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Environment International 31 (2005) 1149 – 1166

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Review article

A review of botanical phytochemicals with mosquitocidal potential


Essam Abdel-Salam Shaalana, Deon Canyonb,*, Mohamed Wagdy Faried Younesc,
Hoda Abdel-Wahaba, Abdel-Hamid Mansour a
a
Zoology Department, Aswan Faculty of Science, South Valley University, Aswan, Egypt
b
Vector Control and Repellent Research Center, School of Public Health and Tropical Medicine, James Cook University,
Townsville Qld 4811, Australia
c
Zoology Department, Faculty of Science, Menoufia University, Shebin El-Kom, Egypt

Received 12 October 2004; accepted 20 February 2005


Available online 20 June 2005

Abstract

Identification of novel effective mosquitocidal compounds is essential to combat increasing resistance rates, concern for the environment
and food safety, the unacceptability of many organophosphates and organochlorines and the high cost of synthetic pyrethroids. An increasing
number of researchers are reconsidering botanicals containing active phytochemicals in their efforts to address some of these problems. To be
highly competitive and effective, the ideal phytochemical should possess a combination of toxic effects and residual capacity. Acute toxicity
is required at doses comparable to some commercial synthetic insecticides while chronic or sub-chronic toxicity is required to produce
growth inhibition, developmental toxicity and generational effects. In this article, we review the current state of knowledge on larvicidal plant
species, extraction processes, growth and reproduction inhibiting phytochemicals, botanical ovicides, synergistic, additive and antagonistic
joint action effects of mixtures, residual capacity, effects on non-target organisms, resistance, screening methodologies, and discuss
promising advances made in phytochemical research.
D 2005 Elsevier Ltd. All rights reserved.

Keywords: Larvicide; Bioinsecticide; Mosquito

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1150
2. Larvicidal phytochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151
2.1. Extraction and fractionation effects on toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151
2.2. Mode and site of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1154
3. Growth and reproduction inhibiting phytochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155
3.1. Effects on growth and reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1159
4. Joint action and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1160
5. Field evaluation of phytochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161
5.1. Residual capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161
5.2. Effect on non-target organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161
5.3. Development of resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162
5.4. Screening methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162

* Corresponding author. Tel.: +61 7 4781 5556; fax: +61 7 4781 5254.
E-mail address: Deon.Canyon@jcu.edu.au (D. Canyon).

0160-4120/$ - see front matter D 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envint.2005.03.003
1150 E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164

1. Introduction nogenic inhibitors. Some of the active compounds


of interest are (5E)-ocimenone from Tagetes minuta
One can speculate that people controlled and killed (Maradufu et al., 1978), rotenone from Derris elliptica
mosquitoes and other domestic insect pests by physically (Ameen et al., 1983), azadirachtin from Azadirachta indica
removing them or by using plant parts and plant derivatives (Schmutterer, 1981), capillin from Artemisia nilagirica
before the advent of synthetic chemicals. In all probability, (Banerji et al., 1990), quassin from Quassia amara (Evans
these plants contained insecticidal phytochemicals that were and Raj, 1991), neolignans from Piper decurrens (Chauret
predominantly secondary compounds produced by plants to et al., 1996), arborine, a new bioactive compound related to
protect themselves against herbivorous insects. However, quinazolone alkaloid, from Glycosmis pentaphylla
there is a little other than anecdotal, traditional or cultural (Muthukrishnan et al., 1999) and goniothalamin from
evidence on this topic (Grodner, 1997; Casida and Quistad, Bryonopsis laciniosa (Kabir et al., 2003).
1998). In the first century AD, Pliny the Elder, the Greek This review would not perhaps be complete unless it
philosopher, wrote ‘‘Natural History’’ in which he recorded mentioned some of the controversy in the field. In general,
all the known pest control methods. At the same time the botanicals interfering with the function of insect nervous
Chinese recorded their use of powdered chrysanthemum as systems have very low toxic effects on dogs, cats and
an insecticide. Pyrethrum, derris, quassia, nicotine, helle- humans, but are toxic to most fish, birds, reptiles and
bore, anabasine, azadirachtin, d-limonene camphor and amphibians. Varma and Dubey (1998) stated that insect
turpentine were some of the more important phytochemical growth regulators are ‘‘generally selective in their toxicity
insecticides widely used in developed countries before the and safe to both the environment and the natural enemies of
introduction of synthetic organic insecticides (Wood, 2003). insect pests’’. However, some antagonists (Anon, 2003)
The discovery of DDT’s insecticidal properties in 1939 and have stated that people falsely believe that these Fnatural
the subsequent development of organochlorine and organo- products (plant secondary compounds) are safer than other
phosphate insecticides suppressed natural product research pesticides because few phytochemicals have undergone
since the answers to insect control were thought to have toxicological testing and many have ‘‘striking adverse
been found. However, in the field of mosquitocidal affects on mammals.’’ This is a good point since many
insecticides, few new effective and economical insecticides Fgreen phytochemicals considered to be natural or organic
have been developed since the introduction of synthetic are more toxic than some synthetic insecticides (Ciccia et
pyrethroids. Examples of these are larvicidal insect growth al., 2000; Yang et al., 2002; Kimball, 2003).
regulators such as methoprene, pyriproxyfen, diflubenzuron Very few extensive review articles have been published
and endotoxins originating from Bacillus thuringiensis on the insecticidal characteristics of botanical chemicals and
israelensis and B. sphaericus. Neem is one of the few the review by Sukumar et al. (1991), who listed 344
natural products that has gained wide acceptance in some insecticidal botanical agents, is the only one that is relevant
countries as an antifeedant. Isman (1997) argued that the to this paper. Other reviews by Schmutterer (1988, 1990)
recent approval of neem in the USA has stimulated research and Mulla and Su (1999) were limited to the biological
into other potential botanicals. Other issues prompting effects of neem products against arthropods of medical and
researchers to enter the field are the high cost of synthetic veterinary importance. Although Sukumar et al. (1991)
pyrethroids, environment and food safety concerns, the primarily discussed alternative mosquitocidal insecticides,
unacceptability and toxicity of many organophosphates and they did not cover significant topics such as the effect of
organochlorines, and increasing insecticide resistance on a different fractions on toxicity of botanical chemicals, mode
global scale. and site of action, development of resistance, joint action of
A brief delve into the literature reveals many laboratory botanical extracts with other phytochemicals and/or syn-
and applied investigations (Perrucci et al., 1997; Roth et al., thetic insecticides, and field application of plant products in
1998; Momin and Nair, 2002) into the biological activity of mosquito control, including non-target effects.
many plant components against a large number of pathogens The lack of reviews in this area is somewhat surprising
and arthropods. An old review with an agricultural focus by since much effort been invested in locating mosquitocidal
Roark (1947) described around 1200 plant species that have compounds from plant extracts (phytochemicals) from
been listed in the literature as having potential insecticidal edible crops, ornamental plants, herbs, grasses, tropical
value. These studies have exposed an array of botanical and subtropical trees and marine angiosperms. This review
insecticides containing a wide spectrum of bioactive was thus written to shed light on these important areas of
fungicides, nematicides, acaricides, insecticides and carci- relevance to botanical extracts and mosquito control.
E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166 1151

