: gabriel.golczer@tufts.edu 

² Departamento de Estudios Ambientales, Universidad Simón Bolívar, Caracas, Venezuela 1080

SUMMARY

The subfamily Phlebotominae is principally composed of the Lutzomyia and Phlebotomus genera: the main vectors of several protozoan, bacterial and viral pathogens. Since the 1990’s molecular markers have enabled us to effectively address many issues concerning this taxon by, for example, solving systematic conflicts, increasing our understanding of speciation and host-parasite co-evolution, and determining the genetic structure of populations. In this paper we review the research undertaken using molecular markers in this taxonomic group. We hope that this will make it easier for scientists to identify markers and data analyses appropriate to their particular research interests. The principal trends we found are a move towards the use of mitochondrial DNA as molecular markers, DNA sequencing as the characterization method of choice, and phylogenetic analysis for analyzing the data. Most of the studies reviewed center on Lutzomyia longipalpis, the main vector for visceral leishmaniasis in the American tropics and Phlebotomus papatasi, the main vector for cutaneous leishmaniasis in Europe, Asia and Africa. Taxonomic problems and the description of genetic structure are the issues most addressed by researchers, followed by resolving systematic conflicts. Future research using molecular markers in the study of sandflies should be aimed towards: a) the development of genetic barcoding as a complementary tool for morphological identification and b) genome sequencing to increase our understanding of host-parasite interactions.

Key words: Molecular markers, sandflies, leishmaniasis, Lutzomyia, Phlebotomus.

Usos y tendencias de marcadores moleculares en flebotomíneos (Diptera: Psychodidae)

RESUMEN

La subfamilia Phlebotominae está compuesta principalmente por los géneros Lutzomyia y Phlebotomus, vectores principales de patógenos virales, bacterianos y protozoarios. Desde los años 90 marcadores moleculares han ayudado a abordar problemas dentro del taxón, como por ejemplo: determinar la estructura genética, resolver conflictos sistemáticos, especiación, co-evolución de parasito y vector. Esta revisión pretende crear un compendio de la investigación realizada con marcadores moleculares en este grupo taxonómico, para así facilitar el trabajo de investigadores que pretenda identificar marcadores y el análisis de datos apropiados para responder sus preguntas. La tendencia principal encontrada fue el uso de ADN mitocondrial como marcador molecular, la secuenciación de ADN como técnica de caracterización y el análisis filogenético como método de análisis predilecto. La mayoría de los estudios revisados se centran en la especie Lutzomyia longipalpis, vector principal de leishmaniosis visceral en las regiones tropicales de América, y Phlebotomus papatasi vector principal de leishmaniosis cutánea en Europa, Asia y América. Los problemas taxonómicos y las descripciones de estructura genética fueron los problemas más abordados por los investigadores, seguidos por la resolución de conflictos sistemáticos. La investigación a futuro empleando marcadores moleculares en flebotomíneos deben apuntar hacia: el desarrollo de barcoding genético como técnica complementaria a la identificación morfológica y la secuenciación de genomas para así avanzar en el área de relaciones parasito-vector.

Palabras clave: Marcadores moleculares, flebotomíneos, leishmaniosis, Lutzomyia, Phlebotomus.

Recibido el 11/02/2015 Aceptado el 03/05/2015

INTRODUCTION

Sandflies of the Phlebotominae sub-family Rondani 1840 are vectors of leishmaniasis, bacterial and viral diseases. The genres Phlebotomus Loew 1845 and Lutzomyia França 1924 are mainly involved in the transmission of several species of the Leishamania genus (Young & Duncan, 1994). The distribution of the genus Phlebotomus is mainly restricted to the old world (Europe, African and Asia continents) and contains few species, in contrast with the genus Lutzomyia which is restricted to the Americas and around 400 described species (Ready, 2000; Young & Duncan, 1994). Mostly all taxonomic descriptions of sandflies are based on morphologic characters. The genus Lutzomyia contains a lot of species complex and groups of cryptic species, a few with lower variability and phenotypical plasticity, blurring the systematic organization and taxonomic separation (Bauzer et al., 2002; Maingon et al., 2007; Uribe, 1999; Yin et al., 2000). The taxonomy of the group has a great epidemiological importance since not all species are proven vectors, and the ones that have been proven so far possess differences in bite behavior or host preference (Rabinovich & Feliciangeli, 2004). Therefore, the clarification of the taxonomic and systematic classification of the group is the main purpose of mostly all studies involving sandflies.

Over the past 30 years, the study of sandflies has been mainly enriched using molecular markers, mostly isozymes and DNA markers (using techniques ranging from RAPD-PCR since 1994 to sequences analysis nowadays), allowing researchers to overcome the limitations of morphology and classic ecology studies, opening new insights specially on taxonomy and systematic of the group (Adamson et al., 1991; Arrivillaga et al., 1995; Mahamat et al., 1992; R. Ward & Miles, 1978). At the present time, the great importance that molecular markers have in the study of sandflies is clear. More than 100 articles have been published using molecular markers allowing for the collection of more than 4,500 DNA sequences stored in GenBank (Benson et al., 2005), providing thousands of characteristics to relate or separate populations, species, groups or genera; this is obviously more than morphologic characteristics can provide.

However, the use of molecular markers doesn’t assure a robust systematic classification or definition of the taxonomic status, i.e. some genes used in systematic studies have been demonstrated to be better than others for reconstructing phylogenies among insect taxa (Simon et al., 1994), while other genes have been proven useless on a taxonomic level even though they have been used in several articles (Golczer & Arrivillaga, 2010). Consequently, a careful evaluation of the molecular marker used or the analysis technique applied is needed at the beginning and end of a study, especially on sandflies, due to the small amount of tissue available to isolate DNA or enzymes to reproduce or correct findings (Golczer & Arrivillaga, 2008).

This article is part a review and part an original synthesis of more than 89 original articles (Table I), first reviewing the use and classification of molecular markers using sandflies articles as examples, then discussing their use and the analysis of the data employed and analyzing the limitations of use, and finally presenting a compilation of PCR primers used in the articles (Table II). The first part is written to allow those familiar with molecular markers to skip it and read the subsequent discussion. Researchers studying sandflies using molecular markers will find this marker compilation useful to facilitate the selection process and assess which markers have been developed, and which of those meet the requirement to answer the question or test the hypothesis stablished.

MOLECULAR MARKERS

A molecular marker is a molecular characteristic of an organism that can be viewed or measured, directly or indirectly using a technique, that provides genotypic information that enable researchers to monitor, differentiate, classify and establish genealogical or phylogenetic relationships; these markers can be classified into biochemical or genetic (molecular) categories and their use is dependent of the question that researchers want to address with them (Avise, 1994; Parker et al., 1998; Walker & Rapley, 2000) .

