Reduced competence to arboviruses following the sustainable invasion of wolbachia into native aedes aegypti from southeastern brazil

ABSTRACT Field release of _Wolbachia_-infected _Aedes aegypti_ has emerged as a promising solution to manage the transmission of dengue, Zika and chikungunya in endemic areas across the

globe. Through an efficient self-dispersing mechanism, and the ability to induce virus-blocking properties, _Wolbachia_ offers an unmatched potential to gradually modify wild _Ae. aegypti_

populations turning them unsuitable disease vectors. Here we describe a proof-of-concept field trial carried out in a small community of Niterói, greater Rio de Janeiro, Brazil. Following

the release of _Wolbachia_-infected eggs, we report here a successful invasion and long-term establishment of the bacterium across the territory, as denoted by stable high-infection indexes

(> 80%). We have also demonstrated that refractoriness to dengue and Zika viruses, either thorough oral-feeding or intra-thoracic saliva challenging assays, was maintained over the

adaptation to the natural environment of Southeastern Brazil. These findings further support _Wolbachia_’s ability to invade local _Ae. aegypti_ populations and impair disease transmission,

and will pave the way for future epidemiological and economic impact assessments. SIMILAR CONTENT BEING VIEWED BY OTHERS QUANTIFYING THE IMPACT OF _WOLBACHIA_ RELEASES ON DENGUE INFECTION IN

TOWNSVILLE, AUSTRALIA Article Open access 11 September 2023 SUPPRESSION OF _AEDES AEGYPTI_ MAY NOT AFFECT SYMPATRIC _AEDES ALBOPICTUS _POPULATIONS: FINDINGS FROM TWO YEARS OF ENTOMOLOGICAL

SURVEILLANCE IN SINGAPORE Article Open access 17 January 2025 FITNESS COMPATIBILITY AND DENGUE VIRUS INHIBITION IN A BANGLADESHI STRAIN OF _AEDES AEGYPTI_ INFECTED WITH THE _WOLBACHIA_

STRAIN _W_ALBB Article Open access 18 April 2025 INTRODUCTION The mosquito _Aedes aegypti_ (= _Stegomyia aegypti_) holds a core status among tropical disease vectors, being able to host and

transmit a broad variety of viruses, such as those causing dengue, Zika and chikungunya1,2. Dengue virus (DENV) is certainly the most prevalent, with a global distribution spectrum including

128 countries3 and approximately 400 million infections annually4. Brazil accounts for a large fraction of these cases, with more than 1.5 million infections only in 20195. In spite of

being historically less prevalent, chikungunya (CHIKV) and Zika (ZIKV) viruses, restricted to Africa and Asia until the early 2000’s, were recently emerging in new territories, with

significant impact on public health6,7,8,9. The introduction of CHIKV in Central and South America, for example, led to about one million suspected disease cases between 2013 and 201410.

Similarly, ZIKV first records in America date to the end of 2013, after which it rapidly spread through the continent11. In 2015, the World Health Organization (WHO) declared a Public Health

Emergency of International Concern following a serious ZIKV outbreak in Northeast Brazil, associated with high rates of microcephaly in newborns12. Since effective vaccines or therapeutic

drugs are still under development13,14,15 and not currently available to fight outbreaks of Zika, dengue and chikungunya, public health initiatives rely entirely on vector control.

Traditionally, this is achieved by the mechanical elimination of breeding sites and the use of chemical insecticides to reduce _Ae. aegypti_ populations. However, both methods have proven

inefficient and unsustainable for the long term, mostly due to the myriad artificial breeding sites used by this species in urban landscapes16,17 and the advent of naturally insecticide

resistant allelic variants18,19,20. In face of these challenges, the development of new solutions is, therefore, a critical need for a more efficient control of _Ae. aegypti_ populations

and/or disease transmission. In recent years, an innovative approach using the endosymbiont _Wolbachia pipientis_ to block arbovirus transmission has been proposed and successfully tested in

_Ae. aegypti_21,22,23,24, gaining momentum as a viable and sustainable alternative to traditional control methods. Naturally found in around 40% of all arthropods25, _Wolbachia_ are

maternally-inherited intracellular bacteria which exploits host reproductive biology to increase its transmission rates and dispersal in nature. For some bacterial strains, this is achieved

by triggering a phenomenon called cytoplasmic incompatibility (CI), which turns the progeny unviable when an infected male copulates with an uninfected female26. Remarkably, _Wolbachia_-host

association can also lead to pathogen interference (PI) phenotypes27,28, particularly relevant to vector control applied studies. In view of this, stable and heritable lines, harboring

different _Wolbachia_ strains, have been created following artificial transinfection of _Ae. aegypti_21,29,30,31,32. Through pathways involving the modulation of host immune system33 and

metabolite sequestration34,35, _Wolbachia_-harboring lines exhibit high-levels of refractoriness to DENV, CHIKV, ZIKV, and other medically relevant arboviruses30,36,37,38. As such, releasing

some of these lines in the field to gradually replace virus-susceptible wild populations could potentially reduce transmission and human infection rates, as recently reported39. Successful

field release trials using _Ae. aegypti_ lines infected with _Wolbachia w_Mel strain have been reported in Northern Australia, Indonesia and more recently in Southeastern Brazil22,40,41. In

the latter, a small community in Rio de Janeiro (RJ) was subject to a rolling out strategy based on adult releases, which led to the invasion and long-term establishment of the bacterium in

the field. Nonetheless, important vector competence data following the invasion is still lacking and needs to be assessed in order to provide evidence of virus-blocking maintenance.

