Revisão sistemática rápida: Efetividade da estratégia Wolbachia para enfrentamento às arboviroses / Rapid review: effectiveness of the Wolbachia strategy for arbovirus control / Revisión sistemática rápida: efectividad de la estrategia de control con Wolbachia en la lucha contra las arbovirosis

[RESUMO]. Objetivo. Identificar e sintetizar evidências sobre efetividade e custo-efetividade da metodologia Aedes com Wolbachia para redução dos casos de arboviroses. Método. Revisão sistemática rápida, com busca realizada em fevereiro de 2022 em cinco portais de busca e bases de dados bibliográficas online. As etapas de seleção foram conduzidas por dois revisores, com resolução de conflitos por um terceiro. A extração dos dados foi feita por um revisor e conferida posteriormente por outro. Os critérios de inclusão foram estudos com descrição de custo-efetividade e efetividade da metodologia Aedes com Wolbachia, a partir de intervenções em campo, publicados nos idiomas português, inglês e espanhol, sem restrição de data de publicação. Resultados. Foram incluídos quatro estudos, sendo um ensaio randomizado em cluster, dois quaseexperimental e um antes-depois, publicados entre 2019 e 2021 em quatro países. Os estudos encontrados tiveram resultados efetivos em alguns dos desfechos avaliados, como na diminuição da incidência de casos de dengue, Zika e chikungunya. Apesar de não terem sido identificados estudos experimentais abordando custo-efetividade da estratégia Aedes com Wolbachia, alguns estudos de modelagem demonstraram um potencial custo-benefício desta estratégia. Conclusão. Considerando os resultados apresentados, conclui-se que a Wolbachia apresenta potencial para ser uma estratégia economicamente efetiva e que leva à redução da incidência de casos de dengue, Zika e chikungunya. Apesar disso, não é possível, todavia, recomendar a estratégia como política pública, pois ainda são necessários mais estudos de grande porte e de alta qualidade metodológica para subsidiar a tomada de decisão política.

[ABSTRACT]. Objective. To identify and synthesize evidence of the effectiveness and cost-effectiveness of the Wolbachia- Aedes methodology for reducing cases of arboviral infection. Methodology. Rapid systematic review. A search of five online portals and bibliographic databases was conducted in February 2022. Two reviewers conducted the selection stages, with any disagreements resolved by a third investigator. Data were extracted by one reviewer and subsequently checked by another. The inclusion criteria were studies describing the cost-effectiveness and effectiveness of the Wolbachia-Aedes methodology, based on field interventions, published in Portuguese, English, and Spanish, with no restrictions on date of publication. Results. Four studies were included: one cluster randomized trial, two quasi-experimental studies, and one pre-post study, published between 2019 and 2021 in four countries. The included studies reported effectiveness for some of the outcomes of interest, such as a reduction in the incidence of dengue, Zika, and chikungunya cases. Although no experimental studies addressing the cost-effectiveness of the Wolbachia- Aedes strategy were identified, some modeling studies have demonstrated potential cost-benefit of this methodology. Conclusion. Considering the results of the included studies, Wolbachia has the potential to be an economically effective strategy that leads to reductions in the incidence of dengue, Zika, and chikungunya. Nevertheless, this strategy cannot yet be recommended as a public policy; additional large-scale studies with high methodological quality are still needed to inform political decision-making.