The most-used biochemical markers are isozymes. These enzymes vary in structural patterns but not on function per se. They are isolated from individuals in an electrophoresis gel and used as Mendelian characteristics or identity patterns, allowing researchers to make inference based on allele frequency, genetic flow and population genetics (Arrivillaga et al., 2003; Avise, 1994; Dujardin et al., 1999; Hillis et al., 1996; Mazzoni et al., 2002).

Genetic markers are more versatile than isozymes because they are evaluated by a variety of techniques such as RFLP (Restriction Fragment Length Polymorphism) (Aransay et al., 1999), DNA Strand Hybridization (Maingon et al., 1993), RAPD-PCR (Random Amplification of Polymorphic DNA) (Adamson et al., 1993; Dvorak et al., 2006; Maingon et al., 1993), SSCP (Single Strand Conformation Polymorphism) (Arrivillaga et al., 2003), Microsatellites (Aransay et al., 2003; Day & Ready, 1999; Hamarsheh et al., 2006; Maingon et al., 2003; Watts et al., 2005) or DNA Sequencing (Vivero et al., 2007). The genetic markers can be classified according to the location of the genome analyzed, such as the mitochondrial genome or nuclear genome. The difference of inheritance patterns can be very useful in taxonomic, systematic, phylogenetic or phylogeographic studies (Avise et al., 1987; Beati et al., 2004; Esseghir et al., 1997; Kambhampati & Smith, 1995; Simon et al., 1994; Togerson et al., 2003).

DISCUSSION OF MOLECULAR MARKERS USE IN SANDFLY RESEARCH

It’s clear that molecular markers (DNA & RNA) have been mainly used by researchers investigating questions about sandflies: 76 of 89 (85%) articles reviewed in this work show this tendency towards the use of DNA and RNA, with isozyme or protein sequences accounting for the rest (13 articles reviewed, 14%). Regarding the use of mitochondrial DNA, only 31% (28 articles) of the articles reviewed use exclusively mitochondrial sequences as markers. The rest (48 of 89) employ a at least a form of nuclear marker such as: nuclear DNA sequences, RAPD, RFLP, SSCP and microsatellites.

There has been a recent increase in the number of articles describing the use nuclear sequences in sandflies, especially in the per and cac regions (Bauzer et al., 2002; Bauzer et al., 2002; Lins et al., 2002; Mazzoni et al., 2002; Mazzoni et al., 2006). These markers, called "clock genes", are involved in the circadian rhythm and temporal regulation of processes, specifically the mating process (Ritchie et al., 1999). These genes have been proven non-informative in phylogenies at a taxonomic level on the L. longipalpis complex, showing no divergence and thus showing no evidence of fixation which will be the signature for an isolation process between taxa (even at a geographic scale) (Golczer & Arrivillaga, 2010). Although these clock genes have also been used on other species such as Lutzomyia intermedia and Lutzomyia whitmani, the authors highlight the low bootstrap values on the nodes of the phylogenetic reconstruction (Mazzoni et al., 2006), and it is important to remark that the method use was Minimum Evolution were the data is transformed into pairwise distance which has been proven to be less robust than other methods such as Maximum Likelihood (Yang, 2006). Other nuclear genes such as 28Sr, 18Sr and ITS are most often used at the level of species, groups or genera, mostly in articles describing research with a systematic focus due to the low mutation rate (Caterino, et al., 2000) .

The most common technique used on phylogenetic studies is DNA sequencing. In comparison with isozymes and RNA markers, this technique has more precision for detecting variability, similarity and phylogeny between species or groups (Caterino et al., 2000); in contrast with isozymes which have a high mutational rate (Hillis et al., 1996).

The questions addressed by researchers have been in relation to genetic structures and species identification (30 of 89 articles reviewed, 44%), beta taxonomy and systematics (13 of 89 articles, 15%) and phylogenetic reconstruction and species delimitation (46 of 89 articles, 51%). The preference for molecular marker used has a tight link between the problems addressed and the limitations of such markers. The questions regarding genetic structures can be explored using various kinds of markers (DNA sequence, isozymes, etc.) and the choice made by the researchers tends to depend on the experience and the light that the marker shed on similar questions in other systems. However, the systematic inquiries are restricted to DNA sequences of low mutation rate, avoiding the risk of saturations and homoplasy (McDowall, 1973).

The species studied most in-depth within the sandflies has been L. longipalpis (32% of articles reviewed). This trend is based in its epidemiological importance, its widespread presence (from Central America to Argentina) and its conflictive taxonomy. Some research declares it as a species complex (Arrivillaga et al., 2000) whereas others differentiate some populations declaring them as different species (Arrivillaga & Feliciangeli, 2001).

Those facts reveal two main goals of sandfly researchers: The need to link their results to epidemiological records and applications, and the need to clarify the taxonomy and systematic classification of the group. The use of molecular markers to develop epidemiological applications arises from the hypothesis that links the vectorial capacity to transmit some Leishmania species and the genetic structure of the sandfly populations (Arrivillaga et al., 2003; Ishikawa et al., 1999). The need for resolution of the taxonomic and systematic issues within the group becomes such an important issue due to the great amount of species within the Lutzomyia genus, or the great similarity between the morphology of the genus that belongs to the Phlebotominae sub-family, which doesn’t allow for a clear identification of the supra-specific and sub-specific groups (Bejarano, 2001; Depaquit et al., 1998; Depaquit et al., 2000).

The countries in which research of sandflies using molecular markers is held reveals a clear tendency in which UK and Brazil are leading the field with 40% and 30% of publications, respectively. Only 12% of articles are produced solely in developing countries. This latter fact reveals the great cost needed to invest on structure and reagents used in molecular techniques, accounting for why 73% of the articles reviewed are produced in collaboration between European and/or North American authors with Latin American authors.

DISCUSSION OF DATA ANALYSIS TECHNIQUES

The use of a molecular marker alone (in any field) doesn’t assure the success of a research project. The posterior analysis of the data provided by those markers reveals the results, giving the same importance to the choice of a molecular marker as to the choice of an analytical tool. Based on this review, phylogenetic analysis was predominant over population genetics analysis.

Isozyme markers provide a kind of information that could be used in population genetics, distance and phylogenetic analysis. But with the latter, the kind of data produced requires special treatments to compare DNA sequences (Nei & Kumar, 2000). In consequence, the analysis of this kind of data in the articles reviewed is biased to distance analysis rather than in phylogenetic analysis.

Distance analysis (UPGMA and Neighbor Joining methods) can be employed with data from RAPD and microsatellites using the pattern shown in the electrophoresis gel as characters (Khuner & Felsenstein, 1994). This kind of data was also used by de Azevedo et al. (2000) and Cárdenas et al. (2001).

