Low-input PacBio sequencing generates high-quality individual fly genomes and characterizes mutational processes.

Long-read sequencing, exemplified by PacBio, revolutionizes genomics, overcoming challenges like repetitive sequences. However, the high DNA requirement ( > 1 µg) is prohibitive for small organisms. We develop a low-input (100 ng), low-cost, and amplification-free library-generation method for PacBio sequencing (LILAP) using Tn5-based tagmentation and DNA circularization within one tube. We test LILAP with two Drosophila melanogaster individuals, and generate near-complete genomes, surpassing preexisting single-fly genomes. By analyzing variations in these two genomes, we characterize mutational processes: complex transpositions (transposon insertions together with extra duplications and/or deletions) prefer regions characterized by non-B DNA structures, and gene conversion of transposons occurs on both DNA and RNA levels. Concurrently, we generate two complete assemblies for the endosymbiotic bacterium Wolbachia in these flies and similarly detect transposon conversion. Thus, LILAP promises a broad PacBio sequencing adoption for not only mutational studies of flies and their symbionts but also explorations of other small organisms or precious samples.

Population genetic structure of Culex tritaeniorhynchus in different types of climatic zones in China.

BACKGROUND: Culex tritaeniorhynchus is widely distributed in China, from Hainan Island in the south to Heilongjiang in the north, covering tropical, subtropical, and temperate climate zones. Culex tritaeniorhynchus carries 19 types of arboviruses. It is the main vector of the Japanese encephalitis virus (JEV), posing a serious threat to human health. Understanding the effects of environmental factors on Culex tritaeniorhynchus can provide important insights into its population structure or isolation patterns, which is currently unclear. RESULTS: In total, 138 COI haplotypes were detected in the 552 amplified sequences, and the haplotype diversity (Hd) value increased from temperate (0.534) to tropical (0.979) regions. The haplotype phylogeny analysis revealed that the haplotypes were divided into two high-support evolutionary branches. Temperate populations were predominantly distributed in evolutionary branch II, showing some genetic isolation from tropical/subtropical populations and less gene flow between groups. The neutral test results of HNQH (Qionghai) and HNHK(Haikou) populations were negative (P < 0.05), indicating many low-frequency mutations in the populations and that the populations might be in the process of expansion. Moreover, Wolbachia infection was detected only in SDJN (Jining) (2.24%), and all Wolbachia genotypes belonged to supergroup B. To understand the influence of environmental factors on mosquito-borne viruses, we examined the prevalence of Culex tritaeniorhynchus infection in three ecological environments in Shandong Province. We discovered that the incidence of JEV infection was notably greater in Culex tritaeniorhynchus from lotus ponds compared to those from irrigation canal regions. In this study, the overall JEV infection rate was 15.27 per 1000, suggesting the current risk of Japanese encephalitis outbreaks in Shandong Province. CONCLUSIONS: Tropical and subtropical populations of Culex tritaeniorhynchus showed higher genetic diversity and those climatic conditions provide great advantages for the establishment and expansion of Culex tritaeniorhynchus. There are differences in JEV infection rates in wild populations of Culex tritaeniorhynchus under different ecological conditions. Our results suggest a complex interplay of genetic differentiation, population structure, and environmental factors in shaping the dynamics of Culex tritaeniorhynchus. The low prevalence of Wolbachia in wild populations may reflect the recent presence of Wolbachia invasion in Culex tritaeniorhynchus.

Targeting a microbiota Wolbachian aminoacyl-tRNA synthetase to block its pathogenic host.

The interplay between humans and their microbiome is crucial for various physiological processes, including nutrient absorption, immune defense, and maintaining homeostasis. Microbiome alterations can directly contribute to diseases or heighten their likelihood. This relationship extends beyond humans; microbiota play vital roles in other organisms, including eukaryotic pathogens causing severe diseases. Notably, Wolbachia, a bacterial microbiota, is essential for parasitic worms responsible for lymphatic filariasis and onchocerciasis, devastating human illnesses. Given the lack of rapid cures for these infections and the limitations of current treatments, new drugs are imperative. Here, we disrupt Wolbachia's symbiosis with pathogens using boron-based compounds targeting an unprecedented Wolbachia enzyme, leucyl-tRNA synthetase (LeuRS), effectively inhibiting its growth. Through a compound demonstrating anti-Wolbachia efficacy in infected cells, we use biophysical experiments and x-ray crystallography to elucidate the mechanism behind Wolbachia LeuRS inhibition. We reveal that these compounds form adenosine-based adducts inhibiting protein synthesis. Overall, our study underscores the potential of disrupting key microbiota to control infections.

