Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida, USA

Abstract The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two‐step PCR assay to detect Wolbachia in wild‐collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop‐mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia‐positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.

60%-70% of all insect species harbor Wolbachia, including some mosquito species (Hilgenboecker, Hammerstein, Schlattmann, Telschow, & Werren, 2008). Wolbachia can be a powerful reproductive manipulator, inducing cytoplasmic incompatibility (CI), parthenogenesis, feminization of males, and male killing in various host species . These properties have been exploited for the development of Wolbachia as a novel strategy for vector mosquito control.
Wolbachia-induced CI favors the reproductive success and spread of colonized females in populations, which can be used to drive desirable traits, including resistance to infection with vector-borne pathogens, into a population. On the other hand, infected mosquito males can cause CI in a population with the presence of different Wolbachia strains or no infection, which can be used for sterile insect technique (SIT) to decrease vector populations (Flores & O'Neill, 2018).
Establishing the prevalence of Wolbachia in Ae. aegypti is critical to public health, because over the past decade, Ae. aegypti transinfected with Wolbachia have been generated with the goal of blocking transmission of dengue virus (Bian, Xu, Lu, Xie, & Xi, 2010;Bull & Turelli, 2013;Hoffmann et al., 2011;McMeniman et al., 2009;O'Neill, 2018;Walker et al., 2011). This approach was initially aimed at shortening mosquito life span below the extrinsic incubation period of the virus (McMeniman et al., 2009), but in the course of these experiments it was discovered that transinfection of Ae. aegypti with Wolbachia strain wMelPop also blocks dengue and chikungunya virus infections of the mosquito (Moreira et al., 2009). A successful large field trial in Australia showed a stable establishment and slow but steady spread of released Ae. aegypti transinfected with wMel in the study area (Schmidt et al., 2017).
However, if a population of Ae. aegypti were to harbor an autochthonous strain of Wolbachia, then this native strain would have a high potential to prevent invasion of a virus-blocking strain that exhibits incompatibility with the native strain (Hoffmann, Ross, & Rasic, 2015). This effect was demonstrated in a study of Ae. albopictus, wherein the wMel transinfected line produced complete bidirectional incompatibility with a wild-type line carrying wAlbA and wAlbB, with 0% hatch rate from crossing between females of either strain with males of the other strain (Blagrove, Arias-Goeta, Failloux, & Sinkins, 2012

| DNA isolation, bacterial 16S rDNA PCR, cloning, and sequencing
Mosquito specimens from traps were desiccated in most cases.
For each mosquito specimen, the abdomen was separated from the thorax by pulling gently with tweezers that were cleaned with 75% ethanol between samples. Abdomens were used for detecting associated microbiota. Metagenomic DNA was isolated individually from each abdomen using DNAzol following the manufacturer's protocol (Thermo Fisher Scientific). Briefly, one abdomen was homogenized in 100 µl DNAzol and centrifuged at 12,000 g for 10 min. Supernatant was transferred to a new tube, 50 µl ethanol was added, and the tube was centrifuged at 12,000 g for 10 min for DNA precipitation. The DNA pellet was dissolved in 30 µl H 2 O.

| Wolbachia PCR assays and sequencing
The PCR assays, using primer sets for the Wolbachia gatB and ftsZ gene from Baldo et al. (2006) (Table S1), were used for detecting Wolbachia in mosquitoes. PCR was run using 2× PCR master mix (MCLAB) with a primer concentration of 0.2 µM and the following cycling parameters: 35 cycles of denaturing at 94°C for 15 s, annealing at a temperature optimal for the amplicon (Table S1)  To validate the Wolbachia detection in Ae. aegypti, we designed strain-specific primers to amplify fragments from two Wolbachia genes encoding phosphoesterase (PE) and diaminopimelate epimerase (DE) based on the draft genomes of wAlbB (Mavingui et al., 2012) and wAlbA (GenBank accession NWVK00000000.1). The sequences of the two genes have distinctive interstrain differences, enabling the design of strain-specific primers (Table S1). A subset of specimens that were positive from the first or second PCR were subjected to the validation PCR and sequencing. The products were sequenced at MCLAB, and the sequences were deposited in GenBank; the accession numbers are presented in Table S2.

| Loop-mediated isothermal amplification (LAMP) assay
Loop-mediated isothermal amplification (LAMP) was developed as an additional assay for the detection of Wolbachia in mosquitoes.

| Maternal transmission of Wolbachia in Las Cruces strain
In August 2017, a colony of Ae. aegypti was established from pupae (n = 8) collected from a larval habitat in a residential area in Las

| Prevalence of Wolbachia in Ae. aegypti and Ae. albopictus populations in New Mexico
Aedes aegypti and Ae. albopictus occur in the state of New Mexico (Hahn et al., 2017(Hahn et al., , 2016

| Prevalence of Wolbachia in Ae. aegypti and Ae. albopictus in southeastern Texas
In 2018

| LAMP assays
The LAMP assay is a sensitive method for detecting target DNA with low abundance in a sample (Notomi et al., 2000).

Wolbachia strain A & B (%) A (%) B (%) Total no. (%)
Male (51) Ae. albopictus specimen could be detected at 100 times dilution of template, but not at 500 times dilution, while the infected Ae. aegypti specimen could not be detected at 20 times dilution. Figure 2 shows a representative result of LAMP assay on the Wolbachia-positive and Wolbachia-negative specimens from New Mexico, Florida, and Texas.

| Maternal transmission of Wolbachia occurs in Las Cruces strain
Wolbachia are known to be transmitted vertically from female to the offspring  As shown in Figure

| D ISCUSS I ON
Wolbachia is commonly associated with wild Ae. albopictus around the world. However, Wolbachia has not been detected in wild Ae. aegypti in previous surveys until recently. In the study by Coon et al. of Wolbachia in the Ae. aegypti population was not investigated in that study (Coon et al., 2016). Similarly, Wolbachia 16S OTUs were detected in two pools of 10 and 25 specimens of Ae. aegypti, respectively, collected in the suburb and urban areas of Thailand (Thongsripong et al., 2017). In a study by Hegde et al. (2018) So cross contamination was not a concern in our study. shown). Wolbachia titer shows striking variation in infected individuals of Ae. albopictus (Ahantarig, Trinachartvanit, & Kittayapong, 2008;Calvitti, Marini, Desiderio, Puggioli, & Moretti, 2015).  Hoffmann et al., 2011;Schmidt et al., 2017). The wAlbB has been successfully introduced into Ae. aegypti to form a line with inherited infection (Xi, Khoo, & Dobson, 2005). Interestingly, the Toll and IMD pathways favor establishment and maintenance of wAlbB infection in the line; the knockdown of Toll and IMD by RNA interference reduces the wAlbB load, while the transgenic activation of Toll and IMD increases the load (Pan et al., 2018). It appears that transinfected Wolbachia can exploit host immunity for a symbiotic association. Our survey revealed the prevalence of wAlbB in Ae. aegypti natural populations in New Mexico, and an infected colony has been established from wild-collected mosquitoes. This provides an opportunity to study the natural Ae. aegypti -Wolbachia association and its impact on various mosquito life traits, such as reproductive manipulation as well as interference with viral transmission.

ACK N OWLED G M ENTS
We

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.