Immunometabolic crosstalk in Aedes fluviatilis and Wolbachia pipientis symbiosis.

Wolbachia pipientis is a maternally transmitted symbiotic bacterium that mainly colonizes arthropods, potentially affecting different aspects of the host's physiology, e.g., reproduction, immunity, and metabolism. It has been shown that Wolbachia modulates glycogen metabolism in mosquito Aedes fluviatilis (Ae. fluviatilis). Glycogen synthesis is controlled by the enzyme GSK3, which is also involved in immune responses in both vertebrate and invertebrate organisms. Here we investigated the mechanisms behind immune changes mediated by glycogen synthase kinase ß (GSK3ß) in the symbiosis between Ae. fluviatilis and W. pipientis using a GSK3ß inhibitor or RNAi-mediated gene silencing. GSK3ß inhibition or knockdown increased glycogen content and Wolbachia population, together with a reduction in Relish2 and gambicin transcripts. Furthermore, knockdown of Relish2 or Caspar revealed that the immunodeficiency pathway acts to control Wolbachia numbers in the host. In conclusion, we describe for the first time the involvement of GSK3ß in Ae. fluviatilis immune response, acting to control the Wolbachia endosymbiotic population.

The re-emerging arboviral threat: Hidden enemies: The emergence of obscure arboviral diseases, and the potential use of Wolbachia in their control.

Mayaro, Oropouche, and O'Nyong-Nyong share many traits with more prominent arboviruses, like dengue and yellow fever, chikungunya, and Zika. These include severe clinical symptoms, multiple animal hosts, and widespread vector species living in close proximity to human habitats, all of which constitute significant risk factors for more frequent outbreaks in the future, greatly increasing the potential of these hidden enemies to follow Zika and become the next wave of global arboviral threats. Critically, the current dearth of knowledge on these arboviruses might impede the success of future control efforts, including the potential application of Wolbachia pipientis. This bacterium inherently possesses broad anti-pathogen properties and a means of genetic drive that allows it to eliminate or replace target vector populations. We conclude that control of obscure arboviruses with Wolbachia might be possible, but successful implementation will be critically dependent on the ability to transinfect key vector species.

pH control in the midgut of Aedesaegypti under different nutritional conditions.

Aedes aegypti is one of the most important disease vectors in the world. Because their gut is the first site of interaction with pathogens, it is important to understand A. aegypti gut physiology. In this study, we investigated the mechanisms of pH control in the midgut of A. aegypti females under different nutritional conditions. We found that unfed females have an acidic midgut (pH âˆ¼6). The midgut of unfed insects is actively maintained at pH 6 regardless of the ingestion of either alkaline or acidic buffered solutions. V-ATPases are responsible for acidification after ingestion of alkaline solutions. In blood-fed females, the abdominal midgut becomes alkaline (pH 7.54), and the luminal pH decreases slightly throughout blood digestion. Only ingested proteins were able to trigger this abrupt increase in abdominal pH. The ingestion of amino acids, even at high concentrations, did not induce alkalinisation. During blood digestion, the thoracic midgut remains acidic, becoming a suitable compartment for carbohydrate digestion, which is in accordance with the higher alpha-glucolytic activity detected in this compartment. Ingestion of blood releases alkalising hormones in the haemolymph, which induce alkalinisation in ex vivo preparations. This study shows that adult A. aegypti females have a very similar gut physiology to that previously described for Lutzomyia longipalpis It is likely that all haematophagous Nematocera exhibit the same type of physiological behaviour.

High capacity of Brazilian Aedes aegypti populations to transmit a locally circulating lineage of Chikungunya virus.

The global incidence of chikungunya has surged in recent decades, with South America, particularly Brazil, experiencing devastating outbreaks. The primary vector for transmitting CHIKV in urban areas is the mosquito species Aedes aegypti, which is very abundant in Brazil. However, little is known about the impact of locally circulating CHIKV genotypes and specific combinations of mosquito populations on vector competence. In this study, we analyzed and compared the infectivity and transmissibility of a recently isolated CHIKV-ECSA lineage from Brazil among four Ae. aegypti populations collected from different regions of the country. When exposed to CHIKV-infected mice for blood feeding, all mosquito populations showed high infection rates and dissemination efficiency. Moreover, using a mouse model to assess transmission rates in a manner that better mirrors natural cycles, we observed that these populations exhibit highly efficient transmission rates of CHIKV-ECSA. Our findings underscore the robust capability of Brazilian Ae. aegypti populations to transmit the locally circulating CHIKV-ECSA lineage, potentially explaining its higher prevalence compared to the Asian lineage also introduced in Brazil.