The effect of repeat feeding on dengue virus transmission potential in Wolbachia-infected Aedes aegypti following extended egg quiescence.

As Wolbachia pipientis is more widely being released into field populations of Aedes aegypti for disease control, the ability to select the appropriate strain for differing environments is increasingly important. A previous study revealed that longer-term quiescence in the egg phase reduced the fertility of mosquitoes, especially those harboring the wAlbB Wolbachia strain. This infertility was also associated with a greater biting rate. Here, we attempt to quantify the effect of this heightened biting behavior on the transmission potential of the dengue virus using a combination of assays for fitness, probing behavior, and vector competence, allowing repeat feeding, and incorporate these effects in a model of R0. We show that Wolbachia-infected infertile mosquitoes are more interested in feeding almost immediately after an initial blood meal relative to wild type and Wolbachia-infected fertile mosquitoes and that these differences continue for up to 8 days over the period we measured. As a result, the infertile Wolbachia mosquitoes have higher virus prevalence and loads than Wolbachia-fertile mosquitoes. We saw limited evidence of Wolbachia-mediated blocking in the disseminated tissue (legs) in terms of prevalence but did see reduced viral loads. Using a previously published estimate of the extrinsic incubation period, we demonstrate that the effect of repeat feeding/infertility is insufficient to overcome the effects of Wolbachia-mediated blocking on R0. These estimates are very conservative, however, and we posit that future studies should empirically measure EIP under a repeat feeding model. Our findings echo previous work where periods of extensive egg quiescence affected the reproductive success of Wolbachia-infected Ae. aegypti. Additionally, we show that increased biting behavior in association with this infertility in Wolbachia-infected mosquitoes may drive greater vector competence. These relationships require further exploration, given their ability to affect the success of field releases of Wolbachia for human disease reduction in drier climates where longer egg quiescence periods are expected.

Update to: Assessing the efficacy of male Wolbachia-infected mosquito deployments to reduce dengue incidence in Singapore.

BACKGROUND: This trial is a parallel, two-arm, non-blinded cluster randomised controlled trial that is under way in Singapore, with the aim of measuring the efficacy of male Wolbachia-infected Aedes aegypti deployments in reducing dengue incidence in an endemic setting with all four dengue serotypes in circulation. The trial commenced in July 2022 and is expected to conclude in September 2024. The original study protocol was published in December 2022. Here, we describe amendments that have been made to the study protocol since commencement of the trial. METHODS: The key protocol amendments are (1) addition of an explicit definition of Wolbachia exposure for residents residing in intervention sites based on the duration of Wolbachia exposure at point of testing, (2) incorporation of a high-dimensional set of anthropogenic and environmental characteristics in the analysis plan to adjust for baseline risk factors of dengue transmission, and (3) addition of alternative statistical analyses for endpoints to control for post hoc imbalance in cluster-based environmental and anthropogenic characteristics. DISCUSSION: The findings from this study will provide the first experimental evidence for the efficacy of releasing male-Wolbachia infected mosquitoes to reduce dengue incidence in a cluster-randomised controlled trial. The trial will conclude in 2024 and results will be reported shortly thereafter. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT05505682. Registered on 16 August 2022. Retrospectively registered. Last updated 11 November 2023.

Comparative analysis of Wolbachia maternal transmission and localization in host ovaries.

