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British Journal of Healthcare and Medical Research - Vol. 10, No. 2

Publication Date: April 25, 2023

DOI:10.14738/jbemi.102.14189.

Tawatsin, A., & Thavara, U. (2023). Development and Application of Biopesticides for the Management of Disease Vectors and

Pests of Public Health Importance. British Journal of Healthcare and Medical Research, Vol - 10(2). 28-43.

Services for Science and Education – United Kingdom

Development and Application of Biopesticides for the

Management of Disease Vectors and Pests of Public Health

Importance

Apiwat Tawatsin

Medical Science Technical Office, Department of Medical Sciences,

Ministry of Public Health, Nonthaburi 11000, Thailand

Usavadee Thavara

Consultant, Department of Medical Sciences,

Ministry of Public Health, Nonthaburi 11000, Thailand

Abstract

The development and practical use of natural products occurred in the late

1970s when a spore-forming bacterium producing enterotoxins was

discovered. This organism was designated as Bacillus thuringiensis ssp.

israelensis (Bti). By 1985, large quantities of Bti formulations were employed

in mosquito and black fly control programs globally. As research efforts in

this area continued, a second bacterium Bacillus sphaericus (Bsph) was

isolated and found to be highly effective against most mosquito species. This

entomopathogen found its way into mosquito control around 1997. Today,

both these (Bti and Bsph) microbial control agents are produced by large- scale fermentation processes in many countries. Recent advances in

formulation technology of both bacteria have increased their usefulness in

vector control programs. Another soil bacterium known as

Saccharopolyspora spinosa (Actinomycetes), was discovered, producing

bioactive components known as spinosyn A and spinosyn D. Large-scale

fermentation technology has been developed and the product known as

spinosad was produced and developed in agriculture, and recently for use in

public health. However, spinosad has not been used to any great extent in

public health as yet. All three microbial control agents have a good margin of

safety for mammals, birds, and wildlife and are environmentally friendly.

Considerable efforts were directed toward developing plant extracts for

vector control, especially mosquitoes. In this context, plant-based extracts

were formulated for the control of larval and adult mosquitoes. Some of the

plant parts were used in manufacturing mosquito coils which number into

the billions on a worldwide basis; however, only a small proportion of coils

are manufactured with plant-derived products. A major area for the

development of plant-based products is the use of plant essential oils which

are formulated in various ways for personal protection from the attack of

hematophagous insects. A number of such insect-repellent products are

commercialized in many countries. The public has the perception that plant- based and other natural products are environmentally friendly and safer to

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British Journal of Healthcare and Medical Research (BJHMR) Vol 10, Issue 2, April- 2023

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use for vector control or apply to human skin as personal protectants than

synthetic chemicals. In this paper, we will dwell upon the research and

development efforts leading to the development, production, and

application of microbial control agents, and phytochemicals for the control

of adult and preimaginal stages of disease vectors, as well as the

development and use of plant essential oils for personal protection from

anthropophilic insects.

Keywords: Biopesticides, phytochemicals, entomopathogens, microbial control

agents, pest control

INTRODUCTION

In recent times, many terms and expressions have been coined to describe a new and improved

breed of pesticides now known as “Biopesticides.” Equivalent terms describing this group of

agents are biorational pesticides, biopesticides, biocides, biological pest control agents, natural

products, botanicals, and naturalists (Menn 1999, Mulla 1997). The two terms that have greater

use and acceptance are “biopesticides” and “natural products.” We have been promoting and

holding “The International Conference on Biopesticides” for some 14 years and this conference

here in Delhi, India is the 5th such one. These conferences have been quite successful and

attendance and research presentations have been increasing. Proceedings of the past four

conferences have been published.

Since biopesticides in the broad sense include tools and strategies for many pests and disease

problems caused by pathogens and insects, the subject now covers insect pest management,

weed control, semiochemicals, antagonistic bacteria and fungi and finally transgenic crop

plants or mosquitoes. The core disciplines of biopesticides research have been bearing on the

management of crop and public health insects, fungal and bacterial diseases of crops and other

injurious agents through the use of “biopesticides” or natural products. This subject covers

many other themes as you can gather from thematic subjects in this conference.

