ABSTRACT

Objective.

To determine the feeding behavior of Aedes aegypti in dengue outbreaks in two rural areas of Peru during the Yaku cyclone and El Niño phenomenon of 2023.

Material and methods.

Eight blood samples (8 pools) were obtained from the abdomen of 80 Aedes aegypti specimens captured in the rural districts of Querecotillo and Marcavelica during the Yaku cyclone and El Niño dengue outbreaks. DNA was extracted from the analyzed samples, then a PCR was directed at the CytB gene as a genetic marker and the PCR products were enzymatically digested with the restrictases Hae III and Mwo I. The PCR-RFLP products were visualized by agarose gel electrophoresis at 4%.

Results.

DNA was obtained from all samples and a 358 bp amplicon was obtained as a PCR product. Likewise, the only RFLP found in Hae III was from Homo sapiens sapiens (233 and 125 bp). RFLP was not found in Hae III of Gallus gallus and RFLP in Mwo I of Canis familiaris and Mus musculus.

Conclusion.

Aedes aegypti showed conserved anthropophilic feeding behavior in dengue outbreaks in rural areas during the Yaku cyclone and El Niño.

Keywords:
Aedes aegypti; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; DNA

KEY MESSAGES

Motivation for the study. Dengue epidemics caused by A. aegypti occur during climatic events in tropical countries such as Peru; however, the feeding behavior of the mosquito usually goes unnoticed.

Main findings. A. aegypti populations in Marcavelica and Querecotillo showed anthropophilic feeding behavior during cyclone Yaku and in the 2023 El Niño. However, populations with different feeding patterns are not ruled out.

Implications. The PCR-RFLP technique of the blood cell cytochrome B gene could be implemented in vector control policies through an entomo-virological surveillance plan.

Keywords:
Aedes aegypti; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; DNA

INTRODUCTION

A. aegypti usually feeds on humans (anthropophilia), a behavior demonstrated by studies on mosquitoes raised under laboratory conditions and mosquitoes captured in the wild ¹1. McBride CS, Baier F, Omondi AB, Spitzer SA, Lutomiah J, Sang R, et al. Evolución de la preferencia de los mosquitos por los humanos vinculada a un receptor de olores. Nature. 2014;515:222-227. doi: 10.1038/naturaleza13964.
https://doi.org/10.1038/naturaleza13964... ^,22. Ponlawat A, Harrington LC. Blood feeding patterns of Aedes aegypti and Aedes albopictus in Thailand. J Med Entomol. 2005;42(5):844-849. doi: 10.1093/jmedent/42.5.844.
https://doi.org/10.1093/jmedent/42.5.844... . Only female A. aegypti mosquitoes can become infected with pathogens and transmit them to the host since they require blood to lay their eggs ³3. Ramírez AL. Patrones de alimentación de zancudos Culex (Melanoconion taeniopus Dyar y Knab (Diptera: Culicidae) colectados de 2007 a 2008 en Puerto Barrios, Izabal. [tesis de licenciatura]. Guatemala: Facultad de Ciencias y Humanidades, Universidad del Valle de Guatemala; 2010. Disponible en: https://repositorio.uvg.edu.gt/handle/123456789/1676.
https://repositorio.uvg.edu.gt/handle/12... .

Having an anthropophilic behavior, A. aegypti does not depend on other vertebrates as food sources, therefore it is very predominant in urban areas since it does not need to feed on glycosidic compounds of plants ⁴4. Marques CA, Marques RA, Degallier N. Is Aedes albopictus only a pest mosquito or also a vector of arboviruses in Brazil? [Internet]. Montpellier: Health Department; 1986 [consultado el 19 de marzo del 2024]. pág. 248-260. Disponible en: https://horizon.documentation.ird.fr/exl-doc/pleins_textes/pleins_textes_6/b_fdi_49-50/010018366.pdf.
https://horizon.documentation.ird.fr/exl... ^,55. Harrington LC, Edman JD, Scott TW. ¿Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood?. J Med Entomol. 2001;38(3): 411-422. doi: 10.1603/0022-2585-38.3.411.
https://doi.org/10.1603/0022-2585-38.3.4... . Because of this, it is the most efficient mosquito species for transmitting dengue virus ⁶6. Lambrechts L, Scott TW, Gubler DJ. Consequences of the expanding global distribution of Aedes albopictus for dengue virus transmission. PLoS Negl Trop Dis. 2010;4(5):e646. doi: 10.1371/journal.pntd.0000646.
https://doi.org/10.1371/journal.pntd.000... .

