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Article

Wolbachia Surface Protein (wsp) Gene Sequencing of Strains A and B in Native Aedes albopictus of Mérida, Yucatán

by
Henry Puerta-Guardo
1,2,*,
Yamili Contreras-Perera
2,
Silvia Perez-Carrillo
2,3,
Azael Che-Mendoza
2,
Karina Jacqueline Ciau-Carrillo
1,
Manuel Parra-Cardeña
1,
Iram Rodriguez-Sanchez
4,
Mayra A. Gomez-Govea
4,
Anuar Medina-Barreiro
2,
Guadalupe Ayora-Talavera
1,
Norma Pavia-Ruz
1,
Abdiel Martin-Park
2,* and
Pablo Manrique-Saide
2
1
Centro de Investigaciones Regionales, Dr. Hideyo Noguchi, Universidad Autonoma de Yucatán (UADY), Mérida C.P. 97315, Yucatán, Mexico
2
Unidad Colaborativa de Bioensayos Entomológicos (UCBE) y del Laboratorio de Control Biológico (LCB) para Ae. aegypti, Universidad Autónoma de Yucatán (UADY), Campus de Ciencias Biológicas y Agropecuarias, Km. 15.5 Carr. Mérida-Xmatkuil s.n., Mérida C.P. 97315, Yucatán, Mexico
3
Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle (MIVEGEC), Research Institute for Development (IRD), Université Montpellier, 34090 Montpellier, France
4
Laboratorio de Fisiología Molecular y Estructural, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza C.P. 66455, Nuevo León, Mexico
*
Authors to whom correspondence should be addressed.
Biology 2025, 14(10), 1399; https://doi.org/10.3390/biology14101399
Submission received: 16 July 2025 / Revised: 14 September 2025 / Accepted: 22 September 2025 / Published: 13 October 2025
(This article belongs to the Special Issue Genomics and Bioinformatics in Microorganism from the Class Insecta)
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Simple Summary

The mosquito Aedes (Stegomyia) albopictus is an invasive species that has spread across nearly all continents and is increasingly associated with the transmission of Aedes-borne human viruses (ABVs), such as dengue (DENV), chikungunya (CHIKV), yellow fever (YFV), and Zika (ZIKV). Wolbachia-based strategies are currently among the most innovative alternative biological methods to control vector populations. Wolbachia is an endosymbiotic bacterium known to interfere with the reproductive mechanisms of vectors and, consequently, with virus replication. Previous studies have demonstrated that Ae. albopictus is naturally associated with two Wolbachia strains, A and B. This study provides additional wsp gene DNA sequencing evidence confirming that native populations of Ae. albopictus in Yucatán—an arbovirus-endemic region—naturally harbor both Wolbachia strains A and B. These findings present an opportunity to strategically plan future surveillance and control programs targeting Ae. albopictus through Wolbachia strain B-based approaches, with the potential to lower the burden of arbovirus diseases in human populations.

Abstract

Aedes (Stegomyia) albopictus (Skuse), a mosquito vector with increasing capacity to transmit human arboviral infections worldwide, naturally harbors the intracellular bacterium Wolbachia spp. This relationship has been observed in native mosquito populations of Ae. albopictus around the world and, more recently, in Mérida, Yucatán, México (abbreviated hereafter as MID). This study provides additional evidence confirming the presence of Wolbachia A (wAlbA) and B (wAlbB) strains in field-collected Ae. albopictus of Mérida, based on wsp gene DNA sequencing analyses of 14 positive PCR samples. Maximum likelihood (ML) analyses of the wsp gene showed high nucleotide sequence homology between Wolbachia from Merida and the globally reported strains A and B, suggesting that these two Wolbachia strains have remained stable in Ae. albopictus over time, regardless of the geographical area. To the best of our knowledge, this is the first report of Wolbachia gene sequencing in native populations of Ae. albopictus in Yucatán, Mexico. Currently many Aedes mosquitoes (e.g., Ae. aegypti) harboring intracellular Wolbachia B bacteria are being released as part of arbovirus and mosquito control programs in Yucatan and globally. Understanding the presence and stability of Wolbachia strains across different Aedes host backgrounds is crucial to ensuring the long-term effectiveness of biological interventions aimed at controlling mosquito populations and arbovirus transmission in endemic areas.

1. Introduction

Wolbachia is a diverse genus of obligate intracellular Gram-negative α-Proteobacteria (order: Rickettsiales) that can be maternally transmitted. Wolbachia have been suggested to infect at least 20% of arthropod species, where an estimated 40–65% are insects, among which 28% are mosquito species, as well as filarial nematodes of mammals and plants [1,2]. All Wolbachia strains are classified as a single species, Wolbachia pipientis, phylogenetically divided into 16 clades named supergroups, denoted from A to Q, mainly based on multilocus sequence typing (MLST) analysis as well as on amino acid sequence analyses of the Wolbachia Surface Protein (wsp) [3,4,5].
Wolbachia displays tropism for somatic and reproductive tissues of arthropod hosts, where they can manipulate their reproductive abilities by inducing cytoplasmic incompatibility (CI) [6,7]. CI confers a reproductive advantage to Wolbachia-infected females over uninfected females, with subsequent persistence and spread of Wolbachia in mosquito populations [8]. This reproductive manipulation has attracted significant interest as it plays a critical role in host biology, ecology, and evolution, as well as in the development of a symbiont-based Wolbachia-based method termed the Incompatible Insect Technique (IIT) for the control of insects of medical and agricultural importance [9].
Additionally, Wolbachia endosymbionts have been evaluated for their ability to suppress Aedes-borne human diseases (ABDs), including dengue, Zika, and chikungunya [10,11,12,13]. Along with Aedes (Stegomyia) aegypti, considered the main vector for many of these arboviral diseases, the commonly known Asian Tiger mosquito Ae. albopictus is a competent vector of ABDs [14,15]. Ae. albopictus is an aggressive biting mosquito that has invaded and colonized many countries in the Americas, Europe, Africa, and the Pacific [16,17,18].
Unlike Ae. aegypti, Ae. albopictus has been shown to naturally harbor Wolbachia, primarily strains from supergroups A and B [19,20,21,22]. Worldwide, although various programs using the release of Wolbachia-infected mosquitoes to suppress/replace natural mosquito populations have been carried out in several countries, including Australia, Brazil, Colombia, Mexico, Indonesia, and Vietnam [23,24,25], in the Americas, there are a handful of studies describing the presence of Wolbachia in wild populations of Aedes mosquitoes, including Ae. albopictus [26,27,28,29,30]. Wolbachia strains and their subgroups present in Ae. albopictus can serve as markers for inferring the geographic origin of mosquito populations, since both the Aedes spp. reservoir and bacteria strains often have specific geographic distributions, reflecting dispersal patterns and evolutionary retreats [1,2,5,19,20]. Therefore, analyzing the distribution of these strains can identify phylogenetic relationships and trace the biogeographic history of Ae. albopictus populations in different regions.
The wsp gene is commonly used as a marker for screening Wolbachia presence/infection, as well as for strain typing and phylogenetic analyses [3,4,31]. In this study, we obtained nucleotide sequences of the wsp gene obtained from Ae. albopictus that were positive for Wolbachia strains A and B according to PCR, and conducted DNA sequencing analyses to confirm the presence of these two strains and their relationship to other Wolbachia strains previously reported in Ae. albopictus and other mosquito species worldwide. In Mexico, the Ministry of Health has incorporated a Wolbachia-based strategy for the replacement of Aedes aegypti populations as part of the national dengue and arboviral disease control plan. It is anticipated that the use of Wolbachia-infected Aedes mosquitoes, including Ae. aegypti and Ae. albopictus, will expand in the coming years, not only in Mexico but also in other Latin American countries and globally. Therefore, it is essential to expand the understanding of the Wolbachia bacteria circulating in local mosquito populations, as this will facilitate the design of future Wolbachia-based intervention strategies for the control and management of mosquito-borne diseases in endemic areas.

