Deteksi Gen Kdr pada Nyamuk Anopheles di Kabupaten Maluku Tenggara Barat

Malaria is still a health problem in Indonesia, particularly in Eastern part of Indonesia. The use of LLIN insecticide bed nets is one of the efforts to reduce the malaria morbidity rate by protecting human from malaria vector bites. The Anopheles flavirostris, Anopheles barbirostris, and Anopheles subpictus mosquitoes are three of the species reported as malaria vectors in West-Southeast Maluku Regency. The aim of this research was to detect the kdr gene in An. flavirostris, An. barbirostris, and An.subpictus mosquitoes collected from Alusi Kelaan village, West-Southeast Maluku Regency. The research was conducted at the Papua Biomedical Research and Development Center, in June 2016. A total of six An. flavirostris, 42 An. barbirostris, and 24 An. subpictus were pooled separately for genomic DNA extraction. The sample used was the An. flavirostris, An. barbirostris, and An. subpictus that survived after the impregnated paper test. The kdr gene detection was carried out using quantitative PCR (qPCR) focused on points V1010 and L1014. The results showed that there were no kdr mutant strains in the An. flavirostris, An. barbirostris, and An. subpictus. These results indicated that the sensitivity of pyrethroid insecticides contained in LLIN mosquito nets to An. flavirostris, An. barbirostris, and An. subpictus mosquitoes was not decreased in West Southeast Maluku Regency.

Characterizations of Larval Gut Bacteria of Anopheles Subpictus Grassi and Their Role in Mosquito Development in Hooghly, West Bengal, India.

Malaria is a serious vector borne disease transmitted by different species of Anopheles mosquitoes. The present study was aimed to isolate & characterize the bacterial flora from the gut of An. subpictus larvae prevalent in Hooghly and explore their roles in host survival and development. Mosquito larvae and adults were collected from field and were maintained in laboratory. Bacterial load in the larval midgut was determined, predominant strains were isolated and characterized by polyphasic approach. Role of these bacteria in larval survival & development were assayed. Bacterial load in the gut of larvae was found to vary in field collected and lab reared mosquitoes in different seasons. Morphological, bio-chemical and molecular analysis explored four common bacterial isolates namely Bacillus subtilis, Bacillus pumilus, Bacillus cereus & Proteus vulgaris in the larval gut throughout the year. Larval survival rate was greatly reduced (0.06) & time of pupation was prolonged (17.8±0.57) in absence of their gut bacteria. Total tissue protein (7.78±0.56), lipid (2.25±0.19) & carbohydrate (16.5±0.79) contents of larvae and body weight & wing length of adult male (0.17±0.02 & 1.74±0.43) & female (0.19±0.02 & 1.99±0.46) mosquitoes were also found to be greatly reduced in the absence of gut bacteria. Developmental characteristics were restored with the introduction of culture suspension of all four resident gut bacterial isolates. Present study indicates that the mosquitoes solely depend on their gut bacteria for their survival & development. So, manipulation or control of this gut bacterial communities might inhibit survival & development of vector mosquitoes.

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.

Compensatory Base Changes Reveal Sexual Incompatibility among Members of the Anopheles subpictus Sensu Lato (Diptera: Culicidae) Species Complex in Sri Lanka

The mosquito Anopheles (Cellia) subpictus sensu lato (s.l.) is a major secondary vector of malaria in Sri Lanka. The sibling species composition in this species complex in Sri Lanka remains debatable. Compensatory base changes (CBCs) in the secondary structures of internal transcribed spacer 2 (ITS2) are reliable sources to predict sexual incompatibility among closely related species. The objective of the present study was to investigate the An. subpictus s.l. populations in Sri Lanka using the CBC analysis. Mosquito DNA was amplified and sequenced for the ITS2 region. The sequences were annotated using ITS2 Database. ITS2 secondary structures were constructed and analyzed for CBCs using various bioinformatics tools. The ITS2 regions consisted of two different lengths, 575 bp and 480 bp. The two CBCs and three hemi CBCs identified in the present study suggest that there may be at least two sexually incompatible sibling species. In conclusion, it is likely that there may be only two reproductively isolated sibling species in the An. subpictus species complex in Sri Lanka. However, due to high divergence of ITS2 in these species, it is reasonable to assume that they may be undergoing a speciation event to separate as a distinct species.

