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Article

The Molecular Detection, Characterization, and Temperature Dependence of Wolbachia Infections in Field Populations of Aedes albopictus (Diptera: Culicidae) Mosquitoes in Greece

by
Michail Misailidis
1,
Nikolaos Kotsiou
1,
Aristotelis Moulistanos
1,
Sandra Gewehr
2,
Antonios A. Augustinos
3,
Spiros Mourelatos
2,
Spiros Papakostas
4,*,† and
Elena Drosopoulou
1,*,†
1
Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
2
Ecodevelopment S.A., Mezaria Place, 57010 Filiro, Greece
3
Department of Plant Protection Patras, Institute of Industrial and Forage Crops, Hellenic Agricultural Organization ‘DIMITRA’, 26442 Patras, Greece
4
Department of Science and Technology, International Hellenic University, 57001 Thermi, Greece
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Diversity 2024, 16(1), 43; https://0-doi-org.brum.beds.ac.uk/10.3390/d16010043
Submission received: 16 December 2023 / Revised: 3 January 2024 / Accepted: 4 January 2024 / Published: 9 January 2024
(This article belongs to the Special Issue Molecular and Microbial Ecology: Plastic Responses and Eco-Evolutionary Interactions with Environmental Stressors and Climate Change)
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Abstract

:
We investigated the prevalence and genetic diversity of Wolbachia pipientis strains in Aedes albopictus populations in Greece. Using a combination of PCR and Sanger sequencing techniques, we genotyped Wolbachia strains in 105 mosquitoes collected across eight different administrative regions in 2021. We found a high prevalence of Wolbachia in both male (90%) and female (97%) mosquitoes. Among the infected samples, 84% had double infections with both wAlbA and wAlbB strains, while 16% had infections with only wAlbB. Our comparison of the Multi-Locus Sequence Typing (MLST) profile, employing gatB–coxA–hcpA–ftsZ–fbpA genotyping, revealed a single MLST profile for each wAlbA and wAlbB strain in Greek populations. The same MLST profiles were also reported in populations from China, Russia, and Argentina, suggesting low levels of global diversity in wAlbA and wAlbB strains. Furthermore, our results indicated a significant association between temperature and the prevalence of single infections (p = 6.498 × 10−7), with higher temperatures correlating with an increased likelihood of single infections. Although male bias showed a tendency towards single infections, the effect was marginally non-significant (p = 0.053). These results were confirmed using a bootstrap-with-replacement analysis approach. Overall, our findings offer novel insights into the distribution and species diversity of Wolbachia strains in Greek Ae. albopictus populations, emphasizing the importance of understanding the short-term plastic and adaptive responses of these organisms to environmental stressors and rapid climate change.