A striking aspect of the articles reviewed is the preference for distance analysis and it’s definition as a phylogenetic method as Mazzoni et al. (2006), Bauzer et al. (2002; 2002), Lins et al. (2002), and Parvizi & Assmar (2007), Seblova et al. (2012), Sacarpassa & Alengcar (2013), Nzelu et al. (2015) among others. The misinterpretation of the data reflects the doubt about the phylogenetic principles used to analyze the data, leading to conclusions not based in any phylogenetic principle (Depaquit et al., 1998).

Also, it is observed that there is a recurring error employing the bootstrap technique (used for statistical support). Aransay et al. (2000) use 100 bootstrap replicates, when a minimum of 500 replicates is established as statistically robust (Soltis & Soltis, 2003). When applying phylogenetic or distance analysis, these articles sometimes don’t report the parameters used in their computer analysis (Bauzer et al., 2002; Bauzer et al., 2002) or the statistical support on the nodes or branch of the trees, affecting the ability to replicate and verify the analysis. This issue affects the reliability of the article at the same degree as if the analysis was performed with 100 bootstrap replicates.

The aforementioned issues regarding the analytical tools employed by various authors reside in the superficial knowledge of their parameters and assumptions (Yang, 2006). Authors should explore the seminal articles and basic literature of the techniques they wish to employ, resulting in a better understanding of the input options given to the software that will perform the calculations instead of just repeating the same steps of the latest article in the field.

LIMITATIONS EMPLOYING MOLECULAR MARKERS IN SANDFLIES

The main restrictions that sandflies pose as a system for molecular studies is the size of the tissue/body, as it is so little that it is very difficult to isolate enough DNA to amplify microsatellite regions, nuclear regions with specific primers or RAPD (Golczer & Arrivillaga, 2008). Other restrictions, such as preservation of tissue, arise in molecular studies with sandflies (Depaquit et al., 2000). Only 8 articles make note of the preservation method: storage with isopropanol (Surendran et al., 2005); storage with ethanol between concentrations of 70% and 100% (Beati et al., 2004; Testa et al., 2002; Watts et al., 2005); and dry storage in a liquid nitrogen tank (Cárdenas et al., 2001; G. Lanzaro et al., 1998; Meneses et al., 2005). This preference is important if the studies want to use isoenzymes as a marker, given that a sandfly preserved in ethanol can't be used for studies involving isoenzymes (Testa et al., 2002). The small size of sandflies affects the quantity of markers used in a given individual, as an example the average weight of a sandfly is 70 μg, and using Golczer

and Arrivillaga (2008) protocol 373 ng of DNA in average can be isolated, since 20 to 100 ng of DNA necessary to run a PCR reaction (depending of the quality, fragmentation of DNA and number of copies of the loci) only a few markers can be amplified using PCR techniques. This affects the ability to combine different markers with different resolution to support the conclusion of an article, helping to explain why only 10% of the articles reviewed employ two kinds of markers (nuclear and mitochondrial sequences).

FUTURE PERSPECTIVES

As the use of molecular markers keeps growing, the molecular tool’s costs decrease, and computational tools increase in complexity and power, researchers should use more than one molecular marker to address one question, thus increasing the robustness of the result. The use of a lot of molecular markers in a study isn’t good enough by itself; it must be accompanied by a proper analysis that is fitting to the nature of the marker, the question addressed and the sample size.

The developing process of new primers could be improved by using new techniques like double digest RADseq (Peterson et al., 2012), which has been proven useful to develop markers for population genetics or genetic mapping without a reference genome. Another sequencing technique that might improve our understanding of genetic variation within a species or genus of sand flies is parallel sequencing of pooled DNA samples (Futschik & Schlötterer, 2010), which overcomes the limitation of tissue size of these.

FINAL REMARKS AND SUGGESTIONS

In our opinion, researchers that want to provide DNA barcoding tools for identification purposes should first validate the taxonomic status of the species, and as a subsequent step evaluate different genetic regions as barcodes. Without proof of the validity or cohesiveness of the taxonomic group in question, the process of testing the potential barcodes could be obscured by the presence of a species complex or groups of different morphological species which lack genetic validity (e.g. product of an incomplete speciation process) (Meyer and Paulay, 2005).

To test the validity of a species the use of more than one molecular marker is necessary. At least one mitochondrial and one nuclear marker as these two types could reveal different evolutionary events (Moore, 1995). An example of the latter is Testa et al. (2002); although the goal wasn’t barcoding per se, the use of two types of markers enriched the conclusions at which the authors arrived. On the other hand, if the hypothesis to test is phylogeographic, mitochondrial markers should be used without the addition of nuclear markers due to the maternal inheritance. An example of the latter case is shown in the research of Arrivillaga et al. (2002).

While the COI (cytochrome oxidase I) region has been widely proven in insects as an useful genetic barcode (Pentinsaari et al., 2014, Porter et al., 2014), where it has been employed in various Phlebotomine species (17 articles of 89 reviewed), its accuracy decreases if the species included haven’t been previously validated by genetic markers. As an example, Contreras-Gutiérrez et al. (2014) couldn’t differentiate between L. youngi and L. spinicrassa, since their taxonomic status is still not clear (Golczer, 2011).

In order to perform systematic studies, different markers with different rates of evolution resolve relationships at different levels. Faster evolving sequences (such as NAD, EF, COI and cytb) work better at resolving low level relationships (species, sister taxa and some sub-generic relationships). More conserved regions (18S, 28S, among others) perform best at resolving higher level relationships (at family and genus level) (Nei and Kumar, 2000). An excellent article to reference Lutzomyia systematic research is Beati et al. (2004), not only because of the variety of markers employed, but also for the appropriate analysis of the data using different phylogenetic methods, adding more robustness to the result by showing congruence between different methods.

Lastly, in order to asses if phlebotomine species are still in the process of speciation or, if the have already completed this process, the phylogenetic analysis (using markers with different rates) should be accompanied by genetic structure analysis based on markers with Mendelian characteristics (SNPs, Microsatellites, Isozymes). These can provide robust evidence of gene flow and migration (or the lack of) between populations of different species. Naturally, the size limitation mentioned above confines the development of such markers, but this be counteracted by next generation sequencing techniques, which provide the necessary tools for developing and increasing sample size of future studies.

ACKNOWLEDGEMENTS

This work was funded by Fondo Nacional de Ciencia y Tecnología, Proyecto Mision Ciencia- Fonacit - 2008000911-2 granted to Jazzmin Arrivillaga.