2. Larvicidal phytochemicals and Aedes larvae to botanical derivatives and insecticides.


Aedes aegypti is commonly used in insecticide screening
Larvae from the three medically important mosquito trials because it is usually less susceptible and because it is
genera Aedes, Anopheles and Culex are all susceptible to a easy to colonize in the laboratory.
greater or lesser extent to some phytochemicals. In this Several observations on interactions between mosquito
review, LC50 values for crude extracts were found to range larvae and aged aquatic vegetation in breeding sites have
widely from promising to very unpractical doses. The lowest confirmed that leaf litter can possess toxic properties (Table
and most promising dose for a crude extract was 0.69 mg/L 1). In an investigation into the toxicity of leaf litter to
recorded for a steam distilled extract of Callitris glanco- nematocerous dipteran larvae (Aedes spp., Chironomus
phylla against Aedes aegypti by Shaalan et al. (2003). If the annularius, Cx. pipiens and Simulium variegatum) by
efficacy of an active raw botanical extract is not reliant on David et al. (2000), toxicity was found to vary with leaf
synergistic or additive effects, the compounds or fractions of age. Green and recently decayed leaves were non-toxic
an extract can be expected to be more purified and thus more while leaves that had decayed for 10 months were the most
potent. The lowest LC50 described for a botanical compound toxic. Accordingly, mosquito larvae were insensitive to
was 0.004 mg/L recorded for pipercide, a constituent of green leaves of Alnus glutinosa, Quercus robur and Populus
Piper nigrum fruits, against Cx. pipiens pallens larvae by nigra and responded with some mortality to old leaf litter
Park et al. (2002). These low doses are comparable to many with expected variations due to mosquito species and plant
synthetic insecticides. taxa. The toxicity of leaf litter was possibly the result of
The bioactivity of phytochemicals against mosquito alternations in leaf chemistry due to abiotic (hydrology,
larvae can vary significantly depending on plant species, climate, UV) and biotic components (microorganisms) that
plant part, age of plant part, solvent used in extraction and transform compounds toxic to dipteran larvae (David et al.,
mosquito species (Table 1). Most studies on phytochemicals 2000). Rey et al. (1999a, 2001a,b) indicated that species
focus on herbs and other medicinal plants. This is because from herbaceous habitats are usually more sensitive to leaf
historical experiential knowledge and some scientific studies litter than those from woody environments and this
have shown them to be particularly active against certain sensitivity usually depends on the extent of exposure, i.e.
organisms. Several studies have focused specifically on density of vegetation and number of vegetative species,
medicinal plants in different geographical regions. Com- from corresponding breeding sites. Moreover, the acquis-
monly a connection is extrapolated between plant activity ition of resistance to leaf litter chemicals by some larvae in
against disease agents based on traditional experience and such breeding sites appears to be a dynamic adaptation to
insecticidal activity against mosquitoes. A wide selection of the environment rather than a genetic trait.
trees and shrubs has been found to contain phytochemicals Almost no research on the effect of phytochemicals on
that may be of use in the botanical control of mosquitoes. adult mosquitoes has been conducted. Results from a study
Some trees and shrubs have also been tested more frequently by Perich et al. (1994) suggest that adulticidal action is a
due to observations indicating a degree of resistance to pests potential and untapped field of research. Their bioassays on
such as termites or herbivorous insects. Studies have ranged extracts from the various parts of T. minuta revealed extracts
in focus from ornamentals to fruit trees and from leguminous of floral parts to have the greatest biocidal effect on Ae.
to eucalyptus and other timber trees. Fewer highly toxic aegypti and An. stephensi adults with impressive LC90s of
species have been found in trees and shrubs in comparison to 0.4% and 0.45%, respectively.
those found in herbal and medicinal plants. The reason for
this may simply be that relatively more known medicinal 2.1. Extraction and fractionation effects on toxicity
plants have been screened for insecticidal properties.
Phytochemicals can be extracted from either the whole After thorough drying and grinding or wet maceration of
plant, as is often the case with herbs and small bushes such raw plant material, extraction of active agents can com-
as Melaleuca alternifolia, or from specific parts of the plant mence. The choice of extraction method, however, can have
known to contain a concentration of the desired active an effect on the efficacy of active plant constituents. Gfrerer
chemical. Concentrations can occur when bioactive chem- et al. (2004) compared different extraction methods for the
icals accumulate in the various parts of the plant, such as determination of chlorinated pesticides in animal feed.
leaves, stems, bark, flowers, fruits, seeds and roots. Several Accelerated solvent extraction and microwave-assisted
studies illustrated in Table 1 demonstrate this phenomenon. extraction were significantly more efficient (127% and
Mosquito larvae of different species display different 115%) when compared to the classical Soxhlet extraction
susceptibilities to the same phytochemicals. In general, Aedes method, while fluid-bed extraction provided the best
larvae are more robust and less susceptible to insecticides and reproducibility. Thus, when Chahad and Boof (1994)
botanical extracts than Culex larvae. The generalization made evaluated the effect of Piper nigrum extracts with Cx.
with Aedes and Culex larvae does not always hold with quinquefasciatus larvae, the extract obtained using the
Anopheles species. The susceptibility of Anopheles larvae Soxhalet method produced higher mortality and reduced
can vary since they can be more or less susceptible than Culex lethal time than the extract obtained by maceration.
1152 E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166

Table 1
Efficacy of botanical extracts and their fractions as larvicides
Plant species Plant part Solvent/fraction/ Mosquito mg/L(LC) Source
compound
Abuta grandifolia Fruit Dichloro methane Ae. aegypti 2.6 Ciccia et al. (2000)
Acorus calamus Rhizome Steam distillate Cx. quinquefasciatus 3.6 – 7.7 Ranaweera (1996)
Ae. aegypti
Ae. albopictus
An. tessellates
An. subpictus
Acorus calomus Commercial oils Ae. aegypti 52 – 74 Sharma et al. (1994)
Cx. fatigans 30 – 38
Agave sisalana Sisal fibre na Ae. aegypti 322 Pizarro et al. (1999)
Cx. quinquefasciatus 183
Allium sativa Bulb Crushed in water Cx. pipiens 160 (42) Thomas and Callaghan
(1999)
Alnus glutinosa Old litter Tannic acid Cx. pipiens >20,000 Rey et al. (1999a)
Ae. rusticus >20,000
Ae. albopictus 15,300
Ae. aegypti 7650
Alnus glutinosa Old litter Tannic acid Aedes & Culex 13,000 Rey et al. (2001b)
Coquillettidia 30,000
Alnus glutinosa Old litter Polyphenols Ae. aegypti 200 – 400 Rey et al. (2001a,b)
Ae albopictus 200 – 400
Cx. pipiens 25
Alnus glutinosa Old litter Different aged Cx. pipiens 25 David et al. (2000)
litter Ae. aegypti 382
Ae. albopictus 429
Ae. rusticus 63,211
Angelico glauca Commercial oils Ae. aegypti 52 – 74 Sharma et al. (1994)
Cx. fatigans 30 – 38
Annona squamosa Leaf Water Ae. aegypti 2400 Monzon et al. (1994)
Cx. quinquefasciatus 4700
Anthemis nobilis Flower na Cx. pipiens 34 Soliman and El-Sherif
(1995)
Argemone mexicana Leaf Benzene Cx. quinquefasciatus 30 – 55 Karmegam et al. (1997)
Azadirachta indica Leaf Water Ae. aegypti 4800 Monzon et al. (1994)
Cx. quinquefasciatus 44,400
Azadirachito indica Seed Crushed seeds Ae. aegypt; 100 (59) Sinniah et al. (1994)
Cx. quinquefasciatus 100 (63)
Bryonopsis laciniosa Whole Goniothalamin Cx. quinquefasciatus 21.5 Kabir et al. (2003)
Cassia obtusifolia Seed Methanol Ae. aegypti 40 (51) Jang et al. (2002)
Cx. pipiens pallens 40 (69)
Cassia tora Seed Methanol Ae. aegypti 20 (59) Jang et al. (2002)
Cx. pipiens pallens 20 (78)
Callitris glaucophylla Wood Steam distilled Ae. aegypti 0.69 Shaalan et al. (2003)
Cx. annulirostris 0.23
Calophyllum inophyllum Leaf Ethyl-acetate Cx. quinquefasciatus 16 Pushpalatha and
fraction An. stephensi 12.9 Muthukrishnan (1999)
Ae. aegypti 35
Calophyllum inophyllum Seed Ethyl-acetate Cx. quinquefasciatus 7.1 Pushpalatha and
fraction An. stephensi 3.9 Muthukrishnan (1999)
Ae. aegypti 8.2
Calotropis procera Latex Aqueous An. labranchiae 28 Markouk et al. (2000)
Root Ethanolic extract 215
Cannabis sativa Leaf Ethanol An. stephensi 1000 Jalees et al. (1993)
Cx. quinquefasciatus 1400
Ae. aegypti 5000
Caulerpa scalpelliformis Whole Acetone Ae. aegypti 53.7 Thangam and Kathiresan
(1991a)
Cedrus deodara Different Steam distillation An. stephensi 63.2 Kumar and Dutta (1987)
parts
Citrus limon Peel Crushed in water Cx. pipiens 160 (80) Thomas and Callaghan
(1999)
E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166 1153