Accumulating evidence suggest that eggs can also be released in the field, thus providing an alternative carrier for _Wolbachia_ introduction into wild _Ae. aegypti_ populations41,42,43.

When compared to adult-driven methods, egg releases thrive on its simpler, more natural approach. Replacing a large, all-at-once, pulse of mosquitoes by a slow and gradual release from

Mosquito Release Containers (MRCs) tends to alleviate undesirable social effects and increase community acceptance41. In this study, we report the results of a trial in which

_Wolbachia_-harboring eggs were released in Jurujuba, a small suburb of Niterói (Rio de Janeiro State). Besides evaluating _Wolbachia_’s ability to invade mosquito populations, we

investigated the bacterium density level and the vector competence for ZIKV and DENV in post-release field samples, contributing to a better characterization of targeted populations in

Southeastern Brazil. RESULTS EGG RELEASES SUCCESSFULLY PROMOTE _WOLBACHIA_ INVASION IN JURUJUBA, NITERÓI (RJ) To evaluate the efficiency of egg releases as a method of _Wolbachia_ field

deployment in Brazil, we carried out a pilot study in Jurujuba, a suburban neighborhood of Niterói (Rio de Janeiro State). Using MRCs, _Wolbachia_-infected eggs (_w_MelRio _Ae. aegypti_

strain40) were distributed over all seven Jurujuba’s sectors (i.e. sub-areas within the neighborhood) (Fig. 1, Supplementary Fig. S1, Supplementary Table S1). BG-sentinels were spatially

distributed (Supplementary Fig. S2, Supplementary Table S2) to collect _Ae. aegypti_ field specimens for _Wolbachia_ molecular diagnosis, before calculation of prevalence rates (percent

infected individuals). Situated at the furthermost housing area of Jurujuba’s peninsula, Ponto Final was selected the starting sector for field release, from which adjacent ones would derive

following a rollout strategy. As such, all related activity planning, including community engagement, territorial mapping, release and monitoring sites allocation, and mass-rearing

production scale was initially tuned to Ponto Final. Here, _Wolbachia_-infected eggs were released over 25 consecutive weeks, from August 2015 to February 2016, during which prevalence rates

rapidly increased from basal to high levels (> 80%) (Fig. 2). In fact, by week 17, infection rates hit 81% and, by the end of the release period, 88%. Post-release prevalence rates were

maintained at high levels in subsequent months, suggesting a successful invasion at this particular sector and paving the way to cover others, fulfilling our initial design. Following new

planning and schedules, the release of _Wolbachia_-infected eggs was extended to Cascarejo, in June 2016, and to Brasília, Salinas, Várzea, Peixe-Galo and Praia de Adão e Eva, in September

2016 (Fig. 2). The release period duration and number of MRCs allocated in each sector varied slightly (Supplementary Table S1), due to their intrinsic properties (i.e. housing area and

human population density). By mid-January 2017, egg release ceased in whole Jurujuba, with most sectors recording mid-to-high rates of _Wolbachia_ infection (60–90%). Mimicking Ponto Final,

the post-release phase generally followed the positive trends of the release period and featured increasing infection rates towards near fixation levels (90–100%) (Fig. 2). This was clearly

the case for Cascarejo, Brasília and Salinas. A notable exception was Várzea, which recorded an erratic invasion profile, with high infection rates by the end of the release period (i.e. ~ 

80% in eight weeks) and a gradual decrease over the following months, when rates hit less than 40%. Here, a complete recovery and stability at high rates was achieved only after 10 months

into the post-release phase. Peixe Galo also revealed a slightly erratic profile but, unlike Várzea, it did not resemble a negative trend. Instead, mid-to-high level oscillation was

sustained over the post-release phase, and part of the noise might be attributed to low sampling. Lastly, Praia de Adão e Eva exhibited consistently high rates over the release period and

the following few weeks, suggesting a standard invasion profile. However, given its low _Ae. aegypti_ abundance, as well as a relatively small area and human population, post-release

monitoring was suspended, yielding no long-term data and preventing a more assertive analysis for this sector. By aggregating weekly infection rates from different sectors, an overall

post-release profile of Jurujuba was revealed (Fig. 3). Confirming previous data analysis, in which sectors were individually treated, the overall profile revealed that Jurujuba consistently

recorded high infection rates (80–100%) along the post-release phase, from mid-January 2017 until December 2019. Data encompassing this period, of almost three years, suggest that

_Wolbachia_ successfully invaded Jurujuba, being able to sustain infection rates in the long-term. _WOLBACHIA_ DENSITY IS HIGHER IN POST-RELEASE FIELD SAMPLES To investigate whether

_Wolbachia_ whole-body density (i.e. titer) was affected over time in Jurujuba’s environment, field samples were tested a few months after releases were finalized (March–May 2017) and one

year later (March–May 2018). Counterpart _w_MelRio colony samples (i.e. collected at equivalent time periods) were also tested to assess data from individuals reared under laboratory

environmental conditions and control generation-dependent variation in density, which could possibly mask evolutionary changes in this trait. Density data were plotted to reveal possible

differences between field and colony samples, as well as details of their distribution (Fig. 4). Indeed, data suggest that _Wolbachia_ density vary among groups, which was further

corroborated by Kruskal–Wallis statistical test (_H_ = 340.2, _P_ < 0.0001). Subsequent multiple comparisons revealed that ‘field’ densities are higher than in ‘colony’ originating

mosquitoes, in samples both from the beginning (_P_ < 0.0001) or from ~ one year into the post-release phase (_P_ < 0.0001). Moreover, when field samples are compared, a significant

increase in density over time is detected (_P_ < 0.0001), suggesting an evolving _Wolbachia_-host relationship in Jurujuba’s environment. _WOLBACHIA_ INHIBITS DENV E ZIKV REPLICATION IN

THE HEAD AND THORAX OF FIELD SAMPLES Following the invasion and long-term stability of _Wolbachia_ in Jurujuba, _Ae. aegypti_ field samples were submitted to vector competence assays.