REFERENCES

1. Absavaran A., Rassi Y., Parvizi P., Oshaghi M., Abaie M., Rafizadeh S. & Javadian E. (2009). Identification of sand flies of the subgenus Larroussius based on molecular and morphological characters in north western Iran. Journal of Arthropod-Borne Diseases (Formerly: Iranian Journal of Arthropod-Borne Diseases). 3(2): 22-35.        [ Links ]

2. Adamson R., Chance M., Ward R., Feliciangeli M. D. & Maingon R. (1991). Molecular approaches applied to the analysis of sympatric sandfly populations in endemic areas of western Venezuela. Parassitologia. 55(1): 45-53.        [ Links ]

3. Adamson R. E., Ward R. D., Feliciangeli M. D. & Maingon R. (1993). The application of random amplified polymorphic DNA for sandfly species identification. Med. Vet. Entomol. 7(3): 203-207.        [ Links ]

4. Aransay A., Scoulica E., Chaniotis B. & Tselentis Y. (1999). Typing of sandflies from Greece and Cyprus by DNA polymorphism of 18S rRNA gene. Insect. Mol. Biol., 8(2): 179-184.        [ Links ]

5. Aransay A., Scoulica E., Tselentis Y. & Ready P. (2000). Phylogenetic relationships of phlebotomine sandflies inferred from small subunit nuclear ribosomal DNA. Insect. Mol. Biol. 9(2): 157-168.        [ Links ]

6. Aransay A. M., Ready P. D. & Morillas-Marquez F. (2003). Population differentiation of Phlebotomus perniciosus in Spain following postglacial dispersal. Heredity. 90: 316-325.        [ Links ]

7. Arrivillaga J. & Feliciangeli D. (2001). Lutzomyia pseudo longipalpis: The first new species within the longipalpis (Diptera: Psychodidae: Phlebotominae) complex from la Rinconada, Curarigua, Lara state, Venezuela. J. Med. Entomol. 38: 783-790.        [ Links ]

8. Arrivillaga J., Hamilton J., Ward R., & Maingon R. (1995). RAPD-PCR to distinguish between sandfly populations (Lutzomyia longipalpis). British Society for Parasitology. Trypanosomiasis and Leishmaniasis Proceedings of the Seminar at the Meeting of the British Society for Parasitology, University of Glasgow, Glasgow, UK. 1: 32.        [ Links ]

9. Arrivillaga J., Mutebi J., Piñango H., Norris D., Alexander B., Feliciangeli M. & Lanzaro G. (2003). The taxonomic status of genetically divergent populations of Lutzomyia longipalpis (diptera: Psychodidae) based on the distribution of mitochondrial and isozyme variation. J. Med. Entomol. 40: 615-627.        [ Links ]

10. Arrivillaga J., Norris D., Feliciangeli M. D. & Lanzaro G. (2002). Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences. Infection, Genetics and Evolution. 2: 83–95.        [ Links ]

11. Arrivillaga J., Rangel Y., Oviedo M. & Feliciangeli M. D. (2000). Genetic divergence among venezuelan populations of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae). J. Med. Entomol. 37: 325-30.        [ Links ]

12. Avise J. (1994). Molecular markers, natural history and evolution. New York: Chapman & Hall.        [ Links ]

13. Avise J., Arnold J., Ball M., Bermingham E., Lamp T., Neigel J. & Saunders N. (1987). INTRASPECIFIC PHYLOGEOGRAPHY: The mitochondrial DNA bridge between population genetics and systematics. Ann. Rev. Ecol. Syst. 18: 489-522.        [ Links ]

14. Bauzer L., Gesto J., Souza N., Ward R., Hamilton J., Kyriacou C. & Peixoto A. (2002). Molecular divergence in the period gene between two putative sympatric species of the Lutzomyia longipalpis Complex. Mol. Biol. Evol. 19: 1624-1627.        [ Links ]

15. Bauzer L., Souza N., Ward R., Kyriacou C. & Peixoto A. (2002). The period gene and genetic differentiation between three Brazilian populations of Lutzomyia longipalpis. Insect Molecular Ecology. 11: 315-323.        [ Links ]

16. Beati L., Cáceres A., Lee J. & Munstermann L. (2004). Systematic relationships among Lutzomyia sand flies (Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA sequences. Int. J. Parasitol. 34: 225-234.        [ Links ]

17. Bejarano E. (2001). Nuevas herramientas para la clasificación taxonómica de los insectos vectores de leismaniosis: Utilidad de los genes mitocondriales. Biomédica. 21: 182-191.        [ Links ]

18. Belen A., Alten B. & Aytekin A. (2004). Altitudinal variation in morphometric and molecular characteristics of Phlebotomus papatasi populations. Med. Vet. Entomol. 18: 343-350.        [ Links ]

19. Belen A., Kucukyildirim S. & Alten B. (2011). Genetic structures of sand fly (Diptera: Psychodidae) populations in a leishmaniasis endemic region of turkey. J. Vector Ecol. 3: 32-48.        [ Links ]

20. Benson D., Karsch-Mizrachi I., Lipman D., Ostell J., & Wheeler D. (2005). GenBank. Nucleic Acids Research. 33: 34-38.        [ Links ]

21. Bottechia M., Oliveira S., Bauzer L., Souza N., Ward R., Garner K. & Peixoto A. (2004). Genetic divergence in the cacophony IVS6 intron among five Brazilian populations of Lutzomyia longipalpis. Journal of Molecular Evolution. 58: 754-761.        [ Links ]

22. Boudabous R., Bounamous A., Jouet D., Depaquit J., Augot D., Ferté H. & Babba H. (2009). Mitochondrial DNA differentiation between two closely related species, Phlebotomus (paraPhlebotomus) chabaudi and Phlebotomus (paraPhlebotomus) riouxi (Diptera: Psychodidae), based on direct sequencing and polymerase chain reaction-restriction fragment length polymorphism. Annals of the Entomological Society of America. 102: 347-353.        [ Links ]

23. Cárdenas E., Munstermann L., Martínez O., Corredor D. & Ferro C. (2001). Genetic variability among populations of Lutzomyia (psathyromyia) shannoni (Dyar 1929) (Diptera: Psychodidae: Phlebotominae) in Colombia. Mem. Inst. Oswaldo Cruz. 96: 189-196.        [ Links ]

24. Caterino M., Cho S. & Sperling F. (2000). The current state of insect molecular systematics: A thriving tower of babel. Annual Review of Entomology. 45: 1-54.        [ Links ]

25. Cohnstaedt L., Beati L., Abraham C., Ferro C. & Munstermann L. (2011). Phylogenetics of the phlebotomine sand fly group verrucarum (Diptera: Psychodidae: Lutzomyia). American Journal of Tropical Medicine and Hygiene. 84: 913-922.        [ Links ]

26. Contreras-Gutiérrez M., Vivero R., Vélez I., Porter C. & Uribe S. (2014). DNA barcoding for the identification of sand fly species (Diptera, Psychodidae, Phlebotominae) in Colombia. PloS one, 9: e85496.        [ Links ]

27. Coutinho-Abreu I., Sonoda I., Fonseca J., Melo M., Balbino V. & Ramalho-Ortigão M. (2008). Lutzomyia longipalpis s.l. in Brazil and the impact of the Sao Francisco river in the speciation of this sand fly vector. Parasite & Vectors. 1: 1-16.        [ Links ]