Can Wolbachia be used to control malaria?

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.

The influence of different sources of blood meals on the physiology of Aedes aegypti harboring Wolbachia wMel: mouse blood as an alternative for mosquito rearing.

BACKGROUND: Aedes aegypti control programs have failed to restrain mosquito population expansion and, consequently, the spread of diseases such as dengue, Zika, and Chikungunya. Wolbachia infection of mosquitoes is a new and promising complementary tool for the control of arbovirus transmission. The use of Wolbachia-infected mosquitoes, mass reared using human blood, is currently being tested in several countries. However, the use of human blood for mass rearing mosquitoes, and thus expansion of this strategy, is problematic. With the aim of overcoming this problem, we tested the effect of different types of blood source on the fitness parameters of female Ae. aegypti and the Wolbachia titer over generations to be able to guarantee the suitability of an alternative source to human blood for mass rearing Wolbachia-infected mosquitoes. METHODS: We investigated and compared essential parameters of the vector capacity of laboratory strains of Ae. aegypti with and without Wolbachia that fed on blood of different types of host (human, guinea pig, and mouse). The parameters analyzed were fecundity, fertility, pupation dynamics, and adult survival. Also, we tested whether it is possible to maintain mosquitoes with Wolbachia on mouse blood over generations without losing the bacterium titer. RESULTS: The average number of eggs per female, egg viability and pupation dynamics in the Wolbachia-infected mosquito (wMelBr) strain were similar, regardless of the blood source. The F1 progenies of females that fed on mouse blood or human blood were analyzed. The longevity of males was lower than that of females. F1 female survival differed depending on the presence of Wolbachia in the mother. In subsequent generations analyzed up until F35, the relative Wolbachia density was even higher when mosquitoes fed on mouse blood in comparison to human blood. CONCLUSIONS: Taken together, our results provide no evidence that the different types of blood influenced the fitness of the Wolbachia-infected mosquitoes. The presence of the bacterium in the colonies of Wolbachia-infected Ae. aegypti after 35 generations under the conditions evaluated indicates that they can be maintained on mouse blood. Based on these results, we show that it is possible to use mouse blood to feed female mosquitoes when using human blood for this purpose is problematic.

Mayaro Virus: The Potential Role of Microbiota and Wolbachia.

The Mayaro virus (MAYV) is an arbovirus that circulates mainly in tropical forests or rural areas in Latin America and is transmitted mainly by Haemagogus mosquitoes. The objective of this study was to evaluate the vector competence, microbiome, and the presence of Wolbachia in three Aedes albopictus populations infected with MAYV. The vector competence was assessed based on viral infection and transmission by RT-qPCR. In addition, the microbiome was evaluated by amplification of the 16S rRNA V4 region and PCR to detect the presence of Wolbachia (strain wAlbA/wAlbB). Our results show that all three populations were susceptible to MAYV infection. The potential transmission of the MAYV was consistent in all populations of naïve mosquitoes injected (more than 50%). The microbiome analysis revealed 118 OTUs (operational taxonomic unit) from the three populations, 8 phyla, 15 classes, 26 orders, 35 families, 65 genera, and 53 species. All populations had Pseudomonas and Wolbachia as predominant genera. There was no difference between the variables for MAYV and Wolbachia (wAlbA or wAlbB) in the abdomen. However, in the head + thorax samples at 14 dpi, there was a difference between the two populations, indicating a possible correlation between the presence of Wolbachia (wAlbB) and infection. Overall, we show evidence that Ae. albopictus displays significant infection and transmission competence for the MAYV in the laboratory, and its bacterial microbiota play an important role in the host, mainly the strains of Wolbachia. The influence of the intestinal microbiota of Ae. albopictus is poorly known, and a better understanding of these interactions would open new perspectives for disease control through the manipulation of microbial communities. The exact contribution of this mosquito species to the transmission of the MAYV in the field remains to be confirmed.

Reduced competence to arboviruses following the sustainable invasion of Wolbachia into native Aedes aegypti from Southeastern Brazil.

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.

Aedes fluviatilis cell lines as new tools to study metabolic and immune interactions in mosquito-Wolbachia symbiosis.

In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions.

Large-Scale Deployment and Establishment of Wolbachia Into the Aedes aegypti Population in Rio de Janeiro, Brazil.

