Habitat Partitioning of Malaria Vectors in Nchelenge District, Zambia

Smita Das, Mbanga Muleba, Jennifer C. Stevenson, and Douglas E. Norris* for the Southern Africa International Centers of Excellence for Malaria Research Team W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland; Tropical Disease Research Centre, Ndola Central Hospital, Ndola, Zambia; Macha Research Trust, Choma, Zambia

Abstract. Nchelenge District in Luapula Province, northern Zambia, experiences holoendemic malaria despite implementation of vector control programs. The major Anopheles vectors that contribute to Plasmodium falciparum transmission in this area had not previously been well defined. Three collections performed during the 2012 wet and dry seasons and the 2013 wet season revealed Anopheles funestus sensu stricto and Anopheles gambiae sensu stricto as the main vectors, where 80–85% of each collection was composed of An. funestus. Both vectors were found to be highly anthropophilic, and An. funestus has higher sporozoite infection rates (SIRs) and entomological inoculation rates (EIRs) year-round compared with An. gambiae:SIRsof1.8–3.0% and 0–2.5%, respectively, and EIRs of 3.7–41.5 infectious bites per 6-month period (ib/p/6mo) and 0–5.9 ib/p/6mo, respectively. Spatial and temporal changes in each vector’s dynamics and bionomics were also observed. Anopheles funestus was the predominant vector in the villages near Kenani Stream in both wet and dry seasons, whereas An. gambiae was found to be the main vector in areas near Lake Mweru during the wet season. The vector data illustrate the need for broader temporal and spatial sampling in Nchelenge and present unique opportunities to further our understanding of malarial transmission and implications for malarial control in high-risk areas.

INTRODUCTION major vector species.8 In addition, there was pronounced spatial heterogeneity in vector composition and transmission Anopheles funestus sensu stricto and Anopheles gambiae rates in the lowland areas.8,9 Differences in vector composi- sensu stricto are the most notorious vectors of Plasmodium tion, biting behaviors, and transmission intensities among falciparum malarial transmission in sub-Saharan Africa. Each study sites that also vary temporally and spatially suggest ’ of these species distributions, their abilities to transmit the that there is unequal risk of malarial transmission within an malarial parasite, and other demographic and environmental endemic area. Heterogeneity in malarial risk has implica- factors are heterogeneous and influence the local intensity of tions for developing and targeting interventions because a 1 malarial transmission. The relative composition of the vec- single vector control measure such as indoor residual spraying tors in areas where both An. funestus and An. gambiae are (IRS) may successfully interrupt transmission in a low trans- sympatric and their respective feeding behaviors vary from mission setting, whereas multiple vector control measures region to region in Africa. When both An. funestus and such as IRS, long-lasting insecticide-treated nets (LLINs), An. gambiae are present in a community, transmission can be larviciding, and/or larval habitat source reduction may be – exceedingly high.1 This often occurs because these vector spe- needed to have an impact in high transmission settings.10 21 cies exploit different breeding habitats and, although sym- Therefore, it is necessary to identify and characterize malarial patric, may stagger their peak densities, which can prolong vectors at a local scale to inform successful control strategies. the transmission season.1 Anopheles gambiae tends to oviposit Nchelenge District in Luapula Province, northern Zambia, in temporary breeding sites such as puddles and animal foot experiences holoendemic transmission of P. falciparum.22 – prints, which are abundant during the rainy season.2 4 In con- Despite clinical evidence of high transmission rates, the mos- trast, An. funestus prefers more permanent breeding sites and quito vectors and their behaviors that influence transmis- populations tend to peak toward the end of the rainy season sion have not been well characterized. Preliminary mosquito – and into the first part of the dry season.2 4 Both vectors have collections by the southern Africa International Centers of been shown to be highly anthropophilic, expressing a ten- Excellence for Malaria Research (ICEMR) in-country part- dency to feed on humans, as well as being endophagic and ner, the Tropical Disease Research Center (Ndola, Zambia), – endophilic, feeding and resting indoors, respectively.2,4 7 suggested that An. funestus s.s. and An. gambiae s.s. are the In communities where both vector species reside, there primary and secondary vectors, respectively, and together may be differences in the vector composition depending on contribute to the maintenance of year-round Plasmodium the season and geography. From June 2003 to 2004 in west- transmission in Nchelenge (M. Muleba, unpublished data). ern Kenya, it was found that An. gambiae s.s. was the major The aims of this study were to confirm and characterize vector species driving transmission at one site in the low- An. funestus s.s. and An. gambiae s.s., and their spatial and lands and three sites in the highlands.8 However, one of the temporal patterns in two ecologically distinct habitats within villages in the lowland site had experienced recent larval Nchelenge District. This study provides fundamental and habitat changes and An. funestus s.s. was found to be the requisite information on vector dynamics of An. funestus s.s. and An. gambiae s.s. populations and their roles in malarial transmission in Nchelenge District.

