Nerio et al. (2010) reviewed the repellent activity of essential oils which contributed to some metabolites, such as monoterpenes (α-pinene, cineole, eugenol, limonene, terpinolene, citronellol, citronellal, camphor, and thymol) against Mosquitoes (Yang et al., 2004) sesquiterpenes, β-caryophyllene, repellent against *A. aegypti*; phytol, a linear diterpene alcohol, against *Anopheles gambiae*; and phenylethyl alcohol, β-citronellol, cinnamyl alcohol, geraniol, and α-pinene, isolated from the essential oil of *Dianthus caryophyllum*, aginst ticks *(Ixodes ricinus).* In addition, cineole, geraniol and piperidine found in bay leaves (*Laurus nobilis*, Lauraceae) possess repellent properties towards cockroaches. Repellents may have an increasingly important role in eliminating insects from certain environments and essential oils could play a major role in new repellent technology. Valuable review on the repellent activity of essential oils are those of Tripathi et al. (2009), Isman (2010), Kumar et

Today, The used fumigants, for instance, phosphine, methyl bromide, and DDVP (2,2 dichlorovinyl dimethyl phosphate) do have adverse effets. Phosphine is the major cause of suicidal deaths in India. Methyl bromide has ozone-depleting potential (UNEP, 2000) and DDVP has a possible human carcinogen potential (Lu, 1995). Thus, there is an urgent need for development of safe alternative that have the potential to replace the toxic fumigants against pests attacking grains, dry stored food, and other agricultural products. The active principles are monoterpenes, sesquiterpenes and their biogenically related phenols. In addition to direct toxicity to insects, many of these substances are eterrents or repellents. Essential oils of *Artemisia species, Anethum sowa, Curcuma long,* and *Lippia alba*. Clove, rosemary, thyme, eucalyptus and various mint species have demonstrated contact and fumigant toxicity to a wide spectrum of insects, including human head lice (Toloza et al., 2008). Isolates like d-limonene, carvones and 1,8-cineole have been well documented as fumigants. The exact mode of action of these oils as fumigant is unknown, but the oils mainly act in the vapour phase via respiratory system. Physical properties of essential oils such as high boiling point, high molecular weight and low vapor pressure are barriers for application in large scale fumigation. For more details about fumigants, see Tripathi et al.,

exposure to the applied oils (Khater et al., 2009). *<IP_ADDRESS>.2 Metabolites reliable for repellent activity*

**<IP_ADDRESS> Fumigant**

(2009), Isman (2010), and Dubey (2011).

al. (2010), Nerio et al. (2010), Dubey (2011), and Maia & Moore (2011).

ingested or skin absorbed by insects. The rapid action against some pests is indicative of a neurotoxic mode of action, and there is evidence for interference with the neuromodulator octopamine (Enan, 2005) or GABA-gated chloride channels (Priestley et al., 2003).

Several essential oil compounds have been demonstrated to act on octopaminergic system of insects. Octopamine is a neurotransmitter, neurohormone, and circulating neurohormone – neuromodulator (Hollingworth, et al., 1984) and its disruption results in total break down of nervous system in insects. The lack of octopamine receptors in vertebrates likely accounts for the profound mammalian selectivity of essential oils as insecticides. Eugenol mimicked octopamine in increasing intracellular calcium levels in cloned cells from the brain of *Periplaneta americana* and *Drosophila melanogaster* (Enan, 2005). Consequently, octopaminergic system of insects represents a biorational target for insect control. Plant volatile oils have long been known to affect the behavioural responses of pests, with the monoterpenoid components appearing most useful as insecticides or antifeedants (Palevitch & Craker, 1994). LMW terpenoids may be too lipophilic to be soluble in the haemolymph after crossing the cuticle, and proposed a route of entry through the tracheae (Veal, 1996). Most insecticides bind to receptor proteins in the insect and, in doing so; they interrupt normal neurotransmission, which lead to paralysis and subsequently death. Recent evidence suggests that low-molecular-weight (LMW) terpenoids may also bind to target sites on receptors that modulate nervous activity. Ionotropic, γ-aminobutyric acid, GABA receptors, the targets of organochlorine insecticides lindane and dieldrin, are modulated by LMW terpenoids with vastly different structures (Priestley et al., 2006). Valuable appraisals about the mode of action are those of Price & Berry (2006), Isman (2006, 2010); Tripathi et al. (2009); and Dubey (2011). Some essential oils have larvicidal effect and the capacity to delayed development and suppress adults emergences and induce abnormalities during development of insects of medical and veterinary importance, Fig (1-5) (Khater, 2003; Shalaby & Khater, 2005; Khater & Shalaby, 2008; Khater & Khater (2009); Khater et al., 2009, 2011).

