Blood feeding patterns of malaria mosquitoes collected using pit shelters and clay pots in the West Gojjam Zone of Ethiopia

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Therefore, this study investigated the species composition, blood meal sources, and infection rates of Anopheles mosquitoes in the rural village of Dilamo, West Gojjam, Ethiopia. Methods Ten households were randomly selected in a malaria-endemic village. Two clay pots were placed under shade outside each house within a radius of 10 meters, one in front of the house and the other at the back. Two other clay pots were placed inside the same household. Ten pit shelters were built, one at a distance of 10 meters from each house, under shade. Mosquito sampling was done twice a month for four months. Anopheles mosquitoes were identified to species using morphological keys. The An. gambiae complex species were molecularly identified. Testing for blood meal sources and circumsporozoite proteins (CSPs) was conducted using an enzyme-linked immunosorbent assay (ELISA). Results A total of 319 female Anopheles mosquitoes were collected, including the species An. demilloni, An. gambiae complex, An. garnhami , An. pretoriensis , and An. cinereus . Among these, An. demilloni was the dominant species, comprising 90.9% of the collection (290/319). The An. gambiae complex represented only 4.4% of the total (14/319). Out of 89 freshly fed Anopheles mosquitoes, 77 (86.5%) were examined for the source of their blood meal. Of these, 74 (96%) tested positive for blood from cattle, humans, or both. The overall human blood meal index was 43% (95% CI: 31.1–55.3), while the bovine blood meal index was 6% (95% CI: 1.6–13.9). The prevalence of Anopheles mosquitoes feeding on humans and cattle (mixed feeding) was 57% (95% CI: 44.8–68.9). None of the Anopheles mosquitoes tested positive for Plasmodium falciparum or P. vivax -210 CSPs. Conclusion The human blood meal index reflects human exposure to mosquito bites. Future research could investigate whether mosquitoes, along with human behaviours and activities, prefer to bite humans indoors or outdoors. Anopheles species composition Blood meal sources CSP rates West Gojjam Ethiopia Figures Figure 1 Figure 2 Figure 3 Background Over the years, initiatives such as insecticide-treated nets (ITNs), indoor residual spraying (IRS), and improved case treatment have significantly decreased malaria cases and deaths, notably until 2015 [ 1 ]. However, the situation has changed, and malaria is now on the rise in many African countries, especially in Ethiopia [ 2 ]. Several potential factors contribute to this resurgence, including the spread of insecticide-resistant mosquitoes [ 3 ] and drug-resistant malaria parasites [ 4 ], as well as changes in mosquito behaviour, which have shifted from indoor to outdoor activities [ 5 ]. Additionally, the emergence of the Anopheles stephensi mosquito [ 6 ] poses a new challenge. Non-biological factors such as climate change, a controversial topic, along with the strain on healthcare systems caused by the COVID-19 pandemic and instability in malaria-endemic countries, may contribute to the increasing rates of malaria [ 2 ]. Anopheles species that transmit malaria exhibit complex biting and resting behaviors, as well as varying rates of Plasmodium sporozoite infection, which determine the type of intervention to be planned and implemented. It is concerning that some malaria vectors are not responding well to current interventions, and an urgent mitigation approach is needed [ 7 ]. To effectively implement intervention strategies, we must deepen our understanding of both primary and secondary vectors and local mosquitoes' resting and feeding patterns. Certain species of Anopheles prefer to bite outdoors at night and avoid contact with insecticide-based interventions [ 8 ]. It is essential to consider these factors when planning our interventions. Mosquito collection should provide detailed information on species composition and Plasmodium sporozoite infection rates, which may be influenced by the sampling methods used [ 9 ]. Different sampling methods yield varying results regarding species composition, feeding patterns, and infection rates. Outdoor collection techniques, such as pit shelters, are particularly effective for gathering fed mosquitoes to analyze the sources of their blood meals among the mosquitoes that typically avoid resting indoors [ 10 ]. Clay pots are an effective tool for collecting resting mosquitoes outdoors [ 11 ]. They are user-friendly, affordable, and readily accessible for large areas, requiring minimal cleaning and maintenance. The data collected through these methods provides valuable insights into transmission risks and mosquito behaviour in outdoor environments, which can guide the development of targeted interventions. Furthermore, it also highlights shifts in mosquito behaviour, indicating a trend toward resting outdoors as a reaction to indoor interventions. Therefore, this study aimed to analyse the species composition, blood meal origins, and infection rates of outdoor resting malaria mosquitoes in West Gojjam, Ethiopia. Materials and methods Description of the study area The study was conducted in Dilamo, a rural village in the South Achefer district, West Gojjam Zone, Ethiopia (Fig. 1 ). South Achefer is 501 km from Addis Ababa and 60 km from Bahir Dar, the capital of the Amhara Regional State. The district is geographically located at 11°50′N latitude and 37°10′E longitude, and its elevation ranges from 1,500 to 2,500 meters above sea level (masl). The district covers an area of 1,183.05 Km 2 and is characterised by high plateaus, mountains and broad valleys suitable for agriculture [ 12 ]. Its annual mean temperature is 23°C, and the mean yearly rainfall is 1,522 mm. South Achefer is endemic to malaria [ 13 ]. Study design Dilamo village was divided into five clusters, each consisting of 30 houses. For this study, two clusters were randomly selected for