Naturally Occurence Of Wolbachia Spp. Infection In Populations Of Aedes Albopictus In The City of Valencia, Spain

Background: The presence of Aedes albopictus was rst reported in Valencia (Eastern Spain) in 2015, having a high sanitary and social impact. Innovative tools for its control include the use of the endosymbiotic bacterium Wolbachia pipientis, which suppress Ae. albopictus populations. The release of mosquito males infected with the bacteria has proven effective as a control strategy in similar urban areas in Rome, Italy. Before this strategy could be implemented in Valencia, it is crucial to know whether natural local populations of Ae. albopictus are infected with Wolbachia pipientis and if so, identifying the bacteria strains/supergroups. Methods: Eggs were collected from water basins from the 19 districts of the city of Valencia between May and October 2019. A total of 50 Ae. albopictus reared mosquito adults were processed and analyzed for the presence Wolbachia by selected markers for 16S rDNA and surface protein (wsp) genes, under optimized PCR conditions and sequenced. Results: Our study reveal that 94,7% of the samples carried the endosymbiont, with no differences among sexes. In relation to the Wolbachia strains, both wAlbA and wAlbB strains were identied in the mosquitoes analyzed, with some samples carrying mixed infections. Conclusions: These data provide the rst characterization of endosymbionts present in natural populations of Ae. albopictus in the Mediterranean area of Spain, and offers relevant information to evaluate the potential use of Wolbachia strains in order to achieve mosquito populations suppression through massive releases of males articially infected. respectively (19). Pioneering studies carried out in Italy, described the replacement of those Wolbachia strains by the one obtained from Culex pipiens (wPip) (20), and very recently, the same group has reported the rst eld experiment in Europe to assess the capacity of those Ae. albopictus populations (in which the natural endosymbiont Wolbachia has been replaced with the Wolbachia strain from the mosquito Culex pipiens), to sterilize wild females (21). These studies provide further support to the IIT as suitable to reduce Ae. albopictus populations in the Mediterranean area. In the present study, as a rst step in the evaluation of IIT as suitable in Valencia, Spain, we have investigated the presence and identication of endosymbionts in natural populations of Ae. albopictus in the city. Our data reveal that 94,7% of the analyzed samples carried the endosymbiont naturally, with wAlbA and wAlbB strains and some samples with mixed infections.


Introduction
The invasion of Aedes (Stegomyia) albopictus (Diptera: Culicidae), known as Asian tiger mosquito, has been reported worldwide (1,2). Its recognized clinical importance lies on its potential to transmit several diseases like diro lariosis, and different exotic anthroponotic arbovirosis including dengue (DENV),

