Abstract
Mosquito control prevails as the most efficient method to protect humans from the dengue virus, despite recent efforts to find a vaccine for this disease. We evaluated insecticide resistance and genetic variability in natural populations of Aedes aegypti (Linnaeus, 1762) from Colombia. This is the first Colombian study examining kdr mutations and population structure. Bioassays with larvae of three mosquito populations (Armenia, Calarcá and Montenegro) were performed according to the World Health Organization (WHO) guidelines, using Temephos. For the analysis of the Val1016Ile mutation and genetic diversity, we sampled recently-emerged adults from four mosquito populations (Armenia, Calarcá, Montenegro and Barcelona). Following the WHO protocol, bioassays implemented with larvae showed resistance to Temephos in mosquito populations from Armenia (77% ± 2) and Calarcá (62% ± 14), and an incipient altered susceptibility at Montenegro (88% ± 8). The RR95 of mosquito populations ranged from 3.7 (Montenegro) to 6.0 (Calarca). The Val1016Ile mutation analysis of 107 genotyped samples indicates that 94% of the specimens were homozygous for the wild allele (1016Val) and 6% were heterozygous (Val1016Ile). The 1016Ile allele was not found in Barcelona. Genetic variability analysis found three mitochondrial lineages with low genetic diversity and gene flow. In comparison with haplotypes from the American continent, those from this study suggest connections with Mexican and North American populations. These results confirm that a continuous monitoring and managing program of A. aegypti resistance in the state of Quindío is required.
Bioassays; gene flow; knockdown resistance; mitochondrial DNA; ND4 gene
Dengue, a viral disease transmitted by a mosquito, has the greatest epidemic potential in
the world (Who 2013WHO (2013) Sustaining the drive to overcome the global impact of
neglected tropical diseases. Available online at:
http://apps.who.int/iris/bitstream/10665/77950/1/9789241564540_eng.pdf [Accessed: 2
April 2013]
http://apps.who.int/iris/bitstream/10665...
). Given that there is no
effective vaccine, in order to control dengue outbreaks it is necessary to control the
vector, Aedes (Stegomyia) aegypti
(Linnaeus, 1762) (Urdaneta-Marquez & Failloux
2011Urdaneta-Marquez L, Anna-Bella F (2011) Population genetic structure of
Aedes aegypti, the principal vector of dengue viruses. Infection, Genetics and
Evolution 11(2): 253-261. doi: 10.1016/j.meegid.2010.11.020
https://doi.org/10.1016/j.meegid.2010.11...
). Vector control strategies in Colombia are mostly dependent on community
participation through education campaigns to reduce potential larval breeding sites.
However, in epidemic situations, insecticides are applied on a large scale (Ministerio de la Protección Social 2011Ministerio de la Protección Social (2011) Guía de Vigilancia
Entomológica y Control de Dengue. Colombia, Ministerio de la Protección Social,
Instituto Nacional de Salud, Organización Panamericana de la Salud (OPS/OMS).
Available online at: http://new.paho.org/col/index.php?option = com_docman&task =
doc_download&gid = 1215&Itemid [Accessed: 8 April 2013]
http://new.paho.org/col/index.php?option...
). In Colombia,
two main classes of insecticides are used: the organophosphates (OP) Temephos since 1970
(Motta-Sanchez et al. 1976Motta-Sanchez A, Tonn R, Uribe L, Calheiros L (1976) A comparison of
methods of application of several insecticides for the control of Aedes aegypti in
villages in Colombia. WHO/VBC 76(23): 1-33.) and Malathion, since
1980 (Ocampo et al. 2011Ocampo C, Salazar-Terreros M, Mina N, McAllister J, Brogdon W (2011)
Insecticide resistance status of Aedes aegypti in 10 localities in Colombia. Acta
Tropica 118(1): 37-44. doi: 10.1016/j.actatropica.2011.01.007
https://doi.org/10.1016/j.actatropica.20...
), and pyrethroids (PY),
used since 1990 (Maestre 2012Maestre R (2012) Susceptibility Status of Aedes aegypti to Insecticides
in Colombia, p. 203-209. In: Farzana P (Ed.) Insecticides - Pest Engineering.
Croatia, InTech Press.).
Currently, resistance to OP and PY is present on a large scale and has been reported to
occur in most regions where A. aegypti is established (Who 2013WHO (2013) Sustaining the drive to overcome the global impact of
neglected tropical diseases. Available online at:
http://apps.who.int/iris/bitstream/10665/77950/1/9789241564540_eng.pdf [Accessed: 2
April 2013]
http://apps.who.int/iris/bitstream/10665...
). Despite the existence of a national network
for the evaluation of insecticide resistance to malaria and dengue vectors in Colombia
(Ministerio de la Protección Social 2011Ministerio de la Protección Social (2011) Guía de Vigilancia
Entomológica y Control de Dengue. Colombia, Ministerio de la Protección Social,
Instituto Nacional de Salud, Organización Panamericana de la Salud (OPS/OMS).
Available online at: http://new.paho.org/col/index.php?option = com_docman&task =
doc_download&gid = 1215&Itemid [Accessed: 8 April 2013]
http://new.paho.org/col/index.php?option...
), the
insecticide resistance status of A. aegypti has not been systematically
monitored. The first report of resistance was to DDT, an insecticide that is no longer used
in Colombia for dengue control, in a mosquito population from Cucuta, near the border with
Venezuela (Gast-Galvin 1961Gast-Galvis A (1961) Una década de labor del Instituto Carlos Finlay de
Colombia. Boletín de la Oficina Sanitaria Panamericana 50(1): 44-58.). Resistance to OP
Temephos was first documented in Cali, Valle del Cauca (Suárez et al. 1996Suárez M, González R, Morales C (1996) Temefos resistance to Aedes
aegypti in Cali, Colombia. American Journal of Tropical Medicine and
HygieneSupplements 55(2): 257.), followed by the states of Norte de Santander, Sucre,
Antioquia, Huila, Nariño, Cundinamarca, Santander, Caquetá, Meta, Guaviare and Atlántico
(Maestre et al. 2009Maestre R, Rey G, Salas J, Vergara C, Santacoloma L, Goenaga O (2009)
Susceptibilidad de Aedes aegypti (Diptera: Culicidae) a temephos en Atlántico -
Colombia. Revista Colombiana de Entomologia 35(2): 54-57., Ocampo 2011Ocampo C, Salazar-Terreros M, Mina N, McAllister J, Brogdon W (2011)
Insecticide resistance status of Aedes aegypti in 10 localities in Colombia. Acta
Tropica 118(1): 37-44. doi: 10.1016/j.actatropica.2011.01.007
https://doi.org/10.1016/j.actatropica.20...
, Santacoloma et al.
2012Santacoloma L, Chaves B, Brochero H (2012) Estado de la susceptibilidad
de poblaciones naturales del vector del dengue a insecticidas en trece localidades de
Colombia. Biomédica 32(3): 333-343. doi: 10.7705/biomedica.v32i3.680
https://doi.org/10.7705/biomedica.v32i3....
, Grisales et al. 2013Grisales N, Poupardin R, Gomez S, Fonseca-Gonzalez I, Ranson H, Lenhart
A (2013) Temephos resistance in Aedes aegypti in Colombia compromisos dengue vector
control. PLOS Neglected Tropical Diseases 7(9): 1-10. doi:
10.1371/journal.pntd.0002438
https://doi.org/10.1371/journal.pntd.000...
). Regarding the
evaluations of Malathion, the populations are not yet resistant to adulticide (Ocampo et al. 2011Ocampo C, Salazar-Terreros M, Mina N, McAllister J, Brogdon W (2011)
Insecticide resistance status of Aedes aegypti in 10 localities in Colombia. Acta
Tropica 118(1): 37-44. doi: 10.1016/j.actatropica.2011.01.007
https://doi.org/10.1016/j.actatropica.20...
