Exploring the uncharted: Novel potential brugian filariasis vectors unveiled in Sri Lanka

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Nimlarathna, Hiruni Harischandra, Nilmini Chandrasena, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7911163/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Mar, 2026 Read the published version in Parasites & Vectors → Version 1 posted 10 You are reading this latest preprint version Abstract Background Sri Lanka is experiencing a re-emergence of brugian filariasis post-Lymphatic Filariasis elimination. A comprehensive understanding of the mosquito species that can facilitate the development of the brugian parasite is essential for implementing targeted surveillance and control measures. This study evaluated the vector potentiality of field-caught mosquitoes for brugian parasites across endemic districts within the filarial transmission belt in Sri Lanka. Methods Mosquito surveillance was conducted at six sites across five districts with the highest reported brugian cases during 2021–2022. Mosquitoes were collected within a 500m buffer zone surrounding identified human cases using dog-baited, window and gravid traps to maximize species diversity in the sample. Mosquitoes were identified morphologically, and randomly selected mosquitoes were molecularly confirmed via a PCR targeting the CO1 region. Vector potentiality was evaluated through observation of nematode parasites upon dissection, molecular confirmation via PCR amplification and sequencing of the Brugia sp. specific Hha 1 region. Mosquitoes harboring brugian parasites were tested for the presence of human blood to investigate their involvement in human brugian filariasis transmission. Statistical analyses were performed using generalized linear mixed models to account for site-specific factors. Results Of 794 mosquitoes from 15 species examined, 10.05% (77 out of 766 mosquitoes dissected) carried potentially infective L3 larvae molecularly confirmed as Brugia spp. in their head and thorax. Nine species across four genera demonstrated competence for parasite development: Mansonia annulifera, Ma. indiana, Ma. uniformis , Culex. lopoceraomyia, Cx. tritaeniorhynchus , Cx. quinquefasciatus , Cx. vishnui , Armigeres subalbatus , and Coquillettidia crassipes. Notably, Ma. indiana which has previously not been identified as a potential vector for brugian filariasis in Sri Lanka showed the highest weighted infectivity at S 1 site. Site-based risk assessment identified the S 1 site as having the highest risk of brugian filariasis followed by S 6 . Conclusions Many mosquito genera demonstrate competence for Brugia spp. development, expanding beyond the previously known Mansonia vectors. The diversity of potentially infective species indicates complex transmission dynamics requiring integrated surveillance approaches. Experimental vector competence studies are needed to confirm natural transmission capability and inform evidence-based control strategies. Brugian filariasis Mosquito surveillance Vector competence Brugia malayi Post-elimination Sri Lanka Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Lymphatic Filariasis (LF) in humans is a mosquito-borne Neglected Tropical Disease caused primarily by Wuchereria bancrofti , Brugia malayi and B. timori. Globally, over 657 million people are threatened across 39 endemic countries, including Sri Lanka [ 1 ]. Commonly observed symptoms include hydrocele and lymphoedema, which can develop into elephantiasis, characterized by massive swelling of extremities. These symptoms are attributed to the inflammatory reaction caused by the adult parasites residing within the lymphatic vessels, leading to damage and dysfunction of lymphatic vessels, and predisposing individuals to lymphoedema. This makes LF one of the leading causes of permanent disfigurement and the second leading cause of long-term disability [ 2 , 3 ]. The severe morbidity of this disease poses a significant socioeconomic burden, hindering the development of affected communities in developing countries [ 4 – 8 ]. Consequently, the estimated financial burden exceeds $ 5.8 billion annually, covering treatments, healthcare costs, and potential income losses [ 9 ]. In the past, both bancroftian filariasis and brugian filariasis (nocturnal periodic strain) were prevalent in Sri Lanka [ 10 ]. Before the LF elimination programme, one-tenth of the inhabitants in the endemic regions of Sri Lanka were at risk of being affected [ 11 – 14 ]. In the late 1960s, this anthroponotic strain was eliminated through the removal of aquatic vegetation required for the breeding of the known vector for the prevalent B. malayi strain [ 10 ]. Following five successful implementations of Mass Drug Administrations (MDA) from 2002 to 2006, Sri Lanka received the certificate for eliminating LF as a public health problem in 2016 from the World Health Organization (WHO) [ 15 ]. However, post-elimination surveillance revealed a concern of re-emergence of brugian filariasis caused by what appears to be a variant of B. malayi [ 16 , 17 ]. This variant exhibits nocturnal sub-periodic behavior, differing from the previously eliminated anthroponotic periodic strain [ 16 , 17 ]. Molecular analysis based on the internal transcribed spacer region 2 (ITS2) suggests it may represent a novel genetic variant or hybrid strain of B. malayi and B. pahangi [ 17 ]. Alarmingly, surveillance reports indicate that brugian infections outnumbered bancroftian infections in 2023 (personal communication with Anti-Filariasis Campaign, Head Quarters), highlighting the urgency of understanding transmission dynamics to prevent the re-establishment of endemic transmission [ 18 ]. Previous studies in Sri Lanka identified Ma. uniformis and Ma. annulifera as competent vectors for the variant brugian strain [ 15 ], but a comprehensive assessment of the vector competence of the re-emerged variant is lacking, with only one study conducted in the Gampaha district, Sri Lanka [ 15 , 16 ]. Understanding vector competence for this variant is critical for several reasons; different parasite strains may have different vector competence patterns, post-elimination environmental changes may have altered vector populations, and effective vector surveillance and control strategies require knowledge of all competent vector species. This study aimed to assess the vector potentiality of mosquito species for brugian parasites across endemic districts in Sri Lanka, providing essential data for evidence-based surveillance and control strategies in the post-elimination era. Methods Study design and ethical consideration This cross-sectional study was conducted across six sites in five districts with the highest number of brugian cases. The study was approved by the Ethics Review Committee of the University of Sri Jayewardenepura (ASP/01/RE/SCI/2022/31), Sri Lanka and Institute of Biology (ERC-IOBSL) (Reg no: ERC-IOBSL 206 02 2020). Study site selection Five districts along Sri Lanka’s LF belt with the highest brugian cases detected by the AFC through Thick Blood Smears (TBSs) observations (2002–2021) were selected: Puttalam (53 cases), Kalutara (32 cases), Gampaha (23 cases), Galle (10 cases), and Colombo (6 cases). The most recently reported human brugian case in each district at the time of the study (April 2022) is referred to as the index case hereafter. Study sites were established within a 500 m radius of the index case in each district, encompassing the flight range of 350 m of Mansonia species [ 25 ]. Two sites were chosen from the Puttalam district due to high case incidence. The selected study sites were Puruduwella- Puttalam District (S 1 ), Maggona- Kalutara District (S 2 ), Wattala- Gampaha District (S 3 ), Induruwa- Galle District (S 4 ), Boralesgamuwa- Colombo District (S 5 ) and Mahawewa- Puttalam District (S 6 ) (Fig. 1 ). Mosquito collection Mosquitoes were collected at S 1 in October 2021, at S 2 in December 2021, at S 3 in February 2022, at S 4 and S 5 in June 2022, and at S 6 in September 2022. Mosquitoes were collected using dog-baited traps, gravid traps, and window traps to maximize species diversity of the mosquito samples to ensure a true representation of the potential vector mosquito population at each site at the time of the study. Mosquito collections were carried out over at least two consecutive nights at each location between 18:00 and 04:00 Sri Lankan Standard Time. Two of each trap type were installed at each study site to ensure consistency across study sites and to minimize sampling bias. A dog was tied overnight within a white, rectangular nylon mesh, tent-like trap (4 m × 3 m × 3 m), which was raised about 15 cm from the ground to allow mosquitoes to enter the trap. The following morning, the dog was released, and the mosquitoes were collected using a mouth aspirator. Window traps measuring 56 cm x 56 cm x 46 cm [ 17 ] were placed outside the windows to collect anthropophilic mosquitoes exiting after a blood meal. The rest of the window was obstructed to ensure mosquitoes exited only through the opening leading to the trap. The trap was left in place overnight, and mosquitoes were collected using mouth aspirators the following morning. Gravid traps with attractants for Culex spp. were used to ensure their representation in the sample. An infusion of water and organic material (300 g fresh cow manure, 150 g of Gliricidia leaves, 10 g of yeast, and 5 L of water) fermented for 1–4 weeks was placed in a plastic dishpan of 20 cm x 39 cm x 32 cm with a motorized suction trap and left overnight. The mosquitoes were collected using a mouth aspirator the following morning. The species of all the collected mosquitoes were identified using standard morphological keys [ 18 ]. A subset of randomly selected mosquitoes was molecularly identified using the Cytochrome c Oxidase 1 (CO1) region to validate the morphological identification. Detection of infected and infective mosquitoes Mosquito dissection The head and thorax regions of female mosquitoes were dissected to identify those harboring nematode parasites. Each mosquito was placed on a slide under a binocular dissecting microscope (Leica LED3000, Germany), and the legs and wings were removed. The head and thorax regions were separated from the abdomen, placed in a separate drop of saline and dissected to identify potentially infective mosquitoes. Larval stages of the parasites were detected based on the morphological features and their movements (Table 1 ). The heads and thoraces of parasite-positive mosquitoes were stored separately for the molecular analysis of the brugian parasites, while the abdomens were stored separately for blood meal analysis at -20 o C. Table 1 Features of the brugian parasites used for the differentiation of the larval stages (19). Features Larval stage L2 L3 Body size Shorter and stouter Longer and slender Body shape More curved or coiled Mostly strait Tail Bluntly ended, not elongated Pointed, elongated Head Less defined Well defined Body movements Sluggish Active DNA extraction from parasite-positive mosquitoes DNA was extracted from the heads and thoraces, and abdomens separately of the parasite-positive mosquitoes using the Blood and Tissue DNA extraction kit (Qiagen, Germany) following the manufacturer’s guidelines with some modifications: the samples were homogenized using a vortex for about 2 min at 2, 000 rpm (Scientific Industries/ SI-A546). Next, 180 µL of buffer ATL and 20 µL of proteinase K were added to the sample and vortexed for 2 min at 2,000 rpm. Subsequently, 200 µL of buffer AL was added, and the samples were vortexed for 1 min at 2,000 rpm before being incubated at 56 o C for 10 min. Then 200 µL of ethanol (90–100%) was added, and the