Mulla (1997) at the first International Conference on “Biopesticides” defined and characterized

“biopesticides”. A couple of years later, a book edited by Hall and Menn (1999) titled

“Biopesticides” presents a comprehensive treatment of this subject. Various experts in the area

of “biopesticides” research and development provided chapters and critical reviews of this

subject matter. This volume covers topics related to the discovery, isolation, safety, toxicology,

development, registration, labeling, commercialization, and application of biopesticides in pest

control programs. Although the earlier attempts were on finding and developing biopesticides

for plant pest control, recent worldwide research efforts have come up with effective and viable

“biopesticides” for the control of insects of public health importance.

In this paper, we will discuss the development and use of microbial control agents and their

toxins and plant-based products for the management of insects of public health importance. In

the past 30 years or so, great strides have been made in discovering and developing entities

with a novel mode of action that provides viable strategies for solving the major resistance

problems encountered in the management and control of medical insects.

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Tawatsin, A., & Thavara, U. (2023). Development and Application of Biopesticides for the Management of Disease Vectors and Pests of Public Health

Importance. British Journal of Healthcare and Medical Research, Vol - 10(2). 28-43.

URL: http://dx.doi.org/10.14738/jbemi.102.14189

Entomopathogenic Bacteria – Their Fermentation Products Employed in Vector Control

At the advent of synthetic insecticides after World War II, DDT and related compounds were

largely used in vector control programs until the emergence of resistance in many target

species. The organophosphorus group of insecticides provided substitutes for the

organochlorine compounds. Synthetic pyrethroids and insect growth regulators were the next

generations of insecticides developed for pest control. Resistance to some or all of these groups

appeared soon after use in some areas of the world, but at a much slower rate than IGRs. Also,

the inherent mammalian and wildlife toxicity and environmental contamination problems of

synthetics caused the disuse of some of these once highly effective insecticides.

By the early 1980s, vector control programs in some areas of the world felt the impact of

resistance to insecticides. The World Health Organization at the end of the 1970s launched a

worldwide effort to find and develop biological control agents with special emphasis on the

isolation, characterization, and development of microbial control agents for use in public

health. As a result of this international effort, many entomopathogenic organisms were isolated

and evaluated against disease vectors. In addition to the WHO global initiative, industrial firms

in many countries launched their own search to isolate and characterize entomopathogens. At

the end of the 1970s, at the peak of resistance in mosquitoes to most synthetic insecticides, the

spore-forming bacterium Bacillus thuringiensis ssp. israelensis (Bti) was discovered. Rapid

development, registration, and labeling of this microbial agent promoted advances in

fermentation, production, and formulation technologies. Within 5 years after its discovery, its

formulations were employed in large-scale mosquito and black fly control programs. The

development of Bti was soon followed by the discovery of another spore-forming bacterium

Bacillus sphaericus (Bsph), which was isolated from black flies from Africa. This bacterium

showed exceptional activity against most mosquitoes. Bti was labeled for mosquito control in

1980, while Bsph was registered in 1997. These two microbial control agents and their

formulations are used at present in large quantities in mosquito and other vector control

programs.

Recently, another soil bacterium Saccharopolyspora spinosa (actinomycete) was isolated and

studied for activity against insects in the agriculture and public health. This bacterium on

fermentation produces two bioactive compounds known as spinosyn A and spinosyn D. This

product is named spinosad, which is currently used in pest control in agriculture, but it will be

launched for use in public health insect control programs in 2009/ 2010. All three bacterial

products are developed for the control of larval mosquitoes and other public health insects. We

will discuss the development and efficacy of various formulations of the three microbial control

agents (Bti, Bsph, and spinosad) with details of the efficacy and evaluation of tailor-made

formulations for use in a variety of habitats supporting immature stages of mosquitoes. It has

been documented that in mosquitoes there is no cross-resistance to these biological agents and

further that no resistance has emerged to Bti. In some polluted habitats, some mosquito species

have developed resistance to B. sphaericus after six to twenty applications. A solution to this

resistance problem was found quickly, by using a mixture of Bsph and Bti, the latter used in

small proportions.

