Variabilidad gentica del Aedes aegyptideterminada

mediante el anlisis del gen mitocondrial Nd4 en once

reas endmicas para dengue en el Per
Genetic variability of Aedes Aegypti determined by mitochondrial gene ND4
analysis in eleven endemic areas for dengue in Peru

Pamela Yez1, a, Enrique Mamani2, a, Jorge Valle3, a, Mara Paquita Garca2,

b
, Walter Len3, a, Pablo Villaseca3, a, Dina Torres2, a, Csar Cabezas4, c

1 Universidad Nacional de San Antonio Abad del Cusco. Cusco, Per.

2 Laboratorio de Metaxnicas Virales, Instituto Nacional de Salud. Lima, Per.

3 Laboratorio de Entomologa, Instituto Nacional de Salud. Lima, Per.

4 Instituto Nacional de salud. Lima. Per

a Bilogo, b tecnlogo mdico, c mdico cirujano.

SUMMARY: In order to establish the genetic variability of Aedes aegypti

determined by the analysis of the MT-ND4 gene, in eleven endemic regions for
dengue in Peru, 51 samples of Ae. Aegypti were tested. The genetic variability was
determined through the amplification and sequencing of a fragment of 336 base-
pairs of MT ND4, the analysis of intra-specific phylogeny was conducted with the
Network Ver. 4.6.10 program; and the phylogenetic analysis, with the Neighbor
Joining distance method. The presence of five haplotypes of Ae. Aegypti grouped in
two lineages was identified: the first one includes haplotypes 1, 3 and 5, and the
second one comprises haplotypes 2 and 4. The geographic distribution of each of
the haplotypes found is also shown. It is concluded that this variability is caused by
the active migration of this vector and the human activity-mediated passive
migration.

Key words: Aedes; Haplotypes; Pedigree; DNA, Mitochondrial; Peru (source: MeSH
NLM).

RESUMEN: Con el objetivo de establecer la variabilidad gentica de Aedes aegypti

determinada por el anlisis del gen mitocondrial ND4, se analizaron 51 especmenes
de Ae. aegypti en once regiones endmicas para dengue en el Per. La variabilidad
gentica se determin mediante la amplificacin y secuenciacin de un fragmento
de 336 pares de bases del gen mitocondrial ND4. El anlisis de filogenia
intraespecfica se realiz con el programa Network Ver. 4.6.10; y el anlisis
filogentico, con el mtodo de distancia Neighbor Joining. Se identific la presencia
de cinco haplotipos de Ae. aegypti agrupados en dos linajes: el primero agrupa a
los haplotipos 1, 3 y 5 y el segundo agrupa los haplotipos 2 y 4, se muestra
adems la distribucin geogrfica de cada uno de los haplotipos encontrados. Se
concluye que esta variabilidad se debe tanto a la migracin activa de este vector
como a la migracin pasiva mediada por la actividad humana.
Palabras clave: Aedes; Haplotipos; Linaje; ADN Mitocondrial; Per (fuente: DeCS
BIREME).

INTRODUCTION

Aedes aegypti is the vector of the four serotypes of dengue virus (DENV) is found in nearly 100
tropical countries and it is estimated that 2.5 billion people live in areas where there is a risk of
transmission of the epidemic. It is considered one of the most efficient vectors in the
transmission of dengue because it is highly anthropophilic.

In Peru, the Aedes aegypti was first detected in 1852, it has entered through the northern border
from the region of Guayaquil (Ecuador), progressively extending along the coast of Peru. In 1938,
the results of a national survey showed that 11 of the 24 departments of Peru were infested
with Aedes aegypti, announcing its eradication by 1958, as a result of intensive control campaign
carried out by the Ministry of Health of Peru, with support the Pan American Health
Organization. However, a reinfestation of Aedes aegypti was detected in Loreto in 1984, from
which expanded to various departments of Peru. In 2001 it was reported his presence in the city
of Lima and this was associated, in 2005, an epidemic of dengue in the district of Comas. In 2012,
the General Directorate of Epidemiology of Peru reported that Aedes aegypti was present in 17
departments of Peru and the DENV circulating in 15 of them.