28. Curler G. & Moulton J. (2012). Phylogeny of psychodid subfamilies (Diptera: Psychodidae) inferred from nuclear DNA sequences with a review of morphological evidence for relationships. Systematic Entomology. 37: 603-616.        [ Links ]

29. Day J. & Ready P. (1999). Relative abundance, isolation and structure of phlebotomine microsatellites. Insect Molecular Ecology. 8: 575-580.        [ Links ]

30. de Azevedo A., Monteiro F., Cabello P., Souza N., Goreti M. & Rangel E. (2000). Studies on populations of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Brazil. Mem. Inst. Oswaldo Cruz, 95: 305-322.        [ Links ]

31. de Queiroz Balbino V., Coutinho-Abreu I., Sonoda I., Melo M., de Andrade P., de Castro J. & Ramalho-Ortigão M. (2006). Genetic structure of natural populations of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) from the Brazilian northeastern region. Acta Tropica, 98: 15-24.        [ Links ]

32. De Souza L., Falqueto A., Biral C., Grimaldi G. & Cupolillo E. (2007). Genetic structure of Lutzomyia (nyssomyia) intermedia Populations from two ecologic regions in Brazil where transmission of Leishmania (viannia) braziliensis reflects distinct eco-epidemiologic features. American Journal of Tropical Medicine and Hygiene, 76: 559-565.        [ Links ]

33. Depaquit, J., Ferté, H., & Léger, N. (2000). Revision du sous-genre ParaPhlebotomus (Phlebotomus- Phlebotominae - Psychodidae - Diptera) approches morphologique et moléculaire. Ann. Pharm. Française, 58: 333-340.        [ Links ]

34. Depaquit J., Ferté H., Léger N., Killick-Kendrick R., Rioux J., Killick-Kendrick M. & Gobert, S. (2000). Molecular systematics of the phlebotomine sandflies of the subgenus ParaPhlebotomus (Diptera, Psychodidae, Phlebotomus) based on ITS2 rDNA sequences. Hypotheses of dispersion and speciation. Insect. Mol. Biol., 9: 293-300.        [ Links ]

35. Depaquit J., Ferté H., Léger N., Lefranc F., Alves-Pires C., Hanafi H. & Volf P. (2002). ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera, Psychodidae): Possible consequences in their ability to transmit Leishmania tropica. Int. J. Parasitol. 32: 1123-1131.        [ Links ]

36. Depaquit J., Lienard E., Verzeaux-Griffon A., Ferté H., Bounamous A., Gantier J. & Léger N. (2008). Molecular homogeneity in diverse geographical populations of Phlebotomus papatasi (Diptera, Psychodidae) inferred from ND4 mtDNA and ITS2 rDNA epidemiological consequences. Infection, Genetics and Evolution, 8: 159-170.        [ Links ]

37. Depaquit J., Perrotey S., Lecointre G., Tillier A., Tillier S., Ferté H. & Léger N. (1998). Systématique moléculaire des phlebotominae: Étude pilote. Paraphylie du genre Phlebotomus. CR Académie Des Sciences Paris, 321: 849-855.        [ Links ]

38. Depaquit J., Torsten N., Schmitt C., Ferté H. & Léger N. (2005). A molecular analysis of the subgenus TransPhlebotomus Artemiev, 1984 (Phlebotomus, Diptera, Psychodidae) inferred from ND4 mtDNA with new northern records of Phlebotomus mascittii Grassi, 1908. Parasitol Res, 95: 113-116.        [ Links ]

39. Di Muccio T., Marinucci M., Frusteri L., Maroli M., Pesson B. & Gramiccia M. (2000). Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, Psychodidae) by ITS2 rDNA sequences. Insect Biochem Mol Biol. 30: 387-393.        [ Links ]

40. Dias E., Fortes-Dias C., Stiteler J., Perkins P., & Lawyer P. G. (1998). Random amplified polymorphic DNA (RAPD) analysis of Lutzomyia longipalpis laboratory populations. Rev. Inst. Med. Trop. S. Paulo. 40: 49-54.        [ Links ]

41. Dujardin J., Le Pont F. & Martinez E. (1999). Quantitative phenetics and taxonomy of some phlebotomine taxa. Mem. Inst. Oswaldo Cruz, 94: 735-741.        [ Links ]

42. Dvorak V., Votypka J., Aytekin A., Alten B. & Volf P. (2011). Intraspecific variability of natural populations of Phlebotomus sergenti, the main vector of Leishmania tropica. J. Vector Ecol., 36(s1): S49-S57.        [ Links ]

43. Dvorak V., Aytekin A., Alten B., Skarupova S., Votypka J. & Volf P. (2006). A comparison of the intraspecific variability of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae). J. Vector Ecol. 31: 229-238.        [ Links ]

44. Esseghir S., Ready P. & BEN-ISMAIL R. (2000). Speciation of Phlebotomus sandflies of the subgenus Larroussius coincided with the late Miocene-Pliocene aridification of the mediterranean subregion. Biol. J. Linn. Soc. 70: 189-219.        [ Links ]

45. Esseghir S., Ready P., Killick-Kendrick R. & Ben-Ismail R. (1997). Mitochondrial haplotypes and phylogeography of Phlebotomus vectors of Leishmania major. Insect. Mol. Biol. 6: 211-225.        [ Links ]

46. Futschik A. & Schlötterer C. (2010). The next generation of molecular markers from massively parallel sequencing of pooled DNA samples. Genetics. 186: 207-218.        [ Links ]

47. Florin D. A., Lawyer P., Rowton E., Schultz G., Wilkerson R., Davies S. J. & Keep L. (2010). Morphological anomalies in two Lutzomyia (Psathyromyia) shannoni (Diptera: Psychodidae: Phlebotominae) specimens collected from fort Rucker, Alabama, and Fort Campbell, Kentucky. J. Med. Entomol. 47: 952-956.        [ Links ]

48. Golczer G. & Arrivillaga J. (2008). Modificación de un protocolo estándar de extracción de ADN para especies pequeñas de flebotomínos (Phlebotominae: Lutzomyia). Rev. Colomb. Entomol. 34: 199-202.        [ Links ]

49. Golczer G. & Arrivillaga J. (2010). Gen periodo no construye filogenias dentro del complejo de especie, Lutzomyia longipalpis (Diptera: Phlebotominae). MES. 5: 10-26.        [ Links ]

50. Golczer G. (2011). Diferenciación y ivergencia filogenética en tres especies crípticas del grupo Verrucarum (Diptera: Psychodidae). Master Thesis, Universidad Simón Bolívar. Sartenejas, Venezuela.        [ Links ]

51. Hamarsheh O. & Amro A. (2011). Characterization of simple sequence repeats (SSRs) from Phlebotomus papatasi (Diptera: Psychodidae) expressed sequence tags (ESTs). Parasit. Vectors. 4: 189.        [ Links ]

