Impacts of land use type on sampling methods for human and simian malaria vectors

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Reliable, epidemiologically relevant, logistically feasible, and cost-effective collection methods are needed to define the transmission risk to humans and the efficacy of vector control for both simian and human malarias. Alternative and/or complementary representative mosquito sampling methods to the labour-intensive human landing collections (HLC) and animal-baited tent traps (ABT) currently used in Indonesia are needed. Methods Carbon dioxide gas from a compressed cylinder and from yeast/sugar fermentation were used as lures in Passive Box, CDC Light and BG Sentinel 2 mosquito traps and compared in Latin square trials to human landing catches (HLCs) and animal baited traps (ABTs) for sensitivity and specificity for anopheline vectors across three land use types (oil palm plantation, mixed-crop agricultural area and village) in North Sumatra. Results The ABT with cow as bait was the most sensitive sampling method for all anopheline species in a direct comparison to HLC and the BG-Sentinel 2 trap in North Sumatra. The Passive Box and CDC Light traps were not sensitive for anophelines regardless of the CO 2 source. The HLC showed high specificity for members of the An. dirus complex, and also captured An. kochi , An. barbirostris complex, and An. maculatus Group mosquitoes. Anopheles leucosphyrus Group Anopheles dirus complex Anopheles maculatus Group Anopheles barbirostris complex land-use malaria vector human landing catch animal-baited tent trap Figures Figure 1 Figure 2 Background Human malaria cases caused by Plasmodium falciparum and Plasmodium vivax are diminishing in many countries in Southeast Asia including Indonesia ( 1 ), while simian malaria cases in humans are increasing in Southeast Asia ( 2 – 13 ). Improved vector surveillance is needed to better understand transmission to humans of both human and simian malaria species ( 14 – 16 ), as well as to define areas of transmission risk and to evaluate the efficacy of vector control strategies against both human and simian malarias. Nine species within the Anopheles leucosphyrus Group, of the Anopheles leucosphyrus and Anopheles dirus species complexes, are implicated as vectors of simian and human malarias ( 13 , 17 – 19 ), but little is known about the behaviours of these vectors ( 20 ). Identifying the transmission risk to humans requires incriminating the vector species, as well as determining where and when contact between the vectors and humans occurs, a function of vector density, human biting rates (HBR) in different habitats, biting time, seasonality of the vectors, and vector species richness ( 21 ). Quantifying these parameters requires reliable, epidemiologically relevant, and cost-effective mosquito collection methods that are operable in the often-remote areas where transmission of simian and human malarias to humans occurs ( 22 ). Commonly used collection methods for anophelines in Indonesia are human landing catches (HLCs) and animal-baited traps (ABTs) ( 23 – 26 ). Although both methods are effective in sampling anophelines, these methods are labour-intensive and expensive, attributes that often limit their deployment. Mechanical mosquito traps could overcome these limitations as many require less labour to set up and monitor than HLCs and ABTs, which would allow increased deployment. For example, the Mosquito Magnet trap was statistically comparable to HLCs for sampling An. leucosphyrus Group members in Malaysia ( 27 ). Thus, there is a need to evaluate more mechanical traps to collect malaria vectors ( 27 – 29 ). This study evaluated the sensitivity and specificity together with the ease and cost of deployment of collection methods for anopheline malaria vectors that are logistically operable in representative areas of Sumatra where simian and human malarias are transmitted (e.g., forests and forest fringes) ( 30 ). Methods Study sites The study was conducted in Ujung Bandar village, subdistrict Salapian, Langkat Regency, North Sumatra Province, Indonesia, in July 2022 and October 2022. The climate is tropical, with over 3000 mm rain/year and mean daytime temperatures ranging between 29–34°C and 19–24°C at night in the study period. The hilly landscape (from 400–650 meters above sea level) is dominated by large-scale oil palm plantations interspersed with small-scale, mixed-crop agriculture areas, and small patches of disturbed forest. Ujung Bandar village consists of nine sub-villages or dusun , in two of which (Dusun 2 and Dusun 5) the study took place. Long-tailed macaques ( Macaca fascicularis ) are common, with pig-tailed macaques ( Macaca nemestrina ) also inhabiting the area. Domestic animals (mainly cows, chickens, and dogs) roam free in and around both dusun . Study design This Latin square study consisted of three experiment rounds conducted in Dusun 2 in July 2022, and repeated in Dusun 5 in October 2022. Mosquito collections were conducted at fixed sampling stations in two concurrent Latin squares per experiment round from 18.30 to 06.00. Each collection method was assigned to one sampling station for one night, and collection methods were rotated to a different station each night within a Latin square round until each collection method was tested in each sampling station. Sampling stations were at least 50 meters apart and fixed in location over the study. Outputs were numbers and species of mosquitoes collected. The first Latin square (3x3 design) round compared the human landing catch (HLC) to two Passive Box traps (PBTs) ( 31 ). Teams of two collectors concurrently conducted HLC by collecting all mosquitoes that landed on their lower legs with mouth aspirators. Passive Box traps were hung with the entrance 1.5m above the ground, one baited with CO 2 from a gas cylinder (flow rate: 250 mL/minute) with the second PBT baited with CO 2 generated from yeast/sugar fermentation. Carbon dioxide flow rates were measured daily during trap setup by a gas flow meter (Kytola Instruments, Muurame, Finland). The yeast/sugar fermentation mixture consisted of 500g sugar and 20g dry yeast in 2L water, in a 5L jerrycan, made daily at 4PM, 2 hours before the start of the experiment. Carbon dioxide gas was delivered to the PBTs via 4mm diameter tubing. The second Latin square round (4x4 design) compared the HLC (as described above) to three mechanical traps (the PBT, the BioGents Sentinel 2 trap [BioGents AG, Germany] (BG-Sentinel 2 trap) and the CDC Miniature Light trap [John W. Hock Company, Gainesville FL, USA]), each baited with CO 2 . Because of the limited samples from Latin square round 1, this mechanical trap comparison was conducted concurrently in duplicates, varying only in the CO 2 source in each replicate (one Latin square had traps baited with CO 2 from a gas cylinder at a flow rate of 250 mL/minute and the other simultaneously had traps baited with CO 2 from yeast/sugar fermentation). The CDC light trap and the PBT were suspended with trap entrances 1.5m above the ground; the CDC light trap was also baited with an incandescent light bulb. The BG-Sentinel 2 trap was placed on the ground under a large umbrella to protect against rain. After four collection nights, the best mechanical trap was selected based on maximum numbers and species of anophelines collected. The third Latin square round (3x3 design) compared the best mechanical trap (from Latin square round 2) baited with CO 2 from a tank source to the HLC, as described in Latin square round 1, and an animal-baited tent trap (ABT) using a cow as bait. The ABT placed a cow with food and water inside a rectangular mosquito net (L*W*H = 3m * 3m * 2m) lifted approximately 50cm above the ground to allow mosquitoes to enter under the net. The inside of the net was searched hourly for 10 minutes by trained collectors with mouth aspirators. The cow owner accompanied the mosquito collectors to minimise stress to the cow and to intervene in the event of stress. All mosquitoes collected during the three Latin square rounds were transferred to paper cups covered with mesh netting, sedated with chloroform, and identified by morphology to genus and to species/species complex for Anopheles and Aedes , when possible ( 32 ). Afterwards, all mosquitoes were stored dry, on cotton wool and silica gel beads, in 1.5mL Eppendorf tubes. Samples of morphologically identified female anophelines were confirmed to species by PCR amplification of the ITS2 gene. Mosquito DNA was extracted using the DNEasy Blood & Tissue kit (Qiagen, Germany), after which the ITS2 gene was amplified using the ITS2A and ITS2B primers ( 33 ). The cycling parameters were initial denaturation at 95°C for 120 sec, then 35 cycles of denaturation (60 sec, 95°C), annealing (30 sec, 51°C), and extension (60 sec, 72°C), followed by a final extension at 72°C for 10 min. The amplified product was run on a 1.5% agarose gel at 90 V/400 mA for 90 minutes and visualised under ultraviolet light. A random subset of the An. leucosphyrus Group samples were analysed by Sanger sequencing to identify species within the An. leucosphyrus Group (Macrogen Indonesia). Statistical analyses Nightly catch numbers of An. leucosphyrus Group and An. maculatus Group