2. Materials and Methods

2.1. Mosquito Sample Collection and Identification

In 2019 from April to December, a total of 45 Ae. albopictus adult mosquitoes were collected in distinct suburban areas of Mérida, Yucatán (abbreviated hereafter as MID) using outdoor BG-sentinel traps, as previously described in [30,32]. The identity of all mosquitoes was established based on standard taxonomic keys [33]. Field collections were performed every week from April to December of 2019 in three suburban areas located at the periphery of the city of Merida in the Peninsula of Yucatan: San Pedro Chimay (20°51′55″ N 89°34′46″ O), Hacienda Tahdzibichen (20°53′06″ N 89°35′52″ O), and Tekik de Regil (hereafter Tekik; 20°48′59″ N 89°33′39″ O). The average altitude of the localities is 9 m above sea level, with annual average temperatures ranging from 26 °C to 27 °C (36 °C max–18 °C min), relative humidity of 70–75%, and two distinct climate phases during the year: a rainy season, from May/June to October, with a rainfall of 882.5 mm, and a dry season, from November to April, with rainfall of 167.9 mm. The sociodemographic features of these localities include an average of 1200 inhabitants per locality with an average of 6 households and 31 inhabitants per hectare. These areas share similar urban and ecological characteristics, such as type of housing, and share large vegetated backyards with vegetation (coverage > 60%) [30].

2.2. DNA Extraction and Wolbachia PCR Amplification

Total genomic DNA was extracted from individual mosquitoes (n = 19, female = 11; male = 8) using a Blood and Tissue DNeasy Kit© (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, with some in house modifications, as previously described in [30]. Total extracted DNA was eluted using nuclease-free water and quantified by spectrophotometry using a Nanodrop One Microvolume UV-Vis system (Thermo Scientific, Madison, WI, USA). PCR amplification of the Wolbachia DNA genome was performed in PCR reactions of 15 µL containing a DNA template (200 ng per reaction), PCR buffer (10×), MgCl2 (50 mM), dNTPs mix (10 mM), Taq DNA polymerase (5 U/µL), RNAse/DNAse-free water, and forward and reverse primers (10 µM) to amplify the DNA genome from Wolbachia strains A (wAlbA) and B (wAlbB) as follow: forward primers named 328F (5′-CCAGCAGATACTATTGCG-3′) and 183F (5′-AAGGAACCGAAGTTCATG-3′) for the wAlbA and wAlbB strains, respectively. For both strains, the only reverse primer 691R (5′-AAAAATTAAACGCTACTCCA-3′) was used as previously described in [30,34,35]. The PCR amplification program was performed using the following parameters: initial denaturation at 95 °C for 5 min; 40 cycles of denaturation at 95 °C for 1 min, Tm annealing at 55 °C for 1 min and extension at 72 °C for 1 min; and final extension at 72 °C for 3 min. All PCR amplifications were performed using a Mastercycler epgradient S PCR thermal cycler (Eppendorf AG, Hamburg, Germany). The presence of Wolbachia in Ae. albopictus was screened based on the amplification of 300–600 bp fragments. PCR amplicons were separated on agarose gel at 1.5% and visualized using a ChemiDocTM MP Imaging system with Image Lab software V 2.0.1 9 (Bio-Rad Laboratories, Hercules, CA, USA).