Bacterial Larvicides

About three decades ago, worldwide research was launched to find, isolate, and develop

entomopathogenic bacteria for the control of disease vectors, especially mosquitoes. As a result

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of this initiative and intensive search, several isolates and strains of two bacterial species which

produced parasporal protein toxins showing high levels of activity against mosquito larvae

were discovered (de Barjac 1978, 1990a, 1990b, de Barjac and Sutherland 1990, Mulla 1991,

Weiser 1984, WHO 1985). Although Bacillus thuringiensis Berliner had been discovered as an

entomopathogen several decades ago, the strains discovered up to the 1970s showed activity

against herbivorous insects only. It was not until the late 1970s, that a highly mosquitocidal

strain of this bacterium was found. This pathogen later described and designated as Bacillus

thuringiensis spp israelensis (deBarjac 1978, 1990a) was isolated by Goldberg and Murgalit

(1977). This pathogen was studied intensively and developed quickly for the control of

mosquito and black fly larvae (de Barjac and Sutherland 1990, Mulla 1990).

Bacillus Thuringiensis Israelensis (Bti) Evaluations Against Mosquitoes

Soon after its discovery, research on production and formulation technology was initiated by

academic institutions, research institutes, and the industry. Tremendous advances were made

in this regard and a variety of formulations (liquid, powder, WDG, granules, briquettes, pellets,

and others) were developed for different habitats and various species. A comprehensive tome

on Bti (deBarjac and Sutherland 1990) was published on the very basic and applied aspects of

bacterial pathogens. There are many publications covering research on laboratory and field

evaluations of Bti and its formulations against various species of mosquitoes and black flies

around the world. This information is all contained in this volume of “Bacterial Control of

Mosquitoes and Black Flies” (deBarjac and Sutherland 1990). Information on laboratory

activity and field efficacy is contained in this volume compiled by Mulla (1990). We will decline

from reviewing or analyzing this published information, except to say that significant advances

were made in the application of Bti and other microbes for the control of medically important

insects. It should also be mentioned that a great deal of research has been conducted on the

safety and environmental aspects of Bti (deBarjac and Sutherland 1990, Mulla 1995, Su and

Mulla 1999b). For details, one is referred to these publications. We will, however, present and

discuss our research findings on the evaluation of Bti formulations against Aedes aegypti

(dengue vector) larvae breeding in water-storage containers commonly used in developing

countries. Most of the other testing and evaluations have been published and contained in

deBarjac and Sutherland 1990).

Bti formulations were rigorously subjected to field evaluation in water-storage containers

where Aedes aegypti, a dengue vector breeds. This entomopathogen when tested in open bodies

of water and impoundments as WDG formulation, showed short residual activity (Mulla 1990).

However, controlled-release formulations were developed which increased the longevity of Bti

against mosquito larvae. These formulations consisted of briquettes (Fansiri et al 2006),

donuts, pellets, and tablets. The tablet formulation was tested and evaluated in water-storage

containers (200 L of water) against Ae. aegypti larvae (Figure 1). The 200L earthen jar is the

most common jar used for water storage in Thailand and elsewhere. This formulation showed

exceptional activity and longevity, giving prolonged control (>80% inhibition of emergence of

adults) for about 112 days at the rate of 1 tablet (0.37 g 2700 ITU/mg) per 50L water (Mulla et

al 2004). Under the experimental conditions in water-storage containers, various factors such

as sunlight, wind, water use practices (removal and addition), and water pollution were

controlled, thus obtaining longevity for 90-112 days. Under normal use conditions; however,

we expect that the duration of efficacy will be shorter; most likely lasting for 2 months or so, a

period which is still adequate from the standpoint of operational control programs. We can get

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Tawatsin, A., & Thavara, U. (2023). Development and Application of Biopesticides for the Management of Disease Vectors and Pests of Public Health

Importance. British Journal of Healthcare and Medical Research, Vol - 10(2). 28-43.

URL: http://dx.doi.org/10.14738/jbemi.102.14189

this inference from village trials of a temephos granular formulation applied to water-storage

containers where its longevity was 2-3 months (Thavara et al 2004) as compared to the

longevity of 6-8 months, using the same formulations in the current experimental set up (Mulla

et al 2004).

In open bodies of water and impoundments, there are a number of other habitat factors which

adversely affect the efficacy and longevity of Bti as well as other mosquito larvicides. Depth of

water can reduce effectiveness against surface frequenting larvae. Vegetation and plant

standing in water also reduce effectiveness and longevity. Exposure of habitat to sunlight and

wind can also reduce the efficacy and longevity of larvicides. Flowing water with currents will

require higher dosages as the movement of water dilutes the concentration. Mosquito breeding

sources with these factors will require higher dosages of Bti and others than habitats not

possessing these features. On account of these considerations applications of Bti (WDG)

formulations produced mosquito larval control for only 7-12 days with .027-.0.53 lbs/acre of

WDG in open sunlit tubs (Su and Mulla 1999a). Granular formulations similarly used yielded

only short-term control.