#### **<IP_ADDRESS> Repellent effect**

Repellents are substances that provide a vapor barrier deterring the arthropod from coming into contact with the surface or flying to, landing on or biting human or animal skin. The use of insect repellent compounds dates back to ancient times as plant oils, smokes, tars, etc. were used to displace or kill insects. The use of repellents by travelers may reduce infection with local diseases in temperate areas. DEET (N,N-diethyl-m-toluamide) is a broad spectrum repellent and the most effective and persistent on skin. Unfortunately, it may cause environmental and human health risks (Pitasawat et al., 2003). Therefore, there has been an increase in search efforts for natural and eco-friendly repellents.

#### *<IP_ADDRESS>.1 Plant- based repellents*

Some plant-based repellents are comparable to, or even better than synthetics; however, essential oil repellents tend to being short-lived in their effectiveness due to their volatility. Nerio (2010) review some splendid ideas for improvement of repellency of essential oils. Repellency assays with essential oils were done for Diptera species, especially mosquitoes and to a lesser extent to coleopteran insects related to losses in stored food. Plants with strong smell, such as French marigold and coriander act as repellents and can protect the corps nearby. Several essential oil- producing plants have been widely studies, such as *Cymbopogon* spp., *Eucalyptus* spp., *Ocimum* spp., the osage orange (hedgeapple) (*Maclura pomifera*), and catnip (*Nepeta cataria*). Several plant oils or their constituents have been commercialized as insect repellents in the past decade, such as soybean, lemon grass, cinnamon, and citronella. Neem oil, from *A. indica*, when formulated as 2% in coconut oil, provided complete protection (i.e. no confirmed bites) for 12 hours from *Anopheles* mosquitoes (Sharma et al., 1993). Essential oils have pronounced *In vitro* and *In vivo* pediculicidal activity as the number of lice infesting water buffaloes in Egypt was significantly reduced 3, 6, 4, and 6 days after treatment with the essential oils of camphor (*Cinnamomum camphora*), peppermint (*Mentha piperita),* chamomile (*Matricaria chamomilla*), and onion (*Allium cepa*), respectively. Surprisingly, the same oils repelled flies (*Musca domestica, Stomoxys calcitrans, Haematobia irritans,* and *Hippobosca equine*) infecting buffaloes for almost 6 days posttreatment. No adverse effects were noted on either animals or pour-on operators after exposure to the applied oils (Khater et al., 2009).

#### *<IP_ADDRESS>.2 Metabolites reliable for repellent activity*

Nerio et al. (2010) reviewed the repellent activity of essential oils which contributed to some metabolites, such as monoterpenes (α-pinene, cineole, eugenol, limonene, terpinolene, citronellol, citronellal, camphor, and thymol) against Mosquitoes (Yang et al., 2004) sesquiterpenes, β-caryophyllene, repellent against *A. aegypti*; phytol, a linear diterpene alcohol, against *Anopheles gambiae*; and phenylethyl alcohol, β-citronellol, cinnamyl alcohol, geraniol, and α-pinene, isolated from the essential oil of *Dianthus caryophyllum*, aginst ticks *(Ixodes ricinus).* In addition, cineole, geraniol and piperidine found in bay leaves (*Laurus nobilis*, Lauraceae) possess repellent properties towards cockroaches. Repellents may have an increasingly important role in eliminating insects from certain environments and essential oils could play a major role in new repellent technology. Valuable review on the repellent activity of essential oils are those of Tripathi et al. (2009), Isman (2010), Kumar et al. (2010), Nerio et al. (2010), Dubey (2011), and Maia & Moore (2011).