1. Introduction

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito endemic to Southeast Asia, and its adaptation to urban environments has led to its global spread [1]. The first detection of this mosquito in Greece occurred in Corfu and Thesprotia in 2003/4, and since then, it has been found in nearly every administrative region [2,3]. Due to its ability to transmit many important human pathogens, such as Zika, Chinkungunya (CHIKV), Dengue (DENV-1, 2, 3, and 4), and West Nile (WNV) viruses, the Asian tiger mosquito has attracted substantial research interest [4,5]. With the traditional use of chemical insecticides raising serious concern about toxic effects on organisms and ecosystems, as well as about increasing mosquito resistance, alternative environmentally friendly methods for the control of mosquito populations have arisen [6]. The Sterile Insect Technique (SIT), a genetic control method that has been used against agricultural pests for decades, has been reconsidered as a promising approach against disease-transmitting mosquito species, including Ae. albopictus, exhibiting considerable progress towards this goal in recent years [7]. Furthermore, symbiont-based approaches are gaining more interest as promising tools for vector control [8].
Wolbachia pipientis is a Gram-negative, intracellular, endosymbiotic bacterium found in several arthropod species and filarial nematodes. It is classified into 17 supergroups from A–R (except G), based on genomic diversity [9]. Supergroups A and B are the most prevalent, found in arthropod species [10]. Initial reports proposed that 20% of arthropod species are infected by Wolbachia [11,12], but a later meta-analysis suggested that the number is closer to 60% [13]. The empirical evidence regarding the intraspecific prevalence of Wolbachia infection seems to follow an “all or nothing” pattern, with either very low (<10%) or very high (>90) infection rates in each species [13]. The spread of Wolbachia in arthropod species is closely linked to the manipulations and phenotypes that the bacterium induces in its hosts. The most frequently observed reproductive phenotype is cytoplasmic incompatibility (CI). In general, CI is observed when the male harbors an infection type that is not present in the female mate, resulting in inviable offspring [10,14,15]. In mosquitoes, another important Wolbachia-associated phenotype observed is pathogen interference (PI). Certain Wolbachia strains can reduce the replication and transmission of specific viruses and other pathogens. This phenotype has been observed in natural infections and has been most successfully demonstrated through transfections [10,16,17]. In recent years, Wolbachia has emerged as a promising tool for reducing mosquito-transmitted diseases, through the introduction of appropriately infected mosquitoes that can exert CI or PI effects in natural populations [8]. It is widely accepted that careful consideration and thorough research on the effects of a particular Wolbachia strain in a specific mosquito species must precede the release of mosquitoes in nature to prevent unintended consequences. Furthermore, an extensive screening of natural populations for Wolbachia prevalence and strain composition is an equally important step before any Wolbachia-based control effort can be considered [8].
In the natural populations of the Ae. albopictus tested, mainly in Asia and South America, Wolbachia infection is highly prevalent (>95%) [18,19,20,21]. Two commonly found Wolbachia strains in Ae. albopictus are wAlbA and wAlbB, belonging to supergroups A and B, respectively [22]. These strains often co-infect the same individual, although single infections with wAlbB, more commonly observed in males, have also been reported [23]. Environmental factors have been shown to affect the density of the Wolbachia strains in Ae. albopictus, with higher temperature and lower nutrition resulting in lower Wolbachia density [24]. Genetic diversity of the wAlbA and wAlbB strains has been studied by the MLST (Multi-Locus Sequence Typing) system [25], a typing system based on the polymorphism of five conserved genes (gatB, coxA, hcpA, ftsZ, and fbpA), in natural populations [25]. Both strains were found to be stable, with wAlbA presenting a unique profile and wAlbB presenting two very similar ones [18,25,26,27].
To date, the prevalence and genetic diversity of Wolbachia strains in Ae. albopictus populations in Europe remain poorly understood. To our knowledge, only one study reports a prevalence of 79% in the field population of the Iberian Peninsula, with no information provided about the type of stains (wAlbA or wAlbB) or genotypic diversity [28]. The frequency of Ae. albopictus-transmitted diseases is expected to increase in Europe in the coming years, driven by climate change [29,30,31], and, thus, increased efforts should be made to study and control the mosquito populations, including the use of Wolbachia-based methods.
In this study, our aim was to elucidate the unknown prevalence of Wolbachia in Ae. albopictus mosquitoes in Greece. As mentioned, Ae. albopictus was first reported in Greece relatively recently (around 2003), and in subsequent years, its populations have expanded throughout the country [3]. We also aimed to evaluate the occurrence of each strain, wAlbA or wAlbB, in field-collected populations of Ae. albopictus. Additionally, we sought to shed light on the genetic diversity of wAlbA or wAlbB strains in Northeastern Europe by comparing our MLST genotypes with those of other studies. Furthermore, we recorded the sex of the mosquitoes and the temperature environment of the studied populations to provide empirical evidence regarding the association between these factors and the propensity for double or single infections. Our experimental design involved a field sampling campaign across eight different administrative regions in Greece between July and September 2021, during which we collected a total of 114 adult Ae. albopictus mosquitoes. The sex of each mosquito was recorded, and we employed PCR and Sanger sequencing techniques to detect and genotype the Wolbachia strains. To assess the impact of environmental temperature, we collected temperature recordings four times a day for 30 days prior to the sampling date. We then analyzed this data in relation to the observed patterns of the occurrence of double/single infections. Overall, our findings provide valuable insights into the distribution and genetic diversity of Wolbachia in Greek Ae. albopictus populations, Furthermore, they highlight the importance of understanding the organisms’ short-term plastic and adaptive responses to environmental stressors and rapid climate change. We anticipate that these results can contribute to the development of effective Wolbachia-based approaches for controlling mosquito-borne diseases in Ae. albopictus in Greece but also in other regions.