longitudinal entomological sampling. Ten houses were selected randomly in each cluster. For each household, two clay pots (10–15 lit capacity) were placed in shaded areas outside the house, within a 10-meter radius. One pot was placed in front of the house, while another was located at the back. Additionally, two clay pots were positioned inside each household, one at each opposite corner. Each pot has a hole with a diameter of 2 cm in the Centre of the base. The hole prevents the pot from retaining water and reduces the possibility of theft. Ten pits were dug under shaded areas within a 10 m radius of the selected houses. Each pit was approximately 1.5 m deep, 1.5 m long, and 1.5 m wide. At a height of 1 m from the bottom of each pit, horizontal holes of about 30 cm deep were made in the pit's wall. Anopheles mosquito collection Mosquito collections were conducted from clay pots and pit shelters twice a month from December 2019 to March 2020. The collection from the clay pots took place between 6:00 and 8:00 a.m. The collectors first placed a mosquito cage over the opening of the pot. Then, they lifted the pot, exposing the opening to the light. By stirring the mosquitoes inside and blowing air through a small hole at the bottom, the mosquitoes would escape the pot and enter the cage. The remaining mosquitoes in the pot were aspirated and transferred to the cage. All debris and organisms in the pot were carefully removed each time. Mosquitoes were collected from constructed pits using handheld aspirators and a torch as a light source [ 14 ]. Their mouths were covered with non-impregnated mosquito nets to prevent mosquitoes from escaping the pit shelters during collection. Species identification The Anopheles mosquitoes caught from clay pots and pit shelters were later identified morphologically as species using a dissecting microscope, following the morphological key [ 15 ]. Anopheles gambiae complexes were identified molecularly using primers that target the Internal Transcribed Spacer (ITS) region in Ribosomal DNA (rDNA) to distinguish the species complex [ 16 ]. Detection of blood meal sources and circumsporozoite proteins The circumsporozoite protein (CSP) was detected using the Enzyme-Linked Immunosorbent Assay (ELISA) method using the head and thorax of female Anopheles mosquitoes [ 17 ]. A direct ELISA was performed according to the procedure outlined by Beier et al. [ 18 ] to identify the blood meal sources of freshly fed mosquitoes. Data Analysis The monthly counts of Anopheles mosquitoes were represented as percentages, along with descriptive statistics. Fisher's exact test (Clopper-Pearson) was conducted using a two-by-two table to compare the proportions of Anopheles mosquitoes that were fed on human and bovine blood sources. The Human Blood Index (HBI) and Bovine Blood Index (BBI) were calculated as the ratio of blood-sucking mosquitoes that fed on humans and cattle, respectively, to the total number tested. Circum-sporozoite protein level is the number of mosquitoes positive for CSP antigen divided by the total number of mosquitoes tested, expressed as a percentage. SPSS software version 25.0 (SPSS, Chicago, IL, USA) was utilised for data entry, cleaning, and analysis. Results Anopheles species composition A total of 319 female Anopheles mosquitoes, comprising five species, were collected from Dilamo village in South Achefer District (Table 1 ). Among these, An. demilloni accounted for 90.9% (290/319), and An. gambiae complex 4.4% (14/319). Anopheles species versus collection method About 30% of the Anopheles mosquitoes (96/319) were collected from clay pots, while 70% (223/319) were found in pit shelters (Table 1 ). Notably, no mosquitoes were sampled from clay pots placed indoors. Table 1 Anopheles mosquito species collected using pit shelters and clay pots in Dilamo village, South Achefer District, West Gojjam Zone, Ethiopia, December 2019 to March 2020. Anopheles species Pit shelter (%) Outdoor clay pots (%) Total (%) An. demilloni 206 (71) 84 (29) 290 (90.9) An. gambiae complex 10 (71) 4 (29) 14 (4.4) An. cinereus 4 (44) 5 (56) 9 (2.8) An. garnhami 4 (80) 1(20) 5 (1.6) An. pretoriensis 0 1(100) 1 (0.3) Total 223 (70) 96 (30) 319 (100) Monthly variation of Anopheles species collections The highest number of female Anopheles species was collected in December from clay pots and pit shelters (Fig. 2 ). After that, the number of collections decreased and reached its lowest point in March. Blood meal origins of Anopheles species Out of 89 freshly fed Anopheles mosquitoes, 77 (86.5%) were examined for their blood meal origins (Table 3). Among these, 74 mosquitoes (96%) tested positive for either bovine or human blood. The overall human blood meal index was 43% (95% Confidence Interval (CI): 31–55), while the bovine blood meal index was only 6% (95% CI: 1.6–14). Additionally, the proportion of Anopheles mosquitoes that fed on both humans and bovines (mixed) was 57% (95% CI: 45–69). Anopheles demilloni primarily fed on humans (39%) more than on bovine (4%). Additionally, the species had a good proportion of mixed-blood meals (54%) (Table 2 ). The proportion of Anopheles mosquitoes that fed on human blood was significantly higher than those that fed on bovine blood (Fisher's exact test: χ² = 25.3; P < 0.0001). Table 2 Overall blood meal origins of Anopheles mosquitoes collected using clay pots and pit shelters in Dilamo, South Achefer District, West Gojjam, Ethiopia, December 2019 to March 2020. Anopheles species Total tested Human fed (index; 95%CI) Bovine fed (index; 95%CI) Human and bovine fed (index; 95%CI) Unidentified (%) An. demilloni 70 27 (39: 28–50) 3 (4: 1.0–12) 38 (54: 42–66) 2 (2.8) An. gambiae s.l. 3 1 (33: 1.0–90) 1 (33.3:1.0–90) 1 (33: 1.0–90) 0 (0) An. garnhami 3 2 (67: 9–99) 0 (0) 1 (33.0: 1.0–90) 0 (0) An. cinereus 1 0 (0) 0 (0) 0 (0) 1 (100) Total 77 30 (43: 31–55) 4 (6: 1.6–14) 40 (57: 45–69) 3 (4.3) In clay pot and pit shelters, the percentage of Anopheles mosquitoes feeding on