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Captures of eggs were done through the employment of standard ovitraps (4). Black plastic bowls (0.4 L volume) lled with water (2/3 of capacity), and supplemented with a wooden stick as an oviposition support, were used. Samplings were carried out between May and October, 2019, in 19 districts of the city of Valencia, Spain ( Fig. 1; Table 1).
According to published methodological information in similar studies (22), eggs collected in the eld were reared in laboratory conditions until adult emergence, and once adult populations reached to 7-10 days of life both males and females were frozen at -20ºC. Species identi cation and sex separation was de nitively con rmed under binocular microscope. Finally, all 50 adult specimens (25 females and 25 males) were maintained individually in a -80ºC freezer until the Wolbachia genotyping analyses.
Replicates of all samples are available upon request.
Wolbachia genotyping: DNA extraction, PCR, sequencing and DNA analyses Individual adult mosquitoes were lysed and homogenized in 200 µL of phosphate buffered saline (PBS) with the help of sterile toothpicks. After centrifugation at low speed (800xg) for 20 min, total genomic DNA of each mosquito was extracted using a commercial kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany) following the modi ed protocol, and using a nal elution volume of 100 µL.
Presence of endosymbiotic bacteria of the genus Wolbachia was assessed using speci c single polymerase chain reaction (PCR) and two molecular markers as previously described (19,23). Brie y, the presence of the endosymbiont was tested by ampli cation of a fragment of the WSP (Wolbachia outer Surface Protein) gene delimited by primers wsp 81F and wsp 691R as described previously (19,23,24). We also performed a PCR to amplify a fragment of the 16S rDNA (Wolbachia-speci c primers) with primers WolbF and Wspecr as recently described by Carvajal et al. (2019) (23).
For the wsp gene ampli cation, we followed the standard wsp protocol (24), with some modi cations; the number of cycles was increased to 45. In the end, a 25 µL nal reaction volume was used consisting of 1X KAPA2G Robust HotStart ReadyMix PCR Kit® (Kapabiosystems, Boston, USA), 0.5 µM of forward and reverse primers, 2 µL of template DNA and PCR-grade water up to nal volume. The nal thermal pro le was as follows: initial denaturation at 95 °C for 3 min; 45 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min; nal extension at 72 °C for 3 min.
For the 16S rDNA gene ampli cation, we used a 25 µL nal reaction volume was used consisting of 1X KAPA2G Robust HotStart ReadyMix PCR Kit® (Kapabiosystems, Boston, USA), 0.5 µM of forward and reverse primers, 2 µL of template DNA and PCR-grade water up to nal volume. Thermal pro les followed the protocol of Simões et al. 2011 (25), but annealing temperature was reduced to 55 ºC, and the number of cycles was increased to 45 cycles. The nal thermal pro le, in accordance with the one used for wsp, was as follows: initial denaturation at 95 °C for 3 min; 45 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min; nal extension at 72 °C for 3 min.
All PCR ampli cation experiments included positive and negative controls. As a positive control we used a Wolbachia-infected Ae. albopictus, as well as two Wolbachia DNAs (A + B strains) kindly provided by Nuria Busquets and Sandra Talavera, CReSA, IRTA, Universidad Autónoma de Barcelona, Spain. Water was used as negative controls. The products size of each fragment was checked by 2% agarose gel electrophoresis set at 60V for 30 min. The wsp primers amplify a DNA fragment ranging from 590-632 bp, depending on the individual Wolbachia strain, while the 16S rDNA fragment is approximately 900 bp. The PCR ampli cation process underwent two replicates to validate the results. Positive samples of each marker were subjected to sequencing, carried out at Servicio Central de Soporte a la Investigación Experimental (SCSIE)-Universitat de València.

Identity of Wolbachia strains and their positions in phylogroups
All sequences were subjected to the Nucleotide Basic Local Alignment Search Tool (BLAST) (26), and compared to deposited Wolbachia sequences in GenBank. Selected sequences of Wolbachia de ned subgroups were used (sequences M84691.1 and KX155506.1 for subgroups A and B in 16S gene, respectively; and sequences MF999264.1 and KJ140127.1, for subgroups A and B in the wsp gene, respectively) (23). Sequences underwent multiple alignment using MUSCLE in MEGA X (27).
After editing, nal lengths used for phylogenetic inference analyses were 512 and 897 bp for wsp and 16S rDNA, respectively.
Maximum likelihood (ML) methods in MEGA X were used to analyze phylogenetic relationships using pre-selected parameters, which included Tamura 3parameter model + Gamma distributed (27). The ML trees were constructed with 1000 bootstrap replicates.
All newly generated sequences were submitted to the GenBank database with accession numbers MT510204-MT510238 for 16S sequences, and MT569490-MT569515 for wsp sequences.

Prevalence of Wolbachia infections
A total of 50 adults (25 females and 25 males) reared in the laboratory were investigated for the presence of Wolbachia infections. Of these, 38 samples (20 females and 18 males) were nally analyzed, and 94.7% (36/38) were PCR positives for Wolbachia using 16S primers (23) ( Table 1). From the 36 samples, 35 gave good quality sequences to be analyzed.
When using a PCR for a fragment of the wsp gene (19), 28/38 (73.7%) of samples showed Wolbachia infection, and good quality sequences were obtained from 25 samples, with another sample from district 12 (named H2) ( Table 1).
Phylogeny of Wolbachia through 16S rDNA sequences All sequenced DNA fragments showed high degree of similarity (> 98%) with 16S rDNA Wolbachia sequences in GenBank. Control DNA sequences for markers of wAlbA and B strains were used. As shown in Fig. 2, most of the 16S rDNA sample sequences (n = 25) belonged to supergroup B, with 6 samples belonging to supergroup A, with the remaining 4 samples localized as intermediate forms.