*Address correspondence to Douglas E. Norris, W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns METHODS Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD Study area. This study was carried out in association with 21205. E-mail: [email protected] the Johns Hopkins Southern Africa ICEMR project. The focus 1234 HABITAT PARTITIONING OF MALARIA VECTORS 1235 of the research reported here is Nchelenge District, Luapula LLINs and IRS with pyrethroids have been implemented in Province, in northern Zambia (9°19′115″S, 28°45′070″E) at an Nchelenge District since 2006 and 2007, respectively. elevation of approximately 807 m above sea level and in a Mosquito collection and handling. Mosquitoes were col- marsh ecotype (Figures 1 and 2). The district lies along the lected by Center for Disease Control light trap (CDC LT) eastern perimeter of Lake Mweru, which serves as the border and pyrethroid spray catch (PSC) in the three lakeside vil- between the southeastern part of the Democratic Republic of lages (Katuna, Yenga, and Malulu) and two inland villages Congo and the northwestern part of Zambia. Flowing from (Kapande B and Mutepuka) during the periods March 24– south to north into Lake Mweru, Kenani Stream is also found April 10, 2012 (wet), August 27–September 9, 2012 (dry), in the area. There is a single rainy season from November and March 5–April 25, 2013 (wet). Collection methods were to May, followed by a cool dry season from May to August approved by the Johns Hopkins Bloomberg School of Public and a hot dry season from August to November (Figure 3). Health Institutional Review Board (no. 00003467) and in Rainfall follows a seasonal pattern, with a peak of 2700 mm Zambia (TDRC/ERC/2010/14/11). during the rainy months and close to 0 mm during the dry CDC LTs and PSCs were set once per household in each months (Figure 3). Longitudinal and cross-sectional house- sampling season, ensuring a 5-day gap between the two col- holds with prior enrollment in the ICEMR program and lection methods in the same household. In the wet 2012 col- located within two defined 1-km2 grids along both Lake lection, 11 lakeside and 11 streamside households were Mweru (grid cell r34c5: 9°18′0.36″S, 28°44′25.08″E; gird cell sampled, totaling 22 CDC LT and 22 PSC collections. During r34c6: 9°18′2.16″S, 28°44′57.84″E) and Kenani Stream (grid the 2012 dry collection, 21 lakeside and 15 streamside house- cell r26c11: 9°22′19.74″S, 28°47′42.72″E; grid cell r29c10: holds were sampled using CDC LTs and PSCs, totaling 36 col- 9°20′42.29″S, 28°47′9.6″E) were chosen for mosquito sam- lections for each trapping method. In the 2013 wet collection, pling. Mosquito collections were conducted at three villages 39 lakeside and 38 streamside households were sampled, total- (Katuna, Yenga, and Malulu) along Lake Mweru and two ing 77 CDC LTs and 77 PSC collections. villages (Kapande B and Mutepuka) near Kenani Stream CDC LTs were hung indoors next to sleeping persons, (Figure 2). These two collection areas are approximately approximately 1 m above the floor, and would typically run 7 km apart and are representative of the local demography from 6:00 PM to 6:00 AM. PSCs were performed in the morn- and landscape. People move between the two sites within ing (6:00 AM–10:00 AM). Briefly, white sheets were placed the year as they switch from fishing to farming livelihoods over all surfaces and a 100% synthetic aerosol pyrethroid and the two localities have high malarial incidence. Both was applied on the exterior eaves to prevent mosquito exit,

FIGURE 1. Nchelenge District field site is in northern Zambia and represents a site with unsuccessful malarial control. 1236 DAS AND OTHERS

2 FIGURE 2. Satellite image of the study area in Nchelenge District. The 1-km grids for study collections are highlighted in blue. Katuna, Yenga, and Malulu villages are located in grids r34c5 and r34c6, and Kapande B village is located in grid r29c10. The white area on the left side of the image represents Lake Mweru. The yellow arrows point to Kenani Stream that flows into Lake Mweru.

the ceiling, indoor eaves, and walls, and then the home abdomen was performed using a modified salt extraction.24 closed. After approximately 15 minutes, the sheets were taken The morphological identification of anopheline mosqui- out of each household and knocked down mosquitoes were toes was confirmed using a PCR specific for members collected. All field-caught mosquitoes were killed by freezing. of the An. gambiae or An. funestus species complexes as – The female anopheline mosquitoes were then morphologi- described previously.25 28 cally identified to species using a dichotomous key3,23 and If a mosquito sample could not be molecularly confirmed dissecting microscope (both vectors and nonvectors). Up to as either An. gambiae or An. funestus, then an internal tran- three mosquitoes were placed in each labeled 0.6 mL micro- scribed spacer 2 (ITS2) PCR was used.26 The ITS2 PCR is centrifuge tube containing silica gel desiccant and cotton robust and has a range of base pair sizes that are specific to wool, and stored either at room temperature or frozen at other anopheline species common to sub-Saharan Africa.26 −20°C until laboratory processing, which took place at Johns Briefly, the forward primer was ITS2A: 5′-TGT GAA CTG Hopkins University Bloomberg School of Public Health in CAG GAC ACA T-3′ and the reverse primer was ITS2B: Baltimore, MD. 5′-TAT GCT TAA ATT CAG GGG GT-3′26 The 25 μLreac- DNA preparation and polymerase chain reaction (PCR). tion mixture contained 2.5 μL 10× reaction buffer, 1 mM For all collected anophelines, the head and thoraces were MgCl2, 2.5 mM dNTPs, 30 pmol of each primer, 2.0 units separated from the abdomen, and DNA extraction of the Taq polymerase, 1 μL extracted DNA, and sterile water.26 HABITAT PARTITIONING OF MALARIA VECTORS 1237

FIGURE 3. The proportion of Anopheles species caught per household that were sampled during the (A) March 24–April 10, 2012 (wet) collection, (B) August 27–September 9, 2012 (dry) collection, and (C) the March 5–April 25, 2013 (wet) collection. Species are denoted by color: Anopheles funestus sensu stricto (green), Anopheles gambiae sensu stricto (yellow), and Anopheles leesoni (purple).

