GRANT PROPOSAL:

PROJECT TITLE: Anopheles larval control for malaria suppression in The Gambia by rotating Bacillus thuringiensis israelensis and Bacillus sphaericus briquette and water-dispersible granule (WDG) applications.

March 17, 2008

BIOL 448 – Tropical Diseases

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INTRODUCTION:

Each year, the burden of malaria causes disease in 515 million people and is responsible

for 3 million deaths, most of which are young children living in Sub-Saharan Africa (Snow et al.,

2005). With the advent and widespread use of the insectidal spray, dichlorodiphenyl-

trichloroethane (DDT) and multi-sector involvement in malaria eradication campaigns in the

1950s and 1960s, the elimination of malaria carrying adult female mosquito, Anopheles gambiae

from countries such as Brazil and Egypt have proven successful (WHO, 1982). However,

worldwide eradication efforts were abandoned in 1969 due to the emergence of insecticide

resistance by mosquitoes including DDT, as well as logistical and financial constraints to

eradication efforts for countries where malaria burden was high (WHOPES 2006; Walker and

Lynch 2007). Primary control methods have always focused on reducing human to adult

mosquito contact but efficacy of insecticides has been met with increasing resistance (WHO,

1982). Instead, the need for an Integrated Vector Management (IVM) programme, which

includes a focus on larval control for the purpose of reducing malaria transmission indirectly by

controlling the malaria vector, is desperately needed.

The two most effective vectors of malaria, Anopheles arabiensis and Anopheles gambiae,

primarily reside in tropical Africa and alternate in abundance seasonally (WHO, 2006). Shifting

away from the use of chemical pesticides, which have acute or chronic toxic effects on a wide

range of non-target organisms including mammals (Lambert & Peferoen, 1992), and tapping into

the potential value of using bio-larvicides for the effective control of malaria carrying mosquitoes

would be of great significance in the effort to indirectly decrease the incidence of malaria in

human populations. Addressing the need for an environmentally friendly, species-specific,

efficient and inexpensive alternative can be answered by our proposal to use natural bio-

larvicides.

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EXECUTIVE SUMMARY:

We have identified the potential large-scale efficacy of implementing the use of microbial

bio-larvicides: Bacillus thuringiensis var. israelensis (Bti) serotype H-14 and Bacillus sphaericus

(Bs) serotype H5a5b, as a part of an Integrated Vector Management (IVM) to control major

malaria vectors in The Gambia. Using this methodology provides a non-chemical alternative to

chemical residual sprays currently being, where increasing resistance is being reported (Sharma et

al., 1991). The ecological integrity of the environment can be retained as Bti and Bs are

extremely species-specific and toxic only to the stomachs of filter-feeding mosquitoe larvae

(Culicidae) and blackflies (Simuliidae), so threat to human health from handling or being

indirectly exposed to these bacteria carries minimal to non-existent risk according to the U.S.

EPA (1998). These strains naturally occur in the environment and have shown not to affect non-

target organisms including other insects, fish, birds, mammals or plants (PMRA; Lambert &

Peferoen, 1992). Other studies have also shown that direct ingestion or application of these

products into the water supply have no adverse health effects (PMRA). In fact, Canada, the U.S.

and many countries in Europe use different varieties of Bacillus thuringiensis, including Bti, as a

part of pest management control measures as well as for insect control plans in agriculture

(PMRA). Initial field and pilot projects on the use of different formulations of Bti and Bs for the

control of malaria in tropical Africa have already taken place with encouraging results (Walker &

Lynch, 2007; Ulrike and Lindsay, 2007; Majambere et al., 2007; Fillinger et al., 2003; Seyoum

and Abate, 1997). However, despite the efficacy being reported, problems of potential resistance,

short duration of action, and thus the need for continual re-application, are obstacles for Bti and

Bs from being implemented on a full-scale basis as noted in previous studies. Given the results

from initial field and pilot projects on the use of different formulations of Bti and Bs for the

control of malaria in tropical Africa as summarized by Walker & Lynch (2007), we believe that

funding put towards our project will address the following problems currently faced in the field:

I.) Addressing resistance:

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Although Bt resistance has been reported in other insect orders, especially in

Lepidopterans, Bti resistance has not yet been reported despite being used for more than 10 years

in Africa, USA and Germany (Glare & O’Callaghan, 2005). This may be attributed to the

complex mode of action of Bti on target organisms (Glare & O’Callaghan, 2005). Furthermore,

because Bti and Bs bind to different classes of midgut brush border membrane receptors we see

little cross-resistance between both species (Nielsen-LeRoux, Charles, 1992; Poopathi and Tyagi,

2006). To delay the opportunity of developing resistance among mosquito populations, we

propose that cyclic application of Bti and Bs be employed. Cyclic selection of different chemical

herbicide regimes is used by farmers in agriculture as a mechanism to slow down evolution of

resistance (Palumbi, 2001). This can be applied to our project because rapid alteration of selection

pressure for resistant mosquito mutants by alternating the use of Bti and Bs will discourage the

selection and propagation of resistant mosquitoes. Previous studies have evaluated the efficacy of

both bacteria strains together or independently. However, we believe that alternating applications

would be a more cost effective strategy.