Jurujuba specimen eggs were collected in Ponto Final over three months, from April to June 2017, which correspond to 14–16 months into the post-release phase of this particular sector.

Specimens eggs from Urca, a _Wolbachia_-free area in the neighboring city, Rio de Janeiro, were collected at the same time period and served as experimental controls. F1 adult females, from

Jurujuba and Urca, were orally challenged with ZIKV or DENV, and viral titers were assessed 14 days post infection (dpi) in head/thorax individual extracts. Two independent assays were

performed for each virus. Our results revealed that oral challenging with ZIKV could promote infection and high viral titers in most samples from Urca, but could not elicit a similar outcome

in samples from Jurujuba, which were mostly negative (Fig. 5a). Mann–Whitney U tests corroborate the significant reduction of ZIKV titers in Jurujuba samples, in both first (_U_ = 47.5, _P_

 < 0.0001) and second assays (_U_ = 36.5, _P_ < 0.0001). The oral challenging with DENV led to an almost identical picture, with most samples from Urca being infected with high viral

titers, whilst in samples from Jurujuba only a few were infected (Fig. 5b). Once again, significant differences between Urca and Jurujuba were found in the first (_U_ = 74, _P_ < 0.0001)

and second assays (_U_ = 20.5, _P_ < 0.0001). In addition, we double-checked the _Wolbachia_ status of the same F1 samples, further confirming its large presence in Jurujuba (~ 88% rate;

55 out of 62 individuals tested positive for _Wolbachia_) and complete absence in Urca (Supplementary Fig. S3). Altogether, our data suggest that the oral exposure with ZIKV or DENV is less

prone to trigger and disseminate infection in _Wolbachia_-harboring Jurujuba specimens than in _Wolbachia_-negative Urca correlates, and that this effect is bacterium-driven. _WOLBACHIA_

LARGELY ATTENUATES SALIVA TRANSMISSION OF DENV AND ZIKV BY FIELD SAMPLES To further understand the extent of _Wolbachia_-mediated refractoriness to ZIKV and DENV, we investigated the

infective potential of saliva from orally-challenged Urca and Jurujuba samples. At 14 dpi, saliva samples were harvested and intrathoracically injected into groups of naïve Urca specimens (n

 = 8) to evaluate the degree of which infective particles could be transmitted (Fig. 6). Infected individual counts were assessed at 5 dpi, for ZIKV, or at 7 dpi, for DENV, and their percent

representation in each group was the metric used for comparisons, along with an overall intrathoracic saliva infection index (OISI; see “Methods” for more details). We also measured ZIKV

and DENV titers in the head/thorax of which saliva were harvested, and plotted the values at the top of each infected group. As previously observed in extracts following oral-infection, ZIKV

and DENV titers were high in head/thorax samples from Urca, but virtually undetectable in those from Jurujuba (except for one sample with low titer). However, it is important to note that

these titers reflect the background infection status, not necessarily translating to the saliva. Our results revealed that saliva samples from orally-infected Urca individuals were carrying

ZIKV and DENV infectious particles. The infection rates for ZIKV and DENV, however, seemed to differ. While three out of the eight groups challenged with ZIKV-infected saliva had at least

one individual positive for the virus (OISI = 26.56 ± 45.53) (Fig. 6a), all eight groups challenged with DENV-infected saliva elicited this response (OISI = 98.44 ± 4.42) (Fig. 6b). This

evidence suggests that despite being susceptible to both viruses, Urca population might be less competent to transmit ZIKV than DENV. In contrast, saliva samples from orally-infected

Jurujuba individuals (_Wolbachia_+) were usually not carrying ZIKV and DENV infectious particles. In fact, all seven groups challenged with saliva from ZIKV-infected individuals did not

elicit a single infection (OISI = 0) (Fig. 6a), whilst only two out of seven groups challenged with saliva from DENV-infected individuals were positive for the virus (a single infection in

each group) (OISI = 3.57 ± 6.10) (Fig. 6b). It is curious, though, that these specific saliva samples had no detectable titers in their corresponding head/thorax extracts, once again

indicating that saliva and background infection status do not always correlate. Here, one could speculate that such _Wolbachia_+ head/thorax extracts harbor very low titers, often below qPCR

sensitivity threshold, and which could have been depleted to an even lower level by preceding saliva harvesting. Overall, our findings support the view that the _Wolbachia_-harboring

