Entomological and virological surveillance for dengue virus in churches in Merida, Mexico

Keywords: Aedes aegypti, Flavivirus, Mosquitoes, Dengue


This study was designed to assess whether churches in endemic dengue districts in Merida, Mexico provide suitable breeding habitats for mosquitoes and are potential sites for dengue virus (DENV) transmission. Churches were inspected for immature and adult mosquitoes once every week from November 2015 to October 2016. A total of 10,997 immatures of five species were collected. The most abundant species were Aedes aegypti (6,051) and Culex quinquefasciatus (3,018). The most common source of immature Ae. aegypti were buckets followed by disposable containers. Adult collections yielded 21,226 mosquitoes of nine species. The most common species were Cx. quinquefasciatus (15,215) and Ae. aegypti (3,902). Aedes aegypti were found all year long. Female Ae. aegypti (1,380) were sorted into pools (166) and assayed for flavivirus RNA by RT-PCR and Sanger sequencing. Two pools were positive for DENV (DENV-1 and 2). In conclusion, we demonstrated that some churches in Merida are infested with mosquitoes all year long and they potentially serve as sites for DENV transmission and should therefore be considered for inclusion in mosquito and arboviruses control and surveillance efforts.


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Baak-Baak, C., Cigarroa-Toledo, N., Pech-May, A., Cruz-Escalona, G., Cetina-Trejo, R., Tzuc-Dzul, J., Talavera-Aguilar, L., FloresRuiz, S., Machain-Williams, C., Torres-Chable, O., Blitvich, B., Mendez-Galvan, J., & Garcia-Rejon, J. (2019). Entomological and virological surveillance for dengue virus in churches in Merida, Mexico. Revista Do Instituto De Medicina Tropical De São Paulo, 61, e9. https://doi.org/10.1590/S1678-9946201961009
Original Articles


Dengue virus (DENV; Flaviviridae, Flavivirus) represents an enormous problem in terms of human morbidity and economic burden. Globally, DENV is responsible for approximately 1.14 million disability-adjusted life-years annually1. In Mexico, an estimated 139,000 symptomatic DENV infections occur each year, with a mean of 65 disability-adjusted life years per million individuals2. There are four serotypes of DENV (DENV-1 to 4). The serotypes are genetically distant from each other and can be further divided into different genotypes3. Some serotypes are more virulent than others and have been associated with increased disease severity (e.g., DENV-2 American/Asian genotype)4. The introduction of distinct viral genotypes can trigger epidemics; thus, it is important that sequencing studies are performed so that viruses are identified and critical information on virus emergence and persistence is acquired5.

DENV is primarily transmitted to humans through the bite of Aedes aegypti3 infected females which feed almost exclusively on humans and are commonly found in households and other sites where people spend much of their time6. DENV is hyperendemic in Yucatan State and elsewhere in the Yucatan Peninsula in Mexico6,7, and chikungunya virus (CHIKV) also circulates in this region8. Previous studies performed inside the houses in Merida, Yucatan State, resulted in the detection of DENV- and CHIKV-infected Ae. aegypti6-8 females. Virus-infected mosquitoes have also been detected in frequently visited public locations. For example, DENV-infected Ae. aegypti were collected in schools in Merida9. In the State of San Luis Potosi, ZIKV-infected Ae. aegypti were detected in cemeteries10. It is therefore important to monitor public sites for potential arboviruses transmission.

Churches regularly receive a large influx of visitors, both locals and tourists, who could potentially become infected during their visits and introduce the virus into new areas. We have previously revealed that engorged Ae. aegypti often occur inside churches in Merida11. Despite this, studies have not been performed to determine whether churches located in neighborhoods with a high incidence of DENV provide suitable breeding habitats for immature mosquitoes and are potential sites for virus transmission. To address these gaps in our knowledge, longitudinal entomological and virological surveillance were performed in selected churches in three endemic dengue districts of Merida. Churches were inspected regularly for the presence of mosquitoes breeding sites, the abundance and species composition of immature and adult mosquitoes were determined, and Ae. aegypti females were assayed for evidence of DENV infection.