females were compared between the different collection methods in a General Linear Mixed Model (GLMM) with Poisson distribution and log-link function. In this model, ‘collection method’ and ‘ dusun ’ were independent variables, while ‘collection night’ and ‘sampling station’ were random variables to account for variability between nights and stations. Overdispersion was addressed by fitting a second, negative binomial GLMM with quasipoisson distribution with the same explanatory variables to the data. Hereafter, the model that best fitted the data was determined by an ANOVA. Because the three Latin square rounds were set up similarly, the statistical analysis described here was applied to all experimental rounds. The effect of different land-use types on the relative abundance of mosquitoes was tested by comparing the proportions of mosquitoes in a Chi-square test of independence. All data analyses were performed in R (R version 4.1.1; R Foundation for Statistical Computing, Vienna, Austria) with the most recent version of the ‘tidyverse’ and ‘lme4’ packages. Results Forty-two percent (n = 72) of morphologically identified An. leucosphyrus Group mosquitoes (n = 171) were identified by sequencing the ITS2 gene as being in the Anopheles dirus complex. As all samples were identified as being in the An. dirus complex, the mosquitos identified as being in the An. leucosphyrus Group will be subsequently referred to as being in the An. dirus complex. Mechanical trap + different CO 2 sources compared to HLC Across both replicates in Latin square round 1 comparing CO 2 sources to HLC, 33 Anopheles dirus complex, 3 Anopheles maculatus Group and 1 Anopheles barbirostris complex females were collected (Table 1 ). In addition, 13 Aedes albopictus , 1 Aedes finlaya , 33 Culex spp., and 7 Armigeres spp. were collected. Differences between collection methods were highly significant, with HLC yielding the highest mean nightly catch of An. dirus complex females (GLMM, p < 0.001). The PBT was less attractive for mosquitoes, regardless of the CO 2 source. Two An. dirus complex females were collected, one by each CO 2 source, and one Culex spp. was collected in a PBT baited with the yeast/sugar mixture while one Armigeres spp. was found in a PBT baited with CO 2 from a gas cylinder. The proportion of An. dirus complex mosquitoes in the HLC collections was 0.356, indicating a relatively high specificity (Fig. 1 a). Mechanical traps compared to HLC In the mechanical trap comparison experiment (Latin square round 2), 67 An. dirus complex, 3 An. maculatus Group and 1 An. barbirostris complex mosquitoes were collected (Table 1 ), as well as 50 Ae. albopictus , 5 Aedes spp., 80 Culex spp., 30 Armigeres spp., and 2 unidentified mosquitoes. The mean nightly catch of An. dirus complex by HLC was significantly higher than the numbers collected by any of the mechanical traps (GLMM, p < 0.001). The number of An. dirus complex or other Anopheles collected did not differ significantly between the three mechanical traps, and the CO 2 source did not significantly affect the number of Anopheles mosquitoes trapped (GLMM, n.s.). The relative abundance of An. dirus complex mosquitoes in HLC collections was 0.311 (Fig. 1 b). HLC, ABT and BG-Sentinel 2 comparison The BG-Sentinel 2 trap baited with CO 2 from a gas cylinder was compared to the HLC and ABT in the third and final experimental round. A total of 71 An. dirus complex, 224 An. maculatus Group, 17 Anopheles kochi , 8 An. barbirostris complex, and 4 Anopheles vagus were collected (Table 1 ). Furthermore, 27 Ae. albopictus , 23 other Aedes spp., 146 Culex spp., 329 Armigeres spp., and 4 Toxorhynchitis spp. were collected. Highly significant differences in the mean nightly catch numbers of An. dirus complex mosquitoes were found with the ABT collecting three and five-fold greater numbers than the BG-Sentinel 2 and HLC, respectively (GLMM, p < 0.001). Collection numbers of An. maculatus Group mosquitoes were also significantly higher in the ABT than in the other two collection methods (GLMM, p < 0.0001). Collection numbers of other anophelines were insufficient for statistical analysis. Additionally, the Anopheles species richness was highest in the ABT, which collected five Anopheles species and was more sensitive, capturing much higher numbers of anophelines than the other methods. In contrast, the BG-Sentinel 2 collected An. dirus complex (n = 18), An. maculatus Group (n = 9), and An. barbirostris complex (n = 5), while the HLC collected An. dirus complex (n = 7), An. maculatus Group (n = 13), and An. kochi (n = 3). The relative abundance of An. dirus complex mosquitoes was much lower than in the previous experiment rounds, with 0.038 in the HLC, 0.281 in the BG-Sentinel 2 trap, and 0.051 in the ABT (Fig. 1 c). Land-use type effects Across the different land-use types, large differences in mosquito catches were observed. There was a significant effect of land-use type on the proportions of collected mosquito females by species across the full experiment (Χ 2 = 840.92, df = 14, p < 0.001). Collections in the small scale, mixed-crop agricultural area captured greater numbers of An. dirus complex and An. barbirostris complex than expected, while the relative abundances of An. maculatus Group, An. kochi , and An. vagus mosquitoes were greater than expected in the oil palm plantation. In the village, the proportion of Armigeres spp. was much greater than expected, while the relative abundance of Culex spp. was less than expected. Analysis of the residuals showed that there was a significant positive correlation between An. dirus complex and the mixed crop agricultural area, as well as a strong positive correlation between An. maculatus Group and the oil palm plantation. In contrast, most mosquito species were negatively correlated with the village residences, except for Armigeres spp., which comprised > 95% of the non-anophelines, Ae. albopictus , or Culex collected (Fig. 2 ). The remaining 5% of the other mosquitoes consisted of Aedes spp. (n = 31) and Toxorhynchites spp. (n = 4). Discussion Mechanical collection methods successfully sampled anophelines including simian malaria vectors in this and other studies (37,47–49). However, the sensitivity of sampling methods for anopheline mosquitoes in this study was greatest when using humans or cattle as lures compared to mechanical mosquito collection methods, including the Passive Box trap, the CDC Miniature light trap, and the BG-Sentinel 2 trap, baited with CO 2 . While numbers of collected mosquitoes are often fewer in traps baited with CO 2 from yeast/sugar fermentation compared to CO 2 from dry ice, other studies reported representative sampling of mosquitoes using yeast/sugar fermentation ( 34 , 35 ). Additionally, studies in Africa ( 36 , 37 ) and Australia ( 38 , 39 ) reported high numbers of mosquitoes including anophelines collected in traps with CO 2 produced by yeast/sugar fermentation, but no direct comparisons to HLC were made. The CO 2 generated from yeast fermentation in the experiments reported here may not have produced amounts of CO 2 sufficient to attract large numbers of mosquitoes. However, CO 2 released from a tank at recommended rates (e.g., 250ml/min) was equally ineffective in attracting large numbers of mosquitoes. Another explanation for the low number of mosquitoes in the mechanical traps might be that the evaluated traps were either not efficient in capturing mosquitoes lured to them or were not attractive to mosquitoes as deployed. Adding an extra odour bait or heat source, or placement at a different elevation, might improve catch numbers ( 36 , 40 , 41 ). Mechanical traps like the CDC Light trap and the BG-Sentinel traps have collected mosquitoes for decades. In sub-Sahara Africa, CDC Light traps and other mechanical traps supplemented or replaced HLCs for malaria vector collections to provide rough estimates of human biting rates, despite their lower sensitivity ( 42 ). In Asia, however, mechanical traps have not been as promising. In Peninsular Malaysia, collection numbers of An. leucosphyrus Group mosquitoes in the Mosquito Magnet were lower, but not statistically different from HLCs, while CDC Light traps yielded much lower numbers of these and other mosquitoes ( 27 ). However, the setup and establishment costs of the Mosquito Magnet are logistically and financially challenging. Smaller mosquito surveys in Indonesia showed that An. leucosphyrus Group mosquitoes were collected by HLC but not CDC light traps in a direct comparison ( 43 , 44 ). In this study, a similar result was observed across the study sites, with CDC light traps and other mechanical traps collecting low numbers of mosquitoes compared to HLCs. The BG-Sentinel 2 trap had not, to our knowledge, been evaluated for sampling Anopheles in Indonesia before this study. However, a previous study in northern Queensland, Australia showed that a BG-Sentinel 2 trap baited with tank CO 2 collected Anopheles females ( 39 ). The third Latin Square round of this study provides some evidence that the BG-Sentinel 2 trap, baited with CO 2 from a gas cylinder, could be at least comparable in sensitivity to the HLC to collect Anopheles . Especially when HLCs are logistically challenging to establish (e.g., in deep