Jurujuba population likely has low susceptibility to ZIKV and DENV infection, with reduced viral multiplication and dissemination within key tissues, not being able to efficiently transmit

the virus through saliva inoculation. DISCUSSION The global burden of dengue, Zika and chikungunya places _Ae. aegypti_ at the top of the list encompassing medically relevant mosquito

vectors1,4,44. Since human immunization is not an option to date, public health authorities focus their efforts on vector suppression campaigns using long-standing protocols with major

constraints. Mechanical removal of breeding sites, for instance, are labor intensive and usually leave some hotspots untouched, in which dry quiescent eggs remain viable until more favorable

conditions resume45,46,47. Deployment of chemical pesticides have also proven inefficient, given the lack of precision and the surge of resistant variants19,48. To tackle some of these

constraints and fulfil the urgent need for more efficient strategies, possible solutions have emerged in recent years22,40,41,42,49,50,51. One promising solution lies on the field-release of

lab-reared _Wolbachia_-infected individuals to gradually replace wild uninfected populations. The concept is fundamentally based on both the CI and PI bacterium-driven effects, arising from

complex interactions with the mosquito host52. While the first favors bacterium inheritance towards fixation, the second confers refractoriness to several arboviruses. Should these effects

translate to wild populations, then _Wolbachia_ offers an unprecedented strategy, both natural and sustainable, to control mosquito-borne disease transmission. Over the last decade, the

World Mosquito Program (formerly ‘Eliminate Dengue: Our Challenge’), field-release trials have been performed to assess whether _Wolbachia_ can live up to expectations in diverse real-world

scenarios22,40,41,42,53. Interestingly, trials have revealed several challenges for a successful _Wolbachia_ invasion and long-term stability in natural populations. As highlighted by pilot

studies in Northern Australia and Vietnam, choosing a bacterium strain associated with high fitness costs to the host, like the virulent _w_MelPop, may impair a population replacement

strategy54. Even though it could still be used in alternative strategies to transiently suppress _Ae. aegypti_ populations and local transmission of arboviruses55,56, its field application

is not sustainable and presumes continuous release of large quantities of individuals57. In contrast, the utilization of strains associated with low-to-mild fitness costs, albeit with

generally lower PI, allow fewer individuals to be released in the field in order to promote an efficient invasion. Fulfilling these criteria, the strain _w_Mel has been advocated as a choice

for field release, collecting successful trials in Australia, Brazil and Indonesia22,23,39,40,41,42,43. Nonetheless, due to its beneficial attributes in warmer climates, keeping density

levels high and stable, _w_AlbB has proven a second option and suitable alternative to _w_Mel, as shown by a recent trial held in Malaysia53. Interestingly, a superinfected line hosting both

_w_Mel and _w_AlbB could also be an alternative for future interventions, as judged by preliminary analysis pointing to a higher PI while not increasing fitness costs58. Thus, the

investigation and field application of new strains or combinations of existing ones has been encouraged to broaden the available options, and help building a _Wolbachia_ toolset that suits

diverse needs32,58. In addition to the _Wolbachia_ strain, other determining factors impacting the invasion dynamics, hence the success of a field trial, include the genetic background of

the host, the rear and release method per se (space–time release schedule, quantity and quality/fitness of released individuals) and the density of local _Ae. aegypti_ populations40,42. Of

particular importance, the genetic background of the host not only establishes unique interactions with _Wolbachia_ strains, but also harbors bacterium-independent fitness traits that may

dictate the adaptation to wild environments. Therefore, background homogenization was key to revert a failed attempt to deploy _w_Mel in Tubiacanca, a small community of Rio de Janeiro (RJ),

Southeastern Brazil40. Here, mimicking prior successful trials held in Australia22,23 was not enough to drive a sustainable invasion, and soon after adult release ceased _Wolbachia_

prevalence dropped. Neither increasing the quantity nor the quality of released individuals (i.e. larger, longer-living individuals) could revert this outcome, suggesting that

fitness-related nuances could be limiting _Wolbachia_’s spread. After a thorough investigation of the infected line, ruling out putative variations in traits affecting mating and

reproduction success40,59,60, or _Wolbachia_’s maternal transmission rate, it was revealed that its genetic footprint of insecticide resistance had been largely attenuated over laboratory

adaptation, becoming particularly less fit to survive in areas with high insecticide usage like those found in Southeastern Brazil40. With the re-introduction of resistant alleles, matching

the frequency found in local populations, the reformed line was then able to switch the negative trend to a successful invasion in a second trial40. Thus, by narrowing disparities between

lab-reared infected lines and wild populations, genetic background homogenization has been perceived as a good practice prior to current field release efforts. In this study, we report the

successful introduction and long-term establishment of _Wolbachia_ into _Ae. aegypti_ populations from Jurujuba, a suburban community of Niterói (RJ). Sitting by the shores of Guanabara bay,

Jurujuba represents an additional site for _Wolbachia_ release trials in Rio de Janeiro (RJ) and surrounding areas, launched some years before in Tubiacanga40. Jurujuba is enclosed in a

greener landscape with softly connected housing clusters (Fig. 1), and Tubiacanga in a more organized and uniform housing display. Contrary to the latter, in which adult-releases were

undertaken, Jurujuba held an alternative egg-release method, providing a chance to evaluate challenges and outcomes of each strategy, and trace future perspectives of trials in the country.

The relatively small distance between both sites (19 km on a straight line over water) also proved valuable by allowing the field release of the same _Wolbachia_-infected line (i.e.

_w_MelRio)40, disregarding the need for a whole genetic background swap. Instead, we carried out only minor quality control (e.g. genetic monitoring of insecticide resistance alleles) and

re-introduction of local genetic variability in every few generations (e.g. addition of wild-caught males to the colony) to secure background homogeneity. Following a rollout strategy,

_Wolbachia_-containing eggs were deployed across all seven sectors of Jurujuba, revealing an overall invasion trend characterized by sustained high indexes at the end of the release period.

