Correction: Anopheles sundaicus complex and the presence of Anopheles epiroticus in Indonesia.

[This corrects the article DOI: 10.1371/journal.pntd.0008385.].

Anopheles sundaicus complex and the presence of Anopheles epiroticus in Indonesia.

Anopheles sundaicus s.l. is an important malaria vector primarily found in coastal landscapes of western and central Indonesia. The species complex has a wide geographical distribution in South and Southeast Asia and exhibits ecological and behavioural variability over its range. Studies on understanding the distribution of different members in the complex and their bionomics related to malaria transmission might be important guiding more effective vector intervention strategies. Female An. sundaicus s.l. were collected from seven provinces, 12 locations in Indonesia representing Sumatra: North Sumatra, Bangka-Belitung, South Lampung, and Bengkulu; in Java: West Java; and the Lesser Sunda Islands: West Nusa Tenggara and East Nusa Tenggara provinces. Sequencing of ribosomal DNA ITS2 gene fragments and two mitochondrial DNA gene markers, COI and cytb, enabled molecular identification of morphologically indistinguishable members of the complex. Findings allowed inference on the distribution of the An. sundaicus s.l. present in Indonesia and further illustrate the phylogenetic relationships of An. epiroticus within the complex. A total of 370 An. sundaicus s.l specimens were analysed for the ITS2 fragment. The ITS2 sequence alignment revealed two consistent species-specific point mutations, a T>C transition at base 479 and a G>T transversion at base 538 that differentiated five haplotypes: TG, CG, TT, CT, and TY. The TG haplotype matched published An. epiroticus-indicative sequences from Thailand, Vietnam and peninsular Malaysia. The previously described insertion event (base 603) was observed in all identified specimens. Analysis of the COI and cytb genes revealed no consistent nucleotide variations that could definitively distinguish An. epiroticus from other members in the Sundaicus Complex. The findings indicate and support the existence of An. epiroticus in North Sumatra and Bangka-Belitung archipelago. Further studies are recommended to determine the full distributional extent of the Sundaicus complex in Indonesia and investigate the role of these species in malaria transmission.

Using barrier screens to characterize mosquito composition, flight activity, and abdominal status in South Lampung, Indonesia.

BACKGROUND: Mosquito sampling methods target different aspects of mosquito behavior and are subject to trap and location specific biases. The barrier screen sampling method was developed and tested to sample free-flying, blood-fed, and host-seeking mosquitoes. During a pilot study, this method was useful in obtaining an unbiased sample of mosquitoes flying between outdoor larval habitats, and sites where blood meals were obtained. However, a relatively small number of blood-fed Anopheles mosquitoes were collected in Indonesia during the pilot study. The sampling method was extended in South Lampung, Indonesia, to enable the collection of blood-fed mosquitoes. This study aimed to intercept mosquitoes flying between human habitations and larval habitats with a barrier screen and to characterize mosquito composition, flight characteristics (direction, height and time), abdominal status, and parity. RESULTS: Barrier screens intercepted 15 different mosquito species in South Lampung: eight Anopheles spp. and seven Culex spp. Species compositions varied among the villages in South Lampung. About 15% of Anopheles spp. caught were blood-fed, of which 28.2% of those tested had fed on humans. This is the first time human blood-fed anophelines have been collected in Indonesia using barrier screens. Blood meals identified included cow, dog, goat, and human, as well as mixed blood meals. Activity of unfed An. subpictus, the primary vector collected, flying towards human habitations peaked between 20:00-12:00 h, with a slow decline in activity until 18:00 h. Unfed and fed An. sundaicus, had a different activity profile compared to An. subpictus. Other species demonstrated varied peak activity times, with earlier activity occurring as a general trend. For the Anopheles mosquitoes collected, 55.5% were collected below 0.5 m and 83.9% were captured resting < 1 m from the ground. Parity dissections enabled age structure by species, which revealed species-specific traits such as nulliparous An. subpictus being more active early in the night relative to An. sundaicus. CONCLUSIONS: This study demonstrates that barrier screens are an effective mosquito sampling method that can be used to gain insights into local mosquito species composition, flight characteristics (direction, height and time), abdominal status, and parity.

Comparative evaluation of anopheline sampling methods in three localities in Indonesia.