2.3. Wolbachia wsp Gene DNA Sequencing Analyses

Double-positive PCR amplicons [n = 30; walbA(+) = 15; wAlbB(+) = 15] were enzymatically cleaned up using ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Scientific) and then submitted for standard Sanger DNA sequencing (500 ng per sample) to PSOMAGEN, Inc. (formerly Macrogen Corp., Rockville, MD, USA), using the two sets of forward primers 328F and 182F, and one reverse primer, 691R (5 pmol/μL), as described above. A total of 60 linear DNA sequences were obtained from 19 analyzed samples using three sets of primers. The obtained partial sequences were processed for editing and analyzed using Geneious software version 6.1 (Biomatters. available at http://www.geneious.com accessed on 1 June 2023). Alignment between reference sequences and sequences in the study group was edited and aligned using the MUSCLE tool (v5.0-5.3, https://drive5.com/software.html accessed on 1 June 2023) supported by Geneious software version 6.1. To do so, a pairwise MUSCLE alignment analysis of all raw experimental nucleotide sequences [328F vs. 691R (n = 30), and 182F vs. 691R (n = 30), (n = 60 total sequences)] was performed against a group of reference sequences (n = 100), obtained from the GenBank dataset of the National Center for Biotechnology Information (NCBI), for the wsp gene of the wAlbA and wAlbB strains. This process allowed us to remove poorly aligned positions and to obtain non-ambiguous sequence alignments to be used in further analyses. A final consensus sequence was generated for each group of samples (n = 28), hereafter identified as wAlbA-MID (n = 14) and wAlbB-MID (n = 14). These consensus sequences were processed through the Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/) to identify regions of similarities between biological sequences existing in the GenBank. Sequencing analyses were performed to compare Wolbachia sequences in the study group with those nucleotide sequences of Wolbachia strains A (n = 78) and B (n = 58) reported for Ae. albopictus in the GenBank database.
Rooted and unrooted maximum likelihood phylogenetic trees of the wsp gene were built using Geneious software version 6.1 to show the relationship between the wsp gene of representative Wolbachia strains detected in Ae. albopictus of Yucatán and distinct Wolbachia strains representing different supergroups described to infect Ae. albopictus worldwide. The Jukes–Cantor genetic distance substitution model was used for Bayesian analysis to infer the evolutionary history of the Wolbachia nucleotide sequences included in the study [36]. The bar indicates a branch length of 0.01. Bootstrap values were obtained from 1000 replicates (Figure 1). The GenBank accession numbers for each nucleotide sequence used in the study are included in each figure (Figure 2 and Figure 3). Finally, to provide complementary information to the wsp gene DNA sequencing data and allow a more detailed description of the potential intra-strain variability of Wolbachia strains A and B in Ae. albopictus from Yucatán, we analyzed distinct genetic diversity indices, including haplotype richness (number of haplotypes), haplotype diversity (Hd), nucleotide diversity (π), and mean genetic distance (RStudio. V4.5.1, https://cran.r-project.org/bin/windows/base/ accessed on 1 June 2023).

3. Results and Discussion

3.1. Detection of Wolbachia Strains A and B in Field-Collected Aedes albopictus from Yucatán

In previous studies, we demonstrated the occurrence of Wolbachia infection (~42%, 19/45) in field-caught Ae. albopictus collected in the suburban areas of the municipality of Mérida, Yucatán [30]. In this study, 15 out of 19 Ae. albopictus specimens showed PCR amplifications for both set of primers specific to the molecular detection of Wolbachia strains A and B of Wolbachia (molecular weights between 400 and 600 bp) (Figure 1A). These fragments belonged to supergroups A and B (Table 1). The analyzed sequences belonging to the Wolbachia strain group A (wAlbA-MID) had variable lengths of 185 and 314 nucleotides (n = 14). On the other hand, the length of the sequences belonging to the Wolbachia strain group B (wAlbB-MID) varied from 252 to 377 nucleotides length (n = 14) (Supplementary Materials).
Furthermore, multiple alignment of several representative nucleotide sequences of the study group [wAlbA-MID (n = 4), wAlbB-MID (n = 4)] and the nucleotide sequence of the wsp gene of the reference strain Wolbachia pipientis (accession: AF020070.1) confirmed these similarities and the location of the amplified nucleotide sequences from Ae. albopictus of Yucatán within the wsp gene of Wolbachia (Supplementary Figure S1A,B). Additionally, these results confirmed that Ae. albopictus harbors two Wolbachia strains from supergroups A and B.
Based on the sequence of the wsp gene, other studies have identified high variability between different Wolbachia isolates, which can be used to resolve the sequencing relationships of different Wolbachia strains [37,38]. Here, we performed a pairwise alignment (MUSCLE) analysis of nucleotide sequences from the wsp gene fragments amplified by PCR in Ae. albopictus of Yucatán. This analysis identified significant similarities within each study group: the wAlbA-MID group (n = 14) showed identity percentages ranging from 98.9 to 100%, and the wAlbB-MID group (n = 14) showed identities ranging from 96.9 and 100% (Supplementary Tables S1 and S2). The results of the sequence alignment, as well as consensus sequences for wAlbA-MID and wAlbB-MID, are presented in Supplementary Figures S2 and S3.
As expected, a comparison between the two groups (wAlbA-MID vs. wAlbB-MID) revealed limited similarities, ranging from 69.4% to less than 78% (Supplementary Table S3), clearly indicating the presence of two Wolbachia strain supergroups in Ae. albopictus. Further analysis using the tree builder tool (Geneious software) generated a cladogram that distinctly shows two separated clusters, corresponding to the wAlbA-MID group and the wAlbB-MID group (Figure 1B).
These results together support and confirm that the wAlbA-MID and wAlbB-MID strains represent two related (in terms of sequencing) but distinct groups of Wolbachia strains circulating in wild-caught Ae. albopictus of Yucatán [19,20,22,30,34]. A preliminary analysis of distinct genetic diversity indices revealed that the number of haplotypes (haplotype richness) was higher among Wolbachia strain A (7 of 14) compared to strain B (4 of 14), indicating that strain A is richer in variants. In terms of haplotype diversity (Hd), which reflects the probability that two randomly chosen sequences differ, Wolbachia strain A showed high diversity (Hd = 0.81), whereas strain B showed moderate diversity (Hd = 0.63). Similarly, nucleotide diversity (π), which measures the average number of nucleotide differences per site between two sequences, was slightly higher in Wolbachia strain A (π = 0.0063) compared to strain B (π = 0.0039), indicating that both strains are highly conserved, with strain A being slightly more variable. However, this observed genetic variability within Wolbachia strains A and B of Ae. albopictus from Yucatan must be interpreted with caution. This study was limited by the collection of mosquitoes from a single location, resulting in a small sample size (n = 45) and a limited number of nucleotide sequences (n = 19). These constraints likely reduced the resolution of intra-population genetic variability. To better understand the extent of Wolbachia genetic diversity, future studies should involve more comprehensive field collection. This includes increasing the number of sampled mosquitoes and expanding sampling efforts to include additional urban and suburban locations, ideally with replication across ecological and geographic gradients.
Biology 14 01399 g001
Figure 1. Molecular characterization and nucleotide sequence relationship between Wolbachia strains in Ae. albopictus of Yucatan. (A) PCR amplification and sequencing of a DNA fragment of the wsp gene of Wolbachia strains A and B in Ae. albopictus. Amplicons with lengths of ~300–400 and ~500–600 base pairs (bp) were separated by electrophoresis on agarose gel (1.5%) (lanes 1–19). DNA Marker: 100 bp (lane 1). (B) Rooted maximum likelihood phylogenetic tree of the wsp gene of 28 Wolbachia strains detected in Ae. albopictus of Yucatan. Strains are designated as wAlbA-MID (n = 14) and wAlbB-MID (n = 14), which include the initial for Wolbachia (w) followed by the abbreviated names of their host species (Ae. albopictus: Alb), the Wolbachia supergroup that these strains may belong to (A and B), an assigned sample number (1–14), and the collection site (Merida: MID). The tree was inferred using Neighbor-Joining consensus tree Nucleotide alignment (MUSCLE) of all edited Wolbachia nucleotide sequences included in the study group and rooted with the Wolbachia endosymbiont outer surface protein precursor (wsp) gene of the Ostrinia furnacalis strain (wfurA) used as an outgroup (accession: EU294311.1). The Jukes–Cantor genetic distance substitution model was used for Bayesian analysis, built using Geneious Tree Builder (Geneious Sofware v6.1.8). The numbers on the branches indicate percentage bootstrap support for major branches obtained from 1000 replicates. Only bootstrap values above 60% that support greedy clustering are shown. The bar indicates a branch length of 0.01.
Figure 1. Molecular characterization and nucleotide sequence relationship between Wolbachia strains in Ae. albopictus of Yucatan. (A) PCR amplification and sequencing of a DNA fragment of the wsp gene of Wolbachia strains A and B in Ae. albopictus. Amplicons with lengths of ~300–400 and ~500–600 base pairs (bp) were separated by electrophoresis on agarose gel (1.5%) (lanes 1–19). DNA Marker: 100 bp (lane 1). (B) Rooted maximum likelihood phylogenetic tree of the wsp gene of 28 Wolbachia strains detected in Ae. albopictus of Yucatan. Strains are designated as wAlbA-MID (n = 14) and wAlbB-MID (n = 14), which include the initial for Wolbachia (w) followed by the abbreviated names of their host species (Ae. albopictus: Alb), the Wolbachia supergroup that these strains may belong to (A and B), an assigned sample number (1–14), and the collection site (Merida: MID). The tree was inferred using Neighbor-Joining consensus tree Nucleotide alignment (MUSCLE) of all edited Wolbachia nucleotide sequences included in the study group and rooted with the Wolbachia endosymbiont outer surface protein precursor (wsp) gene of the Ostrinia furnacalis strain (wfurA) used as an outgroup (accession: EU294311.1). The Jukes–Cantor genetic distance substitution model was used for Bayesian analysis, built using Geneious Tree Builder (Geneious Sofware v6.1.8). The numbers on the branches indicate percentage bootstrap support for major branches obtained from 1000 replicates. Only bootstrap values above 60% that support greedy clustering are shown. The bar indicates a branch length of 0.01.
Biology 14 01399 g001

