Wolbachia infection in natural mosquito populations from Argentina

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Rodriguero, Ana C. Alonso, Marina Stein, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4361303/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Parasitology Research → Version 1 posted 9 You are reading this latest preprint version Abstract The increasing spread of mosquito vectors has made mosquito-borne arboviral diseases a global threat to public health, leading to the urgent need for effective control of such populations. Methods based in the intracellular bacterium Wolbachia Hertig, 1936 are considered environmentally friendly, human-safe, and potentially cost-effective biocontrol strategies to control arboviral diseases. In order to minimize undesirable side effects, it is relevant to understand if Wolbachia ranges in the area and the diversity associated to native infections before implementation. With this purpose, we investigated Wolbachia infection status, diversity and prevalence in populations of Aedes albifasciatus (Macquart, 1838), Aedes fluviatilis (Lutz, 1904), and hybrids of the Culex pipiens (Linnaeus, 1758) complex from Argentina. Additionally, we preliminarily explored the influence of environmental temperature on the Wolbachia prevalence in Ae. fluviatilis . Aedes albifasciatus and Cx. pipiens complex samples were collected in the province of Buenos Aires, and Ae. fluviatilis in the province of Misiones. Aedes albifasciatus was uninfected and infections with strains w Flu and w Pip were revealed in Ae. fluviatilis and hybrids of the Cx. pipiens complex, respectively. All strains were fixed or close to fixation and clustered within supergroup B. No effects of environmental temperature on Wolbachia prevalence of Ae. fluviatilis larvae were revealed, at least with the current design. These results provide valuable information on Wolbachia strains found in natural populations of mosquitoes from Argentina that might be used in heterologous infections in the future or must have taken into account when designing control strategies based on Wolbachia infection. Wolbachia Mosquitos Arboviruses MLST Biocontrol Neglected Diseases Figures Figure 1 Figure 2 Figure 3 Introduction In recent years, there has been an increase in the distribution of mosquito-borne diseases. Once confined to tropical and subtropical regions, vectors are expanding into temperate regions around the world (e.g. López et al. 2021 ; Roberts et al. 2019). This is a consequence of the increase of international trips, rapid urbanization with no proper planning, deforestation, climate change and insufficient or inefficient sanitary measures (Cabrera et al. 2022 ; Da Silva et al. 2023 ; Díaz et al. 2013 ; Hubert et al. 2018). For instance, a population burst of Aedes albifasciatus (Macquart, 1838) because of El Niño-induced climate change, led to an invasion of several urban cities from Argentina (Buenos Aires province). This caused an epidemic break of Western Equine Encephalitis Virus (WEEV) in horses, and subsequently, after more than 30 years without cases, 12 people were infected, and one casualty (WHO 2023 ). Thus, there is urgency for increasing effective control measures against vectors. Nowadays, Wolbachia infection mosquito represents a promising new bio-control strategy (Minwuyelet et al. 2023 ). Wolbachia Hertig, 1936 is the most successful pandemics since the origin of life. Since its discovery in the germ line and somatic tissues of the Culex pipiens (Linnaeus, 1758) mosquito in 1924 (Hertig and Wolbach 1924 ), the number of Wolbachia strains and associated hosts continues to increase (Landmann 2019 ). Evidence shows that the key to its success is mainly its capacity for vertical transmission through the egg driven by reproductive manipulations such as cytoplasmic incompatibility (CI) and for horizontal transfer between host species (Rodriguero 2013 ). Since the discovery of Wolbachia in the mosquito Cx. Pipiens (Hertig and Wolbach 1924 ), scientific reports demonstrate the presence of this bacterium in various mosquitoes genera such as Armigeres (Leicesteria), Mansonia Blanchard, 1901, Coquillettidia Dyar, 1904, Culiseta Felt, 1904, Hodgesia , Theobald, 1904, Tripteroides Giles, 1904 and Uranotaenia Lynch Arribálzaga, 1891 (Nugapola et al. 2017 ; Raharimalala et al. 2016 ; Wiwatanaratanabutr 2013), most of these species without epidemiological relevance. Nevertheless, infection is also known in important species of disease-transmitting mosquitoes in the genera Culex Linnaeus 1758, Aedes Meigen 1818 and Anopheles Meigen 1818, for example (Bian et al. 2013 ; Hoffman et al. 2014; Johnson 2015 ). Most of the strains identified in mosquitoes are within supergroups A or B of Wolbachia , and even superinfections (i.e. infections with two or more strains) can occur (Werren et al. 1995 ; Zou et al. 1998). The similarities between the strains that infect different mosquito species suggest a simple pathway by which these insects find and acquire new infections (horizontal transfer). Within each species there is variation in infection status between populations, from low frequencies to fixation (Charlesworth et al. 2019 ; Hilgenboecker et al. 2008 ; Inácio da Silva et al. 2012). However, the prevalence of Wolbachia infections in these insects may be underestimated because often they can occur at very low densities that are not detectable by conventional PCR (Mee et al. 2015 ). Since the discovery of some strains having the ability to interfere or block the transmission of certain arboviruses of public health importance such as dengue and zika (Landmann 2019 ), a widespread interest in the use of experimentally generated Wolbachia -infected mosquitoes for biological control of infectious diseases (Iturbe-Ormaetxe et al. 2011 ) arose. With this purpose, two approaches were proposed: population replacement and population suppression (Atyame et al. 2014 ; Iturbe-Ormaetxe et al. 2011 ). Both Wolbachia -based biocontrol methods require the field release of transinfected individuals with strains that induce CI during mating with the wild mosquito populations (Ross et al. 2020 ). For this reason, prior to release it is crucial to know which strains of Wolbachia are present in wild populations because: i) pre-existing natural infections can interact and alter the dynamics of introduced strains, making population replacement or suppression a challenging obstacle to overcome (Rasgon and Scott 2004 ); ii) the dynamics of Wolbachia spread can become complicated as the number of incompatible strains present in the population increases (Turelli and Hoffman 1995). Furthermore, understanding the similarities and differences between Wolbachia strains that infect different mosquito species is critical for estimating how frequently (over evolutionary times) mosquitoes experience horizontal transmission events of this bacterium in nature (Rasgon and Scott 2019). On the other hand, the study of Wolbachia infection in vector and non-vector mosquito species aids the discovery of novel strains of these bacteria with desirable characteristics that can be used to artificially transinfect other species of interest (Bourtzis et al. 2014 ). Many factors can influence both the infection dynamics and the extent of bacteria-induced host phenotypes in host-symbiont relationships. Of all of them, symbiotic population density is one of the most relevant (López Madrigal and Duarte 2019). Although both bacterial and host genetic backgrounds are involved in density regulation, environmental factors, like temperature, may also affect bacterial population density (Mouton et al. 2007). Environmental temperature shows a great impact on Wolbachia load and host phenotypes induced by the bacteria. Temperatures above 30°C have been shown to reduce Wolbachia density or even eliminate the infection, while temperatures below 20°C compromise the proliferation rate (López Madrigal and Duarte 2019). Thus, temperatures within the range tolerated by hosts can be extreme for Wolbachia (Ross et al. 2020 ). The effects caused by environmental temperature can be strain-specific (Ross et al. 2017 ). This is an important aspect to consider for evaluation of Wolbacia as a biocontrol tool, since low densities can lead to a reduction in virus blocking efficiency, weaker CI and/or failure of maternal transmission (Ye et al. 2016 ). The works accomplished on Wolbachia in mosquito populations in Argentina are scarce and mainly based on Culex quinquefasciatus Say, 1823 and recently in Aedes albopictus Skuse, 1895 (Alonso et al. 2022 ; Chuchuy et al. 2018 ; Díaz-Nieto et al. 2021 ; Micieli and Glaser 2014 ; Morais et al. 2012 ). Thus, in the present work, the occurrence of this endosymbiont in mosquito populations in natural systems of Argentina was investigated. Our surveys focused on Aedes fluviatilis (Lutz, 1904), a potential vector of yellow fever (Baton et al. 2013 ; Rey and Lounibos 2015 ), Ae. albifasciatus which is considered a pest throughout the south and center of the country (García and Micieli 2000 ; Gleiser et al. 2000 ), and hybrid populations of the C. pipiens complex, which are cosmopolitan vectors of pathogens of medical and veterinary importance (Farajollahi et al. 2011 ). The relationship between environmental temperature and the prevalence of Wolbachia in Ae. fluviatilis from Eldorado (Misiones province) was also investigated. Materials and methods Sampling area Samplings were carried out in four localities located in two provinces of Argentina. Specimens of the complex Cx. pipiens were collected in Berisso (34°52’00’’S − 57°52’ 00’’W) and the city of La Plata (34°55’07” S − 57°57’15” W), province of Buenos Aires (Fig. 1 a); individuals of Ae. albifasciatus were sampled in Ensenada (34°51’S − 57°54’W) and La Plata, province of Buenos Aires (Fig. 1 