BACKGROUND: The effectiveness of vector control efforts can vary based on the interventions used and local mosquito behaviour and adaptability. In many settings, biting patterns of Anopheles mosquitoes can shift in response to interventions targeting indoor-biting mosquitoes, often resulting in higher proportions of mosquitoes feeding outside or at times when people are not protected. These behaviourally resistant mosquitoes have been shown to sustain residual malaria transmission and limit control efforts. Therefore, it is important to accurately sample mosquitoes to understand their behaviour. METHODS: A variety of traps were evaluated in three geographically diverse sites in malaria-endemic Indonesia to investigate local mosquito feeding behaviour and determine effective traps for surveillance. RESULTS: Eight traps were evaluated in three sites: Canti village, Lampung, Kaliharjo village, Purworejo, and Saketa village, Halmahera, Indonesia, including the gold standard human landing collection (HLC) and a variety of traps targeting host-seeking and resting mosquitoes both indoors and outdoors. Trapping, using indoor and outdoor HLC, the Ifakara tent trap C, goat and human-occupied tents, resting pots and boxes, and CDC miniature light traps was conducted for 16 nights in two sites and 8 nights in a third site, using a Latin square design. Trap efficacy varied by site, with outdoor HLC yielding the highest catch rates in Canti and Kaliharjo and a goat-baited tent trap proving most effective in Saketa. In Canti village, anthropophilic Anopheles sundaicus were caught indoors and outdoors using HLCs, peaking in the early morning. In Kaliharjo, a variety of mosquitoes were caught, mostly outdoors throughout the night. HLC was ineffective in Saketa, the only site where a goat-baited tent trap was tested. This trap was effective in catching zoophilic vectors outdoors before midnight. CONCLUSIONS: Different trapping methods were suitable for different species, likely reflecting differences in behaviour among species. The three villages, each located on a different island in the Indonesian archipelago, contained mosquito populations with unique behaviours. These data suggest that the effectiveness of specific vector monitoring and control measures may vary by location.

Host attraction and biting behaviour of Anopheles mosquitoes in South Halmahera, Indonesia.

BACKGROUND: Indonesia is home to a variety of malaria vectors whose specific bionomic traits remain largely uncharacterized. Species-specific behaviours, such as host feeding preferences, impact the dynamics of malaria transmission and the effectiveness of vector control interventions. METHODS: To examine species-specific host attraction and feeding behaviours, a Latin square design was used to compare Anopheles mosquitoes attracted to human, cow, and goat-baited tents. Anopheles mosquitoes were collected hourly from the inside walls of each baited tent. Species were morphologically and then molecularly identified using rDNA ITS2 sequences. The head and thorax of individual specimens were analysed for Plasmodium DNA using PCR. Bloodmeals were identified using a multiplex PCR. RESULTS: A total of 1024, 137, and 74 Anopheles were collected over 12 nights in cow, goat, and human-baited tents, respectively. The species were identified as Anopheles kochi, Anopheles farauti s.s., Anopheles hackeri, Anopheles hinesorum, Anopheles indefinitus, Anopheles punctulatus, Anopheles tessellatus, Anopheles vagus, and Anopheles vanus, many of which are known to transmit human malaria. Molecular analysis of blood meals revealed a high level of feeding on multiple host species in a single night. Anopheles kochi, An. indefinitus, and An. vanus were infected with Plasmodium vivax at rates comparable to primary malaria vectors. CONCLUSIONS: The species distributions of Anopheles mosquitoes attracted to human, goat, and cow hosts were similar. Eight of nine sporozoite positive samples were captured with animal-baited traps, indicating that even predominantly zoophilic mosquitoes may be contributing to malaria transmission. Multiple host feeding and flexibility in blood feeding behaviour have important implications for malaria transmission, malaria control, and the effectiveness of intervention and monitoring methods, particularly those that target human-feeding vectors.

Existence of the rdl mutant alleles among the anopheles malaria vector in Indonesia.