Bacillus Sphaericus (Bsph) Evaluations Against Mosquitoes

The second entomopathogenic bacterium discovered was Bacillus sphaericus strain 2362 from

black flies in Nigeria (Weiser 1984). Other strains namely 2297 (Sri Lanka), 1593 (Indonesia),

and C3-41 (China) were also isolated (deBarjac 1990b). All these strains exhibited similar levels

of activity against mosquito larvae, with strain 2362 in general showing somewhat higher level

of activity (deBarjac and Sutherland 1990, Mulla 1991). None of the strains showed activity

against Aedes aegypti (breeding in artificial containers) and Aedes albopictus breeding in

natural containerized water such as tree holes, bromeliads, leaf axles, etc. Therefore, B.

sphaericus (Bsph) strains were not evaluated against the two container-breeding mosquitoes.

B. sphaericus formulations were extensively tested against culicine and anopheline species in

impoundments and open bodies of water. This entomopathogen proved highly effective against

culicine mosquitoes in polluted water (deBarjac and Sutherland 1990, Mulla et al 1988, 1997,

1999b, 2001a, 2003 Nicolas et al 1987, Skovemond and Bauduin 1997). It was evaluated against

Culex and Anopheles species throughout the world. The formulations tested were corn grit

granules (VectoLex CG) and water dispensable granules (VectoLex WDG) and others. In the

volume of deBarjac and Sutherland (1990), there are 3 chapters dealing with the evaluation

and practical application of B. sphaericus formulations. The chapter by L.A. Lacey (10 pages)

with persistence and formulations of B. sphaericus, and the chapter by JM Hougard (12 pages)

covers formulations and persistence of Bsph in Culex larval sites in tropical Africa. Another

chapter by HH Yap (14 pages) compiles information on the efficacy of Bsph formulations

against Aedes, Anopheles, Culex Mansonia, and Psoropora species. We will not evaluate and

analyze this information but present some recent studies of our own on Bsph.

We evaluated Bsph formulations against the southern house mosquito Culex quinquefasciatus

breeding in highly polluted waters in urban areas in Bangkok, Thailand. In surface drainage

klongs (with slow-moving water in the absence of rain), both GR and WDG (water disposable

granules) yielded 14-28 days of control of larvae at the dosages of 2 g/m2 of GR and 0.1-0.25

g/m2 of WDG. Heavy precipitation increased the volume of water through the klongs and

washed away mosquito larvae, resulting in longer control of larvae (Mulla et al 1997). In some

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stagnant polluted water habitats, Bsph provided Culex quinquefasciatus larval control for

almost one to two months, a performance not shown by other larvicides, with successive

treatments of Bsph (Mulla et al 2001a, 2003). The efficacy and longevity declined due to high

levels of resistance and this high level of resistance was managed by using a mixture of Bti and

Bsph against resistant larvae (Mulla et al 2003).

The efficacy and longevity of B. sphaericus depended on the quality of formulations, species of

mosquitoes, and factors of habitats. In general, it provided persistent control of Culex species

in highly polluted water and water not exposed to sunlight and winds. Other factors of habitat

that influenced efficacy and longevity were vegetation, dilution by incoming water, and solid

wastes. The two formulations of Bsph commonly used are corn grit granules and water

dispensable granules (WDG). The effective rate of application is 2-5 lb/acre of the GR

formulation and 0.25 – 1.0 g/m2 of the WDG formulations. These rates may have to be increased

in habitats with unfavorable features or where less susceptible species is prevalent.

Saccaropolyspora Spinosa – Fermentation Products

Spinosad is a metabolic and fermentation product of the actinomycete bacterium

Saccharopolyspora spinosa. This naturally occurring bacterium was isolated from the soil in

1988. It was identified as a new species (Mertz and Yao 1990, Thompson et al 1997).

Actinomycete bacteria exhibit fungus-like characteristics and they are responsible for the

decomposition of complex organic materials. In 1989, the most bioactive metabolites from the

spinosad formulation broth were identified and designated as spinons A & D (Sparks et al

1998). In the mixture, spinosyn A and spinosyn D constitute 85 and 15 % respectively (Kirst et

al 1992). Both spinons are non-volatile, slightly soluble in water, and stable under a wide range

of temperatures and pH (Dow AgroSciences Tech Information).