II.) Addressing lack of residual activity of Bs and Bti and its short lifespan:

Multiple studies have reported the optimistic potential of Bti and Bs application for use in

the field as a successful means to control and kill Anopheles mosquito larvae (Walker and Lynch,

2007). However, the duration of control for both methods varied from several days to a week for

Bti and several days to a few weeks for Bs, therefore requiring frequent re-application of the

larvicide to be effective (Walker and Lynch, 2007). The lack of residual activity is in part due to

the fact that current formulations of Bti and Bs have a tendency to sink in water, away from the

surface from which anopheline mosquito larvae feed (Kroeger et al., 1995). The economic cost

and man-power needed to sustain this kind of persistent application is therefore, a major obstacle.

Given the established efficacy and slightly longer residual effect of Bs water-dispersible

granules (WDG) toxicity on anophelines in lab and field trials in Western Kenya (Fillinger et al.,

2003), we propose that this Bs formulation be used along with a slow-release formulation of

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floating Bti briquettes (Figure 1). The use of briquettes would address the problem for the need to

constantly re-apply treatment on a weekly treatment as its efficacy has been reported to last for

several months, providing ecologically safe sustained release of the larvicidal Bs toxin into the

environment (Brar et al., 2006).

To our knowledge, wide-spread application of Bti formulated briquette has never been

applied to The Gambia or any region in Sub-Saharan Africa. Its efficacy in controlling

mosquitoes as a part of pest control management programs in Canada and the U.S.A. is

established and accepted where commercial formulations such as “Mosquito Dunks®” (formerly

known as Bactimos™ briquette; Figure 1) for domestic use are also available (Kase and Branton,

1986). The use of Mosquito Dunks® for controlling malaria vectored mosquitoes has never been

evaluated but has shown to be effective against the dengue fever carrying mosquito, Aedes

aegypti. (Fansiri et al., 2006). With funding, we believe that potential widespread use of this Bti

briquette formulation may be a revolutionary answer towards addressing the need for a sustained

slow release formulation for the effective control of malaria carrying anophelines, which

indirectly decreases the devastating incidence of malaria in The Gambia and sub-Saharan regions

of Africa.

Figure 1. “Mosquito Dunks®” Bti briquettes

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BACKGROUND:

About Bacillus thuringiensis israelensis (Bti) and Bacillus sphaericus (Bs)

Bacillus thuringiensis (Bt) and Bacillus sphaericus (Bs) are naturally occurring soil bacterium

found all over the world (Lambert & Peferoen, 1992). Part of its growth cycle may include a

resilient spore phase when environmental conditions are not favourable, enabling it to survive

adverse conditions for long periods of time. These strains are unique because of their ability to

overproduce a small number of proteins which crystallize separately from the spore, forming

parasporal crystals or insecticidal crystal proteins (ICP) (Baumann et al., 1991). Ingestion of

these crystals by certain insect orders has a toxic effect which can lead to death. Due to the

inherent stability, specificity and inert formation of ICP crystals, humans have isolated and

produced successful biopesticides targeting specific insects. Bt and Bs differ slightly in

specificity of binding to specific host gut epithelial receptors. Furthermore, application of Bti to

clean water is more effective whereas Bs tends to be more effective in water that contains some

organic pollution (Walker, 2002).

The mode of action of both strains of bacilli is similar in that larval ingestion of Bti or Bs

ICP leads to its conversion into toxic fragments as a result of both protein digesting enzymes and

the alkaline conditions in the stomach of the insect (Lambert & Peferoen, 1992). Specific receptor

binding of Bti or Bs toxic fragments results in a structural deformation of the midgut epithelial

cells along with corresponding disintegration of the microvillar membrane, ultimately destroying

the insect midgut and leading to death. Due to this specificity of receptor binding found in

specific insects, ingestion of Bti and Bs ICP by other non-target organisms are safe and non-

harmful.

HYPOTHESIS:

Alternating application of Bti (briquette) and Bs (WDG) over the course of the rainy season (June

to October) will decrease the incidence of new malaria infections indirectly as a result of

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decreased survival of mosquito larvae and dramatically reduced population of adult female

mosquito vectors of malaria..

METHODS:

Study Area: The Gambia is largely dominated by a slow moving river, the River Gambia where

tidal movements of the river flood surrounding sedge and other grass species during rainy season,

which lasts from June to October, creating sites for breeding among malaria vectors (Majambere

et al. 2006). The study site we propose will be a rural area made up of about 20 small villages

(8,440 population estimate based on 2002 data) near the town of Farafenni, which is an area

under demographic surveillance by the UK Medical Research Council (MRC). Our site is

roughly 20 km2 and will be adjacent to the site used by Majambere et al. (Figure 2) (2006). Rates

of malaria infection and rainfall data will be determined based on information provided by MRC

before, during and after microbial application.

