ABSTRACT

This study aimed to determine the coexistence and food sources of adult mosquitoes (Diptera: Culicidae) in a rural health center in Piura, Peru by using a descriptive cross-sectional design. Entomological techniques were used to capture and identify mosquitoes, and molecular biotechnology techniques were used to identify food sources. A total of 793 specimens of the Culex and Aedes genera were found coexisting, 789 (99.5%) were Culex quinquefasciatus, 607 (76.9%) were males and 182 (23.1%) were females. Likewise, 4 (100%) corresponded to Aedes aegypti females. The food sources of Aedes aegypti were Homo sapiens sapiens, and Homo sapiens sapiens and Canis familiaris were the food sources of Culex quinquefasciatus. This study provides evidence that rural health centers could be acting as foci of arbovirosis, with the risk that people who come for different ailments could contract diseases transmitted by C. quinquefasciatus and A. aegypti.

Keywords:
Mosquitoes; Sexual Dimorphism; Cytochrome B; PCR; RFLP

KEY MESSAGES

Motivation for the study. Rural health facilities could be potential foci of transmission and scenarios of zoonosis during epidemic outbreaks of dengue and other arbovirosis due to the coexistence of mosquito species that feed on different vertebrates.

Main findings. Aedes aegypti feeds on Homo sapiens sapiens. Culex quinquefasciatus feeds on Homo sapiens sapiens and Canis familiaris. Both coexist in health care areas of the Querecotillo health center.

Implications. Molecular techniques should be integrated into vector control to understand feeding patterns in natural conditions and information on probable reservoirs.

Keywords:
Mosquitoes; Sexual Dimorphism; Cytochrome B; PCR; RFLP

INTRODUCTION

Mosquitoes (Diptera: Culicidae) are the main vectors of tropical diseases, being responsible for causing millions of deaths in urban and rural environments ¹1. Lopez-Solis AD, Solis-Santoyo F, Saavedra-Rodriguez K, Sanchez-Guillen D, Castillo-Vera A, Gonzalez-Gomez R, et al. Aedes aegypti, Ae. albopictus and Culex quinquefasciatus Adults Found Coexisting in Urban and Semiurban Dwellings of Southern Chiapas, Mexico. Insects. 2023;14(6):565. doi: 10.3390/insects14060565.
https://doi.org/10.3390/insects14060565... . However, despite the attention given to them, their feeding behavior is still not fully understood ²2. Fritz ML, Walker ED, Miller JR, Severson DW, Dworkin I. Preferencias divergentes de hospedadores de los mosquitos Culex pipiens aéreos y subterráneos y su descendencia híbrida. Med Vet Entomol. 2015;29(2):115-123. doi: 10.1111/mve.12096.
https://doi.org/10.1111/mve.12096... , since there are species that feed on a wide range of vertebrates and in different degrees of specificity ³3. Wolff GH, Riffell JA. Olfato, experiencia y mecanismos neuronales que subyacen a la preferencia del huésped por los mosquitos. J Exp Biología. 2018;221(4):jeb157131. doi: 10.1242/jeb.157131.
https://doi.org/10.1242/jeb.157131... .

Some of the most representative species are A. albimanus (Wiedemann, 1821) transmitter of the P. falciparum parasite causing malaria ⁴4. Griffing S, Gamboa D, Udhayakumar V. The history of 20th century malaria control in Peru. Malar J. 2013;12:1-7 doi: 10.1186/1475-2875-12-303.
https://doi.org/10.1186/1475-2875-12-303... ; C. quinquefasciatus (Say, 1823) transmitter of Rift Valley fever virus, St. Louis encephalitis virus, West Nile virus, filarial and avian malaria parasites ⁵5. Guagliardo SJ, Levine RS. Etimología: Culex quinquefasciatus. Emerg Infect Dis. 2021;27(8):2041. doi: 10.3201/eid2708.et2708.
https://doi.org/10.3201/eid2708.et2708... ; and A. aegypti transmitter of dengue (DENV), chikungunya (CHIKV), and zika (ZIKV) viruses ⁶6. Bergero P, Guisoni N. Modelo matemático de coinfección de dengue y COVID-19: una primera aproximación. Rev Argent salud pública [Internet]. 2021 [consultado el 27 de enero de 2024];13(1). Disponible en: http://www.scielo.org.ar/scielo.php?pid=S1853-810x2021000200015 Script=sci_arttext.
http://www.scielo.org.ar/scielo.php?pid=... ^,77. Franco Salazar JP. Vigilancia entomovirológica de arbovirus en el Distrito de Santa Marta, Colombia 2018-2019 [tesis de Maestría]. Antioquia: Corporación Académica de Ciencias Básicas Biomédicas, Universidad de Antioquia; 2022. Disponible en: https://bibliotecadigital.udea.edu.co/bitstream/10495/32905/4/FrancoJuan_2022_Arboviruses_Flavivirus_Surveillance.pdf.
https://bibliotecadigital.udea.edu.co/bi... .