2. Materials and Methods

2.1. Mosquito Sampling

Adult mosquitoes were collected between July and September of 2021, in the frame of a standard entomological surveillance of mosquito control projects run in four regions of Greece (Central Macedonia, Western Macedonia, Western Greece, and Thessaly). For the entomological surveillance, CDC-style traps were used, which were supplied with CO2 and UV light to attract mosquitoes with a constant CO2-release rate of 0.5 L/min. The traps are used for wide-area mosquito control projects across the country since 2010 and were designed, fabricated, and operated by Ecodevelopment S.A. (https://ecodev.gr/en/ecodev-english/, accessed on 2 January 2024). The traps were operated fortnightly, and the trapping period was 24 h per trapping night. Random individuals were selected from the hundreds of mosquitoes captured in the traps. These individuals were morphologically identified to a species level by scientific personnel of the entomological laboratory of Ecodevelopment using dichotomous identification keys [32,33,34]. Overall, samples from 8 Greek administrative regions were obtained: 10 from Achaea, 15 from Aetolia-Acarnania, 9 from Imathia, 8 from Thessaloniki, 27 from Karditsa, 19 from Kozani, 13 from Pieria, and 4 from Serres (Table 1). A total of 105 adult Ae. albopictus were analyzed, comprising 74 females (70.5%) and 31 males (29.5%) (Supplementary Table S1). We clarify that, by sample, we mean the content of mosquitoes of one CDC-style trap per night. Only the samples that contained specimens of Ae. albopictus were used.

2.2. DNA Extraction and Wolbachia Detection

DNA was extracted from single Ae. albopictus mosquitoes using a modified DNA extraction CTAB protocol [35]. To detect Wolbachia infection, PCR was performed using the wspecF/wspecR primer pair (5′-YATACCTATTCGAAGGGATAG-3′/5′-AGCTTCGAGTGAAACCAATTC-3′), which amplifies a 438 bp fragment of the 16S rRNA gene [11]. The PCR cycling protocol was as follows: 95 °C for 5 min, followed by 30 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by 72 °C for 5 min [11,36]. The DNA from a Wolbachia-infected Drosophila suzukii laboratory strain was used as a positive control in each reaction. For DNA quality control, the 12SC primer pair (12SCFR: 5′-GAGAGTGACGGGCGATATGT-3′/12SCRR: 5′-AAACCAGGATTAGATACC-CTATTAT-3′), which amplifies a 377 bp fragment of the mosquito 12S rRNA gene, was used. The PCR cycling protocol for this control reaction was 95 °C for 5 min, followed by 30 cycles of 95 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min, followed by 72 °C for 5 min [37,38,39]. Both PCR reactions were carried out in a final volume of 15 μL, containing 1× reaction buffer, 2.5 mM MgCl2, 1 Unit of Taq DNA polymerase (BIOTAQ, meridian BIOSCIENCE, Cincinnati, OH, USA), 0.2 mM dNTPs (Jena Bioscience, Jena, Germany), 0.5 mM of each primer, and 1 μL of the template DNA. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel using the 100 bp DNA ladder PLUS BLUE (GeneOn, Ludwigshafen am Rhein, Germany). The samples were characterized as negative for Wolbachia only when they repeatedly failed to give an amplification signal for wspec (at least two amplification tests) despite producing a strong positive signal for 12S rRNA.