Table 1 (continued)
Plant species Plant part Solvent/fraction/ Mosquito mg/L(LC) Source
compound
Cleome viscosa Whole Petroleum ether Cx. quinquefasciatus 10.7 Kalyanasundaram and
Babu (1982)
Codiaeum variegatum Leaf Water Ae. aegypti 37,600 Monzon et al. (1994)
Cx. quinquefasciatus 12,300
Cotula cinerea Whole Ethyl ether An. labranchiae 310 Markouk et al. (2000)
Curcuma domestica Rhizome Petroleum ether An. culicifacies 4.5 Ranaweera (1996)
Cymbopogon flexuosus Different parts Steam distillation An. stephensi 91.4 Kumar and Dutta (1987)
Cymbopogon martini Different parts Steam distillation An. stephensi 100 Kumar and Dutta (1987)
Cymbopogon nardus Grass Petroleum ether Cx.quinquefasciatus 6.3 Ranaweera (1996)
Ae. aegypti 9.3
Cymbopogon nardus Different parts Steam distillation An. stephensi 105.4 Kumar and Dutta (1987)
Daucus carota Commercial oils Ae. aegypti 36 Sharma et al. (1994)
Cx. fatigans 30
Dictyota dichotoma Whole Acetone Ae. aegypti 61.7 Thangam and Kathiresan
(1991a)
Dirca palustris Seed Hexane Ae. aegypti 10 Ramsewak et al. (2001)
Eucalyptus globulus Different parts Steam distillation An. stephensi 98.5 Kumar and Dutta (1987)
Eucalyptus globulus Leaf na Cx. pipiens 35 Soliman and El-Sherif
(1995)
Eugenia caryophyllus Different parts Steam distillation An. stephensi 96.5 Kumar and Dutta (1987)
Jasminum fructicans Leaf na Cx. pipiens 6 Soliman and El-Sherif
(1995)
Jatropha curcus Leaf Benzene Cx. quinquefasciatus 175 – 188 Karmegam et al. (1997)
Khaya senegalensis Seed Hexane Cx. annulirostris 5.1 Shaalan et al. (2003)
Languas galanga Rhizome Petroleum ether Cx.quinquefasciatus 8.3 Ranaweera (1996)
Ae. albopictus 9.3
Lansium domesticum Leaf Water Ae. aegypti 5000 Monzon et al. (1994)
Cx. quinquefasciatus 8300
Lavandula afficinalis Different parts Steam distillation An. stephensi 83.6 Kumar and Dutta (1987)
Melia azadirachta Different parts Steam distillation An. stephensi 88.5 Kumar and Dutta (1987)
Melia azedarach Seed Acetone Cx. pipiens molestus 30 – 40 Al-Sharook et al. (1991)
Melia volkensii Seed Acetone Cx. pipiens molestus 20 – 30 Al-Sharook et al. (1991)
Melia volkensii Fruit Hexane: Ethyl Ae. aegypti 50 Mwangi and Rembold
(48 h exposure) aetate fraction (1:1) (1988)
Melia volkensii Fruit kernel Methanol: water An. arabiensis 5.4 Mwangi and Mukiama
(48 h exposure) (80:20) (1988)
Mentha arvensis Different parts Steam distillation An. stephensi 83.8 Kumar and Dutta (1987)
Minthostachys setosa Whole Dichloro methane Ae. aegypti 9.2 Ciccia et al. (2000)
Ocimum basilicum Whole Petroleum ether Cx. quinquefasciatus 47.3 Kalyanasundaram and
Babu (1982)
Origanum majoranal Leaf na Cx. pipiens 54 Soliman and El-Sherif
(1995)
Piper longum Fruit Hexane fraction Cx. pipiens pallens 0.21 Lee (2000)
(piperonaline)
Piper longum Fruit Piperonaline Cx. pipiens pallens 0.25 Yang et al. (2002)
Piper nigrum Seed Ethanol Cx. pipiens 26 Moawed (1998)
Petroleum ether 2.6
Piper nigrum Fruit Methanol extract Park et al. (2002)
(48 h exposure) containing:
Pellitorine Cx. pipiens pallens 0.86
Guineensine 0.17
Pipercide 0.004
Retrofractamide A 0.028
Pellitorine Ae. aegypti 0.92
Guineensine 0.89
Pipercide 0.1
Retrofractamide A 0.039
Pellitorine Ae. togoi 0.71
Guineensine 0.75
Pipercide 0.26
Retrofractamide A 0.01
Quassia amara Whole Quassin Cx. quinquefasciatus 6.0 Evans and Raj (1991)
(continued on next page)
1154 E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166

Table 1 (continued)
Plant species Plant part Solvent/fraction/ Mosquito mg/L(LC) Source
compound
Rhinocanthus nasutus Leaf Petroleum ether fraction Ae. aegypti 22 Pushpalatha and
Muthukrishnan (1999)
Rhinocanthus nasutus Leaf Petroleum ether fraction An. stephensi 22
Cx. quinquefasciatus 23.5
Ricinus communis Different parts Steam distillation An. stephensi 113 Kumar and Dutta (1987)
Sassurea lappa Commercial oils Ae. aegypti 52 – 74 Sharma et al. (1994)
Cx. fatigans 30 – 38
Several marine Whole Acetone Ae. aegypti 17 – 95.5 Thangam and Kathiresan
angiosperms (1999, 1991b)
Solanum elaeagnifolium Berries Glycoalkaloid extract An. labranchiae 59.8 Markouk et al. (2000)
Solanum sodomaeum Seed Glycoalkaloid extract An. labranchiae 173.8 Markouk et al. (2000)
Sorghum bicolour Seedling Dhurrin Cx. pipiens 1.12 (90) Jackson et al. (1990)
Tagetes minuta Whole Methylene chloride Ae. aegypti 16 Perich et al. (1994)
An. stephensi 19
Thymus capitatus Whole Volatile oils fraction Cx. pipiens 49 Mansour et al. (2000)
Thymol fraction 58
Valarian wallichii Commercial oils Ae. aegypti 52 – 74 Sharma et al. (1994)
Cx. fatigans 30 – 38
Vetiveria zizanioides Rhizome na Cx. pipiens 67 Soliman and El-Sherif
(1995)
Vicia tetrasperma Seed Methanol Ae. aegypti 100 (67) Jang et al. (2002)
Cx. pipiens pallens 50 (76)
Vitex nequrdo Whole Petroleum ether Cx. quinquefasciatus 66.8 Kalyanasundaram and
Babu (1982)
Withania somnifera Leaf Benzene Cx. quinquefasciatus 342 – 368 Karmegam et al. (1997)
Unless stated, all LC50 values for test substances involve a 24 h exposure period. Lethal concentration (LC) is only mentioned if it is not LC50.