On the other hand, there are also other species that feed on a wide range of vertebrates ranging from mammals to amphibians ⁷7. Wolff GH, Riffell JA. Olfaction, experience and neural mechanisms underlying mosquito host preference. J Exp Biol. 2018; 221(4):jeb157131. doi: 10.1242/jeb.157131.
https://doi.org/10.1242/jeb.157131... . However, recent studies indicate that populations of A. aegypti are adopting this behavior in rural areas, since specimens have been found feeding on humans, mongooses, birds, cattle, pigs, cats, rats and chickens ⁸8. Fitzpatrick D, Hattaway L, Hsueh A, Ramos-Niño M, Cheetham S. PCR Based Blood meal Analysis of Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) in St. George Parish, Grenada. J Med Entomol. 2019;56(4):1170-1175. doi: 10.1093/jme/tjz037.
https://doi.org/10.1093/jme/tjz037... .

It has long been demonstrated that A. aegypti has the capacity to colonize rural areas that act as breeding sites due to the large accumulation of containers, tires and other unusable items that provide ideal conditions for the development of the insect ⁹9. Chiaravalloti Neto FA. Description of Aedes aegypti colonization in the region of São José do Rio Preto, São Paulo. Rev Soc Bras Med Trop. 1997;30(4):279-85. doi: 10.1590/S0037-86821997000400002.
https://doi.org/10.1590/S0037-8682199700... , complicating the formulation and adaptation of new strategies for the control of dengue, given that the dispersion and behavior of the vector has been studied very little in these areas ¹⁰10. Cabezas L, Cabanzo W, Santa F, Olano VA, Sarmiento D, Vargas S, et al. Distribución espacial de Aedes aegypti (Diptera: Culicidae) en el área rural de dos municipios de Cundinamarca, Colombia. Biomédica. 2017;37(2):41-49. doi: 10.7705/biomedica.v34i2.3469.
https://doi.org/10.7705/biomedica.v34i2.... , even more so during epidemic outbreaks of dengue framed in climatic phenomena in which there is a relatively high abundance of the mosquito population.

In this context, there are several extrinsic and intrinsic factors in the transmission of arboviruses to humans. Host location, temperature, humidity and food preference are extrinsic factors; and the circadian cycle, immune and tissue barriers that prevent virus replication and dissemination are the intrinsic factors ¹¹11. Foster WA. Mosquito sugar feeding and reproductive energetics. Annu Rev Entomol. 1995; 40:443-74.^,1212. Franco Quiterio I. Competencia vectorial para transmitir virus dengue y virus Zika en los mosquitos Aedes aegypti colectados en Puente de Ixtla, Morelos. [tesis de licenciatura]. Morelos: Facultad de Ciencias Biológicas Universidad Autónoma del Estado de Morelos; 2021. Disponible en: http://riaa.uaem.mx/handle/20.500.12055/3279.
http://riaa.uaem.mx/handle/20.500.12055/... .

It should be noted that studying the feeding behavior of A. aegypti is not only relevant because of the potential new reservoirs of arboviruses that could be found, but also because of significant physiological changes that could be acquired, such as the increase in the number of eggs per oviposition that has been demonstrated in specimens fed with R. rattus (rat) and O. cuniculus (rabbit), in comparison with H. sapiens sapiens (human) ¹³13. González Fernández MI, Vázquez Anzúrez MJ, Sesma Medrano E, Falcón Lezama JA, González Acosta C, Correa Morales F, et al. Efecto de diferentes fuentes de alimentación sanguínea sobre Aedes aegypti (Díptera: Culicidae) en condiciones de insectario. Horiz sanitario. 2019;18(2):177-183. doi: 10.19136/hs.a18n2.2717.
https://doi.org/10.19136/hs.a18n2.2717... . The implementation of molecular methods to understand the feeding behavior of mosquitoes of public health interest is essential for the development of effective vector control strategies. This is the only way to understand the ecological dynamics of arbovirus transmission ¹⁴14. Chena L, Nara E, Sánchez Z, Espínola E, Russomando G. Estandarización de la técnica PCR-RFLP del gen mitocondrial cytb como herramienta para la identificación de fuentes de alimentación de insectos hematófagos. Mem Inst Investig Cienc Salud. 2014;12(2):33-42..