2.3. Strain Characterization and MLST Genotyping

To characterize the Wolbachia subgroup identity in the samples that were positive for Wolbachia presence, two primer pairs were used: wAlbA_328F/wsp691R (5′-CCAGCAGATACTATTGCG-3′/5′-AAAAATTAAACGCTACTC-CA-3′) for the detection of the wAlbA strain, and wAlbB_183F/wsp691R (5′-AAGGAACCGAAGTTCATG-3′/5′-AAAAATTAA-ACGCTACTCCA-3′) for the detection of the wAlbB strain. The primer pairs produce amplicons of 341 bp and 463 bp, respectively. A multiplex PCR was conducted with the following cycling profile: 95 °C for 5 min, followed by 35 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by 72 °C for 3 min [22]. The PCR reaction was performed in a final volume of 15 μL containing 1× reaction buffer, 2.5 mM MgCl2, 1 Unit of Taq DNA polymerase (BIOTAQ, meridian BIOSCIENCE, Cincinnati, OH, USA), 0.2 mM dNTPs (Jena Bioscience, Jena, Germany), 0.5 mM of each primer, and 1 μL of the template DNA. The results were analyzed using a 1.5% agarose gel, and insects were classified as “double-infected” if they exhibited both wAlbA- (341 bp) and wAlbB- (463 bp) specific bands or as “single-infected” if they only showed a single band.
MLST genotyping was conducted on 11 selected samples: four (two females, two males) double-infected from THE, three (two females, one male) double-infected and one (male) single-infected from KAR, and one (male) double-infected and two (one female, one male) single-infected from AET. For each individual, five loci of the MLST system, as developed for Wolbachia by [25] were PCR-amplified and Sanger-sequenced. For four out of the five loci (gatB, hcpA, ftsZ, fbpA), wAlbA- and wAlbB-strain-specific primers were used to amplify the respective alleles [25]. For locus coxA, wAlbA-specific primers were used to produce the A allele, while universal primers in wAlbB single-infected individuals were used to amplify the B allele, since the wAlbB-specific primers failed to produce the expected amplicon. The PCR cycling profile was 94 °C for 2 min, followed by 37 cycles of 94 °C for 30 s, 55–62 °C for 45 s, and 72 °C for 1.5 min, followed by 72 °C for 10 min (https://pubmlst.org/organisms/Wolbachia-spp, accessed on 2 January 2024). The PCR reactions were performed in a final volume of 20 μL, consisting of 1× reaction buffer, 1.5 mM MgCl2, 1 Unit of Taq DNA polymerase (BIOTAQ, meridian BIOSCIENCE, Cincinnati, OH, USA), 0.2 mM dNTPs (Jena Bioscience, Jena, Germany), 1 mM of each primer, and 1 μL of the extracted DNA [40]. The success of each amplification was evaluated using 1.5% agarose gels. The PCR products were cleaned using the ExoSAP-IT™ (New England Biolabs, Ipswich, MA, USA) protocol and Sanger-sequenced in both directions by GENEWIZ (Takeley, UK). The chromatograms were inspected using the Geneious Prime software, version 2023.0.4 (www.geneious.com, accessed on 2 January 2024). The allele number for each locus was determined according to the Wolbachia MLST database (https://pubmlst.org/organisms/Wolbachia-spp, accessed on 2 January 2024). All sequences have been submitted to GenBank (Accession Numbers: OQ985406-OQ985492).

2.4. Temperature Data Collection and Statistical Analysis

For each sampling location, we calculated the mean temperature from the 30 days prior to the date of the sampling. This approach captures the temperature exposure of the mosquito populations over a longer period than the temperature of the day of the sampling. Using the latitude and longitude of each trap, we recovered the temperature readings for each day at four time points: 6 o’clock in the morning and in the evening, midday, and midnight. We thus averaged 120 temperature readings for each trap for each sampling event. We also accounted for the variability in daily temperature; temperature data were collected from the OpenWeather platform (www.openweather.org, accessed on 2 January 2024) via its API using the Python package “requests”. OpenWeather is a widely used weather data provider used by a variety of industries, including agriculture, transportation, and energy.
To explore, per sampling date, the correlation between the mean temperature, the ratio of single to double Wolbachia infections, and the sex (female, male) of the infected mosquitoes, we employed the non-parametric Kruskal–Wallis test [41], given the limited number of independent observations (n = 11). To further examine the effects of different groups, we performed a post hoc Dunn test [42]. Additionally, to validate our findings and assess their robustness, we utilized a bootstrap-with-replacement approach. We generated 10,000 bootstrap resamples by randomly selecting observations from the original dataset, allowing for duplicates (i.e., with replacement) in each resampled dataset. Kruskal–Wallis and Dunn tests were then performed in each bootstrap resample to assess the effects of temperature and sex ratio. By employing this approach, we were able to estimate the confidence intervals of the correlations observed and strengthen the reliability of our findings.

3. Results

3.1. Wolbachia Prevalence

Out of the 105 Ae. albopictus individuals examined, 100 (95%) were found to carry a Wolbachia infection. The prevalence of infection in males was 90% (28/31), and in females, it was 97% (72/74). The infection rates in each district administrative region were as follows: 90% in ACH (9/10), 87% in AET (13/15), 93% in KAR (25/27), and 100% in IMA (9/9), with THE (8/8), KOZ (19/19), PIE (13/13), and SER (4/4) (Table 2; Supplementary Table S1).

3.2. Wolbachia Strain Characterization

The 100 Wolbachia-infected mosquitoes were screened for wAlbA and wAlbB. The PCR essay detected a double infection (both wAlbA and wAlbB) in 84%, while a single infection with the wAlbB strain was found in 16% of the infected samples. No mosquitoes infected solely with the wAlbA strain were found (Supplementary Table S1). Most female mosquitoes were double-infected (68/74 female mosquitoes or 92%), whereas double infection was present in 52% of males (16/31 male mosquitoes). When inspecting the results by administrative region, single-infected individuals were only found in four regions: ACH (11%), AET (46%), KAR (32%), and IMA (11%) (Table 2; Figure 1; Supplementary Table S1).