Of primary consideration is the type of solvent used since same extract always have different larvicidal activity
polar solvents will extract polar molecules and non-polar because they contain different phytochemicals. For instance,
solvents will extract non-polar molecules. The purpose of a Sun et al. (2001) screened ethyl-acetate ( P = 4.4), n-butyl
general screen for bioactivity is to extract as many alcohol ( P = 3.9) and water fractions of alcoholic extract of
potentially active constituents as possible. This is achieved leaves and stems of Vanilla fragrans against Cx. pipiens
by using solvents ranging from water, the most polar with a larvae. Both n-butyl alcohol and ethyl-acetate fractions were
polarity index ( P) of 10.2 to chloroform (relatively non- active in bioassays, while the water fraction appeared to
polar; P = 4.1) and hexane (non-polar P = 0.1) including a contain no substances that inhibited larval growth. Table 2
number of intermediary solvents such as ethyl alcohols. If lists other studies that have fractioned extracts in search of
incomplete screening of botanical material is attempted, the active phytochemicals.
solvents for phytochemical extractions should be carefully Once a fraction has proved to be effective, compounds
selected because different solvent types can significantly can be extracted to isolate the active ingredient. Some
affect the potency of extracted plant compounds (Karmegam compounds loose efficacy when separated since many
et al., 1997). A converse relationship is said to exist between synergistic relations potentially exist in botanical prepara-
extract effectiveness and solvent polarity where efficacy tions which may promote killing activity.
increases with decreasing polarity (Mulla and Su, 1999)
(Table 1). This is not consistent due to differences between 2.2. Mode and site of action
the characteristics of active chemicals among plants. Berry
and Rodriguez (2003) suggested the use of different The mode of action and site of effect for larvicidal
solvents based on the type of molecules targeted for phytochemicals has received little attention. Rey et al.
extraction. Petroleum ether ( P = 0.1) appears to have been (1999a) and David et al. (2000) found that botanical
the solvent of choice for some time (Table 2). derivatives primarily affect the midgut epithelium and
The crude extract described above contains a complex secondarily affect the gastric caeca and the malpighian
mixture of biocidal active compounds. If an exceptionally tubules in mosquito larvae. Histopathological effects were
low lethal concentration is detected, the extract may be observed to differ qualitatively according to their location
fractionated in order to locate the particular chemical along the midgut and quantitatively according to the
constituent causing the lethal effect. The purpose of concentration assayed, duration of the treatment, and
fractionation is thus to produce several simple mixtures of mosquito species used. Rey et al. (1999b) observed that
compounds to reduce the number of compounds which may the overall destructive effects of tannins on the midgut
be identified in further analyses. Fractions isolated from the epithelium of larvae were more efficient than those
E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166 1155

Table 2
Comparative efficacy of botanical extract fractions and compounds as larvicides
Plant species Solvent/fraction Mosquito mg/L(LC) Source
Allium sativum Methanol crude extract Cx. peus 34 Amonkar and Reeves
Cx. tarsalis 25 (1970)
Ae. aegypti 33
Ae. triseriatus 62
Steam distillate oil fraction Cx. peus 2.1
Cx. tarsalis 2.0
Ae. aegypti 5.6
Ae. triseriatus 4.0
Caulerpa scalpelliformis Acetone Ae. aegypti 53.7 Thangam and Kathiresan
Fraction IV (2:8 methanol – 15.9 (1991a)
acetone)
Fractions III 32
Fraction V 23
Curcuma longa Ethyl acetate 5 Roth et al. (1998)
Hexane 1
Dictyota dichotoma Acetone Ae. aegypti 62 Thangam and Kathiresan
Fractions III (1:9 methanol – 26 (1991a)
acetone)
Fraction IV (2:8 methanol – 28
acetone)
Euphorbia peplus Ethanol Cx. pipiens 510 Moawed (1998)
Petroleum ether 235
Haplophyllum tuberculatum Ethanol Cx. pipiens 20 Moawed (1998)
Petroleum ether 66
Kaempferia galanga Hexane fraction Cx. quinquefasciatus 42 Choochote et al. (1999)
Methanol fraction 1052
Melia volkensii (48 h exposure) Standardized fraction An. arabiensis 5.4 Mwangi and Mukiama
Fractions I & II 0.5 (1988)
Fractions III & IV 50 – 100
Fractions V, VI and VII >100
Nerium indicum Petroleum ether (fraction II— Cx. pipiens fatigans 100 (100) Chavan and Nikam
benzene:methanol—80:20) (1983)
Ruta graveolens Ethanol Cx. pipiens 230 Moawed (1998)
Petroleum ether 380
Tagetes minuta Hydrogenated part (fraction 1) Ae. aegypti 9.6 Perich et al. (1995)
(5E)-ocimenone An. stephensi 10.1
Oxygenated part (fraction 2) Ae. aegypti 42.4
An. stephensi 150.1

produced by Bacillus thuringiensis var. israelensis. This reasonable or excellent LC50 values. Thus the growth
suggested that these compounds should be considered as inhibiting activity of a phytochemical may be essential to
alternative insecticide candidates for mosquito control in its uptake by mosquito control industries. Indeed, the
breeding sites, especially in cases where susceptibility is rational application of exceptional phytochemicals may
decreasing. not only lead to new strategies, but may inhibit the
development of insect resistance.
Over one thousand plant species contain bioactive
3. Growth and reproduction inhibiting phytochemicals substance with many of these containing phytoecdysones,
phytojuvenoids and anti-juvenile hormones, which act as
As with toxicity, the effect of a phytochemical on the IGRs (Varma and Dubey, 1998). The use of ajugarins,
inhibition of mosquito growth and reproductive capacity is isolated from Ajuga remota by Marcard et al. (1986) against
governed by mosquito species, plant species, plant parts, mosquitoes is an example. Another example is provided by
solvents used in extraction and fractions of the same solvent. Neraliya and Srivastava (1996), who tested the insect
Botanical extracts, termed Insect Growth Regulators (IGRs), growth regulatory activity of crude petroleum ether – acetone
can have a pronounced effect on the developmental period, extracts of 25 angiosperm plants on Cx. quinquefasciatus
growth, adult emergence, fecundity, fertility and egg larvae. In this case, 40% of the extracts possessed promising
hatching resulting in effective control. The small dose – bioactivity. But progress is slow and no promising botanical
response slope observed for most phytochemicals over 24 h IGRs have been commercialised to replace current synthetic
renders most of them unusable despite them having IGRs such as methoprene.
1156
Table 3
Effects of continuous exposure to botanical extracts on growth, development, reproduction, abnormalities, hatch rates and fertility
Plant species Solvent/fraction/compound Mosquito mg/L Effect Source
Azadirachta indica Water Ae. aegypti 100 80 T 5% pupal – adult intermediates Zebitz (1984)
Methyl-tert.-butylether/water 20 mortality and 3% pupal – adult
intermediates exhibited protruding
wing-sheath and mouthparts
Albizzia lebbeck Ethyl alcohol Ae. aegypti 500 95% reduced adult emergence at day-7 Qureshi et al. (1986)
Annona squamosa Alkaloids An. stephensi 50 52% total mortality (larvae, pupae Saxena et al. (1993)

