Attractive Toxic Sugar Baits (ATSB) for Mosquito Control: Past, Present

PROJECT REPORT

Candidate Number: 105874

MSc: Control of Infectious Diseases

Title: Attractive Toxic Sugar Baits (ATSB) located near mosquito nets for control of pyrethroid resistant Anopheles arabiensis and Culex quinquefasciatus in Northeast Tanzania

Supervisors: Mark Rowland, Rick Oxborough, & Seth Irish

Word Count: 7,981

Project Length: Standard

Submitted in part fulfillment of the requirements for the degree of MSc in CID For Academic Year 2011-2012 Table of Contents

Table of contents……………………………………………………………….…………..…1 Acknowledgements………………………………………....……………...………….……..2 Abstract..……………………………………………………………………………………...... 3

1. Background………………………………………………………………………...... …….4 1.1 Managing Species Shift...... 5 1.2 Potential for Resistance Management...... 5 1.3 ATSB Attractant and Optimization...... 5 1.4 Successful Trials and Limitations of ATSB...... 6 1.5 Potential Future of ATSB...... 7

2. Aims and objectives...... 8

3. Materials and methods………………………...... 9 3.1 Phase 1 Attractant Testing...... 9 3.2 ATSB & ASB Preparation...... 10 3.3 Phase 1 Oral Insecticide Dose Response Cage Bioassays...... 11 3.4 Phase 2 Experimental Hut Trial (ATSB +Net)...... 12 3.4a Study site and hut design...... 12 3.5 Experimental Hut Trial Design...... 13

4. Ethical Clearance...... 15

5. Data analysis...... 15

6. Results…………………………………………………………...... 16 6.1 Phase 1 Attractant Testing...... 16 6.2 Phase 1 Cage Bioassay...... 16 6.3 Phase 2 Experimental Hut Trial...... 18

7. Discussion ……………………………………………...... 21

Reference list………………………………………………………………...... 23

Acknowledgements I would like to thank my advisors Mark Rowland, Rick Oxborough, Seth Irish, Patrick Tungu, and Matt Kirby for their support in formulating the project, connecting me with additional experts in the field, and providing continuous technical support throughout the project. I would also like to thank the insectary and field staff at the National Institute for Medical Research, Amani Medical Research Centre, Muheza, Tanzania and at the Kilimanjaro Christian Medical College, Joint Malaria Program, Moshi, Tanzania for their assistance rearing and scoring mosquitoes. This work was supported through the Innovative Vector Control Consortium and London School of Hygiene and Tropical Medicine Helena Vrbova and Gordon Smith Scholarships.

Abstract

Background: The purpose of this study was to determine if Anopheles arabiensis and Culex quinquefasciatus would feed on attractive toxic sugar baits (ATSB) if diverted from blood feeding by a mosquito net in an experimental hut trial. This setup aimed to replicate what occurs as mosquito resistance increases to contact insecticides on mosquito nets.

Methods: Phase 1: Candidate oral insecticides and attractants were tested on An. arabiensis, An. gambiae, and Cx. quinquefasciatus in cage bioassays to be used in the ATSB in phase 2. Phase 2: Insecticides and attractants with the highest level of induced mortality in phase 1 were delivered in ATSB bait stations in experimental huts in Northeast Tanzania.

Results: Phase 1 cage bioassays: Guava juice was found to be the strongest attractant and chlorfenapyr 0.5%, boric acid 2%, and tolfenpyrad 1% were all highly effective in killing An. arabiensis, An. gambiae, and Cx. quinquefasciatus. An. arabiensis mortalities for chlorfenapyr 0.5%, boric acid 2%, tolfenpyrad 1% were 91.4%, 83.1%, and 88.5%, An. gambiae mortalities were 100%, 84.5% and 86.0%, and Cx. quinquefasciatus mortalities were 94.8%, 77.6%, and

99.4% respectively. Phase 2 experimental hut trial: ATSB stations positioned near nets and windows proved complimentary. Female An. arabiensis mortalities in the experimental huts for chlorfenapyr 0.5%, boric acid 2%, and tolfenpyrad 1% were 48.0%, 41.3%, and 45.0% and for male An. arabiensis 58.2%, 47.6%, and 48.4% respectively. Female Cx. quinquefasciatus mortalities for chlorfenapyr 0.5%, boric acid 2%, and tolfenpyrad 1% were 42.9%, 40.0%, and

35.7% and for male Cx. quinquefasciatus 66.7%, 50.0%, and 43.8% respectively.

Conclusion: ATSB positioned near mosquito nets and windows is a viable option for killing diverted An. arabiensis and Cx. quinquefasciatus and is a possible tool for resistance management and prolonging the effective life of insecticide treated nets.

