Microsoft Word - 7257-Revisi.edited 10.03.2023.SS-manual ref.docx Epidemiology and Society Health Review| ESHR Vol. 5, No. 1, 2023, pp. 41-50 ISSN 2656-6052 (online) | 2656-1107 (print) http://journal2.uad.ac.id/index.php/eshr/index eshr@ikm.uad.ac.id 10.26555/eshr.v5i1.7257 41 Research Article Distribution and behavior of Anopheles maculatus and its potential as a Malaria vector in Indonesia Riyani Setiyaningsih1, Sapto Prihasto1, Fahmay Dwi Ayuningrum1, Arif Suryo Prasetyo1, Mega Tyas Prihatin1, Sekar Negari1, Siti Alfiah1, Lulus Susanti1, Evi Sulistyorini1, Jery Cahyandaru1, Tri wibowo Ambar Garjito2 1 Institute for Disease Vector and Reservoir Research and Development (IVRCRD), Salatiga, Central Java, Indonesia 2 Vector-borne and Zoonotic Diseases Research Group, Research Center for Public Health and Nutrition, National Research and Innovation Agency, Salatiga, Indonesia * Correspondence: riyanisetia@gmail.com. Phone: +62081575529529 Received 06 January 2023; Accepted 11 March 2023; Published 12 March 2023 ABSTRACT Background: Anopheles maculatus is one of the mosquito species that has been confirmed as a malaria vector in Indonesia. The potential of a mosquito as a vector is influenced by its behavior. Information on the distribution and behavior of An. maculatus needs to be carried out to determine the potential for malaria transmission transmitted by the species in an area. The study aimed to obtain information on the distribution, behavior, and potential of maculatus as a malaria vector in several provinces in Indonesia. Method: Mosquito collection was carried out using human-landing collection, animal-baited trap, cattle-bait, light-trap, and resting morning. A survey of mosquitoes was carried out in 29 provinces in Indonesia. Mosquitoes were identified for the species and detected the blood-sucking behavior with an Enzyme-linked immunosorbent assay and the presence of Plasmodium using a Polymerase Chain Reaction. Results: The results showed that An. maculatus was found in the Riau Islands, Lampung, Bangka Belitung, West Java, Central Java, East Java, and Central Sulawesi. Anopheles maculatus has known to suck the blood of humans and animals with a predominance of animals. Anopheles maculatus is also known to suck blood outdoors predominantly. In general, the activity of An. maculatus sucking blood begins around 18.00 in the evening. Central Java Province was the province with the highest density of An. maculatus mosquitoes, thereby increasing the potential for transmission of malaria cases. Conclusion: Anopheles maculatus was spread in Riau Islands, Lampung, Bangka Belitung, West Java, Central Java, East Java, and Central Sulawesi. This species was known to suck the blood of people and humans, and its blood-sucking activity starts around 18.00 in the evening. Keywords: Anopheles maculatus, Malaria, Vector malaria Setiyaningsih et al. (Distribution and behavior of Anopheles maculatus and its potential as a Malaria vector in Indonesia) Vol. 5, No. 1, 2023, pp. 41-50 10.26555/eshr.v5i1.7257 42 INTRODUCTION Anopheles maculatus is a malaria vector species in Indonesia found in some provinces such as Central Java, West Java, DIY, East Java, Bali, Lampung, North Sumatra, West Sumatra, Riau, Jambi, and South Sumatra. 1–4 Anopheles maculatus was a malaria vector in Thailand, the Philippines, Malaysia 5,6, Afghanistan, Pakistan, China, and India.7 The potential of mosquitoes as malaria vectors can be seen from their behavior. The results of research in Thailand and Purworejo Central Java, Indonesia; An. maculatus was known to suck the blood of animals and humans.5,8 The behavior of sucking mosquito blood significantly affects its potential as a malaria vector. Mosquitoes that have the behavior of sucking human blood have the opportunity to be able to transmit Plasmodium from human to human. Another factor that supports mosquitoes in becoming malaria vectors is their long life and resistance to Plasmodium entering their body.9–11 Studies on the distribution and behavior of An. maculatus as a malaria vector need to be carried out considering the importance of this species as a malaria vector in several provinces in Indonesia. Mosquito behavior that