Larvicides: We require briquette formulations of commercial strains Bti “Mosquito Dunks®”

(7,000 ITU/mg primary powder; Summit Chemical, Baltimore, MD, USA) where 1 briquette

should cover 9.2 m2 of water surface area (Walker, 2002). WDG Bs formulations from

VectoLex® (lot 56-809-PG; potency 650 BsITU/mg) as used in the study conducted by Fillinger

et al. (2003) which have shown to have significant efficacy.

Mosquito larvae monitoring: Location and potential larval breeding sites will be visually

surveyed by foot from April 2008 – May 2009. Access to areas around resident compounds will

continue only after permission by the inhabitant(s). Habitat types will be prescribed according to

definitions outlined by Fillinger et al (2003): swamp, rock pool, puddle, footprint, tyre track,

drain/ditch, pit, cement-lined pit or container. Weekly monitoring at sentinel sites for the

presence of larvae and measuring larval density, using the methods outlined by Fillinger et al.

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(2006) will continue throughout the rainy and dry seasons until the project is over in 2012.

Magnitude of larval reduction will be determined by measuring larval density before, during and

after application of larvicides.

Standardized field trials: Determining optimal dosages of Bti briquette applications will be

determined under standardized field tests at Farafenni Field Station during the rainy (September

to October 2008) and dry season (December 2008 to May 2009). This will also let us examine

the residual and re-treatment intervals required for full-scale implementation in field trials. The

same parameters used in this project will be the same as described by Majambere et al. (2007).

Field Trials: Based on the results from standardized field trials, we will test our hypotheses

under representative field conditions between August 2009 to November 2009 to determine the

efficacy and duration of Bti and Bs action. Bti briquettes will be distributed first in our study via

hand application, preferably from members of the community (Figure 2a) (WHOPES, 1999).

WDG Bs formulation will be applied with handheld or knapsack sprayers (Figure 2b) after Bti

duration of action is over. Application of microbials will continue between August to November

until 2012 when the project ends.

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Figure 2b. Liquid application of microbial larvicides with 15 litres capacity knapsack sprayers on open water surface (edge of floodwater) (Figure 3 from Majambere et. al (2006)). This will be the same method of briquette application

Figure 2. Map of the The Gambia, West Africa (A) and the Majambere et al. (2006) study area (B). The black line encloses the control; the red line encloses the intervention area where the 24 survey sites are marked as stars.

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Figure 2a. Hand application of corn granule formulation in a highly vegetated areas of the floodplains (Figure 4 from Majambere et. al (2006)). This will be the same method used for briquette application

ESTIMATED FUDING COSTS:

Estimated costs for larvicides and customs clearance costs will be under $1100 US based

on Fillinger and Lindsay’s study (2006). Habitat monitoring and larviciding will require

knapsack sprayers and two trained field-workers whose combined salary will be roughly $5150

US per year ($214/month) (Fillinger & Lindsay, 2006). Total cost to protect 8440 people will be

roughly $6250 a year or $0.74 per person a year.

EXPECTED RESULTS:

The total length of our study will run from July 2008 to September 2012. During this

time, we expect to see a prominent reduction in larval survival during the rainy season, similar to

the results seen in Fillinger and Lindsay’s study (Figure 3) (2006), which would support our

hypothesis. We should continue to see this trend every year during the application period as

overall larval density declines, leaving fewer mosquitoes capable of surviving to propagate the

next generation. Further support to our hypothesis can be verified through MRC surveillance

data, where we would expect to see a decrease in the rate of new malaria infections in our

population studied. Should this be the result, the impact of our cyclical application approach and

use of Bti briquettes would be a revolutionary step in helping curb the burden of malaria for

millions of people living in Sub-Saharan Africa.

However, it is important to stress that application of larvicide technology is not the sole

solution for tackling malaria. Ensuring that an Integrated Vector Management (IVM) programme

involving high levels of community participation through public dialogue/education put into

effect through legislation will keep this trend sustainable (WHO, 2006). Traditional IVM control

measures include the use of insecticide-treated nets, repellents, source reduction through small-

scale drainage and killing adult mosquitoes.

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Figure 3. Average monthly mosquito larval densities surveyed at sentinel sites during pre-,post- and intervention times in relation to level of rainfall from Fillinger and Lindsay’s results (2006).

CONCLUDING STATEMENT:

With mounting resistance of malaria-carrying mosquitoes to the current intervention of

chemical pesticides, there is a dire need to see more resources put towards investing in alternative

mosquito control interventions. Targeting the malaria-free larval stage of mosquitoes seems to be

the next logical choice. With clear evidence that significant reductions to larval populations can

be accomplished, implementing the use of naturally occurring, environmentally friendly and

affordable bio-larvicides like Bti and Bs is the next probable frontier to which deserves further

research attention; lest we let the burden of malaria continue to further cripple millions of people

around the world.

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