1. Background

Mosquito vector control through the use of insecticide treated nets (ITN) as well as indoor residual spraying (IRS) has seen substantial success across sub-Saharan Africa. However, the emergence of insecticidal resistance has reduced the effectiveness of both of these tools (N’Guessen et al., 2007; Corbel et al., 2007; Djouaka et al., 2008; WHO, 2011). Early DDT use decimated disease vectors but resulted in the immergence of insecticidal resistance in several of the primary malaria vectors. Following this development, pyrethroids predominated use on ITN, and organophosphates, carbomates, and pyrethroids used for IRS to control resistant mosquitoes but subsequently resulted in further resistance which quickly spread across Africa (Raghavendra et al., 2011). Pyrethroid resistant An. gambaie have been reported in many African countries including Tanzania (Kabula et al, 2012), Côte d’Ivoire (Elissa et al, 1993; Chandre et al., 1999), Kenya (Vulule et al.,1999), Benin (Akogbeto et al., 1999), Burkino faso (Diabate et al., 2002a), Mali (Fanello et al., 2003), Nigeria (Awolola et al., 2002), Cameroon (Etang et al., 2003), and multiple insecticide resistance in both An. gambiae and Cx. quinquefasciatus in Benin (Corbel et al., 2007). Additionally, there have been very high levels of pyrethroid resistanct Cx. quinquefasciatus reported throughout Africa (Chandre et al., 1998). In light of this, novel complimentary techniques are urgently needed to maintain the effective life of these tools especially as control programs push for “universal” ITN coverage of at risk populations and the scaling up of IRS (WHO, 2006). One recently developed tool for malaria vector control is the use of attractive toxic sugar baits (ATSB) which employs a sugar-based olfaction stimulant and food source with an orally ingested insecticide. ATSB have several properties that suggest it could be a plausible tool used in conjunction with ITN and IRS as part of mosquito integrated vector management (IVM). Sugar feeding plays a fundamental role in the life of adult mosquitoes. Both male and female mosquitoes feed on plant sugars, usually floral and extrafloral nectars, honeydew, and fruits juices. Males exclusively feed on sugar where as anautogenous females of some species such as Anopheles gambiae, An. arabiensis, and Culex quinquefasciatus require a blood meal for egg development (Foster, 1995). Though sugar feeding is an essential component of the adult mosquito’s life, it has received little attention as a potential point of intervention for IVM until recently with the development of ATSB. At the moment, the large-scale application of ATSB as a mosquito control tool has been limited to exophagic mosquitoes in arid environments with low levels of sugar source vegetation.

1.1 Managing Species Shift The ATSB’s unique delivery method gives it potential to target a wider subset of mosquito disease vectors. There is a growing realization that IRS and ITN are more effective against some species but not as effective against others. IRS has eliminated localized populations of An. funestus in Tanzania due to its habit of resting indoors after feeding (Smith and Bransby-Williams, 1962; Bruce-Chwatt et al., 1973). The scale-up of insecticide treated nets and long-lasting insecticide treated nets (LLIN) has been highly effective at reducing the density of anthropophagic and endophagic disease vectors such as An. gambiae but less effective at controlling zoophagic and exophagic disease vectors such as An. arabiensis (Russel et al., 2010). This unilateral targeting of vectors has likely been a contributing factor leading to a species shift to more zoophagic and exophagic disease vectors (Kitau et al., 2012). This novel insecticide delivery technique would likely target an additive subset of vector species.

1.2 Potential for Resistance Management The potential of ATSB to kill diverted host seeking mosquitoes makes it a promising candidate for managing insecticide resistant mosquitoes and thereby preserving the long term effective life of ITN. Host seeking mosquitoes which are deterred from feeding by an ITN yet not killed by the insecticide are likely those with resistant mechanisms. These deterred, host seeking mosquitoes may be diverted to obtain a sugar meal if access to a blood meal is restricted (Stone et al., 2012). Additionally, the key advantage of ATSB is that it can deliver novel classes of oral insecticides which are widely available for agricultural pest control but are unsuitable for conventional control of medically important insects which is based on residual contact insecticides applied to ITN or IRS. ITN and IRS rely on pyrethroids, organophosphates, organochlorines, and carbamates, all of which suffer from emerging resistance (Curtis et al.,1996). The ability of the ATSB to deliver novel classes of oral insecticides makes it a strong candidate as a resistance management tool.

1.3 ATSB Attractant and Optimization Past ATSB delivery has depended on its ability to outcompete locally available sugar sources (Muller, et al., 2010b), yet for this study the successful delivery of the solution will likely largely depend on the ATSB’s ability to divert host seeking mosquitoes. Sugars by themselves are not volatiles, instead mosquitoes respond to odors of flowers, raw fruit, honey, and various other plant compounds in wind-tunnel olfactometers (Foster, 1995). Past field trials have each used differing sugar attractants based on the local availability and their relative attractiveness to

5 local mosquito populations (Beier et al., 2012; Muller, Junnila et al., 2010a; Muller, Schlein, 2008; Muller et al., 2010b; Muller et al., 2010c; Muller et al., 2006; Muller and Kravchenko, 2008). For example, in order to test the attractiveness of local sugar sources in Mali, wire cages, covered in glue, were placed around flowering plants, fruits, and seedpods and left for 24 hours. The following day, the quantity and species of mosquitoes were recorded to determine which sugar source attracted the greatest number of the target mosquito species. This trial found guava (Psidiumguajava) and honey melon (Cucumismelo) to be the most attractive and therefore served as the bait in the locally used ATSB (Muller et al., 2010d). This type of site specific field testing of attractants is a key component to optimize the effectiveness of ATSB (Muller et al., 2010d). Further attractants have been successfully used in ATSB such as rotting nectarines (Prunus persica) (Muller and Schlein, 2008; Muller and Kravchenko, 2008) rotting plums (Prunus americana) (Muller et al., 2010c) and rotting prickly pear cactus (Opuntia ficus- indica) (Beier et al., 2012).

1.4 Successful Trials and Limitations of ATSB ATSB for mosquito control was originally developed in Israel in an arid habitat with relatively little sugar source vegetation (Muller et al., 2006). In this setting, it was feasible to cover a high proportion of the local sugar sources with ATSB. This method was very successful in Israel with two reports of An. sergentii population being reduced by over 95% (Beier et al., 2012; Muller et al., 2006). Following this trial, this same concept of applying ATSB in a semi-arid area with low levels of sugar source vegetation was trialed in Mali. Again, the ATSB drastically reduced the local An. gambiae s.l. population by 90% (Muller et al., 2010b). Here too the application of ATSB to a large percentage of the locally available sugar source vegetation was feasible due to low levels of locally competing sugar sources. Additionally, field trials in both Israel and Mali have shown that ATSB not only reduced the total number of mosquitoes but also reduced the longevity of older, more entomologically important mosquitoes. In Israel, ATSB dramatically reduced daily survival rates driving malaria vectorial capacity from 11.2 to 0.0 and from 79.0 to 0.3 (Beier et al., 2012). Similarly, ATSB treatments in Mali reduced the percentage of mosquitoes which had completed three or more gonotrophic cycles from 37% pre-treatment to 6% in a month’s time (Muller et al., 2010b). Though ATSB stations were not as successful as spraying ATSB directly onto vegetation in these settings, ATSB stations have been successful when placed in mosquito resting and breeding sites such as water cisterns in Israel and storm drain systems in Florida, United States (Muller, et al., 2010a; Muller and Schlein, 2008). Mosquitoes emerge to adulthood with critically