forest locations) or when many locations need to be sampled simultaneously, the BG-Sentinel 2 trap could be a viable alternative. Why the BG-Sentinel 2 was more sensitive than the HLC in one of the two experimental rounds in which they were directly compared requires further study. Additionally, running a mechanical trap baited with a CO 2 tank will only be more cost-effective than a single team of HLC collectors when collecting for 16 nights or more in Sumatra, due to its relatively high establishment cost (Supplemental Material 1). The cow-baited tent trap was sensitive, collecting large numbers of anophelines throughout the night. However, the ABT attracted much greater numbers of other mosquito species (e.g., Armigeres spp. and Culex spp.) than the HLC, as well as many zoophilic anophelines. Cow-baited tent traps are likely the best method to collect a wide range of species including vectors of malaria and arboviruses but are less accurate in estimating the biting risk on humans ( 45 ). Animal-baited traps often yield much larger numbers than HLCs, which would require calibration against HLCs to estimate the biting rate on humans. Also, the number of non-target mosquitoes collected in ABTs is significantly greater compared to HLCs, which requires more time to enable mosquito identification in the field. Finally, the ABT is more challenging to deploy than other collection methods. During this study, challenges with the ABTs included finding suitably flat ground to set up the ABT, inadvertently causing stress to the cow despite the presence of the owner, cow escape attempts, and windy weather. Heterogeneity between the first two and the third Latin Square comparison was seen in the relative sensitivity of the BG sentinel 2 trap and the HLC in sampling An. leucosphyrus Group and An. maculatus Group mosquitoes. These results lack a clear reason, since Sanger sequencing of the ITS2 gene showed that all specimens in the random subsample were An. dirus complex, indicating no changes in species occurrence within the An. leucosphyrus Group between the second and third Latin square rounds. The randomised sample size (n = 72) was sufficient to conclude that the only species within the An. leucosphyrus Group occurring in the study area was in the An. dirus complex. Additionally, the sampling sites and individuals conducting the HLC were the same during all three rounds. These individuals were trained and supervised hourly. While the relative abundance of An. dirus complex mosquitoes captured by HLC decreased between rounds two and three, the relative abundance of other anophelines (both An. maculatus Group and An. kochi ) increased for HLC. The hourly mosquito collections patterns were consistent through the night and across all 3 Latin square rounds, suggesting consistent HLC collections. The finding of An. dirus complex in this region of North Sumatra was unexpected, since this species was not previously reported here ( 30 , 46 ). However, a sibling species of An. dirus ( Anopheles cracens ) was reported in Sabang Island, Aceh ( 47 ), and more recently, mosquitoes morphologically identified as An. dirus complex were collected in Central Kalimantan, indicating a wider geographic spread than previously known ( 48 ). Since An. dirus complex mosquitoes are competent vectors of malaria in many areas, further research is required to establish the geographical boundaries of this species in Indonesia, and to estimate the potential risk of malaria transmission to humans in Indonesia. Land-use type strongly affected mosquito collections. Despite the small spatial scale of this study, An. dirus complex mosquitoes were predominantly collected in the mixed-crop agricultural area. The presence of suitable larval habitats (Sebayang, personal communication) could partially explain the high abundance of An. dirus complex mosquitoes in the mixed-crop agricultural area. However, the distance between the village and the larval habitats was well within the flight range of Anopheles females, so it is likely that other, unknown factors are influencing Anopheles densities. Evidence for land-use type associations and An. dirus complex and An. maculatus Group distributions in North Sumatra, Indonesia was found, but longer studies across multiple land-use types are needed to confirm if the land-use type effects observed here are valid across time and space. Table 1 – the number of Anopheles females collected by each collection method in every experimental round. HLC: human landing catch with two collectors per sampling station; PBT: passive box trap; ABT: animal-baited tent trap. An. dirus complex An. barbirostris complex An. kochi An. vagus An. maculatus Group Round 1 HLC 31 1 0 0 3 PBT + tank 1 0 0 0 0 PBT + yeast & sugar 1 0 0 0 0 Total 33 1 0 0 3 Round 2 HLC 61 0 0 0 2 BG-Sentinel 2 + yeast & sugar 2 0 0 0 0 BG-Sentinel 2 + tank 0 1 0 0 1 CDC light trap + yeast & sugar 1 0 0 0 0 CDC light trap + yeast & sugar 0 0 0 0 0 PBT + yeast & sugar 2 0 0 0 0 PBT + tank 1 0 0 0 0 Total 67 1 0 0 3 Round 3 HLC 8 0 3 0 14 ABT 45 4 21 4 212 BG-Sentinel 2 + tank CO 2 18 5 0 0 9 Total 71 9 24 4 241 Conclusions Collection methods using humans or cows were the most sensitive for Anopheles species, including species in the An. leucosphyrus Group, in North Sumatra. Mechanical traps baited with CO 2 were not very efficient, regardless of the CO 2 source or the trap type. The BG-Sentinel 2 trap baited with tank CO 2 showed some potential for sampling anophelines in Sumatra as it showed greater sensitivity for sampling An. dirus complex and An. barbirostris complex than the HLC in one of two direct Latin Square comparisons. Therefore, the BG-Sentinel 2 trap might be useful in large-scale surveillance studies once the basis for the variation in relative sensitivity reported here is understood. Despite the greater absolute number of An. dirus complex captured in the ABT compared to the HLC in a direct comparison, the sensitivity and high specificity of the HLC for An. leucosphyrus complex mosquitoes makes the HLC the preferred sampling method to understand zoonotic malaria transmission to humans and to define high-risk areas of transmission in North Sumatra as it is relatively easy to establish at multiple concurrent sites and directly provides an epidemiologically relevant estimation of the biting risk. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of the Faculty of Medicine, Universitas Sumatera Utara (No. 723/KEP/USU/2021). Meetings were held with community leaders, study participants and village residents, where the aims, the possible risks and potential benefits of the study were explained in Bahasa Indonesia. Mosquito collectors were then recruited from village residents and enrolled in the study after the risks were explained and informed consent was given by collectors performing HLCs. Consent for publication Not applicable. Availability of data and materials The datasets supporting the conclusions of this article are included within the article. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the ZOOMAL project (‘Evaluating zoonotic malaria and agricultural land use in Indonesia’; #LS-2019-116), Australian Centre for International Agricultural Research, Australian Government. BvdS and BS were supported by a James Cook University Postgraduate Research Scholarship. Authors’ contributions BvdS, TRB and TLR conceived the study; TRB, TLR, TAG and IL advised on the fieldwork and laboratory analyses. BvdS and AK conducted the fieldwork, and BvdS and BS performed the molecular analyses. BvdS analysed the data and wrote the initial draft of the manuscript. All authors read and approved the final manuscript. References The World Bank. World Development Indicators. 2023. Setiadi W, Sudoyo H, Trimarsanto H, Sihite BA, Saragih RJ, Juliawaty R, et al. A zoonotic human infection with simian malaria, Plasmodium knowlesi , in Central Kalimantan, Indonesia. Malar J. 2016;15:218. Lubis IND, Wijaya H, Lubis M, Lubis CP, Divis PCS, Beshir KB, et al. 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Jeyaprakasam NK, Pramasivan S, Liew JWK, Van Low L, Wan-Sulaiman WY, Ngui R et al. Evaluation of Mosquito Magnet and other collection tools for Anopheles mosquito vectors of simian malaria. Parasit Vectors [Internet]. 2021;14(184). https://doi.org/10.1186/s13071-021-04689-3 . Hawkes F, Manin BO, Ng SH, Torr SJ, Drakeley C, Chua TH et al. Evaluation of electric nets as means to sample mosquito vectors host-seeking on humans and primates. Parasit Vectors. 2017;10(338). Rohani A, Azahary ARA, Zurainee MN, Najdah WMAW, Zamree I, Hanif MO, et al. Comparative Human Landing Catch and CDC Light Trap in Mosquito Sampling in Knowlesi Malaria Endemic Areas in Peninsula Malaysia. Adv Entomol. 2016;4:1–10. Sinka ME, Bangs MJ, Manguin S, Chareonviriyaphap T, Patil AP, Temperley WH et al. The dominant Anopheles vectors of human malaria in the Asia-Pacific region: Occurrence data, distribution maps and bionomic précis. Parasit Vectors. 2011;4(89). Ritchie SA, Cortis G, Paton C, Townsend M, Shroyer D, Zborowski P et al. A simple non-powered passive trap for the collection of mosquitoes for arbovirus surveillance. J Med Entomol [Internet]. 2013;50(1):185–94. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23427669 . O’Connor C, Soepanto A. Illustrated key to female Anophelines of Indonesia (revised by Atmosoedjono S and Bangs MJ, NAMRU-2). Jakarta: Directorate of Communicable Disease, Ministry of Health Indonesia; 1989. Sum JS, Lee WC, Amir A, Braima KA, Jeffery J, Abdul-Aziz NM et al. Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA. Parasit Vectors. 2014;7(309). Saitoh Y, Hattori J, Chinone S, Nihei N, Tsuda Y, Kurahashi H, et al. Yeast-generated CO2 as a convenient source of carbon dioxide for adult mosquito sampling. J Am Mosq Control Assoc. 2004;20(September):261–4. Oli K, Jeffery J, Vythilingam I. A comparative study of adult mosquito trapping using dry ice and yeast generated carbon dioxide. Trop Biomed. 2005;22(2):249–51. Mweresa CK, Omusula P, Otieno B, van Loon JJA, Takken W, Mukabana WR. Molasses as a source of carbon dioxide for attracting the malaria mosquitoes Anopheles gambiae , and Anopheles funestus . Malar J [Internet]. 2014;13(160). Available from: http://www.ncbi.nlm.nih.gov/pubmed/24767543%5Cnhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4020376%5Cnhttp://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-160%5Cnhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4 . Smallegange RC, Schmied WH, Van Roey KJ, Verhulst NO, Spitzen J, Mukabana WR, et al. Sugar-fermenting yeast as an organic source of carbon dioxide to attract the malaria mosquito Anopheles gambiae . Malar J. 2010;9:292. Steiger DBM, Ritchie SA, Laurance SGW. Land use influences mosquito communities and disease risk on remote tropical Islands: A case study using a novel sampling technique. Am J Trop Med Hyg. 2016;94(2):314–21. van de Straat B, Hiscox A, Takken W, Burkot TR. Evaluating synthetic odours and trap designs for monitoring Anopheles farauti in Queensland, Australia. Malar J [Internet]. 2019;18(299). https://doi.org/10.1186/s12936-019-2923-7 . Hawkes FM, Dabiré RK, Sawadogo SP, Torr SJ, Gibson G. Exploiting Anopheles responses to thermal, odour and visual stimuli to improve surveillance and control of malaria. Sci Rep. 2017;7(17283). Guindo A, Epopa PS, Doumbia S, Millogo AA, Diallo B, Yao FA et al. Improved BioGents® Sentinel trap with heat (BGSH) for outdoor collections of Anopheline species in Burkina Faso and Mali, West Africa. Parasit Vectors [Internet]. 2021;14(82). https://doi.org/10.1186/s13071-020-04527-y . Eckert J, Oladipupo S, Wang Y, Jiang S, Patil V, McKenzie BA, et al. Which trap is best? Alternatives to outdoor human landing catches for malaria vector surveillance: a meta-analysis. Malar J. 2022;21:378. Maekawa Y, Sunahara T, Dachlan YP, Yotoranoto S, Basuki S, Uemura H, et al. First record of Anopheles balabacensis from western Sumbawa Island, Indonesia. J Am Mosq Control Assoc. 2009;25(2):203–5. Barbara KA, Sukowati S, Rusmiarto S, Susapto D, Bangs MJ, Kinzer MH. Survey of Anopheles mosquitoes (Diptera: Culicidae) in West Sumba District, Indonesia. Southeast Asian J Trop Med Public Health. 2011;42(1):71–82. St Laurent B, Burton TA, Zubaidah S, Miller HC, Asih PB, Baharuddin A, et al. Host attraction and biting behaviour of Anopheles mosquitoes in South Halmahera, Indonesia. Malar J. 2017;16:310. Obsomer V, Defourny P, Coosemans M. The Anopheles dirus complex: Spatial distribution and environmental drivers. Malar J. 2007;6:26. Sallum MAM, Peyton EL, Wilkerson RC. Six new species of the Anopheles leucosphyrus group, reinterpretation of An. elegans and vector implications. Med Vet Entomol. 2005;19(2):158–99. Anggraeni Y, Setiyaningsih R, Mujiyanto M, Trapsilowati W, Pujiyanti A, Rahardianingtyas E et al. Molecular detection of Plasmodium spp. in Anopheles and its vector potential in low-endemic areas in Indonesia. Proceeding of the International Conference on Public Health [Internet]. 2022;7(1):107–18. Available from: https://tiikmpublishing.com/data/conferences/doi/icoph/10.1750124246735 .2022.7111.pdf Supplemental Material 1 Cost of one BG-Sentinel 2 trap compared to one HLC team of two collectors. One CO 2 tank can last reliably for 5 collection nights when set at a standard CO 2 release rate of 250 mL/min. The formula to calculate the cost of using a mechanical trap baited with CO 2 is 1). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3857130","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267415343,"identity":"f4df6225-4b8d-460d-9fd0-12ef814909f4","order_by":0,"name":"Bram van de Straat","email":"data:image/png;base64,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","orcid":"","institution":"James Cook University","correspondingAuthor":true,"prefix":"","firstName":"Bram","middleName":"van","lastName":"de Straat","suffix":""},{"id":267415344,"identity":"647fa2c7-aa73-419c-b7e5-2e5388c48ffc","order_by":1,"name":"Ahadi Kurniawan","email":"","orcid":"","institution":"Public Health Laboratory of Medan, the Ministry of Health of Indonesia","correspondingAuthor":false,"prefix":"","firstName":"Ahadi","middleName":"","lastName":"Kurniawan","suffix":""},{"id":267415345,"identity":"345ac5b9-6241-45ee-9ed1-85d65887caa4","order_by":2,"name":"Boni Sebayang","email":"","orcid":"","institution":"James Cook University","correspondingAuthor":false,"prefix":"","firstName":"Boni","middleName":"","lastName":"Sebayang","suffix":""},{"id":267415346,"identity":"bb551f25-8e4f-4836-82f9-595bb3d3318e","order_by":3,"name":"Triwibowo Ambar Garjito","email":"","orcid":"","institution":"National Research and Innovation Agency","correspondingAuthor":false,"prefix":"","firstName":"Triwibowo","middleName":"Ambar","lastName":"Garjito","suffix":""},{"id":267415347,"identity":"c659d977-e347-4ce7-aab8-93bd4c089212","order_by":4,"name":"Inke Nadia D. Lubis","email":"","orcid":"","institution":"Universitas Sumatera Utara","correspondingAuthor":false,"prefix":"","firstName":"Inke","middleName":"Nadia D.","lastName":"Lubis","suffix":""},{"id":267415348,"identity":"7dd54758-c798-4f47-a3b0-6196b6d81bd8","order_by":5,"name":"Tanya L. Russell","email":"","orcid":"","institution":"James Cook University","correspondingAuthor":false,"prefix":"","firstName":"Tanya","middleName":"L.","lastName":"Russell","suffix":""},{"id":267415349,"identity":"43c7865e-c65b-4101-a07a-6572b4c18291","order_by":6,"name":"Thomas R. Burkot","email":"","orcid":"","institution":"James Cook University","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"R.","lastName":"Burkot","suffix":""}],"badges":[],"createdAt":"2024-01-12 13:44:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3857130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3857130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49819829,"identity":"b003d100-9e95-481b-b6bb-73d43500f49d","added_by":"auto","created_at":"2024-01-18 14:29:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49619,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of mosquito species collected during the three experiment rounds, pooled across the two Dusun. a) Experiment round 1, which used Passive Box traps (PBT) to compare CO\u003csub\u003e2\u003c/sub\u003e from a gas tank to CO\u003csub\u003e2\u003c/sub\u003e from yeast/sugar fermentation and human landing catch (HLC); b) Experiment round 2, which compared the HLC to three mechanical traps; c) Experiment round 3, which compared the BG-Sentinel trap with CO\u003csub\u003e2\u003c/sub\u003e from a tank to the HLC and a cow-baited tent trap (ABT).\u003c/p\u003e","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3857130/v1/842ac9fa275a15a78efd8af9.jpeg"},{"id":49819828,"identity":"dad0d6ff-4ebd-45c3-a1da-3324c7d28a0b","added_by":"auto","created_at":"2024-01-18 14:29:05","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37114,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations of different mosquito species with the different land-use types in which the experiment was conducted. Blue dots indicate a positive association, red a negative association, as indicated by the standardised residuals; larger dots mean a stronger association.\u003c/p\u003e","description":"","filename":"groupimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3857130/v1/08b257ac73c2f3e43c8d7a14.jpeg"},{"id":72965589,"identity":"6a890c39-ae5a-4cc1-bc44-61f5448e3e85","added_by":"auto","created_at":"2025-01-04 14:46:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":633839,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3857130/v1/255c0c97-610a-471d-b44e-47cb3c003cd2.pdf"},{"id":49819830,"identity":"973d9aa3-ab0b-4274-8bb6-fa5580d608d8","added_by":"auto","created_at":"2024-01-18 14:29:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54221,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3857130/v1/a999ddd5b89260bec981ff70.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impacts of land use type on sampling methods for human and simian malaria vectors","fulltext":[{"header":"Background","content":"\u003cp\u003eHuman malaria cases caused by \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003ePlasmodium vivax\u003c/em\u003e are diminishing in many countries in Southeast Asia including Indonesia (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), while simian malaria cases in humans are increasing in Southeast Asia (\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Improved vector surveillance is needed to better understand transmission to humans of both human and simian malaria species (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), as well as to define areas of transmission risk and to evaluate the efficacy of vector control strategies against both human and simian malarias.