E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166


and adults) 52% reduced emergence,
reduced total development period,
32% decrease in egg hatching and
32% in egg production
Argemone mexicana Benzene Cx. quinquefasciatus 31.25 83% mortality Karmegam et al. (1997)
Argemone mexicana Benzene Cx. quinquefasciatus 31.25 produced larval – pupal intermediates Karmegam et al. (1997)
and half ecdysed organisms
Callistemon lanceolatus Ethanol Cx. quinquefasciatus 10 – 1000 Cumulative mortality and increased Mohsen et al. (1990)
developmental period
Callistemon lanceolatus Petroleum ether – acetone Cx. quinquefasciatus 48.4 EC50 Neraliya and Srivastava
(1996)
Calophyllum inophyllum Ethyl acetate fraction (seeds) Ae. aegypti 0.22 EC50 for inhibition of adult emergence Pushpalatha and
An. stephensi 0.33 50% increased developmental period Muthukrishnan (1999)
Cx. quinquefasciatus 0.64
Petroleum ether fraction (leaves) Ae. aegypti 2.6
An. stephensi 2.75
Cx. quinquefasciatus 2.9
Calophyllum inophyllum Ethyl acetate fraction (leaf) An. stephensi as above 54% decrease in egg hatching Muthukrishnan and
Ae. aegypti EC50/2 45% decrease in egg hatching Pushpalatha (2001)
Cx. quinquefasciatus 65% decrease in egg hatching
An. stephensi 16.6% reduction in egg production
and 84 – 90% sterility in progeny
Ae. aegypti 20.6% reduction in egg production
and 83 – 91% sterility in progeny
Cx. quinquefasciatus 16.6% reduction in egg production
and 90 – 93% sterility in progeny
Calotropis gigantea Petroleum ether – acetone Cx. quinquefasciatus 134.9 EC50 Neraliya and Srivastava
(1996)
Cassia holosericea Ethyl alcohol Ae. aegypti 1000 Prevented pupation up to day-7 Qureshi et al. (1986)
Citrus reticulata Enriched fraction Cx. quinquefasciatus 50.1 EC50 for inhibition of adult Jayaprakasha et al. (1997)
emergence
Obacounone 6.3
Nomilin 26.6
Limonin 59.6
Clerodendrom inerme (levo)-3-epicaryoptin Cx. quinquefasciatus na Death during larval – pupal molt Pereira and Gurudutt (1990)
and pupal – adult eclosion
Cyperus iria Leaf aged 1 & 2 month Ae. aegypti 1000 100% reduced emergence Schwartz et al. (1998)
Descurainia sophia Ethanol Cx. quinquefasciatus 100 – 1500 Concentration dependent larval Mohsen et al. (1990)
mortality and reduced emergence
Ervatamia coronaria Petroleum ether Ae. aegypti 1000 Prevented pupation up to day-7 Qureshi et al. (1986)
Erythraea roxburghii, Ethyl alcohol Ae. aegypti 1000 100% reduced adult emergence at day-7 Qureshi et al. (1986)
Euphorbia pulcherrima,
Thlaspi arvense, Ocimum Methanol Ae. aegypti 100 Significantly reduced adult emergence Supavarn et al. (1974)
basilicum, Barbarea at day-7
vulgaris, Raphanus sativus,
Brassica nigra, Origanum
majorana, Geranium sp.,
Conium maculatum, Hyptis
sauveoleus, Lithospermum
arvense, Allium

E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166


schoenoprasum and Rumex
crispus
Haplophyllum tuberculatum Ethanol Cx. quinquefasciatus 50 – 2000 Morphogenetic aberrations Mohsen et al. (1989)
Ipomoea carnea fistulosa Acetone An. stephensi 200 Average larval, pupal, developmental Saxena and Sumithra
periods and growth index were 18.93, (1985)
6.36, 25.29 (days) and 0.94 compared
to control 14.44, 3.58, 18.02 (days)
and 5.54.
Melia volkensii Crude An. arabiensis 250 100% reduced emergence and 100% Mwangi and Mukiama
mortality (1988)
Fraction I 10 100% reduced emergence
and 100% mortality
Fraction II 10 74% reduced emergence
and 74% mortality
Melia volkensii Hexane/Ethyl acetate Ae. aegypti 20 100% mortality during molting and Mwangi and Rembold
fraction (1:1) 100% reduced emergence (1988)
Melia volkensii Acetone Cx. pipiens molestus 0.05 94% reduction in adult emergence Al-Sharook et al. (1991)
Momordica charantia Petroleum ether – acetone Cx. quinquefasciatus 29.5 EC50 Neraliya and Srivastava
(1996)
Neem oil Oils from crushed seeds Ae. aegypti 50 Treated larvae developed to pupae Sinniah et al. (1994)
Cx. quinquefasciatus but failed to emerge
Neem oil na An. stephensi 50,000 No pupation occurred and emergence Murugan et al. (1996)
completely inhibited
Nem seed kernel extract na An. stephensi 50,000 80% reduction in emergence Murugan et al. (1996)
Nerium oleander, Syzygium Petroleum ether: ethyl Cx. quinquefasciatus na Larval mortality, interfered with Pushpalatha and
jambotanum acetate (1:1) fraction An. stephensi pupal – adult metamorphosis and Muthukrishnan (1995)
extended duration of larval instars
and of pupation
Oligochaeta ramose Acetone Ae. aegypti 50 74% larval mortality (larval – pupal Saxena and Yadav (1983)
intermediate) and 76% reduced
emergence
Rhazya stricta Partially purified asewerine Ae. aegypti 100 78%, 78% and 86% reduction in Qureshi et al. (1986)
emergence at day-7
(continued on next page)

1157
1158
Table 3 (continued)
Plant species Solvent/fraction/compound Mosquito mg/L Effect Source
Aristolochia bracteate Ethyl alcohol
Valeriana wallichii Ethyl alcohol
Rhinacanthus nasutus Petroleum ether fraction Ae. aegypti 2.9 EC50 for inhibition of adult emergence Pushpalatha and
An. stephensi 2.9 50% increased developmental period Muthukrishnan (1999)
Cx. quinquefasciatus 5.9
Rhinacanthus nasutus, Petroleum ether fraction (leaf) An. stephensi 40% decrease in egg hatching Muthukrishnan and
Ae. aegypti 40% decrease in egg hatching Pushpalatha (2001)
Cx. quinquefasciatus 50% decrease in egg hatching
An. stephensi EC50/2 reduction in egg production and more
than 80% sterility in progeny
Ae. aegypti reduction in egg production and

E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166


more than 80% sterility in progeny
Cx. quinquefasciatus reduction in egg production and
90% sterility in progeny
Samadera indica Ethyl acetate fraction An. stephensi EC50/2 45% decrease in egg hatching Muthukrishnan and
Ae. aegypti 40% decrease in egg hatching Pushpalatha (2001)
Cx. quinquefasciatus 45% decrease in egg hatching
An. stephensi reduced egg production and >80%
sterility in progeny
Ae. aegypti reduced egg production and >80%
sterility in progeny
Cx. quinquefasciatus reduced egg production and 90%
sterility in progeny
Solanum suratense, Ethyl acetate fraction (leaf) An. stephensi 55% decrease in egg hatching Muthukrishnan and
Ae. aegypti 40% decrease in egg hatching Pushpalatha (2001)
Cx. quinquefasciatus 55% decrease in egg hatching
An. stephensi EC50/2 reduced egg production and >80%
sterility in progeny
Ae. aegypti reduced egg production and >80%
sterility in progeny
Cx. quinquefasciatus reduced egg production and 90%
sterility in progeny
Sphaeranthus indicus Sesquiterpene Cx. quinquefasciatus 50 Fecundity and fertility were affected, Sharma (1996)
lactone (isolated from significant decrease in egg hatching
petroleum ether extract) and slight reduction in egg production
Tagetes erectes Petroleum ether An. stephensi Less than 100 Significant effect on mortality and Sharma and Saxena
reduction in the adult emergence (1994)
Vitex negundo Petroleum ether: Cx. quinquefasciatus na Larval mortality, interfered with pupal – adult Pushpalatha and
ethyl acetate An. stephensi metamorphosis and extended duration of Muthukrishnan (1995)
(3:1) fraction larval instars and of pupation
Descurainia sophia Ethanol Cx. quinquefasciatus 1 – 100 Ovicidal activity against freshly laid Mohsen et al. (1990)
eggs and newly hatched larvae
Azadirachta indica Azad wettable powder WP10 Cx. tarsalis and Cx. 1 100% mortality against freshly laid Su and Mulla (1998)
quinquefasciatus eggs after 4 h
Azad emulsifiable concentrate 90 – 100% mortality against freshly laid eggs after 4 h
EC4.5
Technically pure azadirachtin 97 – 99% mortality against freshly laid eggs after 4 h
E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166 1159