[RESUMEN]. Objetivo. Señalar y sintetizar la evidencia sobre efectividad y costo-efectividad de la metodología de control de Aedes con Wolbachia para reducir los casos de arbovirosis. Método. Revisión sistemática rápida, con una búsqueda realizada en febrero del 2022 en cinco portales de búsqueda y bases de datos bibliográficas en línea. Las fases de selección estuvieron a cargo de dos revisores y la resolución de conflictos se asignó a un tercero. Un revisor extrajo los datos y luego otro los verificó. Los criterios de inclusión fueron: estudios que describieran la efectividad y la relación costo-efectividad de la metodología de control de Aedes con Wolbachia, basados en intervenciones sobre el terreno, publicados en portugués, inglés o español, sin restricciones en cuanto a la fecha de publicación. Resultados. Se incluyeron cuatro estudios, un ensayo aleatorizado por conglomerados, dos estudios cuasiexperimentales y uno de tipo antes-después, publicados entre el 2019 y el 2021 en cuatro países. En los estudios se obtuvieron resultados de efectividad en algunos de los criterios de valoración evaluados, como la reducción de la incidencia de casos de dengue, enfermedad de Zika y chikunguña. Aunque no se han encontrado estudios experimentales que aborden la costo-efectividad de la estrategia de control de Aedes con Wolbachia, algunos estudios de modelización han demostrado el posible costo-beneficio de esta estrategia. Conclusión. Teniendo en cuenta los resultados presentados, se puede concluir que la estrategia de control con Wolbachia tiene potencial para ser económicamente efectiva y conducir a una reducción de la incidencia de casos de dengue, enfermedad de Zika y chikunguña. Sin embargo, a pesar de ello, no es posible recomendar la estrategia como política pública, puesto que se requieren todavía más estudios a gran escala y de gran calidad metodológica para apoyar la toma de decisiones en materia de políticas.

Wolbachia infection in natural mosquito populations from Argentina.

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 population control methods. Strategies based in the intracellular bacterium Wolbachia Hertig, 1936 are considered environmentally friendly, safe for humans, and potentially cost-effective for controlling arboviral diseases. To minimize undesirable side effects, it is relevant to assess whether Wolbachia is present in the area and understand the diversity associated with native infections before implementing these strategies. 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. Aedes albifasciatus and C. pipiens complex samples were collected in the province of Buenos Aires, and A. fluviatilis in the province of Misiones. Aedes albifasciatus was found to be uninfected, while infections with strains wFlu and wPip were detected in A. fluviatilis and hybrids of the C. pipiens complex, respectively. All strains were fixed or close to fixation and clustered within supergroup B. These finding provides valuable information on Wolbachia strains found in natural mosquito populations in Argentina that might be used in heterologous infections in the future or be considered when designing control strategies based on Wolbachia infection.

First report of the association between Wolbachia and Cotesia flavipes (Hymenoptera: Braconidae): effect on life history parameters of the parasitoid.

The symbiosis between microorganisms and host arthropods can cause biological, physiological, and reproductive changes in the host population. The present study aimed to survey facultative symbionts of the genera Wolbachia, Arsenophonus, Cardinium, Rickettsia, and Nosema in Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) and Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) in the laboratory and evaluate the influence of infection on the fitness of these hosts. For this purpose, 16S rDNA primers were used to detect these facultative symbionts in the host species, and the hosts' biological and morphological features were evaluated for changes resulting from the infection caused by these microorganisms. The bacterial symbionts studied herein were not detected in the D. saccharalis samples analysed, but the endosymbiont Wolbachia was detected in C. flavipes and altered the biological and morphological aspects of this parasitoid insect. The results of this study may help to elucidate the role of Wolbachia in maintaining the quality of populations/lineages of C. flavipes.

Evaluating quasi-experimental approaches for estimating epidemiological efficacy of non-randomised field trials: applications in Wolbachia interventions for dengue.