Traditional methods of vector control have proven insufficient to reduce the alarming incidence of dengue, Zika, and chikungunya in endemic countries. The bacterium symbiont Wolbachia has emerged as an efficient pathogen-blocking and self-dispersing agent that reduces the vectorial potential of Aedes aegypti populations and potentially impairs arboviral disease transmission. In this work, we report the results of a large-scale Wolbachia intervention in Ilha do Governador, Rio de Janeiro, Brazil. wMel-infected adults were released across residential areas between August 2017 and March 2020. Over 131 weeks, including release and post-release phases, we monitored the wMel prevalence in field specimens and analyzed introgression profiles of two assigned intervention areas, RJ1 and RJ2. Our results revealed that wMel successfully invaded both areas, reaching overall infection rates of 50-70% in RJ1 and 30-60% in RJ2 by the end of the monitoring period. At the neighborhood-level, wMel introgression was heterogeneous in both RJ1 and RJ2, with some profiles sustaining a consistent increase in infection rates and others failing to elicit the same. Correlation analysis revealed a weak overall association between RJ1 and RJ2 (r = 0.2849, p = 0.0236), and an association at a higher degree when comparing different deployment strategies, vehicle or backpack-assisted, within RJ1 (r = 0.4676, p < 0.0001) or RJ2 (r = 0.6263, p < 0.0001). The frequency knockdown resistance (kdr) alleles in wMel-infected specimens from both areas were consistently high over this study. Altogether, these findings corroborate that wMel can be successfully deployed at large-scale as part of vector control intervention strategies and provide the basis for imminent disease impact studies in Southeastern Brazil.

Insect vectors endosymbionts as solutions against diseases.

Viral diseases transmitted by mosquitoes, known as arboviruses, pose a significant threat to human life and are a major burden on many health systems around the world. Currently, arbovirus control strategies rely on insecticides or vector source reduction and, in the absence of effective, accessible and affordable vaccines, mainly on symptomatic based, non-specific treatments. However, insecticides have the potential to interfere with non-target organisms, cause environmental toxicity and insecticide resistance reduces their effectiveness as a sustainable control method. Complementary and sustainable strategies are urgently needed. Wolbachia, an invertebrate endosymbiont, has been used as an alternative strategy for arboviral control, through suppression or modification of mosquito populations. Here we discuss the burden that arboviruses impose on human populations and how Wolbachia can be used as a sustainable strategy for control, in alignment with the United Nations- 2030 Agenda for Sustainable Development.

Vector competence of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus mosquitoes for Mayaro virus.

Newly emerging or re-emerging arthropod-borne viruses (arboviruses) are important causes of human morbidity and mortality worldwide. Arboviruses such as Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), and West Nile virus (WNV) have undergone extensive geographic expansion in the tropical and sub-tropical regions of the world. In the Americas the main vectors of DENV, ZIKV, and CHIKV are mosquito species adapted to urban environments, namely Aedes aegypti and Aedes albopictus, whereas the main vector of WNV is Culex quinquefasciatus. Given the widespread distribution in the Americas and high permissiveness to arbovirus infection, these mosquito species may play a key role in the epidemiology of other arboviruses normally associated with sylvatic vectors. Here, we test this hypothesis by determining the vector competence of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus to Mayaro (MAYV) virus, a sylvatic arbovirus transmitted mainly by Haemagogus janthinomys that has been causing an increasing number of outbreaks in South America, namely in Brazil. Using field mosquitoes from Brazil, female mosquitoes were experimentally infected, and their competence for infection and transmission rates of MAYV was evaluated. We found consistent infection rate for MAYV in Ae. aegypti (57.5%) and Ae. albopictus (61.6%), whereas very low rates were obtained for Cx. quinquefasciatus (2.5%). Concordantly, we observed high potential transmission ability in Ae. aegypti and Ae. albopictus (69.5% and 71.1% respectively), in contrast to Cx. quinquefasciatus, which could not transmit the MAYV. Notably, we found that very low quantities of virus present in the saliva (undetectable by RT-qPCR) were sufficiently virulent to guarantee transmission. Although Ae. aegypti and Ae. albopictus mosquitoes are not the main vectors for MAYV, our studies suggest that these mosquitoes could play a significant role in the transmission of this arbovirus, since both species showed significant vector competence for MAYV (Genotype D), under laboratory conditions.

Historical Perspective and Biotechnological Trends to Block Arboviruses Transmission by Controlling Aedes aegypti Mosquitos Using Different Approaches.