The wide distribution of this vector in Peruvian territory, with the serious consequences that
bring dengue epidemics, necessitate the development and the development of control
strategies. Previous studies indicate that knowledge of the genetic variability of this vector can
contribute to the design of vector control strategies. A viable for the study of genetic variability
of Aedes aegypti alternative is via mitochondrial DNA. Which has numerous advantages for
studying evolutionary relationships, since its length is much smaller than the DNA found in the
chromosomes in the nucleus, it is abundant, has a higher rate of evolution, has great
intraspecific variation and is uniparenteral inheritance . Among the mitochondrial genes, gene
nicotinamide adenine dinucleotide subunit 4 (ND4) has proven to be an excellent marker for the
analysis of the genetic structure of the population and colonization events of Aedes aegypti. The
study of changes in gene ND4 to determine Aedes aegypti haplotypes existing in different
geographic areas, which you could also take specific measures. The aim of this study was to
establish the genetic variability of Aedes aegypti by mitochondrial gene ND4 in some endemic
areas for dengue in Peru.

THE STUDY

A cross-sectional study, for which 49 specimens of Aedes aegypti (larvae or adults) randomly
selected between 2006 and 2013 collected from eleven regions of endemic Peru for dengue,
were included and also have a surveillance system was conducted entomological: Amazonas
(Bagua); Cusco (Quillabamba and Camanti); Loreto (Iquitos); Cajamarca (Jan); Lima (Comas and
Jicamarca); Madre de Dios (Puerto Maldonado); Piura (Sullana and Castilla); Ucayali (Pucallpa);
San Martin (Tarapoto); La Libertad (Trujillo) and Tumbes (La Cruz and Villa Primavera), where
both previously reported the presence of Aedes aegypti and dengue cases. Aedes aegypti
specimens were captured in urban, indoor air and preserved in alcohol area 70 to be
transported to the National Institute of Health (Annex 1). Besides these specimens were
included in the analysis twelve previously reported sequences in GenBank, which were used as
standards of comparison with sequences found in the present study. (Table 1).

Table 1. list of sequences of Aedes aegypti and aedes albopictus that had a previous deposit in
genebanks.

Species geographical year source Accession

origin sequence No

DNA ISOLATION OF TOTAL Aedes aegypti

DNA isolation was performed using silica membranes (DNeasy Blood & Tissue Kit, Qiagen,
Hilden, Germany) protocol designed for the purification of total DNA from insect over 50 mg of
weight.

FRAGMENT AMPLIFICATION ND4 mitochondrial gene of Aedes aegypti

A fragment of 377 bp of the mitochondrial gene Ae ND4. aegypti was amplified by PCR, the
reaction volume was 25 uL, with a final concentration of 1x PCR Buffer (Invitrogen), 3 mM MgCl2
(Invitrogen), 400 uM dNTPs mix (Applied Biosystems), 0.4 uM each first reported previously, 1
U / mL of Taq polymerase (Invitrogen) and 2 uL of the DNA sample.

PCR was performed with an initial cycle at 94 C for 2 min; 40 cycles at 94 C for 1 min; 62 C
for 30 s and 72 C for 1 min; The final extension was 72 C for 7 min; 10 L of the amplification
product was subjected to horizontal electrophoresis in agarose gel with 2 mL 1.5% loading buffer
6x 1x TAE buffer at 110V for 60 min. The gel was stained with 0.5 ug / mL ethidium bromide for
10 min and visualized on a UV light transilluminator. Finally, the images were documented with
BioRad gel fotodocumentador ChemiDoc XRS + model.