Study sites

Merida (population ~ 800,000) is located in the Yucatan Peninsula of Mexico. The city has a distinct rainy (May–October) season and a dry season (November–April). In the rainy season, the mean rainfall is 1,000 mm and the mean temperature is 27.5 °C. In the dry season, the mean rainfall is 300 mm and the mean temperature is 25.1 °C6.

The studied sites were located in three neighborhoods where high DENV transmission has been previously reported6,7,9. The largest church in each neighborhood was selected based on the assumption that it would have more mosquitoes breeding sites and a higher abundance of immature and adult mosquitoes than other churches in the neighborhood. Church leaders provided unlimited access to the entire area, including rooms, offices, kitchens, storage rooms and gardens. It is estimated that each church is visited by at least 900 individuals each week (300 and 600 individuals on weekdays and weekends, respectively), although these numbers are higher when there are important religious holidays, festivities or celebrations (personal communication from church authorities). The neighborhoods are San Jose Tecoh, Bojorquez and Vergel III, located in Southern, Central and Eastern Merida, respectively. The first two churches are located approximately 20 m from the nearest houses. The church in Vergel III is located approximately 80 m from the nearest house. The churches are similar in size (~ 10,000 m2), close to markets and 8 to 10 km apart from each other (Figure 1).

- Location of study sites in Merida, Yucatan, Mexico. Catholic churches were located in three neighborhoods: Bojorquez (Center), San Jose Tecoh (South) and Vergel-III (East).

Figure 1: - Location of study sites in Merida, Yucatan, Mexico. Catholic churches were located in three neighborhoods: Bojorquez (Center), San Jose Tecoh (South) and Vergel-III (East).

Sampling of immature mosquitoes

Mosquitoes were collected at each church once a week from November 2015 to October 2016. Collections were made both indoor and outdoor. Outdoor collections were primarily focused on garden areas and parking lots on church grounds. Containers were classified following the methodology of Garcia-Rejon et al.12. Immatures were removed from water-holding containers using nets, turkey basters and pipettes and placed inside plastic transportation containers labeled according to date, study site and sample identification number. Mosquitoes were individually removed from transportation containers and counted in a white tray. Immatures were allowed to emerge, then adults were identified to species11,12.

Adult mosquito collections

Adult mosquitoes were collected between 8 a.m. and 1 p.m. using a backpack-mounted aspirator (Prokopack Aspirator®, model 1419, John W. Hock Company). Each church was inspected for resting adults once every week. Indoor collections focused primarily on furniture, hanging clothes, curtains as well as dark and humid places. Outdoor collections focused primarily on garden areas and fences. The amount of time actively spent searching for resting mosquitoes at each site was 1-2 h per visit. Both adults and immatures were transported alive to the laboratory of Arbovirologia at Universidad Autónoma de Yucatán and identified according to species using stereomicroscopes and a published identification key13. Female Ae. aegypti were sorted into pools of up to 15 specimens and stored at -80 oC until required.

Mosquitoes homogenizations, RNA extractions and RT-PCR

Pools of female adult Ae. aegypti were placed into eppendorf tubes containing 300 µL of Liebovitz’s L15 medium (Invitrogen, Carlsbad, CA, USA) and mechanically homogenized using sterile pestles. Homogenates were centrifuged at 10,000 × g for 10 min. and supernatants were collected. Total RNA was extracted from an aliquot (100 µL) of each supernatant using an RNeasy kit (Qiagen, Valencia, CA, USA) and tested for flavivirus RNA by reverse transcription-polymerase chain reaction (RT-PCR) using flavivirus-specific primers (MAMD, cFD2 and FS778) which amplify an ~250 nt region of the NS5 gene14. RT-PCRs were performed in 25 µL reaction volumes containing 2.5 µL of total RNA, 2 µL MgCl2 at a concentration of 25mM, 2.5 μL of 5 x reaction buffer, 0.2 μL of dNTPs, 0.5 U of Taq polymerase (Invitrogen), 0.5 μL of each primer at a concentration of 10 mM and 16.15 μL sterile distilled water. Total RNA extracted from DENV-2-infected cells was used as the positive control after reverse transcription. As the negative control, molecular biology grade water was used instead of total RNA. Amplification conditions are as follows: an initial denaturation of 95°C for 1 minute, followed by 35 cycles, each one consisting of 1 min at 95 °C, 1.5 min at 75 °C, and 1 min at 72 °C and then a final extension for 7 min at 72 °C. Amplicons were visualized on 2% agarose gels with 0.5 μg/mL ethidium bromide using a Doc XR+ Gel Documentation System. RT-PCR products were purified using the Zymoclean DNA recovery kit Cat (Zymo Research, Irvine, CA, USA) and sequenced using a 3500xL DNA sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were manually aligned and edited using the Bioedit v.7.0.9 software15 and the Mega v.7 software16.