Continuous climate changes associated with the disorderly occupation of urban areas have exposed Latin American populations to the emergence and reemergence of arboviruses transmitted by Aedes aegypti. The magnitude of the financial and political problems these epidemics may bring to the future of developing countries is still ignored. Due to the lack of effective antiviral drugs and vaccines against arboviruses, the primary measure for preventing or reducing the transmission of diseases depends entirely on the control of vectors or the interruption of human-vector contact. In Brazil the first attempt to control A. aegypti took place in 1902 by eliminating artificial sites of eproduction. Other strategies, such as the use of oviposition traps and chemical control with dichlorodiphenyltrichlorethane and pyrethroids, were successful, but only for a limited time. More recently, biotechnical approaches, such as the release of transgenics or sterile mosquitoes and the, development of transmission blocking vaccines, are being applied to try to control the A. aegypti population and/or arbovirus transmission. Endemic countries spend about twice as much to treat patients as they do on the prevention of mosquito-transmitted diseases. The result of this strategy is an explosive outbreak of arboviruses cases. This review summarizes the social impacts caused by A. aegypti-transmitted diseases, mainly from a biotechnological perspective in vector control aimed at protecting Latin American populations against arboviruses.

Anti-Anopheles darlingi saliva antibodies as marker of Plasmodium vivax infection and clinical immunity in the Brazilian Amazon.

BACKGROUND: Despite governmental and private efforts on providing malaria control, this disease continues to be a major health threat. Thus, innovative strategies are needed to reduce disease burden. The malaria vectors, through the injection of saliva into the host skin, play important role on disease transmission and may influence malaria morbidity. This study describes the humoral immune response against Anopheles (An.) darlingi saliva in volunteers from the Brazilian Amazon and addresses the association between levels of specific antibodies and clinical presentation of Plasmodium (P.) vivax infection. METHODS: Adult volunteers from communities in the Rondônia State, Brazil, were screened in order to assess the presence of P. vivax infection by light microscopy and nested PCR. Non-infected volunteers and individuals with symptomatic or symptomless infection were randomly selected and plasma collected. An. darlingi salivary gland sonicates (SGS) were prepared and used to measure anti-saliva antibody levels. Plasma interleukin (IL)-10 and interferon (IFN)-gamma levels were also estimated and correlated to anti-SGS levels. RESULTS: Individuals infected with P. vivax presented higher levels of anti-SGS than non-infected individuals and antibody levels could discriminate infection. Furthermore, anti-saliva antibody measurement was also useful to distinguish asymptomatic infection from non-infection, with a high likelihood ratio. Interestingly, individuals with asymptomatic parasitaemia presented higher titers of anti-SGS and lower IFN-gamma/IL-10 ratio than symptomatic ones. In P. vivax-infected asymptomatic individuals, the IFN-gamma/IL-10 ratio was inversely correlated to anti-SGS titers, although not for while in symptomatic volunteers. CONCLUSION: The estimation of anti-An. darlingi antibody levels can indicate the probable P. vivax infection status and also could serve as a marker of disease severity in this region of Brazilian Amazon.

Embryonic development and egg viability of wMel-infected Aedes aegypti.

BACKGROUND: Aedes aegypti is a major disease vector in urban habitats, involved in the transmission of dengue, chikungunya and Zika. Despite innumerous attempts to contain disease outbreaks, there are neither efficient vaccines nor definite vector control methods nowadays. In recent years, an innovative strategy to control arboviruses, which exploits the endosymbiotic bacterium Wolbachia pipientis, emerged with great expectations. The success of the method depends on many aspects, including Wolbachia's cytoplasmic incompatibility and pathogen interference phenotypes, as well as its effect on host fitness. In this work, we investigated the influence the Wolbachia strain wMel exerts on embryo development and egg viability and speculate on its field release use. METHODS: Wild-type (Br or Rockefeller) and Wolbachia-harboring specimens (wMelBr) were blood-fed and submitted to synchronous egg laying for embryo development assays. Samples were analyzed for morphological markers, developmental endpoint and egg resistance to desiccation (ERD). Quiescent egg viability over time was also assessed. RESULTS: wMelBr samples completed embryogenesis 2-3 hours later than wild-type. This delay was also observed through the onset of both morphological and physiological markers, respectively by the moments of germband extension and ERD acquisition. Following the end of embryonic development, wMelBr eggs were slightly less resistant to desiccation and showed reduced viability levels, which rapidly decayed after 40 days into quiescence, from approximately 75% to virtually 0% in less than a month. CONCLUSIONS: Our data revealed that the wMel strain of Wolbachia slightly delays embryogenesis and also affects egg quality, both through reduced viability and desiccation resistance. These findings suggest that, although embryonic fitness is somehow compromised by wMel infection, an efficient host reproductive manipulation through cytoplasmic incompatibility seems sufficient to overcome these effects in nature and promote bacterial invasion, as shown by successful ongoing field implementation.