#### **<IP_ADDRESS> Fumigant**

22 Insecticides – Advances in Integrated Pest Management

ingested or skin absorbed by insects. The rapid action against some pests is indicative of a neurotoxic mode of action, and there is evidence for interference with the neuromodulator

Several essential oil compounds have been demonstrated to act on octopaminergic system of insects. Octopamine is a neurotransmitter, neurohormone, and circulating neurohormone – neuromodulator (Hollingworth, et al., 1984) and its disruption results in total break down of nervous system in insects. The lack of octopamine receptors in vertebrates likely accounts for the profound mammalian selectivity of essential oils as insecticides. Eugenol mimicked octopamine in increasing intracellular calcium levels in cloned cells from the brain of *Periplaneta americana* and *Drosophila melanogaster* (Enan, 2005). Consequently, octopaminergic system of insects represents a biorational target for insect control. Plant volatile oils have long been known to affect the behavioural responses of pests, with the monoterpenoid components appearing most useful as insecticides or antifeedants (Palevitch & Craker, 1994). LMW terpenoids may be too lipophilic to be soluble in the haemolymph after crossing the cuticle, and proposed a route of entry through the tracheae (Veal, 1996). Most insecticides bind to receptor proteins in the insect and, in doing so; they interrupt normal neurotransmission, which lead to paralysis and subsequently death. Recent evidence suggests that low-molecular-weight (LMW) terpenoids may also bind to target sites on receptors that modulate nervous activity. Ionotropic, γ-aminobutyric acid, GABA receptors, the targets of organochlorine insecticides lindane and dieldrin, are modulated by LMW terpenoids with vastly different structures (Priestley et al., 2006). Valuable appraisals about the mode of action are those of Price & Berry (2006), Isman (2006, 2010); Tripathi et al. (2009); and Dubey (2011). Some essential oils have larvicidal effect and the capacity to delayed development and suppress adults emergences and induce abnormalities during development of insects of medical and veterinary importance, Fig (1-5) (Khater, 2003; Shalaby & Khater, 2005; Khater & Shalaby, 2008; Khater & Khater

Repellents are substances that provide a vapor barrier deterring the arthropod from coming into contact with the surface or flying to, landing on or biting human or animal skin. The use of insect repellent compounds dates back to ancient times as plant oils, smokes, tars, etc. were used to displace or kill insects. The use of repellents by travelers may reduce infection with local diseases in temperate areas. DEET (N,N-diethyl-m-toluamide) is a broad spectrum repellent and the most effective and persistent on skin. Unfortunately, it may cause environmental and human health risks (Pitasawat et al., 2003). Therefore, there has

Some plant-based repellents are comparable to, or even better than synthetics; however, essential oil repellents tend to being short-lived in their effectiveness due to their volatility. Nerio (2010) review some splendid ideas for improvement of repellency of essential oils. Repellency assays with essential oils were done for Diptera species, especially mosquitoes and to a lesser extent to coleopteran insects related to losses in stored food. Plants with strong smell, such as French marigold and coriander act as repellents and can protect the corps nearby. Several essential oil- producing plants have been widely studies, such as *Cymbopogon* spp., *Eucalyptus* spp., *Ocimum* spp., the osage

been an increase in search efforts for natural and eco-friendly repellents.

octopamine (Enan, 2005) or GABA-gated chloride channels (Priestley et al., 2003).

(2009); Khater et al., 2009, 2011).

*<IP_ADDRESS>.1 Plant- based repellents*

**<IP_ADDRESS> Repellent effect**

Today, The used fumigants, for instance, phosphine, methyl bromide, and DDVP (2,2 dichlorovinyl dimethyl phosphate) do have adverse effets. Phosphine is the major cause of suicidal deaths in India. Methyl bromide has ozone-depleting potential (UNEP, 2000) and DDVP has a possible human carcinogen potential (Lu, 1995). Thus, there is an urgent need for development of safe alternative that have the potential to replace the toxic fumigants against pests attacking grains, dry stored food, and other agricultural products. The active principles are monoterpenes, sesquiterpenes and their biogenically related phenols. In addition to direct toxicity to insects, many of these substances are eterrents or repellents. Essential oils of *Artemisia species, Anethum sowa, Curcuma long,* and *Lippia alba*. Clove, rosemary, thyme, eucalyptus and various mint species have demonstrated contact and fumigant toxicity to a wide spectrum of insects, including human head lice (Toloza et al., 2008). Isolates like d-limonene, carvones and 1,8-cineole have been well documented as fumigants. The exact mode of action of these oils as fumigant is unknown, but the oils mainly act in the vapour phase via respiratory system. Physical properties of essential oils such as high boiling point, high molecular weight and low vapor pressure are barriers for application in large scale fumigation. For more details about fumigants, see Tripathi et al., (2009), Isman (2010), and Dubey (2011).

