Anopheline Diversity in Indonesia: An Evaluation of Animal-Baited Sampling Techniques

Indonesia has rich Anopheline (Diptera: Culicidae) mosquito species living in various types of ecosystems. The study was conducted to profile and compare Anopheles diversity, equitability, and dominance in various ecosystems using different animal-based sampling techniques. The present study analyzed a subset of data collected from a nation-wide vector and animal reservoirs survey in 2016. Analyses were restricted to three ecosystem types (forest, nonforest, and coastal areas) in Java and Sumatera Islands. A total of 5,477 Anopheles were collected by using animal-baited (n = 1,909) and animal-baited trap nets (n = 1,978), consisting of 23 Anopheline species. Overall, Anopheles vagus was the most abundant species, followed by An. subpictus and An. barbirostris. Among the three ecosystems, the forest had a higher diversity index (H′ = 1.98), but each ecosystem has its specific predominant species. Compared with the animal-baited method, the Anopheles abundance collected by animal-baited trap nets was two-fold higher. Ecosystem, elevation, and sampling methods were associated with the abundance of female Anopheles (P-value < 0.001). Our findings revealed that Anopheles were found in a different ecosystem, indicating the potential of malaria transmission. This suggests that improved malaria vector surveillance is essential in all types of ecosystem. Furthermore, the study suggested that animal-baited trap nets could be used as the standard method of outdoor resting sampling in Indonesia in addition to the traditional human landing collection approach.

KARAKTERISASI BERBASIS MARKA MOLEKULER ITS2 TERHADAP SUB-SPESIES KOMPLEKS Anopheles vagus vagus DAN Anopheles vagus limosus

The presence of intraspecies variations of An. vagus later categorized as the subspecies of An. vagus vagus and An. vagus limosus, could be an obstacle to the identification process, which is an important step for malaria vector’s competence characterization. Based on morphological identification, those subspecies could be distinguished by the presences of pale scales in prehumeral and pale bands in proboscis. The objective of this research was to compare subspecies complexes of An. vagus morphologically and molecularly using Internal Transcribed Spacer 2 (ITS2). Anopheles samples were collected from Bangsring, Banyuwangi. Their phylogenetic tree was constructed by using NJ method based on their ITS2 sequences. BLAST result showed that An. vagus vagus and An. vagus limosus were similar to An. vagus FJ654649.1 from East Java Indonesia and East Timor based on its 99% homology and their molecular distance. The Neighbour Joining (NJ) tree grouped those subspecies in one clade with a boostrap value of 82%. This subspeciation might be due to the different rates of evolution. ITS2 sequences of An. vagus vagus and An. vagus limosus were submitted to GenBank with the accession number of MW314227.1 and MW319822.1, respectively. Kemunculan variasi intraspesies An. vagus yang kemudian dikategorikan sebagai subspesies An. vagus vagus dan An. vagus limosus menjadi kendala dalam proses identifikasi yang merupakan langkah penting dalam menentukan kompetensi vektor malaria. Berdasarkan karakter morfologi, subspesies tersebut dibedakan dengan adanya sisik pucat pada bagian prehumeral dan pita pucat pada probosis. Penelitian ini bertujuan untuk membandingkan subspesies An. vagus secara morfologis dan molekuler menggunakan Internal Transcribed Spacer 2 (ITS2). Nyamuk Anopheles didapatkan dari Bangsring, Banyuwangi. Konstruksi pohon filogeni dilakukan berdasarkan sekuen ITS2 yang dianalisis menggunakan metode NJ. Hasil BLAST menunjukkan, ITS2 An. vagus vagus dan An. vagus limosus memiliki tingkat homologi 99% dan jarak evolusi molekuler terendah dengan An. vagus FJ654649.1 dari Jawa Timur Indonesia dan Timor Timur. Pohon NJ mengelompokkan subspesies tersebut dalam satu klade dengan nilai boostrap 82%. Hal ini dapat terjadi karena perbedaan kecepatan evolusi yang memungkinkan terjadinya subspesiasi. Urutan basa ITS2 dari An. vagus vagus dan An. vagus limosus telah didaftarkan ke GenBank dengan nomor aksesi MW314227.1 dan MW319822.1.