6 low energy reserves and tend to take a sugar meal within the first hours after emerging (Foster, 1995). Additionally, some species such as An. claviger and Cx. quinquefasciatus congregate in large numbers in resting sites such as water cisterns and storm drain systems making this an ideal location to use ATSB stations. The use of ATSB stations in this way saw dramatic declines in the population of local mosquitoes. In Israel, the ATSB stations placed in the water cisterns caused a drastic decline of An. claviger reducing the number of human-landing mosquitoes in the area by more than ten-fold (Muller and Schlein, 2008). In Florida, 83.7% of the Cx. quinquefasciatus females were reduced from the storm drains over an eight day period (Muller et al., 2010c). As the method stands now, ATSB are limited to use in environments with low levels of competing sugar source vegetation and to areas with relatively few localized sources of mosquitoes such as in breeding and resting sites. Additionally, ATSB have been found to be less effective where there are high levels of unprotected human hosts (Stone et al., 2012). These major limitations restrict the effective use of the ATSB method from most of malaria burdened sub-Saharan Africa drastically reducing the effective range and overall impact of the intervention. In areas that do not meet these restrictions, it is unlikely that the current ATSB method will be able to produce the same substantial results as past trials unless the bait is much more attractive than it currently is, or the ATSB method is applied in alternative areas where mosquitoes are already focused or drawn in large numbers. ATSB in and of themselves are not the innovation, instead it is how they are applied. The ATSB method applied indiscriminately would likely see little to no effect for mosquito control, yet when ATSB are applied with appropriate entomological insight; it has proven to be a powerful tool for IVM and has strong potential for future large scale application.

1.5 Potential Future of ATSB Though the ATSB method has been limited to the control of exophagic mosquitoes in areas of low sugar source vegetation, ATSB stations could work synergistically with mosquito nets by killing diverted host seeking mosquitoes indoors (Stone et al., 2012). Many species of malaria transmitting mosquitoes are already highly attracted to human hosts indoors, therefore, placing ATSB stations in huts with human inhabitants, protected by a bed net, would target mosquitoes where they are already being attracted and focused. The effective use of a bed net would force mosquitoes to seek alternative food sources such as an ATSB when energy reserves are already low after a night of host seeking. By using ATSB in this method, the bait itself is not the sole attractant but is rather complementary to the already highly attractant

7 human host. Just as in past successful trials, an ATSB, is not the sole attractant but rather is placed where mosquitoes are already being focused. Applying ATSB in this manner would resolve the previously discussed limitations and dramatically expand the effective range of the ATSB method throughout much of malaria endemic sub-Saharan Africa. Yet, much remains unknown about the likelihood of mosquitoes to divert from attempting to take a blood meal to feeding on sugar. One trial reports that glucose pads, placed in experimental huts trialing bed nets, reduced the overall mortality of the mosquitoes in the hut due to starvation (Curtis et al., 1996). Additionally, another report concluded that host seeking mosquitoes, prevented from taking a blood meal throughout a nocturnal feeding period, were more likely to take a sugar meal the following morning than mosquitoes not stimulated by a blood-host (Foster, 1995). Even if a mosquito managed to take a partial blood meal, they have also been shown to take a sugar meal if presented with this additional choice, yet was unlikely to feed on sugar when access to blood was unrestricted (Stone et al., 2012).

2. Aims and Objectives

The aim of this trial is to assess the potential added benefit of using ATSB stations alongside a mosquito net to control wild populations of An. arabiensis, and Cx. quinquefasciatus.

1. Phase one objective: To develop candidate ATSB solutions which are efficacious in both stimulating feeding and killing pyrethroid-resistant and susceptible An. gambiae, An. arabiensis, and Cx. quinquefasciatus under laboratory conditions a. Part 1: To compare differing attractive sugar baits (ASB) with unique fruit attractants b. Part 2: Will use the ASB from part 1 with the best feeding stimulation, in addition to varying doses of chlorfenapyr, boric acid, or tolfenpyrad to optimize mortality in cage bioassays 2. Phase two objective: To use the candidate ATSB from phase 1 alongside an untreated mosquito net in an experimental hut trial to determine the efficacy of the ATSB in killing deterred pyrethroid-resistant and susceptible wild populations of An. arabiensis, and Cx. quinquefasciatus in Pasua village, N.E. Tanzania

3. Materials and Methods

3.1 Phase 1 Attractant Testing The attractant is a key component to the ATSB solution and is a potential point of further advancement. Past ATSB trials have used ripened to rotting fruit juices as the attractant (Muller, Kravchenko, 2008; Muller et al., 2006; Beir et al., 2012; Muller et al., 2006; Muller et al., 2010b; Muller et al., 2010a; Muller, Schlein, 2008; Muller et al., 2010c). Yet, very little research has looked at maximizing the fruit juice attractant in the ATSB except for one trial in Mali which found 48hr ripened guava juice to be the most strongly selected attractant for wild populations of female An. gambiae (Muller et al., 2010d). In attempt to maximize the attractant used in the bait, a brief pilot was performed. Two cages of 50 female An. gambiae and two cages of 50 female Cx. quinquefasciatus were exposed to 4 differing attractants in 30cm square netted cages. After the first replicate, mango juice was the least effective at stimulating feeding and was replaced with beetroot red dye (Dr. Oetker, Leeds, UK) as the fourth attractant for the second replicate.