BACKGROUND: Wolbachia symbiosis in Aedes aegypti is an emerging biocontrol measure against dengue. However, assessing its real-world efficacy is challenging due to the non-randomised, field-based nature of most intervention studies. This research re-evaluates the spatial-temporal impact of Wolbachia interventions on dengue incidence using a large battery of quasi-experimental methods and assesses each method's validity. METHODS: A systematic search for Wolbachia intervention data was conducted via PUBMED. Efficacy was reassessed using commonly-used quasi-experimental approaches with extensive robustness checks, including geospatial placebo tests and a simulation study. Intervention efficacies across multiple study sites were computed using high-resolution aggregations to examine heterogeneities across sites and study periods. We further designed a stochastic simulation framework to assess the methods' ability to estimate intervention efficacies (IE). RESULTS: Wolbachia interventions in Singapore, Malaysia, and Brazil significantly decreased dengue incidence, with reductions ranging from 48.17% to 69.19%. IEs varied with location and duration. Malaysia showed increasing efficacy over time, while Brazil exhibited initial success with subsequent decline, hinting at operational challenges. Singapore's strategy was highly effective despite partial saturation. Simulations identified Synthetic Control Methods (SCM) and its variant, count Synthetic Control Method (cSCM), as superior in precision, with the smallest percentage errors in efficacy estimation. These methods also demonstrated robustness in placebo tests. CONCLUSIONS: Wolbachia interventions exhibit consistent protective effects against dengue. SCM and cSCM provided the most precise and robust estimates of IEs, validated across simulated and real-world settings.

Revealing the microbiome diversity and biocontrol potential of field Aedes ssp.: Implications for disease vector management.

The mosquito Aedes spp. holds important relevance for human and animal health, as it serves as a vector for transmitting multiple diseases, including dengue and Zika virus. The microbiome's impact on its host's health and fitness is well known. However, most studies on mosquito microbiomes have been conducted in laboratory settings. We explored the mixed microbial communities within Aedes spp., utilizing the 16S rRNA gene for diversity analysis and shotgun metagenomics for functional genomics. Our samples, which included Ae. aegypti and Ae. albopictus, spanned various developmental stages-eggs, larvae, and adults-gathered from five semiurban areas in Mexico. Our findings revealed a substantial diversity of 8,346 operational taxonomic units (OTUs), representing 967 bacterial genera and 126,366 annotated proteins. The host developmental stage was identified as the primary factor associated with variations in the microbiome composition. Subsequently, we searched for genes and species involved in mosquito biocontrol. Wolbachia accounted for 9.6% of the 16S gene sequences. We observed a high diversity (203 OTUs) of Wolbachia strains commonly associated with mosquitoes, such as wAlb, with a noticeable increase in abundance during the adult stages. Notably, we detected the presence of the cifA and cifB genes, which are associated with Wolbachia's cytoplasmic incompatibility, a biocontrol mechanism. Additionally, we identified 221 OTUs related to Bacillus, including strains linked to B. thuringiensis. Furthermore, we discovered multiple genes encoding insecticidal toxins, such as Cry, Mcf, Vip, and Vpp. Overall, our study contributes to the understanding of mosquito microbiome biodiversity and metabolic capabilities, which are essential for developing effective biocontrol strategies against this disease vector.

Comparing the long-term persistence of different Wolbachia strains after the release of bacteria-carrying mosquitoes.

This paper proposes a bidimensional modeling framework for Wolbachia invasion, assuming imperfect maternal transmission, incomplete cytoplasmic incompatibility, and direct infection loss due to thermal stress. Our model adapts to various Wolbachia strains and retains all properties of higher-dimensional models. The conditions for the durable coexistence of Wolbachia-carrying and wild mosquitoes are expressed using the model's parameters in a compact closed form. When the Wolbachia bacterium is locally established, the size of the remanent wild population can be assessed by a direct formula derived from the model. The model was tested for four Wolbachia strains undergoing laboratory and field trials to control mosquito-borne diseases: wMel, wMelPop, wAlbB, and wAu. As all these bacterial strains affect the individual fitness of mosquito hosts differently and exhibit different levels of resistance to temperature variations, the model helped to conclude that: (1) the wMel strain spreads faster in wild mosquito populations; (2) the wMelPop exhibits lower resilience but also guarantees the smallest size of the remanent wild population; (3) the wAlbB strain performs better at higher ambient temperatures than others; (4) the wAu strain is not sustainable and cannot persist in the wild mosquito population despite its resistance to high temperatures.

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.