, Santacoloma et al. 2012Santacoloma L, Chaves B, Brochero H (2012) Estado de la susceptibilidad
de poblaciones naturales del vector del dengue a insecticidas en trece localidades de
Colombia. Biomédica 32(3): 333-343. doi: 10.7705/biomedica.v32i3.680
https://doi.org/10.7705/biomedica.v32i3....
).
PY resistance in Colombia was first documented in 2006 in mosquito populations from Santander, Cundinamarca, Meta, Caqueta and Guaviare (Santacoloma et al. 2010Santacoloma L, Chaves B, Brochero H (2010) Susceptibilidad de Aedes aegypti a DDT, deltametrina y lambdacialotrina en Colombia. Revista Panamericana de Salud Publica 27(1): 66-73.). Metabolic resistance and target site insensitivity represent the two major forms of PY resistance (Soderlund & Knipple 1999Soderlund D, Knipple D (1999) Knockdown resistance to DDT and pyrethroids in the house fly (Diptera: Muscidae): from genetic trait to molecular mechanism. Annals of the Entomological Society of America 92(6): 909-915., 2003Soderlund D, Knipple D (2003) The molecular biology of knockdown resistance to pyrethroid insecticides. Insect Biochemistry and Molecular Biology 33(6): 563-577.). Studies have suggested that mutations in the voltage-gated sodium channel (NaV), the target site for PY and DDT, may play a role in PY resistance (Irac 2011Irac (2011) Prevention and Management of Insecticide Resistance in Vectors of Public Health Importance. Atlanta, Insecticide Resistance Action Committee (IRAC), 70p.). NaV is a transmembrane protein present in the neuronal axons and is composed of four homologous domains (I-IV), each with six hydrophobic segments (S1-S6) (Catterall 2000Catterall W (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26(1): 13-25.). 'Knockdown resistance' (kdr) is a generic term applied to insects that fail to lose coordinated activity immediately following PY exposure.
Kdr mutations in the NaV (associated or not with PY resistance) have been
observed in a range of insects, including A. aegypti (Saavedra-Rodriguez et al. 2007Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S, Moulton M,
Flores A, Fernandez-Salas I, Bisset J, Rodriguez M, McCall P, Donnelly M, Ranson H,
Hemingway J, Black W (2007) A mutation in the voltage-gated sodium channel gene
associated with pyrethroid resistance in Latin American Aedes aegypti. Insect
Molecular Biology 16(6): 785-798.). In populations of
A. aegypti from Latin America and Southeast Asia, the mutations
Val1016Ile, Val1016Gly, Phe1534Cys and Asp1794Tyr, all in the IIS6 and IIIS6 segment, are
correlated with insecticide resistance (Brengues et al.
2003Brengues C, Hawkes N, Chandre F, McCarroll L, Duchon S, Guillet P,
Hemingway J (2003) Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated
with novel mutations in the voltage-gated sodium channel gene. Medical and Veterinary
Entomology 17(1): 87-94., Saavedra-Rodriguez et al. 2007Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S, Moulton M,
Flores A, Fernandez-Salas I, Bisset J, Rodriguez M, McCall P, Donnelly M, Ranson H,
Hemingway J, Black W (2007) A mutation in the voltage-gated sodium channel gene
associated with pyrethroid resistance in Latin American Aedes aegypti. Insect
Molecular Biology 16(6): 785-798.,
Chang et al. 2009Chang C, Shen W, Wang T, Lin Y, Hsu E, Dai S (2009) A novel amino acid
substitution in a voltage-gated sodium channel is associated with knockdown
resistance to permethrin in Aedes aegypti. Insect Biochemistry and Molecular Biology
39(4): 272-278. doi: 10.1016/j.ibmb.2009.01.001
https://doi.org/10.1016/j.ibmb.2009.01.0...
, Harris et al. 2010Harris A, Rajatileka S, Ranson H (2010) Pyrethroid resistance in Aedes
aegypti from Grand Cayman. American Journal of Tropical Medicine and Hygiene 83(2):
277-284. doi: 10.4269/ajtmh.2010.09-0623
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, Linss et al.
2014Linss J, Brito L, Garcia G, Saori A, Bruno R, Lima J, Valle D, Martins A
(2014) Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations
in Aedes aegypti Brazilian natural populations. Parasites & Vectors 7(25): 1-12.
doi: 10.1186/1756-3305-7-25
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). However, only one of these, a valine to isoleucine substitution at codon
1016, has been clearly linked to insecticide resistance; selection pressure under
laboratory conditions, bioassays with adults, biochemical assays and molecular screening
have confirmed this finding (Rodpradit et al. 2005Rodpradit P, Boonsuepsakul S, Chareonviriyaphap T, Bangs M, Rongnoparut
P (2005) Cytochrome P450 genes: molecular cloning and overexpression in a
pyrethroid-resistant strain of Anopheles minimus mosquito. Journal of American
Mosquito Control Association 21(1): 71-79. doi:
10.2987/8756-971X(2005)21[71:CPGMCA]2.0.CO;2
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,
Saavedra-Rodriguez et al. 2007 Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S, Moulton M,
Flores A, Fernandez-Salas I, Bisset J, Rodriguez M, McCall P, Donnelly M, Ranson H,
Hemingway J, Black W (2007) A mutation in the voltage-gated sodium channel gene
associated with pyrethroid resistance in Latin American Aedes aegypti. Insect
Molecular Biology 16(6): 785-798., Strode et al. 2008Strode C, Wondji C, David J, Hawkes N, Lumjuan N, Nelson D, Drane D,
Karunaratne S, Hemingway J, Black W, Ranson H (2008) Genomic analysis of
detoxification genes in the mosquito Aedes aegypti. Insect Biochemistry and Molecular
Biology 38(1): 113-123. doi: 10.1016/j.ibmb.2007.09.007
https://doi.org/10.1016/j.ibmb.2007.09.0...
, García et al. 2009García G, Flores A, Fernández-Salas I, Saavedra-Rodríguez K, Reyes-Solis
G, Lozano-Fuentes S, Bond J, Casas-Martínez M, Ramsey J, García-Rejón J,
Domínguez-Galera M, Ranson H, Hemingway J, Eisen L, Black W (2009) Recent rapid rise
of a perrmethrin knock down resistance allele in Aedes aegypti in México. PLOS
Neglected Tropical Diseases 3(10): 531-541. doi:
10.1371/journal.pntd.0000531
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, Martins et al. 2009Martins A, Lima J, Peixoto A, Valle D (2009) Frequency of Val1016Ile
mutations in the voltage-gated sodium channel gene of Aedes aegypti Brazilian
populations. Tropical Medicine & International Health 14(11): 1351-1355. doi:
10.1111/j.1365-3156.2009.02378.x
https://doi.org/10.1111/j.1365-3156.2009...
,
Lumjuan et al. 2011Lumjuan N, Rajatileka S, Changsom D, Wicheer J, Leelapat P,
Prapanthadara L, Ranson H (2011) The role of the Aedes aegypti Epsilon glutathione
transferases in conferring resistance to DDT and pyrethroid insecticides. Insect
Biochemistry Molecular Biology 41(3): 203-209. doi:
10.1016/j.pt.2010.08.004
https://doi.org/10.1016/j.pt.2010.08.004...
