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

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1 2 Journal of Vector Ecology December 29 Bionomics of Anopheles spp. (Diptera: Culicidae) in a malaria endemic region of Sukabumi, West Java, Indonesia Craig A. Stoops 1, Saptoro Rusmiarto 1, Dwiko Susapto 1, Amurl Munif 2, Heri Andris 2, Kathryn A. Barbara 1, and Supratman Sukowati 2 1 United States Naval Medical Research Unit 2, Jakarta, Indonesia 2 National Institute of Health Research and Development, Health Ecology Research and Development Center, Jakarta, Indonesia Received 29 March 29; Accepted 19 June 29 ABSTRACT: A 1-month bionomic study of Anopheles species was conducted in two ecologically distinct villages (coastal and upland) of Sukabumi District, West Java, Indonesia from June 26 to September 27. Mosquitoes were captured using human-landing collections at both sites. During the study, a total of 17,1 Anopheles mosquitoes comprising 13 Anopheles species were caught: 9,11 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 27 and had a peak landing time between 22: and 23:. Anopheles sundaicus was only collected at the coastal site, exophagic, collected most often in October 26, and had a peak landing time between 19: and 2:. Potential malaria vector species such An. aconitus, An. maculatus, and An. sundaicus were present throughout the year. None of the 7,77 Anopheles tested using CSP-ELISA were positive for malaria, although the risk for malaria outbreaks in Sukabumi district remains high. Journal of Vector Ecology 34 (2): Keyword Index: Anopheles, bionomics, malaria vectors, West Java, Indonesia. INTRODUCTION The identification of vector species and knowledge of their ecology and behavior is essential for epidemiologic studies and the design and implementation of vector control strategies. To understand malaria risk in an area, the species of Anopheles mosquitoes present in that area and which of those species are vectors must be known. Collections of anthropophilic Anopheles spp. must be conducted throughout the year to reflect seasonal changes in population size and attack rates (Burkot et al. 1989). It is essential to collect data in several areas within a region due to the microepidemiological nature of malaria where settlements within close proximity can vary dramatically in transmission dynamics (Greenwood 1989). The impact of environmental change, abandonment of vector control strategies, and drug resistance on malaria transmission is dramatically illustrated in the Republic of Indonesia (Takken and Knols 1991). Residual pesticide spraying virtually eliminated malaria from the Indonesian island of Java from 19 to 199 (Atmosoedjono 1991). In Sukabumi District, West Java, the rate of Plasmodium falciparum and P. vivax malaria has increased since In Sukabumi district, one malaria case was reported in 1998, but by 23 that number had increased to 1,79 cases (Indonesian Ministry of Health, unpublished data). In 23 there were 17 deaths attributed to P. falciparum in Simpenan sub-district alone. All of the fatal malaria cases in Sukabumi were in coastal villages. During 26 and 27 Lengkong sub-district reported 11 malaria cases and Simpenan sub-district reported 63 malaria cases (Indonesian MoH, unpublished data). The principal malaria vectors on Java island are reported to be Anopheles aconitus, Anopheles maculatus, and Anopheles sundaicus (Kirnwardoyo 198, Takken and Knols 1991, Baird et al. 1996). Anopheles aconitus is reported to be an upland species associated with terraced rice fields, An. maculatus is associated with hilly areas with larvae found in stream beds and other small pools, and An. sundaicus is a coastal species associated with brackish water breeding sites (Kirnwardoyo 198, Takken and Knols 1991, Baird 1996). The objective of this study was to gain greater insight into malaria transmission dynamics in West Java by conducting bionomics studies of Anopheles species in both coastal and upland regions of Sukabumi. We tested two hypotheses: 1) An. sundaicus will be the most abundant Anopheles species collected in the coastal villages, and 2) An. aconitus will be the most abundant Anopheles species collected in the upland region. MATERIALS AND METHODS Study site The study was carried out in Sukabumi district, West Java, Indonesia (Figure 1) from June 26 to September 27. The district is part of West Java province on the island of Java, Indonesia. The district consists of coastal planes that rise quickly into hills with an average height of 7 m and