52. Hamarsheh O., Presber W., Abdeen Z., Sawalha S., Al-Lahem A. & Schoenian G. (2006). Isolation and characterization of microsatellite loci in the sand fly Phlebotomus papatasi (Diptera: Psychodidae). Mol. Ecol. Notes. 6: 826-828.        [ Links ]

53. Hamarsheh O., Presber W., Abdeen Z., Sawalha S., Al-Lahem A. & Schönian G. (2007). Genetic structure of mediterranean populations of the sandfly Phlebotomus papatasi by mitochondrial cytochrome b haplotype analysis. Med. Vet. Entomol. 21: 270-277.        [ Links ]

54. Hernández C., Ruiz-García M., Munstermann L. & Ferro C. (2008). Estructura genética en cinco especies de flebótomos (Lutzomyia spp.) de la serie townsendi, grupo verrucarum, en Colombia (Diptera: Prychodidae). Rev. Biol. Trop. 56: 1717-1739.        [ Links ]

55. Hillis D., Moritz C. & Mable B. (1996). Molecular systematics (2nd ed.). Sunderland, Massachusetts: Sinauer Associates Inc. 655 pp.        [ Links ]

56. Hodgkinson V., Birungi J., Quintana M., Deitze R. & Munstermann L. (2003). Mitochondrial cytochrome B variation in populations of the visceral leishmaniasis vector Lutzomyia longipalpis across eastern Brazil. Ann. J. Trop. Med. Hyg. 69: 386-392.        [ Links ]

57. Hoyos R., Uribe S. & Vélez I. (2012). Tipificación de especímenes colombianos de Lutzomyia longipalpis (diptera: Psychodidae) mediante "Código de barras". Rev. Colomb. Entomol. 38: 134-140.        [ Links ]

58. Ishikawa E., Ready P., de Souza A., Day J., Rangel E. Davies C. & Shaw J. (1999). A mitochondrial DNA phylogeny indicates close relationships between populations of Lutzomyia whitmani (Diptera: Psychodidae, Phlebotominae) from the rain-forest regions of Amazônia and northeast Brazil. Mem. Inst. Oswaldo Cruz. 94: 339-345.        [ Links ]

59. Kambhampati S. & Smith P. (1995). PCR primers for the amplification of four insect mitochondrial gene fragments. Insect. Mol. Biol. 4: 223-236.        [ Links ]

60. Kato H., Jochim R. C., Gomez E. A., Uezato H., Mimori T., Korenaga M. & Hashiguchi Y. (2012). Analysis of salivary gland transcripts of the sand fly Lutzomyia ayacuchensis, a vector of andean-type cutaneous leishmaniasis. Infect. Genet. Evol. 13: 56-66.        [ Links ]

61. Khalid N., Elnaiem D., Aboud M., Al Rabba F. & Tripet F. (2010). Morphometric and molecular differentiation of Phlebotomus (Phlebotomus) sandflies. Med. Vet. Entomol. 24: 352-360.        [ Links ]

62. Khuner M. & Felsenstein J. (1994). A simulation comparision of phylogeny algorithms under equal and unequal evolutionary rates. Mol. Biol. Evol., 11: 459-468.        [ Links ]

63. Kreutzer R., Palau M., Morales A., Ferro C., Feliciangeli D. & Young D. G. (1990). Genetic relationships among phlebotomine sand flies (Diptera: Psychodidae) in the verrucarum species group. J. Med. Entomol. 27: 1-8.        [ Links ]

64. Lampo M., Togerson D., Márquez L. M., Rinaldi M., García C. & Arab A. (1999). Occurrence of sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) in Venezuela: First evidence from reproductively isolated sympatric populations. Ann. J. Trop. Med. Hyg. 61: 1004-1009.        [ Links ]

65. Lanzaro G., Alexander B., Mutebi J., Montoya-Lerma J. & Warburg A. (1998). Genetic variation among natural and laboratory colony populations of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) from Colombia. Mem. Inst. Oswaldo Cruz. 93: 65-69.        [ Links ]

66. Latrofa M. S., Annoscia G., Dantas-Torres F., Traversa D. & Otranto D. (2012). Towards a rapid molecular identification of the common phlebotomine sand flies in the mediterranean region. Vet. Parasitol. 184: 267-270.        [ Links ]

67. Latrofa M. S., Dantas-Torres F., Weigl S., Tarallo V. D., Parisi A., Traversa D. & Otranto D. (2011). Multilocus molecular and phylogenetic analysis of phlebotomine sand flies (Diptera: Psychodidae) from southern Italy. Acta Tropica. 119: 91-98.        [ Links ]

68. Lins R., Oliveira S., Souza N., de Queiroz R., Justiniano S., Ward R. & Peixoto A. (2002). Molecular evolution of the cacophony IVS6 region in sandflies. Insect. Molecular. Ecology. 11: 117-122.        [ Links ]

69. Lins R. M., Souza N. A. & Peixoto A. A. (2008). Genetic divergence between two sympatric species of the Lutzomyia longipalpis complex in the paralytic gene, a locus associated with insecticide resistance and lovesong production. Mem. Inst. Oswaldo Cruz. 103: 736-40.        [ Links ]

70. Mahamat H., Hassanali A., Morgan H., Mutinga M., Mihok S. & MassambaN. (1992). Isozyme analysis of kenyan phlebotomine sandflies (Diptera : Psychodidae) by isoelectric focusing (IEF) on Pharmacia Phast System. Biochem. Syst. Ecol. 20: 593-596.        [ Links ]

71. Maia C., Parreira R., Cristóvão J., Afonso M. & Campino L. (2015). Exploring the utility of phylogenetic analysis of cytochrome oxidase gene subunit I as a complementary tool to classical taxonomical identification of phlebotomine sand fly species (Diptera, Psychodidae) from southern Europe. Acta Tropica. 144: 1-8.        [ Links ]

72. Maingon R., Feliciangeli M. D., Ward R., Chance M., Adamson R., Rodriguez N. & Segovia, M. (1993). Molecular approaches applied to the epidemiology of leishmaniasis in Venezuela. Arch. Inst. Pasteur Tunis. 70(3-4): 309-324.        [ Links ]

73. Maingon R., Ward R., Hamilton J., Bauzer L. & Peixoto A. (2007). The Lutzomyia longipalpis species complex: Does population substructure matter to Leishmania transmission? Trends Parasitol. 24: 12-17.        [ Links ]

74. Maingon R., Ward R., Hamilton J., Noyes H., Souza N., Kemp S. & Watts P. (2003). Genetic identification of two sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) that produce distinct male sex pheromones in Sobral, Ceará state, Brazil. Mol. Ecol. 12: 1879-1894.        [ Links ]