BACKGROUND: The gamma-aminobutyric acid (GABA) receptor-chloride channel complex is known to be the target site of dieldrin, a cyclodiene insecticide. GABA-receptors, with a naturally occurring amino acid substitution, A302S/G in the putative ion-channel lining region, confer resistance to cyclodiene insecticides that includes aldrin, chlordane, dieldrin, heptachlor, endrin and endosulphan. METHODS: A total of 154 mosquito samples from 10 provinces of malaria-endemic areas across Indonesia (Aceh, North Sumatra, Bangka Belitung, Lampung, Central Java, East Nusa Tenggara, West Nusa Tenggara, West Sulawesi, Molucca and North Molucca) were obtained and identified by species, using morphological characteristic. The DNA was individually extracted using chelex-ion exchanger and the DNA obtained was used for analyses using sequencing method. RESULTS: Molecular analysis indicated 11% of the total 154 Anopheles samples examined, carried Rdl mutant alleles. All of the alleles were found in homozygous form. Rdl 302S allele was observed in Anopheles vagus (from Central Java, Lampung, and West Nusa Tenggara), Anopheles aconitus (from Central Java), Anopheles barbirostris (from Central Java and Lampung), Anopheles sundaicus (from North Sumatra and Lampung), Anopheles nigerrimus (from North Sumatra), whereas the 302 G allele was only found in Anopheles farauti from Molucca. CONCLUSION: The existence of the Rdl mutant allele indicates that, either insecticide pressure on the Anopheles population in these areas might still be ongoing (though not directly associated with the malaria control programme) or that the mutant form of the Rdl allele is relatively stable in the absence of insecticide. Nonetheless, the finding suggests that integrated pest management is warranted in malaria-endemic areas where insecticides are widely used for other purposes.

Survey of Anopheles mosquitoes (Diptera:Culicidae) in West Sumba District, Indonesia.

In August 2007, mosquitoes were collected using four different collection methods at 2 upland interior and 2 coastal villages in West Sumba District, East Nusa Tenggara Province, Indonesia. Methods included human-baited and unbaited tent and malaise traps, human-landing collections (HLC), and unbaited CDC light traps. Mosquitoes were identified to species by morphological characters and all anophelines were tested for malaria circumsporozoite protein (CSP) using an enzyme-linked immunosorbent assay (ELISA). During six trap nights, 4,174 Anopheles mosquitoes belonging to 13 species were captured and identified: An. aconitus, An. annularis, An. barbirostris, An. flavirostris, Hyrcanus Group species, An. indefinitus, An. kochi, An. leucosphyrus group, An. maculatus s.l., An. subpictus s.l., An. sundaicus s.l., An. tessellatus, and An. vagus. Of potential disease vectors, An. annularis, An. subpictus, and An. vagus were the most frequently collected species in the upland interior sites, whereas An. sundaicus, An. subpictus, and An. vagus were most commonly found along the coast. The predominant species from evening human-landing collections (mosquitoes per human) were An. subpictus and An. vagus in the upland interior and An. sundaicus along the coast. All mosquitoes were non-reactive for Plasmodium CSP. One specimen of the An. leucosphyrus group was captured from indoor HLC in Tenateke Village, an upland interior location. This finding appears to represent a new collection record for Sumba Island.

Detection of 1014F kdr mutation in four major Anopheline malaria vectors in Indonesia.

BACKGROUND: Malaria is a serious public health problem in Indonesia, particularly in areas outside Java and Bali. The spread of resistance to the currently available anti-malarial drugs or insecticides used for mosquito control would cause an increase in malaria transmission. To better understand patterns of transmission and resistance in Indonesia, an integrated mosquito survey was conducted in three areas with different malaria endemicities, Purworejo in Central Java, South Lampung District in Sumatera and South Halmahera District in North Mollucca. METHODS: Mosquitoes were collected from the three areas through indoor and outdoor human landing catches (HLC) and indoor restinging catches. Specimens were identified morphologically by species and kept individually in 1.5 ml Eppendorf microtube. A fragment of the VGSC gene from 95 mosquito samples was sequenced and kdr allelic variation determined. RESULTS: The molecular analysis of these anopheline mosquitoes revealed the existence of the 1014F allele in 4 major malaria vectors from South Lampung. These species include, Anopheles sundaicus, Anopheles aconitus, Anopheles subpictus and Anopheles vagus. The 1014F allele was not found in the other areas. CONCLUSION: The finding documents the presence of this mutant allele in Indonesia, and implies that selection pressure on the Anopheles population in this area has occurred. Further studies to determine the impact of the resistance allele on the efficacy of pyrethroids in control programmes are needed.

Bionomics of Anopheles spp. (Diptera: Culicidae) in a malaria endemic region of Sukabumi, West Java, Indonesia.