sample was vortexed for 1 min at 2,000 rpm. The sample mixtures were pipetted into DNeasy Mini spin columns, placed in a 2 ml collection tube and centrifuged (Universal centrifuge, Gemmy, PLC-036H) at ≥ 6,000 g (8,000 rpm) for 1 min. After discarding the flow-through, 500 µL of buffer, AW 1, was added. The sample was centrifuged again at ≥ 6,000 g for 1 min, and the flow-through was discarded. Next, 500 µL of buffer AW 2 was added, and the samples were incubated at room temperature for 3 min before being centrifuged again at ≥ 20,000 g (14,000 rpm) for 1 min. Finally, the DNA was eluted twice into the same tube by adding 20 µL and 15 µL of nuclease-free water, respectively, in two subsequent steps and centrifuging at 6,000 g for 1 min. Molecular confirmation of brugian infections The Brugia spp. -specific Hha1 region was amplified from the DNA extracts of the head and thorax regions of dissection-positive mosquitoes as previously described (20). Briefly, primers 5’-GCGCATAAATTCATCAGC-3’ (Forward) and 5’-GCGCAAAACTTAATTACAAAAGC-3’ (Reverse) were used to amplify the 322 bp Hha1 repeat region of Brugia spp. . The PCR amplification was performed in a 25 µL reaction volume containing 5.0 µL of 5x PCR buffer, 0.5 µL (0.2 mM) dNTP, 10 µM of each primer, 1U Taq polymerase, and 2 µL of the extracted template DNA. The PCR cycle conditions included an initial denaturation step at 94 o C for 5 min, followed by 35 cycles each of denaturation at 94 o C for 1 min, annealing at 59 o C for 1 min, extension at 72 o C for 1 min, with a final extension at 72 o C for 10 min. The products were visualized by agarose gel electrophoresis on a 1.5% agarose gel, and brugian infections were identified based on the presence of a 322 bp band. Positive PCR products from the mosquito head and thorax regions, which had concentrations sufficient for sequencing, were processed at the DNA sequencing facility at Iowa State University, USA, and Macrogen Inc., South Korea. The Basic Local Alignment and Search Tool (BLAST) on the National Centre for Biotechnology Information (NCBI) website was used to confirm the genus of the brugian parasites. Investigating the anthropophilic nature of the of potential vectors of brugian filariasis To determine whether these mosquitoes are involved in the transmission of Brugia spp. to humans, the bloodmeals of the infected and potentially infective mosquitoes were analyzed for human blood. DNA was extracted from the abdomens of parasite-positive mosquitoes obtained from the dissections to analyze the blood meal (21). The 272 bp Cytochrome c region of the mitochondrial genome of humans was amplified using HMNF’- CTCGGCTTACTTCTCTTCC with the universal reverse primer UNVR’- AGTGGGYGRAATATTATGC. The PCR mixture was heated for 5 min at 95 o C, followed by 12 cycles of 94 o C for 30 s, 57 o C for 30 s and 72 o C for 50 s. Two additional sets of 12 cycles each followed, using decreasing annealing temperatures of 56 o C and 55 o C, respectively. A final elongation at 72 o C was performed for 5 min. Statistical Analysis All statistical analyses and visualizations were conducted using R 4.5.1 [ 22 ]. To assess interspecific differences in potential infective probability and presence of human blood in infective mosquitoes, we fitted two binomial generalized linear mixed models (GLMMs) using the “glmmTMB” package with a Gamma distribution and log link function [ 23 ]. In the first model, the response variable was the weighted potentially infective percentage calculated for each mosquito species as the product of its infection status and relative abundance, multiplied by 100. In the second model, the response variable was the probability of potentially infective mosquitoes with human blood, with mosquito species included as a fixed factor in both models. Model significances were evaluated using the ANOVA function in the “car” package [ 24 ]. Post-hoc pairwise comparisons of estimated marginal means (EMMs) were performed using the “emmeans” package with Tukey’s adjustment for multiple testing [ 25 ] and species-specific weighted potential infective percentages and predicted probabilities of infectivity and presence of human blood were extracted. The predicted values with 95% confidence intervals were visualized as bar plots, and the weighted potential infectivity and the weighted potential infectivity of mosquitoes with human blood percentages were illustrated in bubble plots generated with the “ggplot2” package [ 26 ]. Differences in infectivity and infectivity with human blood among mosquito species across sites were analysed using weighted indices. For each species, the percentage relative abundance at each site was calculated as the ratio of the number of individuals of that species to the total number of potential infective mosquitoes identified at the same site. The relative abundance was used as a weighting factor to compute the weighted infectivity and infectivity with human blood indices. Weighted infectivity was obtained by multiplying the proportion of infective individuals by the relative abundance and scaling by 100. The weighted infectivity with human blood was calculated in the same manner using the proportion of human–blood–fed individuals. Zero values were replaced with a constant (1 × 10⁻⁶) prior to modeling to ensure compatibility with the log-link distribution. A generalized linear mixed model was fitted to the “glmmTMB” package to test the effects of mosquito species, site, and their interaction on both weighted indices with a Gamma distribution with a log link function. Model significance was assessed using ANOVA . Estimated marginal means for each species–site combination was obtained using the emmeans package, and pairwise comparisons were adjusted with Tukey’s method. Non-estimable contrasts were excluded prior to post-hoc analysis Results Adult mosquito distributionand species diversity A total of 794 mosquitoes from 15 species were captured throughout the study ( Table 2 ). Of these, 62.7% (n = 498), 27.1% (n = 215), and 10.2% (n = 81) mosquitoes were collected from 12 traps each of dog-baited, gravid and window traps. The highest number of species (n = 14) was recorded from dog-baited traps, while seven and eight species were collected from gravid and window traps, respectively. All three types of traps captured Ma. annulifera, Ma. indiana, Ma. uniformis, Culex quinquefasciatus and Armigeres subalbatus . In contrast, Culex lopoceraomyia, Culex vishnui, Aedes aegypti, Aedes pipersalatus and Anopheles kawani were found only in dog-baited traps, while Culex eumelanomyia brevipalpis was found only in window traps. The highest number of Culex spp. mosquitoes were found in gravid traps. Table 2 Fifteen mosquito species were collected using different traps from October 2021 to September 2022. Type of trap Dog-baited trap Window trap Gravid trap Mosquito species Mansonia annulifera 12 1 2 Ma. indiana 50 6 2 Ma. uniformis 93 5 1 Culex gelidus 1 0 1 Cx. lopoceraomyia 4 0 0 Cx. tritaeniorhynchus 89 0 29 Cx. quinquefasciatus 25 35 172 Cx. vishnui 2 0 0 Cx. eumelanomyia brevipalpis 0 3 0 Aedes aegypti 2 0 0 Ae. albopictus 40 5 0 Ae. pipersalatus 1 0 0 Armigeres subalbatus 167 23 8 Anopheles kawani 5 0 0 Coquillettidia crassipes 7 3 0 Total 498 81 215 Detection of infected and potentially infective mosquito species The ability of brugian parasites to develop into the infective L3 larval stage within the collected mosquito species was assessed to identify potential vectors of brugian filariasis. Mosquitoes harboring L1, L2 or L3 life stages within the head and thorax regions were identified as infected, and of them, those harboring L3 parasites were identified as potentially infective due to the ability to support development to the infective stage within the vector. Of the 15 species of mosquitoes caught, Ma. annulifera, Ma, indiana, Ma. uniformis, Cx. lopoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx. vishnui, Ae. aegypti, Ar. subalbatus a nd Cq. crassipes species (n = 10) were infected and all but Ae. aegypti were potentially infective (Fig. 2 ) suggestive of their potential to serve as vectors of brugian filariasis in Sri Lanka. The weighted binomial GLMM showed significant differences in potential infectivity probability among mosquito species (χ² = 24377, df = 9, p < 0.001). Weighted infective capability varied significantly among mosquito species. Cx. tritaeniorhynchus (n = 22, 19%, mean ± SD = 0.00384 ± 0.00004), followed by Ma. indiana (n = 19, 34%, mean ± SD = 0.00332 ± 0.00005) were identified as the species with the highest predicted weighted potential infectivity percentages. In comparison to other potentially infective species, Ar. subulbatus harbored a higher number of L1 and L2 stage brugian parasites (n = 11, 4.6%) in addition to a considerable number of L3 stage parasites (n = 10, 4%). This could potentially reflect a transitional period where Ar. subulbatus is being manipulated by the brugian parasite to support its development to the infective stage. Ma. annulifera, Cx. lopoceraomyia , and Cx. vishnui exhibited comparatively lower predicted weighted potential infectivity percentages. Mosquito species where no brugian parasites were detected were eliminated from further analysis since there is no evidence to support their potential roles as vector of brugian filariasis. Investigating the anthropophilic nature of potentially infective mosquitoes of brugian filariasis To examine the probable involvement of potentially infective mosquito species in the transmission of human brugian filariasis, they were tested for traces of human DNA. Human blood was detected in all the potentially infective species except Cx. vishnui , suggesting their potential in transmission of human brugian filariasis (Fig. 3 ). Interestingly, the highest potential infectivity percentage with human blood was found in Ma. indiana (n infected = 20, n infective with human DNA= 19, mean ± SD = 0.00338 ± 0.00005) followed by Cx. tritaeniorhynchus (n infected = 13, n infective with human DNA = 11, mean ± SD = 0.00196 ± 0.00002). Being the two species with the highest percentage of potentially infective mosquitoes with human DNA, Ma. indiana and Cx. tritaeniorhynchus could potentially serve as vectors of human brugian filariasis in Sri Lanka in addition to the already established Ma. annulifera and Ma. uniformis vectors. Site-based risk analysis of brugian filariasis Mosquito species in which brugian parasites were detected (Fig. 2 ) were further analyzed to assess their spatial distribution. A site-wise analysis of the abundance of Brugia -positive mosquito species and total percent infectivity was carried out (Fig. 4 A). Further, to identify the dominant species and evaluate the risk of brugian filariasis at each site, the percentages of infected and infective mosquitoes (Fig. 4 B) were calculated. To investigate the risk of transmission of human brugian filariasis at each site, the anthropophilic nature of infected and potentially infective mosquito species was assessed across the six sites (Fig. 4 C). The total abundance of Brugia -positive mosquito species was highest at S 2 (n = 213) and S 3 (n = 150), closely followed by S 1 (n = 132) and S 6 (127) and lowest at S 4 (n = 55), with the highest percentage of potentially infective mosquitoes at S 1 (n = 36, 27.3%) and S 6 (n = 20, 15.7%). Of the nine species identified as potentially infective, S 1 was the most species-rich site (n = 8), followed by S 6 (n = 5). Interestingly, all the species at S 1 and S 6 were potentially infective, with Ma. indiana having the highest number of potentially infective mosquitoes at S 1 (n = 18, 33%) followed by Cx. tritaeniorhynchus (n = 29, 41%). The GLMM results showed significant effects of both mosquito