Detection of Yellow Fever Virus in Sylvatic Mosquitoes during Disease Outbreaks of 2017⁻2018 in Minas Gerais State, Brazil.

Brazil has experienced several arbovirus outbreaks in recent years, among which yellow fever stands out. The state of Minas Gerais faced outbreaks of sylvatic yellow fever in 2017 and 2018, with 1002 confirmed cases and 340 deaths. This work presents the results of survey efforts to detect the yellow fever virus in mosquitoes from two conservation areas in the metropolitan region of Belo Horizonte, Brazil. A total of 867 mosquitoes of 20 species were collected between September 2017 and May 2018, the most abundant being Psorophora (Janthinosoma) ferox (von Humboldt, 1819) (31.3%), Limatus durhamii Theobald, 1901 (19.1%) and Haemagogus (Haemagogus) janthinomys Dyar, 1921 (18.2%). Total RNA was extracted from the mosquitoes for real-time PCR analysis for yellow fever, chikungunya, mayaro, Zika and dengue viruses. The yellow fever infection rate was 8.2% for Hg. janthinomys (13 mosquitoes), which is the main vector of sylvatic yellow fever in Brazil. In addition to surveying the mosquito fauna of these conservation units, this work demonstrates the importance of monitoring the circulation of viruses near large urban centers.

Wolbachia introduction into Lutzomyia longipalpis (Diptera: Psychodidae) cell lines and its effects on immune-related gene expression and interaction with Leishmania infantum.

BACKGROUND: The leishmaniases are important neglected diseases caused by Leishmania spp. which are transmitted by sand flies, Lutzomyia longipalpis being the main vector of visceral leishmaniasis in the Americas. The methodologies for leishmaniasis control are not efficient, causing 1.5 million reported cases annually worldwide, therefore showing the need for development of novel strategies and interventions to control transmission of the disease. The bacterium Wolbachia pipientis is being used to control viruses transmitted by mosquitoes, such as dengue and Zika, and its introduction in disease vectors has been effective against parasites such as Plasmodium. Here we show the first successful establishment of Wolbachia into two different embryonic cell lines from L. longipalpis, LL-5 and Lulo, and analysed its effects on the sand fly innate immune system, followed by in vitro Leishmania infantum interaction. RESULTS: Our results show that LL-5 cells respond to wMel and wMelPop-CLA strains within the first 72 h post-infection, through the expression of antimicrobial peptides and inducible nitric oxide synthase resulting in a decrease of Wolbachia detection in the early stages of infection. In subsequent passages, the wMel strain was not able to infect any of the sand fly cell lines while the wMelPop-CLA strain was able to stably infect Lulo cells and LL-5 at lower levels. In Wolbachia stably infected cells, the expression of immune-related genes involved with downregulation of the IMD, Toll and Jak-Stat innate immune pathways was significantly decreased, in comparison with the uninfected control, suggesting immune activation upon Wolbachia transinfection. Furthermore, Wolbachia transinfection did not promote a negative effect on parasite load in those cells. CONCLUSIONS: Initial strong immune responses of LL5 cells might explain the inefficiency of stable infections in these cells while we found that Lulo cells are more permissive to infection with Wolbachia causing an effect on the cell immune system, but not against in vitro L. infantum interaction. This establishes Lulo cells as a good system for the adaptation of Wolbachia in L. longipalpis.

Pluripotency of Wolbachia against Arboviruses: the case of yellow fever.