3.3. MLST Genotyping

To gain a deeper understanding of the diversity of Wolbachia strains present in Ae. albopictus populations in Greece, 11 samples were genotyped by MLST. We genotyped single- and double-infected samples from both sexes from three different administrative regions, attempting to capture possible genetic variation and different MLST profiles (if any). The MLST profile obtained for the wAlbA strain was identical across all samples. According to the numbering of the Wolbachia MLST database, the identified alleles were 3, 229, 2, 10, and 3 for the loci gatB, coxA, hcpA, ftsZ, and fbpA, respectively, presenting four matches with ST code 2. Similarly, the wAlbB strain exhibited identical profiles across all samples (alleles 242, 229, 166, 210, 27), matching ST code 464. It should be noted that, for double-infected individuals, the allele for the coxA of the wAlbB strain was assumed to be the same as the one in the wAlbB single-infected individuals, given the monomorphic outcome observed for all loci in all samples.

3.4. Temperature and Sex Ratio Effect on Single Wolbachia Infection

The Kruskal–Wallis test revealed a highly significant effect of mean temperature and sex bias on the ratio of single to double Wolbachia infection in the studied mosquito populations (p = 1.09 × 10−6; statistic = 27.46). Further analysis using a post hoc Dunn test distinguished between a highly significant effect of mean temperature (p = 6.50 × 10−7; Figure 2A) and a marginally non-significant effect of sex ratio (p = 0.053). Although the latter may be influenced by the limited number of observations (n = 11), the trend observed appears to be strong (Figure 2B), and the removal of a single outlier value resulted in a significant outcome (p = 0.040). To this end, the bootstrap-with-replacement approach confirmed these observations over 10,000 bootstrap resamples. The mean of p-values for the effect of temperature was p = 2.27 × 10−6, with a high 95% confidence limit of p = 1.69 × 10−5. The mean of p-values for the effect of sex ratio was p = 0.097, with 52.45% of the resamples at p < 0.05 (Figure 3). Overall, these findings suggest a positive relationship between higher environmental temperatures and single Wolbachia infections in Ae. albopictus mosquitoes, with a negative influence from female sex bias (Figure 2).

4. Discussion

4.1. High Wolbachia Infection Rate in Ae. albopictus Population in Greece

The high infection rate (95%) in the natural population of Ae. albopictus in Greece supports previous studies from other regions and confirms that the Wolbachia infection has been fixed in Ae. albopictus worldwide [13,21,43]. However, we observed slight differences in the infection rates among different administrative regions. All the examined administrative regions of Northern Greece exhibited an infection rate of 100% (IMA, KOZ, PIE, SER, THE; Table 2), unlike the administrative regions in Central and Southern Greece (88% mean rate; ACH, AET, KAR; Table 2). Variance in infection rates due to different geographical regions had been previously reported [18]. Understanding the infection dynamics of Wolbachia in Ae. albopictus holds the promise of establishing biologically based management practices, as the density of the endosymbiont Wolbachia plays an important role in crossing sterility (e.g., Werren et al. 1995 [12]).

4.2. Low MLST Profile Diversity for Wolbachia Strains in Greek Populations and Worldwide

The MLST profiles for the wAlbA and wAlbB strains were monomorphic in the Greek populations tested, which is consistent with the low diversity observed in the MLST genes in the Ae. albopictus Wolbachia strains worldwide [18,25,26,27]. Specifically, the sole identified profile for the wAlbB strain (242, 229, 166, 210, and 27, as per the numbering of the MLST database) was also the most common profile identified in almost all the populations studied from China, Russia, and Argentina [18,25,26,27]. There is only one other profile reported for the wAlbB strain in China, which only differs in the gatB allele [18]. The sole identified wAlbA profile (3, 229, 2, 10, and 3) shares identical alleles of four out of the five loci with samples from China and Thailand [18,44]. It only differs in the coxA locus, where we detected the same allele (229) as in the wAlbB strain. Since it has been proposed that the 229 allele has moved from supergroup A to supergroup B through recombination [26], it may be that the supergroup-A-specific primers mis-amplified the 229 allele of the wAlbB strain, as it is expected to be more abundant in the double-infected individuals [45]. Notably, as mentioned in Badieritakis et al. (2018) [3], the Greek populations of Ae. albopictus have been found to have some genetic affinity with those of Thailand [46]. As such, our results seem to align with these earlier findings. The Greek populations of Ae. albopictus have also been established relatively recently. For example, the first reports in Serres, Aetolia-Acarnania, and Thessaloniki were made between 2007 and 2009 [3]. This recent occurrence of populations may have a role in the low MLST diversity observed, due to founder effects and limited time for divergence.