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 25

mechanisms of development, resulting in insects dying before they reach adulthood. IGRs are classified into two general categories based on mode of action: chitin synthesis inhibitors

Chitin synthesis inhibitors (CSIs) affect the ability of insects to produce new exoskeletons when molting. They act on the larval stages by inhibiting or blocking the synthesis of chitin which represent 30-60% of the insect exoskeleton structure. They also increase egg mortality. CSIs include conventional benzoylureas, triazine/pyrimidine derivatives, and buprofezin.

Typical effects benzoylureas or benzoylphenylurea (BPUs) on developing larvae are the rupture of malformed cuticle or death by starvation. BPUs act as ovicides, reducing the egg laying rate or hindering the hatching process by inhibiting embryonic development or failure of hatchability. Commertial porducts of BPUs include diflubenzuron (Dimilin®, Adept®, Micromite®); triflumuron (Alsystin®); teflubenzuron (Nomolt®, Dart®), hexaflumuron (Trueno®, Consult®); chlorfluazuron (Atabron®); flufenoxuron (Cascade®); and flucycloxuron (Andalin®). Among the newer benzoylureas only hexaflumuron (1993) and novaluron (2001) have been registered by EPA. Studies with diflubenzuron, the most investigated BPU, revealed that it alters cuticle composition, especially inhibition of chitin, resulting in abnormal endocuticluar deposition that affects cuticular elasticity and firmness, and cause abortive molting. Diflubenzuron (Dimilin® El –Delta Company, Egypt) is highly effective in controlling mosquitoes, *Culex pipiens* than house flies, *Musca domestica*. LC 50 values were 1.26 and 1000 ppm, respectively. All treated late 3rd and early 4th larvae of *C*. *pipiens* (concetnraions: 0.04 - 40 ppm) were eventually died as Dimilin® prolonged the larval durations (11.9 days vs. 4 days in the control group) and increased larval abnormalities (46.7%). Such abnormalities were larvae with transparent cuticle, splitting of cuticle, and pharate pupae (Fig. 4). It induces pupal abnormalities as well (Fig. 5). Treatment of of *M. domestica* with the same product (at 1ppm) induced larval and pupal malformations reached 23.3 and 56.5%, respectively, and reduce adult emergence (66.7%). Abnormalities of *M. domestica* include small, shrunken, macerated larvae and larvae with week cuticle as well as

and substances interfering with the action of insect hormones.

distorted puparia and failure of adult eclosion (Khater, 2003) (Fig. 1-3).

resistance has yet been detected (Pinto & do Prado, 2001).

Cyromazine (Larvadex ®, Trigard®), a triazine, is a potent CSI and it is selective toward dipterous species and fed to poultry or sprayed to control flies on animals, in manure of broiler and egg producing operations. It controls blowfly infesting sheep and persist for up to 13 weeks (O'Brien & Fahey, 1991) after a single pour-on application, or longer if applied by dip or shower. Moreover, it is used as a leafminers spray in vegetable crops and ornamentals. Cyromazine may inhibit growth or expansion of the body wall (or both) sufficiently to prevent normal internal growth, producing the observed symptoms and leading to abnormal development. The presence of three resistant house fly populations to cyromazine in Brazilian poultry farms strongly suggests that the operational aspects of larvicide use are important for the development of resistance. Cyromazine is applied as a feed-through, both in Brazil and in the USA, where resistance has already been documented. However, in Denmark, where it was approved only as a topical manure spray, no case of