To date, there are no studies on the feeding behavior of A. aegypti during dengue epidemics in rural Peru during climatic events. Therefore, we aimed to determine the feeding behavior of A. aegypti during dengue outbreaks in two rural areas of Peru during the Yaku cyclone and in the El Niño phenomenon of 2023.

MATERIALS AND METHODS

Study design

This is a descriptive in vitro study conducted at the Molecular Biotechnology Laboratory of the Center for Research and Training in Entomology (CICE) of the Luciano Castillo Colonna Subregional Health Directorate, located in the district of Querecotillo, province of Piura, department of Piura, Peru. The study aimed to determine the feeding behavior of A. aegypti from blood samples collected from the abdomen of 80 specimens captured during the Yaku cyclone ¹⁵15. El Peruano. Niño Global comenzó en el mundo, pero aún no en Perú, ¿cuándo se sentirán sus efectos? [Internet]. Lima: El Peruano; 2023 [consultado el 22 de marzo del 2024]. Disponible en: https://www.elperuano.pe/noticia/216933-nino-global-comenzo-en-el-mundo-pero-aun-no-en-peru-cuando-se-sentiran-sus-efectos#:~:text=02%2F07%2F2023%20El%20viernes,se%20dejar%C3%A1n%20sentir%20sus%20efectos%3F. and the 2023 El Niño ¹⁶16. El Comercio. Ciclón Yaku 2023 en el Perú y Ecuador: cómo se origina y por qué afecta estos países [Internet]. Lima: El Comercio; 2023 [consultado el 22 de marzo del 2024]. Disponible en: https://elcomercio.pe/respuestas/por-que/ciclon-yaku-2023-en-el-peru-y-ecuador-como-se-origina-y-por-que-afecta-estos-paises-via-senhami-tdex-noticia/.
https://elcomercio.pe/respuestas/por-que... , inside homes in the rural districts of Querecotillo (4°50′24″S/80°38′57″W) and Marcavelica (4°52′54″S/80°42′12″W) located in Sullana, Piura, Peru (Figure 1).

Analyzed samples

The analyzed samples correspond to eight blood pools from the collections of the molecular biotechnology laboratory of CICE. Of the total number of samples, four belong to the district of Querecotillo (M1, M2, M3 and M4); and four to the district of Marcavelica (M5, M6, M7, M8). In Querecotillo, entomological captures were carried out on March 18; 19; 20; 21; 22 and 23, 2023; and in Marcavelica on June 2; 3; 4; 6 and 8, 2023.

Entomological captures, specimen handling and blood collection

Mosquitoes and blood samples were collected by qualified personnel under strict entomological and conservation protocols for biotechnological applications. Initially, the personnel entered homes in Querecotillo and Marcavelica, then resting A. aegypti specimens were captured using entomological aspirators according to World Health Organization methodology ¹⁷17. Organización Mundial de la Salud. Manual on practical entomology in Malaria. Part.II. [Internet]. Geneva: OMS; 1975 [consultado el 10 de agosto de 2018]. Disponible en: https://iris.who.int/handle/10665/42481.
https://iris.who.int/handle/10665/42481... . Immediately afterwards, the specimens were transferred to the bioassay area of CICE, where they were exposed to ethyl acetate impregnated with cotton for eight minutes, and taxonomically identified according to the Pan American Health Organization ¹⁸18. Organización Panamericana de la Salud. Aedes aegypti: Biología y ecología [Internet]. Washington: OPS; 1986 [consultado el 25 de marzo del 2024]. Disponible en: https://iris.paho.org/handle/10665.2/28513..