3.2. Nucleotide Comparison Analyses with Wolbachia Strains in Aedes albopictus

Consensus tree analyses of the wAlbA-MID (n = 14) and wAlbB-MID (n = 14) strains, along with reference Wolbachia strains from different supergroups (A, B, C, D, and F; see Table 2), revealed distinct phylogenetic clustering. The wAlbA-MID strains grouped closely with members of supergroup A, including the wRi and wHa strains previously identified in Drosophila simulans [39,40], and wMel identified from D. melanogaster [41]. In contrast, wAlbB-MID strains clustered with supergroup B strains such as wAlbB from Ae. albopictus [42], as well as wPip from Culex quinquefasciatus [43] (Figure 2). As shown in Figure 2, both groups showed low sequence identity (<35%) with Wolbachia strains from unrelated supergroups, including the wCle strain from Cimex lectularius (Cimicidae) [44], the wBm strain from Brugia malayi (Filariidae), and the wOo strain from Onchocerca ochengi (Onchocercidae) [45,46]. A detailed comparison of representative nucleotide sequence identities, based on alignment analyses, is provided in Supplementary Table S4. These analyses further confirm the distinct phylogenetic identities of the two Wolbachia strain groups detected in Ae. albopictus of Yucatán.
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Figure 2. The Wolbachia strains in Aedes albopictus of Yucatan belong to supergroups A and B. Unrooted maximum likelihood phylogenetic tree showing the relationship between the wsp gene of Wolbachia strains detected in Ae. albopictus of Yucatan (wAlbA-MID, n = 14; wAlbB- MID, n = 14) (in bold) and distinct Wolbachia strains representing different Wolbachia supergroups described to infect Ae. albopictus elsewhere. Wolbachia supergroup affiliations are given in colored circles and indicated by the letter inside the circle next to the host species names as follows: Drosophila simulants Riverside (wRi) (accession: AF02070.1., NC_012416.1) and Hawaii (wHa) strains (accession: AF020068.1), and D. melanogaster (wMel) strain (accession: NC_002978.6) from supergroup A; Culex quinquefasciatus (wPip) (accession: AF020060.1) and Ae. albopictus (wAlbB) strains from supergroup B (accession: CP041924.1); Onchocerca ochengi (wOo) strain of supergroup C (accession: HE660029.1); Brugia malayi (wBm) strain representing supergroup D (accession: NC_006833.1), and Cimex lectularius (wCle) strain in supergroup F. Taxon labels correspond to Wolbachia strain names as well as Genbank accession numbers. Nucleotide sequences belonging to the study group as well as their species of origin are depicted in bold. Primarily greedy clustering was used as the consensus method. The numbers on clades correspond to bootstrap values, presented as percentages, from 1000 itinerations, as well as consensus support (%) between clades. The scale bar corresponds to inferred evolutionary changes and indicates a branch length of 1.0. The Jukes–Cantor genetic distance substitution model was used for Bayesian analysis. The tree was inferred using Neighbor-Joining consensus tree Nucleotide alignment (MUSCLE) of all edited Wolbachia nucleotide sequences and built using Geneious Tree Builder (Geneious Sofware v6.1.8). The numbers on branches indicate the percentage bootstrap support for major branches obtained from 1000 replicates. Only bootstrap values of 60% or more obtained by the consensus method are shown. The bar indicates a branch length of 0.01.
Figure 2. The Wolbachia strains in Aedes albopictus of Yucatan belong to supergroups A and B. Unrooted maximum likelihood phylogenetic tree showing the relationship between the wsp gene of Wolbachia strains detected in Ae. albopictus of Yucatan (wAlbA-MID, n = 14; wAlbB- MID, n = 14) (in bold) and distinct Wolbachia strains representing different Wolbachia supergroups described to infect Ae. albopictus elsewhere. Wolbachia supergroup affiliations are given in colored circles and indicated by the letter inside the circle next to the host species names as follows: Drosophila simulants Riverside (wRi) (accession: AF02070.1., NC_012416.1) and Hawaii (wHa) strains (accession: AF020068.1), and D. melanogaster (wMel) strain (accession: NC_002978.6) from supergroup A; Culex quinquefasciatus (wPip) (accession: AF020060.1) and Ae. albopictus (wAlbB) strains from supergroup B (accession: CP041924.1); Onchocerca ochengi (wOo) strain of supergroup C (accession: HE660029.1); Brugia malayi (wBm) strain representing supergroup D (accession: NC_006833.1), and Cimex lectularius (wCle) strain in supergroup F. Taxon labels correspond to Wolbachia strain names as well as Genbank accession numbers. Nucleotide sequences belonging to the study group as well as their species of origin are depicted in bold. Primarily greedy clustering was used as the consensus method. The numbers on clades correspond to bootstrap values, presented as percentages, from 1000 itinerations, as well as consensus support (%) between clades. The scale bar corresponds to inferred evolutionary changes and indicates a branch length of 1.0. The Jukes–Cantor genetic distance substitution model was used for Bayesian analysis. The tree was inferred using Neighbor-Joining consensus tree Nucleotide alignment (MUSCLE) of all edited Wolbachia nucleotide sequences and built using Geneious Tree Builder (Geneious Sofware v6.1.8). The numbers on branches indicate the percentage bootstrap support for major branches obtained from 1000 replicates. Only bootstrap values of 60% or more obtained by the consensus method are shown. The bar indicates a branch length of 0.01.
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We also compared the two wAlbA-MID and wAlbA-MID groups of nucleotide sequences (Figure 1) with Wolbachia sequences from supergroups A (n = 78) and B (n = 58) found in Ae. albopictus from various geographical regions worldwide (Figure 3A,B). Pairwise multiple sequence alignment revealed that the wAlbA-MID group exhibits high sequence identities (>98–100%) with Wolbachia strains of supergroup A detected in Ae. albopictus from countries including India (JX476004.1, JX476007.1), the USA (e.g., AF020058.1), Malaysia (e.g., KX573028.1, KC004024.1, MH418426.1), Pakistan (MH503767.1), Taiwan (AY462864.1), México (e.g., MK684349.1, KX118691.1), Italy (EU727139.1), China (KU738324.1), and Sri Lanka (MH777434.1) (Figure 3A, Table 3).