**<IP_ADDRESS> Triazine/pyrimidine derivatives**

*<IP_ADDRESS>.1 Cyromazine*

**2.2.1 Chitin synthesis inhibitors**

**<IP_ADDRESS> Benzoylphenylurea**

#### **<IP_ADDRESS> Commercialization**

Although essential oils are effective when freshly applied, their protective effects usually dissipate relatively quickly. In their review, Nerio et al. (2010) discussed methods to access repellency effects, the synergistic phenomena of such oils and some novel ideas to increase the repellent efficiency. Some fixative materials such as liquid paraffin, vanillin, salicyluric acid, mustard, and coconut oils have been used. Formulations based on creams, polymer mixtures, or microcapsules for controlled release, resulted in an increase of repellency duration. Still, essential oils can be incorporated with polymers into sheets and attractant adhesive films with essential oils were prepared to control insects in agriculture and horticulture. Novel ideas are needed to be explored for better commercialization of essential oil- based pesticides. Several essential oil constituents are already in use as an alternative to conventional insecticides, such as Green Ban® (containing oils of citronella, cajuput, lavender, safrole free sassafrass, peppermint, and bergaptene free bergamot oil); Buzz Away® (containing oils of citronella, cedarwood, eucalyptus, and lemongrass); ValeroTM, a miticide/fungicide for use in grapes, berry crops, citrus, and nuts; and CinnamiteTM, an aphidicide /miticide/fungicide for glasshouse and horticultural crops. The last two products are based on cinnamon oil, with cinnamaldehyde (30% in EC formulations) as the active ingredient. In addition, d-limonene is an active ingredient of commercially available flea shampoos, plus pulegone and citronellal are used as mosquito repellents.

#### **<IP_ADDRESS> Safety of essential oils**

Currently, the US Environmental Protection Agency (US EPA) has registered citronella, lemon, and eucalyptus oils as insect repellent ingredients for application on the skin.  The most attractive aspect of using essential oils and/or their constituents for pest control is their favorable mammalian toxicity because many essential oils and their constituents are commonly used as culinary herbs and spices. Many of the commercial products including essential oils are included on the GRAS list fully approved by FDA and EPA in USA for food and brevarage consumption (EPA, 1993). Some of the puried terpenoid constituents of essential oils are moderately toxic to mammals, but, with few exceptions, the oils themselves or products based on oils are mostly nontoxic to mammals, birds, and sh. Although natural enemies are susceptible via direct contact, predators and parasitoids reinvading a treated crop one or more days after treatment are unlikely to be poisoned by residue contact as often occurs with conventional insecticides. Owing to their volatility, the oils and their constituents are environmentally nonpersistent, with outdoor half lives of\24 h on surfaces, in soil and in water (Isman el al., 2011). There is no harvest restrictions or worker re-entry restrictions for treated crops; they are compatibile with biological control agents and indigenous natural enemies of pests, and they bring about reduce risks to honeybees and other foraging pollinators. For additional information about safety of essential oils, see Isman (2006, 2010), Tripathi et al. (2009), Nerio et al. (2010), and Regnault-Roger (2011). Because many conventional pesticide products fall into disfavour with the public, botanical-based pesticides should become an increasingly popular choice for pest control.

#### **2.2 Insect growth regulators**

Insect growth regulators (IGRs) are chemical compounds that alter growth and development in insects. They don't directly kill insects, but interfere with the normal mechanisms of development, resulting in insects dying before they reach adulthood. IGRs are classified into two general categories based on mode of action: chitin synthesis inhibitors and substances interfering with the action of insect hormones.

#### **2.2.1 Chitin synthesis inhibitors**

Chitin synthesis inhibitors (CSIs) affect the ability of insects to produce new exoskeletons when molting. They act on the larval stages by inhibiting or blocking the synthesis of chitin which represent 30-60% of the insect exoskeleton structure. They also increase egg mortality. CSIs include conventional benzoylureas, triazine/pyrimidine derivatives, and buprofezin.

#### **<IP_ADDRESS> Benzoylphenylurea**

24 Insecticides – Advances in Integrated Pest Management

Although essential oils are effective when freshly applied, their protective effects usually dissipate relatively quickly. In their review, Nerio et al. (2010) discussed methods to access repellency effects, the synergistic phenomena of such oils and some novel ideas to increase the repellent efficiency. Some fixative materials such as liquid paraffin, vanillin, salicyluric acid, mustard, and coconut oils have been used. Formulations based on creams, polymer mixtures, or microcapsules for controlled release, resulted in an increase of repellency duration. Still, essential oils can be incorporated with polymers into sheets and attractant adhesive films with essential oils were prepared to control insects in agriculture and horticulture. Novel ideas are needed to be explored for better commercialization of essential oil- based pesticides. Several essential oil constituents are already in use as an alternative to conventional insecticides, such as Green Ban® (containing oils of citronella, cajuput, lavender, safrole free sassafrass, peppermint, and bergaptene free bergamot oil); Buzz Away® (containing oils of citronella, cedarwood, eucalyptus, and lemongrass); ValeroTM, a miticide/fungicide for use in grapes, berry crops, citrus, and nuts; and CinnamiteTM, an aphidicide /miticide/fungicide for glasshouse and horticultural crops. The last two products are based on cinnamon oil, with cinnamaldehyde (30% in EC formulations) as the active ingredient. In addition, d-limonene is an active ingredient of commercially available flea shampoos, plus pulegone and citronellal