human, bovine, and mixed blood was comparable (Fig. 3 ). Plasmodium CSP rates of Anopheles species A total of 319 female Anopheles mosquitoes were examined for CSPs, which included 290 An. demilloni , 14 An. gambiae complex, nine An. cinereus , five An. garnhami , and one An. pretoriensis . None tested positive for Plasmodium CSPs. Discussion Anopheles demilloni , the An. gambiae complex, An. cinereus , An. garnhami , and An. pretoriensis were collected from Dilamo village of South Achefer district, West-Gojjam, Ethiopia. Among these species, An. demilloni was the dominant species and exhibited a higher human blood meal index than bovine. Clay pots placed outside and pit shelters effectively captured Anopheles mosquitoes for entomological monitoring. However, clay pots placed indoors were unsuccessful in trapping any mosquitoes. Anopheles demilloni was the dominant mosquito species observed, followed by the An. gambiae complex. This species is commonly found in highland areas, and a mosquito infected with the Plasmodium parasite has recently been confirmed [ 19 ]. However, its capacity to transmit the parasite is still under investigation. This species has also been identified in northern Ethiopia, mainly from pit shelters and outdoor clay pots [ 20 ]. In most studies, An. gambiae complex was the dominant species, utilising various collection techniques such as CDC light traps, PSC, pit shelters and clay pots [ 16 , 17 , 18 ]. Regardless of the collection technique and the Anopheles species involved, the human blood meal index was significantly higher than the bovine blood meal index. This study contrasts with a similar one conducted in southern Ethiopia, which found a higher bovine blood meal index in mosquitoes using the same collection method [ 11 ]. In our study, a higher mixed blood meal index was observed in Anopheles mosquitoes. This may be attributed to the proximity of cattle to people's households or situations where cattle share living spaces, resulting in mosquitoes biting indoors [ 23 ]. In the study area, sheep, donkeys, and cattle are the most common livestock. These animals, particularly cattle and sheep, remain outside until nearly midnight. Afterward, sheep and certain selected cattle come inside to share the home with humans for the night. Additionally, the head of the household and school-aged children often remain outside to tend to the cattle. This practice of cohabiting or spending long hours with animals at night may contribute to a higher prevalence of mixed blood meals. Furthermore, cattle near households could increase the density of zoophilic vectors. To help control these malaria-carrying vectors, treating livestock with insecticides and constructing separate enclosures away from human living spaces is advisable. The human blood meal index of Anopheles mosquitoes was high, indicating that humans were exposed to mosquito bites. Anopheles demilloni showed a relative ability to feed on human blood compared to bovine blood. This increased human exposure to mosquito bites demands interventions to protect against it. Despite a high human blood meal index, the mosquitoes tested negative for Plasmodium CSPs. This study aligns with previous research conducted in south-central Ethiopia, where many mosquitoes were documented, but none tested positive for CSPs [ 24 ]. In most cases, the infection rate of sporozoites in mosquitoes is rare despite the high prevalence of malaria. This study has some limitations, including that data were collected over only four months. Consequently, the blood-feeding patterns and infection rates may have been influenced by monthly variations of environmental variables. Additionally, we identified only the blood meal sources of outdoor resting mosquitoes, whereas the blood meal sources of indoor resting mosquitoes and those that bite indoors and outdoors may differ. Conclusion The human blood meal index of Anopheles mosquitoes is high, indicating human exposure to mosquito bites. Further research should explore the biting behaviour of Anopheles mosquitoes and their interactions with humans. This understanding will facilitate the implementation of effective control measures. Declarations Acknowledgements We thank the community for permitting us to collect mosquitoes from their homes and compounds. Author contributions A. A. designed the study and conducted the fieldwork. A. A. critically reviewed and revised the manuscript. F. M. designed the study, supervised the field and laboratory work, and drafted and revised the manuscript. All authors have read and approved the final manuscript submission. Funding This study was financially supported by the School of Graduate Studies at Arba Minch University. Availability of data and materials The data is thoughtfully incorporated within the manuscript. Declarations Ethics approval and consent to participate This study received ethical approval from the Institutional Review Board of Arba Minch University. The homeowner gave consent before installing clay pots and digging pit shelters. Consent for publication Not applicable Competing interests The authors declare that there is no competing interest. Author details 1 Department of Biology, Arba Minch University, Arba Minch, Ethiopia. 2 Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia References Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015; 526: 207–11. WHO. World malaria report. Geneva: World Health Organization; 2023. Moyes CL, Athinya DK, Seethaler T, Battle KE, Sinka M, Hadi MP, et al. Evaluating insecticide resistance across African districts to aid malaria control decisions. PNAS. 2020; 117: 22042–50. Conrad MD, Rosenthal PJ. Antimalarial drug resistance in Africa: the calm before the storm? Lancet Infect Dis. 2019; 19:e338-e351. Mwangangi JM, Mbogo CM, Orindi BO, Muturi EJ, Midega JT, Nzovu J, et al. Shifts in malaria vector species. 