a); individuals of Ae. fluviatilis were collected in Eldorado (26º24’S − 54º38’W), province of Misiones (Fig. 1 b). Mosquitoes and environmental data collection Samples of Ae. albifasciatus were collected during the months of March, April and May 2017. Adult individuals were collected in temporary rainwater puddles using manual battery-powered vacuum cleaners with a person as bait, collecting mainly females and identified using morphological characters by dichotomous keys (Darsie 1985 ). Samples of the Cx. pipiens complex were collected during July and August 2017 (La Plata) and during January and February 2018 (Berisso). Immature stages were collected in household ditches where rainwater and drainage from kitchens and laundry rooms stagnate, to which sewage effluents are usually added. Immature stages were collected using pipettes and fine-mesh aquarium nets. These were conditioned in plastic trays (30 cm x 18 cm x 6 cm) with dechlorinated water and finely ground guinea pig food in the insectary of Centro de Estudios Parasitológicos y de Vectores (CEPAVE, UNLP-CONICET) until adults were obtained. Culex pipiens identification was performed by polymerase chain reaction (PCR) amplification of the AceII gene (nuclear acetylcholinesterase-2 gene), which allows the species to be distinguished between Cx. pipiens / Cx. quinquefasciatus , and the microsatellite locus CQ11, which allows identification of the ecotypes Cx. pipiens , Cx. p. pipiens / Cx. p. molestus , following the protocols of Smith and Fonseca ( 2004 ) and Bahnck and Fonseca ( 2006 ), respectively. Ae. fluviatilis individuals were collected in September 2017 in car tires. Buckets of 400 ml, siphons, pipettes and hand nets were used for the collection of immature stages, which were identified at the specific level in the laboratory under a stereoscopic microscope through the use of dichotomous keys (Consoli and Oliveira 1994 ; Darsie et al. 1985; Mitchell et al. 1985). They were then conditioned in the laboratory in the same way as the Cx. pipiens complex until obtaining adults. Then, adults obtained from all species sampled were cold sacrificed and stored in the freezer at -80°C for subsequent processing in order to detect Wolbachia infection. Additionally, to explore the relationship between environmental temperature and Wolbachia prevalence in Ae. fluviatilis , monthly collections of larvae in different types of sites (tire repair shops, cemeteries and family dwellings) were carried out during 2017 because of the difficulty of obtaining adults from the field. The temperature of the containers where the individuals were obtained (plastic, glass and concrete containers as well as car tires, canvas pools and water tanks) was recorded with a mercury rod thermometer with a temperature range of 0°C to 110°C, which was submerged about 10 cm in the water. DNA extraction from mosquitoes Total genomic DNA extraction from adult mosquitoes was performed with Chelex® 100 (Tecnolab S.A., Bs As, Argentina) according to the manufacturer's instructions. A 5% solution of Chelex resin was prepared. DNA extraction from the larvae was carried out using the Wizard® Genomic DNA Purification kit (Promega, USA) according to the manufacturer's instructions. Molecular Identification of C. pipiens Two multiplex PCRs were accomplished: the first one using the primers ACEquin + ACEpip + B1246s (Smith and Fonseca 2004 ); and the second one using the primers pipCQ11R + molCQ11R + CQ11F2 (Bahnck and Fonseca 2006 ). The PCRs were prepared in a final volume of 12.5 µl containing 6.25 µl of Master Mix (PB-L Productos Bio-Lógicos, Quilmes, Bs. As), 4.25 µl of DNase-free water, 0.5 µl of each primer (10 µM) (Invitrogen, USA) and 1 µl Template DNA. The amplification was carried out in the Eppendorf thermal cycler Mastercycler Nexus (Eppendorf®, USA). The cycling conditions for the first PCR were 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute during 35 cycles, and for the second PCR 94°C for 30 seconds, 54°C for 30 seconds and 72°C for 40 seconds for 40 cycles. Once the reaction was completed, the PCR products were visualized by 1% (w/v) agarose gel electrophoresis using a Mini-Sub CellGT® cell, (BioRad, California), containing 0.5X TBE Buffer, and separated at 90 volts for 40 minutes. Staining was made by adding 0.4 µg/ml of Ethidium Bromide (BrEt) (Promega, USA) to the agarose gel prior to the run. The gels were visualized in a Tm-20 Transilluminator UV light transilluminator (Upland, CA, USA). Detection of Wolbachia Diagnosis of Wolbachia infection was carried out by PCR of a portion of subunit I of the coxA gene (enzyme cytochrome oxidase C) with the primers designed by Baldo et al. ( 2006 ), using Wolbachia DNA obtained from Cx. pipiens as positive control and DNase-free water as negative control. Cycling conditions were 94°C for 15 seconds, 56°C for 45 seconds, 72°C for 1 minute for 37 cycles. To avoid false negative results, the quality of the DNA extraction was verified using the primers S1718 and A2442 (Normark 1996 0 that amplify a portion of the first COI gene (subunit I of the enzyme mitochondrial enzyme Cytochrome Oxidase c) with the conditions specified by Rodriguero et al. ( 2010 ). Wolbachia prevalence was estimated as the proportion of infected individuals over the total number of individuals sampled. Wolbachia typing The strains found were fully typed by MLST (Baldo et al. 2006 ). Amplicons were purified with the Puriprep-GP kit (Inbio Highway, Tandil, Bs. As). Sequences were obtained with an ABI3730 XL sequencer (Macrogen Inc., Korea). Chromatograms were edited using BIOEDIT (Hall 1999 ). Consensus sequences were compared with those deposited in the Gen Bank database ( https://www.ncbi.nlm.nih.gov/genbank/ ) using the BLAST algorithm (Altschul et al. 1997) in order to corroborate identity. Allele number of each locus was assigned through comparison with the Wolbachia MLST database ( https://pubmlst.org/organisms/wolbachia-spp/ ). The strains were characterized by the combination of the allelic numbers (allelic profile or ST). Subsequently, a bayesian phylogenetic analysis was performed including 52 strains retrieved from the Wolbachia MLST database from supergroups A, B, C, D, F, and H and those ones obtained in the present work. The sequences of the coxA , fbpA , ftsZ , gatB , and hcpA genes were concatenated using the Mesquite 2 program (Maddison and Maddison 2007 ) and aligned using the CLUSTALW program (Thompson et al. 1994 ). Our complete data set included 2079 aligned nucleotide positions. To infer the fittest evolutionary model, the jModelTest 2 program (Darriba et al. 2015 ) was used. The GTR + G model was selected for the coxA , ftsZ , gatB and hcpA partitions and the GTR + G + I model was the best fit for the fbpA partition. Bayesian phylogenetic analysis of the concatenated sequences was applied through the “Monte Carlo metropolis-coupled Markov chain” (MC3) algorithm implemented in MrBayes v. 3.2.6 (Huelsenbeck and Ronquist 2001 ; Ronquist et al. 2012 ) using a partitioning algorithm. Two independent analyses were performed with a random-start tree over 2,500,000 generations with a sampling frequency of every 500 trees. Tree space was explored using four strings: one cold and three incrementally heated ones with temperature (T) set to 0.20. The steady state of the Markov cold chain was evaluated for all MrBayes analyses in TRACER v. 1.7 (Rambaut et al. 2018 ), in addition to inspecting the standard deviation of clade cleavage frequencies (< 0.01). All samples prior to burn-in were discarded, in this case the first 500 trees. The remaining trees were taken into account to construct the posterior distribution and to obtain a 50% majority consensus tree and estimates of mean branch lengths. The frequency of all bipartitions was estimated to assess the support of each node (Huelsenbeck et al. 2001). As the tree root of the genus Wolbachia has not yet been determined (Bordenstein et al. 2009 ; Casiraghi et al. 2005 ), no outgroup was included. However, in order to analyze horizontal transfer events, we arbitrarily rooted the tree with a strain of the supergroup H. Results Collection and identification of mosquitoes A total of 191 adult females of Ae. albifasciatus were collected, which were analyzed in 62 pools conformed by 2 to 5 individuals. A total of 118 adults from the Cx. pipiens complex were collected, 64 from the La Plata ditch (34 ♀ and 30 ♂) and 54 from the Berisso ditch (25♀and 29 ♂). PCR-based identification of the members of the Cx. pipiens complex in both ditches showed a pattern of double bands in all individuals for the AceII gene, indicating the presence of hybrids between Cx. pipiens and Cx. quinquefasciatus in both sites studied. In addition, in the Berisso ditch a single band of size 250bp was observed for the amplification the microsatellite locus CQ11, which indicated that in these samples the hybrids result from mating between the form Cx. p. molestus and Cx. quinquefasciatus . A total of 12 adults of Ae. fluviatilis were obtained (6 ♀ and 6 ♂), which were analyzed in three pools of females and four pools of males. A total of 60 larvae of Ae. fluviatilis were obtained during 2017 (1 container/month) and taken into account for prevalence data. Detection of Wolbachia and estimation of prevalence infection All 62 pools of Ae. albifasciatus tested for Wolbachia were negative. Wolbachia detection in the Cx. pipiens complex was performed individually and the percentage of infection was 100% for both males and females at both sampling sites. In these localities, the infection is fixed. Wolbachia prevalence in females of Ae. fluviatilis was 66.6% (only one pool containing a female was negative). In males, an infection prevalence of 100% was reported. The total prevalence was 85.7%. The infection is close to fixation in this population. By the