\u003c/p\u003e \u003cp\u003eNine species within the \u003cem\u003eAnopheles leucosphyrus\u003c/em\u003e Group, of the \u003cem\u003eAnopheles leucosphyrus\u003c/em\u003e and \u003cem\u003eAnopheles dirus\u003c/em\u003e species complexes, are implicated as vectors of simian and human malarias (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), but little is known about the behaviours of these vectors (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Identifying the transmission risk to humans requires incriminating the vector species, as well as determining where and when contact between the vectors and humans occurs, a function of vector density, human biting rates (HBR) in different habitats, biting time, seasonality of the vectors, and vector species richness (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Quantifying these parameters requires reliable, epidemiologically relevant, and cost-effective mosquito collection methods that are operable in the often-remote areas where transmission of simian and human malarias to humans occurs (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCommonly used collection methods for anophelines in Indonesia are human landing catches (HLCs) and animal-baited traps (ABTs) (\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Although both methods are effective in sampling anophelines, these methods are labour-intensive and expensive, attributes that often limit their deployment. Mechanical mosquito traps could overcome these limitations as many require less labour to set up and monitor than HLCs and ABTs, which would allow increased deployment. For example, the Mosquito Magnet trap was statistically comparable to HLCs for sampling \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group members in Malaysia (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Thus, there is a need to evaluate more mechanical traps to collect malaria vectors (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study evaluated the sensitivity and specificity together with the ease and cost of deployment of collection methods for anopheline malaria vectors that are logistically operable in representative areas of Sumatra where simian and human malarias are transmitted (e.g., forests and forest fringes) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy sites\u003c/h2\u003e \u003cp\u003eThe study was conducted in Ujung Bandar village, subdistrict Salapian, Langkat Regency, North Sumatra Province, Indonesia, in July 2022 and October 2022. The climate is tropical, with over 3000 mm rain/year and mean daytime temperatures ranging between 29\u0026ndash;34\u0026deg;C and 19\u0026ndash;24\u0026deg;C at night in the study period. The hilly landscape (from 400\u0026ndash;650 meters above sea level) is dominated by large-scale oil palm plantations interspersed with small-scale, mixed-crop agriculture areas, and small patches of disturbed forest. Ujung Bandar village consists of nine sub-villages or \u003cem\u003edusun\u003c/em\u003e, in two of which (Dusun 2 and Dusun 5) the study took place. Long-tailed macaques (\u003cem\u003eMacaca fascicularis\u003c/em\u003e) are common, with pig-tailed macaques (\u003cem\u003eMacaca nemestrina\u003c/em\u003e) also inhabiting the area. Domestic animals (mainly cows, chickens, and dogs) roam free in and around both \u003cem\u003edusun\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis Latin square study consisted of three experiment rounds conducted in \u003cem\u003eDusun\u003c/em\u003e 2 in July 2022, and repeated in \u003cem\u003eDusun\u003c/em\u003e 5 in October 2022. Mosquito collections were conducted at fixed sampling stations in two concurrent Latin squares per experiment round from 18.30 to 06.00. Each collection method was assigned to one sampling station for one night, and collection methods were rotated to a different station each night within a Latin square round until each collection method was tested in each sampling station. Sampling stations were at least 50 meters apart and fixed in location over the study. Outputs were numbers and species of mosquitoes collected.\u003c/p\u003e \u003cp\u003eThe first Latin square (3x3 design) round compared the human landing catch (HLC) to two Passive Box traps (PBTs) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Teams of two collectors concurrently conducted HLC by collecting all mosquitoes that landed on their lower legs with mouth aspirators. Passive Box traps were hung with the entrance 1.5m above the ground, one baited with CO\u003csub\u003e2\u003c/sub\u003e from a gas cylinder (flow rate: 250 mL/minute) with the second PBT baited with CO\u003csub\u003e2\u003c/sub\u003e generated from yeast/sugar fermentation. Carbon dioxide flow rates were measured daily during trap setup by a gas flow meter (Kytola Instruments, Muurame, Finland). The yeast/sugar fermentation mixture consisted of 500g sugar and 20g dry yeast in 2L water, in a 5L jerrycan, made daily at 4PM, 2 hours before the start of the experiment. Carbon dioxide gas was delivered to the PBTs via 4mm diameter tubing.\u003c/p\u003e \u003cp\u003eThe second Latin square round (4x4 design) compared the HLC (as described above) to three mechanical traps (the PBT, the BioGents Sentinel 2 trap [BioGents AG, Germany] (BG-Sentinel 2 trap) and the CDC Miniature Light trap [John W. Hock Company, Gainesville FL, USA]), each baited with CO\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e. Because of the limited samples from Latin square round 1, this mechanical trap comparison was conducted concurrently in duplicates, varying only in the CO\u003csub\u003e2\u003c/sub\u003e source in each replicate (one Latin square had traps baited with CO\u003csub\u003e2\u003c/sub\u003e from a gas cylinder at a flow rate of 250 mL/minute and the other simultaneously had traps baited with CO\u003csub\u003e2\u003c/sub\u003e from yeast/sugar fermentation). The CDC light trap and the PBT were suspended with trap entrances 1.5m above the ground; the CDC light trap was also baited with an incandescent light bulb. The BG-Sentinel 2 trap was placed on the ground under a large umbrella to protect against rain. After four collection nights, the best mechanical trap was selected based on maximum numbers and species of anophelines collected.\u003c/p\u003e \u003cp\u003eThe third Latin square round (3x3 design) compared the best mechanical trap (from Latin square round 2) baited with CO\u003csub\u003e2\u003c/sub\u003e from a tank source to the HLC, as described in Latin square round 1, and an animal-baited tent trap (ABT) using a cow as bait. The ABT placed a cow with food and water inside a rectangular mosquito net (L*W*H\u0026thinsp;=\u0026thinsp;3m * 3m * 2m) lifted approximately 50cm above the ground to allow mosquitoes to enter under the net. The inside of the net was searched hourly for 10 minutes by trained collectors with mouth aspirators. The cow owner accompanied the mosquito collectors to minimise stress to the cow and to intervene in the event of stress.\u003c/p\u003e \u003cp\u003eAll mosquitoes collected during the three Latin square rounds were transferred to paper cups covered with mesh netting, sedated with chloroform, and identified by morphology to genus and to species/species complex for \u003cem\u003eAnopheles\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e, when possible (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Afterwards, all mosquitoes were stored dry, on cotton wool and silica gel beads, in 1.5mL Eppendorf tubes. Samples of morphologically identified female anophelines were confirmed to species by PCR amplification of the ITS2 gene. Mosquito DNA was extracted using the DNEasy Blood \u0026amp; Tissue kit (Qiagen, Germany), after which the ITS2 gene was amplified using the ITS2A and ITS2B primers (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The cycling parameters were initial denaturation at 95\u0026deg;C for 120 sec, then 35 cycles of denaturation (60 sec, 95\u0026deg;C), annealing (30 sec, 51\u0026deg;C), and extension (60 sec, 72\u0026deg;C), followed by a final extension at 72\u0026deg;C for 10 min. The amplified product was run on a 1.5% agarose gel at 90 V/400 mA for 90 minutes and visualised under ultraviolet light. A random subset of the \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group samples were analysed by Sanger sequencing to identify species within the \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group (Macrogen Indonesia).