SEQUENCING OF GENE FRAGMENT Mitochondrial ND4 Aedes aegypti

The PCR products were purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden,
Germany)). . The sequencing of the amplified fragments was carried out using the kit v3.1 Cycle
Sequencing BigDye Terminator Kit The final reaction volume was 15 uL containing 4 of RR 100
uL BigDye; 2 mL of 5X buffer BygDye; 5 uL molecular grade water; 2 mL of 2 mM and first 2 mL
of purified PCR product. The thermocycler was programmed for initial denaturation step of 94
C for 3 min; followed by 25 cycles of 96 C for 10 s; 50 C for 5 s and 60 C for 4 min. The
sequencing products were purified as technical specifications Centri Sep Kit (Princeton
Separations), then 3500XL sequencer used (Applied Biosystems). The consensus sequences were
obtained through the analysis of sense and antisense sequences of each PCR fragment of 336
bp sequences obtained, using the program BioEdit see. 7.0.9.0 and SeqMan II (DNASTAR Inc.)
software.

Sequence analysis

Sequences were aligned with CLUSTAL X see program. 2.1, along with other previously reported
sequences in GenBank corresponding to Brazil and Peru, in addition to a sequence of Aedes
albopictus, obtained by Costa, et al., 2006 was used as outgroup. To find the values of genetic
diversity (H) and nucleotide (Pi) DNAsp see the program was used. 5.10.01. Phylogenetic analysis
was performed haplotypes found using MEGA program view. 5, with the algorithm Neighbor
Joining (NJ), was made by building a tree with a bootstrap autorplicas 1000, using as outgroup
to Aedes albopictus. The analysis of intraspecific haplotype phylogeny using networks was made
with the Network view program. 4.6.1.0 Median joining the algorithm.

FINDINGS

ND4 gene sequences 49, each of 336 bp, 321 conserved sites, 15 variable positions, all
informative and synonymous mutations were obtained. By analyzing the sequences found for
Peru five haplotypes were found, in Figure 1 the distribution of each in relation to the points
that were collected shown.

Figure 1. collection points aedes

aegypti and their relationship as
haplotypes found by analyzing the
mitochondrial gene ND4 in eleven
endemic areas for dengue in Peru,
2012
Phylogenetic analysis of the haplotypes determined that they are grouped into two lineages.
The first lineage included haplotypes 1, 3 and 5, the haplotype 1 was separated by three
mutations haplotype 3 and 5 was separated haplotype Haplotype 3 by a mutation. The second
lineage separated from the first nine mutations included haplotypes 2 and 4; haplotype 2 was
separated Haplotype 4 three mutations (Figure 2, Annex 2).

Figura 2. Phylogenetic relationships among haplotypes of aedes aegypti in Peru.

Note: bootstrap values greater than 60% is calculated with the model TN93.

DISCUSSION

The discovery of five haplotypes grouped into two lineages and distributed in eleven regions of
Peru described in this study is consistent with previous studies reporting the presence of three
haplotypes, two of them in the Piura region, and one present in Lima and Loreto (Iquitos).
Similarly, another study in 2005 indicated that the genetic variability of Aedes aegypti in America
was 0,82; and the value of nucleotide, 0.02 diversity. Meanwhile, the presence in Peru of two
lineages of Aedes aegypti, consistent with previous studies that divide the population of Aedes
aegypti in two large groups, the coastal variant and the mountain-forest variant. The existence
of these two lineages would be explained by the results of another study that said the
introduction of at least two genetic lineages of Aedes aegypti to Peru; This research to Bracco,
et al adds. who indicate the presence of only two genetic lineages of Aedes aegypti throughout
the Americas. The difference found enters both lineages in nine mutations, is similar to a study
that indicated the existence of three haplotypes, the first of them present in Lima and Iquitos,
which was far from a haplotype present in Piura for four mutations, and a third haplotype (also
in Piura) in ten mutations.

The genetic variability of Aedes aegypti in the geographic regions of Peru is due to active
migration performing female Aedes aegypti looking for where to lay eggs; however, this
migration has geographical and climatic constraints. Then, the genetic variability of Aedes
aegypti would also be explained by a passive or forced migration mediated by human activity,
expressed in its commercial activities and transportation systems. This type of migration
additionally explain the existence of the same haplotype in markedly distant geographic regions,
such as the cases of Lima and Iquitos. Places where previous studies have also indicated that
share a common haplotype.