low number of emerged adults, the prevalence of Wolbachia infection in Ae. fluviatilis larvae was estimated during one year, being 100% every month except in January, which reached 80%, accounting for one negative individual (Fig. 2 ). Wolbachia strains typing and phylogenetic analysis The allelic profiles of the w Flu ( Ae. fluviatilis ) and w Pip ( Culex sp.) strains were obtained (Table 1 ). Sequences are stored at GenBank under Accession Numbers PP062804-8. The w Pip strain is identical to that reported by Baldo et al. ( 2006 ) for Cx. pipiens . The phylogram shows that w Flu belong to supergroup B of the genus Wolbachia (Fig. 3 ). Sequences of the w Flu strain can be retrieved from GenBank (Accession Numbers PP062804-08). Table 1 Allelic profile of the w Flu ( Ae. fluviatilis ) and w Pip ( Culex sp.) strains detected in the present work. Strain ST coxA fbpA ftsZ gatB hcpA w Flu 376 66 27 179 215 229 w Pip 9 3 4 22 4 3 Temperature and prevalence of infection Temperature of the water in the containers with Ae . fluviatilis larvae ranged between 18°C and 37°C, presenting the maximum and minimum peaks in the months of March and October, respectively. The annual average was 25.5°C, without marked seasonal differences (Fig. 2 ). No statistical relationship could be revealed with the current number of replicates. Discussion In this work Wolbachia infection was detected in two of the three species of mosquitoes studied, being Ae. albifasciatus the uninfected. Wolbachia infection was reported for the first time in Ae. fluviatilis from Argentina and detected in hybrid populations of the Cx. pipiens complex. All of them belong to the supergroup B. Indeed, this information is reported for the first time for the w Flu strain, but not for the w Pip strain, which coincided with the already reported in other populations of Cx. pipiens (Baldo et al. 2006 ). Aedes albifasciatus has a wide distribution in Argentina (Mitchell et al. 1985) and lastly, some population explosions driven by climate change turned this vector into a real problem both in economic and health terms. In this work, all the sites studied were free of Wolbachia infection. Though there are few studies in this host, our result coincides with that reported by Díaz-Nieto ( 2013 ) in populations from Mar del Plata, Argentina. Possibly, some type of competitive interaction with other/s component/s of the microbiota might prevent Wolbachia invasion/colonization of this host, as it was observed in Anopheles gambiae (Bourtzis et al. 2014 ; Rossi et al. 2015 ). The strain found in Ae. fluviatilis was identified as w Flu by MLST and placed in supergroup B by phylogenetic analysis. Although this strain was found in the repository of the Wolbachia database ( https://pubmlst.org/organisms/wolbachia-spp ), neither supergroup nor ST were referred in any publication. Instead, Moreira et al. (2009) and Baton et al. ( 2013 ) found this same strain in a colony of Ae. fluviatilis developed at the Oswaldo Cruz Foundation (FIOCRUZ) Minas (Belo Horizonte, Brazil) and in a sample from that locality with no identification up to date. However, they determined that the infection was native to the species and showed that this strain has the ability to colonize host populations due to its high rates of vertical transmission and no apparent fitness cost. Thus, this is the first report of the identification of w Flu in a natural population of Ae. fluviatilis . Although Wolbachia prevalence reported in the present work for this host species should be taken cautiously, since pools were analyzed, we think that it is close to fixation. Among the members of the Cx. pipiens complex, the two most abundant species in the world are Cx. pipiens , with pipiens and molestus ecotypes, and Cx. quinquefasciatus . In La Plata and Berisso, hybrids between Cx. p. F. molestus and Cx. quinquefasciatus were found, in agreement with what had already been reported in previous studies in the area (Cardo et al. 2020 ; Micieli et al. 2013 ). The same strain of Wolbachia , called w Pip, was found in both sites, this being the first report of infection in hybrid populations of the Cx. pipiens complex. This finding coincided with reports in other species of the complex, all of them infected with this same strain from the supergroup B (Díaz-Nieto et al. 2021 ) which strictly corresponds to a set of strains that share the allelic profile (Atyame et al. 2011 ). In both locations of Berisso and La Plata, the infection is fixed. This result agrees with published data on other Cx. pipiens and Cx. quinquefasciatus populations (Duron et al. 2005 ; Rasgon and Scott 2003 ), although there are some reports from populations in South Africa and Russia where the prevalence was lower (Cornel et al. 2003 ; Khrabrova and Sibataev 2019 ). This strain is known to induce partial or complete CI, which can be unidirectional or bidirectional (Duron et al. 2006 ; Guillemaud et al. 1997 ; Rasgon and Scott 2003 ). Phylogenetic analysis shows that all the strains studied in this work belong to the Wolbachia supergroup B. Although the w Flu strains may be closely related the mosquito strain w AlbB, the poor support of the group that contains both and the sampling bias does not allow inferences to be made about the phylogenetic closeness between them. The phylogenetic position of these strains and other mosquito isolates such as w Pip, w Alb_A, Cpip_B, confirms that horizontal transfer events have occurred within this group of insects, as it has been widely documented for this genus of bacteria. Wolbachia is currently used as a tool to reduce the transmission of mosquito-borne arboviruses (Walker et al. 2011 ). It is known that extreme temperatures can affect Wolbachia density within the host, leading to a decrease in the efficiency of arbovirus blocking or in factors relevant for the invasion of new hosts, such as transmission rates and the magnitude of the IC (Ross et al. 2019 ). In this work, the prevalence of Wolbachia strain w Flu was preliminarily explored as a function of the environmental temperature of the mosquito larvae breeding sites. At first sight, both the maximum and the minimum peaks seems not to have affected w Flu prevalence in the population assayed. If confirmed with more replicates, absence of heat stress effects may be due to the fact that Wolbachia strains may respond differently to such stimulus due to different evolutionary histories, or may have been unnoticed in the present work. Indeed, the effect of thermal stress on Wolbachia density during larval development might be manifested in the adult stage (Ulrich et al. 2017). In the present study the measurements were made on the larvae; although the temperature oscillations were within the tolerable range for Wolbachia (20°C to 30°C) (López-Madrigal and Duarte 2019 ), the breeding site of Ae. fluviatilis showed a peak at 37°C during March. While these variations may not have had any effect in the immature stages, they might have it in the adults. Moreover, w Flu density may be a better variable than prevalence to assess heat stress effects on infection (López Madrigal and Duarte 2019). However, adult emergence under laboratory conditions was hard to obtain in the present study and replicates difficult to obtain. Improving rearing conditions of this mosquito species in the lab must be reached before ruling out a relationship between thermal stress and w Flu dynamics. Should this goal be met, further studies should evaluate the effect of heat stress on w Flu density in the adult stage. Finally, because the use of Wolbachia as a biocontrol method has gained great interest, it is necessary to know the current diversity in nature before introducing new strains with desirable characteristics, to avoid future problems such as CI. In this work, two strains were identified in mosquito species from Argentina, one being a new report in the country ( w Flu). Although infection with w Pip was already known in populations from Argentina, this is the first time that it has been reported in hybrid populations of the Cx. pipiens complex. Declarations Acknowledgements : We gratefully acknowledge María Laura Morote for helping in the figures design and Darío Balcazar and Walter Ferrari for technical assistance. Thanks are due to the anonymous reviewers who provided invaluable feedback on the manuscript. Ethical Approval Not applicable Competing interests The authors declare no competing interests. Authors' contributions Ailén Chuchuy, Marcela S. Rodriguero and María V. Micieli contributed to the study conception and design. Material preparation and data collection were performed by Ailén Chuchuy, Ana C. Alonso and Marina Stein. Data analyses were performed by Ailén Chuchuy and Marcela S. Rodriguero. The first draft of the manuscript was written by Ailén Chuchuy. All authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This contribution was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT 2015-0665) and Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 584) to MVM. AC was awarded a doctoral scholarship from Agencia Nacional de Promoción Científica y Tecnológica. MSR, MS and MVM are members of the Research Career of Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. Availability of data and materials The DNA sequences can be retrieved from GenBank. Accession Numbers can be found in the body of the manuscript. References Alonso AC, Stein M, Hisgen CM, Micieli MV (2022) Abiotic factors affecting the prevalence of Wolbachia (Rickettsiaceae) in immature Aedes albopictus (Skuse) (Culicidae). 