Phylogeny of Wolbachia through wsp sequences
All sequenced fragments showed high homology to wsp genes sequences in GenBank (> 94%). We used control markers of wAlbA and B strains, and the tree obtained with the ML method rendered 18 samples in supergroup B, 7 samples in supergroup A, and the remaining sequence as intermediate forms (Fig. 3).
Interestingly, samples ALB12W1H and ALB12W1H2 were located in different clades, being located the former with supergroup A, and the latter in supergroup B.
Comparison of 16S rDNA and wsp for Wolbachia detection A total of 28 individual samples (73.7%) yielded positive ampli cation in both markers (Table 1).
In the 16S rDNA positive samples (n = 36), there were 28 samples with successful wsp ampli cation, while 8 had no successful wsp ampli cation. From those, only good quality sequences were obtained in 25 samples (three samples presented mixed sequences, unable to read: ALB2WOL1M, ALB4WOL1M and ALB7WOL1H) ( Table 1).
Interestingly, six samples were con rmed as belonging to the wAlbA strain with both markers (Figs. 2 and 3; Table 1). From the four sequences considered intermediate with the 16S gene, only one (ALB9WOL1H) was also considered as an intermediate form by the wsp gene sequencing. Two 16S sequences considered to belong to the supergroup B, showed to belong to supergroup A when analyzed for the wsp marker (ALB10WOL1H and ALB13WOL1H), indicating a mixed infection in those samples, as it could be also the case for samples ALB4WOL1M (with no clean sequence in wsp marker, in the only sample analyzed from a male mosquito), and sample ALB12WOL1H (mixed strains in the 16S sequence, and belonging to the A supergroup by the wsp marker) ( Table 1). Two other mixed infections detected by the 16S marker (ALB2WOL1M and ALB8WOL1H) rendered either no sequence or corresponded to supergroup A by the wsp marker, respectively (Table 1).
In relation to sex, most of the mosquito females carried wAlbB strains (50%), with also A strains (27.7%) and mixed A + B infections (22.3%). In the case of mosquito males, 83.3% of the samples carried only wAlbB strains, with two samples carrying A + B mixed infections (11%), and one sample carrying only the wAlbA strain.
Overall, our data con rm the presence of both Wolbachia strains even in the same mosquitoes isolated in Valencia, Spain.