Specimen handling and blood collection was performed by selecting female mosquitoes that showed visible blood on the abdomen and discarding those that did not. The selected females were placed on sterile slides with 100 uL of DNA/RNA Shield Zymo Biomics preservative solution (R1100-250). Then, following an internal laboratory protocol, pressure was exerted with sterile toothpicks on the mosquito abdomen, the blood was expelled, mixed with the preservative solution, then the mixture was aspirated, and transferred to sterile microtubes with 200 uL of the same solution. Finally, the microtubes with the blood samples were stored at -20 ºC in the molecular biotechnology laboratory of the CICE.

DNA extraction

DNA was extracted with the commercial Zymo BIOMICS DNA Miniprep Kit (D4300), the cell lysis step was modified with silica microbeads by a maceration with sterile plastic pistils, and 10,000 RPM centrifugation. Finally, we followed the manufacturer’s instructions.

PCR of the CytB gene

PCR targeting the CytB gene was performed following the protocol proposed by Chena et al. ¹⁴14. Chena L, Nara E, Sánchez Z, Espínola E, Russomando G. Estandarización de la técnica PCR-RFLP del gen mitocondrial cytb como herramienta para la identificación de fuentes de alimentación de insectos hematófagos. Mem Inst Investig Cienc Salud. 2014;12(2):33-42., using the PCR kit GoTaq™ G2 Flexi DNA Polymerase (Promega M7801), and the primers proposed by Oshagi et al. ¹⁹19. Oshaghi MA, Chavshin AR, Vatandoost H. Analysis of mosquito bloodmeals using RFLP markers. Exp Parasitol. 2006;114(4):259-264. doi: 10.1016/j.exppara.2006.04.001.
https://doi.org/10.1016/j.exppara.2006.0... (Cytb 1: 5-CCCCTCAGAATGATATATTTGTCCTCA-3 and Cytb 2: 5́-CCATCATCCAACATCTCACTCAGCATGATGAAA-3). The reaction had a final volume of 50 µL, containing 22.5µL of nuclease-free water, 10 µL of buffer (1X), 3 µL of MgCL (1.5 mM), 1 µL of dNTPs (200 µM), 2.5 µL of Forward cyt b1 (10 uM), 2.5 µL of Reverse cyt b2 (10 uM), 0.5 µL of Gotaq Polymerase enzyme (1 U/reaction) and 8 µL of DNA. Thermal conditions and cycling consisted of an initial denaturation of 95 °C for 5 minutes, followed by 35 cycles with 95 °C for 30 seconds for denaturation, 58 °C for 30 seconds for hybridization, 72 °C for 1 minute for extension, a post-extension of 72 °C for 5 minutes and a storage temperature of 4 °C for up to 24 hours.

Agarose gel electrophoresis

A 4% agarose gel electrophoresis was performed to validate the amplification of the CytB gene by PCR. Then, 3.6 grams of agarose was dissolved in 90 mL of 1X TAE buffer (Tris-Acetate-EDTA) and 4.5 uL of ethidium bromide (Thermo Scientific). A mixture of 4 uL of loading dye (6X DNA loading dye) and 10 uL of sample (PCR product) was loaded into each well of the gel. The electrical conditions for electrophoresis were 80 volts and 200 amps for 30 minutes.

Enzymatic digestion of the CytB gene

Enzymatic digestion of PCR products was performed with Hae III and Mwo I restrictionase enzymes recognizing polymorphic length restriction fragments (RFLP) of the CytB gene in Hae III from H. sapiens sapiens and G. gallus; and RFLP in Mwo I from M. musculus and C. familiaris (Table 1).

For the enzymatic reactions, we chose to follow the protocol described by Chena et al. ¹⁴14. Chena L, Nara E, Sánchez Z, Espínola E, Russomando G. Estandarización de la técnica PCR-RFLP del gen mitocondrial cytb como herramienta para la identificación de fuentes de alimentación de insectos hematófagos. Mem Inst Investig Cienc Salud. 2014;12(2):33-42., adapting the enzyme concentration prescribed in the Hae III and Mwo I enzyme kits from New England Biolabs. We mixed 45 µL of PCR product, 20 µL of Buffer (1X) and 20 µL of restrictase enzyme (10 U/reaction) in the reaction of each enzymeThermal conditions for Hae III were 37 °C for 15 min for enzyme activation and 80 °C for 20 min for inactivation. Thermal conditions for Mwo I were 60 ºC for 15 minutes for enzyme activity. Finally, 35 uL volumes of the enzymatic digestion product were visualized by 4% agarose gel electrophoresis.