Culicid mosquitoes can coexist sharing food from a larval stage ⁸8. Santana-Martínez JC, Molina J, Dussán J. Competencia asimétrica entre Aedes aegypti y Culex quinquefasciatus (Diptera: Culicidae) coexistiendo en sitios de reproducción. Insects. 2017;8(4):111. doi: 10.3390/insects8040111.
https://doi.org/10.3390/insects8040111... , however, they rarely coexist when they reach adulthood, since some species are anthropophilic, others zoophilic and few share both habits ⁹9. Garcia-Rejon JE, Navarro JC, Cigarroa-Toledo N, Baak-Baak CM. An Updated Review of the Invasive Aedes albopictus in the Americas; Geographical Distribution, Host Feeding Patterns, Arbovirus Infection, and the Potential for Vertical Transmission of Dengue Virus. Insects. 2021;12(11):967. doi: 10.3390/insects12110967.
https://doi.org/10.3390/insects12110967... , thus allowing to infer vectorial capacity through feeding patterns and potential reservoirs from molecular markers such as the cytochrome B (CytB) gene of mitochondrial DNA (mtDNA) ¹⁰10. 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 [Internet]. 2014 [consultado el 26 de enero de agosto de 2024];12(2).Disponible en: http://scielo.iics.una.py/scielo.php?pid=S1812-95282014000200007&script=sci_arttext.
http://scielo.iics.una.py/scielo.php?pid... .

The CytB gene is a mtDNA marker widely used in the identification of higher organisms, its choice is based on its small size, its conserved organization, the mutation rate per site per year and the use of universal primers that amplify genes from a wide variety of vertebrates and invertebrates ¹¹11. Brown WM, Prager EM, Wang A, Wilson AC. Mitochondrial DNA sequences of primates: Tempo and mode of evolution. J Mol Evol [Internet]. 1982 [consultado el 13 de marzo de 2024];18:225-239. Disponible en: https://deepblue.lib.umich.edu/bitstream/handle/2027.42/48036/239_2005_Article_BF01734101.pdf?sequence=1&isAllowed=y.
https://deepblue.lib.umich.edu/bitstream... . Although there are studies in which this marker has already been used to identify mosquito food sources ¹⁰10. 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 [Internet]. 2014 [consultado el 26 de enero de agosto de 2024];12(2).Disponible en: http://scielo.iics.una.py/scielo.php?pid=S1812-95282014000200007&script=sci_arttext.
http://scielo.iics.una.py/scielo.php?pid... , it has not yet been used in Peru, particularly in vector species that coexist in rural health centers that could be acting as foci of infection. Therefore, this study aimed to determine the coexistence and food sources of adult mosquitoes (Diptera: Culicidae) in health care areas of the Querecotillo rural health center in the province of Sullana in January 2024.

THE STUDIO

Design and setting

We conducted a quantitative, descriptive, cross-sectional study. The study area corresponds to the Querecotillo health center, located in the rural district of Querecotillo in the province of Sullana, Peru (4° 50' 16.01“ S, 80° 38' 44.02” W) (Figure 1). This facility is characterized by having open infrastructure, and has the following areas: triage, environmental health, febrile, obstetrics hospitalization, obstetrics planning and nursing. The district had up to seven months of continuous rainfall during the El Niño phenomenon of 1993 and 1998. The weather is usually hot, and even hotter during summer, with maximum temperatures of 43.2°C and average humidity of 66%. Rainfall varies between 10 and 200 mm ¹²12. Varona Morante MR. Diseño de losa en el puente carrozable de concreto armado sobre el canal Miguel Checa en el CP Santa Elena Alta km 25+ 770, distrito Querecotillo, provincia Sullana-Piura [tesis de Licenciatura]. Piura: Facultad de Ingeniería Civil, Universidad Nacional de Piura; 2019. Disponible en: https://alicia.concytec.gob.pe/vufind/Record/RUMP_eb30799a1bb0d8bcaad5c44ee1f7da32.
https://alicia.concytec.gob.pe/vufind/Re... .