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Robert, MA, Tinunin DT, Benitez EM et al (2019) Arbovirus emergence in the temperate city of Córdoba, Argentina, 2009–2018. Sci Data 6:276. Rodriguero MS (2013) Wolbachia , una pandemia con posibilidades. Rev Soc Entomol Argentina 72:117–137. Rodriguero MS, Confalonieri VA, Guedes JVC, Lanteri AA (2010) Wolbachia infection in the tribe Naupactini (Coleoptera, Curculionidae): association between thelytokous parthenogenesis and infection status. Insect Mol Biol 19:631–640. Ronquist F, Teslenko M, Van Der Mark P, Ayres DL et al (2012) MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Syst Biol 61:539–542. Ross PA, Axford JK, Yang Q, Staunton KM, Ritchie SA, Richardson KM, Hoffmann AA (2020) Heatwaves cause fluctuations in w Mel Wolbachia densities and frequencies in Aedes aegypti . PLoS Negl Trop Dis 14(1): e0007958. 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Smith JL, Fonseca DM (2004) Rapid Assays for Identification of Members of the Culex (Culex) pipiens Complex,Their Hybrids, and Other Sibling Species (Diptera: Culicidae). Am J Trop Med Hyg 70:339–345. Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence aligment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res Acids Res 22:4673–4680. Turelli M, Hoffmann AA (1995) Cytoplasmic Incompatibility in Drosophila simulans : Dynamics and Parameter Estimates from Natural Populations. Genetics 140:1319–1338. Ulrich JN, Beier JC, Devine GJ, Hugo LE (2016) Heat Sensitivity of w Mel Wolbachia during Aedes aegypti Development. PLoS Negl Trop Dis 10:1–16. Walker T, Johnson PH, Moreira LA, Iturbe-Ormaetxe I et al (2011) The w Mel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature 476:450–455. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4361303","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":301606992,"identity":"646f26bd-36d6-4634-bc3a-07d56e25cf6f","order_by":0,"name":"Ailen Chuchuy","email":"","orcid":"","institution":"CONICET - Centro de Estudios Parasitológicos y de Vectores (CEPAVE-CCT-La Plata- CONICET-UNLP)","correspondingAuthor":false,"prefix":"","firstName":"Ailen","middleName":"","lastName":"Chuchuy","suffix":""},{"id":301606993,"identity":"c9e4c82d-9c42-4de4-b25e-1f9deaf1b321","order_by":1,"name":"Marcela S. Rodriguero","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIie3QMUvDQBTA8RcO6nIh6wstyVe4kKGD4me54OASpy6CYgOFZKnOAfFTFErHSCAuZz9DO7koGHAItYiXq25JyCh4f8i9N+QHlwDodH8zBlxNktWnpXYj6kUGathRL3KIsn5knMyW75td7oyTebn9uAS0ktuMmKtTsO6zRjISxSQN7nJ/JJ4X8gBEsebEFGeAa95IEEMfgnkepHixHNox3DAMGTFjAiCaL/ZLpimGL5/2FyBzX2syBbeL8CrnchkYZSQJ0prkwNoILSYQROdeSgt/CAXKbwnZ44N4ol4bOZotjGp/7MplW1bXJ/KPCW/ztrpynBaiMuLDJBRQLZl8aAeQ7X9o1f2aTqfT/dO+ASDyV+CTfaLQAAAAAElFTkSuQmCC","orcid":"","institution":"Universidad de Buenos Aires","correspondingAuthor":true,"prefix":"","firstName":"Marcela","middleName":"S.","lastName":"Rodriguero","suffix":""},{"id":301606994,"identity":"9ed61a31-9c0c-4e23-a719-1bb2dfabc48b","order_by":2,"name":"Ana C. 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Micieli","email":"","orcid":"","institution":"CONICET - Centro de Estudios Parasitológicos y de Vectores (CEPAVE-CCT-La Plata- CONICET-UNLP)","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"V.","lastName":"Micieli","suffix":""}],"badges":[],"createdAt":"2024-05-03 00:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4361303/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4361303/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00436-024-08352-5","type":"published","date":"2024-10-09T15:57:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56373441,"identity":"0525f8b3-fc60-42fd-9ce2-9602e6454395","added_by":"auto","created_at":"2024-05-13 10:32:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePanel a\u003c/strong\u003e Sampling sites for \u003cem\u003eAedes albifasciatus \u003c/em\u003eand \u003cem\u003eCulex pipiens \u003c/em\u003ecomplex in La Plata city (black squares, \u003cem\u003eAedes albifasciatus\u003c/em\u003e; white squares, \u003cem\u003eCulex Pipiens\u003c/em\u003e), Buenos Aires province, Argentina\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePanel b\u003c/strong\u003e Sampling sites for \u003cem\u003eAedes fluviatilis \u003c/em\u003ein Eldorado, Misiones province, Argentina (white dots, \u003cem\u003eAedes fluviatilis\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4361303/v1/6bc22ecc93e4ed2e334d077a.png"},{"id":56373442,"identity":"453914a8-b3be-40b3-930c-c1fc804024a1","added_by":"auto","created_at":"2024-05-13 10:32:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4719,"visible":true,"origin":"","legend":"\u003cp\u003eMonthly prevalence of \u003cem\u003eWolbachia\u003c/em\u003e infection in \u003cem\u003eAe. fluviatilis\u003c/em\u003eaccording to the temperature of the water in the container\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4361303/v1/736c57a8ac5d94a5e52aa38f.png"},{"id":56373443,"identity":"fbf1cb50-8e6e-4901-8560-60d0c9d4ac29","added_by":"auto","created_at":"2024-05-13 10:32:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":23939,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny based on the genes used for MLST including 52 \u003cem\u003eWolbachia\u003c/em\u003estrains from known supergroups and the strains investigated in this study. Outgroup: \u003cem\u003ew\u003c/em\u003eZang_H. The Bayesian posterior probabilities are indicated above the nodes. Branch lengths indicate number of substitutions per site. Scale bar indicates the number of substitutions per site. The arrows indicate both the \u003cem\u003ew\u003c/em\u003eFlu and \u003cem\u003ew\u003c/em\u003epip strains isolated in the present work\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4361303/v1/38db886655342b2381a17498.png"},{"id":66597860,"identity":"f6d9dda5-9d9a-4373-b10c-29b926e7e508","added_by":"auto","created_at":"2024-10-14 16:11:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":680095,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4361303/v1/d3aedb31-621c-41d8-8fc5-29ced04d1af2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Wolbachia infection in natural mosquito populations from Argentina","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, there has been an increase in the distribution of mosquito-borne diseases. Once confined to tropical and subtropical regions, vectors are expanding into temperate regions around the world (e.g. L\u0026oacute;pez et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Roberts et al. 2019). This is a consequence of the increase of international trips, rapid urbanization with no proper planning, deforestation, climate change and insufficient or inefficient sanitary measures (Cabrera et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Da Silva et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; D\u0026iacute;az et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hubert et al. 2018). For instance, a population burst of \u003cem\u003eAedes albifasciatus\u003c/em\u003e (Macquart, 1838) because of El Ni\u0026ntilde;o-induced climate change, led to an invasion of several urban cities from Argentina (Buenos Aires province). This caused an epidemic break of Western Equine Encephalitis Virus (WEEV) in horses, and subsequently, after more than 30 years without cases, 12 people were infected, and one casualty (WHO \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, there is urgency for increasing effective control measures against vectors. Nowadays, \u003cem\u003eWolbachia\u003c/em\u003e infection mosquito represents a promising new bio-control strategy (Minwuyelet et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eWolbachia\u003c/em\u003e Hertig, 1936 is the most successful pandemics since the origin of life. Since its discovery in the germ line and somatic tissues of the \u003cem\u003eCulex pipiens\u003c/em\u003e (Linnaeus, 1758) mosquito in 1924 (Hertig and Wolbach \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1924\u003c/span\u003e), the number of \u003cem\u003eWolbachia\u003c/em\u003e strains and associated hosts continues to increase (Landmann \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Evidence shows that the key to its success is mainly its capacity for vertical transmission through the egg driven by reproductive manipulations such as cytoplasmic incompatibility (CI) and for horizontal transfer between host species (Rodriguero \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Since the discovery of \u003cem\u003eWolbachia\u003c/em\u003e in the mosquito \u003cem\u003eCx. Pipiens\u003c/em\u003e (Hertig and Wolbach \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1924\u003c/span\u003e), scientific reports demonstrate the presence of this bacterium in various mosquitoes genera such as \u003cem\u003eArmigeres\u003c/em\u003e (Leicesteria), \u003cem\u003eMansonia\u003c/em\u003e Blanchard, 1901, \u003cem\u003eCoquillettidia\u003c/em\u003e Dyar, 1904, \u003cem\u003eCuliseta\u003c/em\u003e Felt, 1904, \u003cem\u003eHodgesia\u003c/em\u003e, Theobald, 1904, \u003cem\u003eTripteroides\u003c/em\u003e Giles, 1904 and \u003cem\u003eUranotaenia\u003c/em\u003e Lynch Arrib\u0026aacute;lzaga, 1891 (Nugapola et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Raharimalala et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wiwatanaratanabutr 2013), most of these species without epidemiological relevance. Nevertheless, infection is also known in important species of disease-transmitting mosquitoes in the genera \u003cem\u003eCulex\u003c/em\u003e Linnaeus 1758, \u003cem\u003eAedes\u003c/em\u003e Meigen 1818 and \u003cem\u003eAnopheles\u003c/em\u003e Meigen 1818, for example (Bian et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hoffman et al. 2014; Johnson \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMost of the strains identified in mosquitoes are within supergroups A or B of \u003cem\u003eWolbachia\u003c/em\u003e, and even superinfections (i.e. infections with two or more strains) can occur (Werren et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Zou et al. 1998). The similarities between the strains that infect different