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Figure 3. Nucleotide sequence comparison of wAlbA and wAlb-B MID with Wolbachia strains belonging to supergroups A and B found in Ae. albopictus worldwide. Unrooted phylogenetic tree layout for comparison of wsp gene sequences obtained from all Wolbachia strains detected in Ae. albopictus of Yucatan: (A) wAlbA-MID (n = 14) and (B) wAlbB-MID (n = 14) (in bold) and other nucleotide sequences of Wolbachia strains detected in Ae. albopictus reported in GenBank [supergroup A (n = 78); supergroup B (n = 58)]. Strains of the study group are designated as wAlbA-MID or wAlbB-MID, as explained in Figure 1B. The tree was inferred using Neighbor-Joining consensus tree Nucleotide alignment (MUSCLE) of all edited Wolbachia nucleotide sequences included in the study group and other Wolbachia sequences of Ae. albopictus worldwide. The Jukes–Cantor genetic distance substitution model was used for Bayesian analysis, and the phylogeny tree was built using Geneious Tree Builder (Geneious Sofware v6.1.8). The taxon labels correspond to the Wolbachia strain names in the study group as well as the GenBank accession numbers. The numbers on branches indicate percentage bootstrap support for major branches obtained from 1000 replicates. Only bootstrap values above 60% or more obtained by the consensus method are shown. The bar indicates a branch length of 2.
Figure 3. Nucleotide sequence comparison of wAlbA and wAlb-B MID with Wolbachia strains belonging to supergroups A and B found in Ae. albopictus worldwide. Unrooted phylogenetic tree layout for comparison of wsp gene sequences obtained from all Wolbachia strains detected in Ae. albopictus of Yucatan: (A) wAlbA-MID (n = 14) and (B) wAlbB-MID (n = 14) (in bold) and other nucleotide sequences of Wolbachia strains detected in Ae. albopictus reported in GenBank [supergroup A (n = 78); supergroup B (n = 58)]. Strains of the study group are designated as wAlbA-MID or wAlbB-MID, as explained in Figure 1B. The tree was inferred using Neighbor-Joining consensus tree Nucleotide alignment (MUSCLE) of all edited Wolbachia nucleotide sequences included in the study group and other Wolbachia sequences of Ae. albopictus worldwide. The Jukes–Cantor genetic distance substitution model was used for Bayesian analysis, and the phylogeny tree was built using Geneious Tree Builder (Geneious Sofware v6.1.8). The taxon labels correspond to the Wolbachia strain names in the study group as well as the GenBank accession numbers. The numbers on branches indicate percentage bootstrap support for major branches obtained from 1000 replicates. Only bootstrap values above 60% or more obtained by the consensus method are shown. The bar indicates a branch length of 2.
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Interestingly, lower sequence similarities (<80%) were observed between Wolbachia strains A from Brazil (e.g., GQ205162.1), India (e.g., JX476005.1), China (e.g., KU738376.1), México (e.g., MK695179.1), and Singapore (e.g., MT645169.1). These strains were separately grouped into a distinct clade.
For the wAlbB-MID group, the multiple alignment analyses revealed high sequence similarity (>98%) when compared Wolbachia strain B previously described in various countries, including India (JX629464.1), Malaysia (JX129186.1, KF781999.1), Pakistan (KX650069.1), the USA (MG765533.1), Italy (EU727140.1), Taiwan (AY462863.1), China (KU738369.1), and Singapore (MT645169.1) (Figure 3B, Table 4). In contrast, fewer sequences, such as those reported in Mexico (e.g., KX118691.1, MK684351.1) and China (e.g., KU738324.1), showed less than 90% sequence similarity (<80%) with the wAlbB-MID group of Merida.
Overall, our results suggest that most of the Wolbachia strains naturally occurring in populations of Ae. albopictus of Mérida are very similar to other Wolbachia strains belonging to supergroups A and B of Ae. albopictus described worldwide. Interesting, nucleotide sequences belonging to the wAlbA-MID group showed lower sequence identities (<80%) compared to Wolbachia strains previously identified as part of supergroup A in Brazil (e.g., GQ205162.1). In contrast, the results for Wolbachia strains in supergroup B were more consistent with previous studies from Asia (Sri Lanka, India, China, Malaysia, and Thailand), which reported that Ae. albopictus and, as well as other Aedes-related species such as Ae. Quadrivittatus, harbored Wolbachia strain B with strong bootstrap support [50,51,52,53,54]. Additional genetic markers such as MLST, including additional Wolbachia genes (e.g., gatB, coxA, hcpA, ftsZ, and fbpA), will enable more in-depth phylogenetic analyses to better define the genetic diversity among Wolbachia strains in Ae. albopictus and other Aedes species. It is well established that Ae. albopictus naturally carries either one strain (wAlbA or wAlbB) or both strains simultaneously (wAlbA and wAlbB) [46,50].
Despite this, Ae. albopictus continues to expand as an important vector for arbovirus transmission [14,15,16,17]. From a vector control perspective, this poses a challenge for the use of Ae. albopictus in Wolbachia-based control programs, which aim to suppress mosquito populations or reduce arbovirus infections in humans. Notably, recent evidence shows that Ae. albopictus artificially infected with four different Wolbachia strains (wMel, wMelPop, wRi, and wPip) have established stable lines with diverse CI patterns and reduced vector capacity for arbovirus transmission [55,56]. These findings support the feasibility of applying mass rearing and integrated SIT to control Ae. albopictus, as has already been implemented for Ae. aegypti worldwide.