Statistical analysis

Entomological indices estimated in this study are as follows: 1) the container index, defined as the percentage of water-filled containers with immature Ae. aegypti and 2) the pupal index, defined as the percentage of containers with Ae. aegypti pupae when compared to containers with Ae. aegypti immatures (larvae and/or pupae). A Mann-Whitney U test was used to compare the number of female Ae. aegypti by season because the data did not meet the assumptions of normality and homogeneity of variances. The statistical analysis was performed using the IBM SPSS Statistics version 22 software for Windows (IBM Corporation, Armonk, NY) and results were considered significant when P ≤ 0.05.


Immature mosquito collections

The total number of containers observed in the entire study was 2,918 (Table 1). This tally is based on the assumption that one container can be counted more than once (i.e. one count for every visit in which the container was observed). Water was detected during 27.5% (804/2,918) of the container observations and 8.1% (65/804) yielded immatures. A total of 10,997 immatures of five species were collected (Table 2). The most abundant species was Ae. aegypti (6,051) followed by Culex quinquefasciatus Say (3,018), Culex nigripalpus Theobald (1,894), Culex thriambus Dyar (26), and Culex interrogator Dyar and Knab (8).

Table 1:
Collections of Ae. aegypti immatures in churches of Merida, Yucatan by season and container type, from November 2015 to October 2016.
Type of container Total no container observations/with water No water-filled containers with immatures Water (L) per container (mean±SD) Total no of immatures
Larvae and pupae b Pupae Larvae Pupae
Rainy season            
Buckets 242/129 25 14 4.18±4.38 2,898 211
Disposable containers 1,099/379 13 7 0.80±4.39 580 30
aLarger containers 97/35 13 4 1.81±3.32 922 25
Vases 57/34 1 1 1.80±0.0 40 8
Flower pots 172/38 1 1 1.15±0.0 115 5
Tires 11/2 1 0 0.35±0.0 88 0
Discarded toilet 6/2 0 0 2.82±1.44 0 0
Storm water-drains 68/45 1 0 78.33±153.57 88 0
Subtotal 1,752/664 55 27 2.63±4.18 4,731 279
Dry season            
Buckets 205/72 6 2 7±6.04 785 33
Disposable containers 692/31 0 0 0.46±0.83 0 0
aLarger containers 52/8 4 1 3.30±1.56 220 3
Vases 3/3 0 0 0.81±0.44 0 0
Flower pots 157/15 0 0 0.95±0.80 0 0
Tires 2/0 0 0 0 0 0
Storm water-drains 55/11 0 0 21.82±18.34 0 0
Subtotal 1,166/140 10 3 5.15±4.61 1,005 36
Total 2,918/804 65 30 3.02±4.31 5,736 315

aWith a capacity >5 liters; bNumber of containers with pupae

Table 2:
Abundance and species composition of immature mosquitoes collected in churches of Merida, Yucatan, from November 2015 to October 2016.
Mosquito species Church Total no of immatures collected
Larvae Pupae
Rainy season      
Aedes aegypti Vergel III 4,257 242
Culex quinquefasciatus Vergel III 2,648 68
Culex nigripalpus Vergel III 1,847 47
Culex interrogator Vergel III 8 0
Aedes aegypti San Jose Tecoh 67 8
Aedes aegypti Bojorquez 407 29
Subtotal   9,234 394
Dry season      
Aedes aegypti Vergel III 994 9
Culex quinquefasciatus Vergel III 291 11
Culex thriambus Vergel III 26 0
Aedes aegypti San Jose Tecoh 11 27
Subtotal   1,322 47
Total   10,556 441