In experimental trials of insect growth regulator (IGR) prolongation of various developmental stages is of little use
efficacy, concentration is not reported in terms of lethal dose for practical vector control since the recruitment rate from
(LC), but as effective concentration (EC). Since reduction in larval to adult populations is of fundamental importance to
adult vectors is of primary importance, Baktharatchagan et the maintenance of disease transmission cycles. The
al. (1993) and Mohsen et al. (1995) argue that the following studies report varying degrees of efficacy using
concentration reducing recruitment by 50% (EC50) is a relatively high concentrations.
more meaningful measure of potential success in the field Identification of phytochemicals with growth inhibition
than the LC50. Few authors, however, go to the extent of properties combined with a considerable capacity to reduce
calculating effective concentrations. adult emergence is the desired endpoint of botanical
Phytochemicals extracted from many plant species show insecticide research. Another desirable quality would be
growth inhibiting effects on the various developmental that a control agent induced IGR effects at less than the
stages of different mosquito species. A range of pre- lethal dose so that recruitment could be reduced over time.
emergent effects can occur; such as prolongation of instar Fortunately, adult emergence is commonly critically affected
and pupae durations, inhibition of larval and pupal molting, by phytochemicals, which are often capable of causing a
morphological abnormalities and mortality especially during combination of acute toxic and chronic growth effects. The
molting and melanization processes. reported results in Table 3 describe the evaluation of a range
Zebitz (1984) calculated the relative developmental stage of botanicals including some promising candidates.
(rDS) of a population to monitor the progress of IGR effects Unfortunately only a limited number of researchers have
as follows: followed WHO protocols for IGR evaluation by going to the
P  extent of determining the effective concentration of bota-
nt Sp  F p
sDS ¼ nical extracts that inhibit 50% of the treated larval
Nt S population from emerging as adults (EC50). The reasons
where n t S p is the number of individuals in a certain for this are not clear but may be due to the protocol being
developmental stage at observation time t, N t S is the total difficult to source or restrictive.
number of stages present at time of observation, and F p is an Despite toxic effects and reductions in adult emergence,
arbitrary multiplication factor calculated by adding the some phytochemicals such as those from Annona squamosa
following numbers 1 (1st instar larvae) or 2 (2nd instar do not alter the larval developmental period. Many
larvae) or 3 (3rd instar larvae) or 4 (4th instar larvae) or 5 botanicals exhibit combined effects on the developmental
(pupae) or 6 (adults). For instance, the presence of 10 2nd, period and adult emergence which occasionally extend to
80 3rd and 10 4th instar larvae gives a rDS of 3 meaning the progeny of exposed larvae. Decreasing mosquito
that the mean developmental stage on day x is the 3rd instar. fecundity caused by exposure of larvae to plant extracts
This evaluation method does not appear to have been has been studied by few researchers. More commonly,
used by any subsequent researchers. It was also not phytochemicals can produce morphological abnormalities in
modified by the author to take other recognized stages into different developmental stages of mosquitoes. Abnormal-
account. There are 10 developmental stages from 4th instar ities, such as lacking of melanization in larval and pupal
to adult that can be affected by IGRs (Busvine, 1978; stages, dead larval– pupal intermediate stage mostly with the
Zebitz, 1984; Mwangi and Rembold, 1988): (1) mature head of a pupa and the abdomen of a larva, dead adults with
larva (2) pupal abdomen retracted (3) pupa partly escaped folded wings in the pupal exuvium and emerged adults
(4) unmelanized pupa (5) partly melanized pupa (6) unable to escape the pupal exoskeleton, and half-ecdysed
melanized pupa (7) pupa with adult form visible (8) adult adults, indicate a metamorphosis-inhibiting effect of the
partly escaped (9) adult largely escaped (10) non-viable or plant extract, which is probably due to disturbance of
viable adults. hormonal control and/or interference in chitin synthesis
Saxena and Sumithra (1985) used a simple formula to during the molting process (Saxena and Yadav, 1983;
calculate a Growth Index (GI) based on emergence and the Saxena and Sumithra, 1985; Mwangi and Rembold, 1988;
duration of the developmental period as follows: Al-Sharook et al., 1991; Saxena et al., 1993; Neraliya and
Ae Srivastava, 1996; Karmegam et al., 1997; Schwartz et al.,
GI ¼ 1998; Pushpalatha and Muthukrishnan, 1999).
Px
Only a few researchers have investigated the effect of
where A e is the percentage of adult emergence and P x is the phytochemicals on egg hatching. The effect of IGRs appears
average developmental period. to persist from exposed larvae through to eggs laid by an
emergent adult with the effect of reducing viability. Saxena
3.1. Effects on growth and reproduction and Sumithra (1985) used a Sterility Index (SI) to calculate
the impact of a toxicant on egg hatch rates as follows:
Exposure of mosquito larvae to sub lethal concentrations  
of active botanical derivatives can often result in an Te  He
SI ¼ 100   100
extension of the duration of development. Obviously simple Ce  He
1160 E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166

where Te is the number of eggs in treated, H e is the have been conducted on agricultural pests rather than pests
percentage of eggs that hatch and C e is the number of of medical importance. The few studies on the mosquito-
control eggs. cidal activity of binary mixtures have investigated the
Ovicidal effects of phytochemicals have not been combined effects of phytochemicals with insecticides or
investigated to any great extent for once mosquito eggs microbial control agents (Table 4).
have hardened they become rather impervious, although this Joint toxicity of various insecticide mixtures was initially
is perhaps truer for aedines than culicines and anophelines. investigated by Sun and Johnson (1960) to differentiate
Studies conducted on freshly laid eggs are of limited use between synergism, antagonism and potentiation. In their
since most eggs exposed to insecticides have already simple equation
hardened.
Co  toxicity factor ¼ ðO  EÞ  100=E
where O is observed % mortality and E is expected %
4. Joint action and toxicity mortality. This factor differentiates the results into three
categories. A positive factor of 20 indicates potentiation, a
Most studies on the synergistic, antagonistic and additive negative factor of 20 indicates antagonism, and the
toxic effects of binary mixtures involving phytochemicals intermediate values of 19 to 19) indicate an additive