, Marcombe et al. 2012Marcombe, S, Blanc R, Pocquet N, Muhammad-Asam R, Poupardin R, Sélior S,
Darriet F, Reynaud S, Yébakima A, Corbel V, Jean-Philippe D, Chandre F (2012)
Insecticide Resistance in the Dengue Vector Aedes aegypti from Martinique:
Distribution, Mechanisms and Relations with Environmental Factors. Plos ONE 7(2):
e30989. doi: 10.1371/journal.pone.0030989
https://doi.org/10.1371/journal.pone.003...
).
Understanding the patterns of genetic structure and gene flow among A.
aegypti populations is pivotal for the development of rational dengue control
programs (Urdaneta-Marquez & Anna-Bella 2011Urdaneta-Marquez L, Anna-Bella F (2011) Population genetic structure of
Aedes aegypti, the principal vector of dengue viruses. Infection, Genetics and
Evolution 11(2): 253-261. doi: 10.1016/j.meegid.2010.11.020
https://doi.org/10.1016/j.meegid.2010.11...
).
The current trend is to use microsatellites (Monteiro et
al. 2014Monteiro F, Shama R, Martins A, Gloria-Soria A, Brown J, Powell J (2014)
Genetic diversity of Brazilian Aedes aegypti: Patterns following an eradication
program. PLos Neglected Tropical Diseases 8(9): e3167. doi:
10.1371/journal.pntd.0003167
https://doi.org/10.1371/journal.pntd.000...
) and/or SNPs (single nucleotide polymorphism) (Rasic et al. 2014Rasic G, Filipoviæ I, Weeks A, Hoffmann A (2014) Genome-wide SNPs lead
to strong signals of geographic structure and relatedness patterns in the major
arbovirus vector, Aedes aegypti. BMC Genomics 15(275): 1-12. doi:
10.1186/1471-2164-15-275
https://doi.org/10.1186/1471-2164-15-275...
). Nevertheless, mitochondrial DNA (mtDNA) has been
widely used in population genetics studies of A. aegypti from different
geographic points and dengue endemic regions (Gonçalves et
al. 2012Gonçalves A, Cunha I, Santos W, Luz S, Ribolla P, Abad-Franch F (2012)
Gene flow networks among American Aedes aegypti populations. Evolutionary
Applications 5(7): 664-676. doi: 10.1111/j.1752-4571.2012.00244.x
https://doi.org/10.1111/j.1752-4571.2012...
). The ND4 mitochondrial gene, which codifies the subunit 4 of the NADH
dehydrogenase enzyme, is an effective tool to analyze genetic population structure and
colonization events in A. aegypti. Such analyses were carried out in
mosquito populations from Brazil (Twerdochlib et al.
2012Twerdochlib A, Dalla A, Leite S, Chitolina R, Westphal B, Navarro-Silva
M (2012) Genetic variability of a population of Aedes aegypti from Paraná, Brazil,
using the mitochondrial ND4 gene. Revista Brasileira de Entomologia 56(2): 249-256.
doi: 10.1590/S0085-56262012005000030
https://doi.org/10.1590/S0085-5626201200...
), Bolivia (Paupy et al. 2012Paupy C, Goff G Le, Brengues C, Guerra M, Revollo J, Barja Z,
Jean-Pierre H, Fontenille D (2012) Genetic structure and phylogeo graphy of Aedes
aegypti, the dengue and yellow-fever mosquito vector in Bolivia. Infection, Genetics
and Evolution 12(6): 1260-1269. doi: 10.1016/j.meegid.2012.04.012
https://doi.org/10.1016/j.meegid.2012.04...
), Peru
(Yáñez et al. 2013Yáñez P, Manami E, Valle J, Garcia M, León W, Villaseca P, Torres D,
Cabezas C (2013) Variabilidad genética del Aedes aegypti determinada mediante el
análisis del gen mitocondrial ND4 en once áreas endémicas para dengue en el Perú.
Revista Peruana de Medicina Experimental y Salud Pública 30(2):
246-250.), Venezuela (Urdaneta-Marquez et al. 2008Urdaneta-Marquez L, Bosio C, Herrera F, Rubio-Palis Y, Salasek M, Black
W (2008) Genetic relationships among Aedes aegypti collections in Venezuela as
determined by mitochondrial DNA variation and nuclear single nucleotide
polymorphisms. American Journal of Tropical Medicine and Hygiene 78(3):
479-491.) and Mexico (Gorrochotegui-Escalante et al. 2002Gorrochotegui-Escalante N, Gomez-Machorro C, Lozano-Fuentes S,
Fernandez-Salas I, Muñoz M, Farfan-Alej J, Garcia-Rejon J, Beaty B, Black W (2002)
Breeding structure of Aedes aegypti populations in Mexico varies by region. American
Journal of Tropical Medicine and Hygiene 66(2): 213-222.). So far, only
RAPDs (Random Amplified Polymorphic DNA) (Ocampo &
Wesson 2004Ocampo C, Wesson D (2004) Population dynamics of Aedes aegypti from a
dengue hyper endemic urban setting in Colombia. American Journal of Tropical Medicine
and Hygiene 71(6): 506-513., Mejía et al. 2011Mejia G, Mora G, Ramos E, Maestre R, Mazenett E, Malambo D, Gómez D
(2011) Identificación genética de subpoblaciones de Aedes aegypti en Cartagena de
Indias - Colombia. Revista Ciencias biomédicas 2(1): 5.) and more
recently the mtDNA (Caldera et al. 2013Caldera S, Jaramillo S, Cochero S, Pérez-Doria A, Bejarano E (2013)
Diferencias genéticas entre poblaciones de Aedes aegypti de municipios del norte de
Colombia, con baja y alta incidencia de dengue. Biomedica 33(1): 89-98. doi:
10.7705/biomedica.v33i0.1573) have been
used to analyze the genetic structure of A. aegypti populations in
Colombia.
Here, we evaluated the insecticide resistance and genetic variability in natural populations of A. aegypti from Colombia. This is the first Colombian study to look at insecticide resistance (OP and PY (kdr mutation)) and population structure. Our findings have shown that insecticide resistance is spreading in the country owing to the 1016Ilekdr allele (in low frequency), and that OP resistance is found in most populations of A. aegypti studied by us. Genetic variability analysis shows that the vector population has low genetic diversity and limited gene flow.
MATERIAL AND METHODS
We collected A. aegypti larvae in 2011, from three municipalities in the state of Quindío: Armenia (4°32'0"N, 75°40'0"W, 1,483 m), Montenegro (4°34'23"N, 75°45'20"W, 1,292 m), Calarcá (4°31'55"N, 75°39'1"W, 1,573 m) and Barcelona (4°25'53", 75°43'26", 1,573 m), a district of Calarcá. We followed the standard methods of the Pan-American Health Organization (PAHO) for determining the infestation rate of A. aegypti (Ops 1995OPS (1995) Dengue y dengue hemorrágico en las Américas: guías para su prevención y control. Washington, DC, Organización Panamericana de la Salud.). At each municipality, we randomly collected immatures from at least 25 different containers located in selected residences in the urban area. This included domestic breeding sites such as water storage vessels, plastic pails, tires, and cans. Each container was located at least 100 m away from the others. Larvae from the same municipality were pooled in the laboratory and stored until adults emerged under controlled conditions (25 ± 1°C, humidity 80 ± 10% and photoperiod 12:12 hours) in the medical entomology laboratory of the Center for the Study of Tropical Diseases (Centro de Investigaciones en Enfermedades Tropicales - CINTROP), Industrial University of Santander (UIS), Santander, Colombia. We collected recently-emerged adults from each population for the analysis of the Val1016Ile mutation and genetic diversity. The mosquitoes were individually placed in absolute ethanol (99.5%) and stored in a freezer at -20°C. The remaining F0 adults were used to produce the F1 generation. Aedes aegypti F1 larvae were used as the source in bioassays to determine Temephos susceptibility. Adults were fed a 10% honey solution and blood meals that were provided by rats, Rattus norvegicus (Berkenhout, 1769), twice a week to induce oviposition.