species and site on the weighted potential infectivity index. The effects of species (χ² = 586.72, df = 8, p < 0.001), site (χ² = 689.32, df = 5, p < 0.001), and their interaction (χ² = 700.13, df = 15, p < 0.001) were all significant. With regard to potential infectivity, Ma. indiana and Cx. tritaeniorhynchus from S 1 and Cx. tritaeniorhynchus from S 6 showed the highest weighted values for potentially infective mosquitoes, where differences were significant ( p < 0.001). In contrast, A subalbatus, Ma uniformis , and Ma. annulifera generally showed lower infectivity across most sites. Human blood was detected in all potentially infective species at S 1 except Cx. vishnui with the highest proportion detected in Ma. indiana (n = 18, 95%) followed by Cx. tritaeniorhynchus (n = 8, 80%,). Significant differences were observed among mosquito species (χ² = 599.59, df = 8, p < 0.001), among sites (χ² = 2000.02, df = 5, p < 0.001), and for the species–site interaction (χ² = 987.76, df = 15, p < 0.001) when considering the weighted number of mosquitoes with human blood. This suggests that the proportion of potential vectors with human blood varied substantially among mosquito species and across sites, with significant spatial heterogeneity in species-specific responses. Pairwise comparisons showed significant variation in both the weighted potential infectivity of mosquitoes with human-blood and across sampling sites. Accordingly, Ma. indiana ( p < 0.001) followed by Cx. tritaeniorhynchus ( p < 0.001) from S 1 can be identified as potential vectors for human brugian filariasis. Human blood was detected in three of the four potentially infective species at S 6 (n = 4, 20%). However, the number of mosquitoes of individual species is insufficient to identify potential vectors at S 6 . Although the total abundance was highest at S 2 and S 3 , the percentage of infectivity was low (S 2 = 5.6%, S 3 = 1.3%) with no human blood being detected in infective mosquitoes at S 2 . These data collectively suggest that S 1 is at the highest risk of transmission of human brugian filariasis, followed by S 6 , with Ma. indiana and Cx. tritaeniorhynchus potentially serving as additional vectors at S 1 . Discussion Brugian filariasis has reemerged in Sri Lanka after four decades of quiescence, with an increase in disease incidence in 2023. The absence of a definitive cure for LF in its advanced stages underscores the critical importance of prevention strategies, particularly through effective vector control and MDA. This study aimed to identify potential vectors of brugian filariasis in Sri Lanka to support targeted vector control measures. Here we present evidence of infection and development of brugian parasites to the infective L3 larval stage in field-caught Ma. annulifera, Ma. indiana, Ma. uniformis, Cx. lopoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx. vishnui, Ar. subalbatus and Cq. crassipes in Sri Lanka. To date, only Ma. annulifera and Ma. uniformis have been reported as potential vectors for brugian filariasis in Sri Lanka [ 15 ]. The presence of infective brugian parasites in field-caught Ma, indiana, Cx. lopoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx. vishnui, Ar. subalbatus and Cq. crassipes species have not been reported in Sri Lanka and Cx. lopoceraomyia, Cx. quinquefasciatus and Cx. vishnui in the world. Interestingly, Ma. indiana was by far the most prevalent and potentially infective mosquito species at the study site of the most recent case and the highest disease incidence of human brugian filariasis at the time of the study, S 1 , closely followed by Cx. tritaeniorhynchus. This study reports a higher number of Ma. indiana , compared to previous studies in Sri Lanka. Three Ma. indiana mosquitoes were reported in a study conducted in the Gampaha district, which were not infected with brugian parasite [ 15 ], and none in a survey conducted over six consecutive months, in which nearly 7000 mosquitoes were analyzed [ 16 ]. However, Ma. indiana has been reported in abundance in filariasis-endemic areas in other countries and has been reported as a potential vector for filariasis in Thailand, Malaysia and Indonesia [ 27 – 29 ]. Studies have shown its capacity to transmit nocturnally sub-periodic B. malayi parasites [ 28 ], supporting its role as a potential vector for brugian filariasis. Cx. tritaeniorhynchus and Ae. albopictus have been reported as vectors of B. malayi in Indonesia [ 30 ], while Armigeres subalbatus has been identified as a vector for zoonotic B. pahangi in Thailand [ 31 ] and Malaysia [ 32 , 33 ] and Cq. crassipes as a vector for B. malayi in Malaysia [ 34 ]. Interestingly, presence of infective L3 Brugia spp. larvae in field-caught Cx. lopoceraomyia, Cx. quinquefasciatus and Cx. vishnui has not been reported elsewhere thus far. In 1995, Bangs et al. reported the susceptibility of Culex tarsalis and Culex erythrothorax to sub-periodic B. malayi through laboratory experiments, thereby providing evidence for the ability of the genus Culex to act as vectors for brugian filariasis [ 35 ]. Interestingly, some laboratory experiments demonstrated the complete refractoriness of Cx. sitiens to sub-periodic B. malayi [ 29 ] and the mid gut was shown to act as a barrier for brugian filarial parasites in Cx. pipiens pipiens [ 36 ]. However, a study done by Erickson et al. reported the presence of filarial DNA within the head region of Cx. pipiens , providing evidence of the potential of Cx. pipiens to act as a vector for brugian filariasis [ 37 ]. Ughasi et al . reported the possibility of mosquito species previously considered non-vectors acting as vectors of filariasis parasites over generations [ 38 ]. This may reflect a parasite with genetic modifications and higher pathogenicity, which requires further investigation. Detection of human blood in potentially infective Ma. annulifera, Ma. indiana, Ma. uniformis, Cx. lopoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Ar. subalbatus and Cq. crassipes suggests a potential role of these species in the transmission of human brugian filariasis. In the current study S 1 and S 6 sites were identified as the most risk areas with regard to human brugian filariasis. Incidentally, S 1 and S 6 are sites in the Puttalam district, the district with the highest disease incidence (53 cases) from 2001 to 2021, as identified through human blood TBSs observations by the AFC. With a high abundance, percent infectivity and presence of human blood within them, Ma. indiana and Cx. tritaeniorhynchus could potentially contribute to the transmission of human brugian filariasis in these areas. Their absence in S 4 and S 5 and low infectivity percentages in S 2 and S 3 could be one reason for the low disease incidence (S 2 = 32, S 3 = 23, S 4 = 10, S 5 = 6) in these areas at the time of the study. The presence of infective mosquitoes and high abundance of potential vector species at S 2 suggests the potential of transmission of brugian filariasis. Absence of human blood within these mosquitoes could be suggestive of a zoonotic brugian filariasis in the area and although human brugian filariasis has not been yet detected within the S 2 study site, presence of the potential vector species indicates the risk of having new cases of human brugian filariasis within this site. In the current study the entire mosquito catch was analyzed for brugian infections by dissection. However, currently, in the national vector surveillance program of the Anti-Filaria Campaign only Mansonia spp. (previously known vector for brugian filariasis) mosquitoes are dissected for brugian filariasis infections and pooled for PCR to detect brugian infections. The identification of new potential vectors suggests the importance of extending the xenomonitoring efforts to other species for a comprehensive analysis of the situation. High abundance of potentially infective, Ma. indiana (S 1 ) and Cx. tritaeniorhynchus (S 1 , S 6 ) and a high percentage of these species positive for human blood indicates a risk of human brugian filariasis transmission within the respective areas. Analysis of the abundance and species richness of Brugia-positive mosquitoes, percentages of potentially infective mosquitoes and the proportion of them with traces of human blood assessed across the six sites revealed S 1 and S 6 as the areas with the greatest risk of human brugian filariasis transmission. However, the current survey is not ideal for comparing the geographical distribution of the vectors, as seasonal variations also play an important role in determining mosquito populations. The survey was not conducted in comparable climatic seasons; mosquitoes were collected during inter-monsoon season at S 1 and during the monsoon season at S 2 -S 6 . Therefore, conducting mosquito collections in comparable seasons would help reveal the true geographical and seasonal patterns of potential vector distribution across the sites. In the present study, mosquitoes carrying L3 larvae were identified as potentially infective due to the ability of those species to support the development of the parasites to the infective stage. However, Yamada et al demonstrated the inability of certain mosquitoes to transmit the disease regardless (39). Therefore, the mosquito species identified in this study as potential vectors of brugian filariasis should undergo further validation through experimental vector competence studies. Such follow-up research is essential to accurately determine the role of these mosquito species in the transmission of brugian filariasis and to guide targeted vector control strategies. Data generated by the current study could be used to refine prevention and control strategies by developing more tailored vector control measures in Sri Lanka and other brugian filariasis endemic countries. Such measures are crucial in preventing the potential resurgence of the disease. The identification of new potential vector species for human brugian filariasis, including several not previously reported in Sri Lanka or globally, reveals significant shifts in the local vector ecology and transmission dynamics. Incorporating multidisciplinary surveillance—combining entomological, ecological, and molecular data—will be essential to detect emerging vector species, assess their public health significance, and design context-specific control strategies. Such integrated efforts are critical for achieving sustainable elimination of human brugian filariasis. Conclusion This study demonstrates that brugian parasites are present in a wide range of mosquito species in post-elimination Sri Lanka, with seven species across four genera capable of supporting parasite development to the infective stage. The identification of Ma. indiana as a highly competent species in Sri Lanka, represents a novel finding with significant epidemiological implications. These findings provide essential baseline data for evidence-based vector control strategies in Sri Lanka’s post-elimination LF programme. Nonetheless, gaps in knowledge remain regarding the natural transmission capacity, seasonal variations, and vector-parasite interactions under field conditions. The diversity of competent species suggests complex transmission dynamics that require comprehensive, adaptable surveillance strategies to prevent the re-establishment of endemic transmission. Future research priorities include experimentally validating vector competence, conducting seasonal surveillance, and assessing natural transmission capacities. Abbreviations LF Lymphatic filariasis MDA mass drug administration AFC Anti-filariasis campaign TBS Thick blood smear ITS2 Internal transcribed spacer region 2 BLAST Basic Local Alignment and Search Tool NCBI National Centre for Biotechnology Information GLMM Generalized linear mixed models EMM Estimated marginal mean. Declarations Ethics approval and consent to participate Ethical approval was obtained from the Ethics Review Committee of Institute of Biology (ERC-IOBSL) (Reg no: ERC-IOBSL 206 02 2020) for animal subjects and Faculty of Medicine, University of Sri Jayewardenepura (03/20) for human subjects of the study. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Funding National Research Council (Grant no: 19–028) and ASP/ 01/RE/SCI/2022/31 grant of the University of Sri Jayewardenepura. Author Contribution Conceptualization: HH, BGDNKdS, NdS, and NC; methodology: SUN, HH, BGDNKdS, and NC; investigation and formal analysis: SUN, HH, TSN; writing-original draft: SUN, and HH; writing- review and editing: HH, BGDNKdS, NC, NdS, MK; funding acquisition: BGDNKdS, and HH; supervision: BGDNKdS, HH, NC, NdS, MK, and CHM. All authors read and approved the final manuscript. 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10:39:13","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132716,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7911163/v1/7fe7cf08b6e10fc0aa3f19e3.html"},{"id":95536158,"identity":"10f8b9f0-48bb-44c1-8268-3b5b9bd247b5","added_by":"auto","created_at":"2025-11-10 10:39:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1536020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of brugian filariasis cases in Sri Lanka and study locations.\u003c/strong\u003e Districts in the filariasis belt includes Puttalam, Gampaha, Colombo, Kalutara, Galle and Matara. Locations of recent positive cases from each district are shown as a black dot, and 500 m buffer zones (colored red) around the recent case were taken as the study areas. Districts are colored based on the human cases reported in between 2006- April 2021.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7911163/v1/43ebdae1f0bbd25a2875e9ac.png"},{"id":95536156,"identity":"308a22ff-ec6e-4ba2-9142-77c58c06221d","added_by":"auto","created_at":"2025-11-10 10:39:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":825263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional mosquito species identified as potential vectors for brugian filariasis. (a)\u003c/strong\u003e Mosquito species that support the development of brugian parasites to L1, L2 and L3 larval stages from the entire collection. The percentage of potentially infective mosquitoes of each species is represented on the bars. \u003cstrong\u003e(b)\u003c/strong\u003e Predicted weighted potential infectivity as a percentage of the total potentially infective mosquito species. The error bars represent SE. The compact letters indicate statistically significant differences among species based on pairwise comparisons (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e Weighted potential infectivity capabilities are calculated considering the weighted potential infectivity and the abundance of each species of potential vector mosquito species identified in this study. Infective capability increases as both are predicted weighted potential infectivity percentage and the abundance of the species increases.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7911163/v1/7b0896a90702c22bde7c6598.png"},{"id":95653973,"identity":"f7111359-5988-46c6-8a71-0b1687331600","added_by":"auto","created_at":"2025-11-11 16:07:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1566599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePotential vectors exhibit an anthropophilic nature.\u003c/strong\u003e (\u003cstrong\u003ea) \u003c/strong\u003ePresence of human blood in infected mosquitoes. Of the infected mosquitoes of each species, potentially infective mosquitoes detected with human blood are represented as a percentage on the bars. \u003cstrong\u003e(b)\u003c/strong\u003e Predicted weighted potential infectivity as a percentage of the total potentially infective mosquito species with human blood. The error bars represent SE. The compact letters indicate statistically significant differences among species based on pairwise comparisons (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(c)\u003c/strong\u003e Weighted potential\u003cem\u003e \u003c/em\u003einfective capability calculated considering the weighted potential infectivity and abundance of each species of potential vector mosquito species for human brugian filariasis identified by this study. Infective capability increases as both the weighted infected percentage and the abundance of the species increases.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7911163/v1/3d29961f9160b9cd5a980ff0.png"},{"id":95654222,"identity":"6779b4da-286f-44d2-86cb-6737b1dae97c","added_by":"auto","created_at":"2025-11-11 16:10:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1556950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRisk analysis between the study sites. (a) \u003c/strong\u003eHeat map of the distribution of the species of mosquitoes in which brugian parasites were detected.\u003cstrong\u003e (b) \u003c/strong\u003eSite-wise species distribution of potentially infective mosquitoes. Potentially infective mosquitoes of each species are represented as a percentage of the dissected mosquitoes on the bars. \u003cstrong\u003e(c) \u003c/strong\u003eDistribution of infected mosquito species with human blood. Potentially infective mosquitoes of each species are represented as a percentage of infected mosquitoes on the bars.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7911163/v1/19e419892a4df536ba0532b4.png"},{"id":104251557,"identity":"fd4c8ae4-fb0c-4fc5-a01e-0f5cadab21cb","added_by":"auto","created_at":"2026-03-09 16:13:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4100464,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7911163/v1/be9f7d93-b978-41f0-8bee-7f731e76cb21.pdf"},{"id":95536157,"identity":"b3b6b785-4873-40c7-8add-751ec7f351ea","added_by":"auto","created_at":"2025-11-10 10:39:12","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1652918,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7911163/v1/c53ed2211ac860f7301d1a82.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the uncharted: Novel potential brugian filariasis vectors unveiled in Sri Lanka","fulltext":[{"header":"Background","content":"\u003cp\u003eLymphatic Filariasis (LF) in humans is a mosquito-borne Neglected Tropical Disease caused primarily by \u003cem\u003eWuchereria bancrofti\u003c/em\u003e, \u003cem\u003eBrugia malayi\u003c/em\u003e and \u003cem\u003eB. timori.\u003c/em\u003e Globally, over 657\u0026nbsp;million people are threatened across 39 endemic countries, including Sri Lanka [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Commonly observed symptoms include hydrocele and lymphoedema, which can develop into elephantiasis, characterized by massive swelling of extremities. These symptoms are attributed to the inflammatory reaction caused by the adult parasites residing within the lymphatic vessels, leading to damage and dysfunction of lymphatic vessels, and predisposing individuals to lymphoedema. This makes LF one of the leading causes of permanent disfigurement and the second leading cause of long-term disability [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The severe morbidity of this disease poses a significant socioeconomic burden, hindering the development of affected communities in developing countries [\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Consequently, the estimated financial burden exceeds \u003cspan\u003e$\u003c/span\u003e5.8\u0026nbsp;billion annually, covering treatments, healthcare costs, and potential income losses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the past, both bancroftian filariasis and brugian filariasis (nocturnal periodic strain) were prevalent in Sri Lanka [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Before the LF elimination programme, one-tenth of the inhabitants in the endemic regions of Sri Lanka were at risk of being affected [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the late 1960s, this anthroponotic strain was eliminated through the removal of aquatic vegetation required for the breeding of the known vector for the prevalent \u003cem\u003eB. malayi\u003c/em\u003e strain [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Following five successful implementations of Mass Drug Administrations (MDA) from 2002 to 2006, Sri Lanka received the certificate for eliminating LF as a public health problem in 2016 from the World Health Organization (WHO) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, post-elimination surveillance revealed a concern of re-emergence of brugian filariasis caused by what appears to be a variant of \u003cem\u003eB. malayi\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis variant exhibits nocturnal sub-periodic behavior, differing from the previously eliminated anthroponotic periodic strain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Molecular analysis based on the internal transcribed spacer region 2 (ITS2) suggests it may represent a novel genetic variant or hybrid strain of \u003cem\u003eB. malayi\u003c/em\u003e and \u003cem\u003eB. pahangi\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Alarmingly, surveillance reports indicate that brugian infections outnumbered bancroftian infections in 2023 (personal communication with Anti-Filariasis Campaign, Head Quarters), highlighting the urgency of understanding transmission dynamics to prevent the re-establishment of endemic transmission [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious studies in Sri Lanka identified \u003cem\u003eMa. uniformis\u003c/em\u003e and \u003cem\u003eMa. annulifera\u003c/em\u003e as competent vectors for the variant brugian strain [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], but a comprehensive assessment of the vector competence of the re-emerged variant is lacking, with only one study conducted in the Gampaha district, Sri Lanka [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Understanding vector competence for this variant is critical for several reasons; different parasite strains may have different vector competence patterns, post-elimination environmental changes may have altered vector populations, and effective vector surveillance and control strategies require knowledge of all competent vector species.\u003c/p\u003e\u003cp\u003eThis study aimed to assess the vector potentiality of mosquito species for brugian parasites across endemic districts in Sri Lanka, providing essential data for evidence-based surveillance and control strategies in the post-elimination era.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy design and ethical consideration\u003c/h2\u003e\u003cp\u003eThis cross-sectional study was conducted across six sites in five districts with the highest number of brugian cases. The study was approved by the Ethics Review Committee of the University of Sri Jayewardenepura (ASP/01/RE/SCI/2022/31), Sri Lanka and Institute of Biology (ERC-IOBSL) (Reg no: ERC-IOBSL 206 02 2020).