Seven-fold more immatures were collected in the rainy season (9,628) compared to the dry season (1,369, Table 2). Analysis of the data at the species level also revealed that immature Ae. aegypti were more common in the rainy season. A total of 1,758 container observations were made in the rainy season. Water was detected in 37.8% (664/1,758) of the container observations and 8.2% (55/664) yielded immatures (Table 1). The pupal index was calculated as 49.1% (27/55). In the dry season, 1,166 container observations were made. Of these, 12.0% (140/1,166) revealed water and 7.1% (10/140) yielded immatures. The pupal index was calculated as 30.0% (3/10) (Table 1). Buckets were the most common source of Ae. aegypti during both seasons and accounted for 64.9% (3,927/6,051) of those collected. Disposable containers and larger containers were the next most common sources of Ae. aegypti at both times of the year. Ae. aegypti were present in every container that held immatures.

When collections were grouped by church, 95.0% (10,448/10,997) of mosquito immatures were collected in Vergel III. Five species were collected in this church. The most abundant species in Vergel III was Ae. aegypti (5,502) followed by Cx. quinquefasciatus (3,018). Sixty breeding sites of Ae. aegypti were registered in Vergel III, with 28 buckets, 16 larger containers and 13 disposable containers yielding 3453, 993 and 755 immatures, respectively. The pupal index was calculated as 43.3% (26/60). In Bojorquez and San Jose Tecoh, one and four breeding sites were registered, respectively.

Adult mosquito collections

In total, 21,226 adult mosquitoes (13,518 males and 7,708 females) of nine species were collected (Table 3). Of the females collected, the most abundant species was Cx. quinquefasciatus (4,686) followed by Ae. aegypti (1,380), Aedes taeniorhynchus (Wiedemann) (1,172) and Cx. nigripalpus (363) which together accounted 98.6% (7,601/7,708) of the adults. Cx. quinquefasciatus was common during both the rainy and dry seasons whereas the other three species were far more abundant in the rainy season.

Table 3:
Abundance and species composition of adult mosquitoes collected in churches of Merida, Yucatan from November 2015 to October 2016.
Species Church Total no of adults collected Blood feeding status
Males Females Unfed Fed Gravid
Rainy season            
Ae. aegypti Vergel-III 2,036 1,029 375 474 180
Ae. taeniorhynchus Vergel-III 272 1,039 410 280 349
Ae. trivittatus Vergel-III 0 32 9 5 18
Cx. coronator Vergel-III 0 1 1 0 0
Cx. interrogator Vergel-III 21 67 53 5 9
Cx. nigripalpus Vergel-III 129 321 185 12 124
Cx. quinquefasciatus Vergel-III 4,766 1,813 974 505 334
Cx. stigmatosoma Vergel-III 0 1 0 1 0
Ps. ferox Vergel-III 0 3 1 1 1
Ae. aegypti San Jose Tecoh 7 26 10 10 6
Cx. quinquefasciatus San Jose Tecoh 87 33 14 7 12
Ae. taeniorhynchus San Jose Tecoh 0 6 4 2 0
Ae. aegypti Bojorquez 193 123 52 52 19
Cx. quinquefasciatus Bojorquez 184 119 55 47 17
Ae. taeniorhynchus Bojorquez 34 115 69 38 8
Ae. trivittatus Bojorquez 0 3 2 0 1
Subtotal   7,729 4,731 2,214 1,439 1,078
Dry season            
Ae. aegypti Vergel-III 281 187 79 76 32
Ae. taeniorhynchus Vergel-III 9 12 6 2 4
Cx. nigripalpus Vergel-III 2 42 35 2 5
Cx. quinquefasciatus Vergel-III 5,421 2,680 1,925 609 146
Ae. aegypti San Jose Tecoh 2 7 5 1 1
Cx. quinquefasciatus San Jose Tecoh 47 23 10 7 6
Ae. aegypti Bojorquez 3 8 5 2 1
Cx. quinquefasciatus Bojorquez 24 18 10 3 5
Subtotal   5,789 2,977 2,075 702 200
Total   13,518 7,708 4,289 2,141 1,278