mosquito species suggest a simple pathway by which these insects find and acquire new infections (horizontal transfer). Within each species there is variation in infection status between populations, from low frequencies to fixation (Charlesworth et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hilgenboecker et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; In\u0026aacute;cio da Silva et al. 2012). However, the prevalence of \u003cem\u003eWolbachia\u003c/em\u003e infections in these insects may be underestimated because often they can occur at very low densities that are not detectable by conventional PCR (Mee et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince the discovery of some strains having the ability to interfere or block the transmission of certain arboviruses of public health importance such as dengue and zika (Landmann \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), a widespread interest in the use of experimentally generated \u003cem\u003eWolbachia\u003c/em\u003e-infected mosquitoes for biological control of infectious diseases (Iturbe-Ormaetxe et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) arose. With this purpose, two approaches were proposed: population replacement and population suppression (Atyame et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Iturbe-Ormaetxe et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Both \u003cem\u003eWolbachia\u003c/em\u003e-based biocontrol methods require the field release of transinfected individuals with strains that induce CI during mating with the wild mosquito populations (Ross et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For this reason, prior to release it is crucial to know which strains of \u003cem\u003eWolbachia\u003c/em\u003e are present in wild populations because: i) pre-existing natural infections can interact and alter the dynamics of introduced strains, making population replacement or suppression a challenging obstacle to overcome (Rasgon and Scott \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e); ii) the dynamics of \u003cem\u003eWolbachia\u003c/em\u003e spread can become complicated as the number of incompatible strains present in the population increases (Turelli and Hoffman 1995). Furthermore, understanding the similarities and differences between \u003cem\u003eWolbachia\u003c/em\u003e strains that infect different mosquito species is critical for estimating how frequently (over evolutionary times) mosquitoes experience horizontal transmission events of this bacterium in nature (Rasgon and Scott 2019). On the other hand, the study of \u003cem\u003eWolbachia\u003c/em\u003e infection in vector and non-vector mosquito species aids the discovery of novel strains of these bacteria with desirable characteristics that can be used to artificially transinfect other species of interest (Bourtzis et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany factors can influence both the infection dynamics and the extent of bacteria-induced host phenotypes in host-symbiont relationships. Of all of them, symbiotic population density is one of the most relevant (L\u0026oacute;pez Madrigal and Duarte 2019). Although both bacterial and host genetic backgrounds are involved in density regulation, environmental factors, like temperature, may also affect bacterial population density (Mouton et al. 2007).\u003c/p\u003e \u003cp\u003eEnvironmental temperature shows a great impact on \u003cem\u003eWolbachia\u003c/em\u003e load and host phenotypes induced by the bacteria. Temperatures above 30\u0026deg;C have been shown to reduce \u003cem\u003eWolbachia\u003c/em\u003e density or even eliminate the infection, while temperatures below 20\u0026deg;C compromise the proliferation rate (L\u0026oacute;pez Madrigal and Duarte 2019). Thus, temperatures within the range tolerated by hosts can be extreme for \u003cem\u003eWolbachia\u003c/em\u003e (Ross et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The effects caused by environmental temperature can be strain-specific (Ross et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This is an important aspect to consider for evaluation of \u003cem\u003eWolbacia\u003c/em\u003e as a biocontrol tool, since low densities can lead to a reduction in virus blocking efficiency, weaker CI and/or failure of maternal transmission (Ye et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe works accomplished on \u003cem\u003eWolbachia\u003c/em\u003e in mosquito populations in Argentina are scarce and mainly based on \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e Say, 1823 and recently in \u003cem\u003eAedes albopictus\u003c/em\u003e Skuse, 1895 (Alonso et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chuchuy et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; D\u0026iacute;az-Nieto et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Micieli and Glaser \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Morais et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thus, in the present work, the occurrence of this endosymbiont in mosquito populations in natural systems of Argentina was investigated. Our surveys focused on \u003cem\u003eAedes fluviatilis\u003c/em\u003e (Lutz, 1904), a potential vector of yellow fever (Baton et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rey and Lounibos \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003eAe. albifasciatus\u003c/em\u003e which is considered a pest throughout the south and center of the country (Garc\u0026iacute;a and Micieli \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Gleiser et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and hybrid populations of the \u003cem\u003eC. pipiens\u003c/em\u003e complex, which are cosmopolitan vectors of pathogens of medical and veterinary importance (Farajollahi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The relationship between environmental temperature and the prevalence of \u003cem\u003eWolbachia\u003c/em\u003e in \u003cem\u003eAe. fluviatilis\u003c/em\u003e from Eldorado (Misiones province) was also investigated.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling area\u003c/h2\u003e \u003cp\u003eSamplings were carried out in four localities located in two provinces of Argentina. Specimens of the complex \u003cem\u003eCx. pipiens\u003c/em\u003e were collected in Berisso (34\u0026deg;52\u0026rsquo;00\u0026rsquo;\u0026rsquo;S \u0026minus;\u0026thinsp;57\u0026deg;52\u0026rsquo; 00\u0026rsquo;\u0026rsquo;W) and the city of La Plata (34\u0026deg;55\u0026rsquo;07\u0026rdquo; S \u0026minus;\u0026thinsp;57\u0026deg;57\u0026rsquo;15\u0026rdquo; W), province of Buenos Aires (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea); individuals of \u003cem\u003eAe. albifasciatus\u003c/em\u003e were sampled in Ensenada (34\u0026deg;51\u0026rsquo;S \u0026minus;\u0026thinsp;57\u0026deg;54\u0026rsquo;W) and La Plata, province of Buenos Aires (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea); individuals of \u003cem\u003eAe. fluviatilis\u003c/em\u003e were collected in Eldorado (26\u0026ordm;24\u0026rsquo;S \u0026minus;\u0026thinsp;54\u0026ordm;38\u0026rsquo;W), province of Misiones (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMosquitoes and environmental data collection\u003c/h2\u003e \u003cp\u003eSamples of \u003cem\u003eAe. albifasciatus\u003c/em\u003e were collected during the months of March, April and May 2017. Adult individuals were collected in temporary rainwater puddles using manual battery-powered vacuum cleaners with a person as bait, collecting mainly females and identified using morphological characters by dichotomous keys (Darsie \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Samples of the \u003cem\u003eCx. pipiens\u003c/em\u003e complex were collected during July and August 2017 (La Plata) and during January and February 2018 (Berisso). Immature stages were collected in household ditches where rainwater and drainage from kitchens and laundry rooms stagnate, to which sewage effluents are usually added. Immature stages were collected using pipettes and fine-mesh aquarium nets. These were conditioned in plastic trays (30 cm x 18 cm x 6 cm) with dechlorinated water and finely ground guinea pig food in the insectary of Centro de Estudios Parasitol\u0026oacute;gicos y de Vectores (CEPAVE, UNLP-CONICET) until adults were obtained. \u003cem\u003eCulex pipiens\u003c/em\u003e identification was performed by polymerase chain reaction (PCR) amplification of the \u003cem\u003eAceII\u003c/em\u003e gene (nuclear acetylcholinesterase-2 gene), which allows the species to be distinguished between \u003cem\u003eCx. pipiens\u003c/em\u003e/\u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, and the microsatellite locus CQ11, which allows identification of the ecotypes \u003cem\u003eCx. pipiens\u003c/em\u003e, \u003cem\u003eCx. p. pipiens\u003c/em\u003e/\u003cem\u003eCx. p. molestus\u003c/em\u003e, following the protocols of Smith and Fonseca (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and Bahnck and Fonseca (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAe. fluviatilis\u003c/em\u003e individuals were collected in September 2017 in car tires. Buckets of 400 ml, siphons, pipettes and hand nets were used for the collection of immature stages, which were identified at the specific level in the laboratory under a stereoscopic microscope through the use of dichotomous keys (Consoli and Oliveira \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Darsie et al. 1985; Mitchell et al. 1985). They were then conditioned in the laboratory in the same way as the \u003cem\u003eCx. pipiens\u003c/em\u003e complex until obtaining adults.