Mosquito-specific viruses (family Flaviviridae, genus Flavivirus) Diisolasi pada Nyamuk Anopheles vagus di Bali

Mosquito-specific viruses (MSVs) adalah virus yang hanya dapat bereplikasi pada sel nyamuk. Virus ini terdiri dari berbagai genus, salah satunya yang paling banyak ditemukan adalah dari famili Flaviviridae, genus Flavivirus. Namun, data keberadaan dan karakteristik MSVs dan vektornya di Bali saat ini sangat terbatas. Oleh karena itu, pengamatan untuk memperluas penemuan keragaman vektor dan filogenetik MSVs famili Flaviviridae, genus Flavivirus di Bali dilakukan pada tahun 2016-2018. Nyamuk-nyamuk dewasa ditangkap menggunakan light trap dan dikelompokkan berdasarkan spesies. Isolasi dan propagasi virus dilakukan pada galur sel C6/36 dan baby hamster kidney-21 (BHK-21). Identifikasi virus dilakukan dengan menggunakan one step reverse-transcriptase polymerase chain reaction (RT-PCR). Terdapat dua pool yang berasal dari nyamuk Anopheles vagus menampakan cythopathic effect (CPE) hanya pada galur sel C6/36 dari total 158 pool. Virus yang diisolasi memiliki persentase identity sekuen nukleotida tertinggi 97% dan sekuen asam amino 96% dengan virus Culex theileri Flavivirus isolat JKT-8650 yang diisolasi pada tahun 1981. Selanjutnya, virus dinamakan Mosquito Flavivirus Isolate Bali (MFB) dengan accession numbers KY995166 dan KY290258. Analisis filogenetik menunjukan bahwa MFB berada satu kluster dengan Culex theileri Flavivirus (CTFV) dari Indonesia, Culex Flavivuruses-Myanmar, Culex theileri Flavivirus-Portugal, dan Mosquito Flavivirus-Turki. Terdapat delapan nukelotida dan enam asam amino yang berbeda antara MFB dan CTFV Indonesia. Pada penelitian ini dapat disimpulkan bahwa MSVs dari famili Flaviviridae, genus Flavivirus berhasil diisolasi dari nyamuk An. vagus di Bali.

Distribution and The Habitat Characteristics of Anopheles vagus (Diptera: Culicidae) Larvae at Paddy Fields in The Vicinity of Dramaga IPB University Campus Dramaga Bogor West Java

Dramaga sub-district is an area that has rice fields close to buffalo stalls and a population of wild Macaca fascicularis as natural hosts for several species of Plasmodium. This causes the Dramaga sub-district to become a potential vector habitat for malaria, including Anopheles vagus. This study aims to analyze distribution of An. vagus larval stage in 4 villages (Cikarawang, Babakan, Margajaya, and Ciherang) in Dramaga subdistrict which have paddy field areas. Larvae were collected using a dipper, and each collection sites of the larvae habitat were marked using Global Positioning System. The physical characteristics of the water habitat measured were temperature, pH, total dissolved solids and the electrical conductivity of the water. Pearson's correlation test was used to analyze the relationship between the number of larvae An. vagus with each of the parameters water physical characteristics. The results of this study obtained 309 An. vagus larvae from the 27 collection points. Larvae of An. vagus was found in 3 villages (i.e. Cikarawang, Babakan and Ciherang), but was not found in Margajaya village. The habitat characteristics of An. vagus in paddy fields showed a water temperature of 26.6-31.7oC, pH 6.40-8.10, the total dissolved solid 36-285 ppm, and the electrical conductivity 72-262 μm/cm. The Pearsons correlation test results did not show a significant difference between the number of An. vagus larvae and each parameter (r= -0.26, 0.13, -0.15 and -0.16, respectively). The presence of An. vagus in this area could be considered in malaria vector control programs.