The PCR was performed with an initial denaturation for vector species. For mixed blood meals, they were counted as 2 minutes at 94°C, followed by 40 cycles at 94°C for 30 sec- a blood meal for each host animal. onds, 50°C for 30 seconds, 72°C for 40 seconds, and then a The abdominal DNA for each anopheline was then tested final extension step was 10 minutes at 72°C.26 for the presence of the P. falciparum parasite by PCR.32 This The mosquitoes that were molecularly confirmed as PCR amplifies a small portion of the cytochrome b gene of An. gambiae s.s. were also tested for molecular M-form (now P. falciparum and is more sensitive than the commonly used recognized as Anopheles coluzzii) or S-form as detailed by Snounou and others32 PCR, producing a fragment with an Favia and others.29 Briefly, the 5′ end of the intergenic recom- expected size of 183 base pairs.33 binant DNA spacer region is amplified to produce a 727 base Circumsporozoite protein enzyme-linked immunosorbent pair band for M-form, 475 base pair band for S-form, and assay (CSP-ELISA) for P. falciparum detection. The CSP- both bands for hybrid specimens.29 ELISA method as described by the Malaria Research and All specimens, regardless of being visually fed or unfed, Reference Reagent Resource Center (MR4) was used to were then tested for host animal blood using the Kent and detect P. falciparum CSP in the mosquito head and thorax. Norris24 multiplex PCR, which amplifies the cytochrome b The values associated with each mosquito that are two times gene of the mitochondrial genome producing a range of mam- the mean of the negative controls on the plate were con- malian host-specific bands from 132 to 680 base pairs.30 A sidered to be P. falciparum positive. CSP-ELISA was per- modification to this PCR was made to better detect a human formed on all Anopheles mosquitoes collected in the wet and host blood meal; the original forward primer to detect a human dry seasons of 2012. However, due to the large numbers of blood meal was removed and replaced with a new forward anophelines caught during the March–April 2013 collection, and reverse primer set that amplifies a human-specific 193 base a subsample of mosquitoes was selected for CSP-ELISA. pair region of the cytochrome B gene. The forward primer The subsample was representative of all collection dates and was FOR16068: 5′-GAC TCA CCC ATC AAC AAC CG-3′, also provided a large enough sample size for each species for and the reverse primer was REV16260: 5′-GGC TTT GGA statistical analyses: 695/2,417 (28.8%) An. funestus s.s. and GTT GCA GTT GA-3′. Amplification conditions were not 520/564 (92.2%) An. gambiae s.s. altered for this primer replacement. Spatial and temporal mapping of malarial vectors. To map Samples that did not produce a band(s) for host blood meal the presence of malarial vectors and their proportions within withthemodifiedmultiplexPCRwerethenscreenedwitha each household in Nchelenge District both spatially and tem- more sensitive PCR, which amplifies a conserved 98 base porally, ArcMap 2.0 (ESRI ArcGIS) software was used to pair region of the cytochrome b gene of the mitochondrial create a layer of vector composition and density per house- genome of the mammalian host, followed by a restriction frag- holds over a satellite image of Nchelenge District. This was ment length polymorphism assay to identify animal host of done for each collection period to visualize changes in vector interest.31 The identification of the mammalian host was used composition and abundances spatially and temporally. to calculate the human blood index (HBI), the proportion of Human biting rate (HBR) and entomological inoculation human blood meals relative to the total blood meals, for each rate (EIR). The average number of bites per person per 1238 DAS AND OTHERS night (HBR) was calculated by dividing the number of PCR- An. gambiae s.s. (henceforth referred to as An. gambiae), confirmed An. funestus s.s. and An. gambiae s.s. vectors from and 7.7% (N =28)An. leesoni (Figure 4). Within the col- CDC LTs by the number of people sleeping in the household lection, the abundance of An. funestus was greater than the night of the collection. Because CDC LT collections have An. gambiae per household per CDC LT night (relative been shown previously in western Kenya and Zambia to be risk [RR] = 5.6, 95% confidence interval [CI] = 2.2, 14.3, a reasonable correlate for estimating the HBR when human P > 0.001) and per PSC trap morning (RR = 19, 95% CI = 6.8, landing catches cannot be performed, EIR calculations from 53.2, P < 0.001). CDC LT collections were regarded as the best measurement In the dry season of 2012, collections from CDC LTs – of transmission intensity.34 37 and PSCs were composed of 1,324 anophelines, of which The seasonal EIR for each vector species per household 99.3% (N =1,315)wereAn. funestus,0.6%(N =8)were was calculated as the product of the HBR, sporozoite infec- An. gambiae, and 0.1% (N =1)wereAn. leesoni.The tion rate (SIR), and 180 days (6 months for each season), as abundance of An. funestus caught per household was higher collections were conducted in either the wet or dry seasons. than An. gambiae in both CDC LT collections per trap night The SIR is defined as the proportion of infectious vectors in (RR = 130, 95% CI = 42, 404, P < 0.001) and PSC collec- a household or total collection. The overall EIR estimated tions per trap morning (RR = 253.2, 95% CI = 54.1, 1187.2, for Nchelenge District was the average of all household P < 0.001). EIRs. For the March–April 2013 collection where the sample A total of 2,989 Anopheles mosquitoes were caught in the size was sufficiently large, overall EIR calculations were fur- 2013 wet season using