Bioassays were carried out with A. aegypti natural populations from Armenia, Montenegro and Calarcá. We were unable to run the bioassay with the population from Barcelona, since it was not possible to establish the colony base because there were few immatures on the field. Bioassays included F1 generation larvae and the insecticide Temephos pestanal 250 mg 97.5% (Sigma-Aldrich) following the World Health Organization (WHO) guidelines (Who 1998WHO (1998) Test procedures for insecticide resistance monitoring in malaria vectors, bioefficacy and persistence of insecticides on treated surfaces. Geneva, World Health Organization, WHO/CDS/CPC/MAL/98.12.). Bioassays were calibrated with Rockefeller, a susceptible strain of A. aegypti (Centers for Disease Control, CDC), using a diagnostic concentration of 0.0162 ppm Temephos. This is twice the LC99 (lethal concentration that kills 99% of the larvae) of the susceptible strain. The results from larvae were expressed as mortality rates 24h after exposure to Temephos. The following criteria proposed by the Who (1998WHO (1998) Test procedures for insecticide resistance monitoring in malaria vectors, bioefficacy and persistence of insecticides on treated surfaces. Geneva, World Health Organization, WHO/CDS/CPC/MAL/98.12.) guidelines were adopted to classify population susceptibility status: susceptible (percentage of mortality > 98%), susceptibility incipiently altered (80-98%), or resistant (< 80%).
Dose-response bioassays followed the WHO guidelines to determine larval susceptibility to Temephos (Who 1981WHO (1981) Instructions for determining the susceptibility resistance of mosquito larvae to insecticides. Geneva, World Health Organization, VBC, 81.806.). In these experiments, third instar or initial fourth instar larvae were exposed to 10 concentrations of the insecticide to determine larval mortality between 5 and 95%. At each concentration and in the control, four replicates containing 20 larvae each were tested. Larval mortality was checked 24 h after exposure. All tests were repeated three times on different days.
Mortality data (expressed as a number of dead specimens per dose) was applied to calculate lethal concentrations to 50 and 95% (LC50 and LC95) of exposed individuals, and were analyzed by the log-probit method of Finney (1971Finney D (1971) Probit Analysis. Cambridge, University Press, 3rd ed.) using the Probit software by Raymond (1993Raymond, M. 1995. PROBIT. France, software.). Resistance ratios (RR50 and RR95) were obtained by dividing the lethal concentration of the population by the equivalent lethal concentration of the Rockefeller population.
DNA extraction followed Bona et al. (2012Bona A, Piccoli C, Leandro A, Kafka R, Twerdochilib A, Navarro-Silva M
(2012) Genetic profile and molecular resistance of Aedes (Stegomyia) aegypti
(Diptera: Culicidae) in Foz do Iguaçu (Brazil), at the border with Argentina and
Paraguay. Zoologia 29(6): 540-548. doi:
10.1590/S1984-46702012000600005
https://doi.org/10.1590/S1984-4670201200...
).
Individual mosquitoes from each locality were genotyped at position1016 of the genomic
DNA using allele-specific PCR (AS-PCR). We used three primers to determine the presence
of the Val1016Ile mutation, one for the 1016Val allele: 5'-GCG GGC AGG GCG GCG GGG GCG
GGG CCA CAA ATTGTT TCC CAC CCG CAC CGG -3', one for the 1016Ile allele: 5'-GCG GGC ACA
AAT TGT TTC CCA CCC GCA CTG A -3', and a third common to both alleles: 5'-GGA TGA ACC
GAA ATT GGA CAA AAG C -3'. PCR reactions followed the protocol described by Saavedra-Rodriguez et al. (2007Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S, Moulton M,
Flores A, Fernandez-Salas I, Bisset J, Rodriguez M, McCall P, Donnelly M, Ranson H,
Hemingway J, Black W (2007) A mutation in the voltage-gated sodium channel gene
associated with pyrethroid resistance in Latin American Aedes aegypti. Insect
Molecular Biology 16(6): 785-798.) and Martins et al. (2009Martins A, Lima J, Peixoto A, Valle D (2009) Frequency of Val1016Ile
mutations in the voltage-gated sodium channel gene of Aedes aegypti Brazilian
populations. Tropical Medicine & International Health 14(11): 1351-1355. doi:
10.1111/j.1365-3156.2009.02378.x
https://doi.org/10.1111/j.1365-3156.2009...
). Amplified PCR products were
checked in 10% polyacrylamide gel. Using the gel results, we calculated genotypic and
allelic frequencies, and Hardy-Weinberg equilibrium (HW) (Salman 2007Salman A (2007) Conceitos básicos de genética de populações. Porto
Velho, Embrapa, 118p., Hartl 2008Hartl D (2008) Princípios de genética de populações. Ribeirão Preto,
FUNPEC, 3rd ed.). The
Rockefeller strain of A. aegypti, a standard for insecticide
susceptibility reared in the laboratory, and life-history trait parameters, were used as
reference for the wild-type alleles (1016Val) of the Nav gene.
After DNA extraction, we used two primers to amplify a segment of the ND4 gene, a
universal ND4R primer: 5'-ATT GCC TAA GGC TCA TGT AG-3' and a reverse NDAR primer:
5'-TCG GCT TCC TAG TCG TTC AT-3 (Costa-da-Silva et al.
2005Costa-da-Silva A, Capurro M, Bracco J (2005) Genetic lineages in the
yellow fever mosquito Aedes (Stegomyia) aegypti (Diptera: Culicidae) from Peru.
Memórias do Instituto Oswaldo Cruz 100(6): 539-544.). PCR reactions and sequencing followed Twerdochlib et al. (2012Twerdochlib A, Dalla A, Leite S, Chitolina R, Westphal B, Navarro-Silva
M (2012) Genetic variability of a population of Aedes aegypti from Paraná, Brazil,
using the mitochondrial ND4 gene. Revista Brasileira de Entomologia 56(2): 249-256.
doi: 10.1590/S0085-56262012005000030
https://doi.org/10.1590/S0085-5626201200...
).
Consensus sequences were obtained using the Staden software version 1.5, and were
aligned with the program BioEdit version 7.0 (Hall
2004Hall T (2004) Bioedit. Carlsbad, Ibis Therapeutics, v.
7.0.), using the ClustalW tool (Thompson et
al. 1994Thompson J, Higgins D, Gibson T (1994) CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22
(22): 4673-4680.). The sequences were compared with others available on GenBank using
Tblastx to verify the amplified fragment. Genetic diversity and neutrality tests were
calculated using the program DnaSP, version 5.0 (Librado
& Rozas 2009Librado P, Rozas J (2009) DnaSP v5: A software for comprehensive
analysis of DNA polymorphism data. Bioinformatics 25(11): 1451-1452. doi:
10.1093/bioinformatics/btp187
https://doi.org/10.1093/bioinformatics/b...
). Molecular variation analysis (AMOVA) was performed with the
program Arlequin version 3.5 (Excoffier & Lischer
2010Excoffier L, Lischer H (2010) Arlequin suite ver 3.5: A new series of
programs to perform population genetics analyses under Linux and Windows. Molecular
Ecology Resources 10(3): 564-567. doi:
10.1111/j.1755-0998.2010.02847.x
https://doi.org/10.1111/j.1755-0998.2010...