Table 4
The synergistic, antagonistic and additive effects of botanical extracts and phytochemicals in combination with other botanicals or synthetic insecticides
Test substance 1 mg/L Test substance 2 mg/L Mosquito Effect Source
Ocimum basilicum, 1 and 5 Phenthoate 6 concentrations Cx. quinquefasciatus 0.34 – 0.75 SF Kalyanasundaram
O. sanctum, Fenthion from each insecticide 0.35 – 0.74 SF and Babu (1982)
Azadirachta indica,
Lantana camara and
Vitex negundo
Ruta graveolens ethanol 175 Cypermethrin 0.0025 Cx. pipiens Synergistic Moawed (1998)
extract petroleum ether 225
ext. Haplophyllum 38
tuberculatum
(water extract)
H. tuberculatum 12.5 Cypermethrin 0.0025 Cx. pipiens Additive Moawed (1998)
Euphorbia peplus 380
(ethanol extracts)
E. peplus 200 Piper nigrum 22.5 Cx. pipiens Antagonistic Moawed (1998)
H. tuberculatum 50 (ethanol extract)
(petroleum ether
extracts)
Neem derivatives na Bacillus thuringiensis na Ae. aegypti Additive or Schmutterer (1990)
var. israelensis synergistic
Neem seed kernel na Methoprene na Ae. aegypti Synergistic Mulla and Su
extract Ae. togoi (1999)
An. stephensi
Cx. quinquefasciatus
Lemon, orange and Different Lemon, orange and Different Cx. quinquefasciatus Synergistic Mwaiko (1992)
bitter orange concentrations bitter orange concentrations
Thymus capitatus Non-lethal Malathion na Cx. pipiens Synergistic Mansour et al.
concentrations Permethrin Additive (2000)
Pirimiphos-methyl Antagonistic
Poa ampla (blue grass) 100 Neotyphodium n/a Cx. pipiens Highly Ju et al. (1998)
ethanol extract typhnium (fungus) synergistic
Caulerpa scalpelliformis 0.1 and 0.5 DDT 12 concentrations Ae. aegypti 0.89 SF Thangam and
BHC from each insecticide 0.71 SF Kathiresan (1991b)
Malathion 0.84 SF
Dictyota dichotoma 0.1 and 0.5 DDT 12 concentrations Ae. aegypti 0.97 SF Thangam and
BHC from each insecticide 0.76 SF Kathiresan (1991b)
Malathion 0.85 SF
Rhizophora apiculata 0.1 and 0.5 DDT 12 concentrations Ae. aegypti 0.97 SF Thangam and
BHC from each insecticide 0.81 SF Kathiresan (1991b)
Malathion 0.80 SF
DDT 0.77 SF
BHC 0.68 SF
Malathion 0.71 SF
E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166 1161

effect. Similarly, Trisyono and Whalon (1999) compared the amount of information on the persistence of such
observed mortalities to expected mortalities based on the compounds after application is limited. For instance,
formula pesticides containing azadirachtin are reputed to have a
residual effect of about 8 days depending on environmental
E ¼ Oa þ Ob ð1  Oa Þ factors such as ultra-violet light and rainfall (Schmutterer,
1988). This may have been an overestimate since Sinniah
where E is expected mortality and O a and O b are observed et al. (1994) found that crude neem oil extract only
mortalities of different compounds at a given concentration. remained effective up to 5 days in ideal laboratory
Mixture effects were declared to be synergistic, antagonistic conditions against Ae. aegypti and Cx. quinquefasciatus.
or additive after v 2 analysis with the formula In reality, an effective range of 4 to 8 days can be expected
in field conditions and in cases of systemic effects some
v2 ¼ ðOm  EÞ2 =E
days longer (Mulla and Su, 1999). Over this brief residual
where O m is observed mortality from the mixture and E is period, activity was observed to increase for a few days
expected mortality. Thus when considering a pair, it is given and then progressively decline. Pushpalatha and Muthuk-
that v 2 with df = 1 and P = 0.05 is 3.84. So, the effect is rishnan (1999) studied the persistence of seed and leaf
considered to be synergistic in the case of a pair with v 2 extracts of Calophyllum inophyllum and leaf extracts of
values >3.84 and more than expected mortality, and additive Rhinacanthus nasutus against Cx. quinquefasciatus juve-
in the case of a pair with v 2 values <3.84. niles in laboratory and semi-field conditions. The activity
Kalyanasundaram and Babu (1982) calculated the of ethyl acetate fraction of C. inophyllum increased over
synergistic factor (SF) with the formula time with the highest activity observed for samples
incubated for 5 days before exposure to larvae. Similarly,
SF ¼ LC50 of mixture LC50 of insecticide alone
a petroleum ether fraction of R. nasutus had the same
where a SF of <1 confirms synergism and the smaller the activity with the duration of incubation of up to 3 days and
value the higher the synergistic activity. gradually declined (but not remarkably). Under semi-field
Synergism between synthetic insecticides and phyto- conditions, these phytochemicals were stable and their
chemicals appears to be more common than between activity persisted in the medium for up to 10 days with
different phytochemicals although this may be the result declining toxicity.
of increased focus in this area. In an attempt to explain
synergistic activity involving phytochemicals, Thangam and 5.2. Effect on non-target organisms
Kathiresan (1991b) surmised that synergism may be due to
phytochemicals inhibiting a mosquito larva’s ability to Very little has been researched and published on
employ detoxifying enzymes against synthetic chemicals. phytochemicals and their effects on non-target organisms.
Mixtures of plant extracts with compounds showing From the conclusions of neem and tannin researchers, it
synergistic or potentiating interactions between them are can be surmised that botanical derivatives are not free of
considered to have a higher and longer-lasting effect risk and further field investigations are necessary. Variable
(Chockalingam et al., 1990). Identifying these synergist results have been found for neem formulations and
compounds within mixtures may lead to the development of extracts ranging from no effect to significant effects on
more effective mosquitocides as well as the use of smaller non-target organisms. In India, Rao et al. (1995) applied
amounts in the mixture to achieve satisfactory levels of A. indica to rice fields and found a marked reduction in
efficacy. the abundance of late instar culicine and anopheline larvae
and pupae. No significant reduction of most non-target
organisms, including different aquatic insects, frog tad-
5. Field evaluation of phytochemicals poles and plant spiders was observed. Minimal non-target
toxicity from two neem formulations was recorded by
5.1. Residual capacity Kreutzweiser (1997) for microinvertebrates. Significant
mortality only occurred in Isonychia bicolor rufa (May
The degradation of natural products under field con- fly) out of eight species and no significant mortality nor
ditions is expected to take place faster than in the antifeedant effects were observed in 3 detritivorous
laboratory because of environmental factors, such as species after a 28-day exposure. Kreutzweiser (1997)
ultraviolet light, temperature, pH and microbial activity. concluded that there was little risk to aquatic macro-
Several botanical derivatives have shown efficacy in invertebrates resulting from contamination of water bodies
mosquito control field trials, but most trials have tested with neem-based insecticides. Kreutzweiser et al. (2000)
neem and few have included investigation into residual and then performed dose studies and found that neem, or
non-target effects. Both degradation and residual effects of perhaps neem formulation materials, can be deleterious at
phytochemicals are important factors since they affect higher doses. Outdoor stream channels were treated with
toxicity and suitability for field applications. Unfortunately, low (0.28 mg/L), medium (0.84 mg/L) and high (2.54 mg/
1162 E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166

L) doses of a commercial azadarachtin formulation called resistant to organophosphate insecticides. No cross-resist-