CDC LTs and PSCs. The majority of the ther partitioned among lakeside and streamside villages. collection was made up of An. funestus (80.9%, N = 2,417), Statistical analysis. Abundances, SIR, and EIR of An. funestus followed by An. gambiae (18.9%, N = 564) and An. leesoni s.s. and An. gambiae s.s. in the March–April 2012 (wet), (0.2%, N = 8). Within the collection, the number of collected August–September 2012 (dry), and March–April 2013 (wet) An. funestus per household was greater than An. gambiae collections were compared using STATA version 11 (Stata per CDC LT per night (RR = 3.9, 95% CI = 2.5, 5.9, P > Corp., College Station, TX). For comparison of vector abun- 0.001) and PSCs per trap morning (RR = 5.1, 95% CI = 3.0, dances and EIRs, the negative binomial regression model 8.8, P <0.001). for overdispersed data was used. Logistic regression model When comparing individual species abundance among was used to compare SIRs. A P value < 0.05 was consid- collection seasons, the 2012 and 2013 wet collections had ered statistically significant. The kappa statistic was calcu- smaller catches of An. funestus per household per CDC LT lated to classify the agreement between CSP-ELISA and trap night (2012 wet collection: RR = 0.14, 95% CI = 0.04, PCR method results as “poor” (< 0.40), “fair” (0.40–0.70), 0.50, P < 0.004; 2013 wet collection: RR = 0.25, 95% CI = and “excellent” (> 0.70). 0.04, 0.52, P = 0.015) and PSC trap morning (2012 wet collection: RR = 0.26, 95% CI = 0.11, 0.63, P = 0.003; 2012 dry RESULTS collection: RR = 0.55, 95% CI = 0.26, 1.17, P = 0.121) when compared with the 2012 dry season collection. In contrast, Seasonal variation. During the 2012 wet season, a total of both the 2012 and 2013 wet season collections had larger 366 Anopheles mosquitoes were collected using CDC LTs and abundances of An. gambiae per household per CDC LT night PSCs and were composed of 83.3% (N =305)An. funestus (2012 wet collection: RR = 3.5, 95% CI = 1.1, 11.1, P = 0.04; s.s. (henceforth referred to as An. funestus), 9.0% (N = 33) 2013 wet collection: RR = 9.2, 95% CI = 3.4, 24.8, P < 0.001)

FIGURE 4. Mean number of Anopheles funestus sensu stricto and Anopheles gambiae sensu stricto collected using Center for Disease Con- trol light trap and pyrethroid spray catch collection methods in lakeside and streamside households in the 2012 wet, 2012 dry, and 2013 wet collection periods. HABITAT PARTITIONING OF MALARIA VECTORS 1239 or PSC trap morning (2012 wet collection: RR = 3.5, 95% 45/1,098 (4.1%) were identified as An. funestus and were CI = 0.64, 19.1, P = 0.15; 2013 wet collection: RR = 27, 95% human fed. The HBIs for An. funestus and An. gambiae were CI = 5.8, 125.9, P < 0.001) than the 2012 dry season collection. 0.99 and 1.0, respectively. The majority of the collected An. gambiae were success- In the wet season collection of 2013, 444/2,417 (18.4%) fully identified as S-form (527/564; 93.4%). A small propor- An. funestus and 100/564 (17.7%) An. gambiae were visu- tion of the collected An. gambiae (37/564; 6.6%) could not ally fed. No visually fed An. leesoni (N = 8) were observed. be identified to molecular form due to failed PCRs that were Of those visually fed, 420 An. funestus and 94 An. gambiae attempted twice. specimens had taken blood meals exclusively from human Spatial variation. In the wet season of 2013, spatial dif- hosts. Mixed human and goat blood meals were detected ferences in vector composition were evident when compar- in 24 An. funestus and six An. gambiae.OneAn. funestus ing collections from villages near Lake Mweru to those mosquito fed on goat exclusively. Of the anophelines that located inland along Kenani Stream. In the lakeside villages were classified as “unfed,” 164/2,445 (6.7%) were found to (Katuna, Yenga, and Malulu), 86 collections from 39 house- contain mammalian blood by PCR; 153 An. funestus and holds resulted in a total of 133 anophelines caught, of which 11 An. gambiae. One hundred and forty-six of the 153 the majority was An. gambiae (85.7%, N = 114), with the An. funestus (95.4%) and 8/11 (72.7%) An. gambiae with remainder An. funestus (14.3%, N = 19). In 99 collections blood meals had fed on human hosts. Seven blood meals from 38 households in the streamside villages (Kapande B, taken by An. funestus (7/153) and three blood meals taken Mutepuka), 2,847 anophelines were collected and were by An. gambiae (3/11) were mixed blood meals of human composed of 2,397 An. funestus (84.2%) and 450 An. gambiae and goat. The HBIs for An. funestus and An. gambiae were (15.8%). The streamside collection revealed higher numbers 0.95 and 0.93, respectively. of both An. funestus and An. gambiae caught per household CSP-ELISA versus PCR detection of P. falciparum. The per CDC trap night than the lakeside collection (An. funestus: CSP-ELISA method of P. falciparum detection for both RR = 42.7, 95% CI = 21.8, 83.7, P < 0.001; An. gambiae: Anopheles vectors was compared with the PCR method RR = 1.7, 95% CI = 1.1, 2.8, P = 0.03). Similarly, greater by calculating the kappa value for each collection. It was numbers of An. gambiae were caught per household per observed that most Anopheles specimens that were positive PSC trap morning in streamside villages compared with by CSP-ELISA were not positive by PCR, and vice versa. As lakeside villages (RR = 2.4, 95% CI = 0.97, 6.1, P = 0.05). a result, the kappa values for the March–April 2012 (wet), No An. funestus were caught in streamside households by August–September 2012 (dry), and March–April 2013 (wet) PSC traps. collections were 0.0, 0.23 (standard error [SE] = 0.073, 95% When species composition was studied within a locality CI = 0.092, 0.380), and 0.20 (SE = 0.067, 95% CI = 0.071, during