The following 4 attractants were compared:

1. Mango Juice 2. Jack Fruit Juice 3. Guava Juice 4. Beetroot Red dye 5. 10% Glucose (Baseline)

These attractants were selected either because of their local availability or their past success in previous trials. Beetroot red dye was also trialed to determine if the dye was serving as an additional attractant or deterring feeding. Juices were ripened outdoors in the sun for 48hrs to increase the volatiles, whereas the dye and 10% glucose solutions were fresh. The mango and jack fruit juices were made fresh whereas the guava juice was from a cartoon (Azam, Bakhresa Food Products LTD, Dar Es Salaam, Tanzania). Three ml of juice was put on cotton pads on plastic trays. The pads were put into each corner of each Image 1 (top view) Cage bioassay attractant testing delivery cage and left for five minutes (see Image 2). After five

9 minutes, the numbers of feeding mosquitoes currently on the bait pads were counted and recorded, the feeding mosquitoes were blown off the bait pads, and the treatments removed from the cages. Those mosquitoes which had landed on the bait pads during the five minute period but had relocated prior to the end of the five minute period were not counted. Five minutes was chosen to count only mosquitoes which were selecting a bait and were then staying on the bait long enough to take a full meal therefore potential a lethal dose of insecticide if an insecticide was present. Five minutes of decontamination period was allowed between each five minute treatment cycle rotation to allow lingering volatiles to pass from the cages. After the five minute decontamination period, the four attractant treatments were put back into the cages the same cage for another five minutes. This cycle was repeated twelve times. Each time the four pads were placed in the cage, they were rotated so that every pad was in each corner three times. The order in which the pads were put into the cages, each treatment cycle, was generated with a random number generator.

3.2 ATSB & ASB Preparation The bait solution used in the cage bioassays and experimental hut trial followed the same base recipe as previous ATSB studies (Muller, Kravchenko, 2008; Muller et al., 2006; Beir et al., 2012; Muller et al., 2006; Muller et al., 2010b; Muller et al., 2010a; Muller, Schlein, 2008; Muller et al., 2010c) consisting of 35% v/v over-ripe to rotting guava juice (Azam, Bakhresa Food Products LTD, Dar Es Salaam, Tanzania), 10% w/v brown sugar, 2% v/v red food dye (Dr. Oetker, Leeds, UK), and 0.5%-2% of varying oral insecticides (boric acid, tolfenpyrad 15SC, chlorfenapyr). The following oral insecticides and doses were trialed in phase 1: boric acid 1% and 2% w/v, Tolfenpyrad 15SC 0.5% and 1% v/v, and chlorfenapyr (21.45%) 0.5% and 1% v/v. Guava juice was ripened for 48hrs before adding to the bait solution. Guava juice was previously found to be highly attractive to An. gambiae (Muller et al., 2010d) and was also found to be highly attractive to An. arabiensis and Cx. quinquefasciatus in observational cage bioassays (Stewart, unpublished). These three oral insecticides were selected based on their local availability (chlorfenapyr & boric acid), novelty (tolfenpyrad), class diversity (all three), potential to kill resistant mosquitoes (all three) (Irish, unpublished; Raghavendra et al, 2011; N’Guessan et al., 2007), or their proven effectiveness in past ATSB studies (boric acid).

3.3 Phase 1 Oral Insecticide Dose Response Cage Bioassays Prior to this final trial, pilot trials were performed with a wide range of doses. The candidate doses from these initial pilot trials were then trialed with replication in the full trial.

The initial pilot dosages were as follows:

Boric Acid 1%, 2%, 3%, and 4% Tolfenpyrad 0.5%, 1% and 2% Chlorfenapyr 0.5%, 1% and 2%

In order to determine which oral insecticides and dosages to use in the phase 2 experimental hut trial, cage bioassays were performed with female An. gambiae sensu strict Kisumu strain originally from Kenya, F1 generation of wild An. arabiensis from Lower Moshi, and Cx. quinquefasciatus, a multiple resistant strain with elevated oxidase and knockdown resistance (kdr) pyrethroid resistance mechanisms. Mosquitoes were 3 to 4 days post immergence and were starved for 4 hours prior to testing. Approximately 50 female mosquitoes were placed in 30cm square cages and were exposed to the ATSB for 11 hours throughout the night aiming to replicate the nightly exposure time in the phase 2 experimental hut trial between bait setting and collection. There were three replicates of each treatment for An. gambaie and Cx. quinquefasciatus testing.

The following 4 treatment arms were compared:

Attractive sugar bait (ASB) No insecticide ASB + Tolfenpyrad 1% ASB + Chlorfenapyr 0.5% ASB + Boric Acid 2%

Approximately 25ml of the ATSB solution was soaked into a cotton pad and placed in the center of the cage on top of an upside down cup (see Image 1). Food dye was used in the bait solution to indicate feeding on the ATSB by observing the dye in the mosquito abdomen. After 11 hours, mosquitoes were scored as live fed (dyed), alive unfed (not dyed), dead fed Image 2 Cage bioassay ATSB delivery 11

(dyed), and dead unfed (not dyed). The scorer was blinded from the treatment and the baits were indistinguishable from one another except for an identification code. Live mosquitoes were transferred to paper cups and given a 10% glucose cotton pad and were kept in a 26°C room with a relative humidity of 70-80%. No more than 10 mosquitoes were kept per cup. After 24 hours, mosquitoes were scored for delayed mortality.

3.4 Phase 2 Experimental Hut Trial (ATSB +Net) 3.4a Study site and hut design The four experimental huts are situated at Pasua village, Kilimanjaro region, NE Tanzania (3°22’46S and 30°20’47E). The experimental hut site is directly adjacent to irrigated rice paddies which were recently flooded and planted at the beginning of the trial (see Image 3). The surrounding area has numerous flowering plants, fruit trees, domestic animals, and unprotected sleepers. The huts were constructed of adobe bricks with a corrugated iron Image 3 Experimental hut adjacent to irrigated rice paddies roof. They are built on plinths and surrounded by water filled moats to prevent entry of scavenging ants and flooding water during the rainy season (see Image 4). White linoleum flooring was installed in order to aid in collecting dead mosquitoes in rooms and verandas. Inner walls were plastered with mud and had a 5cm gap between the top of the wall and the wooden ceiling for mosquito entry. The internal hut Image 4 Experimental Hut Pasua village, Tanzania is surrounded by four verandas two of which were kept open to allow entry of wild mosquitoes and two kept closed to capture exiting mosquitoes from the eaves. Coned baffles were installed in the open eaves which allowed mosquitoes to enter yet prevent mosquitoes from exiting through the open verandas. Additionally, two exit traps were placed in windows on either side of the room.