influences its potential as a malaria vector is resting behavior and blood-sucking behavior. This was also because there were possible differences in mosquito behavior in locations because each region has different environmental conditions. Based on this background, this study aims to discover the development of An: maculatus' distribution behavior and its potential as a malaria vector in Indonesia. METHOD The research was conducted in 29 provinces in Indonesia. We selected three districts in each province and six sampling points in each district. Each point represented forest, non-forest, and coastal ecosystems near and far from settlements. Sampling was conducted for five days at each point, so with six points in one district, the sampling duration was 30 days.2 A list of sampling locations can be found in Table 1. DKI Jakarta Province was an exception because no forest ecosystem was found, so sampling was conducted at locations representing the DKI Jakarta area. The selected locations were Central Jakarta, West Jakarta, South Jakarta, East Jakarta, North Jakarta, and the Thousand Islands. This study was cross-sectional, and the sample was taken at a particular time. The research sample was An. maculatus which was caught using various research methods in all provinces. The study population was mosquitoes caught in all provinces. The research variables were the number of An. maculatus responded every hour in each province, the number of An. maculatus mosquitoes caught in each method in all provinces, and Plasmodium sp positive in An. maculatus mosquitoes in each province. Mosquitoes were caught using the human landing collection method, livestock bait, Animal- baited Trap (ABT), light trap, and morning resting collection. Mosquitoes caught in each method identified their species using a mosquito identification key.12,13 The time used to catch mosquitoes in each technique was different. A list of fishing times and fishing hours per hour can be seen in Table 2. Setiyaningsih et al. (Distribution and behavior of Anopheles maculatus and its potential as a Malaria vector in Indonesia) Vol. 5, No. 1, 2023, pp. 41-50 10.26555/eshr.v5i1.7257 43 Table 1. Sampling location of An. maculatus in several provinces in Indonesia Number Provinsi City/County Name 1 Papua Biak, Merauke , Sarmi 2 Central Java Pati, Pekalongan, Purworejo 3 South Sumatra Banyuasin, Lahat, Organ Komeling Ilir (OKI) 4 Central Sulawesi Parigi Moutong, Tojo Una Una, Toli-toli 5 Aceh Aceh Timur, Aceh Barat, Pidie 6 West Sumatra Pesisir Selatan, Padang Pariaman, Pasaman Barat 7 Lampung Tanggamus, Lampung Selatan, Pasawaran 8 Bangka Belitung Bangka, Belitung, Bangka Tengah 9 West Java Garut, Subang, Pangandaran 10 East Java Malang, Banyuwangi, Pasuruan 11 Banten Pandeglang. Lebak, Serang 12 Nusa Tenggara Barat Lombok Barat, Bima, Lombok Utara 13 Nusa Tenggara Timur Belu. Ende, Sumba Tengah 14 West Kalimantan Sambas, Ketapang, Kayong Utara 15 South Kalimantan Barito Kuala, Kota Baru, Tanah Laut 16 North Sulawesi Minahasa, Kota Manado, Kota Belitung 17 Southeast Sulawesi Muna, Konawe, Bombana 18 Maluku Maluku Tenggara Barat, Maluku Tenggara, Kepulauan Aru 19 North Maluku Halmahera Tengah, Halmahera Selatan, Pulau Morotai 20 Riau Bengkalis, Dumai, Kepulauan Meranti 21 Jambi Bungo, Sarolangun, Tanjung Jabung Barat 22 Special Region of Yogyakarta Kulon Progo, Gunung Kidul, Bantul 23 Bali Jembrana, Badung, Karangasem 24 Central Kalimantan Gunungmas, Pulang Pisau, Murung Raya 25 South Sulawesi Pangkajene Kepulauan, Bulukumba, Luwu Timur 26 West Papua Raja Ampat, Manokwari, Fak-fak 27 Riau Islands Kota Batam, Bintan, Lingga 28 DKI Jakarta Central Jakarta, West Jakarta, South Jakarta, East Jakarta, North Jakarta, the Thousand Islands. 