the wet season of 2013, there were significant dif- 0.334), respectively, suggesting poor agreement between the ferences in the abundance of An. funestus and An. gambiae two methods for all field collections. caught both within the lakeside and the streamside areas. In Comparison of SIR and EIR of vectors within collections. the lakeside collection, more An. gambiae were caught than In the 2012 wet collection, An. funestus hadanSIRof An. funestus (RR = 4.8, 95% CI = 2.5, 9.3, P < 0.001) per 1.8% by CSP-ELISA and a seasonal EIR of 3.7 infectious household per CDC LT night, whereas in the streamside bites per 6-month period (ib/p/6mo) (Table 1). No infectious households, there were more An. funestus than An. gambiae An. gambiae by CSP-ELISA were detected. The 2012 dry col- per CDC LT night (RR = 5.3, 95% CI = 3.9, 7.3, P < 0.001). lection revealed that An. funestus had an SIR of 2.4% and an For PSC collections per trap morning, no An. funestus were EIR of 41.5 ib/p/6mo (Table 1). During the 2012 dry collec- caught lakeside. In contrast, near the stream, An. funestus tion, CDC LTs caught only six An. gambiae and none were was the dominant vector compared with An. gambiae per positive for P. falciparum. Because no infectious An. gambiae PSC trap morning (RR = 5.1, 95% CI = 3.4, 7.7, P < 0.001). were caught in the wet and dry collections of 2012, logistic Blood-feeding behavior. Mammalian host was assessed regression comparing An. gambiae and An. funestus SIRs for all anophelines due to the possibility that there may be could not be performed. There was no statistically significant mosquitoes that appear “unfed,” but are actually fed as deter- difference in both vector’s EIRs within the 2012 wet and 2012 mined by molecular assays.24,31,38 HBI was, therefore, calcu- dry collections (2012 wet: RR = 18.3, 95% CI = −3585.8, lated using PCR-confirmed blood meals. 3622.3, P = 0.99; 2012 dry: RR =19, 95% CI = −1999.4, In the 2012 wet collection, 111/343 An. funestus (32.4%), 9/36 An. gambiae (25%), and 6/32 An. leesoni (18.8%) were visually fed. All blood meals for An. funestus and An. gambiae TABLE 1 were identified as human, whereas five of the An. leesoni Total abundance, SIR, and EIR for Anopheles funestus sensu stricto fed on humans and one An. leesoni had fed on goat. Of and Anopheles gambiae sensu stricto based on CDC LT collec- those anophelines that were visually “unfed,” five were found tions during the 2012 wet, 2012 dry, and 2013 wet seasons in Nchelenge District to have human blood meals: 2/232 An. funestus (0.9%), 1/27 SIR EIR An. gambiae, (3.7%), and 2/26 An. leesoni (7.7%). The HBI Collection Anopheles spp. (%) (ib/p/6mo) for both An. funestus and An. gambiae was 1.0 and 0.88 for Wet 2012 (N = 158) An. funestus s.s. (n = 134) 1.8 3.7 An. leesoni. An. gambiae s.s. (n = 24) 0.0 0.0 In the dry season of 2012, 215/1,315 An. funestus (16.5%), Dry 2012 (N = 787) An. funestus s.s. (n = 781) 2.4 41.5 2/8 An. gambiae (25%), and 0/1 An. leesoni (0%) were visu- An. gambiae s.s. (n =6) 0.0 0.0 Wet 2013 (N = 856) An. funestus s.s. (n = 480) 3.0 39.6 ally fed. With the exception of two An. funestus blood meals An. gambiae s.s. (n = 376) 2.5 5.9 identified as goat, all fed mosquitoes had taken human CDC LT = Center for Disease Control light trap; EIR = entomological inoculation rate; blood meals. Of those anophelines that were visually “unfed,” ib/p/6mo = infectious bites per 6-month period; SIR = sporozoite infection rate. 1240 DAS AND OTHERS

TABLE 2 CI = 0.41, 4.4, P = 0.019). A difference in the proportion Total abundance, SIR, and EIR for Anopheles funestus sensu stricto of infectious An. funestus between the 2013 wet collection and Anopheles gambiae sensu stricto from CDC LT collections in and the 2012 dry collection (χ2 = 7.29, 95% CI = 0.46, 3.2, the lakeside villages (r34c5, r34c6; Katuna, Yenga, and Malulu) and streamside villages (r29c10, r26c11; Kapande B and Mutepuka) P = 0.009) was also observed, but there was no significant during the 2013 wet season in Nchelenge District difference in the EIRs (RR = 0.05, 95% CI = −2.5, 2.6, SIR EIR P = 0.971). There was no statistically significant difference Wet 2013 Anopheles spp. (%) (ib/p/6mo) in the SIRs and EIRs of An. funestus between the 2012 Lakeside An. funestus s.s. (N = 19) 0.0 0.0 and 2013 wet collections (SIR: χ2 = 0.0, 95% CI = −1.5, 1.5, An. gambiae s.s. (N = 98) 1.5 0.60 P = 0.967; EIR: RR = 2.4, 95% CI = −0.69, 5.4, P = 0.129). Streamside An. funestus s.s. (N = 461) 3.9 51.5 No infectious An. gambiae were caught in the 2012 wet An. gambiae s.s. (N = 278) 3.4 10.1 and dry collections. As a result, logistic regression and nega- CDC LT = Center for Disease Control light trap; EIR = entomological inoculation rate; ib/p/6mo = infectious bites per 6-month period; SIR = sporozoite infection rate. tive binomial regression comparing An. gambiae SIRs and EIRs, respectively, between collection periods could not be performed. 