Currently, the US Environmental Protection Agency (US EPA) has registered citronella, lemon, and eucalyptus oils as insect repellent ingredients for application on the skin.  The most attractive aspect of using essential oils and/or their constituents for pest control is their favorable mammalian toxicity because many essential oils and their constituents are commonly used as culinary herbs and spices. Many of the commercial products including essential oils are included on the GRAS list fully approved by FDA and EPA in USA for food and brevarage consumption (EPA, 1993). Some of the puried terpenoid constituents of essential oils are moderately toxic to mammals, but, with few exceptions, the oils themselves or products based on oils are mostly nontoxic to mammals, birds, and sh. Although natural enemies are susceptible via direct contact, predators and parasitoids reinvading a treated crop one or more days after treatment are unlikely to be poisoned by residue contact as often occurs with conventional insecticides. Owing to their volatility, the oils and their constituents are environmentally nonpersistent, with outdoor half lives of\24 h on surfaces, in soil and in water (Isman el al., 2011). There is no harvest restrictions or worker re-entry restrictions for treated crops; they are compatibile with biological control agents and indigenous natural enemies of pests, and they bring about reduce risks to honeybees and other foraging pollinators. For additional information about safety of essential oils, see Isman (2006, 2010), Tripathi et al. (2009), Nerio et al. (2010), and Regnault-Roger (2011). Because many conventional pesticide products fall into disfavour with the public, botanical-based pesticides should become an increasingly

Insect growth regulators (IGRs) are chemical compounds that alter growth and development in insects.  BPUs act as ovicides, reducing the egg laying rate or hindering the hatching process by inhibiting embryonic development or failure of hatchability. Commertial porducts of BPUs include diflubenzuron (Dimilin®, Adept®, Micromite®); triflumuron (Alsystin®); teflubenzuron (Nomolt®, Dart®), hexaflumuron (Trueno®, Consult®); chlorfluazuron (Atabron®); flufenoxuron (Cascade®); and flucycloxuron (Andalin®). Among the newer benzoylureas only hexaflumuron (1993) and novaluron (2001) have been registered by EPA. Studies with diflubenzuron, the most investigated BPU, revealed that it alters cuticle composition, especially inhibition of chitin, resulting in abnormal endocuticluar deposition that affects cuticular elasticity and firmness, and cause abortive molting. Diflubenzuron (Dimilin® El –Delta Company, Egypt) is highly effective in controlling mosquitoes, *Culex pipiens* than house flies, *Musca domestica*. LC 50 values were 1.26 and 1000 ppm, respectively. All treated late 3rd and early 4th larvae of *C*. *pipiens* (concetnraions: 0.04 - 40 ppm) were eventually died as Dimilin® prolonged the larval durations (11.9 days vs. 4 days in the control group) and increased larval abnormalities (46.7%). Such abnormalities were larvae with transparent cuticle, splitting of cuticle, and pharate pupae (Fig. 4). It induces pupal abnormalities as well (Fig. 5). Treatment of of *M. domestica* with the same product (at 1ppm) induced larval and pupal malformations reached 23.3 and 56.5%, respectively, and reduce adult emergence (66.7%). Abnormalities of *M. domestica* include small, shrunken, macerated larvae and larvae with week cuticle as well as distorted puparia and failure of adult eclosion (Khater, 2003) (Fig. 1-3).

#### **<IP_ADDRESS> Triazine/pyrimidine derivatives**

#### *<IP_ADDRESS>.1 Cyromazine*

Cyromazine (Larvadex ®, Trigard®), a triazine, is a potent CSI and it is selective toward dipterous species and fed to poultry or sprayed to control flies on animals, in manure of broiler and egg producing operations. It controls blowfly infesting sheep and persist for up to 13 weeks (O'Brien & Fahey, 1991) after a single pour-on application, or longer if applied by dip or shower. Moreover, it is used as a leafminers spray in vegetable crops and ornamentals. Cyromazine may inhibit growth or expansion of the body wall (or both) sufficiently to prevent normal internal growth, producing the observed symptoms and leading to abnormal development. The presence of three resistant house fly populations to cyromazine in Brazilian poultry farms strongly suggests that the operational aspects of larvicide use are important for the development of resistance. Cyromazine is applied as a feed-through, both in Brazil and in the USA, where resistance has already been documented. However, in Denmark, where it was approved only as a topical manure spray, no case of resistance has yet been detected (Pinto & do Prado, 2001).