). Population structure was determined using the Wright fixation index
(FST, Wright 1921) and gene flow (Nm) was obtained by the program Arlequin
3.5 (Excoffier & Lischer 2010Excoffier L, Lischer H (2010) Arlequin suite ver 3.5: A new series of
programs to perform population genetics analyses under Linux and Windows. Molecular
Ecology Resources 10(3): 564-567. doi:
10.1111/j.1755-0998.2010.02847.x
https://doi.org/10.1111/j.1755-0998.2010...
) followed by
Bonferroni correction.
The Mantel test was used to estimate the correlation between genetic (FST)
and geographic (km) distances. The GenAlEx6 software was used to test isolation by
distance (Peakall & Smouse 2012Peakall R, Smouse P (2012) GenAlEx 6.5: genetic analysis in Excel.
Population genetic software for teaching and research-an update. Bioinformatics
28(19): 2537-2539. doi: 10.1093/bioinformatics/bts460
https://doi.org/10.1093/bioinformatics/b...
).
Geographical distances for this analysis were obtained using Google Earth 6.0. The
software Mega ver. 5.05 (Tamura et al. 2007Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary
Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24(8):
1596-1599. doi: 10.1093/molbev/msm092
https://doi.org/10.1093/molbev/msm092...
) was
used to create a tree with the Neighbor-Joining method, following the Jukes-Cantor
genetic distance model. Bootstrap support was estimated with 1,000 replicates.
Aedes (Stegomyia) albopictus
(Skuse, 1894) (GenBank#EF153761) was used as the external group.
The haplotypes of this study were deposited in the GenBank under accession KF241755 -
KF241757. In order to estimate gene flow among the populations analyzed, they were
compared with the haplotypes available from America, published by Gonçalves et al. (2012Gonçalves A, Cunha I, Santos W, Luz S, Ribolla P, Abad-Franch F (2012)
Gene flow networks among American Aedes aegypti populations. Evolutionary
Applications 5(7): 664-676. doi: 10.1111/j.1752-4571.2012.00244.x
https://doi.org/10.1111/j.1752-4571.2012...
); these haplotypes are free of nuclear
mitochondrial pseudogenes (NUMTs). Samples containing mixtures of mtDNA and NUMT
sequences are expected to significantly affect the outcome of genealogy- and
frequency-based analyses. This is because mtDNA and NUMTs have separate genealogies and
thus different evolutionary histories (Gonçalves et al.
2012Gonçalves A, Cunha I, Santos W, Luz S, Ribolla P, Abad-Franch F (2012)
Gene flow networks among American Aedes aegypti populations. Evolutionary
Applications 5(7): 664-676. doi: 10.1111/j.1752-4571.2012.00244.x
https://doi.org/10.1111/j.1752-4571.2012...
, Ribeiro 2012Ribeiro L (2012) Mitochondrial pseudogenes in insect DNA barcoding:
differing points of view on the same issue. Biota Neotropica 12(3): 301-308. doi:
10.1590/S1676-06032012000300029
https://doi.org/10.1590/S1676-0603201200...
). To reduce the
error caused by NUMTs in the samples two analyses were carried out: 1. We searched for
heterozygous sites in the chromatogram and additional termination codons (Ribeiro 2012Ribeiro L (2012) Mitochondrial pseudogenes in insect DNA barcoding:
differing points of view on the same issue. Biota Neotropica 12(3): 301-308. doi:
10.1590/S1676-06032012000300029
https://doi.org/10.1590/S1676-0603201200...
), and 2. We compared the haplotypes
with a list of NUMTs verified by Black & Bernhardt
(2009Black WC, Bernhardt SA (2009) Abundant nuclear copies of mitochondrial
origin (NUMTs) in the Aedes aegypti genome. Insect Molecular Biology 18(6): 705-713.
doi: 10.1111/j.1365-2583.2009.00925.x
https://doi.org/10.1111/j.1365-2583.2009...
) and Hlaing et al. (2009Hlaing T, Tun-Lin W, Somboon P, Socheat D, Setha T, Min S, Chang M,
Walton C (2009) Mitochondrial pseudogenes in the nuclear genome of Aedes aegypti
mosquitoes: implications for past and future population genetic studies. BMC Genetics
10: 11. doi: 10.1186/1471-2156-10-11
https://doi.org/10.1186/1471-2156-10-11...
). If any
NUMT was found, it was removed from the analysis.
RESULTS
We collected 1976 immatures (NI = Number of immatures) from 77 domestic breeding sites (DB = Domestic breeding sites) of four mosquito populations, Armenia (NI = 739, DB = 25), Calarcá (NI = 628, DB = 25), Montenegro (NI = 531, DB = 19) and Bacelona (NI = 78, DB = 8).
Diagnostic concentration (% mortality)
The results of bioassays with larvae following the WHO protocol showed that A. aegypti populations (Mortality rate ± SD) from Armenia (77% ± 2) and Calarcá (62% ± 14) are resistant to OP Temephos and that the Montenegro population has incipient, altered susceptibility to the insecticide (88% ± 8).
Multiple concentrations (RR)
Dose-response bioassays following the WHO protocol resulted in resistance ratios (RR95) greater than three, and were the highest in the A. aegypti populations from Calarcá. In general, the slope values of the A. aegypti populations studied were lower than those obtained from the Rockefeller strain, confirming their heterogeneity in comparison to the reference strain and the differences in their response to OP Temephos. The LC50 and LC95 of all population studied are presented for comparison in Table I.
Temephos susceptibility profile from Colombian populations of Aedes aegypti, showing means (standard deviations) for slopes, LC and RR.
Genotyping the 1016 fragment of NaV
A total of 107 A. aegypti individuals were genotyped for the Val1016Ile mutation. Of these individuals, 94% were homozygous dominant (Val/Val); 6% were heterozygous (Val/Ile), and homozygous recessive genotypes (Ile/Ile) were not found. In all populations, the genotypic frequencies of Val/Val were greater than the frequencies of Val/Ile and Ile/Ile (Table II). The frequencies of the allele 1016Val were greater than the frequencies of the 1016Ile allele in all populations (Fig. 1). The populations from Armenia, Calarcá and Montenegro are in Hardy-Weinberg equilibrium. Nevertheless, bioassays with adults should be carried out to confirm the susceptibility status in these populations.
Allelic frequencies of 1016Val and 1016Ile in the Nav of four A. aegypti populations from Colombia.
Genetic diversity - fragment of mitochondrial gene ND4
The amplified product of the ND4 gene was 311bp. There were four polymorphic sites and 307 monomorphic sites. The analysis of the amplified fragment of 42 individuals resulted in three haplotypes without NUMTs: H1-Col (GenBank# KF241755), H2-Col (GenBank# KF241756) and H3-Col (GenBank# KF241757). The most frequent of these haplotypes was H2-Col (48%), followed by H3-Col (28%) and H1-Col (24%). Only H3-Col occurred in Montenegro; H1, H2 and H3-Col occurred in Barcelona; and H1 and H2-Col occurred in Armenia and Calarcá (Table III). Haplotypes were determined by three transitions: G↔A (site 48); T↔C (sites 144, 249) and one transversion: A↔T (site 90) (Table IV). The average nucleotide composition was 20% Cytosine, 29% Thymine, 43% Adenine and 8% Guanine. Haplotypes found in this study were similar to the H2 haplotype, which is found in Mexico and North America (Fig. 2).
Number of individuals observed for each haplotype in samples of four populations of Aedes aegypti from Colombia.