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStudy site selection\u003c/h3\u003e\n\u003cp\u003eFive districts along Sri Lanka\u0026rsquo;s LF belt with the highest brugian cases detected by the AFC through Thick Blood Smears (TBSs) observations (2002\u0026ndash;2021) were selected: Puttalam (53 cases), Kalutara (32 cases), Gampaha (23 cases), Galle (10 cases), and Colombo (6 cases). The most recently reported human brugian case in each district at the time of the study (April 2022) is referred to as the index case hereafter. Study sites were established within a 500 m radius of the index case in each district, encompassing the flight range of 350 m of \u003cem\u003eMansonia\u003c/em\u003e species [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Two sites were chosen from the Puttalam district due to high case incidence. The selected study sites were Puruduwella- Puttalam District (S\u003csub\u003e1\u003c/sub\u003e), Maggona- Kalutara District (S\u003csub\u003e2\u003c/sub\u003e), Wattala- Gampaha District (S\u003csub\u003e3\u003c/sub\u003e), Induruwa- Galle District (S\u003csub\u003e4\u003c/sub\u003e), Boralesgamuwa- Colombo District (S\u003csub\u003e5\u003c/sub\u003e) and Mahawewa- Puttalam District (S\u003csub\u003e6\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMosquito collection\u003c/h3\u003e\n\u003cp\u003eMosquitoes were collected at S\u003csub\u003e1\u003c/sub\u003e in October 2021, at S\u003csub\u003e2\u003c/sub\u003e in December 2021, at S\u003csub\u003e3\u003c/sub\u003e in February 2022, at S\u003csub\u003e4\u003c/sub\u003e and S\u003csub\u003e5\u003c/sub\u003e in June 2022, and at S\u003csub\u003e6\u003c/sub\u003e in September 2022. Mosquitoes were collected using dog-baited traps, gravid traps, and window traps to maximize species diversity of the mosquito samples to ensure a true representation of the potential vector mosquito population at each site at the time of the study. Mosquito collections were carried out over at least two consecutive nights at each location between 18:00 and 04:00 Sri Lankan Standard Time. Two of each trap type were installed at each study site to ensure consistency across study sites and to minimize sampling bias. A dog was tied overnight within a white, rectangular nylon mesh, tent-like trap (4 m \u0026times; 3 m \u0026times; 3 m), which was raised about 15 cm from the ground to allow mosquitoes to enter the trap. The following morning, the dog was released, and the mosquitoes were collected using a mouth aspirator. Window traps measuring 56 cm x 56 cm x 46 cm [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] were placed outside the windows to collect anthropophilic mosquitoes exiting after a blood meal. The rest of the window was obstructed to ensure mosquitoes exited only through the opening leading to the trap. The trap was left in place overnight, and mosquitoes were collected using mouth aspirators the following morning. Gravid traps with attractants for \u003cem\u003eCulex spp.\u003c/em\u003e were used to ensure their representation in the sample. An infusion of water and organic material (300 g fresh cow manure, 150 g of \u003cem\u003eGliricidia\u003c/em\u003e leaves, 10 g of yeast, and 5 L of water) fermented for 1\u0026ndash;4 weeks was placed in a plastic dishpan of 20 cm x 39 cm x 32 cm with a motorized suction trap and left overnight. The mosquitoes were collected using a mouth aspirator the following morning. The species of all the collected mosquitoes were identified using standard morphological keys [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A subset of randomly selected mosquitoes was molecularly identified using the Cytochrome c Oxidase 1 \u003cem\u003e(CO1)\u003c/em\u003e region to validate the morphological identification.\u003c/p\u003e\n\u003ch3\u003eDetection of infected and infective mosquitoes\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eMosquito dissection\u003c/h2\u003e\u003cp\u003eThe head and thorax regions of female mosquitoes were dissected to identify those harboring nematode parasites. Each mosquito was placed on a slide under a binocular dissecting microscope (Leica LED3000, Germany), and the legs and wings were removed. The head and thorax regions were separated from the abdomen, placed in a separate drop of saline and dissected to identify potentially infective mosquitoes. Larval stages of the parasites were detected based on the morphological features and their movements (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The heads and thoraces of parasite-positive mosquitoes were stored separately for the molecular analysis of the brugian parasites, while the abdomens were stored separately for blood meal analysis at -20 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFeatures of the brugian parasites used for the differentiation of the larval stages (19).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFeatures\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eLarval stage\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eL3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eShorter and stouter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLonger and slender\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody shape\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMore curved or coiled\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMostly strait\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBluntly ended, not elongated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePointed, elongated\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHead\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLess defined\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWell defined\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody movements\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSluggish\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eActive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDNA extraction from parasite-positive mosquitoes\u003c/h2\u003e\u003cp\u003eDNA was extracted from the heads and thoraces, and abdomens separately of the parasite-positive mosquitoes using the Blood and Tissue DNA extraction kit (Qiagen, Germany) following the manufacturer\u0026rsquo;s guidelines with some modifications: the samples were homogenized using a vortex for about 2 min at 2, 000 rpm (Scientific Industries/ SI-A546). Next, 180 \u0026micro;L of buffer ATL and 20 \u0026micro;L of proteinase K were added to the sample and vortexed for 2 min at 2,000 rpm. Subsequently, 200 \u0026micro;L of buffer AL was added, and the samples were vortexed for 1 min at 2,000 rpm before being incubated at 56 \u003csup\u003eo\u003c/sup\u003eC for 10 min. Then 200 \u0026micro;L of ethanol (90\u0026ndash;100%) was added, and the sample was vortexed for 1 min at 2,000 rpm. The sample mixtures were pipetted into DNeasy Mini spin columns, placed in a 2 ml collection tube and centrifuged (Universal centrifuge, Gemmy, PLC-036H) at \u0026ge;\u0026thinsp;6,000 g (8,000 rpm) for 1 min. After discarding the flow-through, 500 \u0026micro;L of buffer, AW\u003csub\u003e1,\u003c/sub\u003e was added. The sample was centrifuged again at \u0026ge;\u0026thinsp;6,000 g for 1 min, and the flow-through was discarded. Next, 500 \u0026micro;L of buffer AW\u003csub\u003e2\u003c/sub\u003e was added, and the samples were incubated at room temperature for 3 min before being centrifuged again at \u0026ge;\u0026thinsp;20,000 g (14,000 rpm) for 1 min. Finally, the DNA was eluted twice into the same tube by adding 20 \u0026micro;L and 15 \u0026micro;L of nuclease-free water, respectively, in two subsequent steps and centrifuging at 6,000 g for 1 min.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMolecular confirmation of brugian infections\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eBrugia spp.\u003c/em\u003e-specific \u003cem\u003eHha1\u003c/em\u003e region was amplified from the DNA extracts of the head and thorax regions of dissection-positive mosquitoes as previously described (20). Briefly, primers 5\u0026rsquo;-GCGCATAAATTCATCAGC-3\u0026rsquo; (Forward) and 5\u0026rsquo;-GCGCAAAACTTAATTACAAAAGC-3\u0026rsquo; (Reverse) were used to amplify the 322 bp \u003cem\u003eHha1\u003c/em\u003e repeat region of \u003cem\u003eBrugia spp.\u003c/em\u003e. The PCR amplification was performed in a 25 \u0026micro;L reaction volume containing 5.0 \u0026micro;L of 5x PCR buffer, 0.5 \u0026micro;L (0.2 mM) dNTP, 10 \u0026micro;M of each primer, 1U \u003cem\u003eTaq\u003c/em\u003e polymerase, and 2 \u0026micro;L of the extracted template DNA. The PCR cycle conditions included an initial denaturation step at 94 \u003csup\u003eo\u003c/sup\u003eC for 5 min, followed by 35 cycles each of denaturation at 94 \u003csup\u003eo\u003c/sup\u003eC for 1 min, annealing at 59 \u003csup\u003eo\u003c/sup\u003eC for 1 min, extension at 72 \u003csup\u003eo\u003c/sup\u003eC for 1 min, with a final extension at 72 \u003csup\u003eo\u003c/sup\u003eC for 10 min. The products were visualized by agarose gel electrophoresis on a 1.5% agarose gel, and brugian infections were identified based on the presence of a 322 bp band.\u003c/p\u003e\u003cp\u003ePositive PCR products from the mosquito head and thorax regions, which had concentrations sufficient for sequencing, were processed at the DNA sequencing facility at Iowa State University, USA, and Macrogen Inc., South Korea. The Basic Local Alignment and Search Tool (BLAST) on the National Centre for Biotechnology Information (NCBI) website was used to confirm the genus of the brugian parasites.\u003c/p\u003e\n\u003ch3\u003eInvestigating the anthropophilic nature of the of potential vectors of brugian filariasis\u003c/h3\u003e\n\u003cp\u003eTo determine whether these mosquitoes are involved in the transmission of \u003cem\u003eBrugia\u003c/em\u003e spp. to humans, the bloodmeals of the infected and potentially infective mosquitoes were analyzed for human blood. DNA was extracted from the abdomens of parasite-positive mosquitoes obtained from the dissections to analyze the blood meal (21). The 272 bp Cytochrome c region of the mitochondrial genome of humans was amplified using HMNF\u0026rsquo;- CTCGGCTTACTTCTCTTCC with the universal reverse primer UNVR\u0026rsquo;- AGTGGGYGRAATATTATGC. The PCR mixture was heated for 5 min at 95 \u003csup\u003eo\u003c/sup\u003eC, followed by 12 cycles of 94 \u003csup\u003eo\u003c/sup\u003eC for 30 s, 57 \u003csup\u003eo\u003c/sup\u003eC for 30 s and 72 \u003csup\u003eo\u003c/sup\u003eC for 50 s. Two additional sets of 12 cycles each followed, using decreasing annealing temperatures of 56 \u003csup\u003eo\u003c/sup\u003eC and 55 \u003csup\u003eo\u003c/sup\u003eC, respectively. A final elongation at 72 \u003csup\u003eo\u003c/sup\u003eC was performed for 5 min.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses and visualizations were conducted using R 4.5.1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To assess interspecific differences in potential infective probability and presence of human blood in infective mosquitoes, we fitted two binomial generalized linear mixed models (GLMMs) using the \u0026ldquo;glmmTMB\u0026rdquo; package with a Gamma distribution and log link function [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the first model, the response variable was the weighted potentially infective percentage calculated for each mosquito species as the product of its infection status and relative abundance, multiplied by 100. In the second model, the response variable was the probability of potentially infective mosquitoes with human blood, with mosquito species included as a fixed factor in both models. Model significances were evaluated using the \u003cem\u003eANOVA\u003c/em\u003e function in the \u0026ldquo;car\u0026rdquo; package [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Post-hoc pairwise comparisons of estimated marginal means (EMMs) were performed using the \u0026ldquo;emmeans\u0026rdquo; package with Tukey\u0026rsquo;s adjustment for multiple testing [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and species-specific weighted potential infective percentages and predicted probabilities of infectivity and presence of human blood were extracted. The predicted values with 95% confidence intervals were visualized as bar plots, and the weighted potential infectivity and the weighted potential infectivity of mosquitoes with human blood percentages were illustrated in bubble plots generated with the \u0026ldquo;ggplot2\u0026rdquo; package [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDifferences in infectivity and infectivity with human blood among mosquito species across sites were analysed using weighted indices. For each species, the percentage relative abundance at each site was calculated as the ratio of the number of individuals of that species to the total number of potential infective mosquitoes identified at the same site. The relative abundance was used as a weighting factor to compute the weighted infectivity and infectivity with human blood indices. Weighted infectivity was obtained by multiplying the proportion of infective individuals by the relative abundance and scaling by 100. The weighted infectivity with human blood was calculated in the same manner using the proportion of human\u0026ndash;blood\u0026ndash;fed individuals. Zero values were replaced with a constant (1 \u0026times; 10⁻⁶) prior to modeling to ensure compatibility with the log-link distribution.