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 27

Fig. 2. Pupal abnormalities of house flies: I. Larval pupal- intermediates: A. The anterior and posterior ends as pupae (left and right arrows), while the rest of the body as larva (middle arrow). B. Fungal growth anteriorly (left arrow) and cracked pupal case at the middle (lower arrows). II. Pupae: A. Normal pupa. B. Small pupa with visible cephalopharyngeal skeleton. C. Larviform pupa, pigmented with small dark spots at the intersegmental regions. D. Cshaped pupa with anterior constriction and small patches of black pigments (arrow heads). E. Distorted puparia. F. Pupa with a groove (arrow head). G. Blackish posterior end (arrow head). H. Transparent puparia. I. Hyphal growth appears on a small puparia. Adapted form

Khater (2003).

Fig. 1. Morphological malformations of larvae of house flies. I. A. Normal larva. B-E. Malformed larvae, treated with essential oils and insect growth regulators showing signs of pigmentation. C. Macerated larva with week transparent cuticle. II. Larvae infected with fungi. A. Red Pin -point pigments all over the larval body with apparent fungal growth (arrow heads). B. Larva with diffuse blackish pigmentation. C. Larva with an ulcer in the middle. D. Ulcerated and macerated larva with white nodules and fungal growth.

#### *<IP_ADDRESS>.2 Dicyclanil*

Dicyclanil (ZR ®, ComWin ®), a pyrimidine derivative, is highly active against dipteran larvae and available as a pour-on formulation for blowfly control in sheep in Australia and New Zealand providing up to 20 weeks' protection (Bowen et al., 1999). On the whole,

Fig. 1. Morphological malformations of larvae of house flies. I. A. Normal larva. B-E. Malformed larvae, treated with essential oils and insect growth regulators showing signs of pigmentation. C. Macerated larva with week transparent cuticle. II. Larvae infected with fungi. A. Red Pin -point pigments all over the larval body with apparent fungal growth (arrow heads). B. Larva with diffuse blackish pigmentation. C. Larva with an ulcer in the middle. D. Ulcerated and macerated larva with white nodules and fungal growth.

Dicyclanil (ZR ®, ComWin ®), a pyrimidine derivative, is highly active against dipteran larvae and available as a pour-on formulation for blowfly control in sheep in Australia and New Zealand providing up to 20 weeks' protection (Bowen et al., 1999). On the whole,

*<IP_ADDRESS>.2 Dicyclanil*

Fig. 2. Pupal abnormalities of house flies: I. Larval pupal- intermediates: A. The anterior and posterior ends as pupae (left and right arrows), while the rest of the body as larva (middle arrow). B. Fungal growth anteriorly (left arrow) and cracked pupal case at the middle (lower arrows). II. Pupae: A. Normal pupa. B. Small pupa with visible cephalopharyngeal skeleton. C. Larviform pupa, pigmented with small dark spots at the intersegmental regions. D. Cshaped pupa with anterior constriction and small patches of black pigments (arrow heads). E. Distorted puparia. F. Pupa with a groove (arrow head). G. Blackish posterior end (arrow head). H. Transparent puparia. I. Hyphal growth appears on a small puparia. Adapted form Khater (2003).

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 29

Fig. 4. Morphological abnormalities of mosquito larvae: A. Normal larva, B. Pigmented larva, C-F. Larvae with deformed cuticles, G. Larva with an opaque swelling on the thorax and black coloration at the posterior end, H. Pharate pupa (prepupa), I, J. larvae show

symptoms of mycosis. Adapted from Khater (2003).

Fig. 3. Morphological abnormalities of house flies. I. (A-F) Failure of adult eclosion. II. Adults (A. Normal adult, B. Crumpled adult with poorly developed wing and legs, C. Small adult, D. Elongated adult with deformed wing, abdomen, and legs. Adapted from Khater (2003).

Fig. 3. Morphological abnormalities of house flies. I. (A-F) Failure of adult eclosion. II. Adults (A. Normal adult, B. Crumpled adult with poorly developed wing and legs, C. Small adult, D. Elongated adult with deformed wing, abdomen, and legs. Adapted from Khater (2003).