75. Mazzoni C., Araki A., Ferreira G., Azevedo R., Barbujani G. & Peixoto A. (2008). Multilocus analysis of introgression between two sand fly vectors of leishmaniasis. BMC Evolutionary Biology. 8: 141-153.        [ Links ]

76. Mazzoni C., Gomes C., Souza N., de Queiroz R., Justiniano S., Ward R. & Peixoto A. (2002). Molecular evolution of the period gene in sandflies. J. Mol. Evol. 55: 553-562.        [ Links ]

77. Mazzoni C., Souza N., Andrade-Coelho C., Kyriacou C. & Peixoto A. (2006). Molecular polymorphism, differentiation and introgression in the period gene between Lutzomyia intermedia and Lutzomyia whitmani. BMC Evolutionary Biology. 6: 85-96.        [ Links ]

78. McDowall R. (1973). Zoogeography and taxonomy. Tuatara. 20: 88-96.        [ Links ]

79. Meneses C., Cupolillo E., Monteiro F. & Rangel E. (2005). Micro-geographical variation among male populations of the sandfly, Lutzomyia (nyssomyia) intermedia, from an endemic area of American cutaneous leishmaniasis in the state of Rio de Janeiro, Brazil. Med. Vet. Entomol. 19: 38-47.        [ Links ]

80. Meyer C. P. & Paulay G. (2005). DNA barcoding: error rates based on comprehensive sampling. PLoS biology. 3: 2229-2238.        [ Links ]

81. Moin-Vaziri V., Depaquit J., Yaghoobi-Ershadi M., Oshaghi M., Derakhshandeh-Peykar P., Ferté H. & Nadim A. (2007). Intraspecific variation within Phlebotomus sergenti Parrot (1917) (Diptera: Psychodidae) based on mtDNA sequences in Islamic Republic of Iran. Acta Tropica. 102: 29-37.        [ Links ]

82. Moore W. (1995). Inferring phylogenies from mtDNA variation: mitochondrial-gene trees versus nuclear-gene trees. Evolution. 49: 718-726.        [ Links ]

83. Mukhopadhyay J., Kashinath G., Rangel E. & Munstermann L. (1998). Genetic variability in biochemical characters of Brazilian field populations of the Leishmania vector Lutzomyia longipalpis (Diptera: Psychodidae). Ann. J. Trop. Med. Hyg. 59: 893-901.        [ Links ]

84. Mutebi J. P., Alexander B., Sherlock I., Wellington J., Souza A. A., Shaw J. & Lanzaro G. C. (1999). Breeding structure of the sand fly Lutzomyia longipalpis (Lutz & Neiva) in Brazil. American Journal of Tropical Medicine and Hygiene, 61: 149-157.        [ Links ]

85. Mutebi J., Tripet F., Alexander J. & Lanzaro G. (2002). Genetic differentiation among populations of Lutzomyia longipalpis (Diptera: Psychodidae) in central and south America. Ann. Entomol. Soc. Am. 95: 740-752.        [ Links ]

86. Nei M. & Kumar S. (2000). Molecular evolution and phylogenetics. New York: Oxford University Press.        [ Links ]

87. Nzelu C., Cáceres A., Arrunátegui-Jiménez M., Lañas-Rosas M., Yañez-Trujillano H., Luna-Caipo D., Holguín-Mauricci C., Katakura K., et al. (2015). DNA barcoding for identification of sand fly species (Diptera: Psychodidae) from leishmaniasis-endemic areas of Peru. Acta Tropica. 145: 45-51.        [ Links ]

88. Oliveira S., Bottechia M., Bauzer L., Souza N., Ward R., Kyriacou C. & Peixoto A. (2001). Courtship song genes and speciation in sand flies. Mem. Inst. Oswaldo Cruz. 96: 403-405.        [ Links ]

89. Parker P., Snow A., Schug M., Booton G. & Fuerst P. (1998). What molecules can tell us about populations: Choosing and using a molecular marker. Ecology. 79: 361-382.        [ Links ]

90. Parvizi P. & Amirkhani A. (2008). Mitochondrial DNA characterization of Sergentomyia sintoni populations and finding mammalian Leishmania infections in this sandfly by using ITS-rDNA gene. Iran. J. Vet. Res. Shiraz University. 9(1): 9-18.        [ Links ]

91. Parvizi P. & Assmar M. (2007). Nuclear elongation factor-1α gene a molecular marker for iranian sandfly identification. Iran. J. Pub. Health. 36: 25-37.        [ Links ]

92. Parvizi P., Benlarbi M. & Ready P. (2003). Mitochondrial and wolbachia markers for the sandfly Phlebotomus papatasi little population differentiation between peridomestic sites and gerbil burrows in Isfahan province, Iran. Med. Vet. Entomol. 17: 351-362.        [ Links ]

93. Parvizi P. & Ready P. (2006). Molecular investigation of the population differentiation of Phlebotomus papatasi, important vector of L. major, in different habitats and regions of Iran. Iran. Biomed. J. 10: 69-77.        [ Links ]

94. Pentinsaari M., Hebert P. & Mutanen M. (2014). Barcoding beetles: A regional survey of 1872 species reveals high identification success and unusually deep interspecific divergences. PLoS ONE. 9(9): e108651.        [ Links ]

95. Pérez-Doria, A., Bejarano, E., & Vélez, I. (2008). Description of the mitochondrial serine transfer RNA (UCN) of Lutzomyia columbiana (Diptera, Psychodidae). Rev. Bras. Entomol. 52: 591-594.        [ Links ]

96. Pérez-Doria A., Bejarano E. & Vélez I. (2008). Molecular evidence confirms the taxonomic separation of Lutzomyia tihuiliensis from Lutzomyia pia (Diptera: Psychodidae) and the usefulness of pleural pigmentation patterns in species identification. J. Med. Entomol. 45: 653-659.        [ Links ]

97. Pérez-Doria A., Bejarano E. & Vélez I. (2008). Caracteres moleculares para la determinación taxonómica de tres especies de Lutzomyia (Diptera: Psychodidae), vectores potenciales de Leishmania presentes en el valle de Aburrá, Colombia. Rev. Soc. Entomol. Argent. 67(3-4): 99-108.        [ Links ]

98. Pérez-Doria A. & Bejarano E. (2011). tRNASer (UCN) mitocondrial de Lutzomyia hartmanni predicción de la estructura secundaria del tRNASer (UCN) mitocondrial del flebotomíneo Lutzomyia hartmanni (Diptera: Psychodidae). Acta Biol. Colomb. 16: 87-94.        [ Links ]

99. Peterson B. K., Weber J. N., Kay E. H., Fisher H. S., & Hoekstra H. E. (2012). Double digest RADseq: and genotyping in model and non-model species. PloS One, 7(5): e37135.        [ Links ]