DENV-1 Titer Impacts Viral Blocking in wMel Aedes aegypti with Brazilian Genetic Background.

Several countries have been using Wolbachia deployments to replace highly competent native Aedes aegypti populations with Wolbachia-carrying mosquitoes with lower susceptibility to arboviruses such as dengue, Zika, and chikungunya. In Rio de Janeiro, Wolbachia deployments started in 2015 and still present a moderate introgression with a modest reduction in dengue cases in humans (38%). Here, we evaluated the vector competence of wild-type and wMel-infected Ae. aegypti with a Brazilian genetic background to investigate whether virus leakage could contribute to the observed outcomes in Brazil. We collected the specimens in three areas of Rio de Janeiro with distinct frequencies of mosquitoes with wMel strain and two areas with wild Ae. aegypti. The mosquitoes were orally exposed to two titers of DENV-1 and the saliva of DENV-1-infected Ae. aegypti was microinjected into wMel-free mosquitoes to check their infectivity. When infected with the high DENV-1 titer, the presence of wMel did not avoid viral infection in mosquitoes' bodies and saliva but DENV-1-infected wMel mosquitoes produced lower viral loads than wMel-free mosquitoes. On the other hand, wMel mosquitoes infected with the low DENV-1 titer were less susceptible to virus infection than wMel-free mosquitoes, although once infected, wMel and wMel-free mosquitoes exhibited similar viral loads in the body and the saliva. Our results showed viral leakage in 60% of the saliva of wMel mosquitoes with Brazilian background; thus, sustained surveillance is imperative to monitor the presence of other circulating DENV-1 strains capable of overcoming the Wolbachia blocking phenotype, enabling timely implementation of action plans.

The greening-causing agent alters the behavioral and electrophysiological responses of the Asian citrus psyllid to a putative sex pheromone.

The Asian Citrus Psyllid (ACP), Diaphorina citri, is a vector of the pathological bacterium Candidatus Liberibacter asiaticus (CLas), which causes the most devastating disease to the citrus industry worldwide, known as greening or huanglongbing (HLB). Earlier field tests with an acetic acid-based lure in greening-free, 'Valencia' citrus orange groves in California showed promising results. The same type of lures tested in São Paulo, Brazil, showed unsettling results. During the unsuccessful trials, we noticed a relatively large proportion of females in the field, ultimately leading us to test field-collected males and females for Wolbachia and CLas. The results showed high rates of Wolbachia and CLas infection in field populations. We then compared the olfactory responses of laboratory-raised, CLas-free, and CLas-infected males to acetic acid. As previously reported, CLas-uninfected males responded to acetic acid at 1 µg. Surprisingly, CLas-infected males required 50 × higher doses of the putative sex pheromone, thus explaining the failure to capture CLas-infected males in the field. CLas infection was also manifested in electrophysiological responses. Electroantennogram responses from CLas-infected ACP males were significantly higher than those obtained with uninfected males. To the best of our knowledge, this is the first report of a pathogen infection affecting a vector's response to a sex attractant.

Aerial release of Aedes aegypti male mosquitoes using an unmanned aerial vehicle: a novel control strategy.

OBJECTIVE: To development of a methodology for the chilling, handling, transport, and release of male Aedes aegypti mosquitoes, reared in insectary conditions to release in the field with unmanned vehicles to compete sexually with wild males in the field. MATERIALS AND METHODS: A population of Ae. aegypti from different areas in Tapachula, Chiapas, was used. Laboratory tests were conducted: Effect of temperature and cooling time on the knockdown, recovery of males, and copulatory success. RESULTS: The chilling temperature of 3 ± 1ºC for 30 min, was used as a knockdown temperature before handling, packing, transportation, and aerial release. The males subjected to the entire process, including the semi-field aerial release test, showed normal sexual behavior activity, obtaining 100% of females inseminated. CONCLUSION: These results present the feasibility of applying a new control methodology using unmanned aerial vehicle (UAV) as support for the sterile insect release technique (SIT), use of Wolbachia or both, in male Ae. aegypti, for the design of strategies to control their populations.