Mosquito capture and identification

Adult mosquitoes were captured using the World Health Organization (WHO) resting capture methodology ¹³13. Organización Mundial de la Salud. Manual on practical entomology in Malaria. Part.II. [Internet]. Geneva: OMS; 1975 [consultado el 28 de enero de 2024]. Disponible en: https://apps.who.int/iris/handle/10665/42481.; between 4:30 p.m. (dusk) and 7:00 p.m. (night) on January 10, 12, 17, 19, 24 and 26, 2024. They were transferred to the Entomology Research and Training Center (CICE), then exposed to ethyl acetate impregnated on absorbent cotton for five minutes and taxonomically identified using the entomological guideline of the Pan American Health Organization ¹⁴14. 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. for A. aegypti, and of Consoli et al. ¹⁵15. Consoli R, Laureco T, Oliveira L. Principales mosquitos de importancia sanitaria en Brasil. Brasil: Editorial Fiocruz;1994. for C. quinquefasciatus.

Blood collection and DNA extraction

DNA was collected and extracted by separating female mosquitoes that had visible blood residues on the abdomen from those females that did not. Then, the mosquitoes were placed on slides using a homemade protocol, then 100 uL of DNA/RNA Shield Zymo Biomics (R1100-250) preservative solution was added, and pressure was exerted with sterile toothpicks on the abdominal segment, blood was obtained and mixed with 100 uL of the solution, then aspirated and deposited in vials with 200 uL of the same solution. Blood cell DNA was extracted from the collected blood using the Zymo Biomics Kit (D4300), replacing the cell lysis step with silica microbeads with a macerate with sterile plastic pistils, and a 10,000 rpm centrifugation. Finally, we followed the factory protocol.

PCR of the CytB gene

PCR was performed according to the indications by Chena et al. ¹⁰10. 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 [Internet]. 2014 [consultado el 26 de enero de agosto de 2024];12(2).Disponible en: http://scielo.iics.una.py/scielo.php?pid=S1812-95282014000200007&script=sci_arttext.
http://scielo.iics.una.py/scielo.php?pid... , and the protocol of the GoTaq™ G2 PCR kit (Promega M7801), using the primers designed by Oshagi et al. ¹⁶16. 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-CCATCATCCAACATCTCTCAGCATGATGAAA-3). The final volume of 50 µL contained the following: 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 min, followed by 35 cycles with 95 °C for 30 sec for denaturation, 58 °C for 30 sec for hybridization, 72 °C for 1 min for extension, a post-extension of 72 °C for 5 min and a storage temperature of 4 °C for up to 24 hours.

Enzymatic digestion of the CytB gene and agarose gel electrophoresis

We used Hae III and Mwo I enzymes that recognize RFLP in Hae III from H. sapiens sapiens and G. gallus; and RFLP in Mwo I from M. musculus and C. familiaris. PCR products were digested following the factory protocols for each enzyme. Forty-five μL of PCR product, 20 μL of Buffer (1X) and 15 μL of enzyme (10 U/reaction) were mixed. Hae III enzyme was incubated at 37 °C for 15 min followed by 80 °C for 20 min. Mwo I enzyme was incubated at 60 °C for 15 minutes. The reaction products were analyzed by electrophoresis on 3% agarose gels with 2.7 grams of agarose, 90 mL of 1X TAE buffer (Tris-Acetate-EDTA), 4.5 uL of ethidium bromide, 4 uL of loading dye (6X DNA loading dye) and 5 uL of sample (PCR and digestion product). The gel was exposed to 80 volts and 200 Amp for 40 minutes. The 1 kb marker (Opti-DNA Marker, G106) was used for the PCR product and the 100 bp marker (Opti-DNA Marker, G016) was used for the enzymatic digestion products.

Data analysis

Quantitative data were tabulated in Microsoft Excel v.2021 spreadsheets and analyzed with Jamovi v.2.3.28. Molecular data were photoregistered with an Honor X7 CMA-LX3 smartphone camera and analyzed with NEBcutter™ v3.0.

Ethical aspects

We requested consent from the physician on duty in charge of the health center prior to conducting the research, explaining the consistency and implications of the study. No patients or human samples were analyzed in this study, and therefore the approval of an institutional ethics committee was not required.

FINDINGS

Coexistence of mosquitoes

In the areas of the Querecotillo health center, we found a total of 793 coexisting mosquitoes between the Culex and Aedes genera; 789 (99.5%) were C. quinquefasciatus and only 4 (0.5%) were A. aegypti (Table 1). We found that 607 (76.9%) C. quinquefasciatus mosquitoes were male and 182 (23.1%) were female. All 4 (100%) A. aegypti mosquitoes were female.