However, a thorough observation of each sector uncovers distinct invasion profiles (Fig. 2), with some featuring accentuated trends and reaching early high infection indexes, and others

showing less pronounced trends along the release period. The phenomena underlying distinct _Wolbachia_ invasion dynamics could be linked to the density and spatial distribution of local _Ae.

aegypti_ populations and, ultimately, to human occupation51,61. Thus, it is reasonable to speculate that sectors featuring accentuated invasion trends have smaller populations of _Ae.

aegypti_, as a result of better management of breeding sites or even fewer inhabitants. An opposing scenario might explain less pronounced, more resilient, invasion trends. Speculating on

Várzea’s erratic profile, however, is challenging since none of the above causes seem reasonably suited to explain it assertively, leaving room to non-controlled events (e.g. indoors

insecticide-spraying). Interestingly though, Várzea constitutes a stretch of houses surrounded by forest, and connecting three other sectors: Brasília, Cascarejo and Salinas (Fig. 1). Thus,

it is possible that migration from these adjacent sectors could have contributed to Várzea’s profile, first with non-infected individuals and then with infected ones, respectively dampening

and recovering rates over the post-release phase. Our data corroborate to a stable, self-sustaining, and long-term persistence of _Wolbachia_ infection in _Ae. aegypti_ from Jurujuba. We

have shown that over the post-release phase, spanning mid-January 2017 to December 2019, _Wolbachia_ infection of field specimens were sustained at near-fixation indexes with only minor

fluctuation (80–100%) (Fig. 3). Curiously, while experiencing and adapting to the natural habitat, _Wolbachia_’s association with infected hosts seems to have evolved to higher whole-body

densities (Fig. 4), corroborating previous findings of a trial held in Australia62. When a comparison is drawn to colony-reared individuals, a significant increase in this parameter can be

observed after only a few months (i.e. 2–4) in the field, becoming even more pronounced after one year. Considering that density levels has been positively correlated to maternal

transmission rates32,63, as well as to the strength of CI and PI21,64,65,66,67, our findings suggest _Wolbachia_-host association affects have not been alleviated due to co-evolution in the

field, and shall endure in the years to come. Indeed, further supporting this view, our vector competence analysis suggests that PI is maintained in _Wolbachia_-positive Jurujuba samples

collected slightly over one year into the post-release phase (Figs. 5, 6). In both oral and saliva challenging assays, Jurujuba samples were highly refractory to ZIKV or DENV, impairing not

only the replication of viral particles but also its dissemination to tissues playing key roles in transmission to humans (e.g. salivary glands). Since most samples from Jurujuba were

_Wolbachia_-positive (~ 88%) (Supplementary Fig. S3) and those from Urca, a _Wolbachia_-free area, were highly susceptible to both viruses, we could endorse that the refractory effect was

bacterium driven. Despite numerous studies of pathogen interference in _w_Mel-harboring lines, including some on a Brazilian background context36,68, the data presented here account for the

first evidence of ZIKV- and DENV-blocking in samples from Rio de Janeiro and surrounding areas, notably Jurujuba and Tubiacanga, which have been subjected to release trials in recent years.

Most important, it adds an important validation to the undergoing control strategy, leaving to epidemiological analysis the last verdict. Corroborating previous trials in Australia and

Indonesia41,42,43, the release of _Wolbachia_-infected eggs in Jurujuba proved an efficient method to introduce and disseminate the bacterium into Brazilian populations of _Ae. aegypti_.

Volunteers and community members could actively participate, with low-demanding basic training, in field deployment schedules, naturally enhancing engagement and the strategy’s successful

outcome43. Altogether, these beneficial features highly encourage the release of _Wolbachia_-infected eggs as part of control strategies in Brazil and other countries, particularly in those

sites lacking proper infra-structure or financial support. Ultimately, this work adds a new chapter on a successful story of _Wolbachia_ field releases in Southeastern Brazil. When

associated with a local genetic background, and continually monitored for homogeneity, _w_Mel’s costs on fitness could be overridden by its efficient drive mechanism and spread into wild

populations of Rio de Janeiro. Its long-term stability in the field, as shown by persistent high-infection indexes and pathogen interference, further reinforces the method’s sustainability

and constitutes solid grounds to future epidemiological studies. Should we observe a significant impact on humans, then _Wolbachia_’s deployment shall gain momentum in public health

initiatives and pave the way to cover larger areas in the country. METHODS MOSQUITO LINES AND MAINTENANCE To introduce _Wolbachia_ into Brazilian _Ae. aegypti_, an Australian line infected

with the _w_Mel strain21 was backcrossed for 8 generations to a natural mosquito population of Rio de Janeiro, Brazil24. Following the genetic background introgression, additional crosses

and _knockdown resistance_ (_kdr_) screening were undertaken to replicate natural insecticide resistance profiling and generate the line _w_MelRio. To assure a minimal variation in this

profiling overtime, and sustain a homogeneous genetic background, _w_MelRio colony was refreshed with 10% wild males once in every five generations40. To maintain _w_MelRio, immatures (i.e.

larval stages L1 to L4) were reared in dechlorinated water, at 28 °C, and fed Tetramin Flakes (Tetra GmbH, Herrenteich, Germany) until pupal formation. Following adult emersion, groups of

1000 females and 800 males were sorted and kept in BugDorm cages (MegaView Science Co Ltd., Taiwan) at 25 °C, with 10% sucrose solution ad libitum. Every three days, females were fed human

blood (from blood donation centers; see details under ‘ethical considerations’), through Hemotek artificial feeders (Hemotek Ltd, UK). Note that, to avoid arboviral contamination of our

colony, all blood samples were formerly tested negative for DENV, ZIKV, CHIKV, MAYV and YFV by multiplex qPCR assays36,68. Egg-laying was induced by placing dampened strips of filter paper