2037.4, P = 0.99). In the wet season collection of 2013, both An. funestus and An. gambiae had SIRs of 3.0% and 2.5% DISCUSSION (Table 1) and the EIRs were 39.6 ib/p/6mo and 5.9 ib/p/6mo for An. gambiae, respectively. There were no significant dif- In this study, malarial vector transmission dynamics were ferences in SIRs (χ2 = 3.44, 95% CI = 0.07, 2.2, P = 0.065) characterized in two distinct ecologies in Nchelenge District, and EIRs (RR = 0.11, 95% CI = −3.2, 3.4, P = 0.95) between Zambia, over three consecutive seasons: 2012 wet, 2012 dry, An. funestus and An. gambiae. and 2013 wet. The densities of An. funestus and An. gambiae During the 2013 wet collection, spatial variation in vector varied markedly between wet and dry seasons and all col- EIRs and SIRs was studied between the Lake Mweru lections revealed microspatial dynamics of An. funestus and and Kenani Stream villages (Table 2). Along the lakeside, An. gambiae in villages located along Lake Mweru and Kenani P. falciparum was detected by CSP-ELISA in only An. gambiae, Stream. During the wet seasons, An. gambiae was the primary resulting in an SIR of 1.5% and an EIR of 0.60 ib/p/6mo vector found in lakeside villages, whereas An. funestus and (Table 2). In contrast, both An. funestus and An. gambiae An. gambiae were the primary and secondary vectors, respec- near the stream were found to be infectious. Higher infectivity tively, in interior streamside villages. The dry season collec- rates were detected than the anopheline vectors found along tion suggested that An. funestus is the dominant vector of the lakeside: SIRs of 3.9% and 3.4%, respectively (Table 2); transmission in both the lakeside and streamside villages however, the lack of infectious An. funestus inthelakesidevil- during periods of low rainfall, when only a few sympatric lages prohibited logistic regression analysis to statistically com- An. gambiae were caught. It should also be noted that lake- pare the SIRs between the two localities. For An. gambiae, side mosquito densities were a fraction of inland and stream- the SIRs between the villages near the lake and stream were side densities, regardless of season (Figure 4). The abundances not significantly different (χ2 = 3.97, 95% CI = −0.34, 3.9, of An. gambiae in both the 2012 and 2013 wet collections P = 0.10). The seasonal EIRs in the streamside collections were significantly greater than the 2012 dry collection. This is were also higher than the lakeside collections for An. funestus consistent with previous reports of An. gambiae proliferation and An. gambiae, 51.2 ib/p/6mo and 10.1 ib/p/6mo, respec- at the beginning of the wet season, whereas An. funestus tively, but again this difference was not statistically signifi- increases toward the end of the wet season and dominates cant for either An. funestus (RR = 20.1, 95% CI = −2027.7, in the dry season.39,40 The increases in An. gambiae mosqui- 2067.9, P = 0.99) or An. gambiae (RR = 2.9, 95% CI = −0.10, toes are likely due to a wide range of temporary sunlit breed- 5.9, P = 0.06). ing habitats found throughout Nchelenge District such as Spatial differences in SIRs and EIRs of vectors were also animal footprints, puddles, and ground depressions formed investigated within the lakeside and streamside areas in the due to heavy rains during the wet season.3,41,42 In villages 2013 wet collection. Within the lakeside collection, there near Lake Mweru, An. gambiae larvae and pupae in ovipo- was no statistical significance between the An. funestus and sition surveys have been collected in puddles and boats An. gambiae EIRs (RR = 18.9, 95% CI = −10930.4, 10968.3, onshore along the lake, as well as in ditches along the main P = 0.997). No infectious An. funestus were caught near the road (unpublished data). In contrast, An. funestus typically lake. As a result, logistic regression could not be performed prefer vegetated, semipermanent to permanent breeding to compare its SIR with that of An. gambiae. In the stream- sites such as swamps and marshes, which are abundant in side collections, there was a significantly higher proportion Nchelenge, but are likely to be washed out during heavy of infectious An. funestus per trap night than An. gambiae rain.3,41,43 Overall, An. funestus was found throughout all (χ2 = 5.79, 95% CI = 0.19, 2.10, P = 0.019), although there collection seasons at high densities, especially inland near was no statistically significant difference in EIRs between Kenani Stream, which likely serves as a stable habitat with An. funestus and An. gambiae in the streamside villages permanent marsh and swamps along the stream channel that (RR = 1.61, 95% CI = −0.29, 3.52, P = 0.096). remain flooded through the dry season. Comparison of SIR and EIR between collections. The Both An. funestus and An. gambiae were found to be highly SIRs and EIRs for each vector were compared among the anthropophilic, but surprisingly a few mixed human/goat and 2012 and 2013 collections. There was a difference in both goat only blood meals were identified in both vectors, which – the proportion of infectious An. funestus in the 2012 wet col- has been infrequently reported.40,44 46 In Nchelenge District, lection compared with 2012 dry collection (χ2 = 5.19, 95% goats are occasionally kept indoors at night and may serve CI = 0.17, 3.5, P = 0.031) and the EIR (RR = 2.4, 95% as an incidental host to indoor foraging An. funestus and HABITAT PARTITIONING OF MALARIA VECTORS 1241

An. gambiae. Anopheles funestus and An. gambiae are widely to stable breeding habitats and low rainfall, factors that – known for their anthropophilic, as well as endophilic and support survival of the An. funestus population.3,64 71 The endophagic behavior,18,47,48 so it is not unexpected that in longevity of An. funestus is particularly important because Nchelenge District, both vectors have a tendency to feed a higher proportion of malarial parasites can successfully almost exclusively on humans.3 During the study it was develop and, combined with frequent exposure to infections observed that inhabitants often participate in social gatherings still present in the local human population, can continue into the late evening, indoors and outdoors. As a result, transmission into the wet season. However, when compar- we cannot overlook the possibility of indoor and/or outdoor ing the SIR and EIRs of An. funestus from the 2012 dry exposure to these two vectors prior to being under a bed net and 2013 wet periods, the wet collection parameters