(2) Haplotype network among mitochondrial ND4 haplotypes. The rectangle represents the ancestral haplotype. Each black dot indicates a single substitution. The size of the circle is not proportional to the haplotype frequency. The numbers inside the circle indicate the Haplotype number. H1-H3-Col present study; H1-H4, H7, H9, H18, H21-25, H27 (Haplotype present at Mexico and North America), H6 (Brazilian Amazon, Southeastern Brazil, Peru, Mexico and North America), H11, H17, H26, H28 (Brazilian Amazon), H10, H15 (Brazilian Amazon, Southeastern Brazil, Venezuela, Mexico and North America); H12, 14 (Brazilian Amazon, Venezuela), H13 (Brazilian Amazon, Southeastern Brazil), H16 (Brazilian Amazon, Southeast Brazil, Mexico and North America), H19 (Southeastern Brazil, Mexico and North America), H20 (Venezuela); H29, 30 (Southeastern Brazil). (3) Dendrogram for three A. aegypti haplotypes from Colombian populations using the Neighbor-Joining method, according to the Jukes-Cantor model. Bootstrap values are at the nodes of each branch.
Haplotype diversity was 0.67 ± 0.036 (mean ± SD, n = 42), nucleotide diversity was 0.0058 ± 0.0002 and there were 1.81 nucleotide differences on average. The neutral selectivity test results (p > 0.05) were in agreement with the assumptions of the neutral mutation model (p > 0.05, Tajima's D Test 2.24, Fu Fs Test 2.58).
Analysis of molecular variance (AMOVA, Fst = 0.58, p < 0.05) indicated genetic structure. Most of the variation occurred among populations (58%), while 42% occurred within populations. Significant Fst values after Bonferroni correction indicated that mosquito populations from Armenia-Montenegro, Barcelona-Montenegro, Barcelona-Calarcá and Calarcá-Montenegro are genetically structured, and that they are about 11 km apart from one another (Table V). Genetic distance (FST) and geographic distance (km) were correlated (Mantel, R2 = 0.77; p < 0.05) as expected. Two groups were resolved with Neighbor-Joining: Group I comprised of the most frequent haplotypes, H2-Col (48%) and H3-Col (28%), and group II of the least frequent haplotype, H1-Col (24%, Fig. 3).
DISCUSSION
We found that insecticide resistance is present in most studied populations of
A. aegypti and is determined by the 1016Ilekdr allele
(showing low frequency), which confers OP resistance. Even though resistance to Temephos
had been previously documented in Colombia (Grisales et
al. 2013Grisales N, Poupardin R, Gomez S, Fonseca-Gonzalez I, Ranson H, Lenhart
A (2013) Temephos resistance in Aedes aegypti in Colombia compromisos dengue vector
control. PLOS Neglected Tropical Diseases 7(9): 1-10. doi:
10.1371/journal.pntd.0002438
https://doi.org/10.1371/journal.pntd.000...
),our findings on Temephos resistance are important because it has
been the main insecticide used to control the immature stages of natural A.
aegypti populations in the country (Ocampo
et al. 2011Ocampo C, Salazar-Terreros M, Mina N, McAllister J, Brogdon W (2011)
Insecticide resistance status of Aedes aegypti in 10 localities in Colombia. Acta
Tropica 118(1): 37-44. doi: 10.1016/j.actatropica.2011.01.007
https://doi.org/10.1016/j.actatropica.20...
). Additionally, Temephos pressure on larvae may generate
cross-resistance to PY or other OP used in the control of the adult stages (Rodríguez et al. 2002Rodríguez M, Bisset J, Ruiz M, Soca A (2002) Cross-resistance to
pyrethroid and organophosphorus insecticides induced by selection with temephos in
Aedes aegypti (Diptera: Culicidae) from Cuba. Journal of Medical Entomology 39(6):
882-888. doi: 10.1603/0022-2585-39.6.882
https://doi.org/10.1603/0022-2585-39.6.8...
, Tikar et al. 2009Tikar S, Kumar A, Prasad G, Prakash S (2009) Temephos-induced resistance
in Aedes aegypti and its cross-resistance studies to certain insecticides from India.
Parasitology Research 105: 57-63. doi: 10.1007/s00436-009-1362-8
https://doi.org/10.1007/s00436-009-1362-...
).
The application of the OP Temephos on breeding sites is essential to control A.
aegypti immatures worldwide (Who
2013WHO (2013) Sustaining the drive to overcome the global impact of
neglected tropical diseases. Available online at:
http://apps.who.int/iris/bitstream/10665/77950/1/9789241564540_eng.pdf [Accessed: 2
April 2013]
http://apps.who.int/iris/bitstream/10665...
). However, long-term use of this insecticide has caused the emergence of
resistance in several Latin American countries (Rodríguez et al. 2007Rodríguez M, Bisset J, Fernández D (2007) Levels of insecticide
resistance and resistance mechanisms in Aedes aegypti (Diptera: Culicidae) from some
Latin American countries. Journal of the American Mosquito Control Association 23(4):
420-429. doi: 10.2987/5588.1
https://doi.org/10.2987/5588.1...
, Lima et al.
2011Lima E, Paiva M, Araújo A de, Silva E da, Silva U da, Oliveira L de,
Santana A, Barbosa C, Paiva C de, Goulart M, Wilding C, Ayres C, Melo M (2011)
Insecticide resistance in Aedes aegypti population from Ceará, Brazil. Parasites
& Vectors 4(5): 1-12. doi: 10.1186/1756-3305-4-5
https://doi.org/10.1186/1756-3305-4-5...
, Bisset et al. 2013Bisset J, MArin R, Rodríguez M, Severson D, Ricardo Y, French L, Días M,
Perez O (2013) Insecticide resistance in two Aedes aegypti (Diptera: Culicidae)
strains from Costa Rica. Journal of Medical Entomology 50(2):
352-361.). Colombia's
participation in the continental vector control campaign led by OPS (Santacoloma et al. 2010Santacoloma L, Chaves B, Brochero H (2010) Susceptibilidad de Aedes
aegypti a DDT, deltametrina y lambdacialotrina en Colombia. Revista Panamericana de
Salud Publica 27(1): 66-73.), and several dengue
outbreaks in 1970 and in 1980 in most Colombian regions (Boshell et al. 1986Boshell J, Groot H, Gacharná M, Márquez G, González M, Gaitán M, Berlie
C (1986) Dengue en Colombia. Biomédica 6(3-4): 101-106.) has been the cause of intensive insecticide use to
reduce dengue cases, selecting resistant vector populations.
Regarding the weak presence of the 1016Ilekdr allele associated with PY
resistance, the detection of the Val1016Ile mutation in A. aegypti
natural populations could have dire consequences for the continued use of PY, since
studies on selection pressure using PY insecticides under laboratory conditions have
documented fixation of the 1016Ilekdr allele after only five generations
(Irac 2011Irac (2011) Prevention and Management of Insecticide Resistance in
Vectors of Public Health Importance. Atlanta, Insecticide Resistance Action Committee
(IRAC), 70p., Saavedra-Rodriguez et al. 2012Saavedra-Rodriguez K, Salas I, Strode C, Ranson H, Hemingway J, Black W
(2012) Transcription of detoxification genes after permethrin selection in the
mosquito Aedes aegypti. Insect Molecular Biology 21(1): 61-77. doi:
10.1111/j.1365-2583.2011.01113.x
https://doi.org/10.1111/j.1365-2583.2011...
). In the last decade, the
1016Ilekdr allele has rapidly spread in A. aegypti
populations from Mexico and Brazil, simultaneously with the intensification of PY usage
due to the emergence of dengue outbreaks (García et al.
2009García G, Flores A, Fernández-Salas I, Saavedra-Rodríguez K, Reyes-Solis
G, Lozano-Fuentes S, Bond J, Casas-Martínez M, Ramsey J, García-Rejón J,
Domínguez-Galera M, Ranson H, Hemingway J, Eisen L, Black W (2009) Recent rapid rise
of a perrmethrin knock down resistance allele in Aedes aegypti in México. PLOS
Neglected Tropical Diseases 3(10): 531-541. doi:
10.1371/journal.pntd.0000531
https://doi.org/10.1371/journal.pntd.000...