2 Vol. 34, no. 2 Journal of Vector Ecology 21 an average rainfall of 3- mm per year (Figure 1). The study sites were rural villages composed of a mix of human houses, animal pens, rice paddies, and home gardens. Home gardens are gardens planted within or near a village, often consisting of cassava, bananas, corn, cowpeas, kidney beans, and ground nuts (Whitten et al. 1996). Mosquito collections Five villages were selected in Sukabumi district based on elevation, demography, and presence of malaria cases within in the previous year. Three villages in Simpenan sub-district served as study sites: Cibeas (7 o 4 36 S/16 o E), Sangrawayang (7 o 38 S/16 o 3 42 E), and Cisantri (7 4 6 S/ E), and two villages in Lengkong sub-district: Lengsar (7 o 8 12 S/16 o E) and Translok (7 o 6 S/16 o E). The villages of Cibeas, Sangrawayang, and Cisantri are considered coastal villages and Lengsar and Translok are considered upland sites. The villages of Sangrawayang and Cisantri are very close together and to a casual observer would be considered a single village. Villages were made up of 1 to 2 houses, separated at varying distances (<1 km) from one another (Cibeas from Sangrawayang/Cisantri and Lengsar from Translok). Evidence of malaria cases in the previous 12 months was required for a village to be selected. Four houses from each village were selected for collection activities except in Sangrawayang and Cisantri where only two houses were selected for collection activities (for a total of four houses for Sangrawayang/Cisantri). A total of 16 houses served as permanent collection sites. Routine adult mosquito collections took place once a week at each sentinel site. Collections consisted of 12-h (18:-6:) indoor/ outdoor human-landing captures (HLC). Collections were conducted from June 26 to September 27. The same collector conducted the HLCs throughout the 12-h period. Specimens were identified based on morphological criteria (NAMRU-2 unpublished keys) and stored frozen or dried over silica gel. All specimens collected are maintained in the NAMRU-2 insect collection and voucher specimens of each species collected will be made available for future study in coordination with the Ministry of Health, Republic of Indonesia. Differences in endophily, exophily, and number of species and individuals collected between sites were analyzed using one-way ANOVAs. Species abundance and rainfall were analyzed using Spearman s rank correlation. STATA 9 (StataCorp LP, College Station TX) was used to conduct statistical analyses. Malaria sporozoite testing Protocols were performed as outlined in Wirtz et al. (198). Individual mosquitoes were dissected and the Figure 1. Map of Java showing location of collection sites and elevations of an adult Anopheles bionomics study from June 26 to October 27 in Sukabumi district, West Java province, Republic of Indonesia. Stars indicate sub-districts and black dots indicate study villages.

3 22 Journal of Vector Ecology December 29 Table 1. Anopheles species, indoor and outdoor collections, and percent collected at coastal and upland sites in Sukabumi District, West Java, Indonesia, June 26 to October 27. Statistically significant differences (p<.1) are marked in bold type. Upland Coastal Anopheles species Indoor (%) Outdoor (%) Indoor (%) Outdoor (%) Total (%) An. aconitus 931 (19.4) 3,871 (8.6)* 227 (9.1)* 17 (4.9),186 (3.) An. annularis 1 (11.1) 8 (88.9)* 66 (47.1) 74 (2.9) 149 (.8) An. barbirostris 14 (6.4) 2,14 (93.6)* 1,61 (27.9) 2,742 (72.1)* 6,2 (3) An. flavirostris 6 (17.4) 284 (82.6)* 7 (43.8) 9 (6.3) 36 (2.1) An. indefinitus 2 (33.3) 4 (66.7) 6 (.4) An. kochi 1 (1) 38 (44.) 49 (6.3) 88 (.9) An. maculatus 43 (9.4) 414 (9.6)* 797 (46.2) 927 (3.8) 2,181 (12.8) An. minimus 1 (1) 1 (.1) An. peditaeniatus 1 (1) 1 (1) 2 (.1) An. subpictus 9 (6.) 6 (4.) 1 (.1) An. sundaicus 6 (26.) 17 (73.9) 23 (.1) An. tessellatus 2 (4.) 3 (6.) 1 (69.)* 47 (3.9) 17 (.9) An. vagus 9 (11.3) 71 (88.8)* 1,18 (41.4) 1642 (8.6) 2,88 (16.8) Total 1,192 (1) 6,77 (8.) 3,476 (38.),67 (62.) 