Background: Yellow fever outbreaks have re-emerged in Brazil during 2016-18, with mortality rates up to 30%. Although urban transmission has not been reported since 1942, the risk of re-urbanization of yellow fever is significant, as Aedes aegypti is present in most tropical and sub-tropical cities in the World and still remains the main vector of urban YFV. Although the YFV vaccine is safe and effective, it does not always reach populations at greatest risk of infection and there is an acknowledged global shortage of vaccine supply. The introgression of Wolbachia bacteria into Ae. aegypti mosquito populations is being trialed in several countries ( www.worldmosquito.org) as a biocontrol method against dengue, Zika and chikungunya. Here, we studied the ability of Wolbachia to reduce the transmission potential of Ae. aegypti mosquitoes for Yellow fever virus (YFV). Methods: Two recently isolated YFV (primate and human) were used to challenge field-derived wild-type and Wolbachia-infected ( wMel +) Ae. aegypti mosquitoes. The YFV infection status was followed for 7, 14 and 21 days post-oral feeding (dpf). The YFV transmission potential of mosquitoes was evaluated via nano-injection of saliva into uninfected mosquitoes or by inoculation in mice. Results: We found that Wolbachia was able to significantly reduce the prevalence of mosquitoes with YFV infected heads and thoraces for both viral isolates. Furthermore, analyses of mosquito saliva, through indirect injection into naïve mosquitoes or via interferon-deficient mouse model, indicated Wolbachia was associated with profound reduction in the YFV transmission potential of mosquitoes (14dpf). Conclusions: Our results suggest that Wolbachia introgression could be used as a complementary strategy for prevention of urban yellow fever transmission, along with the human vaccination program.

Wolbachia significantly impacts the vector competence of Aedes aegypti for Mayaro virus.

Wolbachia, an intracellular endosymbiont present in up to 70% of all insect species, has been suggested as a sustainable strategy for the control of arboviruses such as Dengue, Zika and Chikungunya. As Mayaro virus outbreaks have also been reported in Latin American countries, the objective of this study was to evaluate the vector competence of Brazilian field-collected Ae. aegypti and the impact of Wolbachia (wMel strain) upon this virus. Our in vitro studies with Aag2 cells showed that Mayaro virus can rapidly multiply, whereas in wMel-infected Aag2 cells, viral growth was significantly impaired. In addition, C6/36 cells seem to have alterations when infected by Mayaro virus. In vivo experiments showed that field-collected Ae. aegypti mosquitoes are highly permissive to Mayaro virus infection, and high viral prevalence was observed in the saliva. On the other hand, Wolbachia-harboring mosquitoes showed significantly impaired capability to transmit Mayaro virus. Our results suggest that the use of Wolbachia-harboring mosquitoes may represent an effective mechanism for the reduction of Mayaro virus transmission throughout Latin America.

In tune with nature: Wolbachia does not prevent pre-copula acoustic communication in Aedes aegypti.

BACKGROUND: Mosquito-borne diseases are rapidly spreading to vast territories, putting at risk most of the world's population. A key player in this scenario is Aedes aegypti, a hematophagous species which hosts and transmits viruses causing dengue and other serious illnesses. Since vector control strategies relying only on insecticides have proven unsustainable, an alternative method involving the release of Wolbachia-harboring individuals has emerged. Its successful implementation vastly depends on how fit the released individuals are in the natural habitat, being able to mate with wild populations and to spread Wolbachia to subsequent generations. In mosquitoes, an important aspect of reproductive fitness is the acoustic communication between males and females, which translates to interactions between harmonic frequencies in close proximity flight. This study aimed to characterize the flight tone produced by individuals harboring Wolbachia, also evaluating their ability to establish stable acoustic interactions. METHODS: Wild-type (WT) and Wolbachia-harboring specimens (wMelBr) were thorax-tethered to blunt copper wires and placed at close proximity to sensitive microphones. Wing-beat frequencies (WBFs) were characterized at fundamental and harmonic levels, for both single individuals and couples. Harmonic interactions in homogeneous and heterogeneous couples of WT and wMelBr variants were identified, categorized and quantified accordingly. RESULTS: In tethered 'solo' flights, individuals harboring Wolbachia developed WBFs, differing slightly, in a sex-dependent way, from those of the WT strain. To test the ability to form harmonic 'duets', tethered couples of wMelBr and WT individuals were shuffled in different sex pairs and had their flight tones analyzed. All couple types, with WT and/or wMelBr individuals, were able to interact acoustically in the frequency range of 1300-1500 Hz, which translates to the convergence between male's second harmonic and female's third. No significant differences were found in the proportions of interacting couples between the pair types. Surprisingly, spectrograms also revealed the convergence between alternative harmonic frequencies, inside and outside the species putative hearing threshold. CONCLUSIONS: Wolbachia infection leads to small sex-dependent changes on the flight tones of Ae. aegypti, but it does not seem to prevent the stereotyped harmonic interaction between males and females. Therefore, when released in the natural habitat to breed with native individuals, Wolbachia-harboring individuals shall be fit enough to meet the criteria of acoustically-related mating behavior and promote bacteria dispersion effectively.