\u003c/p\u003e\u003cp\u003eA generalized linear mixed model was fitted to the \u0026ldquo;glmmTMB\u0026rdquo; package to test the effects of mosquito species, site, and their interaction on both weighted indices with a Gamma distribution with a log link function. Model significance was assessed using \u003cem\u003eANOVA\u003c/em\u003e. Estimated marginal means for each species\u0026ndash;site combination was obtained using the emmeans package, and pairwise comparisons were adjusted with Tukey\u0026rsquo;s method. Non-estimable contrasts were excluded prior to post-hoc analysis\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAdult mosquito distributionand species diversity\u003c/h2\u003e\u003cp\u003eA total of 794 mosquitoes from 15 species were captured throughout the study \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of these, 62.7% (n\u0026thinsp;=\u0026thinsp;498), 27.1% (n\u0026thinsp;=\u0026thinsp;215), and 10.2% (n\u0026thinsp;=\u0026thinsp;81) mosquitoes were collected from 12 traps each of dog-baited, gravid and window traps. The highest number of species (n\u0026thinsp;=\u0026thinsp;14) was recorded from dog-baited traps, while seven and eight species were collected from gravid and window traps, respectively. All three types of traps captured \u003cem\u003eMa. annulifera, Ma. indiana, Ma. uniformis, Culex quinquefasciatus and Armigeres subalbatus\u003c/em\u003e. In contrast, \u003cem\u003eCulex lopoceraomyia, Culex vishnui, Aedes aegypti, Aedes pipersalatus\u003c/em\u003e and \u003cem\u003eAnopheles kawani\u003c/em\u003e were found only in dog-baited traps, while \u003cem\u003eCulex eumelanomyia brevipalpis\u003c/em\u003e was found only in window traps. The highest number of \u003cem\u003eCulex\u003c/em\u003e spp. mosquitoes were found in gravid traps.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFifteen mosquito species were collected using different traps from October 2021 to September 2022.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType of trap\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDog-baited trap\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eWindow trap\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGravid trap\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMosquito species\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMansonia annulifera\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMa. indiana\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMa. uniformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCulex gelidus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCx. lopoceraomyia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCx. quinquefasciatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e172\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCx. vishnui\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCx. eumelanomyia brevipalpis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAe. albopictus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAe. pipersalatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eArmigeres subalbatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAnopheles kawani\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCoquillettidia crassipes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e498\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e215\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eDetection of infected and potentially infective mosquito species\u003c/h2\u003e\u003cp\u003eThe ability of brugian parasites to develop into the infective L3 larval stage within the collected mosquito species was assessed to identify potential vectors of brugian filariasis. Mosquitoes harboring L1, L2 or L3 life stages within the head and thorax regions were identified as infected, and of them, those harboring L3 parasites were identified as potentially infective due to the ability to support development to the infective stage within the vector.\u003c/p\u003e\u003cp\u003eOf the 15 species of mosquitoes caught, \u003cem\u003eMa. annulifera, Ma, indiana, Ma. uniformis, Cx. lopoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx. vishnui, Ae. aegypti, Ar. subalbatus a\u003c/em\u003end \u003cem\u003eCq. crassipes\u003c/em\u003e species (n\u0026thinsp;=\u0026thinsp;10) were infected and all but \u003cem\u003eAe. aegypti\u003c/em\u003e were potentially infective (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) suggestive of their potential to serve as vectors of brugian filariasis in Sri Lanka.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe weighted binomial GLMM showed significant differences in potential infectivity probability among mosquito species (χ\u0026sup2; = 24377, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Weighted infective capability varied significantly among mosquito species. \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;22, 19%, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;0.00384\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00004), followed by \u003cem\u003eMa. indiana\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;19, 34%, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;0.00332\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00005) were identified as the species with the highest predicted weighted potential infectivity percentages. In comparison to other potentially infective species, \u003cem\u003eAr. subulbatus\u003c/em\u003e harbored a higher number of L1 and L2 stage brugian parasites (n\u0026thinsp;=\u0026thinsp;11, 4.6%) in addition to a considerable number of L3 stage parasites (n\u0026thinsp;=\u0026thinsp;10, 4%). This could potentially reflect a transitional period where \u003cem\u003eAr. subulbatus\u003c/em\u003e is being manipulated by the brugian parasite to support its development to the infective stage.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMa. annulifera, Cx. lopoceraomyia\u003c/em\u003e, and \u003cem\u003eCx. vishnui\u003c/em\u003e exhibited comparatively lower predicted weighted potential infectivity percentages. Mosquito species where no brugian parasites were detected were eliminated from further analysis since there is no evidence to support their potential roles as vector of brugian filariasis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eInvestigating the anthropophilic nature of potentially infective mosquitoes of brugian filariasis\u003c/h2\u003e\u003cp\u003eTo examine the probable involvement of potentially infective mosquito species in the transmission of human brugian filariasis, they were tested for traces of human DNA. Human blood was detected in all the potentially infective species except \u003cem\u003eCx. vishnui\u003c/em\u003e, suggesting their potential in transmission of human brugian filariasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Interestingly, the highest potential infectivity percentage with human blood was found in \u003cem\u003eMa. indiana\u003c/em\u003e (n \u003csub\u003einfected\u003c/sub\u003e = 20, n \u003csub\u003einfective with human DNA=\u003c/sub\u003e 19, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;0.00338\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00005) followed by \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e (n \u003csub\u003einfected\u003c/sub\u003e = 13, n \u003csub\u003einfective with human DNA\u003c/sub\u003e = 11, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;0.00196\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00002). Being the two species with the highest percentage of potentially infective mosquitoes with human DNA, \u003cem\u003eMa. indiana\u003c/em\u003e and \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e could potentially serve as vectors of human brugian filariasis in Sri Lanka in addition to the already established \u003cem\u003eMa. annulifera\u003c/em\u003e and \u003cem\u003eMa. uniformis\u003c/em\u003e vectors.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSite-based risk analysis of brugian filariasis\u003c/h2\u003e\u003cp\u003eMosquito species in which brugian parasites were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were further analyzed to assess their spatial distribution. A site-wise analysis of the abundance of \u003cem\u003eBrugia\u003c/em\u003e-positive mosquito species and total percent infectivity was carried out (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Further, to identify the dominant species and evaluate the risk of brugian filariasis at each site, the percentages of infected and infective mosquitoes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) were calculated. To investigate the risk of transmission of human brugian filariasis at each site, the anthropophilic nature of infected and potentially infective mosquito species was assessed across the six sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe total abundance of \u003cem\u003eBrugia\u003c/em\u003e-positive mosquito species was highest at S\u003csub\u003e2\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;213) and S\u003csub\u003e3\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;150), closely followed by S\u003csub\u003e1\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;132) and S\u003csub\u003e6\u003c/sub\u003e (127) and lowest at S\u003csub\u003e4\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;55), with the highest percentage of potentially infective mosquitoes at S\u003csub\u003e1\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;36, 27.3%) and S\u003csub\u003e6\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;20, 15.7%). Of the nine species identified as potentially infective, S\u003csub\u003e1\u003c/sub\u003e was the most species-rich site (n\u0026thinsp;=\u0026thinsp;8), followed by S\u003csub\u003e6\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;5). Interestingly, all the species at S\u003csub\u003e1\u003c/sub\u003e and S\u003csub\u003e6\u003c/sub\u003e were potentially infective, with \u003cem\u003eMa. indiana\u003c/em\u003e having the highest number of potentially infective mosquitoes at S\u003csub\u003e1\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;18, 33%) followed by \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;29, 41%). The GLMM results showed significant effects of both mosquito species and site on the weighted potential infectivity index. The effects of species (χ\u0026sup2; = 586.72, df\u0026thinsp;=\u0026thinsp;8, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), site (χ\u0026sup2; = 689.32, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and their interaction (χ\u0026sup2; = 700.13, df\u0026thinsp;=\u0026thinsp;15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were all significant. With regard to potential infectivity, \u003cem\u003eMa. indiana\u003c/em\u003e and \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e from S\u003csub\u003e1\u003c/sub\u003e and \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e from S\u003csub\u003e6\u003c/sub\u003e showed the highest weighted values for potentially infective mosquitoes, where differences were significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, \u003cem\u003eA subalbatus, Ma uniformis\u003c/em\u003e, and \u003cem\u003eMa. annulifera\u003c/em\u003e generally showed lower infectivity across most sites.