3.5 Experimental Hut Trial Design

The trial ran for 20 nights from July 31 to August 19, 2012. ATSB solutions were presented to mosquitoes soaked on a hanging cotton towel (20x30cm2) with half of a 1L plastic bottle attached to the bottom to catch drips and hold excess solution to wick up the towel (see Image 5). Four bait stations were hung from the ceiling and positioned at the corners of the hanging untreated bed net (see Image 6).

Additionally, baits were positioned near Image 5 ATSB bait station positioning in experimental hut window traps on cotton wool. One bait hung in the center of the window and two bait strips were hung on either side of the window (see Image 7). 400ml of the ATSB solutions were added nightly per hut to keep the bait stations moist. Cotton pads soaked in 10% glucose were also available in window traps and in closed verandas and were changed Image 6 ATSB bait station Image 7 ATSB station positioning nightly. Male volunteers slept under the near window traps bed nets from 19:00hrs to 6:00hrs the following morning which was previously found to be optimum for attracting feeding mosquitoes (Oxborough, unpublished). The ATSB bait stations were rotated through the huts according to a Latin square design so that each treatment was with each sleeper and hut combination an equal number of times. The treatments were rotated between huts every four nights.

The following 4 treatment arms were compared which were previously found optimal in phase 1:

1. 2% boric acid ATSB + untreated net + 10% glucose in veranda and window traps 2. 0.5% chlofenapyr ATSB + untreated net + 10% glucose in veranda and window traps 3. 1% tolfenpyrad ATSB + untreated net + 10% glucose in veranda and window traps 4. ASB (no insecticide) + untreated net + 10% glucose in veranda and window traps

Mosquitoes were collected with aspirators each morning at 6:30hrs. Live mosquitoes in the room were not collected to allow for natural resting times, during which a Image 8 Wild-caught Cx. quinquefasciatus (left) & An. arabiensis (right) females sugar meal could be marked with dye from feeding on ATSB collected from experimental huts taken. Mosquitoes were only collected after exiting the room. Dead and live mosquitoes were collected from the veranda and window traps. Only dead mosquitoes were collected from the room. Ten minutes were spent collecting mosquitoes per veranda and room in order to inhibit over and under searching for mosquitoes. Collections in verandas, window traps, and the room were cross-checked by two individuals. Prior to rotating the bait stations through the huts, mosquito entrance was blocked every fourth morning and live mosquitoes in the room were given an additional 24hrs to feed or exit the room before collection. Every fifth night, when the bait stations were rotated, live mosquitoes in the room were removed and the rooms cleaned. Mosquitoes were immediately scored as live or dead, as bait fed (dyed) (see Image 8) or not bait fed, and identified to species. Live mosquitoes were placed in paper cups with no more than ten per cup and given a 10% glucose cotton pad and held in a 26°C room with a relative humidity of 70-80%. The mosquitoes were scored after 24hrs for delayed mortality. Bait feeding (dyed) was double scored separately by two individuals and cross-checked. Identifying dye in the abdomen became increasingly difficult as mosquitoes desiccated and for those treatments which killed quickly (ie. tolfenpyrad) before mosquitoes could take an identifiable sugar meal especially for Anophelines.

Prior to running the full experimental hut trial, two 4 night pilot trials were conducted to observe mosquito bait feeding behaviors and to make adjustments to the scoring protocol. The pilots had the same experimental setup as the full trial but with a different bait station design. The pilot bait station setup consisted of five hanging plastic dishes (see Image 9) at the corners of the net and one hanging over the center with Image 9 Pilot experimental hut trial ATSB approximately 25mls of ATSB soaked in a cotton wool bait station positioning

14 pad in each dish. The pads were changed nightly with the ATSB. The lead scientist stayed in a hut throughout the pilot trials making observations and improvements to the design. Three observations and improvements were made to the original pilot design:

1. Observation: Mosquitoes were attracted to the bait stations, but landing on the vertical surface of the dish and probing the side of the dish but less willing to enter down into the dish to feed on the solution. Therefore, the vertical surface area of the baits was increased. 2. Observation: A majority of the mosquitoes were exiting the room through the window trap compared to the eaves. Therefore, bait stations were added near the window traps. 3. Observation: The bait stations were dry before the following evening. Therefore, the volume of ATSB solution added per hut was increased from 100ml to 400ml per night.

4. Ethical Clearance

Approval was obtained from the London School of Hygiene and Tropical Medicine ethics review committee approval number: 011/391 and by the Tanzanian National Institute of Medical Research approval number: NIMR/HQ/R.8c/Vol.I/24. Trial participants signed written informed consent forms and were offered free medical services during the trial and up to three weeks after the end of participation in the trial.

5. Data Analysis

Data was analyzed using Stata 12.1 software (Stata Co., College Station TX, USA). The analysis of the phase 1 cage bioassay data was performed using logistical regression for proportional data (proportion dying, marking with dye of the total, and marking with dye of the dead) and adjusted for replicates. The analysis of the phase 2 experimental hut data was performed using logistical regression for proportional data (proportion dying, marking with dye of the total, marking with dye of the dead, and dying of the dyed) and adjusted for the effects of individual sleepers and huts.