(i.e. partially immersed in water-containing cups) inside the cages for 2–3 days, after which they were gradually dried at room temperature. Strips loaded with eggs (i.e. ovistrips) were

kept at room temperature until further use, either for colony maintenance or field release. Eggs older than 40 days were discarded due to a decay in overall quality60. EGG RELEASES

Mass-reared _w_Mel-infected Brazilian _Ae. aegypti_, _w_MelRio, were released as eggs in Jurujuba (22°56′ 00″ S, 43°07′ 00″ W), a lower-middle-class community in the city of Niterói (state

of Rio de Janeiro, Brazil). Located by the shores of Guanabara bay, this community has grown from a typical fisherman settlement, with informal occupancy, to a total population of 2797

residents in 890 houses. Jurujuba encompasses a total area of 2.53 km2, divided into seven smaller sectors (i.e. sub-areas or localities within the neighborhood): Ponto Final, Várzea,

Brasília, Cascarejo, Praia de Adão e Eva, Peixe-Galo and Salinas. _w_MelRio eggs were released in the field through special deployment devices, referred to as mosquito release containers

(MRCs), which consisted of small white plastic buckets (19 cm height × 18 cm top diameter × 15.5 cm base diameter) with four small holes on the side wall, only a few centimeters away from

the top lid. Each MRC was loaded with 1 L of water, 0.45 g of Tetramin Tropical Tablets (i.e. one and a half tablet) (Tetra GmbH, Herrenteich, Germany) and an ovistrip containing

approximately 150–300 eggs. After six to seven days, about 80% of the immatures were pupae, and after 11 to 12 days, most of the adults had already emerged and left the device from the wall

holes. Every 15 days, MRCs were checked and reloaded so that another rearing and release cycle could take place. Release sites were spatially distributed as evenly as possible (Supplementary

Fig. S1), so as to maximize the spread of _Wolbachia_-harboring individuals and promote mating with their wild peers. The release strategy was optimized by splitting the sites into two

groups, A and B, with alternate MRC loading schedules. Thus, while MRCs from group A were releasing adults, those from group B were being loaded with new ovistrips, water and food. In the

following week, an opposite situation occurred, with MRCs from group B releasing adults. The release schedules, as well as the number of allocated MRCs, varied according to each Jurujuba’s

sector (Supplementary Table S1). ETHICAL CONSIDERATIONS All methods were carried out in accordance with relevant guidelines and regulations. Study protocol for _Wolbachia_ field release was

approved by the National Research Ethics Committee (CONEP, CAAE 02524513.0.1001.0008) and three government agencies: IBAMA (Ministry of Environment), Anvisa (Ministry of Health) and MAPA

(Ministry of Agriculture, Livestock and Supply) to obtain the RET (Special Temporary Registry, 25351.392108/2013-96). Prior to mosquito releases, an informed consent was obtained from 70% of

Jujuruba households. Also, a written informed consent was obtained from households that hosted BG-sentinel mosquito traps. For the maintenance and mass-rearing of _Wolbachia_-infected _Ae.

aegypti_, adult females were fed human blood from a donation center (Hospital Antonio Pedro, Rio de Janeiro State University), with supporting regulatory approval (CONEP, CAAE

59175616.2.0000.0008) We only used blood bags which would have been discarded by the donation center, mainly due to insufficient volume to meet their quality assurance policy. Samples had no

information on donor’s identity, sex, age and any clinical condition, but were tested negative for several diseases, including Hepatitis B, Hepatitis C, Chagas disease, syphilis, HIV and

HTLV, as part of the Brazilian Government routine screening. For vector competence assays, human blood was obtained from Fundação Hemominas as part of a research agreement with Instituto

René Rachou (Fiocruz Minas) (OF.GPO/CCO-Nr224/16). _WOLBACHIA_ FIELD MONITORING AND DENSITY LEVEL ASSESSMENT _Ae. aegypti_ field population was monitored with BG-Sentinel traps (Biogents AG,

Regensburg, Germany), spread across Jurujuba in a semi-homogeneous fashion (Supplementary Fig. S2, Supplementary Table S2, Supplementary Datasheet S1). These monitoring sites were chosen

among suitable households who formally agreed with hosting of a trap, and had to be reallocated according to necessity (i.e. household quits hosting the trap). Working traps were checked

weekly by removing the catch bags (e.g. small meshed envelopes placed inside the BG-Sentinels to collect trapped insects) and bringing them to the laboratory for species identification and

_Wolbachia_ screening. Catch bags were barcoded according to the trap ID and site, so as to create a pipeline for field samples. Screening for _Wolbachia_ in _Ae. aegypti_ samples was

undertaken by qPCR. Briefly, individual DNA was extracted by homogenizing head/thorax body parts in Squash Buffer (10 mM Tris–Cl, 1 mM EDTA, 25 mM NaCl, pH 8.2) supplemented with Proteinase

K (200 ug/ml) and incubating at 56 °C for 5 min. Extraction ended by enzyme inactivation at 98 °C for 15 min. DNA amplifications were carried out with FastStart Essential DNA Probes Master

(Roche), using specific primers and probes to _Wolbachia pipientis_ WD0513 and _Ae. aegypti rps17_ markers (Supplementary Table S3). Thermocycling conditions were set on a LightCycler 96