\u003c/p\u003e\u003cp\u003eHuman blood was detected in all potentially infective species at S\u003csub\u003e1\u003c/sub\u003e except \u003cem\u003eCx. vishnui\u003c/em\u003e with the highest proportion detected in \u003cem\u003eMa. indiana\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;18, 95%) followed by \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;8, 80%,). Significant differences were observed among mosquito species (χ\u0026sup2; = 599.59, df\u0026thinsp;=\u0026thinsp;8, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), among sites (χ\u0026sup2; = 2000.02, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and for the species\u0026ndash;site interaction (χ\u0026sup2; = 987.76, df\u0026thinsp;=\u0026thinsp;15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) when considering the weighted number of mosquitoes with human blood. This suggests that the proportion of potential vectors with human blood varied substantially among mosquito species and across sites, with significant spatial heterogeneity in species-specific responses. Pairwise comparisons showed significant variation in both the weighted potential infectivity of mosquitoes with human-blood and across sampling sites. Accordingly, \u003cem\u003eMa. indiana\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) followed by \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) from S\u003csub\u003e1\u003c/sub\u003e can be identified as potential vectors for human brugian filariasis. Human blood was detected in three of the four potentially infective species at S\u003csub\u003e6\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;4, 20%). However, the number of mosquitoes of individual species is insufficient to identify potential vectors at S\u003csub\u003e6\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eAlthough the total abundance was highest at S\u003csub\u003e2\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e, the percentage of infectivity was low (S\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.6%, S\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.3%) with no human blood being detected in infective mosquitoes at S\u003csub\u003e2\u003c/sub\u003e. These data collectively suggest that S\u003csub\u003e1\u003c/sub\u003e is at the highest risk of transmission of human brugian filariasis, followed by S\u003csub\u003e6\u003c/sub\u003e, with \u003cem\u003eMa. indiana\u003c/em\u003e and \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e potentially serving as additional vectors at S\u003csub\u003e1\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBrugian filariasis has reemerged in Sri Lanka after four decades of quiescence, with an increase in disease incidence in 2023. The absence of a definitive cure for LF in its advanced stages underscores the critical importance of prevention strategies, particularly through effective vector control and MDA. This study aimed to identify potential vectors of brugian filariasis in Sri Lanka to support targeted vector control measures.\u003c/p\u003e\u003cp\u003eHere we present evidence of infection and development of brugian parasites to the infective L3 larval stage in field-caught \u003cem\u003eMa. annulifera, Ma. indiana, Ma. uniformis, Cx. lopoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx. vishnui, Ar. subalbatus\u003c/em\u003e and \u003cem\u003eCq. crassipes\u003c/em\u003e in Sri Lanka. To date, only \u003cem\u003eMa. annulifera\u003c/em\u003e and \u003cem\u003eMa. uniformis\u003c/em\u003e have been reported as potential vectors for brugian filariasis in Sri Lanka [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The presence of infective brugian parasites in field-caught \u003cem\u003eMa, indiana, Cx. lopoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Cx. vishnui, Ar. subalbatus\u003c/em\u003e and \u003cem\u003eCq. crassipes\u003c/em\u003e species have not been reported in Sri Lanka and \u003cem\u003eCx. lopoceraomyia, Cx. quinquefasciatus\u003c/em\u003e and \u003cem\u003eCx. vishnui\u003c/em\u003e in the world. Interestingly, \u003cem\u003eMa. indiana\u003c/em\u003e was by far the most prevalent and potentially infective mosquito species at the study site of the most recent case and the highest disease incidence of human brugian filariasis at the time of the study, S\u003csub\u003e1\u003c/sub\u003e, closely followed by \u003cem\u003eCx. tritaeniorhynchus.\u003c/em\u003e This study reports a higher number of \u003cem\u003eMa. indiana\u003c/em\u003e, compared to previous studies in Sri Lanka. Three \u003cem\u003eMa. indiana\u003c/em\u003e mosquitoes were reported in a study conducted in the Gampaha district, which were not infected with brugian parasite [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and none in a survey conducted over six consecutive months, in which nearly 7000 mosquitoes were analyzed [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, \u003cem\u003eMa. indiana\u003c/em\u003e has been reported in abundance in filariasis-endemic areas in other countries and has been reported as a potential vector for filariasis in Thailand, Malaysia and Indonesia [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Studies have shown its capacity to transmit nocturnally sub-periodic \u003cem\u003eB. malayi\u003c/em\u003e parasites [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], supporting its role as a potential vector for brugian filariasis. \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e and \u003cem\u003eAe. albopictus\u003c/em\u003e have been reported as vectors of \u003cem\u003eB. malayi\u003c/em\u003e in Indonesia [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], while \u003cem\u003eArmigeres subalbatus\u003c/em\u003e has been identified as a vector for zoonotic \u003cem\u003eB. pahangi\u003c/em\u003e in Thailand [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Malaysia [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and \u003cem\u003eCq. crassipes\u003c/em\u003e as a vector for \u003cem\u003eB. malayi\u003c/em\u003e in Malaysia [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInterestingly, presence of infective L3 \u003cem\u003eBrugia spp.\u003c/em\u003e larvae in field-caught \u003cem\u003eCx. lopoceraomyia, Cx. quinquefasciatus\u003c/em\u003e and \u003cem\u003eCx. vishnui\u003c/em\u003e has not been reported elsewhere thus far. In 1995, Bangs \u003cem\u003eet al.\u003c/em\u003e reported the susceptibility of \u003cem\u003eCulex tarsalis\u003c/em\u003e and \u003cem\u003eCulex erythrothorax\u003c/em\u003e to sub-periodic \u003cem\u003eB. malayi\u003c/em\u003e through laboratory experiments, thereby providing evidence for the ability of the genus \u003cem\u003eCulex\u003c/em\u003e to act as vectors for brugian filariasis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Interestingly, some laboratory experiments demonstrated the complete refractoriness of \u003cem\u003eCx. sitiens\u003c/em\u003e to sub-periodic \u003cem\u003eB. malayi\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and the mid gut was shown to act as a barrier for brugian filarial parasites in \u003cem\u003eCx. pipiens pipiens\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, a study done by Erickson \u003cem\u003eet al.\u003c/em\u003e reported the presence of filarial DNA within the head region of \u003cem\u003eCx. pipiens\u003c/em\u003e, providing evidence of the potential of \u003cem\u003eCx. pipiens\u003c/em\u003e to act as a vector for brugian filariasis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Ughasi \u003cem\u003eet al\u003c/em\u003e. reported the possibility of mosquito species previously considered non-vectors acting as vectors of filariasis parasites over generations [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This may reflect a parasite with genetic modifications and higher pathogenicity, which requires further investigation. Detection of human blood in potentially infective \u003cem\u003eMa. annulifera, Ma. indiana, Ma. uniformis, Cx. lopoceraomyia, Cx. tritaeniorhynchus, Cx. quinquefasciatus, Ar. subalbatus\u003c/em\u003e and \u003cem\u003eCq. crassipes\u003c/em\u003e suggests a potential role of these species in the transmission of human brugian filariasis.\u003c/p\u003e\u003cp\u003eIn the current study S\u003csub\u003e1\u003c/sub\u003e and S\u003csub\u003e6\u003c/sub\u003e sites were identified as the most risk areas with regard to human brugian filariasis. Incidentally, S\u003csub\u003e1\u003c/sub\u003e and S\u003csub\u003e6\u003c/sub\u003e are sites in the Puttalam district, the district with the highest disease incidence (53 cases) from 2001 to 2021, as identified through human blood TBSs observations by the AFC. With a high abundance, percent infectivity and presence of human blood within them, \u003cem\u003eMa. indiana\u003c/em\u003e and \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e could potentially contribute to the transmission of human brugian filariasis in these areas. Their absence in S\u003csub\u003e4\u003c/sub\u003e and S\u003csub\u003e5\u003c/sub\u003e and low infectivity percentages in S\u003csub\u003e2\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e could be one reason for the low disease incidence (S\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;32, S\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;23, S\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10, S\u003csub\u003e5\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6) in these areas at the time of the study.\u003c/p\u003e\u003cp\u003eThe presence of infective mosquitoes and high abundance of potential vector species at S\u003csub\u003e2\u003c/sub\u003e suggests the potential of transmission of brugian filariasis. Absence of human blood within these mosquitoes could be suggestive of a zoonotic brugian filariasis in the area and although human brugian filariasis has not been yet detected within the S\u003csub\u003e2\u003c/sub\u003e study site, presence of the potential vector species indicates the risk of having new cases of human brugian filariasis within this site.\u003c/p\u003e\u003cp\u003eIn the current study the entire mosquito catch was analyzed for brugian infections by dissection. However, currently, in the national vector surveillance program of the Anti-Filaria Campaign only \u003cem\u003eMansonia\u003c/em\u003e spp. (previously known vector for brugian filariasis) mosquitoes are dissected for brugian filariasis infections and pooled for PCR to detect brugian infections. The identification of new potential vectors suggests the importance of extending the xenomonitoring efforts to other species for a comprehensive analysis of the situation.