Spinosad has a novel mode of action. It alters the formation of nicotine and GABA –gated ion

channels and consistently results in neuronal excitation (Salgado 1998, Watson 2001).

Spinosad with its novel mode of action is an ideal product for the management of resistance. It

has shown no cross-resistance to currently used insecticides and can be rotated with all other

classes of existing mosquito larvicides (Dow AgroSciences Tech Information). The mode of

entry is by ingestion as well as cuticular penetration. Spinosad has been registered for use on

over 250 crops. It has a good margin of safety for mammals. Short-term toxicity as determined

in rats was LC50>3738 to >5000 mg/kg body weight for male and female rats respectively.

Dermal and inhalation tests also showed a high level of safety. It has a good margin of safety for

birds and fish and most aquatic macroinvertebrates. It does, however, impact some groups of

aquatic crustaceans. Therefore, caution should be exercised in making applications in aquatic

habitats where mosquitoes breed and where sensitive macroinvertebrates might prevail.

Spinosad – Evaluation and Efficacy Against Mosquito Larvae

Soon after the isolation of the actinomycete bacterium Saccoropolyspora spinosa (producing

spinosad) from the soil, it was cultured and the products were first tested against mosquito

larvae (Kirst et al 1992). It was noted that the product had considerable activity against

mosquito larvae. Notwithstanding this information, spinosad was first developed for the

control of crop insects. It is now registered for insect pest control of some 250 crops. Interest

in testing and developing this natural product for mosquito control did not materialize until

2003/2005. We conducted preliminary studies on the activity of spinosad against mosquito

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Tawatsin, A., & Thavara, U. (2023). Development and Application of Biopesticides for the Management of Disease Vectors and Pests of Public Health

Importance. British Journal of Healthcare and Medical Research, Vol - 10(2). 28-43.

URL: http://dx.doi.org/10.14738/jbemi.102.14189

larvae in 2000 (Mulla unpublished report). During this period and thereafter numerous studies

were carried out on the activity of spinosad technical and formulated materials against

mosquito larvae in different regions. A flurry of studies followed evaluating various

formulations of spinosad against larvae of mosquitoes: Aedes aegypti, Ae. albopictus, Culex

quinquefasciatus, Cx. pipiens, Anopheles albimanus, An. stephensi, and An. quadrimaculatusin the

laboratory and field (Bond et al 2004, Cetin et al 2005, Darriet et al 2005, Darriet and Corbel

2006, Paul et al 2006, Romi et al 2006, Thompson and Hutchins 1999). In these studies, various

levels of activity were noted in the laboratory against various species. In field studies, efficacy,

and longevity were dose-dependent, species variables, and habitat conditions.

Spinosad Against Culex Mosquito Larvae in California, USA

We initiated detailed studies on the evaluation of technical spinosad and SC 120 emulsifiable

formulation against larvae of Culex quinquefasciatus in the laboratory in 2005 (Jiang and Mulla

2010). We determined in repeated replicated studies that spinosad showed a high level of

activity against the larvae. We determined the LC50 and LC90 (lethal concentrations killing 50%

and 90%) of the exposed larvae. We carried out tests at multiple dosages against 2nd and 4th

instar larvae and the mortality was assessed at 24 and 48 hr post-exposure. Constant

temperature (26-28° C) and photoperiod (14 L:10hr D) were used.

The LC50 of the technical material against 2nd instar larvae for 24 hr exposure was 0.021 mg/L

AI for the 4th instars it was 0.033 mg/L. The LC50 of the SC120 (11.6%) was lower being 0.012

(2nd instar) and 0.014 (4th instar). The LC90 for the technical material was 0.051 (2nd instar) and

0.060 (4th instar) and for the SC120 the LC90 values were 0.026 (2nd instar) and 0.032(4th

instar). The data showed that mortality increased at the 48-hr exposure than the 24-hr

exposure for both the 2nd and 4th instars. These tests showed that larvae of Culex

quinquefasciatus are quite sensitive to both technical and emulsifiable formulations, the latter

being more active than the former. With this data at hand, we got encouraged to take spinosad

to simulated field set-ups using standard microcosms and mesocosms supporting natural

populations of Culex mosquitoes.