Reduced dengue incidence following city-wide wMel Wolbachia mosquito releases throughout three Colombian cities: Interrupted time series analysis and a prospective case-control study.

BACKGROUND: The introduction of Wolbachia (wMel strain) into Aedes aegypti mosquitoes reduces their capacity to transmit dengue and other arboviruses. Randomised and non-randomised studies in multiple countries have shown significant reductions in dengue incidence following field releases of wMel-infected Ae. aegypti. We report the public health outcomes from phased, large-scale releases of wMel-Ae. aegypti mosquitoes throughout three contiguous cities in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Following pilot releases in 2015-2016, staged city-wide wMel-Ae. aegypti deployments were undertaken in the cities of Bello, Medellín and Itagüí (3.3 million people) between October 2016 and April 2022. The impact of the Wolbachia intervention on dengue incidence was evaluated in two parallel studies. A quasi-experimental study using interrupted time series analysis showed notified dengue case incidence was reduced by 95% in Bello and Medellín and 97% in Itagüí, following establishment of wMel at ≥60% prevalence, compared to the pre-intervention period and after adjusting for seasonal trends. A concurrent clinic-based case-control study with a test-negative design was unable to attain the target sample size of 63 enrolled virologically-confirmed dengue (VCD) cases between May 2019 and December 2021, consistent with low dengue incidence throughout the Aburrá Valley following wMel deployments. Nevertheless, VCD incidence was 45% lower (OR 0.55 [95% CI 0.25, 1.17]) and combined VCD/presumptive dengue incidence was 47% lower (OR 0.53 [95% CI 0.30, 0.93]) among participants resident in wMel-treated versus untreated neighbourhoods. CONCLUSIONS/SIGNIFICANCE: Stable introduction of wMel into local Ae. aegypti populations was associated with a significant and sustained reduction in dengue incidence across three Colombian cities. These results from the largest contiguous Wolbachia releases to-date demonstrate the real-world effectiveness of the method across large urban populations and, alongside previously published results, support the reproducibility of this effectiveness across different ecological settings. TRIAL REGISTRATION: NCT03631719.

Large-scale releases and establishment of wMel Wolbachia in Aedes aegypti mosquitoes throughout the Cities of Bello, Medellín and Itagüí, Colombia.

BACKGROUND: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and has been shown to reduce the transmission of dengue and other Aedes-borne viruses. Here we report the entomological results from phased, large-scale releases of Wolbachia infected Ae. aegypti mosquitoes throughout three contiguous cities located in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Local wMel Wolbachia-infected Ae. aegypti mosquitoes were generated and then released in an initial release pilot area in 2015-2016, which resulted in the establishment of Wolbachia in the local mosquito populations. Subsequent large-scale releases, mainly involving vehicle-based releases of adult mosquitoes along publicly accessible roads and streets, were undertaken across 29 comunas throughout Bello, Medellín and Itagüí Colombia between 2017-2022. In 9 comunas these were supplemented by egg releases that were undertaken by staff or community members. By the most recent monitoring, Wolbachia was found to be stable and established at consistent levels in local mosquito populations (>60% prevalence) in the majority (67%) of areas. CONCLUSION: These results, from the largest contiguous releases of wMel Wolbachia mosquitoes to date, highlight the operational feasibility of implementing the method in large urban settings. Based on results from previous studies, we expect that Wolbachia establishment will be sustained long term. Ongoing monitoring will confirm Wolbachia persistence in local mosquito populations and track its establishment in the remaining areas.

Wolbachia dominance influences the Culex quinquefasciatus microbiota.