\u003c/p\u003e \u003cp\u003eThen, adults obtained from all species sampled were cold sacrificed and stored in the freezer at -80\u0026deg;C for subsequent processing in order to detect \u003cem\u003eWolbachia\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eAdditionally, to explore the relationship between environmental temperature and \u003cem\u003eWolbachia\u003c/em\u003e prevalence in \u003cem\u003eAe. fluviatilis\u003c/em\u003e, monthly collections of larvae in different types of sites (tire repair shops, cemeteries and family dwellings) were carried out during 2017 because of the difficulty of obtaining adults from the field. The temperature of the containers where the individuals were obtained (plastic, glass and concrete containers as well as car tires, canvas pools and water tanks) was recorded with a mercury rod thermometer with a temperature range of 0\u0026deg;C to 110\u0026deg;C, which was submerged about 10 cm in the water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction from mosquitoes\u003c/h2\u003e \u003cp\u003eTotal genomic DNA extraction from adult mosquitoes was performed with Chelex\u0026reg; 100 (Tecnolab S.A., Bs As, Argentina) according to the manufacturer's instructions. A 5% solution of Chelex resin was prepared. DNA extraction from the larvae was carried out using the Wizard\u0026reg; Genomic DNA Purification kit (Promega, USA) according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular Identification of\u003c/b\u003e \u003cb\u003eC. pipiens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwo multiplex PCRs were accomplished: the first one using the primers ACEquin\u0026thinsp;+\u0026thinsp;ACEpip\u0026thinsp;+\u0026thinsp;B1246s (Smith and Fonseca \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2004\u003c/span\u003e); and the second one using the primers pipCQ11R\u0026thinsp;+\u0026thinsp;molCQ11R\u0026thinsp;+\u0026thinsp;CQ11F2 (Bahnck and Fonseca \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The PCRs were prepared in a final volume of 12.5 \u0026micro;l containing 6.25 \u0026micro;l of Master Mix (PB-L Productos Bio-L\u0026oacute;gicos, Quilmes, Bs. As), 4.25 \u0026micro;l of DNase-free water, 0.5 \u0026micro;l of each primer (10 \u0026micro;M) (Invitrogen, USA) and 1 \u0026micro;l Template DNA. The amplification was carried out in the Eppendorf thermal cycler Mastercycler Nexus (Eppendorf\u0026reg;, USA). The cycling conditions for the first PCR were 94\u0026deg;C for 30 seconds, 55\u0026deg;C for 30 seconds and 72\u0026deg;C for 1 minute during 35 cycles, and for the second PCR 94\u0026deg;C for 30 seconds, 54\u0026deg;C for 30 seconds and 72\u0026deg;C for 40 seconds for 40 cycles. Once the reaction was completed, the PCR products were visualized by 1% (w/v) agarose gel electrophoresis using a Mini-Sub CellGT\u0026reg; cell, (BioRad, California), containing 0.5X TBE Buffer, and separated at 90 volts for 40 minutes. Staining was made by adding 0.4 \u0026micro;g/ml of Ethidium Bromide (BrEt) (Promega, USA) to the agarose gel prior to the run. The gels were visualized in a Tm-20 Transilluminator UV light transilluminator (Upland, CA, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003eWolbachia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDiagnosis of \u003cem\u003eWolbachia\u003c/em\u003e infection was carried out by PCR of a portion of subunit I of the \u003cem\u003ecoxA\u003c/em\u003e gene (enzyme cytochrome oxidase C) with the primers designed by Baldo et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), using \u003cem\u003eWolbachia\u003c/em\u003e DNA obtained from \u003cem\u003eCx. pipiens\u003c/em\u003e as positive control and DNase-free water as negative control. Cycling conditions were 94\u0026deg;C for 15 seconds, 56\u0026deg;C for 45 seconds, 72\u0026deg;C for 1 minute for 37 cycles. To avoid false negative results, the quality of the DNA extraction was verified using the primers S1718 and A2442 (Normark \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1996\u003c/span\u003e0 that amplify a portion of the first \u003cem\u003eCOI\u003c/em\u003e gene (subunit I of the enzyme mitochondrial enzyme Cytochrome Oxidase c) with the conditions specified by Rodriguero et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003eWolbachia\u003c/em\u003e prevalence was estimated as the proportion of infected individuals over the total number of individuals sampled.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWolbachia\u003c/b\u003e \u003cb\u003etyping\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe strains found were fully typed by MLST (Baldo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Amplicons were purified with the Puriprep-GP kit (Inbio Highway, Tandil, Bs. As). Sequences were obtained with an ABI3730 XL sequencer (Macrogen Inc., Korea). Chromatograms were edited using BIOEDIT (Hall \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Consensus sequences were compared with those deposited in the Gen Bank database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using the BLAST algorithm (Altschul et al. 1997) in order to corroborate identity. Allele number of each locus was assigned through comparison with the \u003cem\u003eWolbachia\u003c/em\u003e MLST database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmlst.org/organisms/wolbachia-spp/\u003c/span\u003e\u003cspan address=\"https://pubmlst.org/organisms/wolbachia-spp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The strains were characterized by the combination of the allelic numbers (allelic profile or ST).\u003c/p\u003e \u003cp\u003eSubsequently, a bayesian phylogenetic analysis was performed including 52 strains retrieved from the \u003cem\u003eWolbachia\u003c/em\u003e MLST database from supergroups A, B, C, D, F, and H and those ones obtained in the present work. The sequences of the \u003cem\u003ecoxA\u003c/em\u003e, \u003cem\u003efbpA\u003c/em\u003e, \u003cem\u003eftsZ\u003c/em\u003e, \u003cem\u003egatB\u003c/em\u003e, and \u003cem\u003ehcpA\u003c/em\u003e genes were concatenated using the Mesquite 2 program (Maddison and Maddison \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and aligned using the CLUSTALW program (Thompson et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Our complete data set included 2079 aligned nucleotide positions.\u003c/p\u003e \u003cp\u003eTo infer the fittest evolutionary model, the jModelTest 2 program (Darriba et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) was used. The GTR\u0026thinsp;+\u0026thinsp;G model was selected for the \u003cem\u003ecoxA\u003c/em\u003e, \u003cem\u003eftsZ\u003c/em\u003e, \u003cem\u003egatB\u003c/em\u003e and \u003cem\u003ehcpA\u003c/em\u003e partitions and the GTR\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I model was the best fit for the \u003cem\u003efbpA\u003c/em\u003e partition.\u003c/p\u003e \u003cp\u003eBayesian phylogenetic analysis of the concatenated sequences was applied through the \u0026ldquo;Monte Carlo metropolis-coupled Markov chain\u0026rdquo; (MC3) algorithm implemented in MrBayes v. 3.2.6 (Huelsenbeck and Ronquist \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Ronquist et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) using a partitioning algorithm. Two independent analyses were performed with a random-start tree over 2,500,000 generations with a sampling frequency of every 500 trees. Tree space was explored using four strings: one cold and three incrementally heated ones with temperature (T) set to 0.20. The steady state of the Markov cold chain was evaluated for all MrBayes analyses in TRACER v. 1.7 (Rambaut et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), in addition to inspecting the standard deviation of clade cleavage frequencies (\u0026lt;\u0026thinsp;0.01). All samples prior to burn-in were discarded, in this case the first 500 trees. The remaining trees were taken into account to construct the posterior distribution and to obtain a 50% majority consensus tree and estimates of mean branch lengths. The frequency of all bipartitions was estimated to assess the support of each node (Huelsenbeck et al. 2001). As the tree root of the genus \u003cem\u003eWolbachia\u003c/em\u003e has not yet been determined (Bordenstein et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Casiraghi et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), no outgroup was included. However, in order to analyze horizontal transfer events, we arbitrarily rooted the tree with a strain of the supergroup H.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCollection and identification of mosquitoes\u003c/h2\u003e \u003cp\u003eA total of 191 adult females of \u003cem\u003eAe. albifasciatus\u003c/em\u003e were collected, which were analyzed in 62 pools conformed by 2 to 5 individuals.\u003c/p\u003e \u003cp\u003eA total of 118 adults from the \u003cem\u003eCx. pipiens\u003c/em\u003e complex were collected, 64 from the La Plata ditch (34 ♀ and 30 ♂) and 54 from the Berisso ditch (25♀and 29 ♂). PCR-based identification of the members of the \u003cem\u003eCx. pipiens\u003c/em\u003e complex in both ditches showed a pattern of double bands in all individuals for the \u003cem\u003eAceII\u003c/em\u003e gene, indicating the presence of hybrids between \u003cem\u003eCx. pipiens\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e in both sites studied. In addition, in the Berisso ditch a single band of size 250bp was observed for the amplification the microsatellite locus CQ11, which indicated that in these samples the hybrids result from mating between the form \u003cem\u003eCx. p. molestus\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA total of 12 adults of \u003cem\u003eAe. fluviatilis\u003c/em\u003e were obtained (6 ♀ and 6 ♂), which were analyzed in three pools of females and four pools of males. A total of 60 larvae of \u003cem\u003eAe. fluviatilis\u003c/em\u003e were obtained during 2017 (1 container/month) and taken into account for prevalence data.