6. Results 6.1 Phase 1 Attractant Testing

48hr ripened guava juice was found to be the strongest attractant for An. gambiae and Cx. quinquefasciatus females as compared to 48hr ripened jack fruit juice, 48hr ripened mango juice, fresh beetroot red dye, and fresh 10% glucose solution (Figure 2). Cx. quinquefasciatus females landed on the guava juice to feed at a rate of 13.5 times that of landing on the 10% glucose solution and An. gambiae females landed on the guava juice to feed at a rate 5.4 times that of landing on the 10% glucose solution. Additionally, the beetroot red dye used in the ATSB was found to be a feeding stimulant. Cx. quinquefasciatus females landed on the beetroot red dye to feed at a rate 1.8 times that of landing on the 10% glucose solution and An. gambiae females landed on the beetroot red dye to feed at a rate of 2.9 times that of landing on the 10% glucose solution. Both 48hr ripened guava juice and beetroot red dye were used in the ATSB trialed in the phase 2 experimental hut trial.

Figure 2

An. gambiae Cx. quinquefasciatus 6 5.4 16 13.5 5 14 12 relative relative 4 10 2.9 8

3 6 choices

10% glucose 10% 2.3 4 1.8 1.0 2 2 0.1 1.0 0 1 0.7 to 10% 10% glucose to 0.0 0 relative to feeding choices of Rate Jack Guava Beetroot Mango 10% Rate offeeding Rate Fruit red dye Glucose

6.2 Phase 1 Cage Bioassay Chlorfenapyr 0.5%, boric acid 2%, and tolfenpyrad 1% ATSB caused significantly mortalities for female An. gambiae (p=0.000, p=0.000 , p=0.000 respectively), An. arabiensis (p=0.000, p=0.000, p=0.000 respectively), and Cx. quinquefasciatus (p=0.000, p=0.000, p=0.000 respectively) when exposed in cage bioassays (Table 1 & Figure 1). The percent feeding on the ATSB as indicated by the percent showing dye was highest for the boric acid 2% ATSB and showed the least inhibition of feeding as compared to the control ASB feeding (Table 2). Of those mosquitoes which were confirmed fed on the ATSB/ showing dye, the boric acid

ATSB always had a few mosquitoes which were still live after 24hrs, whereas for chlorfenapyr and tolfenpyrad ATSB they always proved fatal (Table 3).

Figure 1

An. arabiensis Females Cx. quinquefasciatous An. gambiae Females Females 100 100 90 90 100 80 90 80 70 80 70 60 70 60 50 60 50 100 40 91.4 83.1 88.5 50 94.8 99.4 40 84.5 86 40 77.6 30 3.6 30 30 20 20 3.2 20 12.6 10 10 10

0 0 0

Percent Mortality (%) Mortality Percent (%) Mortality Percent Percent Mortality (%) Mortality Percent

Table 1 Cage bioassay: overall mortality (%)

Control Chlorfenapyr 0.5% Boric Acid 2% Tolfenpyrad 1% Cx. quinquefasciatus 3.2(1.3-7.4)a 94.8(89.9-97.4) b 77.6(70.6-83.3) c 99.4(95.9-99.9) d females N=157 N=153 N=165 N=166 An. arabiensis 3.6(0.9-13.4) a 91.4(80.9-96.4) b 83.1(71.3-90.6) b 88.5(77.8-94.4) b females N=55 N=58 N=59 N=61 An. gambiae 12.6(8.0-19.3) a 100 b 84.5(77.7-89.4) b 86.0(79.3-90.8) b Females N=135 N=147 N=148 N=143 % Mortality(95% Confidence Interval) *Differing superscript letter indicates statistical significance at P<0.05 as compared to the other treatment and control arms. *N=total number of mosquitoes tested

Table 2 Cage bioassay: overall percent showing dye (%)

Control Chlorfenapyr 0.5% Boric Acid 2% Tolfenpyrad 1% Cx. quinquefasciatus 84.7(78.2-89.5)a 81.0(74.1-86.5) a 86.1(79.9-90.6) a 44.0(36.6-51.6) b Females An. arabiensis 89.1(77.8-95.0) a 44.8(32.6-57.7) b 64.4(51.5-75.5) c 41.0(29.4-53.6) b females An. gambiae 74.1(66.0-80.8) a 27.9(21.2-35.7) b 68.2(60.3-75.2) a 25.9(19.4-33.7) b Females % Showing Dye(95% Confidence Interval) *Differing superscript letter indicates statistical significance at P<0.05 as compared to the other treatment and control arms.

Table 3 Cage bioassay: percent of the dyed mosquitoes that died (%)

Control Chlorfenapyr 0.5% Boric Acid 2% Tolfenpyrad 1% Cx. quinquefasciatus 1.5(0.4-5.8)a 100 b 89.4(83.2-93.5) c 100 b Females An. arabiensis 2.0(0.3-13.1) a 100 b 92.1(78.2-97.4) c 100 b females An. gambiae 9.0(4.7-16.4) a 100 b 92.1(85.0-96.0) c 100 b females % Dyed which Died(95% Confidence Interval) *Differing superscript letter indicates statistical significance at P<0.05 as compared to the other treatment and control arms.

6.3 Phase 2 Experimental Hut Trial All three of the experimental huts with the candidate ATSB had significantly higher mortality as compared to the control hut for both An. arabiensis and Cx. quinquefasciatus and both females and males (Table 4 & Figure 3). As indicated by the overall percentages of mosquitoes showing dye, the feeding preference between the ATSB and the control ASB was not significant for most species and treatments (Table 5). However, for both the ATSB and the control ASB, few collected mosquitoes were showing dye (Table 5). Even amongst the dead, the proportion showing dye was limited (Table 6) providing less direct evidence for the ATSB as the cause of the increased mortalities. In light of this, this same trend was observed in the cage bioassays in that a relatively low proportion of the dead were showing the dye. For that reason, the presence of dye was an unreliable indicator of ATSB feeding. This could be due to a small fatal dose of ATSB not being a dose sufficient enough to show the dye. Amongst those mosquitoes which were showing dye, chlorfenapyr 0.5%, boric acid 2%, and tolfenpyrad 1% ATSB solutions caused significantly high levels of mortality as compared with the control for An. arabiensis females (p=0.000, p=0.000, p=0.000 respectively), An. arabiensis males (p=0.000, p=0.000, p=0.000 respectively), and Cx. quinquefasciatus females (p=0.001, p=0.001, p=0.005 respectively) (Table 7).