Discussion
Aedes albopictus is considered a competent vector for at least 23 arboviruses (28). Among them, the situation of DENV is particularly worrying since this arbovirus has emerged as the most important viral mosquito-borne disease globally in recent years. In Southern Europe autochthonous DENV transmission episodes have occurred on several occasions (29), with a recent detection of Ae. albopictus mosquitoes infected with DENV in Spain in 2015 (the rst detection in Europe) (30). Arbovirus surveillance is extremely important to identify possible foci of transmission, and this relies on multidisciplinary activities, including community engagement to early detect the mosquito and control its population, especially in urban areas (30).
There is a great consensus among the scienti c community about the need to search for new, innovative and complementary mosquito control methods in urban environments. Traditional methods employed to reduce Aedes populations include source reduction, public education and insecticide application routinely implemented by hundreds of municipalities across Europe (31). However, in some cases success can be limited due to low levels of communities' participation, lack of coordination between different administrations and bad practices in insecticides applications. Moreover, insecticide resistance cannot be discarded as an additional problem to achieve optimal results on Ae. albopictus control in Europe (32).
Population suppression of Aedes mosquitoes through the implementation of Wolbachia strategy is considered a method of high impact on resistance management (33). Some researches support the use of Wolbachia pipientis (wPip) infected Ae. albopictus strain (ARwP) males for autocidal suppression strategies against the Asian tiger mosquito (34).
Before those initiatives could be implemented, the precise identi cation of the Wolbachia strains present in wild Ae. albopictus populations in those areas is required to prevent unexpected effects like ine cient loss of compatibility (35). With this aim, we collected and analyzed mosquito samples from the 19 districts of the city of Valencia, Spain.
To our knowledge, this is the rst report and characterization of natural Wolbachia infections in wild populations of Ae. albopictus in Spain, and one of the rst studies in the Mediterranean area, including Italy (36). Since the rst report of Ae. albopictus in Spain in 2004 (9), it has been spread from Catalonia to the rest of the Mediterranean area including the Balearic islands (37), as well as other inland and northern regions in Spain such as Extremadura and Basque Country (38).
Our data reveal a high rate of Wolbachia infection in the wild populations of Ae. albopictus (94.7%), which i.s similar to the prevalence recently found in Asian countries like China (93.3%) (39), Thailand (100%) (40), or Korea (99%) (41), as well as in the US (95%) (42), and Brazil (99,3%) (43), and far from the low prevalence found in Mexico (a median of 38%) (44). Very few reports have been published in the Mediterranean countries, with great differences found in different locations in France (metropolitan and Corsica) (35,45), and Greece (35).
Based on our phylogenetic studies, only wAlbA and wAlbB supergroups were found in our analysis, with wAlbB being predominant (25/35, 71.4%), with only few individual samples of wAlbA, con rming previous reports (39). Interestingly, we detected both strains in a single mosquito, con rming previous ndings (19,40). Our phylogenetic analysis also detected "intermediate" (mixed) Wolbachia infections, some of them con rmed when sequences from both markers were analyzed (i.e. samples ALB10WOL1H and ALB13WOL1H belonged to different supergroups depending on the marker used). The presence of different strains in the same geographical location also supports the presence of mixed infections, and suggest possible crossing between mosquitoes carrying different Wolbachia strains.
Regarding to sex, most of the mono-infections were detected in males (88.8%), mostly with wAlbB strain (only one mosquito male showed wAlbA infection). Mosquito females presented more mixed infections than males (22.3% vs 11%), and half of the single infected samples carried wAlbB strains. These results are in agreement with previous reports either in eld or laboratory studies, suggesting a recent invasion and spread of Wolbachia in mosquito species (21,35,36,46). The recent studies by Caputo et al. (2020) (21) using ARwP infected males and released in urban areas of Rome (Italy), have con rmed the feasibility of IIT as way of controlling Ae. albopictus populations (21). They show promising results characterized by only 30% of females collected in the release spots, showing 100% sterility, and 20% showing strongly reduced fertility compared with control sites (21). Regarding to ARwP males longevity, they survived up to 2 weeks after release, which is considered adequate for the preservation of reproductive tness in males, and very similar to wild Asian tiger mosquito males longevity in normal situations in the eld (47). These results are in the same line that those from Calvitti et al. (2009) (48), since they found no differences between uninfected and infected males (ARwP) of Ae. albopictus with respect to longevity, mating rate, sperm capacity and mating competitiveness in laboratory condition and greenhouses.
Finally, it is important to remark which is the current Wolbachia situation according to Biocides regulation in Europe. Any products which is used to control unwanted organisms, including mosquitoes, that are harmful to human or animal health or to the environment, or simply can cause damage to human activities, should be registered in Europe as a biocide according to European Directive of biocides (Directive 98/8/EC) (49). After collecting information about the use of Wolbachia as a potential biocide in Europe, the European Commission has recently approved its use stating as follows: The bacteria of the genus Wolbachia, or any preparation containing those bacteria, used for the purpose of inoculating those bacteria into mosquitoes, with the objective of creating non-naturally infected mosquitoes for vector control purposes, shall be considered a biocidal product; while non-naturally infected mosquitoes, irrespectively of the infection technique used, shall be considered neither a biocidal product nor a treated article" (49). Considering these regulations, now there is a clear route for the introduction process of Wolbachia into the vector control programs of Europe according to European laws. In this context, the City Council of València is supporting an initiative regarding the implementation of Wolbachia strains for the control of Ae. albopictus populations which will be pioneering in Spain.

Declarations
No Ethics approval and consent to participate or consent for publication was required.