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Eshetu T, Eligo N, Massebo F. Cattle feeding tendency of Anopheles mosquitoes and their infection rates in Aradum village, North Wollo, Ethiopia: an implication for animal-based malaria control strategies. Malar J. 2023;22:81 Massebo F, Balkew M, Gebre-Michael T, Lindtjorn B. Entomologic inoculation rates of Anopheles arabiensis in southwestern Ethiopia. Am J Trop Med Hyg. 2013; 89:466–73. Esayas E, Woyessa A, Massebo F. Malaria infection clustered into small residential areas in lowlands of southern Ethiopia. Parasite Epidemiol Control. 2020; 10: e00149. Ndenga BA, Mulaya NL, Musaki SK, Shiroko JN, Dongus S, Fillinger U. Malaria vectors and their blood-meal sources in an area of high bed net ownership in the western Kenya highlands. Malar J. 2016;15: 76 Kenea O, Balkew M, Tekie H, Gebre-Michael T, Deressa W, Loha E, et al. Human-biting activities of Anopheles species in south-central Ethiopia. Parasite Vectors. 2016;9:527 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-5989388","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440126891,"identity":"5802b0b1-abd9-4766-b559-c9596bf1ad48","order_by":0,"name":"Atakilt Arega","email":"","orcid":"","institution":"Arba Minch University","correspondingAuthor":false,"prefix":"","firstName":"Atakilt","middleName":"","lastName":"Arega","suffix":""},{"id":440126892,"identity":"922cc64d-d1b1-40c9-b411-11648f0d4bba","order_by":1,"name":"Abebe Animut","email":"","orcid":"","institution":"Aklilu Lemma Institute of Pathobiology, Addis Ababa 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11:39:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22616,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of female \u003cem\u003eAnopheles \u003c/em\u003emosquitoes collected using clay pots and pit shelters in Delamo, South Achefer District, West Gojjam, Ethiopia, December 2019 to March 2020.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5989388/v1/1e06d4dff5195e618ddae445.png"},{"id":80312321,"identity":"3b1a9dbe-e9bc-4d08-aabc-62a30361f31a","added_by":"auto","created_at":"2025-04-10 11:39:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20992,"visible":true,"origin":"","legend":"\u003cp\u003eThe blood meal origins of \u003cem\u003eAnopheles\u003c/em\u003emosquitoes collected by clay pots and pit shelters in Dilamo, South Achefer District, West Gojjam, Ethiopia, December 2019 to March 2020\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5989388/v1/b33e484f17941e07811c65d9.png"},{"id":83782952,"identity":"095a1970-8013-4a84-91cb-86016e17b97b","added_by":"auto","created_at":"2025-06-02 16:09:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1073729,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5989388/v1/934e0c9d-579d-4cf7-8a64-c0968cecf15d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eBlood feeding patterns of malaria mosquitoes collected using pit shelters and clay pots in the West Gojjam Zone of Ethiopia\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eOver the years, initiatives such as insecticide-treated nets (ITNs), indoor residual spraying (IRS), and improved case treatment have significantly decreased malaria cases and deaths, notably until 2015 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the situation has changed, and malaria is now on the rise in many African countries, especially in Ethiopia [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Several potential factors contribute to this resurgence, including the spread of insecticide-resistant mosquitoes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and drug-resistant malaria parasites [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], as well as changes in mosquito behaviour, which have shifted from indoor to outdoor activities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Additionally, the emergence of the \u003cem\u003eAnopheles stephensi\u003c/em\u003e mosquito [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] poses a new challenge. Non-biological factors such as climate change, a controversial topic, along with the strain on healthcare systems caused by the COVID-19 pandemic and instability in malaria-endemic countries, may contribute to the increasing rates of malaria [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnopheles\u003c/em\u003e species that transmit malaria exhibit complex biting and resting behaviors, as well as varying rates of \u003cem\u003ePlasmodium\u003c/em\u003e sporozoite infection, which determine the type of intervention to be planned and implemented. It is concerning that some malaria vectors are not responding well to current interventions, and an urgent mitigation approach is needed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To effectively implement intervention strategies, we must deepen our understanding of both primary and secondary vectors and local mosquitoes' resting and feeding patterns. Certain species of \u003cem\u003eAnopheles\u003c/em\u003e prefer to bite outdoors at night and avoid contact with insecticide-based interventions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It is essential to consider these factors when planning our interventions.\u003c/p\u003e \u003cp\u003eMosquito collection should provide detailed information on species composition and \u003cem\u003ePlasmodium\u003c/em\u003e sporozoite infection rates, which may be influenced by the sampling methods used [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Different sampling methods yield varying results regarding species composition, feeding patterns, and infection rates. Outdoor collection techniques, such as pit shelters, are particularly effective for gathering fed mosquitoes to analyze the sources of their blood meals among the mosquitoes that typically avoid resting indoors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Clay pots are an effective tool for collecting resting mosquitoes outdoors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. They are user-friendly, affordable, and readily accessible for large areas, requiring minimal cleaning and maintenance. The data collected through these methods provides valuable insights into transmission risks and mosquito behaviour in outdoor environments, which can guide the development of targeted interventions. Furthermore, it also highlights shifts in mosquito behaviour, indicating a trend toward resting outdoors as a reaction to indoor interventions. Therefore, this study aimed to analyse the species composition, blood meal origins, and infection rates of outdoor resting malaria mosquitoes in West Gojjam, Ethiopia.