Instrument (Roche), as follows: 95 °C for 10 min (initial denaturation), and 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Samples were analyzed using absolute quantification, by

comparison to serial dilutions of either gene product, cloned and amplified in the pGEMT-Easy plasmid (Promega). Negative control samples were normalized between plates, and were used as

reference to determine a minimum threshold for positive samples. DENV AND ZIKV ISOLATION AND REPLICATION IN MOSQUITO CELLS ZIKV was kindly provided by Instituto Aggeu Magalhães (IAM,

Fiocruz) through viral isolation of a symptomatic patient sample from Recife (PE, Brazil) in 2015 (ZikV/H.sapiens/Brazil/BRPE243/2015). DENV was sourced following a viral isolate from a

patient sample diagnosed with Dengue type 1 in Contagem (MG, Brazil), also in 2015 (Den1/H.sapiens/Brazil/BRMV09/2015). Both ZIKV and DENV samples were accompanied by patients’ written

consent (CONEP, reference number 862.912), being further catalogued into the national database of genetic patrimony and associated knowledge (SISGEN, access number AA1D462). In vitro culture

of viral particles were done as previously described36. Briefly, ZIKV and DENV were replicated in _Aedes albopictus_ C6/36 cells, grown at 28 °C in Leibovitz L-15 medium (ThermoFisher)

supplemented with 10% fetal bovine serum (FBS) (ThermoFisher). After seven days, supernatants were harvested and virus titers were assessed, first by Reverse Transcription (RT)-qPCR, and

later by plaque assay with VERO cells grown under 37 °C, 5% carbon dioxide, in Dulbecco’s Modified Eagle Medium (DMEM) (ThermoFisher) supplemented with 3% Carboxymethylcellulose (Synth) and

2% FBS. VECTOR COMPETENCE ASSAYS To perform vector competence assays with field samples of _Ae. aegypti_, ovitraps were mounted in both Ponto Final (Jurujuba) and Urca, a _Wolbachia_-free

area in Rio de Janeiro. Ovitraps were collected from the field over 13 weeks, from April to June 2017, which corresponds to the time-frame between 14 and 16 months along the post-release

phase in Ponto Final. Once in the insectary, eggs samples were reared to the adult stage in a controlled insectary environment (refer to ‘mosquito lines and maintenance’ for details). For

virus challenging assays through oral-feeding, young females (4–6 days old) were starved for 20 to 24 h, and subsequently offered culture supernatant containing ZIKV or DENV mixed with human

red blood cells (2:1 ratio), using an artificial membrane feeding system36. It is important to mention that, as for the colony maintenance protocol, blood samples used here were also

submitted to quality control prior to its use in the assays, mainly due to putative arbovirus contaminations which could affect the experimental outputs. Likewise, all samples were tested

negative for DENV, ZIKV, CHIKV, MAYV and YFV by multiplex qPCR assays36,68. Oral-infections were performed twice for each virus. ZIKV was offered first from fresh (initial virus titer of 4.8

 × 108 PFU/mL) and second from frozen culture supernatant (initial virus titer of 7.6 × 106 PFU/mL). In contrast, DENV was offered from fresh supernatants only (virus titers of 2 × 106

PFU/mL and 6.5 × 107 PFU/mL), since frozen versions failed to infect. Specimens were allowed to feed for one hour, after which engorged females were selected and maintained with 10% sucrose

solution ad libitum, during the whole extrinsic incubation period. At 14 days post-infection (dpi), viral loads were assessed in heads/thorax extracts by RT-qPCR (refer to ‘Viral diagnosis’

for more details). For saliva-mediated virus challenging assays, ZIKV and DENV pre-exposed females (14 dpi) from Jurujuba (_Wolbachia_ +) and Urca (_Wolbachia_ −) were starved for about 16 h

(overnight) before being knocked down and kept at 4 °C for wings and legs removal. Salivation was induced by introducing a 10 µL sterile filter tip, pre-loaded with 5 µl of a solution [30%

sucrose (w/v) diluted in 50% fetal bovine serum (FBS) and 50% DMEM medium], into the mosquito proboscis for 30 min. Saliva samples were individually collected, and 276 nL was

intrathoracically injected into young naive females (Urca) using a Nanoject II hand held injector (Drummond), as previously described36,68. Each saliva sample was used to inoculate 8–14

naïve _Wolbachia_-free _Ae. aegypti_ specimens, of which 8 were screened for infective particles. ZIKV and DENV were quantified by RT-qPCR at 5 dpi and 7 dpi, respectively (refer to ‘Viral

diagnosis’ for more details). Overall Intrathoracic Saliva Infection index (OISI) was obtained by averaging the percentages (± SD) of infected individuals in each group. VIRAL DIAGNOSIS To

identify ZIKV and DENV particles in individual samples, whole specimens were processed into head/thorax homogenates for RNA/DNA extraction with the High Pure Viral Nucleic Acid Kit (Roche),

according to manufacturer’s instructions30. Extracted samples were diluted in nuclease-free water to a concentration of 50 ng/μL. ZIKV, DENV and _Wolbachia_ levels, in vector competence

assays, were quantified by RT-qPCR using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) and specific primers and probes (Supplementary Table S3). Reactions were run on a LightCycler 96