Previous studies have indicated the existence in Peru of two variants of Aedes aegypti, the
coastal variant and the mountain-forest variant, separated by the Andes, stating that this would
serve as a natural barrier that would prevent the spread of this vector. Our results show that
this natural barrier has been compromised, then found one haplotype on both sides of the ridge.
This fact, as explained above lines, it should be passive vector migration mediated by human
activity. Consequently, we can not use, to group haplotypes found, the names of variants and
coastal sierra-rainforest.

While the mitochondrial gene is used for the analysis intraspecific Aedes aegypti, it has recently
been reported in this species the presence of nuclear pseudocopias (NUMT) which may alter the
divergence inferred if they are either analyzed as mitochondrial copies. In this regard, it will be
very useful to use in future studies, nuclear markers, in addition to the mitochondrial marker for
discriminating the possibility of NUMT.

The main limitation of our study was that it the time taken for the collection of specimens was
too broad (2006-2012), with which there is a possibility that the variability in the geographical
areas has been changed. Nevertheless, we believe that our study is a contribution to the
development of research designed to evaluate the interaction or association Aedes aegypti with
serotypes circulating dengue, with the resistance of this vector to insecticides, and their levels
dispersion. Thus contributing to the development of intervention measures for the prevention
and control of dengue.

In conclusion, we have described five haplotypes of Aedes aegypti, grouped into two lineages,
in eleven endemic areas of dengue in Peru. This variability can be explained by the active
migration of the vector and its passive migration mediated by human activity both. Similarly,
due to passive migration of Aedes aegypti should reconsider the traditional classification of
coastal and mountain-forest variants, as found one haplotype in both regions.

Acknowledgements: Entomology Laboratory of the National Institute of Health for support in

morphological identification of specimens analyzed, as well as providing us with specimens of
Madre de Dios and Lima. A: Mr. Enrique Purisaca (Amazonas); Dina Torres and Luis Ayma
(Cusco); Milady Gatti (Loreto); Lucinda Troyes (Cajamarca); Edwin Tineo (Mother of God); Miguel
Castro (Piura); Sadith Arvalo (Ucayali); Etty Lopez (San Martin); Bertha Moreno (La Libertad)
and Micsuco Astudillo (Tumbes) for support in sending specimens analyzed in this study.

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INTRODUCCIN

El Aedes aegypti es el vector de los cuatro serotipos del virus de dengue (DENV), se
encuentra en casi 100 pases tropicales y se calcula que 2,5 billones de personas
habitan en reas donde existe el riesgo de transmisin de la epidemia . Es
considerado como uno de los vectores ms eficientes en la transmisin del dengue
debido a que es altamente antropoflico .

En el Per, el Aedes aegypti fue detectado por primera vez en el ao 1852, habra
ingresado por la frontera norte desde la regin de Guayaquil (Ecuador),
extendindose progresivamente a lo largo de la costa del Per. En 1938, los
resultados de una encuesta nacional mostraron que 11 de los 24 departamentos del
Per estaban infestados con Aedes aegypti , anuncindose su erradicacin para
1958, como resultado de la intensa campaa de control ejecutada por el Ministerio
de Salud del Per, con el apoyo de la Organizacin Panamericana de la Salud . Sin
embargo, una reinfestacin de Aedes aegypti fue detectada en Loreto en 1984, a
partir de la cual se expandi a varios departamentos del Per. En el 2001 se
inform de su presencia en la ciudad de Lima y esta se asoci, en 2005, a una
epidemia de dengue en el distrito de Comas . En el 2012, la Direccin General de
Epidemiologia del Per inform que el Aedes aegypti estaba presente en 17
departamentos del Per y que el DENV circulaba en 15 de ellos .