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDescription of the study area\u003c/h2\u003e \u003cp\u003eThe study was conducted in Dilamo, a rural village in the South Achefer district, West Gojjam Zone, Ethiopia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). South Achefer is 501 km from Addis Ababa and 60 km from Bahir Dar, the capital of the Amhara Regional State. The district is geographically located at 11\u0026deg;50\u0026prime;N latitude and 37\u0026deg;10\u0026prime;E longitude, and its elevation ranges from 1,500 to 2,500 meters above sea level (masl). The district covers an area of 1,183.05 Km\u003csup\u003e2\u003c/sup\u003e and is characterised by high plateaus, mountains and broad valleys suitable for agriculture [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Its annual mean temperature is 23\u0026deg;C, and the mean yearly rainfall is 1,522 mm. South Achefer is endemic to malaria [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eDilamo village was divided into five clusters, each consisting of 30 houses. For this study, two clusters were randomly selected for longitudinal entomological sampling. Ten houses were selected randomly in each cluster. For each household, two clay pots (10\u0026ndash;15 lit capacity) were placed in shaded areas outside the house, within a 10-meter radius. One pot was placed in front of the house, while another was located at the back. Additionally, two clay pots were positioned inside each household, one at each opposite corner. Each pot has a hole with a diameter of 2 cm in the Centre of the base. The hole prevents the pot from retaining water and reduces the possibility of theft.\u003c/p\u003e \u003cp\u003eTen pits were dug under shaded areas within a 10 m radius of the selected houses. Each pit was approximately 1.5 m deep, 1.5 m long, and 1.5 m wide. At a height of 1 m from the bottom of each pit, horizontal holes of about 30 cm deep were made in the pit's wall.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003emosquito collection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMosquito collections were conducted from clay pots and pit shelters twice a month from December 2019 to March 2020. The collection from the clay pots took place between 6:00 and 8:00 a.m. The collectors first placed a mosquito cage over the opening of the pot. Then, they lifted the pot, exposing the opening to the light. By stirring the mosquitoes inside and blowing air through a small hole at the bottom, the mosquitoes would escape the pot and enter the cage. The remaining mosquitoes in the pot were aspirated and transferred to the cage. All debris and organisms in the pot were carefully removed each time.\u003c/p\u003e \u003cp\u003eMosquitoes were collected from constructed pits using handheld aspirators and a torch as a light source [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Their mouths were covered with non-impregnated mosquito nets to prevent mosquitoes from escaping the pit shelters during collection.\u003c/p\u003e\n\u003ch3\u003eSpecies identification\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes caught from clay pots and pit shelters were later identified morphologically as species using a dissecting microscope, following the morphological key [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eAnopheles gambiae\u003c/em\u003e complexes were identified molecularly using primers that target the Internal Transcribed Spacer (ITS) region in Ribosomal DNA (rDNA) to distinguish the species complex [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDetection of blood meal sources and circumsporozoite proteins\u003c/h3\u003e\n\u003cp\u003eThe circumsporozoite protein (CSP) was detected using the Enzyme-Linked Immunosorbent Assay (ELISA) method using the head and thorax of female Anopheles mosquitoes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A direct ELISA was performed according to the procedure outlined by Beier et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] to identify the blood meal sources of freshly fed mosquitoes.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eThe monthly counts of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were represented as percentages, along with descriptive statistics. Fisher's exact test (Clopper-Pearson) was conducted using a two-by-two table to compare the proportions of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes that were fed on human and bovine blood sources. The Human Blood Index (HBI) and Bovine Blood Index (BBI) were calculated as the ratio of blood-sucking mosquitoes that fed on humans and cattle, respectively, to the total number tested. Circum-sporozoite protein level is the number of mosquitoes positive for CSP antigen divided by the total number of mosquitoes tested, expressed as a percentage. SPSS software version 25.0 (SPSS, Chicago, IL, USA) was utilised for data entry, cleaning, and analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003especies composition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 319 female \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes, comprising five species, were collected from Dilamo village in South Achefer District (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among these, \u003cem\u003eAn. demilloni\u003c/em\u003e accounted for 90.9% (290/319), and \u003cem\u003eAn. gambiae\u003c/em\u003e complex 4.4% (14/319).