Many insects and other animals carry microbial endosymbionts that influence their reproduction and fitness. These relationships only persist if endosymbionts are reliably transmitted from one host generation to the next. Wolbachia are maternally transmitted endosymbionts found in most insect species, but transmission rates can vary across environments. Maternal transmission of wMel Wolbachia depends on temperature in natural Drosophila melanogaster hosts and in transinfected Aedes aegypti, where wMel is used to block pathogens that cause human disease. In D. melanogaster, wMel transmission declines in the cold as Wolbachia become less abundant in host ovaries and at the posterior pole plasm (the site of germline formation) in mature oocytes. Here, we assess how temperature affects maternal transmission and underlying patterns of Wolbachia localization across 10 Wolbachia strains diverged up to 50 million years-including strains closely related to wMel-and their natural Drosophila hosts. Many Wolbachia maintain high transmission rates across temperatures, despite highly variable (and sometimes low) levels of Wolbachia in the ovaries and at the developing germline in late-stage oocytes. Identifying strains like closely related wMel-like Wolbachia with stable transmission across variable environmental conditions may improve the efficacy of Wolbachia-based biocontrol efforts as they expand into globally diverse environments.

Bacterial Symbionts Contribute to Insecticide Susceptibility of Diaphorina citri via Changing the Expression Level of Host Detoxifying Genes.

Insecticide susceptibility is mainly determined by the insect host, but symbiotic bacteria are also an important affecting factor. In this study, we investigate the relationship between the structure of gut bacterial symbionts and insecticide susceptibility in Diaphorina citri, the important carrier of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). Our results indicated that antibiotic treatment significantly increased the susceptibility of D. citri to bifenthrin and thiamethoxam, and significantly decreased the relative abundance of Wolbachia and Profftella, enzyme activities of CarEs, and expression level of multiple CarE genes. The relative loads of Wolbachia and Profftella were positively correlated with DcitCCE13, DcitCCE14, DcitCCE15, and DcitCCE16. RNAi and prokaryotic expression revealed that DcitCCE15 is associated with bifenthrin metabolism. These results revealed that bacterial symbionts might regulate DcitCCE15 expression, which is involved in the susceptibility of D. citri to bifenthrin.

Wolbachia mediates crosstalk between miRNA and Toll pathways to enhance resistance to dengue virus in Aedes aegypti.

The obligate endosymbiont Wolbachia induces pathogen interference in the primary disease vector Aedes aegypti, facilitating the utilization of Wolbachia-based mosquito control for arbovirus prevention, particularly against dengue virus (DENV). However, the mechanisms underlying Wolbachia-mediated virus blockade have not been fully elucidated. Here, we report that Wolbachia activates the host cytoplasmic miRNA biogenesis pathway to suppress DENV infection. Through the suppression of the long noncoding RNA aae-lnc-2268 by Wolbachia wAlbB, aae-miR-34-3p, a miRNA upregulated by the Wolbachia strains wAlbB and wMelPop, promoted the expression of the antiviral effector defensin and cecropin genes through the Toll pathway regulator MyD88. Notably, anti-DENV resistance induced by Wolbachia can be further enhanced, with the potential to achieve complete virus blockade by increasing the expression of aae-miR-34-3p in Ae. aegypti. Furthermore, the downregulation of aae-miR-34-3p compromised Wolbachia-mediated virus blockade. These findings reveal a novel mechanism by which Wolbachia establishes crosstalk between the cytoplasmic miRNA pathway and the Toll pathway via aae-miR-34-3p to strengthen antiviral immune responses against DENV. Our results will aid in the advancement of Wolbachia for arbovirus control by enhancing its virus-blocking efficiency.

Reproductive manipulation: Wolbachia induce host parthenogenesis using a stolen transformer.

The Wolbachia strain that infects the parasitoid wasp Encarsia formosa induces female-producing parthenogenesis. A new study shows that a Wolbachia-encoded gene has replaced the use of the ancestral wasp homologue that normally controls sexual reproduction, resulting in parthenogenesis.

Reduction of Wolbachia in Diaphorina citri (Hemiptera: Liviidae) increases phytopathogen acquisition and decreases fitness.