Figure 3

100.0 100.0 An. arabiensis females Cx. quinquefasciatous females 90.0 90.0 80.0 80.0 70.0 70.0 60.0 60.0 50.0 50.0 40.0 40.0 30.0 30.0

7.0 Percent Mortality (%) Mortality Percent Percent Mortality (%) Mortality Percent 48.0 20.0 41.3 45.0 20.0 42.9 40.0 35.7 10.0 10.0 17.5 0.0 0.0

100.0 An. arabiensis males 100.0 Cx. quinquefasciatous males 90.0 90.0 80.0 80.0 70.0 70.0 60.0 60.0 50.0 50.0 40.0 40.0

30.0 58.2 30.0 Percent Mortality (%) Mortality Percent Percent Mortality (%) Mortality Percent 48.4 20.0 47.6 20.0 26.7 66.7 50.0 43.8 10.0 10.0 33.3 0.0 0.0

Table 4 Experimental hut: overall mortality (%)

Control Chlorfenapyr 0.5% Boric Acid 2% Tolfenpyrad 1% An. arabiensis females 17.5(11.2-26.4)a 48.0(38.4-57.7) b 41.3(32.3-51.0) b 45.0(35.9-54.4) b N=97 N=100 N=104 N=109 An. arabiensis males 26.7(20.7-33.8) a 58.2(50.5-63.5) b 47.6(40.9-54.4) c 48.4(41.7-55.1) c N=172 N=165 N=210 N=213 Cx. quinquefasciatus 7.0(2.3-19.5) a 42.9(28.9-58.0) b 40.0(25.3-56.7) b 35.7(20.4-54.6) b Females N=43 N=42 N=35 N=28 Cx. quinquefasciatus 33.3(14.6-59.4) a 66.7(33.3-88.9) a 50.0(27.3-72.7) a 43.8(22.5-67.6) a males N=15 N=9 N=16 N=16 % Mortality(95% Confidence Interval) *Differing superscript letter indicates statistical significance at P<0.05 as compared to the other treatment and control arms. *N=total number of mosquitoes tested

Table 5 Experimental hut: overall percent showing dye (%)

Control Chlorfenapyr 0.5% Boric Acid 2% Tolfenpyrad 1% An. arabiensis females 24.7(17.2-34.3)a 17.0(10.8-25.7) a 15.0(9.6-23.6) a 17.4(11.4-25.7) a An. arabiensis males 3.1(0.4-19.1) a 27.8(12.1-51.9) a 75.0(54.4-88.3) b 57.1(36.0-76.0) c Cx. quinquefasciatus 11.6(4.9-25.1) a 21.4(11.5-36.3) b 28.6(16.1-45.4) c 7.1(1.8-24.5) d Females Cx. quinquefasciatus 0.0 a 16.7(2.3-63.1) b 12.5(1.7-53.7) b 0.0 a males % Showing Dye(95% Confidence Interval) *Differing superscript letter indicates statistical significance at P<0.05 as compared to the other treatment and control arms.

Table 6 Experimental hut: percent of the dead mosquitoes which had visible dye (%)

Control Chlorfenapyr 0.5% Boric Acid 2% Tolfenpyrad 1% An. arabiensis females 17.6(5.8-42.7)a 35.4(23.3-49.8) a 34.9(22.2-50.1) a 32.7(21.1-46.8) a An. arabiensis males 10.9(4.6-23.6) a 16.7(10.5-25.5) a 24.0(16.6-33.3) a 19.4(12.9-28.2) a Cx. quinquefasciatus 0.0 a 38.9(19.8-62.1) b 57.1(31.6-79.4) b 20.0(5.0-54.1) b Females Cx. quinquefasciatus 0.0 a 16.7(2.3-63.1) b 12.5(1.7-53.7) b 0.0 a males % Dyed of the Dead(95% Confidence Interval) *Differing superscript letter indicates statistical significance at P<0.05 as compared to the other treatment and control arms.

Table 7 Experimental hut: percent of the dyed mosquitoes that died

Control Chlorfenapyr 0.5% Boric Acid 2% Tolfenpyrad 1% An. arabiensis females 12.5(4.1-32.4) a 100 b 93.7(66.5-99.1) b 84.1(60.8-94.8) b An. arabiensis males 15.6(6.7-32.5) a 88.9(64.8-97.2) b 95.2(72.9-99.3) b 95.2(72.9-99.3) b Cx. quinquefasciatus 0.0 a 77.8(42.1-94.4) b 80.0(45.9-95.0) b 100 c Females Cx. quinquefasciatus N/A 100 a 100 a N/A males % Dyed which Died(95% Confidence Interval) *Differing superscript letter indicates statistical significance at P<0.05 as compared to the other treatment and control arms.