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003especies versus collection method\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAbout 30% of the \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes (96/319) were collected from clay pots, while 70% (223/319) were found in pit shelters (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, no mosquitoes were sampled from clay pots placed indoors.\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\u003cem\u003eAnopheles\u003c/em\u003e mosquito species collected using pit shelters and clay pots in Dilamo village, South Achefer District, West Gojjam Zone, Ethiopia, December 2019 to March 2020.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnopheles\u003c/em\u003e species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePit shelter (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOutdoor clay pots (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. demilloni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e206 (71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e290 (90.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14 (4.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. cinereus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (2.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. garnhami\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1(20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (1.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. pretoriensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1(100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (0.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e223 (70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e319 (100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMonthly variation of\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003especies collections\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe highest number of female \u003cem\u003eAnopheles\u003c/em\u003e species was collected in December from clay pots and pit shelters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After that, the number of collections decreased and reached its lowest point in March.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBlood meal origins of\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOut of 89 freshly fed \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes, 77 (86.5%) were examined for their blood meal origins (Table\u0026nbsp;3). Among these, 74 mosquitoes (96%) tested positive for either bovine or human blood. The overall human blood meal index was 43% (95% Confidence Interval (CI): 31\u0026ndash;55), while the bovine blood meal index was only 6% (95% CI: 1.6\u0026ndash;14). Additionally, the proportion of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes that fed on both humans and bovines (mixed) was 57% (95% CI: 45\u0026ndash;69). \u003cem\u003eAnopheles demilloni\u003c/em\u003e primarily fed on humans (39%) more than on bovine (4%). Additionally, the species had a good proportion of mixed-blood meals (54%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe proportion of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes that fed on human blood was significantly higher than those that fed on bovine blood (Fisher's exact test: χ\u0026sup2; = 25.3; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverall blood meal origins of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes collected using clay pots and pit shelters in Dilamo, South Achefer District, West Gojjam, Ethiopia, December 2019 to March 2020.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAnopheles\u003c/em\u003e species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman fed\u003c/p\u003e \u003cp\u003e(index; 95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBovine fed (index; 95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHuman and bovine fed (index; 95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnidentified (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. demilloni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27 (39: 28\u0026ndash;50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (4: 1.0\u0026ndash;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38 (54: 42\u0026ndash;66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (2.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e s.l.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (33: 1.0\u0026ndash;90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (33.3:1.0\u0026ndash;90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (33: 1.0\u0026ndash;90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. garnhami\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (67: 9\u0026ndash;99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (33.0: 1.0\u0026ndash;90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. cinereus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (43: 31\u0026ndash;55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (6: 1.6\u0026ndash;14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40 (57: 45\u0026ndash;69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3 (4.