Mosquito feeding sources

A total of 184 females were tested, 82 did not contain abdominal blood and 102 had recently ingested blood (2 A. aegypti and 100 C. quinquefasciatus). A. aegypti blood was grouped into a pool of 2 specimens (PA), and C. quinquefasciatus blood into two pools of 10 specimens (PC1 and PC2) and a pool of 3 specimens (PC3), discarding 77 mosquitoes due to coagulation problems during processing. We found PCR products of 358 bp and human RFLP in A. aegypti specimens captured in the obstetrics planning area. Human RFLP was also found in C. quinquefasciatus mosquitoes captured in the triage and obstetrics planning areas; and dog RFLP was found in C. quinquefasciatus specimens from the obstetrics hospitalization area (Table 2 and Figure 2).

DISCUSSION

In this study, we found 793 coexisting mosquitoes between A. aegypti (4 specimens) and C. quinquefasciatus (789 specimens) in the Querecotillo health center. It was not possible to analyze all the captured mosquitoes. However, in those that were analyzed, we found that A. aegypti fed on humans and C. quinquefasciatus fed on dogs and humans.

The coexistence of A. aegypti with C. quinquefasciatus and their difference in quantity in a rural area of Peru has already been reported by Ruiz et al. ¹⁷17. Ruiz-Polo AA, Núñez-Rodríguez CM, Saavedra-Ríos CY, Niño-Mendoza LE, Santillan-Valdivia RE. Coexistencia de mosquitos adultos (Diptera: Culicidae) en el interior de viviendas de una localidad rural durante un brote de dengue en Sullana, Piura, 2023. Rev Peru Med Exp Salud Publica. 2024;41(1):89-90. doi: 10.17843/rpmesp.2024.411.13416.
https://doi.org/10.17843/rpmesp.2024.411... . Salazar and Moncada ¹⁸18. Salazar MJ, Moncada LI. Ciclo de vida de Culex quinquefasciatus Say, 1826 (Diptera: Culicidae) bajo condiciones no controladas en Bogotá. Biomédica [Internet]. 2004 [consultado el 02 de febrero de 2024]; 24(4). Disponible en: http://www.scielo.org.co/scielo.php?pid=S0120-41572004000400007&script=sci_arttext. reported that both species coexist in Colombia. This is explained by adaptation mechanisms by Ruiz et al. ¹⁹19. Ruiz N, Rincón GA, Parra HJ, Duque JE. Dinámica de oviposición de Aedes (Stegomyia) aegypti (Diptera: Culicidae), estado gonadotrófico y coexistencia con otros culícidos en el área Metropolitana de Bucaramanga, Colombia. Universidad Rev Ind Santander. 2018;50(4):308-319. doi: 10.18273/revsal.v50n4-2018004.
https://doi.org/10.18273/revsal.v50n4-20... . The number of both species differs in most cities in tropical countries, with C. quinquefasciatus being approximately 20 times more abundant than A. aegypti²⁰20. Azmi SA, Das S, Chatterjee S. Seasonal prevalence and blood meal analysis of filarial vector Culex quinquefasciatus in coastal areas of Digha, West Bengal, India. J Vector Borne Dis [Internet]. 2015 [consultado el 02 de febrero de 2024];52. Disponible en: https://www.mrcindia.org/journal/issues/523252.pdf.
https://www.mrcindia.org/journal/issues/... . Therefore, our results can be elucidated, given that we found more C. quinquefasciatus specimens than A. aegypti.

The A. aegypti mosquito is usually described as a species that only feeds on humans ²¹21. Degallier N, Filho GC. Mosquitos (Diptera, Culicidae): generalidades, clasificación e importancia vectorial. Brasil: DF; 2000.. In our results, human-feeding specimens were found in the obstetrics planning area, which is consistent with the literature. Reports from Thailand report that A. aegypti populations feeds on humans, cattle, pigs, cats, rats, and chickens ²²22. 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... . In the Caribbean, Fitzpatrick et al. ²³23. 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... reported populations of A. aegypti that fed on humans, mongooses, dogs, domestic cats and wild birds.