RESULTS

Figure 2A shows the DNA (fluorescent band) extracted from the blood cells of all the analyzed samples. Figure 2B shows the amplicons of the DNA extracted from all samples (358 bp). In Figure 2C, RFLP can be found in Hae III with molecular weights 233 and 125 bp corresponding to H. sapiens sapiens. No RFLP was observed in Hae III from G. gallus and no RFLP was observed in MwoI from M. musculus and C. familiaris.

DISCUSSION

The feeding behavior of A. aegypti in dengue outbreaks during climatic phenomena such as the Yaku cyclone and El Niño has not been researched, in spite of its importance influencing the development of the vector and the transmission of the virus. It is important to highlight that the Yaku cyclone emerged and dissipated during the month of March 2023 ¹⁶16. El Comercio. Ciclón Yaku 2023 en el Perú y Ecuador: cómo se origina y por qué afecta estos países [Internet]. Lima: El Comercio; 2023 [consultado el 22 de marzo del 2024]. Disponible en: https://elcomercio.pe/respuestas/por-que/ciclon-yaku-2023-en-el-peru-y-ecuador-como-se-origina-y-por-que-afecta-estos-paises-via-senhami-tdex-noticia/.
https://elcomercio.pe/respuestas/por-que... , followed by El Niño ¹⁵15. El Peruano. Niño Global comenzó en el mundo, pero aún no en Perú, ¿cuándo se sentirán sus efectos? [Internet]. Lima: El Peruano; 2023 [consultado el 22 de marzo del 2024]. Disponible en: https://www.elperuano.pe/noticia/216933-nino-global-comenzo-en-el-mundo-pero-aun-no-en-peru-cuando-se-sentiran-sus-efectos#:~:text=02%2F07%2F2023%20El%20viernes,se%20dejar%C3%A1n%20sentir%20sus%20efectos%3F. in June of the same year. Likewise, during these scenarios, dengue epidemics occurred in March ²⁰20. Ministerio de Salud: Dirección Subregional de Salud Luciano Castillo Colonna [Internet]. Sala situacional diaria de dengue. Piura: MINSA; 2023 [consultado el 26 de marzo del 2024]. Disponible en: https://cdn.www.gob.pe/uploads/document/file/6133598/5418673-sala-dengue-02-de-abril-2024.pdf.
https://cdn.www.gob.pe/uploads/document/... ⁾ and June ²¹21. Ministerio de Salud: Dirección Regional de Salud de Piura [Internet]. Sala situacional de salud. Piura: MINSA; 2023 [consultado el 26 de marzo del 2024]. Disponible en: https://cdn.www.gob.pe/uploads/document/file/4665981/Sala%20situacional%20de%20Dengue%20SE%20023-2023.pdf?v=1686318198.
https://cdn.www.gob.pe/uploads/document/... . Therefore, since there was a difference of two months between March and June (April and May), the impact of the dengue epidemics could have been mitigated, given that the approximate life span of A. aegypti is 4 to 6 weeks ²²22. Organización Panamericana de la Salud [Internet]. OPS; 2024 [consultado el 10 de mayo del 2024]. Disponible en: https://www.paho.org/es/temas/zika.
https://www.paho.org/es/temas/zika... . In addition, its eggs are highly resistant to desiccation and low temperatures for up to one year ²³23. Howard O, Dyar HG, Knab F. The Mosquitoes of North and Central America and the West Indies. Carnegie Institute: Washington; 1912..