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003eWolbachia\u003c/b\u003e \u003cb\u003eand estimation of prevalence infection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAll 62 pools of \u003cem\u003eAe. albifasciatus\u003c/em\u003e tested for \u003cem\u003eWolbachia\u003c/em\u003e were negative.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWolbachia\u003c/em\u003e detection in the \u003cem\u003eCx. pipiens\u003c/em\u003e complex was performed individually and the percentage of infection was 100% for both males and females at both sampling sites. In these localities, the infection is fixed.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWolbachia\u003c/em\u003e prevalence in females of \u003cem\u003eAe. fluviatilis\u003c/em\u003e was 66.6% (only one pool containing a female was negative). In males, an infection prevalence of 100% was reported. The total prevalence was 85.7%. The infection is close to fixation in this population. By the low number of emerged adults, the prevalence of \u003cem\u003eWolbachia\u003c/em\u003e infection in \u003cem\u003eAe. fluviatilis\u003c/em\u003e larvae was estimated during one year, being 100% every month except in January, which reached 80%, accounting for one negative individual (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWolbachia\u003c/b\u003e \u003cb\u003estrains typing and phylogenetic analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe allelic profiles of the \u003cem\u003ew\u003c/em\u003eFlu (\u003cem\u003eAe. fluviatilis\u003c/em\u003e) and \u003cem\u003ew\u003c/em\u003ePip (\u003cem\u003eCulex\u003c/em\u003e sp.) strains were obtained (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Sequences are stored at GenBank under Accession Numbers PP062804-8. The \u003cem\u003ew\u003c/em\u003ePip strain is identical to that reported by Baldo et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) for \u003cem\u003eCx. pipiens\u003c/em\u003e. The phylogram shows that \u003cem\u003ew\u003c/em\u003eFlu belong to supergroup B of the genus \u003cem\u003eWolbachia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Sequences of the \u003cem\u003ew\u003c/em\u003eFlu strain can be retrieved from GenBank (Accession Numbers PP062804-08).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAllelic profile of the \u003cem\u003ew\u003c/em\u003eFlu (\u003cem\u003eAe. fluviatilis\u003c/em\u003e) and \u003cem\u003ew\u003c/em\u003ePip (\u003cem\u003eCulex\u003c/em\u003e sp.) strains detected in the present work.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eST\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecoxA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003efbpA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eftsZ\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003egatB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003ehcpA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ew\u003c/b\u003e\u003cb\u003eFlu\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e229\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ew\u003c/b\u003e\u003cb\u003ePip\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTemperature and prevalence of infection\u003c/h2\u003e \u003cp\u003eTemperature of the water in the containers with \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003efluviatilis\u003c/em\u003e larvae ranged between 18\u0026deg;C and 37\u0026deg;C, presenting the maximum and minimum peaks in the months of March and October, respectively. The annual average was 25.5\u0026deg;C, without marked seasonal differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No statistical relationship could be revealed with the current number of replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work \u003cem\u003eWolbachia\u003c/em\u003e infection was detected in two of the three species of mosquitoes studied, being \u003cem\u003eAe. albifasciatus\u003c/em\u003e the uninfected. \u003cem\u003eWolbachia\u003c/em\u003e infection was reported for the first time in \u003cem\u003eAe. fluviatilis\u003c/em\u003e from Argentina and detected in hybrid populations of the \u003cem\u003eCx. pipiens\u003c/em\u003e complex. All of them belong to the supergroup B. Indeed, this information is reported for the first time for the \u003cem\u003ew\u003c/em\u003eFlu strain, but not for the \u003cem\u003ew\u003c/em\u003ePip strain, which coincided with the already reported in other populations of \u003cem\u003eCx. pipiens\u003c/em\u003e (Baldo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAedes albifasciatus\u003c/em\u003e has a wide distribution in Argentina (Mitchell et al. 1985) and lastly, some population explosions driven by climate change turned this vector into a real problem both in economic and health terms. In this work, all the sites studied were free of \u003cem\u003eWolbachia\u003c/em\u003e infection. Though there are few studies in this host, our result coincides with that reported by D\u0026iacute;az-Nieto (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) in populations from Mar del Plata, Argentina. Possibly, some type of competitive interaction with other/s component/s of the microbiota might prevent \u003cem\u003eWolbachia\u003c/em\u003e invasion/colonization of this host, as it was observed in \u003cem\u003eAnopheles gambiae\u003c/em\u003e (Bourtzis et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rossi et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe strain found in \u003cem\u003eAe. fluviatilis\u003c/em\u003e was identified as \u003cem\u003ew\u003c/em\u003eFlu by MLST and placed in supergroup B by phylogenetic analysis. Although this strain was found in the repository of the \u003cem\u003eWolbachia\u003c/em\u003e database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmlst.org/organisms/wolbachia-spp\u003c/span\u003e\u003cspan address=\"https://pubmlst.org/organisms/wolbachia-spp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), neither supergroup nor ST were referred in any publication. Instead, Moreira et al. (2009) and Baton et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) found this same strain in a colony of \u003cem\u003eAe. fluviatilis\u003c/em\u003e developed at the Oswaldo Cruz Foundation (FIOCRUZ) Minas (Belo Horizonte, Brazil) and in a sample from that locality with no identification up to date. However, they determined that the infection was native to the species and showed that this strain has the ability to colonize host populations due to its high rates of vertical transmission and no apparent fitness cost. Thus, this is the first report of the identification of \u003cem\u003ew\u003c/em\u003eFlu in a natural population of \u003cem\u003eAe. fluviatilis\u003c/em\u003e. Although \u003cem\u003eWolbachia\u003c/em\u003e prevalence reported in the present work for this host species should be taken cautiously, since pools were analyzed, we think that it is close to fixation.\u003c/p\u003e \u003cp\u003eAmong the members of the \u003cem\u003eCx. pipiens\u003c/em\u003e complex, the two most abundant species in the world are \u003cem\u003eCx. pipiens\u003c/em\u003e, with \u003cem\u003epipiens\u003c/em\u003e and \u003cem\u003emolestus\u003c/em\u003e ecotypes, and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e. In La Plata and Berisso, hybrids between \u003cem\u003eCx. p.\u003c/em\u003e F. \u003cem\u003emolestus\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e were found, in agreement with what had already been reported in previous studies in the area (Cardo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Micieli et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The same strain of \u003cem\u003eWolbachia\u003c/em\u003e, called \u003cem\u003ew\u003c/em\u003ePip, was found in both sites, this being the first report of infection in hybrid populations of the \u003cem\u003eCx. pipiens\u003c/em\u003e complex. This finding coincided with reports in other species of the complex, all of them infected with this same strain from the supergroup B (D\u0026iacute;az-Nieto et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) which strictly corresponds to a set of strains that share the allelic profile (Atyame et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn both locations of Berisso and La Plata, the infection is fixed. This result agrees with published data on other \u003cem\u003eCx. pipiens\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e populations (Duron et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Rasgon and Scott \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), although there are some reports from populations in South Africa and Russia where the prevalence was lower (Cornel et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Khrabrova and Sibataev \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This strain is known to induce partial or complete CI, which can be unidirectional or bidirectional (Duron et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Guillemaud et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Rasgon and Scott \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhylogenetic analysis shows that all the strains studied in this work belong to the \u003cem\u003eWolbachia\u003c/em\u003e supergroup B. Although the \u003cem\u003ew\u003c/em\u003eFlu strains may be closely related the mosquito strain \u003cem\u003ew\u003c/em\u003eAlbB, the poor support of the group that contains both and the sampling bias does not allow inferences to be made about the phylogenetic closeness between them. The phylogenetic position of these strains and other mosquito isolates such as \u003cem\u003ew\u003c/em\u003ePip, \u003cem\u003ew\u003c/em\u003eAlb_A, Cpip_B, confirms that horizontal transfer events have occurred within this group of insects, as it has been widely documented for this genus of bacteria.