C. quinquefasciatus is a mosquito with a very varied diet, which includes humans and dogs ²⁰20. Azmi SA, Das S, Chatterjee S. Seasonal prevalence and blood meal analysis of filarial vector Culex quinquefasciatus in coastal areas of Digha, West Bengal, India. J Vector Borne Dis [Internet]. 2015 [consultado el 02 de febrero de 2024];52. Disponible en: https://www.mrcindia.org/journal/issues/523252.pdf.
https://www.mrcindia.org/journal/issues/... . This behavior is demonstrated by our results, since human-fed specimens were found in the triage and obstetrics planning areas. In addition, dog-fed specimens were found in the obstetrics hospitalization area. The C. quinquefasciatus mosquito not only feeds on humans, but also on cats, pigs, cows, horses and even reptiles ²⁴24. Fundación Oswaldo Cruz. Evaluación de la metodología de aspiración de mosquitos adultos para el seguimiento de la infestación por Aedes aegypti en el área endémica del dengue en Recife/PE [Internet]. Lima: Centro de Investigación Aggeu Magalhães; 2013 [consultado el 02 de febrero de 2024]. Disponible en: https://www.arca.fiocruz.br/handle/icict/13309.. The feeding patterns of C. quinquefasciatus in the hemisphere exhibit highly anthropophilic behavior ²⁵25. Barreras R, Machado-Allison CE, Bulla L. Persistencia de criaderos sucesión y regulación poblacional en tres culícidos urbanos (Culex fatigans Wied., Culex corniger Theo. y Aedes aegypti (L). Acta Cient Venez. 1981;32:386-93..

The finding of dog-feeding C. quinquefasciatus mosquitoes in the obstetrics hospitalization area poses a very relevant risk to the health of neonates and puerperal women receiving medical care. Previous studies have found dogs seropositive for Venezuelan equine encephalitis virus (VEEV) ²⁶26. Coffey LL, Crawford C, Dee J, Miller R, Freier J, Vasilakis N, et al. Evidencia serológica de actividad generalizada del virus de los Everglades en perros, Florida. Emerg Infect Dis. 2006;12(12):1873-1879. doi: 10.3201/eid1212.060446.
https://doi.org/10.3201/eid1212.060446... , which causes brain necrosis in fetuses and newborn infants when mothers are infected with VEEV during pregnancy ²⁷27. Wenger F. Necrosis cerebral masiva del feto en casos de encefalitis equina Venezolana. Invest clín.1995;36(2):37-51.. Likewise, some ZIKV strains have the ability to infect the C. quinquefasciatus mosquito ²⁸28. Ayres CFJ, Guedes DRD, Paiva MHS, Morais-Sobral MC, Krokovsky L, Machado LC, et al. zika virus detection, isolation and genome sequencing through Culicidae sampling during the epidemic in Vitória, Espírito Santo, Brazil. Parasit Vectors. 2019;12(1):220. doi: 10.1186/s13071-019-3461-4.
https://doi.org/10.1186/s13071-019-3461-... , which would play a secondary role in ZIKV transmission, since A. aegypti is the most likely vector. However, during dengue epidemics, the flow of febrile patients to the Querecotillo health center would be a source of virus transmission. This is a relevant scenario from the epidemiological point of view, considering that C. quinquefasciatus that fed on dogs and humans were found coexisting with A. aegypti that fed on humans, since there is scientific evidence that serotypes 2 and 3 of the dengue virus have been detected in domestic dogs, which could act as potential reservoirs 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... .

Our study has some limitations. The Querecotillo health center has an open infrastructure and is located in a rural area with a warm climate and continuous rainfall; therefore, our results only apply to this facility. In addition, the number of analyzed pools does not allow us to generalize about food sources. However, this is the first research on coexistence and food sources of arbovirus vectors within a rural health facility in Peru.

In conclusion, our results suggest that the Querecotillo health center represents a risk regarding the transmission of arbovirosis, since specimens of A. aegypti and C. quinquefasciatus were found in healthcare areas, coexisting and feeding on vertebrates other than humans, such as dogs (observed in C. quinquefasciatus). Research is needed to detect dengue, zika and chikungunya arboviruses in adult mosquitoes, in order to understand the transmission dynamics in rural health centers. This study contributes to the development of preventive strategies for arbovirus transmission in Sullana, Peru.

Acknowledgments

To Dr. Nely Ito Vilca, for allowing us to conduct the research at the Querecotillo health center. To Mrs. Milagros Teresa Aguilar Taboada, for her guidance in the areas of medical care. To the assistants Leodán Enrique Saavedra Seminario and Carmela Sofia Arcila Diaz, for their collaboration in the entomological captures. To the bachelor in Biological Sciences Cindy Yuriko Saavedra Rios, for her support in the design and adaptation of protocols for DNA extraction and PCR.

References

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