La amplia distribucin de este vector en el territorio peruano, sumado a las graves

repercusiones que traen las epidemias de dengue, hacen necesario el desarrollo y la
elaboracin de estrategias de control. Estudios previos sealan que el conocimiento
de la variabilidad gentica de este vector puede contribuir en el diseo de
estrategias de control vectorial . Una de alternativas viables para el estudio de la
variabilidad gentica del Aedes aegypti se realiza a travs del ADN mitocondrial. El
cual posee numerosas ventajas para el estudio de las relaciones evolutivas, ya que
su longitud es mucho menor que el ADN hallado en los cromosomas del ncleo, es
abundante, tiene una mayor tasa de evolucin, posee una gran variacin
intraespecfica y es de herencia uniparenteral . Entre los genes mitocondriales, el
gen de la nicotinamida adenn dinucletido subunidad 4 (ND4) ha demostrado ser
un excelente marcador para el anlisis de la estructura gentica de la poblacin y
eventos de colonizacin de Aedes aegypti . El estudio de las variaciones del gen
ND4 permite determinar los haplotipos de Aedes aegypti existentes en diferentes
reas geogrficas, con lo cual se podra adems tomar medidas especficas. El
objetivo del presente estudio fue establecer la variabilidad gentica de Aedes
aegypti mediante el gen mitocondrial ND4 en algunas reas endmicas para dengue
de Per.

EL ESTUDIO

Se realiz un estudio transversal, para el cual se incluyeron 49 especmenes de Aedes

aegypti (larvas o adultos) seleccionados de manera aleatoria entre el 2006 y el 2013
colectados de once regiones del Per endmicas para dengue, y que cuentan adems
con un sistema de vigilancia entomolgica: Amazonas (Bagua); Cusco (Quillabamba
y Camanti); Loreto (Iquitos); Cajamarca (Jan); Lima (Comas y Jicamarca); Madre
de Dios (Puerto Maldonado); Piura (Sullana y Castilla); Ucayali (Pucallpa); San Martin
(Tarapoto); La Libertad (Trujillo), y Tumbes (La Cruz y Villa Primavera), donde
previamente se report tanto la presencia de Aedes aegypti as como casos de
dengue. Los especmenes de Aedes aegypti fueron capturados en el rea urbana,
intradomiciliaria y conservados en alcohol de 70 para ser transportados al Instituto
Nacional de Salud (Anexo 1). Adems de estos especmenes se incluyeron en el
anlisis doce secuencias previamente reportadas en el GenBank, las cuales fueron
utilizadas como patrones de comparacin con las secuencias halladas en el presente
estudio . (Tabla 1).

AISLAMIENTO DEL ADN TOTAL DE Aedes aegypti

El aislamiento de ADN se realiz empleando membranas de slice (DNeasy Blood &

Tissue Kit, Qiagen, Hilden, Germany), protocolo diseado para la purificacin del
ADN total de insectos de ms de 50 mg de peso .

AMPLIFICACIN DEL FRAGMENTO DEL GEN MITOCONDRIAL ND4 DE Aedes aegypti

Un fragmento de 377 pares de bases del gen mitocondrial ND4 de Ae. aegypti fue
amplificado mediante PCR, el volumen de la reaccin fue de 25 L, con una
concentracin final de Buffer PCR 1x (Invitrogen), MgCl 2 3 mM (Invitrogen), mix de
dNTPs 400 M (Applied Biosystems), 0,4 M de cada primer reportado
previamente 1 U/ L de Taq Polimerasa (Invitrogen) y 2 L de la muestra de ADN.

La PCR fue realizada con un ciclo inicial a 94 C por 2 min; 40 ciclos a 94 C por 1
min; 62 C por 30 s y 72 C por 1 min; la extensin final fue de 72 C por 7 min;
10 L del producto de amplificacin fue sometido a electroforesis horizontal en gel
de agarosa al 1,5% con 2 L del buffer loading 6x en buffer TAE 1x a 110v por 60
min. El gel fue teido con 0,5 g/mL de bromuro de etidio por 10 min y visualizado
en un transiluminador de luz UV. Finalmente, se documentaron las imgenes con
fotodocumentador de geles BioRad modelo ChemiDoc XRS+.