4.3. Differential Strain Distribution of Wolbachia by Sex

By examining the double and single infection patterns in relation to the sex of the individual mosquitoes, we noted that almost all the female mosquitoes were double-infected (92%), whereas only about half of the males carried both strains (52%). We observed that, in males, a single infection with the wAlbB strain is more common in a marginally non-significant level (Figure 2B), which is in agreement with the scientific literature [19,20,21,23,47]. There are different hypotheses for the detection of both strains in females and only the wAlbB strain in males. It is believed that male mosquitoes lose their wAlbA infection as they get older, as an evolutionary selection of nuclear countermeasures to Wolbachia manipulation [23]. Female Ae. albopictus carry a fixed infection with both strains, with rare instances of single-infected females [19,20,21,23,47]. The mechanism of CI suggests that females need both strains to reverse the sperm modification each Wolbachia strain induces in males to maximize mating compatibility [10,48]. However, males with a single infection or no infection remain compatible with double-infected females [10]. Male embryos carry both infections since their mothers are almost always double-infected, but they tend to lose the wAlbA infection as they age. However, the same pattern is not observed in the wAlbB strain. If the adaptive decrease of wAlbA in males is accepted, then the persistence of the wAlbB at high density could be explained either by the fact that the infection with the wAlbB strain is more recent [43] or by the possibility of a more effective vertical transmission of the wAlbB strain, which would reduce selective pressure towards the decrease in its density in males [23].

4.4. Differential Strain Distribution of Wolbachia by Environmental Temperature

The administrative region-specific infection rates highlighted the finding that non-infected individuals were only found in the three southernmost administrative regions examined (ACH, AET, KAR). Furthermore, single-infected individuals were also mainly found in these three administrative regions (with one single-infected individual found in Imathia as well) (Table 2). This observation aligns with previous results from China, showing that Wolbachia prevalence in Ae. albopictus natural populations is affected by geographical location and climate type [18]. It is also in agreement with hypotheses that state that higher temperatures lead to decreased Wolbachia density in laboratory populations, affecting both infections but particularly wAlbA [24,49]. Our statistical analysis strongly supported this hypothesis, as the effect of the mean temperature from 30 days prior to the sampling date had a highly significant impact on the ratio of single infections in the mosquito populations (p = 6.50 × 10−7; Figure 2A). The mechanistic underpinnings of this observation are not fully understood. Some insight could be provided by studies concluding that maternal transmission under high temperatures and larval density is more often unsuccessful for the wAlbA than for the wAlbB strain [45,49]. Even so, when scoring single- to double-infected individuals, it should not be disregarded that the density of the wAlbA strain is smaller than that of wAlbB in double-infected individuals [18,45], and, thus, it is possible that what appears to be an absence of the wAlbA infection could, in fact, reflect a reduction beyond PCR detection limits [15,49]. Nevertheless, the findings above raise important points regarding the molecular ecology of the Wolbachia infections, which may also be of applied interest.

5. Conclusions

Altogether, this study provides important insights into the distribution and diversity of Wolbachia strains in Ae. albopictus populations in Greece and the broader Northeastern European region. We report the first MLST genotype information of Wolbachia strains in Ae. albopictus in Europe and identify a high infection rate in the natural population in Greece, confirming that the Wolbachia infection has become fixed in populations worldwide. We found a positive correlation between higher environmental temperatures and single Wolbachia infections in Ae. albopictus mosquitoes, demonstrating that ecological and evolutionary factors play a role in Wolbachia dynamics. Overall, this study contributes to our understanding of Wolbachia dynamics in the natural populations of Ae. albopictus. We anticipate that future research with even denser sampling will evaluate our findings, and it will have profound implications for the potential use of Wolbachia-based approaches in controlling mosquito-borne diseases in the area.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/d16010043/s1, Table S1: Supplementary_Table_S1.xlsx.