29 North Kalimantan Bulungan, Nunukan, Tarakan Table 2. The duration and catchment method of Anopheles maculatus Method Catch time Length of catchment Indoor human bait 6 am - 6 pm 40 minute Outdoor human bait 6 am - 6 pm 40 minute Animal baited trap 6 am - 6 pm 15 minute Cattle bait 6 am - 6 pm 15 minute Morning resting 6 pm - 9 pm 3 hour Anopheles maculatus found in all capture methods was prepared by separating the head and thorax from the abdomen and then analyzing its Plasmodium's presence using PCR. Primers used in detecting Plasmodium were rPLU1 (5’ - TCA AAG ATT AAG CCA TGC AAG TGA – Setiyaningsih et al. (Distribution and behavior of Anopheles maculatus and its potential as a Malaria vector in Indonesia) Vol. 5, No. 1, 2023, pp. 41-50 10.26555/eshr.v5i1.7257 44 3’), rPLU5 (5’ - CCT GTT GTT GCC TTA AAC TCC – 3’), rPLU3 (5’ - TTT TTA TAA TAA GGA TAA CTA CTA CGG AAA AGC TGT – 3’), rPLU4 (5’ - TAC CCG TCA TAG CCA TGT TAG GCC AAT ACC – 3’) 14,15 The blood-fed or half-gravid An maculatus mosquitoes will be tested their blood feed using the direct-ELISA method. RESULTS Based on the results of the study showed that An. maculatus was not found in all provinces in Indonesia. The results of the survey in 29 provinces in Indonesia, An. maculatus was distributed in Riau Islands, Lampung, Bangka Belitung, West Java, Central Java, East Java, and Central Sulawesi (Figure 1). In all seven provinces, 141 An. maculatus mosquitoes were collected. The highest density of mosquitoes An. maculatus was found in Central Java Province. Figure 1. Distribution An. maculatus in Indonesia Anopheles maculatus collection in each province showed different fluctuations and densities. Based on the behavior of sucking blood, An. maculatus suck blood starting at 6:00 p.m. in all provinces. However, the An. maculatus activity of sucking blood generally does not occur all night. An. maculatus in Central Java Province was found sucking blood throughout the night with peak density at 21.00-22.00. Fluctuations in mosquito density based on blood-sucking time in each province showed in Figure 2. Setiyaningsih et al. (Distribution and behavior of Anopheles maculatus and its potential as a Malaria vector in Indonesia) Vol. 5, No. 1, 2023, pp. 41-50 10.26555/eshr.v5i1.7257 45 Figure 2. Density fluctuation An. maculatus based on blood-sucking time in Indonesia. The results of mosquito catching by various methods show that, generally, An. maculatus dominant was found to suck blood in animals (96.67%), and only a small percentage were found to suck people's blood outside the home (3.33% ). The distribution and percentage of An. maculatus behavior in sucking blood in each province can be seen in Table 3. Table 3. Blood-sucking behavior of Anopheles maculatus in various provinces in Indonesia Province name The number of mosquitoes by the method ABT CB IHB OHB MR Riau Islands 0 0 0 1 0 Lampung 0 0 0 1 0 Bangka Belitung 0 0 0 1 0 West Java 0 0 0 1 0 Central Java 68 26 0 0 0 East Java 1 0 0 0 0 Central Sulawesi 21 0 0 0 0 Total 90 26 0 4 0 The total base of blood-sucking behavior 116 4 Percentage of mosquitoes sucking animal blood. 96.67 Percentage of mosquitoes sucking human blood. 3.33 DISCUSSION Our study shows that An. maculatus was found in some provinces in Indonesia, for example, in the Riau Islands, Lampung, Bangka Belitung, West Java, Central Java, East Java, and Central Sulawesi. Based on previous studies, An. maculatus was known as a malaria vector in the provinces of Central Java, West Java, DIY, East Java, Bali, Lampung, North Sumatra, West Sumatra, Riau, Jambi, and South Sumatra.2 The previous study also mentioned that An. maculatus be a malaria vector in Laos with An. rampae, An. sawadwongporni, An. 0 5 10 15 20 25 30 18-19 19-20 20-21 21-22 22-23 23-24 24-01 01-02 02-03 03-04 04-05 05-06 N um be r of m os qu ito A n. m ac ul at us (t ai l) Time of collection Riau Islands Lampung Bangka Belitung West Java Central Java East Java Central Sulawesi Information: ABT: Animal-baited trap; CB: Cattle-bait; IHB: Indoor Human-bait; OHB: Outdoor human-bait; MR: Morning resting Setiyaningsih et al. (Distribution and behavior of Anopheles maculatus and its potential as a Malaria vector in Indonesia) Vol. 5, No. 1, 2023, pp. 41-50 10.26555/eshr.v5i1.7257 46 pseudowillmori, An. dravidicus, An. minimus, An. aconitus, An. pampanai, An. harrisoni, An. dirus, An. baimaii, An. nemophilou,16 Peninsula Malaysia, along with An. cracens.17 In addition, An. maculatus was also known as the primary malaria vector in Morong, Bataan, Philippines, along with An. flavirostris.18 The discovery of An. maculatus in several provinces in Indonesia shows the potential for transmission and an