were – that may also contribute to malarial transmission.49 54 higher than the dry collection suggesting that a larger sam- Each vector’s role in transmission was further defined by pling, especially during the dry season, would help better determining the SIR for both An. funestus and An. gambiae. define the seasonal transmission dynamics. A comparison The agreement between SIRs determined by CSP-ELISA of the wet collections suggested a higher An. funestus EIR and PCR methods for each collection was considered “poor” in 2013 than 2012. During the 2013 wet collection, only for all collections. There have been several studies that have infected An. gambiae werefoundinthelakesiderelative reported false CSP-ELISA positive results compared with to An. funestus, and a higher, but not significant, SIR was – microscopy and PCR.55 59 Other studies have been unable to observed in streamside An. gambiae. Due to the low number identify the cross-reacting antigens suspected of leading to of infectious specimens, sample size may have limited the false CSP-ELISA results, but have shown that the unknown ability to detect significance, particularly along Lake Mweru, antigen is heat unstable and can be removed by heating the where low densities of mosquitoes reside compared with areas CSP-ELISA homogenate to 100°C for 10 minutes.55,60 Because inland and near Kenani Stream. In the streamside villages, this method of heating CSP-ELISA homogenate was not the An. funestus SIR was greater than that of An. gambiae. performed for this study, it is essential that future research The large numbers of both An. funestus and An. gambiae in the confirm initial CSP-ELISA results with a second-heated inland streamside collections helped define patterns in SIRs CSP-ELISA or confirming infection with another diagnostic and EIRs, whereas differences in numbers of the two species method. The secondary method used in this study, detecting in the relatively small lakeside collection were not signifi- Plasmodium spp. in mosquitoes by PCR can detect as little cant. The trend suggests that An. gambiae is the predominant as 10 sporozoites, whereas CSP-ELISA requires at least vector lakeside. In the streamside villages, the An. funestus 100 sporozoites for detection.55 The PCR performed in this EIR was higher than that for An. gambiae. study detects all stages of Plasmodium spp., and due to ineffi- General trends for An. funestus and An. gambiae EIRs cient recovery of DNA from the ELISA homogenate of across the collection periods show that transmission by mosquito head and thorax was carried out on the mosquito An. funestus is highest during the dry season when An. gambiae abdomen only. A positive result by PCR from the abdomen, has either low abundance or is absent. Throughout the wet therefore, does not necessarily imply salivary gland infection seasons, An. funestus remains the dominant vector of trans- and could lead to overestimation of the SIR.55 CSP-ELISA mission, but at a lower intensity than in the dry season, likely is often the preferred method as it detects only the human- due to larval habitat washout during the rains. Anopheles infectious stage of the parasite. However, a sample that is gambiae contributes very little to transmission during the dry positive by CSP-ELISA and by PCR of the abdomen would season and then serves as a secondary vector with an EIR strongly indicate infection with P. falciparum. In this study, similar to that of An. funestus in the wet season. As a result, only 15 of 99 An. funestus and 0 of 26 An. gambiae were posi- malarial transmission in Nchelenge District is maintained tive by both methods (Table 3), suggesting that there is very year-round primarily by An. funestus. Spatially, transmission little agreement between CSP-ELISA and PCR. intensity of malaria is much higher in the inland streamside The SIRs and subsequent EIRs reported in this study villages with high EIRs by both An. funestus and An. gambiae. are based on the CSP-ELISA method, which is more spe- In the lakeside villages, An. gambiae appears to be the cific to salivary gland infection compared with PCR and dominant malarial vector, as no infectious An. funestus were – more widely used for EIR estimations,55,61 63 allowing for detected in this study, likely an artifact of the small sample comparison with other studies. The An. funestus SIR and size. As Nchelenge endures a high burden of malaria year- EIR in the 2012 dry collection was higher than the 2012 wet round (J. Pinchoff, unpublished data), it is likely that these collection. The large proportion of infectious An. funestus two vectors drive transmission across the district. Here, found within households during the dry season are likely due both vectors are highly resistant to pyrethroids (M. Muleba,

TABLE 3 Comparison of Anopheles funestus sensu stricto and Anopheles gambiae sensu stricto sporozoite infection rates by CSP-ELISA, PCR, and both CSP-ELISA and PCR combined for the 2012 wet, 2012 dry, and 2013 wet collections Anopheles spp. CSP-ELISA PCR CSP-ELISA and PCR Wet 2012 An. funestus s.s. (N = 343) 8 (2.3%) 0 (0%) 0 (0%) An. gambiae s.s. (N = 36) 0 (0%) 0 (0%) 0 (0%) Dry 2012 An. funestus s.s. (N = 1315) 31 (2.4%) 32 (2.4%) 8 (0.61%) An. gambiae s.s. (N =8) 0 (0%) 0 (0%) 0 (0%) Wet 2013 An. funestus s.s. (N = 676) 19 (2.8%) 24 (3.6%) 7 (1%) An. gambiae s.s. (N = 505) 16 (3.2%) 10 (1.98%) 0 (0%) CSP-ELISA = circumsporozoite enzyme-linked immunosorbent assay; PCR = polymerase chain reaction. 1242 DAS AND OTHERS unpublished data), which may explain why previous IRS RE, Hay SI, 2010. The dominant Anopheles vectors of human campaigns have shown little effect in controlling malaria malaria in Africa, Europe and the Middle East: occurrence data, in Nchelenge.59 distribution maps and bionomic precis. Parasit Vectors 3: 117. 