A 15-month bionomic study of Anopheles species was conducted in two ecologically distinct villages (coastal and upland) of Sukabumi District, West Java, Indonesia from June 2006 to September 2007. Mosquitoes were captured using human-landing collections at both sites. During the study, a total of 17,100 Anopheles mosquitoes comprising 13 Anopheles species were caught: 9,151 at the coastal site and 7,949 at the upland site. Anopheles barbirostris, Anopheles maculatus, and Anopheles vagus were the predominant species caught at the coastal site, and Anopheles aconitus, Anopheles barbirostris, and An. maculatus predominated in the upland site. Overall, species were exophagic at both sites, but there was variation between species. Anopheles aconitus was endophagic at the coastal site, exophagic at the upland site, collected most often in April 2007 and had a peak landing time between 22:00 and 23:00. Anopheles sundaicus was only collected at the coastal site, exophagic, collected most often in October 2006, and had a peak landing time between 19:00 and 20:00. Potential malaria vector species such An. aconitus, An. maculatus, and An. sundaicus were present throughout the year. None of the 7,770 Anopheles tested using CSP-ELISA were positive for malaria, although the risk for malaria outbreaks in Sukabumi district remains high.

Remotely-sensed land use patterns and the presence of Anopheles larvae (Diptera: Culicidae) in Sukabumi, West Java, Indonesia.

Land use patterns and the occurrence of Anopheles species larvae were studied in Sukabumi District, West Java, Indonesia, from October 2004 to September 2005. Two land use maps derived using remote sensing were used. One map derived from Quickbird satellite images of 150 km2 of the Simpenan and Ciemas subdistricts (106 degrees 27' 53"-106 degrees 38' 38" E and 6 degrees 59' 59"-7 degrees 8' 46" S) in Sukabumi and one using ASTER images covering 4,000 km2 of Sukabumi District from 106 degrees 22' 15"-107 degrees 4' 1" E and 6 degrees 42' 50" - 7 degrees 26' 13" S. There was a total of 11 Anopheles spp. collected from 209 sampling locations in the area covered by the Quickbird image and a total of 15 Anopheles spp. collected from 1,600 sampling locations in the area covered by the ASTER map. For the area covered by the land use maps, ten species were found to have statistically positive relationships between land use class and species presence: Anopheles aconitus, An. annularis, An. barbirostris. An. flavirostris, An. insulaeflorum, An. kochi, An. maculatus, An. subpictus, An. sundaicus, and An. vagus. Quickbird and ASTER satellite images both produced land maps that were adequate for predicting species presence in an area. The land use classes associated with malaria vector breeding were rice paddy (An. aconitus, An. subpictus), plantation located near or adjacent to human settlements (An. maculatus), bush/shrub (An. aconitus, An. maculatus, An. sundaicus), bare land, and water body land use on the coast located < or = 250 m of the beach (An. sundaicus). Understanding the associations of habitat and species in one area, predictions of species presence or absence can be made prior to a ground survey allowing for accurate vector survey and control planning.

Environmental factors associated with spatial and temporal distribution of Anopheles (Diptera: Culicidae) larvae in Sukabumi, West Java, Indonesia.

A 12-mo ecological study of the spatial-temporal distribution of immature stages of Anopheles species was conducted in Sukabumi District, West Java, Indonesia. The study characterized 1,600 sites from a contiguous coastal and hill zone (0-800-m elevation) of which 64% contained Anopheles larvae. Principal component and multiple logistic regression analyses identified ecological parameters associated with presence of nine [Anopheles aconitus Doenitz, Anopheles annularis Van de Wulp, Anopheles barbirostris Van der Wulp, Anopheles flavirostris (Ludlow), Anopheles insulaeflorum (Swellengrebel and Swellengrebel de Graaf), Anopheles kochi Doenitz, Anopheles maculatus Theobald, Anopheles sundaicus (Rodenwaldt), and Anopheles vagus Doenitz] of 15 Anopheles species collected. Combined data for all nine species showed increased Anopheles presence associated with wet season periods and higher elevation habitats exhibiting reduced tree canopy coverage, higher water temperatures, and shallower water depths. Habitat variables measured included topography (elevation), water conditions (temperature, pH, salinity depth, and velocity), habitat characteristics (substrate and canopy cover), density and type of aquatic vegetation coverage (riparian, floating, and emergent), and distance from nearest human habitation. Significant relationships were found for nine species when using all habitats in the analysis. Habitat characteristics for three species were refined. An. aconitus and An. barbirostris were associated with higher elevation rice, Oryza savita L., paddies with relatively shallow water depths, higher water temperatures, higher acidity and salinity concentrations, and a greater average distance from human habitation. An. vagus presence in rice paddies was associated with lower elevation fields, deeper and cooler water, less acidic and saline conditions, and habitats closer to human dwellings. Overall, the distribution of Anopheles species in Sukabumi was found to be nonrandom and predictable on the basis of habitat characteristics.