17,1 (1) head-thorax portion was ground in a BSA-casein grinding solution (blocking buffer: NP-4). The ELISA was performed in a standard sequence with appropriate incubation times between steps. First, species-specific anti-sporozoite monoclonal antibody (MAb) was absorbed onto the surface of individual wells (U-bottom 96-well microtitration plates), active binding sites were then blocked with blocking buffer, mosquito triturate added to the well, aspirated and washed, followed by the addition of an peroxidase-linked (labeled) species-specific anti-sporozoite MAb to complete the ELISA. A peroxidase substrate solution was added and the reaction read at 4 nm using an ELISA plate reader at 3 and 6 min post-reaction. Positive and negative controls were run on each plate for each parasite species tested. Samples were tested for Plasmodium falciparum, P. vivax 21, and P. vivax 247 sporozoites. RESULTS Mosquito collections A total of 17,1 Anopheles mosquitoes was collected from June 26 to September 27: 9,11 at the coastal site and 7,949 at the upland site. Thirteen species were collected during 1,24 person-night collections (12 nights at two study sites) on humans (Table 1). Table 1 provides the number and percent of each species collected at each site and total collected for both study sites. There were no statistical differences in the total number of Anopheles collected between the upland and coastal sites (F 1,3122 =.1, P=.6968), but a significantly greater number of An. aconitus (p<.1) and An. flavirostris (p<.) were collected at the upland site vs the coastal site. A significantly greater number of An. barbirorstris (p<.1), An. maculatus (p<.1), and An. vagus (p<.1) were collected at the coastal site vs the upland site. No other species were collected significantly more often in either the upland or coastal site. Anopheles aconitus was significantly exophagic at the upland site (p<.1). Anopheles aconitus and An. tessellatus were significantly endophagic at the coastal site (p<.1). Anopheles baribirostris was strongly exophagic at both sites with >7% of the collections outdoors at both sites (p<.1). Anopheles maculatus was strongly exophilic at the upland site (9.6%) (p<.1), but almost half (46%) of the collections at the coastal site were collected indoors. Only 26% of An. sundaicus were collected indoors. Anopheles annularis, An. flavirostris, An. kochi, and An. vagus were collected more often indoors at the coastal site with between 4 and % of each collected indoors. At the coastal site the mean number of bites per human per night was.9 for An. barbirostris, 4. for An. vagus, and 2.26 for An. maculatus. For the upland site the mean number of bites per human per night was 6.26 for An. aconitus, 2.9 for An. barbirostris, and.6 for An. maculatus. For the remaining species the mean number of bites was <1 bite per human night at both sites. Seasonality Figure 2 provides rainfall graphed with the abundance per month of the three most often collected species at the coastal and upland sites. The following summarizes the results of the collection records for species not depicted in Figure 2. Anopheles sundaicus was collected between October 26 to September 27 with the highest single month collection in October 26 (n=9). Anopheles annularis and An. tessellatus

4 Vol. 34, no. 2 Journal of Vector Ecology 23 Coastal (Simpenan) # bites/human/night JUN6 JUL AUG SEP OCT NOV DEC JAN7 FEB MAR APR MAY JUN JUL AUG SEP Rainfall (mm) An.barbirostris An.maculatus An.vagus Upland (Lengkong) Rainfall (mm) 2 7 # bites/human/night Rainfall (mm) JUN6 JUL AUG SEP OCT NOV DEC JAN7 FEB MAR APR MAY JUN JUL AUG SEP Rainfall An.aconitus An.barbirostris An.maculatus Figure 2. Monthly density of An. barbirostris, An. maculatus, An. vagus, and An. aconitus in relation to rainfall at coastal and upland sites from June 26 to October 27 in Sukabumi District, West Java, Indonesia.

5 24 Journal of Vector Ecology December 29 Figure 3. Total number of Anopheles aconitus, An. barbirostris, An. maculatus, and An. vagus collected during each hour of 12-h Human Landing Collections at both the coastal and upland sties in Sukabumi, West Java, Indonesia. Anopheles annularis Anopheles sundaicus Number Collected Time Time Anopheles flavirostris Anopheles tessellatus 4 3 Number Collected Time Time Figure 4. Total number of Anopheles annularis, An. flavirostris, An. sundaicus and An. tessellatus collected during each hour of 12-h Human Landing Collections at both the coastal and upland sties in Sukabumi West Java, Indonesia.