Total DNA extraction was achieved in all analyzed samples. mDNA was extracted from the white cells of all blood samples. White cells are nucleated and have organelles such as mitochondria ²⁴24. Riera MA, Rojas ME, Zapata PD. Protocolo de extracción de DNA por salting-out para pequeños volúmenes de sangre. Rev Cien Tecnol. 2010;(14):4-7.. White blood cells are targets for molecular biotechnology applications in the medical field ²⁵25. López-Hurtado M, Cuevas-Recillas KN, Flores-Salazar VR, Guerra-Infante FM. ADN de Chlamydia trachomatis en leucocitos de sangre periférica de neonatos ADN de Chlamydia trachomaatis en leucocitos de sangre periférica de neonatos. Enferm Infecc Microbiol Clin. 2015;33(7):458-463. doi: 10.1016/j.eimc.2014.09.019.
https://doi.org/10.1016/j.eimc.2014.09.0... . The PCR products that we obtained were 358 bp amplicons, which is in agreement with the reports by Oshagi et al. ¹⁹19. Oshaghi MA, Chavshin AR, Vatandoost H. Analysis of mosquito bloodmeals using RFLP markers. Exp Parasitol. 2006;114(4):259-264. doi: 10.1016/j.exppara.2006.04.001.
https://doi.org/10.1016/j.exppara.2006.0... and Chena et al. ¹⁴14. Chena L, Nara E, Sánchez Z, Espínola E, Russomando G. Estandarización de la técnica PCR-RFLP del gen mitocondrial cytb como herramienta para la identificación de fuentes de alimentación de insectos hematófagos. Mem Inst Investig Cienc Salud. 2014;12(2):33-42..

Regarding food sources, RFLPs were found in Hae III from H. sapiens sapiens (Figure 2C); which determined that A. aegypti maintained an anthropophilic diet during the epidemic scenarios and climatic events in which they were captured. These findings are consistent with previous literature describing A. aegypti as a hematophagous and anthropophilic mosquito ²⁶26. Degallier N, Filho GC. Mosquitos (Diptera, Culicidae): generalidades, clasificación e importancia vectorial. Brasil: DF; 2000.. According to the Pan American Health Organization ²⁷27. Organización Panamericana de la Salud [Internet]. OPS; 2024 [consultado el 27 de marzo del 2024]. Disponible en: https://www.paho.org/es/temas/dengue.
https://www.paho.org/es/temas/dengue... , A. aegypti is characterized as a species with a strict diet of humans. Recent laboratory studies have reported that, due to the unavailability of humans as a food source, A. aegypti has fed directly on other vertebrates such as rats and rabbits ¹⁴14. Chena L, Nara E, Sánchez Z, Espínola E, Russomando G. Estandarización de la técnica PCR-RFLP del gen mitocondrial cytb como herramienta para la identificación de fuentes de alimentación de insectos hematófagos. Mem Inst Investig Cienc Salud. 2014;12(2):33-42., a behavior possibly caused by the availability of the anthropogenic food source. Other studies indicate that A. aegypti is a mosquito that also has a blood-feeding acceptance for vertebrates such as pigs, dogs, or chickens ²⁸28. Colindres DM, Cerna EF. Organización de una colonia de Aedes aegypti (Diptera: Culicidae) para estudios de resistencia a pesticidas. Revista Portal de la Ciencia [Internet]. 2012 [consultado el 28 de marzo del 2024];2. Disponible en: https://www.academia.edu/download/78607650/Organizacin_de_una_colonia_de_Aedes_aegy20220113-29494-1dikx7m.pdf.
https://www.academia.edu/download/786076... . In Thailand, A. aegypti populations have been found feeding on cattle, pigs, cats, rats, and chickens ⁶6. Lambrechts L, Scott TW, Gubler DJ. Consequences of the expanding global distribution of Aedes albopictus for dengue virus transmission. PLoS Negl Trop Dis. 2010;4(5):e646. doi: 10.1371/journal.pntd.0000646.
https://doi.org/10.1371/journal.pntd.000... . Recent scientific evidence indicates that A. aegypti also feeds on domestic dogs, since dogs in rural and urban areas have been found infected with dengue virus (serotypes DENV2 and DENV3) ²⁹29. Thongyuan S, Kittayapong P. First evidence of dengue infection in domestic dogs living in different ecological settings in Thailand. PLoS ONE. 2017;12(8):e0180013. doi: 10.1371/journal.pone.0180013.
https://doi.org/10.1371/journal.pone.018... . In Puerto Rico, two rural localities with A. aegypti populations fed on dog, cat, horse, and chicken blood have been reported ³⁰30. Barrera B, Bingham AM, Hassan HK, Amador M, Mackay A, Unnasch TR, et al. Vertebrate Hosts of Aedes aegypti and Aedes mediovittatus (Diptera: Culicidae) in Rural Puerto Rico. J Med Entomol. 2012;49(4):917-921. doi: 10.1603/ME12046.
https://doi.org/10.1603/ME12046... .