Microorganisms present in mosquitoes and their interactions are key factors affecting insect development. Among them, Wolbachia is closely associated with the host and affects several fitness parameters. In this study, the bacterial and fungal microbiota from two laboratory Culex quinquefasciatus isolines (wild type and tetracycline-cured) were characterized by metagenome amplicon sequencing of the ITS2 and 16S rRNA genes at different developmental stages and feeding conditions. We identified 572 bacterial and 61 fungal OTUs. Both isolines presented variable bacterial communities and different trends in the distribution of diversity among the groups. The lowest bacterial richness was detected in sugar-fed adults of the cured isoline, whereas fungal richness was highly reduced in blood-fed mosquitoes. Beta diversity analysis indicated that isolines are an important factor in the differentiation of mosquito bacterial communities. Considering composition, Penicillium was the dominant fungal genus, whereas Wolbachia dominance was inversely related to that of Enterobacteria (mainly Thorsellia and Serratia). This study provides a more complete overview of the mosquito microbiome, emphasizing specific highly abundant components that should be considered in microorganism manipulation approaches to control vector-borne diseases.

wMel Wolbachia alters female post-mating behaviors and physiology in the dengue vector mosquito Aedes aegypti.

Globally invasive Aedes aegypti disseminate numerous arboviruses that impact human health. One promising method to control Ae. aegypti populations is transinfection with Wolbachia pipientis, which naturally infects ~40-52% of insects but not Ae. aegypti. Transinfection of Ae. aegypti with the wMel Wolbachia strain induces cytoplasmic incompatibility (CI), allows infected individuals to invade native populations, and inhibits transmission of medically relevant arboviruses by females. Female insects undergo post-mating physiological and behavioral changes-referred to as the female post-mating response (PMR)-required for optimal fertility. PMRs are typically elicited by male seminal fluid proteins (SFPs) transferred with sperm during mating but can be modified by other factors, including microbiome composition. Wolbachia has modest effects on Ae. aegypti fertility, but its influence on other PMRs is unknown. Here, we show that Wolbachia influences female fecundity, fertility, and re-mating incidence and significantly extends the longevity of virgin females. Using proteomic methods to examine the seminal proteome of infected males, we found that Wolbachia moderately affects SFP composition. However, we identified 125 paternally transferred Wolbachia proteins, but the CI factor proteins (Cifs) were not among them. Our findings indicate that Wolbachia infection of Ae. aegypti alters female PMRs, potentially influencing control programs that utilize Wolbachia-infected individuals.

Dengue Exposure and Wolbachia wMel Strain Affects the Fertility of Quiescent Eggs of Aedes aegypti.

(1) Background: The deployment of the bacterium Wolbachia to reduce arbovirus transmission is ongoing in several countries worldwide. When Wolbachia-carrying Aedes aegypti are released and established in the field, females may feed on dengue-infected hosts. The effects of simultaneous exposure on life-history traits of Ae. aegypti to Wolbachia wMel strain and dengue-1 virus DENV-1 remain unclear. (2) Methods: We monitored 4 groups (mosquitoes with either DENV-1 or Wolbachia, coinfected with DENV-1 and Wolbachia, as well as negative controls) to estimate Ae. aegypti survival, oviposition success, fecundity, collapsing and fertility of quiescent eggs for 12 weeks. (3) Results: Neither DENV-1 nor Wolbachia had a significant impact on mosquito survival nor on mosquito fecundity, although the last parameter showed a tendency to decrease with ageing. There was a significant decrease in oviposition success in individuals carrying Wolbachia. Wolbachia infection and storage time significantly increased egg collapse parameter on the egg viability assay, while DENV-1 had a slight protective effect on the first four weeks of storage. (4) Conclusions: Despite limitations, our results contribute to better understanding of the tripartite interaction of virus, bacteria and mosquito that may take place in field conditions and aid in guaranteeing the Wolbachia strategy success.

Molecular detection of Wolbachia and Bartonella as part of the microbiome of phlebotomine sand flies from Chiapas, Mexico.