7. Discussion

The similar success of each of the three tested yet diverse classes of oral insecticides highlights the potential of the ATSB+Net method to be a promising tool for insecticide resistance management throughout malaria endemic sub-Saharan Africa. One suggested method for insecticide resistance management is through delivering unrelated classes of insecticides either in mixtures or rotations but due to the limited number of available contact insecticides has been rather unachievable to this point with conventional ITN and IRS methods (Curtis et al., 1998). However, the ATSB’s ability to effectively deliver diverse classes of oral insecticides makes it ideal for insecticide rotations or mixtures for resistance management in the future. Further, the supply of safe and cost-effective contact insecticides for the use in public health is diminishing and the development of new contact insecticides is limited (Zaim and Guillet, 2002). However, the ATSB method provides a novel route for delivering oral insecticides developed through the strong agricultural pipeline largely focused on the development of oral insecticides for crop protection from chewing pests as compared to contact insecticides currently widely used for medically important vector control. Additionally, there are already further classes of oral insecticides which could be delivered through the ATSB+Net method as described by Allen et al. (Allen, 2010). The high mortalities seen in the phase 1 cage bioassays and the high mortality of those mosquitoes showing dye in both phase 1 and 2 highlights the effectiveness of stomach poisons used to kill mosquitoes. Nevertheless, there is much improvement that can be made in delivery and stimulating feeding. Though guava juice has been described in this study and others (Muller et al., 2010d) as being an effective feeding stimulant, there is an urgent need for more exhaustive studies looking to identify stronger attractants and feeding stimulants for wild populations of mosquitoes. The optimization of the ATSB+Net method largely depends on stimulating feeding. As shown, the future success of this method depends more on the baits ability to stimulate feeding than to kill those mosquitoes which have fed. Further testing to optimize feeding by wild populations of vector mosquito species is a key component for the future development of this method and should be of significant focus for future trials (Muller et al., 2010d). In conjunction with the attractant optimization studies, in depth insecticide dosage studies are needed to maximize mortality yet limit feeding inhibition. Additionally, bait station design and positioning within the hut were also both observed as playing significant roles in mortality optimization and should not be overlooked.

In both the cage bioassays and experimental hut trial, the dye was a poor indicator of ATSB feeding. Though few mosquitoes showed dye in the experimental hut trial, there is still strong evidence that the increase in mortality was due to the ATSB as this same trend was observed in the cage bioassays where there were no other sources of significant mortality as compared to the Image 10 Dead An. gambiae mosquitoes on tolfenpyrad 1% ATSB feeding pad (only 1 of the 6 displayed visible dye) control. It was observed that chlorfenapyr and tolfenpyrad killed much quicker than boric acid and even those mosquitoes which had died while still feeding on the ATSB were not showing dye (see Image 10). It is likely that there was a significant amount of mosquitoes which were consuming a lethal dose of chlorfenapyr and tolfenpyrad ATSB, but a sub dyeing dose of ATSB. Boric acid ATSB killed slower and likely allowed for longer feeding and therefore the dye served as a more accurate indicator of feeding. An alternative marker for ATSB feeding would be of significant use in future trials to provide stronger direct evidence linking the increased mortality to the ATSB. The lack of visible dye seen in mosquitoes may also have been due to residual properties and not ingestion. Chlorfenapyr has been described as a new residual insecticide against pyrethroid resistant mosquito for vector control (Raghavendra et al., 2011). This alternative mode of action may have been responsible for an added portion of the mortalities besides ingestion. Tolfenpyrad, however, has been described as being rather ineffective as a residual insecticide (Irish, unpublished) yet still showed a similar mortality to chlorfenapyr with known residual properties. On the other hand, Chlorfenapyr killed significantly greater proportions than boric acid for Cx. quinquefasciatus and An. gambiae females (p= 0.000 & p=0.000 respectively) in the phase 1 cage bioassays. In light the scaling up of ITN and LLIN use, the use of untreated nets in this trial may seem to have reduced relevance. However, the use of an untreated mosquito net was decided upon to more accurately quantify the mortality solely caused by the ATSB and not diluted by the mortality caused by a treated net. The use of an untreated net also sheds light on the effectiveness of the ATSB method as the efficacy of treated nets decreases with the emergence and progression of vector resistance. Nevertheless, future development of the ATSB+Net method will need to assess the potential added mortality to a treated mosquito net and nets with holes.

References

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15. Muller, G., J. Beier, et al. (2010d). "Field experiments of Anopheles gambiae attracation to local fruits/seedpods and flowering plants in Mali to optimize strategies for malaria vector control in Africa using attractive toxic sugar bait method." Malaria Journal 9:262.

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Faculty of Infectious and Tropical Diseases Student’s Questionnaire

Candidate No: 105874 MSc: Control of Infectious Diseases

Project Supervisor: Mark Rowland, Rick Oxborough, & Seth Irish

Project Title: Attractive Toxic Sugar Baits (ATSB) near mosquito nets for control of pyrethroid resistant Anopheles arabiensis and Culex quinquefasciatus in Northeast Tanzania

As part of our assessment procedure for student projects we are asking you to complete the following short questionnaire. Please tick the most appropriate statements in each section and bind it into your project. A copy of this questionnaire must be bound into your finished project report.

(Please ensure you tick the correct box)

Who initiated the project? My supervisor Me

How much help did you get in developing the project? none: I decided on the design alone some: I used my initiative but was helped by suggestions from my supervisor substantial: My supervisor had most say, but I added ideas of my own maximal: I relied on the supervisor for ideas at all stages not applicable: the nature of the project was such that I had minimal opportunity to contribute to the design

How much help did you get in carrying out the work for the project? none: I worked alone with no supervisor input minimal: I worked alone with very little supervisor input appropriate: I asked for help when needed substantial: the supervisor gave me more assistance than expected excessive: the supervisor had to give me excessive assistance to enable me to get data

What was the degree of technical difficulty involved? slight: data easily obtained moderate: data were moderately difficult to obtain substantial: data were difficult to obtain

How much help were you given in the analysis and interpretation of any results? none standard: My supervisor discussed the results with the me and advised on statistics and presentation

substantial: My supervisor pointed out the significance of the data and told me how to analyse it

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*if too little or too much, were there any reasons for it, e.g. unforeseen technical problems, lack of materials, etc.? No technical or material problems. I thoroughly enjoyed the topic I was researching and was therefore willing to put in long hours daily (5AM-8:30PM) and through the weekends.

During the course of the work was your contact with your supervisor Daily Weekly Monthly Varied but at regular intervals Never Was this contact with your supervisor too infrequent infrequent but sufficient frequent but not excessive excessive

Please comment on your experiences during the project

I thoroughly enjoyed the topic I was researching, felt I had the appropriate amount of technical support for planning and carrying out the project, and felt I achieved my learning objectives for my future career goals.

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