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eNightly catch numbers of \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group and \u003cem\u003eAn. maculatus\u003c/em\u003e Group females were compared between the different collection methods in a General Linear Mixed Model (GLMM) with Poisson distribution and log-link function. In this model, \u0026lsquo;collection method\u0026rsquo; and \u0026lsquo;\u003cem\u003edusun\u003c/em\u003e\u0026rsquo; were independent variables, while \u0026lsquo;collection night\u0026rsquo; and \u0026lsquo;sampling station\u0026rsquo; were random variables to account for variability between nights and stations. Overdispersion was addressed by fitting a second, negative binomial GLMM with quasipoisson distribution with the same explanatory variables to the data. Hereafter, the model that best fitted the data was determined by an ANOVA. Because the three Latin square rounds were set up similarly, the statistical analysis described here was applied to all experimental rounds. The effect of different land-use types on the relative abundance of mosquitoes was tested by comparing the proportions of mosquitoes in a Chi-square test of independence. All data analyses were performed in R (R version 4.1.1; R Foundation for Statistical Computing, Vienna, Austria) with the most recent version of the \u0026lsquo;tidyverse\u0026rsquo; and \u0026lsquo;lme4\u0026rsquo; packages.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eForty-two percent (n\u0026thinsp;=\u0026thinsp;72) of morphologically identified \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group mosquitoes (n\u0026thinsp;=\u0026thinsp;171) were identified by sequencing the ITS2 gene as being in the \u003cem\u003eAnopheles dirus\u003c/em\u003e complex. As all samples were identified as being in the \u003cem\u003eAn. dirus\u003c/em\u003e complex, the mosquitos identified as being in the \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group will be subsequently referred to as being in the \u003cem\u003eAn. dirus\u003c/em\u003e complex.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMechanical trap\u0026thinsp;+\u0026thinsp;different CO\u003csub\u003e2\u003c/sub\u003e sources compared to HLC\u003c/h2\u003e \u003cp\u003eAcross both replicates in Latin square round 1 comparing CO\u003csub\u003e2\u003c/sub\u003e sources to HLC, 33 \u003cem\u003eAnopheles dirus\u003c/em\u003e complex, 3 \u003cem\u003eAnopheles maculatus\u003c/em\u003e Group and 1 \u003cem\u003eAnopheles barbirostris\u003c/em\u003e complex females were collected (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, 13 \u003cem\u003eAedes albopictus\u003c/em\u003e, 1 \u003cem\u003eAedes finlaya\u003c/em\u003e, 33 \u003cem\u003eCulex\u003c/em\u003e spp., and 7 \u003cem\u003eArmigeres\u003c/em\u003e spp. were collected. Differences between collection methods were highly significant, with HLC yielding the highest mean nightly catch of \u003cem\u003eAn. dirus\u003c/em\u003e complex females (GLMM, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The PBT was less attractive for mosquitoes, regardless of the CO\u003csub\u003e2\u003c/sub\u003e source. Two \u003cem\u003eAn. dirus\u003c/em\u003e complex females were collected, one by each CO\u003csub\u003e2\u003c/sub\u003e source, and one \u003cem\u003eCulex\u003c/em\u003e spp. was collected in a PBT baited with the yeast/sugar mixture while one \u003cem\u003eArmigeres\u003c/em\u003e spp. was found in a PBT baited with CO\u003csub\u003e2\u003c/sub\u003e from a gas cylinder. The proportion of \u003cem\u003eAn. dirus\u003c/em\u003e complex mosquitoes in the HLC collections was 0.356, indicating a relatively high specificity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMechanical traps compared to HLC\u003c/h2\u003e \u003cp\u003eIn the mechanical trap comparison experiment (Latin square round 2), 67 \u003cem\u003eAn. dirus\u003c/em\u003e complex, 3 \u003cem\u003eAn. maculatus\u003c/em\u003e Group and 1 \u003cem\u003eAn. barbirostris\u003c/em\u003e complex mosquitoes were collected (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), as well as 50 \u003cem\u003eAe. albopictus\u003c/em\u003e, 5 \u003cem\u003eAedes\u003c/em\u003e spp., 80 \u003cem\u003eCulex\u003c/em\u003e spp., 30 \u003cem\u003eArmigeres\u003c/em\u003e spp., and 2 unidentified mosquitoes. The mean nightly catch of \u003cem\u003eAn. dirus\u003c/em\u003e complex by HLC was significantly higher than the numbers collected by any of the mechanical traps (GLMM, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The number of \u003cem\u003eAn. dirus\u003c/em\u003e complex or other \u003cem\u003eAnopheles\u003c/em\u003e collected did not differ significantly between the three mechanical traps, and the CO\u003csub\u003e2\u003c/sub\u003e source did not significantly affect the number of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes trapped (GLMM, n.s.). The relative abundance of \u003cem\u003eAn. dirus\u003c/em\u003e complex mosquitoes in HLC collections was 0.311 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHLC, ABT and BG-Sentinel 2 comparison\u003c/h2\u003e \u003cp\u003eThe BG-Sentinel 2 trap baited with CO\u003csub\u003e2\u003c/sub\u003e from a gas cylinder was compared to the HLC and ABT in the third and final experimental round. A total of 71 \u003cem\u003eAn. dirus\u003c/em\u003e complex, 224 \u003cem\u003eAn. maculatus\u003c/em\u003e Group, 17 \u003cem\u003eAnopheles kochi\u003c/em\u003e, 8 \u003cem\u003eAn. barbirostris\u003c/em\u003e complex, and 4 \u003cem\u003eAnopheles vagus\u003c/em\u003e were collected (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, 27 \u003cem\u003eAe. albopictus\u003c/em\u003e, 23 other \u003cem\u003eAedes\u003c/em\u003e spp., 146 \u003cem\u003eCulex\u003c/em\u003e spp., 329 \u003cem\u003eArmigeres\u003c/em\u003e spp., and 4 \u003cem\u003eToxorhynchitis\u003c/em\u003e spp. were collected. Highly significant differences in the mean nightly catch numbers of \u003cem\u003eAn. dirus\u003c/em\u003e complex mosquitoes were found with the ABT collecting three and five-fold greater numbers than the BG-Sentinel 2 and HLC, respectively (GLMM, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Collection numbers of \u003cem\u003eAn. maculatus\u003c/em\u003e Group mosquitoes were also significantly higher in the ABT than in the other two collection methods (GLMM, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Collection numbers of other anophelines were insufficient for statistical analysis. Additionally, the \u003cem\u003eAnopheles\u003c/em\u003e species richness was highest in the ABT, which collected five \u003cem\u003eAnopheles\u003c/em\u003e species and was more sensitive, capturing much higher numbers of anophelines than the other methods. In contrast, the BG-Sentinel 2 collected \u003cem\u003eAn. dirus\u003c/em\u003e complex (n\u0026thinsp;=\u0026thinsp;18), \u003cem\u003eAn. maculatus\u003c/em\u003e Group (n\u0026thinsp;=\u0026thinsp;9), and \u003cem\u003eAn. barbirostris\u003c/em\u003e complex (n\u0026thinsp;=\u0026thinsp;5), while the HLC collected \u003cem\u003eAn. dirus\u003c/em\u003e complex (n\u0026thinsp;=\u0026thinsp;7), \u003cem\u003eAn. maculatus\u003c/em\u003e Group (n\u0026thinsp;=\u0026thinsp;13), and \u003cem\u003eAn. kochi\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3). The relative abundance of \u003cem\u003eAn. dirus\u003c/em\u003e complex mosquitoes was much lower than in the previous experiment rounds, with 0.038 in the HLC, 0.281 in the BG-Sentinel 2 trap, and 0.051 in the ABT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLand-use type effects\u003c/h2\u003e \u003cp\u003eAcross the different land-use types, large differences in mosquito catches were observed. There was a significant effect of land-use type on the proportions of collected mosquito females by species across the full experiment (Χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;840.92, df\u0026thinsp;=\u0026thinsp;14, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Collections in the small scale, mixed-crop agricultural area captured greater numbers of \u003cem\u003eAn. dirus\u003c/em\u003e complex and \u003cem\u003eAn. barbirostris\u003c/em\u003e complex than expected, while the relative abundances of \u003cem\u003eAn. maculatus\u003c/em\u003e Group, \u003cem\u003eAn. kochi\u003c/em\u003e, and \u003cem\u003eAn. vagus\u003c/em\u003e mosquitoes were greater than expected in the oil palm plantation. In the village, the proportion of \u003cem\u003eArmigeres\u003c/em\u003e spp. was much greater than expected, while the relative abundance of \u003cem\u003eCulex\u003c/em\u003e spp. was less than expected. Analysis of the residuals showed that there was a significant positive correlation between \u003cem\u003eAn. dirus\u003c/em\u003e complex and the mixed crop agricultural area, as well as a strong positive correlation between \u003cem\u003eAn. maculatus\u003c/em\u003e Group and the oil palm plantation. In contrast, most mosquito species were negatively correlated with the village residences, except for \u003cem\u003eArmigeres\u003c/em\u003e spp., which comprised\u0026thinsp;\u0026gt;\u0026thinsp;95% of the non-anophelines, \u003cem\u003eAe. albopictus\u003c/em\u003e, or \u003cem\u003eCulex\u003c/em\u003e collected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The remaining 5% of the other mosquitoes consisted of \u003cem\u003eAedes\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;31) and \u003cem\u003eToxorhynchites\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMechanical collection methods successfully sampled anophelines including simian malaria vectors in this and other studies (37,47\u0026ndash;49). However, the sensitivity of sampling methods for anopheline mosquitoes in this study was greatest when using humans or cattle as lures compared to mechanical mosquito collection methods, including the Passive Box