Instrument (Roche), using the following thermocycling conditions: 50 °C for 5 min (initial RT step), 95 °C for 20 s (RT inactivation/DNA initial denaturation), and then 40 cycles of 95 °C

for 3 s and 60 °C for 30 s. Each RNA/DNA sample was used in two reactions, one with ZIKV, DENV or _Wolbachia_ primers, and another with _Ae_. _aegypti rps17_ endogenous control30. Absolute

quantification was achieved by comparing amplification profiles with standard curves generated by serial dilutions of their respective gene products, amplified from a cloned sequence in

pGEM-T Easy vector (Promega). Negative control samples (no virus RNA) served as reference to fix a minimum threshold for positive ones. ZIKV and DENV loads were defined as their copy number

per sample (head/thorax or saliva), while _Wolbachia_ loads were relative quantifications to the _rps17_ reference gene. Here, it is worth noting that, while _Wolbachia_ titer is naturally

variable and dependent on its whole-body density, the overall expression of _rps17_ is stable and particularly suitable for internal controls in assays with adult females69, as demonstrated

previously by us and others30,62,68. MAP CREATION AND SOURCE CODES The satellite image map of Jurujuba was created with ArcGIS Desktop 10.7 (Esri Inc.,

https://www.esri.com/en-us/arcgis/products/arcgis-desktop/overview) using Google Earth (Google LLC) source code, under the license and in accordance with the fair use described in

‘https://about.google/brand-resource-center/products-and-services/geo-guidelines/’. Maps with geotagged MRCs and BG-Sentinel traps were created with ArcGIS Desktop 10.7 and OpenStreetMap

source code (OpenStreetMap contributors), under the license CC-BY-SA 2.0. STATISTICAL ANALYSES Graphs and statistical analyzes were performed in GraphPad Prism 8 (GraphPad Software Inc.,

https://www.graphpad.com). Kruskal–Wallis test followed by Dunn’s post-hoc multiple comparisons were used to analyze _Wolbachia_ density data from field-collected and colony samples. ZIKV

and DENV loads in head/thorax extracts, from both oral and saliva-challenging samples, were compared using the Mann–Whitney U test. For all statistical inferences, ⍺ was set to 0.05. DATA

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quantitative real-time PCR in _Aedes aegypti_. _Sci. Rep._ 7, 43618 (2017). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We would like to

thank all members of the Mosquitos Vetores research group, for critical and helpful reviews, as well as past and present team members of the World Mosquito Program, whose dedication was key

to the positive outcome of this study. Special thanks to Flavia Teixeira, for ethical and regulatory compliance, Roberto Peres and Catia Cabral, for field deployment and mass-rearing

supervision and Simon Kutcher (WMP Global) for overall technical inputs, during early days of our implementation. We are also grateful for the field assistance provided by public agents of

the Health Municipality of Niterói, and for the incredible support by Jurujuba community members. FUNDING LAM is a fellow of CNPq. This work was funded by the Brazilian Ministry of Health

(SVS and SCTIE), and a grant to Monash University from the Bill and Melinda Gates Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or

preparation of the manuscript. AUTHOR INFORMATION Author notes * These authors contributed equally: João Silveira Moledo Gesto, Gabriel Sylvestre Ribeiro and Marcele Neves Rocha. AUTHORS AND

AFFILIATIONS * Grupo Mosquitos Vetores: Endossimbiontes e Interação Patógeno Vetor, Instituto René Rachou, Fiocruz Minas, Belo Horizonte, MG, Brazil João Silveira Moledo Gesto, Gabriel

Sylvestre Ribeiro, Marcele Neves Rocha, Fabiano Duarte Carvalho, Thiago Nunes Pereira & Luciano Andrade Moreira * Fiocruz Ceará, Fortaleza, CE, Brazil Fernando Braga Stehling Dias *

World Mosquito Program, Fiocruz, Rio de Janeiro, RJ, Brazil João Silveira Moledo Gesto, Gabriel Sylvestre Ribeiro, Marcele Neves Rocha, Fernando Braga Stehling Dias, Julia Peixoto & 

Luciano Andrade Moreira Authors * João Silveira Moledo Gesto View author publications You can also search for this author inPubMed Google Scholar * Gabriel Sylvestre Ribeiro View author

publications You can also search for this author inPubMed Google Scholar * Marcele Neves Rocha View author publications You can also search for this author inPubMed Google Scholar * Fernando

Braga Stehling Dias View author publications You can also search for this author inPubMed Google Scholar * Julia Peixoto View author publications You can also search for this author

inPubMed Google Scholar * Fabiano Duarte Carvalho View author publications You can also search for this author inPubMed Google Scholar * Thiago Nunes Pereira View author publications You can

also search for this author inPubMed Google Scholar * Luciano Andrade Moreira View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS G.S.R.,

M.N.R., F.B.S.D. and L.A.M. conceived the study. J.S.M.G., G.S.R., M.N.R., F.B.S.D., J.P., F.D.C. and T.N.P. performed the investigation, data curation and analysis. L.A.M. managed the

project supervision, validation and funding. J.S.M.G. and L.A.M. drafted the manuscript, and all authors reviewed and approved its final version. CORRESPONDING AUTHOR Correspondence to

Luciano Andrade Moreira. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE Springer Nature remains neutral with

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G.S., Rocha, M.N. _et al._ Reduced competence to arboviruses following the sustainable invasion of _Wolbachia_ into native _Aedes aegypti_ from Southeastern Brazil. _Sci Rep_ 11, 10039

(2021). https://doi.org/10.1038/s41598-021-89409-8 Download citation * Received: 16 October 2020 * Accepted: 20 April 2021 * Published: 11 May 2021 * DOI:

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