4. Conclusions

Here, we confirmed the presence of two Wolbachia strains (supergroups A and B) in field-collected Ae. albopictus from suburban areas of Mérida, Yucatán, through wsp gene sequencing. These results align with previous reports of Ae. albopictus naturally harboring Wolbachia A and B worldwide, showing high sequence homology with strains from Asia and North America, but lower similarity to strains from Brazil, suggesting regional differentiation. Historical records indicate that Ae. albocpictus was introduced in the Americas in 1983 via used-tire shipments to the USA, and later detected in Brazil (1986) and Mexico (1988) [57]. The distribution of related and unrelated Wolbachia strains across distant regions likely reflects mosquito dispersal through natural migration or human activities, such as trade. These findings suggest historical connectivity among mosquito populations, with phylogenetic analysis offering insights into the biogeographic history of Ae. albopictus.
In Merida, Ae. albopictus has only recently been identified in urban and periurban areas of this municipality [32], limiting specimen availability for this study. Despite this constraint, our work provides the first description of Wolbachia gene sequencing in Aedes mosquitos of Yucatán. These results establish a foundation for future research with larger sample sizes and broader collection areas to assess the abundance, distribution, and diversity of Wolbachia strains in Aedes populations across Yucatán but also Mexico.
Although wsp genes are widely used in phylogenetic studies [58], single-gene analyses may be limited by recombination among Wolbachia strains [59]. Our complementary nucleotide diversity (π) analyses showed that strain A was slightly more diverse than strain B (π = 0.0063 vs. 0.0039), though both exhibited low variability, consistent with closely related endosymbionts such as Wolbachia A and B. Future studies incorporating additional markers (e.g., gltA, groEL, ftsZ) and multilocus sequence typing (MLST) [60,61] will better characterize Wolbachia diversity in Yucatán and México.
In conclusion, our results provide baseline evidence for the presence of Wolbachia strains A and B in wild Ae. albopictus populations of Yucatán. These findings can inform future Wolbachia-based intervention strategies, leveraging locally circulating Wolbachia strains to reduce mosquito populations and mitigate arboviral transmission in endemic regions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology14101399/s1, All supplementary material has been submitted along with the main manuscript. Figure S1. (A) Multiple nucleotide sequence alignment between representative wAlbA- MID (n = 4) and wAlbB-MID (n = 4) sequences and the wsp gene sequence of a reference Wolbachia strain (accession AF020070.1) (MUSCLE alignment: Geneious software version 6.1). Maximum number of iterations: 10. Agreement between DNA nucleotide sequences are shown in bright colors: adenine (A, red), thymine (T, green), cytosine (C, blue), and guanine (G, yellow). Strains are designated as wAlbA- MID and wAlbB-MID which includes the initial for Wolbachia (w) followed by the abbreviated name of their host species (Ae. albopictus: Alb), the Wolbachia supergroup that these strains may belong to (A and B), a sample designed number (1–14), and the collection site (Merida: MID). (B) Schematic representation of Wolbachia complete DNA genome depicting the nucleotide sequence location of the gene encoding for the outer membrane protein precursor (wsp) (~779 bp) (accession: AF020070). Figure S2. Multiple nucleotide sequence alignment view of the Wolbachia nucleotide sequences amplified from Ae. albopictus of Yucatan. A total of 28 DNA sequences corresponding to the wsp gene of the Wolbachia endosymbionts detected in Ae. albopictus of Yucatan were separately analyzed through the multiple alignment tool (MUSCLE) of the Geneious software version 6.1. Strains in the study group are designated as (A) wAlbA-MID and (B) wAlbB-MID which include the initial for Wolbachia (w) followed by the abbreviated name of their host species (Ae. albopictus: Alb), the Wolbachia supergroup that these strains may belong to (A and B), a sample designed number (1–14), and the collection site (Merida: MID). Nucleotide sequence length is indicated by the number above the set of sequences. Wolbachia endosymbiont outer surface protein precursor (wsp) gene of Ostrinia furnacalis strain (wfurA) was used as an outgroup control (accession: EU294311.1). Nucleotide alignment (MUSCLE) of all edited Wolbachia nucleotide sequences was performed using the Neighbor-joining consensus method. Maximum number of iterations used: 10. DNA nucleotide agreement between all sequences are shown in bright colors: adenine (A, red), thymine (T, green), cytosine (C, blue), and guanine (G, yellow). Agreements inside each study group are shown in gray. Bases matching at least 99% of the sequences. The percentage of sequence coverage and identity as well as the generated consensus sequence after multiple alignment analyses are shown at the top as indicated in the figure. Figure S3. Multiple nucleotide sequence alignment view of the Wolbachia nucleotide sequences amplified from Ae. albopictus of Yucatan. A total of 28 DNA sequences corresponding to the wsp gene of the Wolbachia endosymbionts detected in Ae. albopictus of Yucatan were analyzed through the multiple alignment tool (MUSCLE) of the Geneious software version 6.1. Strains in the study group are designated as (A) wAlbA-MID and (B) wAlbB-MID which include the initial for Wolbachia (w) followed by the abbreviated name of 568 their host species (Ae. albopictus: Alb), the Wolbachia supergroup that these strains may belong to (A and B), a sample designed number (1–14), and the collection site (Merida: MID). Nucleotide sequence length is indicated by the number above the set of sequences. Wolbachia endosymbiont outer surface protein precursor (wsp) gene of Ostrinia furnacalis strain (wfurA) was used as an outgroup control (accession: EU294311.1). Nucleotide alignment (MUSCLE) of all edited Wolbachia nucleotide sequences was performed using the Neighbor-joining consensus method. Maximum number of iterations used: 10. DNA nucleotide agreement between all sequences are shown in bright colors: adenine (A, red), thymine (T, green), cytosine (C, blue), and guanine (G, yellow). Agreements inside each study group are shown in gray. Bases matching at least 99% of the sequences. A small view of the phylogenetic organization of these sequences into a tree format is shown in blue (left side). Supplementary Table S1. Pairwise sequence comparison of the nucleotide sequences of wAlbA-MID strains (n = 14) detected in Aedes albopictus of Yucatan. Table S2. Pairwise sequence comparison of the nucleotide sequences of wAlbB-MID strains (n = 14) detected in Aedes albopictus of Yucatan. Table S3. Heatmap of the nucleotide sequence identity similarities (%) between all wAlbA-MID and wAlbB-MID strains found in Ae. albopictus of Yucatan. Table S4. Pairwise sequence comparison of representative nucleotide sequences of the wAlb-MID strains (n = 8) detected in Aedes albopictus of Yucatan and reference sequences belonging to multiple Wolbachia serogroups.