100. Porter T., Gibson J., Shokralla S., Baird D., Golding G. & Hajibabaei M. (2014). Rapid and accurate taxonomic classification of insect (class Insecta) cytochrome c oxidase subunit 1 (COI) DNA barcode sequences using a naïve Bayesian classifier. Mol. Ecol. Resour. 14: 929-942.        [ Links ]

101. Rabinovich J. E. & Feliciangeli M. D. (2004). Parameters of Leishmania braziliensis transmission by indoor Lutzomyia ovallesi in Venezuela. Am. J. Trop. Med. Hyg. 70: 373-382.        [ Links ]

102. Raja B., Jaouadi K., Haouas N., Mezhoud H., Bdira S., Amor S. & Hamouda B. (2012). Mitochondrial cytochrome b variation in populations of the cutaneous leishmaniasis vector Phlebotomus papatasi across eastern Tunisia. Int. J. Biodivers. Conserv. 4: 189-196.        [ Links ]

103. Ramalho-Ortigao J. M., Pitaluga A., Telleria E., Marques C., Souza A. & Traub-Cseko Y. (2007). Cloning and characterization of a V-ATPase subunit C from the American visceral leishmaniasis vector Lutzomyia longipalpis modulated during development and blood ingestion. Mem. Inst. Oswaldo Cruz. 102: 509-515.        [ Links ]

104. Ready P. (2000). Sand fly evolution and its relationship to Leishmania transmission. Mem. Inst. Oswaldo Cruz. 95: 589-590.        [ Links ]

105. Ritchie M. G., Halsey E. J. & Gleason J. M. (1999). Drosophila song as a species-specific mating signal and the behavioral importance of kyriacou & hall cycles in D. melanogaster song. Animal Behavior. 58: 649-657.        [ Links ]

106. Zapata S., Mejía L., Le Pont F., León R., Pesson B., Ravel C. & Depaquit J. (2012). A study of a population of Nyssomyia trapidoi (Diptera: Psychodidae) caught on the pacific coast of Ecuador. Parasit. Vectors. 5: 1-8.        [ Links ]

107. Scarpassa V. & Alencar R. (2012). Lutzomyia umbratilis, the main vector of Leishmania guyanensis, represents a novel species complex? PloS one, 7(5): 1-10.        [ Links ]

108. Scarpassa, V., & Alencar, R. (2013). Molecular taxonomy of the two Leishmania vectors Lutzomyia umbratilis and Lutzomyia anduzei (Diptera: Psychodidae) from the Brazilian Amazon. Parasit. Vectors. 6: 258.        [ Links ]

109. Seblova V., Volfova V., Dvorak V., Pruzinova K., Votypka J., Kassahun A., et al. (2013). Phlebotomus orientalis sand flies from two geographically distant Ethiopian localities: biology, genetic analyses and susceptibility to Leishmania donovani. PLoS neglected tropical diseases. 7(4): e2187.        [ Links ]

110. Silva M., Nascimento M., Leonardo F., Rebêlo J. & Pereira S. (2011). Genetic differentiation in natural populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae) with different phenotypic spot patterns on tergites in males. Neotrop. Entomol. 40: 501-506.        [ Links ]

111. Simon C., Frati F., Beckenbach A., Crespi B. J., Liu H. & Flook P. (1994). Evolution, weighting and phylogenetic utility of mitochondrial gene sequence and compilation of conserved polimerase chain reaction primers. Ann. Entomol. Soc. Am. 87: 651-701.        [ Links ]

112. Soltis P. & Soltis D. (2003). Applying the bootstrap in phylogeny reconstruction. Statical Science. 18: 256-267.        [ Links ]

113. Surendran S., Karunaratne S., Adams Z., Hemingway J. & Hawkes N. (2005). Molecular and biochemical characterization of a sand fly population from Sri Lanka: Evidence for insecticide resistance due to altered esterases and insensitive acetylcholinesterase. Bull. Entomol. Res. 95: 371-380.        [ Links ]

114. Testa J., Montoya-Lerma J., Cadena H., Oviedo M. & Ready P. (2002). Molecular identification of vectors of Leishmania in Colombia: Mitochondrial introgression in the Lutzomyia townsendi series. Acta Tropical. 84: 205-218.        [ Links ]

115. Togerson D., Lampo M., Velázquez Y. & Woo, P. (2003). Genetic relationships among some species groups within the genus Luztomyia (Diptera: Psychodidae). Ann. J. Trop. Med. Hyg. 69: 484-493.        [ Links ]

116. Uribe S. (1999). The status of the Lutzomyia longipalpis species complex and possible implications for Leishmania transmission. Mem. Inst. Oswaldo Cruz. 94: 729-734.        [ Links ]

117. Uribe S., Lehmann T., Rowton E., Vélez I. & Porter C. (2001). Speciation and population structure in the morphospecies Lutzomyia longipalpis (Lutz & Neiva) as derived from the mitochondrial ND4 gene. Mol. Phylogenet. Evol. 18: 84-93.        [ Links ]

118. Vivero R., Contreras-Gutiérrez M. & Bejarano E. (2007). Análisis de la estructura primaria y secundaria del ARN de transferencia mitocondrial para serina en siete especies de Lutzomyia. Biomédica. 27: 429-438.        [ Links ]

119. Walker J. & Rapley R. (2000). Molecular biology and biotechnology. Royal Society of Chemistry.        [ Links ]

120. Ward R. & Miles M. (1978). Preliminary isoenzyme studies on phlebotomine sandflies (Diptera: Psychodidae). Ann. Trop. Med. Parasitol. 72: 398-400.        [ Links ]

121. Watts P. C., Hamilton J. G., Ward R. D., Noyes H. A., Souza N. A., Kemp S. J. & Maingon R. D. (2005). Male sex pheromones and the phylogeographic structure of the Lutzomyia longipalpis species complex (Diptera: Psychodidae) from Brazil and Venezuela. Am. J. Trop. Med. Hyg. 73: 734-43.        [ Links ]

122. Yahia H., Ready P., Hamdani A., Testa J. & Guessous-Idrissi N. (2004). Regional genetic differentiation of Phlebotomus sergenti in three Moroccan foci of cutaneous leishmaniasis caused by Leishmania tropica. Parasite. 11: 189-199.        [ Links ]

123. Yang Z. (2006). Computational Molecular Evolution. New York, Oxford University Press.        [ Links ]

124. Yin H., Norris D. & Lanzaro G. (2000). Sibling species in the Lutzomyia longipalpis complex differ in levels of mRNA expression for the salivary peptide, Maxadilan. Insect. Molecular Ecology. 9: 309-314.        [ Links ]

125. Young D. G. & Duncan M. A. (1994). Guide to the identification and geographic distribution of Lutzomyiasand flies in Mexico, the West Indies, central and south America (Diptera: Psychodidae). Gainsville, FL.: Associated Publishers American Entomological Institute.        [ Links ]