Phlebotomine sand flies are dipterans of relevance due to their role as vectors of several pathogens worldwide. Bacteria in the gut of sand flies possibly affect their vectorial capacity and competence to transmit parasites. A retrospective study was performed in sand fly specimens that had previously been collected in four localities of the state of Chiapas during the period 2009-2011 to detect Wolbachia and Bartonella and their possible coinfection with Leishmania. For the molecular detection of bacteria, we used primers and conditions that had previously been reported. A total of 531 sand fly specimens of 10 species were analyzed. Four Wolbachia strains were detected in five sand fly species, showing a prevalence of 8.6%. All the Wolbachia strains had previously been reported in other taxa. In one sand fly species, we also detected a new lineage of Bartonella evidenced by a phylogenetic analysis. No sand fly specimens showed coinfections of these bacteria and Leishmania. The bacteria found in the phlebotomine sand flies are possibly transmitted by plant-mediated horizontal transmission and during blood meal feeding.

First molecular evidence of Wolbachia occurrence in Amblyomma sculptum (Acari: Ixodidae).

As the main vector for the bacterium Rickettsia rickettsii in Brazil, the tick Amblyomma sculptum is a parasite of great public health importance in this country. Wolbachia is an endosymbiont bacterium highly widespread among invertebrates and because of its impact on its hosts' biology, form a powerful alternative for pests and disease control. The aim of this study was to investigate the occurrence of this bacterium in A. sculptum. For this, 187 adult ticks collected in two municipalities in the interior of the state of São Paulo, Brazil, were analyzed using molecular techniques and bioinformatics tools. A total of 15 ticks were positive for the presence of Wolbachia. Phylogenetic analysis on the 16S rRNA gene indicated that the Wolbachia DNA sequences obtained in this investigation belonged to different clades, probably in supergroups B and F. This was the first study to report the occurrence of Wolbachia in A. sculptum and it enriches knowledge about the susceptibility of ticks to this bacterium. Now that we know that Wolbachia can be found in A. sculptum, the objective for a next study must be to investigate Wolbachia's possible origin in this tick.

First report of natural Wolbachia infections in mosquitoes from Cuba.

Mosquitoes are extensively responsible for the transmission of pathogens. Novel strategies using Wolbachia could transform that scenario, since these bacteria manipulate mosquito reproduction, and can confer a pathogen transmission-blocking phenotype in culicids. Here, we screened the Wolbachia surface protein region by PCR in eight Cuban mosquito species. We confirmed the natural infections by sequencing and assessed the phylogenetic relationships among the Wolbachia strains detected. We identified four Wolbachia hosts: Aedes albopictus, Culex quinquefasciatus, Mansonia titillans, and Aedes mediovittatus (first report worldwide). Knowledge of Wolbachia strains and their natural hosts is essential for future operationalization of this vector control strategy in Cuba.

Monotonicity properties arising in a simple model of Wolbachia invasion for wild mosquito populations.

In this paper, we propose a simplified bidimensional Wolbachia infestation model in a population of Aedes aegypti mosquitoes, preserving the main features associated with the biology of this species that can be found in higher-dimensional models. Namely, our model represents the maternal transmission of the Wolbachia symbiont, expresses the reproductive phenotype of cytoplasmic incompatibility, accounts for different fecundities and mortalities of infected and wild insects, and exhibits the bistable nature leading to the so-called principle of competitive exclusion. Using tools borrowed from monotone dynamical system theory, in the proposed model, we prove the existence of an invariant threshold manifold that allows us to provide practical recommendations for performing single and periodic releases of Wolbachia-carrying mosquitoes, seeking the eventual elimination of wild insects that are capable of transmitting infections to humans. We illustrate these findings with numerical simulations using parameter values corresponding to the wMelPop strain of Wolbachia that is considered the best virus blocker but induces fitness loss in its carriers. In these tests, we considered multiple scenarios contrasting a periodic release strategy against a strategy with a single inundative release, comparing their effectiveness. Our study is presented as an expository and mathematically accessible tool to study the use of Wolbachia-based biocontrol versus more complex models.