Author Contributions

Conceptualization, H.P.-G., Y.C.-P., A.M.-P. and P.M.-S.; methodology, H.P.-G., Y.C.-P., S.P.-C., K.J.C.-C., M.P.-C., A.M.-B., A.C.-M. and A.M.-P.; software, H.P.-G.; validation, H.P.-G., Y.C.-P., A.M.-P., S.P.-C. and A.M.-P.; formal analysis, H.P.-G., Y.C.-P., S.P.-C. and A.M.-P.; investigation, H.P.-G., Y.C.-P., S.P.-C., K.J.C.-C., M.P.-C., A.M.-P. and P.M.-S.; resources, H.P.-G., Y.C.-P., A.M.-P. and P.M.-S.; data curation, H.P.-G., Y.C.-P., S.P.-C., K.J.C.-C., M.P.-C., I.R.-S., M.A.G.-G., G.A.-T., N.P.-R. and A.M.-P.; writing—original draft preparation, H.P.-G., Y.C.-P., A.M.-P. and P.M.-S.; writing—review and editing, H.P.-G., Y.C.-P., I.R.-S., M.A.G.-G., G.A.-T., N.P.-R., A.M.-P. and P.M.-S.; visualization, H.P.-G., Y.C.-P. and A.M.-P.; supervision, H.P.-G., Y.C.-P. and A.M.-P.; project administration, H.P.-G., Y.C.-P., A.M.-P. and P.M.-S.; funding acquisition, A.M.-P. and P.M.-S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Fondo Mixto Consejo Nacional de Ciencia y Tecnología (CONACYT) (México)—Gobierno del Estado de Yucatán (project YUC-2017-03-01-556). Abdiel Martin-Park was supported by the Investigadoras e Investigadores por México program—Secihti. Yamili Contreras-Perera Postdoc.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study is included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We would like to thank the entomological teams at the Collaborative Unit for Entomological Bioassays (UCBE) and the Laboratory for Biological Control of Aedes aegypti (LCB) for their valuable technical support, and the Virology Lab at CIR-Biomedicas for providing the facilities to perform the molecular biology experiments. We also thank Juan Pirod Alayola at LCB for performing the haplotype analyses of the Wolbachia DNA sequences using RStudio.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
wAlbAWolbachia strain A of Ae. albopictus.
wAlbBWolbachia strain B of Ae. albopictus.
wspWolbachia surface protein
MIDMérida
ABVsAedes-borne human viruses
DENVDengue virus
ZIKVZika virus
CHIKVChikungunya virus
ABDAedes-borne human diseases