SECUENCIACIN DEL FRAGMENTO DEL GEN MITOCONDRIAL ND4 DE Aedes aegypti

Los productos de PCR fueron purificados mediante la utilizacin del QIAquick PCR
Purification Kit (Qiagen, Hilden, Germany) . La secuenciacin de los fragmentos
amplificados se llev a cabo utilizando el kit BigDye Terminator v3.1 Cycle
Sequencing Kit. El volumen final de la reaccin fue de 15 L conteniendo 4 L de
RR 100 BigDye; 2 L de buffer 5X BygDye; 5 L agua grado molecular; 2 L del
primer 2 mM y 2 L de producto de PCR purificado. El termociclador se program
para un paso de desnaturalizacin inicial de 94 C por 3 min; seguido por 25 ciclos
de 96 C por 10 s; 50 C por 5 s y 60 C por 4 min. Los productos de secuenciacin
fueron purificados segn especificaciones tcnicas del Kit Centri Sep (Princeton
Separations) , utilizndose luego el secuenciador 3500xL (Applied Biosystems). Las
secuencias consenso se obtuvieron a travs del anlisis de las secuencias sentido y
antisentido de cada fragmento de PCR obtenindose secuencias de 336 pb,
mediante el uso del programa BioEdit ver. 7.0.9.0 y el software SeqMan II (Dnastar
Inc).

ANLISIS DE SECUENCIAS

Las secuencias fueron alineadas con el programa CLUSTAL X ver. 2.1, junto con
otras secuencias previamente reportadas en el GenBank que corresponde a Brasil y
Per, adems de una secuencia de Aedes albopictus, obtenida por Costa, et al.,
2006 (15) que se utiliz como grupo externo . Para hallar los valores de diversidad
gentica (H) y nucleotdica (Pi) se utiliz el programa DNAsp ver. 5.10.01. El
anlisis filogentico de los haplotipos encontrados se realiz mediante la utilizacin
del programa MEGA ver. 5, con el algoritmo de Neighbor Joining (NJ), se hizo
mediante la construccin de un rbol con un bootstrap de 1000 autorplicas,
utilizando como grupo externo a Aedes albopictus (16). El anlisis de filogenia
intraespecfica utilizando redes de haplotipos fue realizado con el programa Network
ver. 4.6.1.0 con el algoritmo Median joining.
El anlisis filogentico de los haplotipos determin que ellos se encuentran
agrupados en dos linajes. El primer linaje incluy a los haplotipos 1, 3 y 5, el
haplotipo 1 estuvo separado por tres mutaciones del haplotipo 3 y el haplotipo 5
estuvo separado del haplotipo 3 por una mutacin. El segundo linaje, separado del
primero por nueve mutaciones, incluy los haplotipos 2 y 4; el haplotipo 2 estuvo
separado del haplotipo 4 por tres mutaciones (Figura 2, Anexo 2).
DISCUSIN

El hallazgo de cinco haplotipos agrupados en dos linajes y distribuidos en once

regiones del Per descrito en el presente estudio, coincide con estudios previos que
informan de la presencia de tres haplotipos, dos de ellos en la regin Piura, y uno
presente en Lima y Loreto (Iquitos) (17). De igual manera, otro estudio en el 2005
seal que la variabilidad gentica de Aedes aegypti en Amrica era 0,82; y el valor
de diversidad nucleotdica, 0,02(9). Por su parte, la presencia en Per de dos linajes
de Aedes aegypti, concuerdan con estudios previos que dividen a la poblacin de
Aedes aegypti en dos grandes grupos, la variante costera y la variante sierra-
selva(18, 19). La existencia de estos dos linajes estara explicada por los resultados de
otra investigacin que seal la introduccin de, al menos, dos linajes genticos de
Aedes aegypti al Per (17); a ello se suma la investigacin de Bracco, et al. quienes
sealan la presencia de solo dos linajes genticos de Aedes aegypti en todo el
continente americano (9). La diferencia encontrada entra ambos linajes en nueve
mutaciones, es similar a un estudio que indic la existencia de tres haplotipos, el
primero de ellos presente en Lima e Iquitos, que distaba de un haplotipo presente
en Piura por cuatro mutaciones, y de un tercer haplotipo (tambin en Piura) en diez
mutaciones (17).