Author Contributions

Conceptualization, E.D., S.P., S.M., A.A.A. and S.G.; methodology, E.D., S.P., S.M., A.A.A., S.G., N.K. and M.M.; software, S.P. and M.M.; validation, S.P. and M.M.; formal analysis, E.D., S.P., N.K. and M.M.; investigation, E.D., S.P., N.K. and M.M.; resources, E.D., S.P., S.M. and S.G.; data curation, E.D., S.P., S.G., N.K. and M.M.; writing—original draft preparation, E.D., S.P., A.A.A., S.G. and M.M.; writing—review and editing, E.D., S.P., S.M., A.A.A., S.G., A.M., N.K. and M.M.; visualization, S.P. and M.M.; supervision, E.D. and S.P.; project administration, E.D., S.P. and S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All sequences have been submitted to GenBank (Accession Numbers: OQ985406-OQ985492). Python code associated with the statistical and simulation analyses has been deposited to GitHub (https://github.com/spirospapakostas/wolbachia-Ae_albopictus-2023).

Acknowledgments

We thank Alexander Triantafyllidis and Nikoleta Karaiskou from the Department of Biology of the Aristotle University of Thessaloniki for hosting the molecular work conducted in this study.

Conflicts of Interest

Authors Sandra Gewehr and Spiros Mourelatos are employed by the company Ecodevelopment S.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

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Figure 1. The distribution of the Wolbachia proportion of single (wAlbB) to double infection in the natural populations of Ae. albopictus at each of the Greek administrative regions studied (colored areas). Administrative region abbreviations are as in Table 1 and Table 2. Administrative region colors depict the different single infection proportion levels as shown in the legend. Administrative region boundaries’ color indicates average temperature as cyan (23.067–25.341 °C), pink (25.341–27.615 °C), or red (27.615–29.888 °C). These ranges represent equidistant bins across the observed minimum–maximum temperature range. When samples from multiple time points were taken from an administrative region, the overall temperature average was used for this visualization. Additional information is provided in Supplementary Table S1.
Figure 1. The distribution of the Wolbachia proportion of single (wAlbB) to double infection in the natural populations of Ae. albopictus at each of the Greek administrative regions studied (colored areas). Administrative region abbreviations are as in Table 1 and Table 2. Administrative region colors depict the different single infection proportion levels as shown in the legend. Administrative region boundaries’ color indicates average temperature as cyan (23.067–25.341 °C), pink (25.341–27.615 °C), or red (27.615–29.888 °C). These ranges represent equidistant bins across the observed minimum–maximum temperature range. When samples from multiple time points were taken from an administrative region, the overall temperature average was used for this visualization. Additional information is provided in Supplementary Table S1.
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Figure 2. Boxplots showing the distribution of the proportion of mosquitoes infected with single Wolbachia strains across (A) three temperature bins and (B) six female-to-male sex ratio bins in the studied populations of mosquitoes. The three bins in each of the cases represent equal splits across the observed min–max range (N = 1, 3, and 8 in temperature bins from lower to higher °C and N = 7, 3, and 2 in sex ratio bins from lower to higher female bias). The lower and upper hinges of the boxes represent the first and third quartiles, while the line inside the box represents the median. Whiskers extend to the lowest and highest values within 1.5 times the interquartile range, and points beyond this range are shown as individual data points. The plot (A) shows that the proportion of single-infected mosquitoes increases as temperature increases, and the plot (B) shows that the same proportion decreases towards more female-biased sex ratios.
Figure 2. Boxplots showing the distribution of the proportion of mosquitoes infected with single Wolbachia strains across (A) three temperature bins and (B) six female-to-male sex ratio bins in the studied populations of mosquitoes. The three bins in each of the cases represent equal splits across the observed min–max range (N = 1, 3, and 8 in temperature bins from lower to higher °C and N = 7, 3, and 2 in sex ratio bins from lower to higher female bias). The lower and upper hinges of the boxes represent the first and third quartiles, while the line inside the box represents the median. Whiskers extend to the lowest and highest values within 1.5 times the interquartile range, and points beyond this range are shown as individual data points. The plot (A) shows that the proportion of single-infected mosquitoes increases as temperature increases, and the plot (B) shows that the same proportion decreases towards more female-biased sex ratios.