Neemix 4.5 and a neem extract to determine effects on ance was apparent since lethal values for these phytochem-
aquatic insect communities. No significant difference in icals were identical between strains. Another study using the
abundance and species richness of aquatic insects was same phytochemical revealed a counter cross-resistance
found between control and Neemix treated stream effect (Thomas and Callaghan, 1999). Garlic was found to
channels at the low dose, but medium and high doses be more toxic to a mosquito strain that was resistant to
reduced the abundance and richness of aquatic insect organophosphate insecticides than to a susceptible strain. It
communities compared with controls. When the neem was concluded that the use of botanical larvicidal com-
extract (0.9 mg/L azadirachtin) was applied, no significant pounds would not necessarily avoid problems posed by
effect on community structure was observed, but signifi- some existing mechanisms of resistance to synthetic
cant effects were detected at 3.0 mg/L. These results compounds.
suggest that additive or synergistic formulation ingredients
in Neemix may be contributing to the adverse effects on 5.4. Screening methodology
non-target insect communities, and that neem is a risk to
non-target organisms at higher doses. As can be seen from the studies described, the extraction,
Vegetable tannins have also been shown to exhibit isolation and formulation procedures are not standardised.
biocidal properties among crustacean taxa representative Insecticide efficacy methods are also not uniform and vary
of the non-target fauna associated with Alpine mosquito considerably despite the provision of WHO standard
breeding sites (Pautou et al., 2000). Bioassays distinguished protocols (WHO, 1981) over several decades for the
the crustacean taxa into sensitive species (Diaptomus castor, determination of lethal and effective concentrations. Units
Chydorus sphaericus and Eucypris fuscata) and insensitive should be standardized to mg/L, which is the most popular
species (Daphnia pulex, Eucypris virens and Acanthocy- form of dose expression. It is unfortunate that a primary
clops robustus). Rey et al. (1999a) noted that, sensitivity to screening method for potential larvicides is not universally
tannic acid among non-target organisms is variable. S. used before time, funds and energy are expended in detailed
variegatum larvae were observed to be much more sensitive examinations of phytochemicals that are useless to vector
than C. annularius larvae and Chaoborus crysttallinus control from a practical point of view. The diagnostic doses
larvae were relatively insensitive. David et al. (2000) also provided by the WHO for adult testing are an example of a
noted that S. variegatum larvae were less sensitive to 10- primary screen. This has limited but not negated the
month-old leaf litter of A. glutinosa than C. annularius usefulness of comparisons between studies. For the purpose
larvae. of efficiently testing large numbers of samples within a
biodiscovery process, mosquitocidal bioassays may occur at
5.3. Development of resistance five stages (Fig. 1).
Stage 1 screening should be undertaken to determine
Unlike synthetic insecticides, the literature on mosqui- the general potency of a crude botanical extract as a
tocidal botanical agents does not contain any evidence to larvicide. All extracts should be produced using a limited
suggest that resistance to these substances has emerged. range of polar to non-polar solvents or via other methods
This is most likely not due to any reason of consequence such as steam distillation. Larvae should be subjected to a
but because botanical agents are not often used in vector single dose of 10 mg/L of the crude botanical extract
control. In their review article on the biological effects of where 100% mortality is indicative of potential efficacy.
neem products against medically and veterinary important Commonly, a descending series of concentrations from the
arthropods, Mulla and Su (1999) mentioned that no cases same plant extract (e.g. 1000, 500, 100, 10, 5 mg/L) are
of resistance to neem products have been recorded. used, however, this constitutes a waste of extract material
Similarly, Schmutterer (1988) stated that attempts to select for if there is no efficacy above 10 mg/L, the extract
for resistance against neem products in the laboratory should be discarded. As can be seen from the many studies
were not successful. The absence of resistance to these listed, too much research effort has been wasted on in-
products among mosquitoes might be because they are depth investigation of products that should have been
mixtures of various related compounds with different discarded at this stage.
modes of action and hence the development of resistance Stage 2 screening involves determination of lethal and
to such products is somewhat difficult (Schmutterer, 1988; effective concentrations for 50% of the test population
Mulla and Su, 1999). (LC50 and EC50) and is carried out using a range of doses to
Cross-resistance is always a concern when new insecti- determine the exact efficacy of potential larvicides as acute
cides are developed since their mode of action may be or chronic toxicants. At this stage, other effects may be
similar to well established insecticides. Amonkar and investigated on developmental time, reproduction, fertility,
Reeves (1970) evaluated a methanol crude extract and an fecundity, ovicidal action and potential for resistance.
oil fraction of Allium sativum against strains of Ae. Standard WHO methods (WHO, 1981) should be followed
nigromaculis that were highly susceptible and highly to enable accurate study comparisons.
E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166 1163

Natural Organisms
High priority extracts Taxonomy

Create crude botanical extracts


from a range of solvents Chemical screening LC-UV-MS-NMR
Stage 3
Priority extracts selected Unknown compound Mosquitocidal
Stage 1 bioassay
Conduct mosquitocidal guided
bioscreening at 10 mg/L Shortlist structures
Differences fractionation

Known compound
Similarities
Priority extracts selected

Search natural products Leads identified


databases and literature
Inactive Active

Purification and High priority Low priority


Stage 2
structure elucidation
Mosquitocidal bioassays
LC50 (toxicity) & EC50 (IGR)
Stage 4
Simulated field trials, small-scale field
Priority extracts selected trials and formulation resolution

Non-selective Economic priority setting based on:


1. Purification
Active 2. Large-scale supply Stage 5
3. Drug development Mosquitocidal bioassays
4. Field application rates on joint action
Highly Active

Fig. 1. Required strategies and entomological stages in the biodiscovery process from extract selection to identification and isolation of highly effective
insecticide leads.

Stage 3 screening involves mosquitocidal bioassay activity. In this case the use of the mosquito Cx.
guided fractionation to identify highly active fractions and quinquefasciatus or a close relative is preferable.
compounds isolated from the crude extract. This step should
only be carried out on the most promising phytochemicals
such as those showing an LC50 of <1 mg/L or those 6. Conclusions
exhibiting a wide range of combined effects.
Stage 4 involves the determination of effective field A large number of different plant species representing
application rates in simulated field trials and/or small-scale different geographical areas around the world have been
field trials. Since technical insecticides are rarely used and shown to possess phytochemicals that are capable of
formulation can significantly affect control efficacy, various causing a range of acute and chronic toxic effects. Not
formulations must be tested to determine how best to only have many botanical extracts been shown to cause
‘‘enhance stability, reduce toxicity, improve efficacy or remarkable deleterious effects on the fecundity and
facilitate handling of the product’’ (Chavasse and Yap, hatchability of mosquito eggs, but they have been shown
1997). to have significant and promising larvicidal properties that
Stage 5 involves consideration of possible joint-action include growth regulating effects. Any one of these effects
effects and aims to identify synergistic mixtures that can taken alone is usually not impressive, but the combined
enhance control activities and minimise the development of ovicidal, larvicidal and IGR effects possessed by many
insecticide resistance. It quite possible that a less active phytochemicals can produce impressive results. When
phytochemical, potentially discarded in the previous stages, joint-action is considered, the application possibilities for
could possess exceptional synergistic qualities in combina- vector control increase significantly. Indeed, joint-action
tion with other synthetic or natural mosquitocidal agents. may well prolong the usefulness of synthetic insecticides
However, this is unlikely and further investigation of these that will eventually be unusable due to resistance.
agents should be justified. Several issues remain to be researched and improved
Selection of mosquito species for testing is also of upon including residual efficacy (degradation and persis-
fundamental importance since great variations exist in tence), preservation of non-target organisms, development
responses between the genera and species. Since aedines of resistance, and standardization of testing protocols. These
are usually less susceptible to insecticides and since Ae. should not be seen as major obstacles however, since B.
aegypti is the most commonly colonized mosquito, it should thuringiensis israelensis has been used in tidal mangrove
be used for comparative screening. Obviously this species areas where the residual effect is limited to 2 days. The
will not be the species of choice if a polluted environment paucity in field trials suggests that most phytochemicals do
involving decomposed leaf litter is being evaluated for not get past initial screening. The onus is thus placed upon
1164 E.A.-S. Shaalan et al. / Environment International 31 (2005) 1149 – 1166

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