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn clay pot and pit shelters, the percentage of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes feeding on human, bovine, and mixed blood was comparable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmodium\u003c/b\u003e \u003cb\u003eCSP rates of\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 319 female \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were examined for CSPs, which included 290 \u003cem\u003eAn. demilloni\u003c/em\u003e, 14 \u003cem\u003eAn. gambiae\u003c/em\u003e complex, nine \u003cem\u003eAn. cinereus\u003c/em\u003e, five \u003cem\u003eAn. garnhami\u003c/em\u003e, and one \u003cem\u003eAn. pretoriensis\u003c/em\u003e. None tested positive for \u003cem\u003ePlasmodium\u003c/em\u003e CSPs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eAnopheles demilloni\u003c/em\u003e, the \u003cem\u003eAn. gambiae\u003c/em\u003e complex, \u003cem\u003eAn. cinereus\u003c/em\u003e, \u003cem\u003eAn. garnhami\u003c/em\u003e, and \u003cem\u003eAn. pretoriensis\u003c/em\u003e were collected from Dilamo village of South Achefer district, West-Gojjam, Ethiopia. Among these species, \u003cem\u003eAn. demilloni\u003c/em\u003e was the dominant species and exhibited a higher human blood meal index than bovine. Clay pots placed outside and pit shelters effectively captured \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes for entomological monitoring. However, clay pots placed indoors were unsuccessful in trapping any mosquitoes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnopheles demilloni\u003c/em\u003e was the dominant mosquito species observed, followed by the \u003cem\u003eAn. gambiae\u003c/em\u003e complex. This species is commonly found in highland areas, and a mosquito infected with the \u003cem\u003ePlasmodium\u003c/em\u003e parasite has recently been confirmed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, its capacity to transmit the parasite is still under investigation. This species has also been identified in northern Ethiopia, mainly from pit shelters and outdoor clay pots [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In most studies, \u003cem\u003eAn. gambiae\u003c/em\u003e complex was the dominant species, utilising various collection techniques such as CDC light traps, PSC, pit shelters and clay pots [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Regardless of the collection technique and the \u003cem\u003eAnopheles\u003c/em\u003e species involved, the human blood meal index was significantly higher than the bovine blood meal index. This study contrasts with a similar one conducted in southern Ethiopia, which found a higher bovine blood meal index in mosquitoes using the same collection method [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In our study, a higher mixed blood meal index was observed in \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes. This may be attributed to the proximity of cattle to people's households or situations where cattle share living spaces, resulting in mosquitoes biting indoors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the study area, sheep, donkeys, and cattle are the most common livestock. These animals, particularly cattle and sheep, remain outside until nearly midnight. Afterward, sheep and certain selected cattle come inside to share the home with humans for the night. Additionally, the head of the household and school-aged children often remain outside to tend to the cattle. This practice of cohabiting or spending long hours with animals at night may contribute to a higher prevalence of mixed blood meals. Furthermore, cattle near households could increase the density of zoophilic vectors. To help control these malaria-carrying vectors, treating livestock with insecticides and constructing separate enclosures away from human living spaces is advisable.\u003c/p\u003e \u003cp\u003eThe human blood meal index of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes was high, indicating that humans were exposed to mosquito bites. \u003cem\u003eAnopheles demilloni\u003c/em\u003e showed a relative ability to feed on human blood compared to bovine blood. This increased human exposure to mosquito bites demands interventions to protect against it. Despite a high human blood meal index, the mosquitoes tested negative for \u003cem\u003ePlasmodium\u003c/em\u003e CSPs. This study aligns with previous research conducted in south-central Ethiopia, where many mosquitoes were documented, but none tested positive for CSPs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In most cases, the infection rate of sporozoites in mosquitoes is rare despite the high prevalence of malaria.\u003c/p\u003e \u003cp\u003eThis study has some limitations, including that data were collected over only four months. Consequently, the blood-feeding patterns and infection rates may have been influenced by monthly variations of environmental variables. Additionally, we identified only the blood meal sources of outdoor resting mosquitoes, whereas the blood meal sources of indoor resting mosquitoes and those that bite indoors and outdoors may differ.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe human blood meal index of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes is high, indicating human exposure to mosquito bites. Further research should explore the biting behaviour of Anopheles mosquitoes and their interactions with humans. This understanding will facilitate the implementation of effective control measures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank the community for permitting us to collect mosquitoes from their homes and compounds.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eA. A. designed the study and conducted the fieldwork. A. A. critically reviewed and revised the manuscript. F. M. designed the study, supervised the field and laboratory work, and drafted and revised the manuscript. All authors have read and approved the final manuscript submission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the School of Graduate Studies at Arba Minch University.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data is thoughtfully incorporated within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis study received ethical approval from the Institutional Review Board of Arba Minch University. The homeowner gave consent before installing clay pots and digging pit shelters.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no competing interest.\u003c/p\u003e\n\u003cp\u003eAuthor details\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Biology, Arba Minch University, Arba Minch, Ethiopia.\u0026nbsp;\u003csup\u003e2\u003c/sup\u003eAklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. The effect of malaria control on \u003cem\u003ePlasmodium falciparum\u003c/em\u003e in Africa between 2000 and 2015. Nature. 