\u003c/p\u003e\u003cp\u003eHigh abundance of potentially infective, \u003cem\u003eMa. indiana\u003c/em\u003e (S\u003csub\u003e1\u003c/sub\u003e) and \u003cem\u003eCx. tritaeniorhynchus\u003c/em\u003e (S\u003csub\u003e1\u003c/sub\u003e, S\u003csub\u003e6\u003c/sub\u003e) and a high percentage of these species positive for human blood indicates a risk of human brugian filariasis transmission within the respective areas. Analysis of the abundance and species richness of Brugia-positive mosquitoes, percentages of potentially infective mosquitoes and the proportion of them with traces of human blood assessed across the six sites revealed S\u003csub\u003e1\u003c/sub\u003e and S\u003csub\u003e6\u003c/sub\u003e as the areas with the greatest risk of human brugian filariasis transmission. However, the current survey is not ideal for comparing the geographical distribution of the vectors, as seasonal variations also play an important role in determining mosquito populations. The survey was not conducted in comparable climatic seasons; mosquitoes were collected during inter-monsoon season at S\u003csub\u003e1\u003c/sub\u003e and during the monsoon season at S\u003csub\u003e2\u003c/sub\u003e-S\u003csub\u003e6\u003c/sub\u003e. Therefore, conducting mosquito collections in comparable seasons would help reveal the true geographical and seasonal patterns of potential vector distribution across the sites.\u003c/p\u003e\u003cp\u003eIn the present study, mosquitoes carrying L3 larvae were identified as potentially infective due to the ability of those species to support the development of the parasites to the infective stage. However, Yamada \u003cem\u003eet al\u003c/em\u003e demonstrated the inability of certain mosquitoes to transmit the disease regardless (39). Therefore, the mosquito species identified in this study as potential vectors of brugian filariasis should undergo further validation through experimental vector competence studies. Such follow-up research is essential to accurately determine the role of these mosquito species in the transmission of brugian filariasis and to guide targeted vector control strategies. Data generated by the current study could be used to refine prevention and control strategies by developing more tailored vector control measures in Sri Lanka and other brugian filariasis endemic countries. Such measures are crucial in preventing the potential resurgence of the disease.\u003c/p\u003e\u003cp\u003eThe identification of new potential vector species for human brugian filariasis, including several not previously reported in Sri Lanka or globally, reveals significant shifts in the local vector ecology and transmission dynamics. Incorporating multidisciplinary surveillance\u0026mdash;combining entomological, ecological, and molecular data\u0026mdash;will be essential to detect emerging vector species, assess their public health significance, and design context-specific control strategies. Such integrated efforts are critical for achieving sustainable elimination of human brugian filariasis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that brugian parasites are present in a wide range of mosquito species in post-elimination Sri Lanka, with seven species across four genera capable of supporting parasite development to the infective stage. The identification of \u003cem\u003eMa. indiana\u003c/em\u003e as a highly competent species in Sri Lanka, represents a novel finding with significant epidemiological implications. These findings provide essential baseline data for evidence-based vector control strategies in Sri Lanka\u0026rsquo;s post-elimination LF programme. Nonetheless, gaps in knowledge remain regarding the natural transmission capacity, seasonal variations, and vector-parasite interactions under field conditions. The diversity of competent species suggests complex transmission dynamics that require comprehensive, adaptable surveillance strategies to prevent the re-establishment of endemic transmission. Future research priorities include experimentally validating vector competence, conducting seasonal surveillance, and assessing natural transmission capacities.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLymphatic filariasis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emass drug administration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAFC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAnti-filariasis campaign\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThick blood smear\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eITS2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInternal transcribed spacer region 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBLAST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBasic Local Alignment and Search Tool\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNCBI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNational Centre for Biotechnology Information\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGLMM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGeneralized linear mixed models\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEMM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEstimated marginal mean.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003e Ethical approval was obtained from the Ethics Review Committee of Institute of Biology (ERC-IOBSL) (Reg no: ERC-IOBSL 206 02 2020) for animal subjects and Faculty of Medicine, University of Sri Jayewardenepura (03/20) for human subjects of the study.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNational Research Council (Grant no: 19\u0026ndash;028) and ASP/ 01/RE/SCI/2022/31 grant of the University of Sri Jayewardenepura.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: HH, BGDNKdS, NdS, and NC; methodology: SUN, HH, BGDNKdS, and NC; investigation and formal analysis: SUN, HH, TSN; writing-original draft: SUN, and HH; writing- review and editing: HH, BGDNKdS, NC, NdS, MK; funding acquisition: BGDNKdS, and HH; supervision: BGDNKdS, HH, NC, NdS, MK, and CHM. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003e The authors wish to acknowledge the support provided by the staff of the Anti-filariasis Campaign especially Ms. Lakmini, the staff of the Regional Director of Health Services branches at Madampe, Colombo, Kalutara, and Galle, and the staff of the Department of Entomology at the Medical Research Institute, and Mr. Lahiru Herath for supporting the field study. Also, Dr. Dayananda at the University of Sri Jayewardenepura for the support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.parasitesandvectors.com/content/5/1/89\u003c/span\u003e\u003cspan address=\"http://www.parasitesandvectors.com/content/5/1/89\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamada S. An experimental study on twenty-four species of Japanese mosquitoes regarding their suitability as intermediate hosts for Filaria bancroqfti Cobbold. Sci Rep Gov Inst Inf Dis. 1927;6:559\u0026ndash;622.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Brugian filariasis, Mosquito surveillance, Vector competence, Brugia malayi, Post-elimination, Sri Lanka","lastPublishedDoi":"10.21203/rs.3.rs-7911163/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7911163/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eSri Lanka is experiencing a re-emergence of brugian filariasis post-Lymphatic Filariasis elimination. A comprehensive understanding of the mosquito species that can facilitate the development of the brugian parasite is essential for implementing targeted surveillance and control measures. This study evaluated the vector potentiality of field-caught mosquitoes for brugian parasites across endemic districts within the filarial transmission belt in Sri Lanka.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eMosquito surveillance was conducted at six sites across five districts with the highest reported brugian cases during 2021\u0026ndash;2022. Mosquitoes were collected within a 500m buffer zone surrounding identified human cases using dog-baited, window and gravid traps to maximize species diversity in the sample. Mosquitoes were identified morphologically, and randomly selected mosquitoes were molecularly confirmed via a PCR targeting the \u003cem\u003eCO1\u003c/em\u003e region. Vector potentiality was evaluated through observation of nematode parasites upon dissection, molecular confirmation via PCR amplification and sequencing of the \u003cem\u003eBrugia sp.\u003c/em\u003e specific \u003cem\u003eHha\u003c/em\u003e1 region. Mosquitoes harboring brugian parasites were tested for the presence of human blood to investigate their involvement in human brugian filariasis transmission. Statistical analyses were performed using generalized linear mixed models to account for site-specific factors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOf 794 mosquitoes from 15 species examined, 10.05% (77 out of 766 mosquitoes dissected) carried potentially infective L3 larvae molecularly confirmed as \u003cem\u003eBrugia spp.\u003c/em\u003ein their head and thorax. Nine species across four genera demonstrated competence for parasite development: \u003cem\u003eMansonia annulifera, Ma. indiana, Ma. uniformis\u003c/em\u003e, \u003cem\u003eCulex. lopoceraomyia, Cx. tritaeniorhynchus\u003c/em\u003e, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, \u003cem\u003eCx. vishnui\u003c/em\u003e, \u003cem\u003eArmigeres subalbatus\u003c/em\u003e, and \u003cem\u003eCoquillettidia crassipes.\u003c/em\u003e Notably, \u003cem\u003eMa. indiana\u003c/em\u003e which has previously not been identified as a potential vector for brugian filariasis in Sri Lanka showed the highest weighted infectivity at S\u003csub\u003e1\u003c/sub\u003e site. Site-based risk assessment identified the S\u003csub\u003e1\u003c/sub\u003e site as having the highest risk of brugian filariasis followed by S\u003csub\u003e6\u003c/sub\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eMany mosquito genera demonstrate competence for \u003cem\u003eBrugia spp.\u003c/em\u003e development, expanding beyond the previously known \u003cem\u003eMansonia\u003c/em\u003e vectors. The diversity of potentially infective species indicates complex transmission dynamics requiring integrated surveillance approaches. Experimental vector competence studies are needed to confirm natural transmission capability and inform evidence-based control strategies.\u003c/p\u003e","manuscriptTitle":"Exploring the uncharted: Novel potential brugian filariasis vectors unveiled in Sri Lanka","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 10:39:08","doi":"10.21203/rs.3.rs-7911163/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-04T14:47:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-28T20:24:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-26T21:36:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19218226970344177641502356545854859717","date":"2025-11-06T01:43:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21546521950488039319434437970114313820","date":"2025-11-03T06:35:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77223110201101135929038644009561754430","date":"2025-10-29T23:08:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-28T20:00:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-24T07:53:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-24T04:59:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-10-21T06:31:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2e34ea9f-9922-49af-bbd6-4d148a598760","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:09:01+00:00","versionOfRecord":{"articleIdentity":"rs-7911163","link":"https://doi.org/10.1186/s13071-026-07264-w","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2026-03-06 15:58:10","publishedOnDateReadable":"March 6th, 2026"},"versionCreatedAt":"2025-11-10 10:39:08","video":"","vorDoi":"10.1186/s13071-026-07264-w","vorDoiUrl":"https://doi.org/10.1186/s13071-026-07264-w","workflowStages":[]},"version":"v1","identity":"rs-7911163","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7911163","identity":"rs-7911163","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}