Spesies sibling merupakan spesies –spesies yang memiliki kemiripan ciri morfologi serta terjadi isolasi reproduksi dalam suatu populasi. Identifikasi spesies sibling sangat penting dilakukan karena setiap spesies memiliki perbedaan kapasitas vektorial.Pengetahuan yang komprehensif mengenai identitas vektor Anophelespenting dalam upaya pengendalian penyakit malaria berbasis vektor. Tujuan dari penelitian ini adalah untuk mengidentifikasi karakter morfologi An.vagus vagusdan An. vagus limosusasal Desa Bangsring Banyuwangi. Identifikasi morfologi dilakukan dengan mengamati bagian kaki, palpus, proboscis dan sayap dengan menggunakan mikroskop stereo berdasarkan beberapa buku kunci identifikasi. Hasil identifikasi menunjukkan bahwa An. vagusvagusmemiliki karakteristik kaki tidak berbercak, proboscis dengan gelang pucat dan prehumeral sayap dengan pita pucat diantara dua pita gelap. Sedangkan An. vagus limosus memiliki karakteristik kaki tidak berbercak, proboscis keseluruhan gelap dan prehumeral sayap dengan pita gelap seluruhnya.
Since the malaria outbreak in 2011, the breeding place of Anopheles in Bangsring Village on Banyuwangi District has been monitored by District Public Health Office as part of a vector surveillance program. Morphological identification is still a standard tool to observe Anopheles occurrence and diversity, but the presence of cryptic species made it unreliable. In this study, a molecular approach called DNA barcoding technique was used to assist the morphology-based techniques to identify Anopheles species found in Bangsring. The internal transcribed spacer 2 (ITS2) sequence was used as molecular marker. Based on the morphological features, we were able to identify Anopheles (An.) vagus, An. subpictus, An. sundaicus and An. aconitus. ITS2 sequences from the four identified species were then analyzed simultaneously with eighteen reference sequences from NCBI which had a high similarity of 98-100%. The NJ phylogenetic tree formed three major clades, where the two clades as monophyletic clades were An. vagus and An. aconitus. Another clade was formed as polyphyletic clade containing An. subpictus and An. sundaicus. Although An. subpictus and An. sundaicus were placed in the same clade, seven nucleotide differences were observed in their ITS2 sequence. The intra-specific variation of those two species was 0.08 and 0.49%, respectively, while the interspecific variation was 1.39%. Interspecific variation which was higher than the mean intra-specific variation might indicate that An. sundaicus and An. subpictus were a distantly species. However, the value of interspecific variation lower than 3% might also indicate that those species were classified as a complex species. All ITS2 sequences from morphologically identified species had similar results with molecular-based techniques. This result showed that molecular identification using the ITS2 sequence was reliable in supporting morphological identification among closely related anopheline mosquitoes and gave further information about their evolutionary divergence.
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.
The salivary gland proteins of the Anopheles (An.) female mosquito play vital roles in the transmission of Plasmodium into the human host. A comprehensive understanding of Anopheles identity and its related salivary proteins is a key for vector-based malaria control. This research aims to analyse the protein profile and immunogenicity of Anopheles salivary gland proteins from 2 potential Anopheles vectors, An. vagus and An. sundaicus. Female Anopheles are collected from Bangsring village, Banyuwangi Regency by direct-landing collection. Each species are identified by morphological features and internal transcribed spacer 2 (ITS2). Immunogenic proteins were determined by Western Blotting with IgG from people living in the endemic area as primary antibodies. Immunoblotting results showed the presence of 3 immunogenic protein bands with a molecular weight of 34, 46, and 66 kDa in both species. Furthermore, the 99 kDa protein band was present merely in An. sundaicus. Moreover, the result showed that two distinct species have different profiles and immunogenic proteins.
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