Microcosms and Mesocosm Studies on Spinosad Against Culex Mosquito Larvae in

California, USA

For the simulated field studies, we used the emulsifiable (SC 120, 11.6% AI) formulation. The

studies were carried out first in fiberglass tubs (24 tubs, 1 m2, water depth 30 cm containing

240 l water), followed by experiments in earthen ponds (38 non-vegetated and 64 vegetated;

27 or 37.5 m2 surface, water depth 30 cm) filled with 8100 L (non-vegetated) or 11250 L

(vegetated) of reservoir water. The SC120 was diluted in distilled water and aliquots were

sprayed on the water’s surface. The material was applied to obtain multiple dosages, 0.05, 0.1,

0.25 and 0.5 mg/L AI in microcosms and 0.025, 0.05, and 0.1 mg/L for the mesocosms (Figure

2).

Treated and untreated units (tubs) and ponds (mesocosms) replicated 4 times were sampled

for larvae using a standard dipper before and at intervals after treatments. The samples were

categorized into early instar larvae (1st and 2nd), late instar, (3rd and 4th), and pupae (Jiang and

Mulla 2010).

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There are numerous publications containing information on plant-based repellents and

protectants and it will not be possible to list and review them here. Three publications offer an

enormous amount of information on the research and development of personal protectants and

repellents (Debboun et al 2007, Moore et al 2007, Sukumar et al 1991). These publications have

an impressive list of published works on this subject. A considerable amount of research has

been carried out on the neem tree (Azadiracta indica) and its various products for the control

of medically important insects, including reports on the usefulness of neem products as

repellents, protectants and deterrents (Mulla and Su 1999). The recent volume published on

the neem tree by the Neem Foundation (Schmuterer 2002) contains valuable information on

the neem tree and the unique products derived from this tree which are used in pest control

and many industrial, medicinal, and agricultural enterprises. Bioactive materials from neem

trees have multiple modes of action, such as repellency, deterrence, sterility, growth regulation,

and toxicity (Isman 1999, Mulla and Su 1999, Schmuterer 2002).

The repellent chemicals found in different parts of plants have been identified in some plant

extracts. These constituents not only act as repellents but also as feeding deterrents, toxicants,

growth regulators, etc. The major groups of chemical substances, identified are categorized as

alkaloids, phenols, terpenoids, and others. Different plant species and their parts yield different

groups of chemicals in varying quantities. Some plants may have one or a few phytochemicals

possessing repellent properties while others may have quite a few principles. The citronella

group of grasses originating in India has a widespread distribution. These plants have varying

amounts of repellent chemicals (mostly terpenoids), but the most abundant repellent chemicals

contained are citronella, citronellol, and geraniol. The grass Cymbopogon nardus or citronella is

the most common species used in the commercial blending of repellent products. Several other

species of Cymbopogon are used in commercially available plant products.

Osimum species (basilcum in particular) contains a number of repellent compounds which also

act as mosquito larvicides. Species of Hyptis, Mentha, and Thymus also contain insect-repellent

principles and they are used in various ways by the local communities. Tagetes species have

exhibited larvicidal and insecticidal properties, their essential oils act as repellents in some

species and not others. Artimisia spp has both essential oils which act as repellent and other

principles acting as larvicides. The neem tree Azadiracta indica has attracted a great deal of

attention as a source of agricultural biopesticides. In India, neem tree parts have been used for

thousands of years as insect repellents and protectants. In recent years, materials from this

plant have been developed for the control of medical insects. The neem tree has about 35

bioactive compounds which show toxicity, causing sterility, and decreased fecundity as well as

repellency and deterrence. Proper formulations can increase the toxicity and repellency of

neem and other plant products. Bioactivity is related to the concentration of bioactive

principles and the extent of release and absorption (Isman 1999, Mulla and Su 1999,

Schmuterer 2002).

Lemon eucalyptus extracts from Corymbia citriodora (from China), have been the subject of

many tests. The essential oil extract was noted to have repellency to mosquitoes, slightly better

than most Eucalyptus spp. The compound p-menthane-3.8-diol (PMD) was identified as a

byproduct. This material was proven to be highly repellent, equaling the repellency of deet

(commercial repellent). The Chinese name of this repellent is Quwenling “effective repellent of

mosquitoes.”