increase in cases of malaria transmitted by An. maculatus. East Java Province was a malaria-receptive area because An. maculatus was found, previously confirmed as a malaria vector despite receiving a malaria elimination certificate. Meanwhile, Riau, Lampung, Bangka Belitung, West Java, Central Java, and Sulawesi provinces can transmit malaria because the provinces were still found in malaria-endemic areas.19 The potential of An. maculatus as a malaria vector can be seen from its blood-sucking and resting behavior. Based on the results of studies showing that An. maculatus was known to be dominantly found to suck the blood of livestock, but it was also found to suck human blood. Although it was not dominantly found to suck human blood, contact with humans allows malaria transmission in the local area. This was because most of the regions found by An. maculatus were malaria-endemic areas.19 Studies on China's Hainan Island also show An. maculatus was found to suck human and animal blood. Anopheles maculatus in the area was known to have blood-sucking behavior several times during one gonotrophic cycle. This condition increases the possibility of malaria transmission.20 Results of research in Bangladesh An. maculatus was also known to suck human and animal blood.21 Study in Laos An. maculatus was the dominant species whose behavior was sucking animal blood (98%).16 Study in Pu Teuy Village, Sai Yok Region, Kanchanaburi Province, west-central Thailand, and Morong, Bataan Pilipina An. maculatus was also found to be predominantly sucking the blood of livestock compared to human blood.05,18 Study in Vietnam An. maculatus tends to be zoophilic and predominantly sucks the blood of buffaloes and cows.22 The potential of An. maculatus as a vector was also influenced by its ability to survive when Plasmodium enters and develops in its body. Although the results showed no discovery of Plasmodium in the An. maculatus examination, considering that the species has been confirmed as a malaria vector, the potential for malaria transmission transmitted by the species can occur again if in the environment there are still cases of malaria. Results of studies in Thailand and Myanmar An. maculatus was known to be positive for Plasmodium with a sporozoite index of 1.1%. Anopheles maculatus was known to be the dominant species in the area alongside An. sawadwongporni.23 Studies in Nepal also show An. maculatus was known to be sporozoite positive with An. fluviatile during the spring and found in the vicinity of cages in the forests of Churia Hills and during the summer in the village without spraying.24 Study in northern Central Vietnam. An. maculatus with An. sinensis, An. aconitus, An. harrisoni, An. sawadwongporni, An. peditaeniatus, and An. philippinensis found protein circumsporozoite.22 Based on the study of An. maculatus blood-sucking activity starting at 18:00 in all provinces, with different fluctuations. In general, blood-sucking activities do not occur throughout the night. In contrast to An. maculatus in Central Java Province, it was found that blood-sucking activity occurred throughout the night. This will enlarge contact with humans allowing the transmission of malaria. The results of a study in the Philippines An. maculatus found that the peak of density occurred at 22.00-00.00. The opportunity of An. maculatus as a malaria vector was also influenced by its density. Central Java had the highest density of mosquitoes compared to other provinces. High density has the potential to increase the occurrence of Setiyaningsih et al. (Distribution and behavior of Anopheles maculatus and its potential as a Malaria vector in Indonesia) Vol. 5, No. 1, 2023, pp. 41-50 10.26555/eshr.v5i1.7257 47 contact with humans, thereby increasing the chances of malaria transmission. The results of the research in Purworejo, Central Java, An. maculatus was found to be dominant compared to other species. Some of the species found together with An. maculatus include An. balabacensis (19.76%), An. vagus (11.74%), An. kochi (5.23%), An. barbirostris (3.53%), An. aconitus (3.40%), An. minimus (1.28%), An. flavirostris (1.00%), An. annularis (0.04%), An. tesselatus (0.01%), and An. koliensis (0.01%).8 Based on studies in Thailand, An. maculatus has a high density in the highlands, and the highest density occurs in the rainy season.25 A survey in Bataan Pilipina An. maculatus revealed that peak density occurs at the end of the dry season.18 Based on studies showing that An. maculatus, in general, was found sucking blood outdoors. This behavior causes the potential for malaria transmission transmitted by An. maculatus tends to occur outdoors. Studies in malaria-endemic areas, umphang valley, Tak Province, Western Thailand An. maculatus was also found predominantly found outdoors.26 Studies in sub-Saharan Africa have the potential for malaria transmission to occur outdoors; this was because malaria vectors were found outdoors. The discovery of mosquito blood-sucking activities outdoors can be caused by vector control in the house with insecticidal mosquito nets and indoor residual spraying (IRS).27 Studies on Guinea's Bioko Island show An. gambie was previously found indoors after prolonged application of vector control in the home with insecticidal mosquito nets caused the mosquito to adapt and change its behavior of sucking blood outdoors.28 Studies in Sub-Saharan Africa show the application of insecticidal mosquito nets and the IRS can reduce cases and deaths caused by malaria. However, the application of vector control in the home impacts changes in vector behavior and vector types that play a role in malaria transmission. This will affect the control methods that should be carried out.27 Studies in Uganda of vector control applications with the IRS and insecticidicide mosquito nets, in addition to decreasing vector populations and the average number of sporozoites, also led to changes in the type of vectors that act as malaria vectors. The mosquito that worked as a malaria vector before the application of vector control in the house was An. gambiae. In contrast, the one that acts as a vector after applying vector control was An. arabiensis. The species was widely found outdoors.29 Due to changes in vector behavior, vector control was also carried out indoors and outdoors.30 Another factor that affects An. maculatus can play the role of a vector was the breeding places. In general, An. maculatus was found in ponds with clear, muddy water containing aquatic plants with an environmental situation of shading. An. maculatus was also found around rivers with slow water flow.5,6 The potential of An. maculatus is a malaria vector because apolipoprotein D and cathepsin D proteins were found in the salivary glands in this species.7 CONCLUSION Anopheles maculatus was distributed in the Riau islands, Lampung, Bangka Belitung, West Java, Central Java, East Java, and Central Sulawesi. Anopheles maculatus predominantly sucks the blood of livestock and partially sucks the blood of people, with most of its activity occurring outdoors. Anopheles maculatus generally sucks blood starting around 18:00, and blood-sucking action typically does not happen overnight. Central Java Province had the highest density of mosquitoes and blood-sucking activities at night. Anopheles maculatus was a potential malaria vector in Central Java compared to several other provinces. Setiyaningsih et al. (Distribution and behavior of Anopheles maculatus and its potential as a Malaria vector in Indonesia) Vol. 5, No. 1, 2023, pp. 41-50 10.26555/eshr.v5i1.7257 48 Acknowledgment The authors would like to thank the directors and all coordinators of IVRCRD who provided direction during the data collection until the article writing process. We also thank the researchers, technicians, and administrative team who have assisted in completing the research and all parties involved in the research process (the coordinators, team leaders, and enumerators). Declarations Authors' contribution RS was the main contributor responsible for a technical research team, supervising the research process, analyzing data, compiling research articles, and improving the results of research article revisions. SP was the main contributor to data analysis and the technical research team. MTP is responsible for the technical team of research and data analysis. 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