Wolbachia pipientis is a maternally inherited intracellular bacterium that infects a wide range of arthropods. Wolbachia can have a significant impact on host biology and development, often due to its effects on reproduction. We investigated Wolbachia-mediated effects in the Asian citrus psyllid, Diaphorina citri Kuwayama, which transmits Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease. Diaphorina citri are naturally infected with Wolbachia; therefore, investigating Wolbachia-mediated effects on D. citri fitness and CLas transmission required artificial reduction of this endosymbiont with the application of doxycycline. Doxycycline treatment of psyllids reduced Wolbachia infection by approximately 60% in both male and female D. citri. Psyllids treated with doxycycline exhibited higher CLas acquisition in both adults and nymphs as compared with negative controls. In addition, doxycycline-treated psyllids exhibited decreased fitness as measured by reduced egg and nymph production as well as adult emergence as compared with control lines without the doxycycline treatment. Our results indicate that Wolbachia benefits D. citri by improving fitness and potentially competes with CLas by interfering with phytopathogen acquisition. Targeted manipulation of endosymbionts in this phytopathogen vector may yield disease management tools.

A survey of Wolbachia infection in brachyceran flies from Iran.

Wolbachia is a maternally inherited intracellular bacterium that is considered to be the most plentiful endosymbiont found in arthropods. It reproductively manipulates its host to increase the chances of being transmitted to the insect progeny; and it is currently used as a means of suppressing disease vector populations or controlling vector-borne diseases. Studies of the dissemination and prevalence of Wolbachia among its arthropod hosts are important for its possible use as a biological control agent. The molecular identification of Wolbachia relies on different primers sets due to Wolbachia strain variation. Here, we screened for the presence of Wolbachia in a broad range of Brachycera fly species (Diptera), collected from different regions of Iran, using nine genetic markers (wsp, ftsZ, fbpA, gatB, CoxA, gltA, GroEL dnaA, and 16s rRNA), for detecting, assessing the sensitivity of primers for detection, and phylogeny of this bacterium. The overall incidence of Wolbachia among 22 species from six families was 27.3%. The most commonly positive fly species were Pollenia sp. and Hydrotaea armipes. However, the bacterium was not found in the most medically important flies or in potential human disease vectors, including Musca domestica, Sarcophaga spp., Calliphora vicinia, Lucilia sericata, and Chrysomya albiceps. The primer sets of 16s rRNA with 53.0% and gatB with 52.0% were the most sensitive primers for detecting Wolbachia. Blast search, phylogenetic, and MLST analysis of the different locus sequences of Wolbachia show that all the six distantly related fly species likely belonging to supergroup A. Our study showed some primer sets generated false negatives in many of the samples, emphasizing the importance of using different loci in detecting Wolbachia. The study provides the groundwork for future studies of a Wolbachia-based program for control of flies.

Updates on traditional methods for combating malaria and emerging Wolbachia-based interventions.

The escalating challenge of malaria control necessitates innovative approaches that extend beyond traditional control strategies. This review explores the incorporation of traditional vector control techniques with emerging Wolbachia-based interventions. Wolbachia, a naturally occurring bacteria, offers a novel approach for combatting vector-borne diseases, including malaria, by reducing the mosquitoes' ability to transmit these diseases. The study explores the rationale for this integration, presenting various case studies and pilot projects that have exhibited significant success. Employing a multi-dimensional approach that includes community mobilization, environmental modifications, and new biological methods, the paper posits that integrated efforts could mark a turning point in the struggle against malaria. Our findings indicate that incorporating Wolbachia-based strategies into existing vector management programs not only is feasible but also heightens the efficacy of malaria control initiatives in different countries especially in Pakistan. The paper concludes that continued research and international collaboration are imperative for translating these promising methods from the laboratory to the field, thereby offering a more sustainable and effective malaria control strategy.

Wolbachia symbionts control sex in a parasitoid wasp using a horizontally acquired gene.