6 Vol. 34, no. 2 Journal of Vector Ecology 2 were collected most often in April 27 (n=92 and n=64, respectively). Anopheles flavirostris was collected most often in February 27 (n=6). Anopheles indefinitus was collected in the months of January 27 (n=2), July 27 (n=1), and June 27 (n=3). Two peaks of An. kochi were observed: one in January 27 (n=2) and one in May 27 (n=19). The single specimen of An. minimus was collected in December 26. One specimen of An. peditaeniatus was collected in April 27 at the coastal site and one specimen was collected in May 27 at the upland site. Four An. subpictus were collected in April, June, and July of 27. When data from both sites were combined, no relationship between all Anopheles species abundance and rainfall was found (p=.47). There was no relationship found when each site was analyzed separately (upland p=.239 and coastal p=.276). When species were analyzed individually, only An. indefinitus (p<.1) and An. vagus (p<.1) were found to have a significant positive relationship between abundance and rainfall. Although not significant, there appears to be a correlation between An. aconitus and rainfall (Figure 2). Hourly collections Data for each species across the study site was combined to provide a detailed picture of biting times. Figure 3 shows the hourly collections for the four most often collected species at the coastal and upland sites: An. aconitus, An. barbirostris, An. maculatus, and An. vagus. Figure 4 shows the hourly collections of the three most often collected species (excluding species represented in Figure 3) at all sites, An. annularis, An. flavirostris, An. tessellatus, and the coastal malaria vector, An. sundaicus. The following are the results for the species not pictured in Figures 3 and 4. One An. indefinitus was collected between 21: and 22:, one was collected between 22: and 23:, two were collected between 23: and 24:, and one was collected between 3: and 4:. The 88 An. kochi were collected steadily throughout the night, but two peaks were observed: 14 specimens collected between 21: and 22: and 14 collected between 24: and 1:. The only specimen of An. minimus was collected between 2: and 21:. One specimen of An. peditaeniatus at the coastal site was collected between 2: and 21: and one An. peditaeniatus was collected at the upland site between 22: and 23:. Six of the 19 (32%) An. subpictus were collected between 21: and 22:, with the remaining 13 collected between 19: and :. Malaria sporozoite testing Following the collections, 7,77 specimens were tested using ELISA for circumsporozoite protein. No specimens of Anopheles tested positive for either Plasmodium falciparum or P. vivax. (P.v. 21 and P.v. 247) sporozoites. DISCUSSION Over the 1 months we found differences between sites in the abundance and species composition of the Anopheles spp. attracted to humans. At both the coastal and upland sites, humans were bitten by Anopheles outdoors throughout the night with possible vector species (An. aconitus, An. maculatus, An. sundaicus) present throughout the year. We hypothesized that the important malaria vector An. sundaicus would be the species most often collected in the HLCs at the coastal site, however, An. barbirostris, a reported zoophilic species, was the most often collected species. At the upland site, we collected the important malaria vector An. aconitus most often, supporting our second hypothesis. We did not find any sporozoite positive mosquitoes. Without direct evidence of sporozoite presence, it is not possible to unequivocally state which Anopheles spp. are the principal malaria vectors at either the upland or coastal site. The malaria eradication program on Java in the 19s and 196s was largely successful at eliminating transmission (Atmosoedjono 1991). The human population in this region of Java is mobile, with local residents travelling to other Indonesian islands for work, such as Kalimantan or Papua, with higher levels of malaria transmission, and returning to live with their families in Sukabumi. People with active malaria infections returning to Sukabmi from these areas could be the reason behind the localized outbreaks, such as the outbreak in 23. Based on our observations that An. aconitus was collected significantly more often at the upland site, our findings support the importance of this species as a malaria vector in upland areas of West Java (Kirnowardoya 198). Our results are similar to those of Chow et al. (196) from East Java, who found that An. aconitus is present throughout the year with a population peak in April and May and exophilic with a peak in biting occurring before midnight. Locations where this species is found in Sukabumi should be targeted for control efforts during malaria epidemics. Anopheles maculatus is important in epidemic malaria transmission in Central Java increasing the concern in West Java (Baird et al 1996). Direct evidence of sporozoites in Sukabumi was not observed during this study; we suggest that at both the upland and coastal sites An. maculatus could play a role in malaria transmission. Anopheles maculatus was one of the three most often collected species at both sites supporting evidence that breeding habitats are common around all of the study villages (Stoops et al. 28). In Sukabumi, An. maculatus is present throughout the year, exophilic with bites peaking between 23:-24:. Upathum et al. (1988) also found the An. maculatus complex in Thailand to be exophilic with peak biting occurring in the first quarter of the night and abundance was positively related to rainfall. A high level of suspicion should surround An. maculatus during malaria outbreaks and this species should also be targeted for control. Surprisingly few An. sundaicus were collected at the coastal site. We had hypothesized that this species would be the most often collected species in the coastal HLCs. The reason why so few individuals of An. sundaicus were collected is unclear. During a large-scale study of larval habitats in Sukabumi, few An. sundaicus larvae were collected across the district, but there were several An. sundaicus habitats