2015; 526: 207\u0026ndash;11.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWHO. World malaria report. 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Cattle feeding tendency of \u003cem\u003eAnopheles\u0026nbsp;\u003c/em\u003emosquitoes and their infection rates in Aradum village, North Wollo, Ethiopia: an implication for animal-based malaria control strategies. Malar J. 2023;22:81\u003c/li\u003e\n \u003cli\u003eMassebo F, Balkew M, Gebre-Michael T, Lindtjorn B. Entomologic inoculation rates of \u003cem\u003eAnopheles arabiensis\u003c/em\u003e in southwestern Ethiopia. Am J Trop Med Hyg. 2013; 89:466\u0026ndash;73.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEsayas E, Woyessa A, Massebo F. Malaria infection clustered into small residential areas in lowlands of southern Ethiopia. Parasite Epidemiol Control. 2020; 10: e00149.\u003c/li\u003e\n \u003cli\u003eNdenga BA, Mulaya NL, Musaki SK, Shiroko JN, Dongus S, Fillinger U. Malaria vectors and their blood-meal sources in an area of high bed net ownership in the western Kenya highlands. Malar J. 2016;15: 76\u003c/li\u003e\n \u003cli\u003eKenea O, Balkew M, Tekie H, Gebre-Michael T, Deressa W, Loha E, et al. Human-biting activities of \u003cem\u003eAnopheles\u003c/em\u003e species in south-central Ethiopia. Parasite Vectors. 2016;9:527\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Anopheles species composition, Blood meal sources, CSP rates, West Gojjam, Ethiopia","lastPublishedDoi":"10.21203/rs.3.rs-5989388/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5989388/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnderstanding the feeding behaviours and infection rates of malaria-transmitting \u003cem\u003eAnopheles \u003c/em\u003especies is essential for designing effective vector control strategies. Therefore, this study investigated the species composition, blood meal sources, and infection rates of \u003cem\u003eAnopheles\u003c/em\u003emosquitoes in the rural village of Dilamo, West Gojjam, Ethiopia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTen households were randomly selected in a malaria-endemic village. Two clay pots were placed under shade outside each house within a radius of 10 meters, one in front of the house and the other at the back. Two other clay pots were placed inside the same household. Ten pit shelters were built, one at a distance of 10 meters from each house, under shade. Mosquito sampling was done twice a month for four months. \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were identified to species using morphological keys. The \u003cem\u003eAn. gambiae\u003c/em\u003e complex species were molecularly identified. Testing for blood meal sources and circumsporozoite proteins (CSPs) was conducted using an enzyme-linked immunosorbent assay (ELISA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 319 female \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were collected, including the species \u003cem\u003eAn. demilloni,\u003c/em\u003e \u003cem\u003eAn. gambiae\u003c/em\u003e complex, \u003cem\u003eAn. garnhami\u003c/em\u003e, \u003cem\u003eAn. pretoriensis\u003c/em\u003e, and \u003cem\u003eAn. cinereus\u003c/em\u003e. Among these, \u003cem\u003eAn. demilloni\u003c/em\u003ewas the dominant species, comprising 90.9% of the collection (290/319). The \u003cem\u003eAn. gambiae\u003c/em\u003e complex represented only 4.4% of the total (14/319). Out of 89 freshly fed \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes, 77 (86.5%) were examined for the source of their blood meal. Of these, 74 (96%) tested positive for blood from cattle, humans, or both. The overall human blood meal index was 43% (95% CI: 31.1–55.3), while the bovine blood meal index was 6% (95% CI: 1.6–13.9). The prevalence of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes feeding on humans and cattle (mixed feeding) was 57% (95% CI: 44.8–68.9). None of the \u003cem\u003eAnopheles \u003c/em\u003emosquitoes tested positive for \u003cem\u003ePlasmodium falciparum\u003c/em\u003e or \u003cem\u003eP. vivax\u003c/em\u003e-210 CSPs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human blood meal index reflects human exposure to mosquito bites. Future research could investigate whether mosquitoes, along with human behaviours and activities, prefer to bite humans indoors or outdoors.\u003c/p\u003e","manuscriptTitle":"Blood feeding patterns of malaria mosquitoes collected using pit shelters and clay pots in the West Gojjam Zone of Ethiopia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 11:39:42","doi":"10.21203/rs.3.rs-5989388/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-14T20:00:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-11T19:25:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279145755588864852413613698152227727782","date":"2025-04-10T15:39:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-08T13:25:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159430930080569351203297855765050424608","date":"2025-04-07T11:43:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-07T09:48:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-02T04:46:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2025-04-01T16:04:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"397dbcad-87a7-4d81-ad8b-1e9c560c996a","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:02:53+00:00","versionOfRecord":{"articleIdentity":"rs-5989388","link":"https://doi.org/10.1186/s12936-025-05408-4","journal":{"identity":"malaria-journal","isVorOnly":false,"title":"Malaria Journal"},"publishedOn":"2025-05-28 15:56:56","publishedOnDateReadable":"May 28th, 2025"},"versionCreatedAt":"2025-04-10 11:39:42","video":"","vorDoi":"10.1186/s12936-025-05408-4","vorDoiUrl":"https://doi.org/10.1186/s12936-025-05408-4","workflowStages":[]},"version":"v1","identity":"rs-5989388","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5989388","identity":"rs-5989388","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}