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWolbachia\u003c/em\u003e is currently used as a tool to reduce the transmission of mosquito-borne arboviruses (Walker et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). It is known that extreme temperatures can affect \u003cem\u003eWolbachia\u003c/em\u003e density within the host, leading to a decrease in the efficiency of arbovirus blocking or in factors relevant for the invasion of new hosts, such as transmission rates and the magnitude of the IC (Ross et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this work, the prevalence of \u003cem\u003eWolbachia\u003c/em\u003e strain \u003cem\u003ew\u003c/em\u003eFlu was preliminarily explored as a function of the environmental temperature of the mosquito larvae breeding sites. At first sight, both the maximum and the minimum peaks seems not to have affected \u003cem\u003ew\u003c/em\u003eFlu prevalence in the population assayed. If confirmed with more replicates, absence of heat stress effects may be due to the fact that \u003cem\u003eWolbachia\u003c/em\u003e strains may respond differently to such stimulus due to different evolutionary histories, or may have been unnoticed in the present work. Indeed, the effect of thermal stress on \u003cem\u003eWolbachia\u003c/em\u003e density during larval development might be manifested in the adult stage (Ulrich et al. 2017). In the present study the measurements were made on the larvae; although the temperature oscillations were within the tolerable range for \u003cem\u003eWolbachia\u003c/em\u003e (20\u0026deg;C to 30\u0026deg;C) (L\u0026oacute;pez-Madrigal and Duarte \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the breeding site of \u003cem\u003eAe. fluviatilis\u003c/em\u003e showed a peak at 37\u0026deg;C during March. While these variations may not have had any effect in the immature stages, they might have it in the adults. Moreover, \u003cem\u003ew\u003c/em\u003eFlu density may be a better variable than prevalence to assess heat stress effects on infection (L\u0026oacute;pez Madrigal and Duarte 2019). However, adult emergence under laboratory conditions was hard to obtain in the present study and replicates difficult to obtain. Improving rearing conditions of this mosquito species in the lab must be reached before ruling out a relationship between thermal stress and \u003cem\u003ew\u003c/em\u003eFlu dynamics. Should this goal be met, further studies should evaluate the effect of heat stress on \u003cem\u003ew\u003c/em\u003eFlu density in the adult stage.\u003c/p\u003e \u003cp\u003eFinally, because the use of \u003cem\u003eWolbachia\u003c/em\u003e as a biocontrol method has gained great interest, it is necessary to know the current diversity in nature before introducing new strains with desirable characteristics, to avoid future problems such as CI. In this work, two strains were identified in mosquito species from Argentina, one being a new report in the country (\u003cem\u003ew\u003c/em\u003eFlu). Although infection with \u003cem\u003ew\u003c/em\u003ePip was already known in populations from Argentina, this is the first time that it has been reported in hybrid populations of the \u003cem\u003eCx. pipiens\u003c/em\u003e complex.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: We gratefully acknowledge Mar\u0026iacute;a Laura Morote for helping in the figures design and Dar\u0026iacute;o Balcazar and Walter Ferrari for technical assistance. Thanks are due to the anonymous reviewers who provided invaluable feedback on the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAil\u0026eacute;n Chuchuy, Marcela S. Rodriguero and Mar\u0026iacute;a V. Micieli contributed to the study conception and design. Material preparation and data collection were performed by Ail\u0026eacute;n Chuchuy, Ana C. Alonso and Marina Stein. Data analyses were performed by Ail\u0026eacute;n Chuchuy and Marcela S. Rodriguero. The first draft of the manuscript was written by Ail\u0026eacute;n Chuchuy. All authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis contribution was supported by grants from Agencia Nacional de Promoci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (PICT 2015-0665) and Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (PIP 584) to MVM. AC was awarded a doctoral scholarship from Agencia Nacional de Promoci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica. MSR, MS and MVM are members of the Research Career of Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas, Argentina.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA sequences can be retrieved from GenBank. Accession Numbers can be found in the body of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlonso AC, Stein M, Hisgen CM, Micieli MV (2022) Abiotic factors affecting the prevalence of \u003cem\u003eWolbachia\u003c/em\u003e (Rickettsiaceae) in immature \u003cem\u003eAedes albopictus\u003c/em\u003e (Skuse) (Culicidae). J Invertebr Pathol 189:107730\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtyame CM, Labbe P, Dumas E, Milesi P, Charlat S, Fort P, Weill M (2014) \u003cem\u003eWolbachia.\u003c/em\u003e Divergence and the Evolution of Cytoplasmic Incompatibility in \u003cem\u003eCulex pipiens\u003c/em\u003e. PLoS One 9:21\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtyame CM, Delsuc F, Pasteur N, Weill M, Duron O (2011) Diversification of \u003cem\u003eWolbachia\u003c/em\u003e Endosymbiont in the \u003cem\u003eCulex pipiens\u003c/em\u003e Mosquito. 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Proc R Soc B Biol Sci 265:509\u0026ndash;515.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Wolbachia, Mosquitos, Arboviruses, MLST, Biocontrol, Neglected Diseases","lastPublishedDoi":"10.21203/rs.3.rs-4361303/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4361303/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe increasing spread of mosquito vectors has made mosquito-borne arboviral diseases a global threat to public health, leading to the urgent need for effective control of such populations. Methods based in the intracellular bacterium \u003cem\u003eWolbachia\u003c/em\u003e Hertig, 1936 are considered environmentally friendly, human-safe, and potentially cost-effective biocontrol strategies to control arboviral diseases. In order to minimize undesirable side effects, it is relevant to understand if \u003cem\u003eWolbachia\u003c/em\u003e ranges in the area and the diversity associated to native infections before implementation. With this purpose, we investigated \u003cem\u003eWolbachia\u003c/em\u003e infection status, diversity and prevalence in populations of \u003cem\u003eAedes albifasciatus\u003c/em\u003e (Macquart, 1838), \u003cem\u003eAedes fluviatilis\u003c/em\u003e (Lutz, 1904), and hybrids of the \u003cem\u003eCulex pipiens\u003c/em\u003e (Linnaeus, 1758) complex from Argentina. Additionally, we preliminarily explored the influence of environmental temperature on the \u003cem\u003eWolbachia\u003c/em\u003e prevalence in \u003cem\u003eAe. fluviatilis\u003c/em\u003e. \u003cem\u003eAedes albifasciatus\u003c/em\u003e and \u003cem\u003eCx. pipiens\u003c/em\u003e complex samples were collected in the province of Buenos Aires, and \u003cem\u003eAe. fluviatilis\u003c/em\u003e in the province of Misiones. \u003cem\u003eAedes albifasciatus\u003c/em\u003e was uninfected and infections with strains \u003cem\u003ew\u003c/em\u003eFlu and \u003cem\u003ew\u003c/em\u003ePip were revealed in \u003cem\u003eAe. fluviatilis\u003c/em\u003e and hybrids of the \u003cem\u003eCx. pipiens\u003c/em\u003e complex, respectively. All strains were fixed or close to fixation and clustered within supergroup B. No effects of environmental temperature on \u003cem\u003eWolbachia\u003c/em\u003e prevalence of \u003cem\u003eAe. fluviatilis\u003c/em\u003e larvae were revealed, at least with the current design. These results provide valuable information on \u003cem\u003eWolbachia\u003c/em\u003e strains found in natural populations of mosquitoes from Argentina that might be used in heterologous infections in the future or must have taken into account when designing control strategies based on \u003cem\u003eWolbachia\u003c/em\u003e infection.\u003c/p\u003e","manuscriptTitle":"Wolbachia infection in natural mosquito populations from Argentina","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-13 10:32:04","doi":"10.21203/rs.3.rs-4361303/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-20T15:30:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-17T19:27:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-15T05:49:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305692802964671321350785670583201014893","date":"2024-05-07T20:38:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189480508344443884225153041912597086246","date":"2024-05-07T17:48:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-07T14:34:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-07T06:27:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-07T02:57:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2024-05-03T00:24:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8190f18e-e4af-4400-8d31-3e544fd06a53","owner":[],"postedDate":"May 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:08:50+00:00","versionOfRecord":{"articleIdentity":"rs-4361303","link":"https://doi.org/10.1007/s00436-024-08352-5","journal":{"identity":"parasitology-research","isVorOnly":false,"title":"Parasitology Research"},"publishedOn":"2024-10-09 15:57:51","publishedOnDateReadable":"October 9th, 2024"},"versionCreatedAt":"2024-05-13 10:32:04","video":"","vorDoi":"10.1007/s00436-024-08352-5","vorDoiUrl":"https://doi.org/10.1007/s00436-024-08352-5","workflowStages":[]},"version":"v1","identity":"rs-4361303","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4361303","identity":"rs-4361303","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}