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Table 1. Nucleotide sequence similarities found between Wolbachia DNA fragments amplified from field-caught Ae. albopictus and nucleotide sequences reported in GenBank (NCBI) using the standard nucleotide BLASTN+2.17.0 (https://blast.ncbi.nlm.nih.gov/ accessed on 1 June 2023).
Table 1. Nucleotide sequence similarities found between Wolbachia DNA fragments amplified from field-caught Ae. albopictus and nucleotide sequences reported in GenBank (NCBI) using the standard nucleotide BLASTN+2.17.0 (https://blast.ncbi.nlm.nih.gov/ accessed on 1 June 2023).
Accession NumberMax ScoreTotal ScorePercentage IdentityWolbachia Strain
(wsp Gene)
MK684349.1494494100A
KY817484.1494494100A
KX573028.1494494100A
KY523670.1494494100A
KJ140127.1494494100A
KF725078.1494494100A
JX476002.1494494100A
KC668278.1494494100A
HM007832.1494494100A
MK684351.149249299A
MF805776.1492492100A
KX118690.1492492100A
KF725079.1492492100A
JX475999.1492492100A
AF020058.1490490100A
KC668284.148848899.63A
EU651894.1481481100A
MK684350.147547598.88A
KU738337.1472472100A
KJ140133.146646698.13A
GQ469985.146646698.5A
MH418437.1457457100A
MN307069.1756756100B
MK695179.1756756100B
MK695177.1756756100B
MK695176.1756756100B
MK695175.1756756100B
CP041924.1756756100B
CP041923.1756756100B
MH418465.1756756100B
MH418464.1756756100B
MH418463.1756756100B
Table 2. Representative Wolbachia strains belonging to distinct Wolbachia supergroups.
Table 2. Representative Wolbachia strains belonging to distinct Wolbachia supergroups.
Host OrganismName of StrainSupergroup Accession NumberReference
D. simulanswRi (Riverside)AAF020070.1
NC_012416.1		Braig et al., 1998 [35]; Baião et al., 2019 [47]
D. simulanswHa (Hawaii) AAF020068.1Braig et al., 1998 [35]
D. melanogasterwMelANC_002978.6Wu et al., 2004 [48]
C. quinquefasciatuswPipBAF020060.1Zhou et al., 1998 [3]
Ae. albopictuswAlbBBCP041924.1Kulkarni et al., 2019 [26]
Onchocerca ochengiwOoCHE660029.1Darby et al., 2012 [49]
Brugia malayiwBmDNC_006833.1Foster et al., 2005 [45]
Cimex lectulariuswCleFAP013028.1Nikoh et al., 2014 [44]
Table 3. Wolbachia strain A of Ae. Albopictus, with high (98–100%) sequence identity compared to the wAlbA-MID group. GenBank accession numbers and places (country) of detection are shown.
Table 3. Wolbachia strain A of Ae. Albopictus, with high (98–100%) sequence identity compared to the wAlbA-MID group. GenBank accession numbers and places (country) of detection are shown.
Accession NumberCountry
JX476004/7.1India (Orissa)
AF020058.1
MG765532.1		USA
MH418426/32/34/37.1
KX573017/18/19/22/23/38
KF782059/60/68/72/81/83/88/90
KF782100/02/05/08.1
KC004024.1		Malaysia
MH503767.1Pakistan
AY462864.1Taiwan
MK684349/50/51
KX118690/91/92.1		Mexico
EU727139.1Italy
KU738324/25.1China
MH777434.1Sri Lanka
Table 4. Wolbachia strain B of Ae. Albopictus, with high (98–100%) sequence identity compared to the wAlbB-MID group. GenBank accession numbers and places (country) of detection are shown.
Table 4. Wolbachia strain B of Ae. Albopictus, with high (98–100%) sequence identity compared to the wAlbB-MID group. GenBank accession numbers and places (country) of detection are shown.
Accession NumberCountry
JX629464/67India (Orissa)
JX129186.1
KX5731/32/37.1
MH418463/65.1
KF781999/06/12/28/33/41/42/45/47.1		USA
KX650069.1Pakistan (Punjab)
MG765533.1
AF020059.1		USA
EU727140.1Italy
AY462863.1Taiwan
KU738369/76/82/83/84/85.1China
MT645169.1Singapore
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Puerta-Guardo, H.; Contreras-Perera, Y.; Perez-Carrillo, S.; Che-Mendoza, A.; Ciau-Carrillo, K.J.; Parra-Cardeña, M.; Rodriguez-Sanchez, I.; Gomez-Govea, M.A.; Medina-Barreiro, A.; Ayora-Talavera, G.; et al. Wolbachia Surface Protein (wsp) Gene Sequencing of Strains A and B in Native Aedes albopictus of Mérida, Yucatán. Biology 2025, 14, 1399. https://doi.org/10.3390/biology14101399

AMA Style

Puerta-Guardo H, Contreras-Perera Y, Perez-Carrillo S, Che-Mendoza A, Ciau-Carrillo KJ, Parra-Cardeña M, Rodriguez-Sanchez I, Gomez-Govea MA, Medina-Barreiro A, Ayora-Talavera G, et al. Wolbachia Surface Protein (wsp) Gene Sequencing of Strains A and B in Native Aedes albopictus of Mérida, Yucatán. Biology. 2025; 14(10):1399. https://doi.org/10.3390/biology14101399

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Puerta-Guardo, Henry, Yamili Contreras-Perera, Silvia Perez-Carrillo, Azael Che-Mendoza, Karina Jacqueline Ciau-Carrillo, Manuel Parra-Cardeña, Iram Rodriguez-Sanchez, Mayra A. Gomez-Govea, Anuar Medina-Barreiro, Guadalupe Ayora-Talavera, and et al. 2025. "Wolbachia Surface Protein (wsp) Gene Sequencing of Strains A and B in Native Aedes albopictus of Mérida, Yucatán" Biology 14, no. 10: 1399. https://doi.org/10.3390/biology14101399

APA Style

Puerta-Guardo, H., Contreras-Perera, Y., Perez-Carrillo, S., Che-Mendoza, A., Ciau-Carrillo, K. J., Parra-Cardeña, M., Rodriguez-Sanchez, I., Gomez-Govea, M. A., Medina-Barreiro, A., Ayora-Talavera, G., Pavia-Ruz, N., Martin-Park, A., & Manrique-Saide, P. (2025). Wolbachia Surface Protein (wsp) Gene Sequencing of Strains A and B in Native Aedes albopictus of Mérida, Yucatán. Biology, 14(10), 1399. https://doi.org/10.3390/biology14101399

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