Host reproduction can be manipulated by bacterial symbionts in various ways. Parthenogenesis induction is the most effective type of reproduction manipulation by symbionts for their transmission. Insect sex is determined by regulation of doublesex (dsx) splicing through transformer2 (tra2) and transformer (tra) interaction. Although parthenogenesis induction by symbionts has been studied since the 1970s, its underlying molecular mechanism is unknown. Here we identify a Wolbachia parthenogenesis-induction feminization factor gene (piff) that targets sex-determining genes and causes female-producing parthenogenesis in the haplodiploid parasitoid Encarsia formosa. We found that Wolbachia elimination repressed expression of female-specific dsx and enhanced expression of male-specific dsx, which led to the production of wasp haploid male offspring. Furthermore, we found that E. formosa tra is truncated and non-functional, and Wolbachia has a functional tra homolog, termed piff, with an insect origin. Wolbachia PIFF can colocalize and interact with wasp TRA2. Moreover, Wolbachia piff has coordinated expression with tra2 and dsx of E. formosa. Our results demonstrate the bacterial symbiont Wolbachia has acquired an insect gene to manipulate the host sex determination cascade and induce parthenogenesis in wasps. This study reveals insect-to-bacteria horizontal gene transfer drives the evolution of animal sex determination systems, elucidating a striking mechanism of insect-microbe symbiosis.

Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito species-specific virome.

In arthropod-associated microbial communities, insect-specific viruses (ISVs) are prevalent yet understudied due to limited infectivity outside their natural hosts. However, ISVs might play a crucial role in regulating mosquito populations and influencing arthropod-borne virus transmission. Some studies have indicated a core virome in mosquitoes consisting of mostly ISVs. Employing single mosquito metagenomics, we comprehensively profiled the virome of native and invasive mosquito species in Belgium. This approach allowed for accurate host species determination, prevalence assessment of viruses and Wolbachia, and the identification of novel viruses. Contrary to our expectations, no abundant core virome was observed in Culex mosquitoes from Belgium. In that regard, we caution against rigidly defining mosquito core viromes and encourage nuanced interpretations of other studies. Nonetheless, our study identified 45 viruses of which 28 were novel, enriching our understanding of the mosquito virome and ISVs. We showed that the mosquito virome in this study is species-specific and less dependent on the location where mosquitoes from the same species reside. In addition, because Wolbachia has previously been observed to influence arbovirus transmission, we report the prevalence of Wolbachia in Belgian mosquitoes and the detection of several Wolbachia mobile genetic elements. The observed prevalence ranged from 83% to 92% in members from the Culex pipiens complex.IMPORTANCECulex pipiens mosquitoes are important vectors for arboviruses like West Nile virus and Usutu virus. Virome studies on individual Culex pipiens, and on individual mosquitoes in general, have been lacking. To mitigate this, we sequenced the virome of 190 individual Culex and 8 individual Aedes japonicus mosquitoes. We report the lack of a core virome in these mosquitoes from Belgium and caution the interpretation of other studies in this light. The discovery of new viruses in this study will aid our comprehension of insect-specific viruses and the mosquito virome in general in relation to mosquito physiology and mosquito population dynamics.

Reanalysis of cluster randomised trial data to account for exposure misclassification using a per-protocol and complier-restricted approach.

The intention-to-treat (ITT) analysis of the Applying Wolbachia to Eliminate Dengue (AWED) trial estimated a protective efficacy of 77.1% for participants resident in areas randomised to receive releases of wMel-infected Aedes aegypti mosquitoes, an emerging dengue preventive intervention. The limiting assumptions of ITT analyses in cluster randomised trials and the mobility of mosquitoes and humans across cluster boundaries indicate the primary analysis is likely to underestimate the full public health benefit. Using spatiotemporally-resolved data on the distribution of Wolbachia mosquitoes and on the mobility of AWED participants (n = 6306), we perform complier-restricted and per-protocol re-examinations of the efficacy of the Wolbachia intervention. Increased intervention efficacy was estimated in all analyses by the refined exposure measures. The complier-restricted analysis returned an estimated efficacy of 80.7% (95% CI 65.9, 89.0) and the per-protocol analysis estimated 82.7% (71.7, 88.4) efficacy when comparing participants with an estimated wMel exposure of ≥ 80% compared to those with <20%. These reanalyses demonstrate how human and mosquito movement can lead to underestimation of intervention effects in trials of vector interventions and indicate that the protective efficacy of Wolbachia is even higher than reported in the primary trial results.

Pan-Genome Analysis of Wolbachia, Endosymbiont of Diaphorina citri, Reveals Independent Origin in Asia and North America.