Significant statistical difference in the number of Ae. aegypti females per season was observed (Z=-0.57, p=0.001). Approximately six-fold more Ae. aegypti females were collected in the rainy season compared to the dry season (Table 3). Of the 1,178 Ae. aegypti females collected during the rainy season, 536 were identified as fed, 437 as unfed and 205 as gravid (Table 4). In the dry season, 202 Ae. aegypti females were collected with 89 identified as unfed, 79 as fed and 34 as gravid.

Table 4:
Detection of dengue virus RNA in adult Ae. aegypti females in churches of Merida from November 2015 to October 2016.
Area within Merida city Neighborhoods No total females No total unfed No total fed No total gravid No of pools tested No of positive pools for DENV
Rainy season              
East Vergel-III 1,029 375 474 180 125 2
South San Jose Tecoh 26 10 10 6 3 0
Center Bojorquez 123 52 52 19 14 0
Subtotal   1,178 437 536 205 142 2
Dry season              
East Vergel-III 187 79 76 32 22 0
South San Jose Tecoh 7 5 1 1 1 0
Center Bojorquez 8 5 2 1 1 0
Subtotal   202 89 79 34 24 0
Total   1,380 526 615 239 166 2

Vergel III was the most common source of adult mosquitoes; 95% (12,937 males and 7,227 females) of adults of nine species were collected (Table 3). Of the 7,227 females collected, the most abundant species was Cx. quinquefasciatus (4,493) followed by Ae. aegypti (1,216), Ae. taeniorhynchus (1,051), Cx. nigripalpus (363), Cx. interrogator (67), Ae. trivittatus (32), Cx. thriambus (26), Ps. ferox (3), Cx. coronator (1) and Cx. stigmatosoma (1). Eighty-eight percent (1,216/1,380) of Ae. aegypti females was collected in this site.

Detection of DENV in Ae. aegypti

Adult Ae. aegypti females were sorted into 166 pools and analyzed for flavivirus RNA by RT-PCR and Sanger sequencing. Two (1.2%) pools were positive (Table 4). One pool contained DENV-1 and the other contained DENV-2 (GenBank accession number KU232287 and KJ189370, respectively). The DENV minimal infection rate (MIR) for Ae. aegypti females (expressed as the number of positive mosquito pools per 1,000 mosquitoes tested) was 1.5. Both DENV-positive pools were collected inside the church in Vergel III. The DENV-1-positive pool consisted of mosquitoes collected in a storage room. The DENV-2-positive pool consisted of mosquitoes collected inside the nave, the main area of the church where the congregation is held. Both pools comprised of mosquitoes were collected in October 2016.


Vergel III represented a higher potential entomological risk than the other investigated churches. The pupal index in this church over the entire study period was 43.3%, peaking at 47.0% in the rainy season. Pupae are considered a better proxy than other life stages for estimating mosquitoes abundance17. Based on the operative criteria of the Mexican Ministry of Health, a pupal index greater than 5% is regarded as an emergency level status that requires immediate action (i.e. treatment or elimination of breeding sites)18. Adult Ae. aegypti were also more abundant at Vergel III compared to the other churches in this study. It has previously been reported that greater mosquitoes abundance increases the risk of human DENV transmission7. Both pools of DENV-infected Ae. aegypti were detected in the church of Vergel III. Similarly, in Iquitos, Peru, abundance of Ae. aegypti females were better proxies for DENV risk19.

DENV-infected Ae. aegypti have been detected inside schools and houses in Merida6,7,9. Water-filled containers are common in Merida and contribute to the high abundance of mosquitoes12,20. It should also be noted that Ae. aegypti with Ile 1,016 and Cys 1,534 mutations in their voltage gated sodium channel gene have been detected in Merida. These mutations are associated with pyrethroid resistance21,22. Thus, increased vector control is required in this region. One way to achieve this goal is through increased community intervention. In this study, we educated church workers on mosquitoes (i.e. their life cycle and role in arbovirus transmission) and recommended that church visitors should use personal protection against mosquitoes (e.g. repellents).