, Linss et al. 2014Linss J, Brito L, Garcia G, Saori A, Bruno R, Lima J, Valle D, Martins A
(2014) Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations
in Aedes aegypti Brazilian natural populations. Parasites & Vectors 7(25): 1-12.
doi: 10.1186/1756-3305-7-25
https://doi.org/10.1186/1756-3305-7-25...
). Therefore,
enhanced surveillance for resistance should be a priority in localities where the
1016Ilekdr allele is found, before new adaptive alleles can be selected
for decreasing the deleterious effects of kdr (Brito et
al. 2013Brito L, Linss J, Lima-Camara T, Belinato T, Peixoto A, Lima J, Valle D,
Martins A (2013) Assessing the Effects of Aedes aegypti kdr Mutations on Pyrethroid
Resistance and Its Fitness Cost. PLOS One 8(4): e60878. doi:
10.1371/journal.pone.0060878
https://doi.org/10.1371/journal.pone.006...
). Consequently, bioassays with adults should be performed to confirm
the susceptibility status of these populations.
Our results suggest that alternative control strategies need to be found before Temephos
resistance compromises operational control. An alternative to reduce selection pressure
for resistance is to devise an insecticide swapping program implementing the criteria
proposed by the Brazilian Ministry of Health. According to these guidelines, Temephos
needs to be replaced with another insecticide with a different action mechanism in
populations with RR95 ≥ 3 (Ministério da
Saúde 2006Ministério da Saúde (2006) Reunião técnica para discutir status de
resistência de Aedes aegyptia inseticidas. Brasília, Ministério da Saúde, Secretaria
de Vigilância em Saúde, Coordenação Geral do Programa Nacional de Controle da
Dengue.). In studies conducted on populations of A.
aegypti from Brazil, it was observed that when the application of Temephos
is interrupted in locations where RR95 is greater than 10, resistance
declines only gradually, and several years are needed for Temephos to be effective again
(Montella et al. 2007Montella I, Martins A, Viana-Medeiros P, Lima J, Braga I, Valle D (2007)
Insecticide Resistance Mechanisms of Brazilian Aedes aegypti Populations from 2001 to
2004. American Journal of Tropical Medicine and Hygiene 77(3):
467-477.). On the other hand,
despite the fact that Colombia has its own vector control program, the susceptibility
status of the populations we evaluated should be considered as "populations with
susceptibility loss to OP Temephos"; hence continuous monitoring of susceptibility
status is recommended in order to determine the accurate moment to change the active
substance (Ministerio de la Protección Social
2011Ministerio de la Protección Social (2011) Guía de Vigilancia
Entomológica y Control de Dengue. Colombia, Ministerio de la Protección Social,
Instituto Nacional de Salud, Organización Panamericana de la Salud (OPS/OMS).
Available online at: http://new.paho.org/col/index.php?option = com_docman&task =
doc_download&gid = 1215&Itemid [Accessed: 8 April 2013]
http://new.paho.org/col/index.php?option...
). Nevertheless, the RR95 values in all populations were
greater than three.
Chemical measures used in vector control programs could affect the genetic diversity of
A. aegypti populations, and as a result, induce genetic changes
through bottleneck and genetic drift effects (Urdaneta-Marquez & Anna-Bella 2011Urdaneta-Marquez L, Anna-Bella F (2011) Population genetic structure of
Aedes aegypti, the principal vector of dengue viruses. Infection, Genetics and
Evolution 11(2): 253-261. doi: 10.1016/j.meegid.2010.11.020
https://doi.org/10.1016/j.meegid.2010.11...
). Low genetic diversity is most likely
a result of a decline in population size caused by insecticide use, as it was observed
in vector populations from Trinidad and Tobago, and Venezuela (Yan et al. 1998Yan G, Chadee D, Severson D (1998) Evidence for genetic hitchhiking
effect associated with insecticide resistance in Aedes aegypti. Genetics 148(2):
793-800., Herrera et al.
2006Herrera F, Urdaneta L, Rivero J, Zoghbi N, Ruiz J, Carrasquel G,
Martínez J, Pernalete M, Villegas P, Montoya A, Rubio-Palis Y, Rojas E (2006)
Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela.
Memórias Instituto Oswaldo Cruz 101(6): 625-633.). However, some studies have revealed the presence of greater genetic
diversity in areas that are frequently treated with insecticides, as shown for
A. aegypti populations from French Polynesia and Brazil (Paupy et al. 2000Paupy C, Vazeille-Falcoz M, Mousson L, Rodhain F, Failloux A (2000)
Aedes aegypti in Tahiti and Moorea (FrenchPolynesia): isoenzyme differentiation in
the mosquito population according to human population density. American Journal of
Tropical Medicine and Hygiene 62(2): 217-224., Ayres et al. 2004Ayres C, Melo-Santos M, Prota J, Solé-Cava A, Furtado A (2004) Genetic
structure of natural populations of Aedes aegypti at the micro- and macro geographic
levels in Brazil. Journal of the American Mosquito Control Association 20(4):
350-356.). In our results, genetic diversity (Hd), number of
haplotypes (N) and nucleotide diversity (π) were lower (N = 3, Hd = 0.67 and π = 0.006)
than in other studies on the ND4 gene of A. aegypti. For example, 36
locations in the Americas, Asia and Africa (N = 20, Hd = 0.82 and π = 0.020) (Bracco et al. 2007Bracco J, Capurro M, Lourenço-de-Oliveira R, Sallum M (2007) Genetic
variability of Aedes aegypti in the Americas using a mitochondrial gene: evidence of
multiple introductions. Memórias Instituto Oswaldo Cruz 102(5):
573-580.), five states in Brazil (N = 24,
Hd = 0.80 and π = 0.017) (Paduan & Ribolla
2008Paduan K, Ribolla P (2008) Mitochondrial DNA Polymorphism and
Heteroplasmy in Populations of Aedes aegypti in Brazil. Journal of Medical Entomology
45(1): 59-67. doi: 10.1603/0022-2585(2008)45[59:MDPAHI]2.0.CO;2
https://doi.org/10.1603/0022-2585(2008)4...
) and two populations from Colombia (N = 10, Hd = 0.068 and π = 0.009)
(Caldera et al. 2013Caldera S, Jaramillo S, Cochero S, Pérez-Doria A, Bejarano E (2013)
Diferencias genéticas entre poblaciones de Aedes aegypti de municipios del norte de
Colombia, con baja y alta incidencia de dengue. Biomedica 33(1): 89-98. doi:
10.7705/biomedica.v33i0.1573).
The studied Colombian A. aegypti populations were genetically
structured, a trend also found in other populations from South America and Central
America (Gorrochotegui-Escalante et al. 2002Gorrochotegui-Escalante N, Gomez-Machorro C, Lozano-Fuentes S,
Fernandez-Salas I, Muñoz M, Farfan-Alej J, Garcia-Rejon J, Beaty B, Black W (2002)
Breeding structure of Aedes aegypti populations in Mexico varies by region. American
Journal of Tropical Medicine and Hygiene 66(2): 213-222.,
Costa-da-Silva et al. 2005Costa-da-Silva A, Capurro M, Bracco J (2005) Genetic lineages in the
yellow fever mosquito Aedes (Stegomyia) aegypti (Diptera: Culicidae) from Peru.