Wolbachia, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus (CLas) that causes citrus greening disease, is naturally infected with Wolbachia (wDi). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 wDi genomes to gain a comprehensive understanding of wDi. Based on average nucleotide identity (ANI) analysis, we classified the wDi strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed wDi genomes. The variation analysis between Asia and North America wDi showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of wDi contributes to our understanding of the geographical population of wDi, the origin of hosts of D. citri, and the interaction between wDi and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB).

Mosquito Microbiomes of Rwanda: Characterizing Mosquito Host and Microbial Communities in the Land of a Thousand Hills.

Mosquitoes are a complex nuisance around the world and tropical countries bear the brunt of the burden of mosquito-borne diseases. Rwanda has had success in reducing malaria and some arboviral diseases over the last few years, but still faces challenges to elimination. By building our understanding of in situ mosquito communities in Rwanda at a disturbed, human-occupied site and at a natural, preserved site, we can build our understanding of natural mosquito microbiomes toward the goal of implementing novel microbial control methods. Here, we examined the composition of collected mosquitoes and their microbiomes at two diverse sites using Cytochrome c Oxidase I sequencing and 16S V4 high-throughput sequencing. The majority (36 of 40 species) of mosquitoes captured and characterized in this study are the first-known record of their species for Rwanda but have been characterized in other nations in East Africa. We found significant differences among mosquito genera and among species, but not between mosquito sexes or catch method. Bacteria of interest for arbovirus control, Asaia, Serratia, and Wolbachia, were found in abundance at both sites and varied greatly by species.

Rapid spread of a vertically transmitted symbiont induces drastic shifts in butterfly sex ratio.

The causes and consequences of sex-ratio dynamics constitutes a pivotal subject in evolutionary biology1. Under conditions of evolutionary equilibrium, the male-to-female ratio tends to be approximately 1:1; however, this equilibrium is susceptible to distortion by selfish genetic elements exemplified by driving sex chromosomes and cytoplasmic elements2,3. Although previous studies have documented instances of these genetic elements distorting the sex ratio, studies specifically tracking the process with which these distorters spread within populations, leading to a transition from balanced parity to a skewed, female-biased state, are notably lacking. Herein, we present compelling evidence documenting the rapid spread of the cytoplasmic endosymbiont Wolbachia within a localized population of the pierid butterfly Eurema hecabe (Figure 1A). This spread resulted in a shift in the sex ratio from near parity to an exceedingly skewed state overwhelmingly biased toward females, reaching 93.1% within a remarkably brief period of 4 years.

Diversity of Wolbachia infections in Sri Lankan mosquitoes with a new record of Wolbachia Supergroup B infecting Aedes aegypti vector populations.

Wolbachia bacteria are common endosymbionts of insects and have recently been applied for controlling arboviral vectors, especially Aedes aegypti mosquito populations. However, several medically important mosquito species in Sri Lanka were present with limited information for the Wolbachia infection status. Therefore, the screening of Wolbachia in indigenous mosquitoes is required prior to a successful application of Wolbachia-based vector control strategy. In this study, screening of 78 mosquito species collected from various parts of the country revealed that 13 species were positive for Wolbachia infection, giving ~ 17% infection frequency of Wolbachia among the Sri Lankan mosquitoes. Twelve Wolbachia-positive mosquito species were selected for downstream Wolbachia strain genotyping using Multi Locus Sequencing Type (MLST), wsp gene, and 16S rRNA gene-based approaches. Results showed that these Wolbachia strains clustered together with the present Wolbachia phylogeny of world mosquito populations with some variations. Almost 90% of the mosquito populations were infected with supergroup B while the remaining were infected with supergroup A. A new record of Wolbachia supergroup B infection in Ae. aegypti, the main vectors of dengue, was highlighted. This finding was further confirmed by real-time qPCR, revealing Wolbachia density variations between Ae. aegypti and Ae. albopictus (p = 0.001), and between males and females (p < 0.05). The evidence of natural Wolbachia infections in Ae. aegypti populations in Sri Lanka is an extremely rare incident that has the potential to be used for arboviral vector control.