Aedes aegypti as potential vector of filariasis in Pekalongan, Central Java Province, Indonesia

Background: The filariasis elimination program in Indonesia has been conducted, but new cases and some chronic cases are still often found. Objective: This study aims to determine levels of endemicity and to identify filarial worm species in filariasis cases and s and their surrounding communities by using microscopic examination, polymerase chain reaction (PCR), and to examine levels of infection in vectors mosquito by surgery and PCR. Also to to determine that Ae. aegypti can act as vector of filariasis. Methods: This study was conducted at 10 locations in Pekalongan Regency, Central Java Province, with a cross sectional design. Intravenous blood sampling was conducted on 102 respondents consisting of 10 elephantiasis patients and 92 non-elephantiasis patients at night, starting at 8 pm, then examined microscopically and PCR. Mosquitoes in this study were collected by using a human landing collection method for 12 hours from 6 pm to 6 am by volunteers. Artificial infection of microfilaria W. bancrofti was held against Cx. quinquefasciatus and Ae.aegypti from laboratory collection. Results: Results of this study found that there were 5.729 of mosquitos, consisting of 8 species, namely Culex quinquefasciatus, Culex vishnui, Culex tritaeniorhynchus, Aedes aegypti, Aedes albopictus, Anopheles subpictus, Anopheles vagus, and Armigeres kesseli. Microfilarial (mf) rate was 0.89%, and and the blood PCR showed infection rate of 3.92% and the blood PCR showed infection rate of 3.92%. No larva was found in female mosquito dissection. The PCR results showed that the infection rate was 9.10% in Ae. aegypty pool respectively. Artificial infection results was negative both dissecting microscopis and PCR. Conclusion: This study revealed that the locations were low of filariasis endemicity. The mf rate was less than 1%, and there was a moderate density to high density of microfilaria in the patients. The low level of infection rates in mosquito is suggested as an alert to its potential transmission.

Anopheles Mosquito Species in Betung Bedarah, Tebo Regency, Jambi Province

Background: Indonesia is a tropical country which is a good breeding ground for mosquitoes. Mosquitoes are still one of arthropods that contribute to occurrence of infectious diseases, including malaria, which is a disease that is a health problem in Jambi Province.Objective: To determine types of Anopheles mosquito spesies in Betung Bedarah Tebo, Jambi.Methods: A discriptif study to identifed distribution of mosquitoes in Betung Bedarah, Tebo Regency used human landing collection and resting collection methods. Collection of mosquito samples used an aspirator and light trap. Analysis used tabulation.Results: Of the 64 mosquitoes collected, 25% were Anopheles mosquitoes which consisted of 43.7% Anopheles barbirostris, 25% Anopheles vagus, 18.7% Anopheles aconitus, and 12.6% Anopheles kochi.Conclusion: Most of mosquito species collected at Betung Berdarah Tebo were Anopheles barbirostris.

Activity of Cytochrome P450 Monooxygenase (CYPs) Metabolic Enzymes as Markers of Insecticide Resistance in Anopheles vagus Muara Enim Mosquitoes, Indonesia

A B S T R A C TThe use of synthetic pyrethroids in insecticide-treated bed nets (LLINs) and sprayform (IRS) has been carried out since 2012 in Muara Emil Village and since 2016 inall villages in Tanjung Agung District, Muara Enim Regency. Biochemical resistancecan occur to the enzymes that play a role in detoxifying the insecticide. This studyaims to identify whether there is an increase in the levels of the enzyme CytochromeP450 monooxygenase (CYPs) through the enzyme biochemical test on the malariavector mosquito Anopheles vagus originating from Pagar Dewa Village and MuaraEmil Village, Muara Enim Regency. The sample of female Anopheles mosquitoes wasnot full. Blood was taken at night using the resting collection method. Mosquitoesthat have been identified as Anopheles vagus species are used to check CYPs enzymelevels using the ELISA method. The results of the biochemical test showed thatenzyme levels increased from the Muara emil and Pagar Dewa village mosquitoeswith a percentage of 71% and 61.53% (with cut off point OD> 0.165). The value ofCYPs enzyme levels for mosquitoes from Muara Emil village was higher than that ofthe mosquitoes from Pagar Dewa Village (mean 0.005027 ± 0.007). The increasedactivity of the CYPs enzyme plays a role in the detoxification of synthetic pyrethroidinsecticides that can cause resistance. The high levels of enzymes from the villagesof Muara Emil and Desa Farah Dewa are in line with intensive insecticide exposurein eradicating malaria vectors in the highest malaria transmission areas in MuaraEnim Regency.

Effect of morphology on larvicidal activity of chemically synthesized zinc oxide nanoparticles against mosquito vectors

We report the larvicidal impacts of four different morphologies of zinc oxide nanoparticles (ZnO NPs) [star-shaped (S), needle-like (N), plate-like (P), and cubical (C)] on mosquito larvae of Aedes albopictus and Anopheles vagus.