trap, the CDC Miniature light trap, and the BG-Sentinel 2 trap, baited with CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eWhile numbers of collected mosquitoes are often fewer in traps baited with CO\u003csub\u003e2\u003c/sub\u003e from yeast/sugar fermentation compared to CO\u003csub\u003e2\u003c/sub\u003e from dry ice, other studies reported representative sampling of mosquitoes using yeast/sugar fermentation (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Additionally, studies in Africa (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and Australia (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) reported high numbers of mosquitoes including anophelines collected in traps with CO\u003csub\u003e2\u003c/sub\u003e produced by yeast/sugar fermentation, but no direct comparisons to HLC were made. The CO\u003csub\u003e2\u003c/sub\u003e generated from yeast fermentation in the experiments reported here may not have produced amounts of CO\u003csub\u003e2\u003c/sub\u003e sufficient to attract large numbers of mosquitoes. However, CO\u003csub\u003e2\u003c/sub\u003e released from a tank at recommended rates (e.g., 250ml/min) was equally ineffective in attracting large numbers of mosquitoes. Another explanation for the low number of mosquitoes in the mechanical traps might be that the evaluated traps were either not efficient in capturing mosquitoes lured to them or were not attractive to mosquitoes as deployed. Adding an extra odour bait or heat source, or placement at a different elevation, might improve catch numbers (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMechanical traps like the CDC Light trap and the BG-Sentinel traps have collected mosquitoes for decades. In sub-Sahara Africa, CDC Light traps and other mechanical traps supplemented or replaced HLCs for malaria vector collections to provide rough estimates of human biting rates, despite their lower sensitivity (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In Asia, however, mechanical traps have not been as promising. In Peninsular Malaysia, collection numbers of An. \u003cem\u003eleucosphyrus\u003c/em\u003e Group mosquitoes in the Mosquito Magnet were lower, but not statistically different from HLCs, while CDC Light traps yielded much lower numbers of these and other mosquitoes (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). However, the setup and establishment costs of the Mosquito Magnet are logistically and financially challenging. Smaller mosquito surveys in Indonesia showed that \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group mosquitoes were collected by HLC but not CDC light traps in a direct comparison (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). In this study, a similar result was observed across the study sites, with CDC light traps and other mechanical traps collecting low numbers of mosquitoes compared to HLCs. The BG-Sentinel 2 trap had not, to our knowledge, been evaluated for sampling \u003cem\u003eAnopheles\u003c/em\u003e in Indonesia before this study. However, a previous study in northern Queensland, Australia showed that a BG-Sentinel 2 trap baited with tank CO\u003csub\u003e2\u003c/sub\u003e collected \u003cem\u003eAnopheles\u003c/em\u003e females (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The third Latin Square round of this study provides some evidence that the BG-Sentinel 2 trap, baited with CO\u003csub\u003e2\u003c/sub\u003e from a gas cylinder, could be at least comparable in sensitivity to the HLC to collect \u003cem\u003eAnopheles\u003c/em\u003e. Especially when HLCs are logistically challenging to establish (e.g., in deep forest locations) or when many locations need to be sampled simultaneously, the BG-Sentinel 2 trap could be a viable alternative. Why the BG-Sentinel 2 was more sensitive than the HLC in one of the two experimental rounds in which they were directly compared requires further study. Additionally, running a mechanical trap baited with a CO\u003csub\u003e2\u003c/sub\u003e tank will only be more cost-effective than a single team of HLC collectors when collecting for 16 nights or more in Sumatra, due to its relatively high establishment cost (Supplemental Material 1).\u003c/p\u003e \u003cp\u003eThe cow-baited tent trap was sensitive, collecting large numbers of anophelines throughout the night. However, the ABT attracted much greater numbers of other mosquito species (e.g., \u003cem\u003eArmigeres\u003c/em\u003e spp. and \u003cem\u003eCulex\u003c/em\u003e spp.) than the HLC, as well as many zoophilic anophelines. Cow-baited tent traps are likely the best method to collect a wide range of species including vectors of malaria and arboviruses but are less accurate in estimating the biting risk on humans (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Animal-baited traps often yield much larger numbers than HLCs, which would require calibration against HLCs to estimate the biting rate on humans. Also, the number of non-target mosquitoes collected in ABTs is significantly greater compared to HLCs, which requires more time to enable mosquito identification in the field. Finally, the ABT is more challenging to deploy than other collection methods. During this study, challenges with the ABTs included finding suitably flat ground to set up the ABT, inadvertently causing stress to the cow despite the presence of the owner, cow escape attempts, and windy weather.\u003c/p\u003e \u003cp\u003eHeterogeneity between the first two and the third Latin Square comparison was seen in the relative sensitivity of the BG sentinel 2 trap and the HLC in sampling \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group and \u003cem\u003eAn. maculatus\u003c/em\u003e Group mosquitoes. These results lack a clear reason, since Sanger sequencing of the ITS2 gene showed that all specimens in the random subsample were \u003cem\u003eAn. dirus\u003c/em\u003e complex, indicating no changes in species occurrence within the \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group between the second and third Latin square rounds. The randomised sample size (n\u0026thinsp;=\u0026thinsp;72) was sufficient to conclude that the only species within the \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group occurring in the study area was in the \u003cem\u003eAn. dirus\u003c/em\u003e complex. Additionally, the sampling sites and individuals conducting the HLC were the same during all three rounds. These individuals were trained and supervised hourly. While the relative abundance of \u003cem\u003eAn. dirus\u003c/em\u003e complex mosquitoes captured by HLC decreased between rounds two and three, the relative abundance of other anophelines (both \u003cem\u003eAn. maculatus\u003c/em\u003e Group and \u003cem\u003eAn. kochi\u003c/em\u003e) increased for HLC. The hourly mosquito collections patterns were consistent through the night and across all 3 Latin square rounds, suggesting consistent HLC collections.\u003c/p\u003e \u003cp\u003eThe finding of \u003cem\u003eAn. dirus\u003c/em\u003e complex in this region of North Sumatra was unexpected, since this species was not previously reported here (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). However, a sibling species of \u003cem\u003eAn. dirus\u003c/em\u003e (\u003cem\u003eAnopheles cracens\u003c/em\u003e) was reported in Sabang Island, Aceh (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), and more recently, mosquitoes morphologically identified as \u003cem\u003eAn. dirus\u003c/em\u003e complex were collected in Central Kalimantan, indicating a wider geographic spread than previously known (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Since \u003cem\u003eAn. dirus\u003c/em\u003e complex mosquitoes are competent vectors of malaria in many areas, further research is required to establish the geographical boundaries of this species in Indonesia, and to estimate the potential risk of malaria transmission to humans in Indonesia.\u003c/p\u003e \u003cp\u003eLand-use type strongly affected mosquito collections. Despite the small spatial scale of this study, \u003cem\u003eAn. dirus\u003c/em\u003e complex mosquitoes were predominantly collected in the mixed-crop agricultural area. The presence of suitable larval habitats (Sebayang, personal communication) could partially explain the high abundance of \u003cem\u003eAn. dirus\u003c/em\u003e complex mosquitoes in the mixed-crop agricultural area. However, the distance between the village and the larval habitats was well within the flight range of \u003cem\u003eAnopheles\u003c/em\u003e females, so it is likely that other, unknown factors are influencing \u003cem\u003eAnopheles\u003c/em\u003e densities. Evidence for land-use type associations and \u003cem\u003eAn. dirus\u003c/em\u003e complex and \u003cem\u003eAn. maculatus\u003c/em\u003e Group distributions in North Sumatra, Indonesia was found, but longer studies across multiple land-use types are needed to confirm if the land-use type effects observed here are valid across time and space.\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\u003e\u0026ndash; the number of Anopheles females collected by each collection method in every experimental round. HLC: human landing catch with two collectors per sampling station; PBT: passive box trap; ABT: animal-baited tent trap.