Successful application of Wolbachia in insect vector control depends on its ability to invade at high frequency under field conditions. The transfection of Wolbachia in mosquitoes can cause resistance for a variety of pathogens.^1,2 In this study, we examined the field infection frequency of native Wolbachia in the vector Aedes spp. mosquitoes of the Soconusco region, Chiapas, Mexico. Aedes albopictus and Ae. aegypti mosquitoes were sampled during the rainy season of 2015 (June to November) in six selected cemeteries in the central part of the Soconusco, Chiapas (15°19’ N, 92°44’ W) (figure 1).

Figure 1. Study area. Cemeteries: 1) Jardín de Tapachula, 2) Raymundo Enríquez, 3) Cacahoatán, 4) Tuxtla Chico, 5) Mazatán, and 6) Huehuetán. These sites were sampled for mosquitoes from May to July, 2015 

Wild females of both species were preserved separately at -20 °C. Larvae and pupae obtained from the breeding sites were maintained until adult emergence; newly emerged females were preserved as previously described. Genomic DNA was extracted using DNAzol kit and analyzed by PCR to determine the presence of Wolbachia by means of amplifying the surface protein gene (wsp) reported previously.³ Integrity of the DNA was analyzed using primers to amplify the actin gene of Ae. aegypti and Ae. albopictus.⁴ To confirm the presence of Wolbachia, PCR products from the wsp gene were purified using the QIAquick PCR purification kit (QIAGEN) for sequencing. The sequences were used for Blast analysis using the GenBank database (http://blast.ncbi.nlm.nih.gov).

A total of 215 mosquitoes were collected, 49.8% of which were identified as Ae. aegypti and 50.2% as Ae. albopictus. Adults were collected only in the cemeteries Jardín de Tapachula (13% Ae. albopictus, and 56% Ae. aegypti), and Raymundo Enríquez (26% Ae. albopictus, and 5% Ae. aegypti). Larvae and pupae sampling was performed in all cemeteries (55% Ae. albopictus and 45% Ae. aegypti) (table I). Positive samples for Wolbachia infection were detected by PCR amplification of a 600 bp fragment corresponding to wsp gene (figure 2, upper panel), showing that Wolbachia infections only occurred in Ae. albopictus (lanes 3, 4, 6, 7, 8, 9, 11, and 12) and is absent in Ae. aegypti (lane 5). Not all the Ae. albopictus were infected with Wolbachia (lane 10). Actin gene was used as quality control for DNA and PCR reactions (figure 2, lower panel).

Figure 2. Wolbachia wsp gene amplification in mosquitoes Aedes spp. Gene wsp was amplified by PCR. In the lower panel the actin gene amplification of the same DNA samples is shown as control. 1) 100 bp ladder; 2) negative control; 3) and, 4) Ae. albopictus collected using an entomological net (Jardín de Tapachula); 5) Ae. aegypti collected using an entomological net (Jardín de Tapachula); 6), 7), 8), 9), and 10) Ae. albopictus larvae (Huehuetán); 11), 12) Ae. albopictus larvae (Cacahoatán). Collections were performed from May to July, 2015 

In general, Wolbachia infection rate was 38% in Ae. albopictus. Wolbachia infection in collected adult mosquitoes from Jardín de Tapachula and Raymundo Enríquez cemeteries was 12.5 and 25%, respectively. In addition, Wolbachia infection in mosquitoes from collected larvae was greater in Ae. albopictus from Huehuetán cemetery (81%), followed by 32% of infected mosquitoes from Raymundo Enríquez (table I). Sequencing results of three randomly selected PCR amplicons were aligned using Clustal W algorithm. Two samples, named W5 (accession no. KX118690) and W83 (accession no. KX118691) showed 100% identity with Wolbachia strain previously reported in Ae. albopictus; and W4 (accession no. KX118692) showed 99% identity.

This is the first study in the Soconusco region, Chiapas, that shows the infection of Wolbachia in Ae. albopictus and its absence in Ae. aegypti local populations. This evidence may be relevant from the epidemiological point of view because Wolbachia infection in Ae. albopictus has different effects in the mosquito life cycle as well as a blocking effect on the development of pathogens, which strengthens the idea of using Wolbachia as a biological control strategy for emerging and reemerging arboviruses in the country.