6. Githeko AK, Adungo NI, Karanja DM, Hawley WA, Vulule The present study demonstrates that An. funestus and JM, Seroney IK, Ofulla AV, Atieli FK, Ondijo SO, Genga IO, An. gambiae are the major vectors of malarial transmission Odada PK, Situbi PA, Oloo JA, 1996. Some observations in Nchelenge District, Zambia. During the wet season, both on the biting behavior of Anopheles gambiae s.s., Anopheles vectors contribute to P. fal ci par um transmission, whereas arabiensis, and Anopheles funestus and their implications for malaria control. Exp Parasitol 82: 306–315. during the dry season, only An. funestus appears to main- 7. Curtis CF, 1996. An overview of mosquito biology, behaviour tain transmission. Moreover, the villages found inland along and importance. Ciba Found Symp 200: 3–7. Kenani Stream endure much higher biting rates of An. funestus 8. Ndenga B, Githeko A, Omukunda E, Munyekenye G, Atieli H, and An. gambiae than the areas along Lake Mweru, where Wamai P, Mbogo C, Minakawa N, Zhou G, Yan G, 2006. An. gambiae appears to be the primary vector during the Population dynamics of malaria vectors in western Kenya highlands. J Med Entomol 43: 200–206. rainy season. It is especially alarming that despite significant 9. Githeko AK, Ayisi JM, Odada PK, Atieli FK, Ndenga BA, spatial differences in vector composition and transmission Githure JI, Yan G, 2006. Topography and malaria transmis- intensity at these two representative sites in Nchelenge, high sion heterogeneity in western Kenya highlands: prospects for year-round transmission in maintained across these habitats. focal vector control. Malar J 5: 107. The characterization of major Anopheles vectors in Nchelenge 10. Fontaine RE, Pull JH, Payne D, Pradhan GD, Joshi GP, Pearson JA, Thymakis MK, Camacho ME, 1978. Evalua- in three consecutive seasons and across habitats will further tion of fenitrothion for the control of malaria. Bull World guide development and implementation of effective vector Health Organ 56: 445–452. control strategies in this area. 11. Gimnig JE, Kolczak MS, Hightower AW, Vulule JM, Schoute E, Kamau L, Phillips-Howard PA, ter Kuile FO, Nahlen BL, Hawley WA, 2003. Effect of permethrin-treated bed nets on Received October 9, 2015. Accepted for publication February 7, 2016. the spatial distribution of malaria vectors in western Kenya. Published online March 21, 2016. Am J Trop Med Hyg 68: 115–120. 12. Gimnig JE, Vulule JM, Lo TQ, Kamau L, Kolczak MS, Phillips- Acknowledgments: We gratefully acknowledge the Southern Africa Howard PA, Mathenge EM, ter Kuile FO, Nahlen BL, Hightower International Centers of Excellence for Malaria Research field teams AW, Hawley WA, 2003. Impact of permethrin-treated bed nets in Nchelenge for their logistical support and participation in field on entomologic indices in an area of intense year-round malaria collections and laboratory analysis. We are also very grateful to the transmission. Am J Trop Med Hyg 68: 16–22. communities in Zambia in whose households collections were made. 13. Killeen GF, Fillinger U, Knols BG, 2002. Advantages of larval Financial support: This work was supported in part, through funding control for African malaria vectors: low mobility and behav- from the Southern Africa International Centers of Excellence for ioural responsiveness of immature mosquito stages allow high Malaria Research (U19AI089680-01) to Douglas E. Norris. Smita effective coverage. Malar J 1: 8. Das was supported by a National Institutes of Health T32 Grant 14. Killeen GF, McKenzie FE, Foy BD, Schieffelin C, Billingsley PF, (2T32AI007417-16) and the Martin Frobisher Fellowship Fund from Beier JC, 2000. The potential impact of integrated malaria the W. Harry Feinstone Department of Molecular Microbiology and transmission control on entomologic inoculation rate in highly Immunology, a Johns Hopkins Malaria Research Institute Fellow- endemic areas. Am J Trop Med Hyg 62: 545–551. ship from the Johns Hopkins Malaria Research Institute, and a 15. Shililu J, Ghebremeskel T, Mengistu S, Fekadu H, Zerom M, Johns Hopkins Global Health Established Field Placement Award Mbogo C, Githure J, Novak R, Brantly E, Beier JC, 2003. from the Johns Hopkins Center for Global Health, Johns Hopkins High seasonal variation in entomologic inoculation rates in University Bloomberg School of Public Health. Eritrea, a semi-arid region of unstable malaria in Africa. Am J Trop Med Hyg 69: 607–613. Authors’ addresses: Smita Das and Douglas E. Norris, W. Harry 16. Utzinger J, Tanner M, Kammen DM, Killeen GF, Singer BH, Feinstone Department of Molecular Microbiology and Immunology, 2002. Integrated programme is key to malaria control. Nature The Johns Hopkins Malaria Research Institute, Johns Hopkins 419: 431. Bloomberg School of Public Health, Baltimore, MD, E-mails: smita 17. Norris LC, Norris DE, 2013. Heterogeneity and changes in [email protected] and [email protected]. Mbanga Muleba, inequality of malaria risk after introduction of insecticide-treated Tropical Disease Research Centre, Ndola Central Hospital, Ndola, bed nets in Macha, Zambia. Am J Trop Med Hyg 88: 710–717. Zambia, E-mail: [email protected]. Jennifer C. Stevenson, W. Harry 18. Norris LC, Fornadel CM, Hung WC, Pineda FJ, Norris DE, Feinstone Department of Molecular Microbiology and Immunology, The 2010. Frequency of multiple blood meals taken in a single Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg gonotrophic cycle by Anopheles arabiensis mosquitoes in Macha, School of Public Health, Baltimore, MD, and Macha Research Trust, Zambia. Am J Trop Med Hyg 83: 33–37. 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