The feeding preference of A. aegypti for humans is the result of an evolutionary process, since they were originally from African forests where they fed on the blood of wild animals and developed immature stages in trees ³¹31. Tabachnick WJ. Evolutionary genetics and arthropod-borne disease: the yellow fever mosquito. Amer Entomologist. 1991;37(1):14-26. doi: 10.1093/ae/37.1.14.
https://doi.org/10.1093/ae/37.1.14... ^,3232. Eisen L, Moore CG. Aedes (Stegomyia) aegypti in the continental United States: a vector at the cool margin of its geographic range. J Med Entomol. 2013;50(3):467-478. doi: 10.1603/ME12245.
https://doi.org/10.1603/ME12245... . The anthropophilic habit of A. aegypti is due to its diurnal hematophagic activity and interaction with the host environment ³³33. Lozovei AL. Culicídeos (Mosquitos). 1ª ed. São Paulo: Atheneu; 2001.. The mosquito presents temporary genetic changes at the microgeographic level during dengue epidemic events, giving rise to genetic variability and eventual formation of subpopulations and subspecies with biological changes such as anthropophilic levels, biting behavior, insecticide resistance, vectorial capacity and competence ³⁴34. Cadavid JM, Rúa G, Campo O, Bedoya G, Rojas W. Cambios genéticos temporales y microgeográficos de Aedes aegypti en Medellín, Colombia. Biomédica. 2015;35(1):53-61. doi: 10.7705/biomedica.v35i1.2343.
https://doi.org/10.7705/biomedica.v35i1.... ^,3535. Jaimes-Dueñez J, Arboleda S, Triana-Chávez O, Gómez-Palacio A. Spatio-Temporal Distribution of Aedes aegypti (Diptera: Culicidae) Mitochondrial Lineages in Cities with Distinct Dengue Incidence Rates Suggests Complex Population Dynamics of the Dengue Vector in Colombia. PLoS Negl Trop Dis. 2015; 9(4) e0003553. doi: 10.1371/journal.pntd.0003553.
https://doi.org/10.1371/journal.pntd.000... . Feeding on vertebrates other than humans is influenced by hormone levels that vary throughout the life of the female mosquito ³⁶36. Alto BW, Lounibos LP, Juliano SA. Age-dependent bloodfeeding of Aedes aegypti and Aedes albopictus on artificial and living hosts. J Am Mosq Control Assoc. 2003;19(4):347-52..

This study has certain limitations. First, the number of analyzed vertebrates does not allow us to rule out the possibility that A. aegypti may have shown changes in feeding behavior during the studied climatic phenomena, since the methodology of the study does not describe RFLP for other species. Then, acrylamide gels were not used for the clear visualization of RFLPs with low molecular weight, being replaced by very concentrated agarose gels, causing that the RFLPs with higher molecular weight agglutinate and are more visible than those with low weight during electrophoresis. However, our study achieved its objective, being the first studying the feeding behavior of A. aegypti using molecular markers of blood cells during climatic phenomena in Peru.

In conclusion, A. aegypti maintained a conserved anthropophilic feeding behavior in Querecotillo and Marcavelica during the dengue outbreaks that occurred during the Yaku cyclone and during El Niño 2023, having found that humans were its only food source. However, feeding changes at some point in time and in another part of the country are not ruled out. Complementary research should focus on detecting dengue virus in all component phases of the biological cycle, and in domestic animals during climatic events, in order to determine vertical infections and potential reservoirs in massive populations of the mosquito. This study contributes to the validation of intradomiciliary strategies proposed and currently implemented for vector control in the analyzed rural areas, since changes in the feeding pattern of A. aegypti imply a variation in its intradomiciliary behavior.

Acknowledgments

To the Subregional Directorate of Health Luciano Castillo Colonna for the support provided through the acquisition of inputs for the development of the research. To Biologist Leslie Diana Luis Arismendiz, for her support in the photoregistration of the research.

References

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History