Environmental conditions may influence the mosquitoes population size. High populations of Ae. aegypti in the rainy season have been previously reported in Yucatan State6,9,20. Seven-fold more immature Ae. aegypti were collected in the rainy season compared to the dry season presumably due to an increase in the number of water-filled containers. A high number of breeding sites and immature Ae. aegypti were identified in Vergel III. Buckets followed by disposable containers were the most common breeding sites for Ae. aegypti and most were filled by rain water. Disposable containers were also common breeding sites for Ae. aegypti in and around houses in Merida6,12. Previous studies in Merida have also shown that tires and flower pots are common breeding sites in vacant lots and parking lots, respectively20. Storm-water drains along streets and sidewalks harbor many species of mosquitoes throughout the year23.

It is important to highlight two characteristics of Vergel III: 88% of Ae. aegypti females were collected in this church and it is located approximately 80 m from the nearest house. We speculate that most Ae. aegypti in Vergel III had not traveled there from the houses. Aedes aegypti has a limited flight range; it is generally accepted that females do not fly more than 50-100 m in their entire lifetime24 and that they remain close to human hosts and oviposition sites12,20. Therefore, humans potentially contribute more than the vector for the movement of DENV to new areas. To the best of our knowledge, this is the first study to detect DENV-infected Ae. aegypti in church environments. Mosquito-based surveillance for arboviruses can be used to detect viruses prior to the occurrence of outbreaks as human infections are often asymptomatic2,3. The DENV MIR in this study was 1.5 which is considerably lower than the 4.6 reported in Merida schools9. However, our results are similar to earlier studies performed inside the houses of dengue patients6,7.

Nine mosquito species were collected in Vergel III, and the most common species was Ae. aegypti, consistent with previous studies performed in Merida. Diversity of mosquito species represented 17.3% (9/52) of mosquito fauna identified in Yucatan State25. Other mosquito species involved in arbovirus disease transmission were also identified including Cx. interrogator, Cx. quinquefasciatus, Cx. nigripalpus, Cx. stigmatosoma and Cx. thriambus which are competent vectors of West Nile virus and St. Louis encephalitis virus26,27. West Nile virus has been detected in Cx. quinquefasciatus in Nuevo Leon, Northern Mexico28 and was abundant in the present study. West Nile virus has also been detected in Cx. nigripalpus and Cx. interrogator in Chiapas, Southern Mexico27. The alphavirus, Venezuelan equine encephalitis virus, was detected in Cx. coronator in Veracruz State29. Several orthobunyaviruses, including Cache Valley virus were isolated from Ae. taeniorhynchus in Yucatan State30. It is therefore important that mosquito control efforts in Merida do not focus only on Ae. aegypti.

Our study provides additional evidence that public sites harbor infected disease vectors. Previous studies performed in Merida identified DENV-infected Ae. aegypti in schools9 and a cemetery31. DENV- and ZIKV-infected Ae. aegypti and Ae. albopictus were identified in cemeteries in San Luis Potosi in Central-Northern Mexico10. In Belo Horizonte, Brazil, both DENV and ZIKV were detected in Ae. aegypti collected at a university32. Furthermore, La Crosse virus was detected in Aedes triseriatus in cemeteries in Tennessee, USA33. However, it is important to note that our study has several limitations. For example, a small number of churches were surveyed and those from areas with low arbovirus transmission were not included. Therefore, it is not known if our findings are typical for churches in Merida or elsewhere in Mexico. Nevertheless, the findings from this study and previous reports demonstrate the need to monitor public sites to control disease transmission.



We thank the church leaders for their permission to work, providing unlimited access to the entire area for mosquitoes collections.


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The study was supported in part by the Consejo Nacional de Ciencia y Tecnologia de México, Grant Problemas Nacionales (PDCPN 2014-247005).