7 26 Journal of Vector Ecology December 29 found near the coastal villages within the reported flight range (1.6 to 9 km) of this species (Dusfour et al. 24, Stoops et al. 27). During a malaria outbreak in Central Java, Kirnowardoya and Yoga (1987) found An. sundaicus to be the principal malaria vector but observed population size to vary between sites and years. It is possible that we conducted our study when habitats were not widespread or persistent enough to produce large numbers of adults. All of the 17 fatalities in the 23 Sukabumi P. falciparum outbreak were located in the coastal subdistrict. So even though fewer than expected individuals and no sporozoite positive specimens of An. sundaicus were collected in our HLCs, this species should still be considered an important malaria vector in coastal areas of Sukabumi based on reported vector competence (Kirnowardoya 198, Dusfour et al. 24). An interesting finding was that two reportedly zoophilic species, An. barbirostris and An. vagus, were among the most often collected species in the HLCs at both the coastal and upland sites (Chow et al. 196). Direct comparisons of HLCs with animal catches and analyses of blood meal sources in Sukabumi should be conducted before a definitive answer on the feeding behavior of An. barbirostris and An. vagus can be reached. The importance of An. vagus requires more indepth study due to earlier findings during adult Anopheles surveys which found P. falciparum sporozoite positive An. vagus in the study area (NAMRU-2, unpublished data). We found no positive relationship between abundance of Anopheles adults captured in the HLCs and rainfall. Residents of Sukabumi are being bitten by potential malaria vectors throughout the year. When species were analyzed individually we did find a positive relationship with An. vagus and rainfall. Chow et al. (196) observed An. vagus was more often collected in HLCs during the rainy season in East Java. Even though it is a zoophilic species, An. vagus during the rainy season may be important in malaria transmission due to high biting populations. Anopheles spp. presence and abundance will vary from site to site so dependence on the amount of rainfall will also depend on the site. In areas with high levels of rice cultivation, suitable habitat will be present throughout the year for An. aconitus, An. maculatus, and An. vagus (Stoops et al. 28). In areas without rice, the availability of habitats available for oviposition will increase only if there is enough rain. Large amounts of rain will cause the number of habitats, such as pools in streams and irrigation canals, available to decrease (Stoops et al. 27). During an outbreak, the environment and potential Anopheles larval habitats (rice paddies or tidally-influenced pools), around the village where the outbreak is occurring should be considered when planning anti-anopheles control strategies. The observation that the majority of Anopheles spp. were collected outdoors, including the three important malaria vectors An. aconitus, An. maculatus, and An. sundaicus, has implications in the control of malaria epidemics because it may limit the effectiveness of Indoor Residual Spraying (IRS). IRS is an important part of malaria control programs worldwide and was responsible for largely eliminating malaria transmission on Java (Atmosoedjono 1991). Based on our results, IRS should remain a part of an epidemic control strategy, at least in the upland areas, because 6% of An. aconitus and 4-% of several other Anopheles spp., including An. maculatus, were collected indoors. Epidemic malaria control programs that include IRS may need to be supplemented with larval control and space spraying around settlements to achieve effective control of an outbreak (Sudomo et al. 198). Species identifications were based on morphological characters and several of the species identified may be sibling species complexes. Identification of siblings that are more or less refractory to malaria infection will be important in ultimately understanding the risk of malaria transmission in Sukabumi. Although there is evidence that species such as An. barbirostris s.l., An. maculatus s.l., and An. sundaicus s.l. are complexes of sibling species across their distribution in Asia, there is no evidence that any of the species collected during this study consist of sibling complexes living in sympatry in Sukabumi (Dusfour et al. 24, Stoops et al. 27). Specimens are stored at the Naval Medical Research Unit-2, Jakarta, Indonesia, and are available for further study in coordination with the Indonesian Ministry of Health. Malaria epidemics are a high risk to residents of Sukabumi throughout the year. Mosquito surveillance efforts should be continued to monitor changes in malaria vector abundance and distribution due to habitat modification such as the increasing number of palm oil plantations in the region and the effects of global climate change. Acknowledgments This work could not have been done without the strong support of D. Supardi, Malaria Field Coordinator for Sukabumi District, and H. B. Thahadibrata, Head of Ministry of Health for Sukabumi District. We also thank A. Rachmat for making the map for Figure 1. This study was supported by the Department of Defense Global Emerging Infections Surveillance and Response System (DoD- GEIS). 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