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAn. dirus\u003c/em\u003e complex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAn. barbirostris\u003c/em\u003e complex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAn. kochi\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eAn. vagus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eAn. maculatus\u003c/em\u003e Group\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRound 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePBT\u0026thinsp;+\u0026thinsp;tank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePBT\u0026thinsp;+\u0026thinsp;yeast \u0026amp; sugar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e33\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e0\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003e0\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRound 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBG-Sentinel 2\u0026thinsp;+\u0026thinsp;yeast \u0026amp; sugar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBG-Sentinel 2\u0026thinsp;+\u0026thinsp;tank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDC light trap\u0026thinsp;+\u0026thinsp;yeast \u0026amp; sugar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDC light trap\u0026thinsp;+\u0026thinsp;yeast \u0026amp; sugar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePBT\u0026thinsp;+\u0026thinsp;yeast \u0026amp; sugar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePBT\u0026thinsp;+\u0026thinsp;tank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e67\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e0\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003e0\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003e3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRound 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eABT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e212\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBG-Sentinel 2\u0026thinsp;+\u0026thinsp;tank CO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e71\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e24\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003e241\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCollection methods using humans or cows were the most sensitive for \u003cem\u003eAnopheles\u003c/em\u003e species, including species in the \u003cem\u003eAn. leucosphyrus\u003c/em\u003e Group, in North Sumatra. Mechanical traps baited with CO\u003csub\u003e2\u003c/sub\u003e were not very efficient, regardless of the CO\u003csub\u003e2\u003c/sub\u003e source or the trap type. The BG-Sentinel 2 trap baited with tank CO\u003csub\u003e2\u003c/sub\u003e showed some potential for sampling anophelines in Sumatra as it showed greater sensitivity for sampling \u003cem\u003eAn. dirus\u003c/em\u003e complex and \u003cem\u003eAn. barbirostris\u003c/em\u003e complex than the HLC in one of two direct Latin Square comparisons. Therefore, the BG-Sentinel 2 trap might be useful in large-scale surveillance studies once the basis for the variation in relative sensitivity reported here is understood. Despite the greater absolute number of \u003cem\u003eAn. dirus\u003c/em\u003e complex captured in the ABT compared to the HLC in a direct comparison, the sensitivity and high specificity of the HLC for \u003cem\u003eAn. leucosphyrus\u003c/em\u003e complex mosquitoes makes the HLC the preferred sampling method to understand zoonotic malaria transmission to humans and to define high-risk areas of transmission in North Sumatra as it is relatively easy to establish at multiple concurrent sites and directly provides an epidemiologically relevant estimation of the biting risk.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Faculty of Medicine, Universitas Sumatera Utara (No. 723/KEP/USU/2021). Meetings were held with community leaders, study participants and village residents, where the aims, the possible risks and potential benefits of the study were explained in Bahasa Indonesia. Mosquito collectors were then recruited from village residents and enrolled in the study after the risks were explained and informed consent was given by collectors performing HLCs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the ZOOMAL project (\u0026lsquo;Evaluating zoonotic malaria and agricultural land use in Indonesia\u0026rsquo;; #LS-2019-116), Australian Centre for International Agricultural Research, Australian Government. BvdS and BS were supported by a James Cook University Postgraduate Research Scholarship.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBvdS, TRB and TLR conceived the study; TRB, TLR, TAG and IL advised on the fieldwork and laboratory analyses. BvdS and AK conducted the fieldwork, and BvdS and BS performed the molecular analyses. BvdS analysed the data and wrote the initial draft of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThe World Bank. World Development Indicators. 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSetiadi W, Sudoyo H, Trimarsanto H, Sihite BA, Saragih RJ, Juliawaty R, et al. A zoonotic human infection with simian malaria, \u003cem\u003ePlasmodium knowlesi\u003c/em\u003e, in Central Kalimantan, Indonesia. 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Six new species of the \u003cem\u003eAnopheles leucosphyrus\u003c/em\u003e group, reinterpretation of \u003cem\u003eAn. elegans\u003c/em\u003e and vector implications. Med Vet Entomol. 2005;19(2):158\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnggraeni Y, Setiyaningsih R, Mujiyanto M, Trapsilowati W, Pujiyanti A, Rahardianingtyas E et al. Molecular detection of \u003cem\u003ePlasmodium\u003c/em\u003e spp. in \u003cem\u003eAnopheles\u003c/em\u003e and its vector potential in low-endemic areas in Indonesia. Proceeding of the International Conference on Public Health [Internet]. 2022;7(1):107\u0026ndash;18. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tiikmpublishing.com/data/conferences/doi/icoph/10.1750124246735\u003c/span\u003e\u003cspan address=\"https://tiikmpublishing.com/data/conferences/doi/icoph/10.1750124246735\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.2022.7111.pdf Supplemental Material 1 \u003cem\u003eCost of one BG-Sentinel 2 trap compared to one HLC team of two collectors.\u003c/em\u003e One CO\u003csub\u003e2\u003c/sub\u003e tank can last reliably for 5 collection nights when set at a standard CO\u003csub\u003e2\u003c/sub\u003e release rate of 250 mL/min. The formula to calculate the cost of using a mechanical trap baited with CO\u003csub\u003e2\u003c/sub\u003e is 1).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anopheles leucosphyrus Group, Anopheles dirus complex, Anopheles maculatus Group, Anopheles barbirostris complex, land-use, malaria, vector, human landing catch, animal-baited tent trap","lastPublishedDoi":"10.21203/rs.3.rs-3857130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3857130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMalaria transmission to humans is increasing in complexity in Indonesia with simian malaria cases in humans increasing as human malaria incidence diminishes. Reliable, epidemiologically relevant, logistically feasible, and cost-effective collection methods are needed to define the transmission risk to humans and the efficacy of vector control for both simian and human malarias. Alternative and/or complementary representative mosquito sampling methods to the labour-intensive human landing collections (HLC) and animal-baited tent traps (ABT) currently used in Indonesia are needed.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eCarbon dioxide gas from a compressed cylinder and from yeast/sugar fermentation were used as lures in Passive Box, CDC Light and BG Sentinel 2 mosquito traps and compared in Latin square trials to human landing catches (HLCs) and animal baited traps (ABTs) for sensitivity and specificity for anopheline vectors across three land use types (oil palm plantation, mixed-crop agricultural area and village) in North Sumatra.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe ABT with cow as bait was the most sensitive sampling method for all anopheline species in a direct comparison to HLC and the BG-Sentinel 2 trap in North Sumatra. The Passive Box and CDC Light traps were not sensitive for anophelines regardless of the CO\u003csub\u003e2\u003c/sub\u003e source. The HLC showed high specificity for members of the \u003cem\u003eAn. dirus\u003c/em\u003e complex, and also captured \u003cem\u003eAn. kochi\u003c/em\u003e, \u003cem\u003eAn. barbirostris\u003c/em\u003e complex, and \u003cem\u003eAn. maculatus\u003c/em\u003e Group mosquitoes.\u003c/p\u003e","manuscriptTitle":"Impacts of land use type on sampling methods for human and simian malaria vectors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 14:29:00","doi":"10.21203/rs.3.rs-3857130/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa44934d-26f6-4a3a-9018-4e10689e8282","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-04T14:38:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 14:29:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3857130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3857130","identity":"rs-3857130","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}