La variabilidad gentica de Aedes aegypti en las regiones geogrficas de Per se

debera a la migracin activa que realiza la hembra del Aedes aegypti en busca de
donde poner los huevos (2); sin embargo, este tipo de migracin presenta
restricciones geogrficas y climticas. Entonces, la variabilidad gentica de Aedes
aegypti tambin estara explicada por una migracin pasiva o forzada mediada por
la actividad del ser humano, expresada en su actividades comerciales y en los
sistemas de trasporte. Este tipo de migracin, adicionalmente, explicara la
existencia de un mismo haplotipo en regiones geogrficas marcadamente distantes,
como los casos de Lima e Iquitos. Lugares en los cuales estudios previos tambin
han sealado que comparten un mismo haplotipo (17).

Estudios previos han sealado la existencia en Per de dos variantes de Aedes

aegypti, la variante costera y la variante sierra-selva, separadas por la cordillera de
los Andes, afirmando que esta servira como una barrera natural que evitara la
dispersin de este vector. Nuestros resultados muestran que esta barrera natural
ha sido vulnerada, pues se ha encontrado un mismo haplotipo a ambos lados de la
cordillera. Este hecho, como se explic lneas arriba, se debera a la migracin
pasiva del vector mediada por la actividad humana. En consecuencia, no podemos
usar, para agrupar los haplotipos encontrados, las denominaciones de variantes
costera y de sierra-selva (17, 19, 20).

Si bien el gen mitocondrial es utilizado para el anlisis intraespecfico de Aedes

aegypti, recientemente se ha reportado en esta especie la presencia de
pseudocopias nucleares (NUMT) que pueden alterar la divergencia inferida si son
indistintamente analizados como copias mitocondriales (20). En tal sentido, ser de
gran utilidad utilizar en futuros estudios, marcadores nucleares, adems del
marcador mitocondrial para discriminar la posibilidad de NUMT

La principal limitacin de nuestro estudio radic en que el tiempo empleado para la

colecta de especmenes fue demasiado amplio (2006-2012), con lo cual existe la
posibilidad que la variabilidad en las zonas geogrficas haya sufrido modificaciones.
A pesar de ello, consideramos que nuestro estudio constituye un aporte en el
desarrollo de lneas de investigacin destinadas a evaluar la interaccin o
asociacin del Aedes aegypti con los serotipos de dengue circulantes, con la
resistencia de este vector a insecticidas, y con sus niveles de dispersin. Para as
contribuir con el desarrollo de medidas de intervencin para la prevencin y control
del dengue.

En conclusin, se han descrito cinco haplotipos de Aedes aegypti, agrupados en dos

linajes, en once zonas endmicas de dengue en el Per. Esta variabilidad se
explicara tanto por la migracin activa del vector como por su migracin pasiva,
mediada por la actividad humana. De igual manera, debido a la migracin pasiva
del Aedes aegypti se debe reconsiderar la clasificacin tradicional de variantes
costera y sierra-selva, ya que se ha encontrado un mismo haplotipo en ambas
regiones.

Agradecimientos: al Laboratorio de Entomologa del Instituto Nacional de Salud

por el apoyo en la identificacin morfolgica de los especmenes analizados, as
como proveernos de especmenes de Madre de Dios y Lima. A los seores: Enrique
Purisaca (Amazonas); Dina Torres y Luis Ayma (Cusco); Milady Gatti (Loreto);
Lucinda Troyes (Cajamarca); Edwin Tineo (Madre de Dios); Miguel Castro (Piura);
Sadith Arvalo (Ucayali); Etty Lopez (San Martin); Bertha Moreno (La Libertad) y
Micsuco Astudillo (Tumbes) por el apoyo en el envo de los especmenes analizados
en este estudio.

Contribuciones de autora: PY diseo el estudio; PY y WC recolectaron los

especmenes; PY, EM, JV y DT analizaron e interpretaron los datos; PY, EM, DT y CC
redactaron; PY, EM y MPG intervinieron para la aprobacin de la versin final; JV y
PV aportaron con material de estudio; EM y MP obtuvieron el financiamiento; todos
contribuyeron para la revisin critica

Fuentes de financiamiento: Instituto Nacional de Salud. Lima, Per.

Conflictos de inters: los autores declaran no tener conflictos de inters en la

publicacin del presente estudio.
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