Identification of Anopheles Mosquito Species as Malaria Vector In Riau, Indonesia

Malaria merupakan penyakit infeksi yang sering terjadi di negara tropis dan sub tropis, termasuk Indonesia. Provinsi Riau termasuk dalam kategori insidensi rendah dimana 58% kabupaten telah menyandang status eliminasi malaria tahun 2016. Namun demikian daerah tersebut masih berpotensi menjadi daerah reseptif karena keberadaan vektor malaria yaitu nyamuk Anopheles yang terdiri dari beragam spesies. Penelitian ini bertujuan mengidentifikasi spesies nyamuk Anopheles dari 4 kabupaten di Provinsi Riau yaitu Kabupaten Indragiri Hilir, Rokan Hilir, Dumai dan Indragiri Hulu. Sampel diambil pada malam hari yang hinggap pada hewan maupun manusia yang dilanjutkan dengan identifikasi spesies secara morfologis. Observasi lingkungan dilakukan pada pagi hari. Terdapat tiga spesies Anopheles yang ditemukan yaitu dari sejumlah 219 ekor nyamuk terdapat 119 Anopheles sundaicus (54,34 %), 99 Anopheles subpictus (45,20%)and 1 Anopheles balabacensis (0,46 %). Terdapat dua lokasi yang menjadi tempat perindukan utama yaitu rawa dan genangan air.

Molecular forms of Anopheles subpictus and Anopheles sundaicus in the Indian subcontinent

Background Anopheles subpictus and Anopheles sundaicus are closely related species, each comprising several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of these species complexes. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance. Methods DNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II (COII) of morphologically identified specimens of two nominal species, An. subpictus sensu lato (s.l.) and An. sundaicus s.l., collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses. Results Molecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. and three molecular forms of An. subpictus s.l. (provisionally designated as Form A, B and C) in the Indian subcontinent. Phylogenetic analyses revealed two distinct clades: (i) subpictus clade, with a single molecular form of An. subpictus (Form A) prevalent in mainland India and Sri Lanka, and (ii) sundaicus clade, comprising of members of Sundaicus Complex, two molecular forms of An. subpictus s.l. (Form B and C), prevalent in coastal areas or islands in Indian subcontinent, and molecular forms of An. subpictus s.l. reported from Thailand and Indonesia. Based on the number of float-ridges on eggs, all An. subpictus molecular Form B were classified as Species B whereas majority (80%) of the molecular Form A were classified as sibling species C. Fixed intragenomic sequence variation in ITS2 with the presence of two haplotypes was found in molecular Form A throughout its distribution. Conclusion A total of three molecular forms of An. subpictus s.l. and two molecular forms of An. sundaicus s.l. were recorded in the Indian subcontinent. Phylogenetically, two forms of An. subpictus s.l. (Form B and C) prevalent in coastal areas or islands in the Indian subcontinent and molecular forms reported from Southeast Asia are members of Sundaicus Complex. Molecular Form A of An. subpictus is distantly related to all other forms and deserve a distinct specific status.

Molecular forms of Anopheles subpictus s.l. and Anopheles sundaicus s.l. in the Indian subcontinent

BackgroundAnopheles subpictus s.l. and Anopheles sundaicus s.l. are closely related species, each comprising of several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of An. subpictus complex. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance.MethodsDNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II of morphologically identified specimens of two nominal species, An. subpictus s.l. and An. sundaicus s.l. collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses.ResultsMolecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. (identified as An. epiroticus s.s. and An. sundaicus D) and three molecular forms of An. subpictus s.l. (provisionally designated as Form A, B and C) in the Indian subcontinent. Phylogenetic analyses revealed two distinct clades: (i) subpictus clade, with a single molecular form of An. subpictus (Form A) prevalent in mainland India and Sri Lanka, and (ii) sundaicus clade, comprising of members of Sundaicus Complex., two molecular forms of An. subpictus s.l., (Form B and C) prevalent in coastal areas or islands, and molecular forms reported from Thailand and Indonesia. Based on the number of float-ridges on eggs, all An. subpictus molecular Form B were classified as Species B whereas majority (80%) of the molecular Form A were classified as sibling species C. Fixed intragenomic sequence variation in ITS2 with the presence of two haplotypes was found in molecular Form A throughout its distribution.ConclusionA total of three molecular forms of An. subpictus s.l. and two molecular forms of An. sundaicus s.l. were recorded in the Indian subcontinent. Phylogenetically, two forms of An. subpictus s.l., (Form B and C) prevalent in coastal areas or islands in the Indian subcontinent and molecular forms reported from Southeast Asia are members of Sundaicus Complex. Molecular Form A of An. subpictus is distantly related to all other forms and deserve a distinct specific status. Presence of An. epiroticus in Indian territory is recorded for the first time.