Memórias do Instituto Oswaldo Cruz 100(6): 539-544., Urdaneta-Marquez et al. 2008Urdaneta-Marquez L, Bosio C, Herrera F, Rubio-Palis Y, Salasek M, Black
W (2008) Genetic relationships among Aedes aegypti collections in Venezuela as
determined by mitochondrial DNA variation and nuclear single nucleotide
polymorphisms. American Journal of Tropical Medicine and Hygiene 78(3):
479-491., Paupy et al. 2012Paupy C, Goff G Le, Brengues C, Guerra M, Revollo J, Barja Z,
Jean-Pierre H, Fontenille D (2012) Genetic structure and phylogeo graphy of Aedes
aegypti, the dengue and yellow-fever mosquito vector in Bolivia. Infection, Genetics
and Evolution 12(6): 1260-1269. doi: 10.1016/j.meegid.2012.04.012
https://doi.org/10.1016/j.meegid.2012.04...
, Twerdochlib et
al. 2012Twerdochlib A, Dalla A, Leite S, Chitolina R, Westphal B, Navarro-Silva
M (2012) Genetic variability of a population of Aedes aegypti from Paraná, Brazil,
using the mitochondrial ND4 gene. Revista Brasileira de Entomologia 56(2): 249-256.
doi: 10.1590/S0085-56262012005000030
https://doi.org/10.1590/S0085-5626201200...
, Caldera et al. 2013Caldera S, Jaramillo S, Cochero S, Pérez-Doria A, Bejarano E (2013)
Diferencias genéticas entre poblaciones de Aedes aegypti de municipios del norte de
Colombia, con baja y alta incidencia de dengue. Biomedica 33(1): 89-98. doi:
10.7705/biomedica.v33i0.1573). The
genetic structure of the studied Colombian populations occurred between regions
separated by less than 17 km, similar to the patterns observed in Venezuela (distance
less than 15 km between them), which suggest that gene flow is restricted (Herrera et al. 2006Herrera F, Urdaneta L, Rivero J, Zoghbi N, Ruiz J, Carrasquel G,
Martínez J, Pernalete M, Villegas P, Montoya A, Rubio-Palis Y, Rojas E (2006)
Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela.
Memórias Instituto Oswaldo Cruz 101(6): 625-633.). Geographic barriers associated
with Andean mountains in the state of Quindío probably obstruct gene flow. However, in
Quindio state, where tourism is intense, there is more road traffic between the studied
populations (Gobernación del Quindío 2013Gobernacion Del Quindío (2013) Página oficial del departamento del
Quindío. Available on line at: http://www.quindio.gov.co [Accessed: 8 April
2013]
http://www.quindio.gov.co...
, Invias 2013Invias (2013) Resumen del estado de la red vial con criterio técnico
2013. Available online at:
www.invias.gov.co/index.php/hechos-de-transparencia/informacion-financiera-y-contable/doc_download/916-resumen-del-estado-de-la-red-vial-con-criterio-tecnico-2013
[Accessed: 8 April 2013]
www.invias.gov.co/index.php/hechos-de-tr...
), which ties the gene flow in
populations of A. aegypti to human transport (Huber et al. 2004Huber K, Loan L, Chantha N, Failloux A (2004) Human transportation
influences Aedes aegypti gene flow in Southeast Asia. Acta Tropica 90(1): 23-29. doi:
10.1016/j.actatropica.2003.09.012
https://doi.org/10.1016/j.actatropica.20...
, Costa-da-Silva et
al. 2005Costa-da-Silva A, Capurro M, Bracco J (2005) Genetic lineages in the
yellow fever mosquito Aedes (Stegomyia) aegypti (Diptera: Culicidae) from Peru.
Memórias do Instituto Oswaldo Cruz 100(6): 539-544.).
Overall, two mitochondrial lineages were present in the studied Colombian populations,
and the most frequent haplotypes came from group I. This pattern is in agreement with
previous studies (Gorrochotegui-Escalante 2002Gorrochotegui-Escalante N, Gomez-Machorro C, Lozano-Fuentes S,
Fernandez-Salas I, Muñoz M, Farfan-Alej J, Garcia-Rejon J, Beaty B, Black W (2002)
Breeding structure of Aedes aegypti populations in Mexico varies by region. American
Journal of Tropical Medicine and Hygiene 66(2): 213-222.,
Bosio et al. 2005Bosio C, Harrington L, Jones J, Sithiprasasna R, Norris D, Scott T
(2005) Genetic structure of Aedes aegypti populations in Thailand using mitochondrial
DNA. American Journal of Tropical Medicine and Hygiene 72(4):
434-442., Herrera et al. 2006Herrera F, Urdaneta L, Rivero J, Zoghbi N, Ruiz J, Carrasquel G,
Martínez J, Pernalete M, Villegas P, Montoya A, Rubio-Palis Y, Rojas E (2006)
Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela.
Memórias Instituto Oswaldo Cruz 101(6): 625-633., Bracco et al.
2007Bracco J, Capurro M, Lourenço-de-Oliveira R, Sallum M (2007) Genetic
variability of Aedes aegypti in the Americas using a mitochondrial gene: evidence of
multiple introductions. Memórias Instituto Oswaldo Cruz 102(5):
573-580., Paduan & Ribola 2008Paduan K, Ribolla P (2008) Mitochondrial DNA Polymorphism and
Heteroplasmy in Populations of Aedes aegypti in Brazil. Journal of Medical Entomology
45(1): 59-67. doi: 10.1603/0022-2585(2008)45[59:MDPAHI]2.0.CO;2
https://doi.org/10.1603/0022-2585(2008)4...
). The
haplotypes found in this study indicate a relationship between the Mexican and North
American populations. These connections result from passive dispersal of A.
aegypti among different countries, and passive vector dispersal is likely to
be the most common pattern world-wide (Gorrochotegui-Escalante et al. 2002Gorrochotegui-Escalante N, Gomez-Machorro C, Lozano-Fuentes S,
Fernandez-Salas I, Muñoz M, Farfan-Alej J, Garcia-Rejon J, Beaty B, Black W (2002)
Breeding structure of Aedes aegypti populations in Mexico varies by region. American
Journal of Tropical Medicine and Hygiene 66(2): 213-222., Huber et
al. 2004Huber K, Loan L, Chantha N, Failloux A (2004) Human transportation
influences Aedes aegypti gene flow in Southeast Asia. Acta Tropica 90(1): 23-29. doi:
10.1016/j.actatropica.2003.09.012
https://doi.org/10.1016/j.actatropica.20...
, Bosio et al. 2005Bosio C, Harrington L, Jones J, Sithiprasasna R, Norris D, Scott T
(2005) Genetic structure of Aedes aegypti populations in Thailand using mitochondrial
DNA. American Journal of Tropical Medicine and Hygiene 72(4):
434-442., Gonçalves et al. 2012Gonçalves A, Cunha I, Santos W, Luz S, Ribolla P, Abad-Franch F (2012)
Gene flow networks among American Aedes aegypti populations. Evolutionary
Applications 5(7): 664-676. doi: 10.1111/j.1752-4571.2012.00244.x
https://doi.org/10.1111/j.1752-4571.2012...
). Further studies are
necessary to ascertain how the vector entered Colombia, since the connection among the
populations in this study is clear. We conclude that continuous monitoring and managing
programs are needed to control A. aegypti populations in Colombia.
Given that insecticide resistance could potentially compromise vector control programs,
a threshold of RR95 ≥ 3.0 should be established for swapping among
insecticides with different modes of action.
ACKNOWLEDGEMENT
We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process 140224/2013-0).
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Publication Dates
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Publication in this collection
Jan-Feb 2015
History
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Received
15 July 2014 -
Reviewed
24 Oct 2014 -
Accepted
23 Dec 2014