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Figure 3. Histograms illustrating the distribution of negative log10-transformed p-values from the simulation analysis (10,000 bootstrap resamples) for the effects of (A) temperature and (B) female-to-male ratio. Vertical lines indicate the observed significance (in blue), while the 95% confidence intervals of the significance from the simulations are represented in purple. Bars colored in pink correspond to simulations falling below the 5% significance threshold [−log10(0.05) = 1.3]. Non-significant values are indicated with grey bars.
Figure 3. Histograms illustrating the distribution of negative log10-transformed p-values from the simulation analysis (10,000 bootstrap resamples) for the effects of (A) temperature and (B) female-to-male ratio. Vertical lines indicate the observed significance (in blue), while the 95% confidence intervals of the significance from the simulations are represented in purple. Bars colored in pink correspond to simulations falling below the 5% significance threshold [−log10(0.05) = 1.3]. Non-significant values are indicated with grey bars.
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Table 1. Information about the studied Ae. albopictus sites, the coordinates of the traps, the sampling dates, and the studied sample sizes. More detailed information is provided in the Supplementary Table S1.
Table 1. Information about the studied Ae. albopictus sites, the coordinates of the traps, the sampling dates, and the studied sample sizes. More detailed information is provided in the Supplementary Table S1.
Administrative Region
(Abbreviation)		Trap Coordinates
(Latitude/Longitude)		Sampling DatesCollected
Mosquitoes
Achaea (ACH)38.314695/21.81423730/0810
Aetolia-Acarnania (AET)38.61610755/21.3825769;
38.39032018/21.85072874		12/07; 11/0815
Imathia (IMA)40.536217/22.2022726/089
Karditsa (KAR)39.397834/22.070087;
39.37078032/21.93258031		27/7; 7/9; 21/927
Kozani (KOZ)40.312092/21.82230416/8; 27/919
Pieria (PIE)40.237544/22.58214830/813
Serres (SER)41.205796/23.07492113/74
Thessaloniki (THE)40.64858/22.95406716/98
Table 2. Results of the sex, the Wolbachia infection, and the type of infection (single or double) in the studied mosquitoes. More detailed information, such as the date of the samplings and the average temperature, is provided in the Supplementary Table S1. Male and female samples are indicated with the symbols ♂ and ♀, respectively.
Table 2. Results of the sex, the Wolbachia infection, and the type of infection (single or double) in the studied mosquitoes. More detailed information, such as the date of the samplings and the average temperature, is provided in the Supplementary Table S1. Male and female samples are indicated with the symbols ♂ and ♀, respectively.
Administrative
Region (Abbreviation)		No. of
Samples/Sex		Wolbachia
Prevalence		Double Infection (wAlbA + wAlbB)Single Infection (wAlbB)
Achaea
(ACH)		5♂, 5♀90% (9/10)89% (4♂, 4♀)11% (1♂)
Aetolia-Acarnania (AET)9♂, 6♀87% (13/15)54% (5♂, 2♀)46% (3♂, 3♀)
Imathia
(IMA)		9♀100% (9/9)89% (8♀)11% (1♀)
Karditsa
(KAR)		14♂, 13♀93% (25/27)68% (4♂, 13♀)32% (8♂)
Kozani
(KOZ)		19♀100% (19/19)100% (19♀)0%
Pieria
(PIE)		13♀100% (13/13)100% (13♀)0%
Serres
(SER)		4♀100% (4/4)100% (4♀)0%
Thessaloniki (THE)3♂, 5♀100% (8/8)100% (3♂, 5♀)0%
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Misailidis, M.; Kotsiou, N.; Moulistanos, A.; Gewehr, S.; Augustinos, A.A.; Mourelatos, S.; Papakostas, S.; Drosopoulou, E. The Molecular Detection, Characterization, and Temperature Dependence of Wolbachia Infections in Field Populations of Aedes albopictus (Diptera: Culicidae) Mosquitoes in Greece. Diversity 2024, 16, 43. https://0-doi-org.brum.beds.ac.uk/10.3390/d16010043

AMA Style

Misailidis M, Kotsiou N, Moulistanos A, Gewehr S, Augustinos AA, Mourelatos S, Papakostas S, Drosopoulou E. The Molecular Detection, Characterization, and Temperature Dependence of Wolbachia Infections in Field Populations of Aedes albopictus (Diptera: Culicidae) Mosquitoes in Greece. Diversity. 2024; 16(1):43. https://0-doi-org.brum.beds.ac.uk/10.3390/d16010043

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Misailidis, Michail, Nikolaos Kotsiou, Aristotelis Moulistanos, Sandra Gewehr, Antonios A. Augustinos, Spiros Mourelatos, Spiros Papakostas, and Elena Drosopoulou. 2024. "The Molecular Detection, Characterization, and Temperature Dependence of Wolbachia Infections in Field Populations of Aedes albopictus (Diptera: Culicidae) Mosquitoes in Greece" Diversity 16, no. 1: 43. https://0-doi-org.brum.beds.ac.uk/10.3390/d16010043

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