Fig. 4. Morphological abnormalities of mosquito larvae: A. Normal larva, B. Pigmented larva, C-F. Larvae with deformed cuticles, G. Larva with an opaque swelling on the thorax and black coloration at the posterior end, H. Pharate pupa (prepupa), I, J. larvae show symptoms of mycosis. Adapted from Khater (2003).

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 31

larvae become moribund, slip their head capsule, and the hind gut may be extruded in extreme cases. Synthesis of new cuticle begins and apolysis of the new cuticle form the old one takes place. The new cuticle is not sclerotized or tanned. Consequently, the food intake by larva is prevented as the mouthparts become soft and mushy. Larval death is due to incomplete molting, starvation, and desiccation due to hemorrhage. Both Tebufenozide and methoxyfenozide, selectively toxic to Lepidopteran pests, have been classified by US EPA as reduced risk pesticides. It worth to mention that azadirachtin in neem tree acts as ecdysone blocker and disrupt insect growth, see botanical insecticides above. In general, BSHs have insect selectivity, reduced risk ecotoxicology, and mammalian profile as they have new

The juvenile hormone analogues (JHA) mimic the activity of naturally occurring JHs and prevent metamorphosis to the adult stage. JH is one of the most pleiotropic hormones known and functions in various aspects such as embryogenesis, molting and metamorphosis, reproduction, diapause, communication, migration/ dispersal, caste differentiation, pigmentation, silk production, and phase transformation. The major function of JH is the maintenance of larval status or the so- called juvenilizing effect, See Tunaz (2004). JHAs are highly effective at the beginning stage of metamorphosis and embryogenesis, for instance, freshly ecdysed last larval instars, freshly ecdysed pupal instars, and deposited eggs. Thus embryogenesis is disrupted when young eggs are treated with JHAs. Application to early last instar larvae would result in the development of supernumerary instars, whereas treatment at the later stage would result in abnormal pupation and development of larval-pupal mosaics or intermediates (Khater, 2003). Treatment of of *M. domestica* and *C. pipiens* with pyriproxyfen (Sumilarv ® Somitomo Co.) interferes with normal metamorphosis and results in various developmental abnormalities including larval pupal intermediates that do not survive (Fig. 1-5); Lower concentrations significantly prolonged larval and papal durations, 10.5 and 11 days (at conc. 0.1 ppm) and 9.9 and 15.1 days (at conc. 4 ppm), respectively, than those of the control (3, 5.7 days and 3.9, 1.1 days, respectively) (Khater, 2003). Methoprene (Altosid ®, Apex ®, Pharorid ®, Precor ®), the first compound introduced into the market, is a terpenoid compound with very low mammalian toxicity and is regularly used for flea control. It is sensitive to light. Thus, It has been used extensively and successfully in indoor environments and on pets in the form of collars, shampoos, sprays, and dips and also as a feed through larvicide for hornfly, *Haematobia,* control on cattle (Graf, 1993). Other IGRs available for use against household and agricultural pests are pyriproxyfen and fenoxycarb. Fenoxycarb (Logic®, Award®, Comply®, Torus®) is a carbamate stomach insecticide that has also JH-type effects when contacted or ingested by various arthropod pests, e.g., ants, roaches, ticks, chiggers and many others. Some JHAs are of plant origin, such as Juvenoids, isolated form plants, the "paper factor" form the balsam fir (*Abies balsamea*) and Juvocimenes from the sweet basil plant (*Ocimum basilicum*) (Bowers & Nishida, 1980). Morphogenetic control is effective for controlling insects that are pests as adults, like most insects of medical and veterinary importance, ex. mosquitoes, nuisance flies, and fire ants. They are not effective against lepidopteran agricultural pests because the larval stage is responsible for plant destruction. Many JH analogs (or mimics) (JHAs) are attractive candidates for pest control because of the ease of their synthesization and their pest selectivity than those of other peptide and steroid

mode of action, therefore, they are ideally integrated in IPM programs.

**<IP_ADDRESS> Juvenile hormone analogues**

hormones (Eto, 1990).

Fig. 5. Pupal abnormalities of mosquitoes: A. Normal pupa. B. Albino pupa. C. Elephantoid pupa, with enlarged cephalic region and extended abdomen. D. Black cephalothorax (lower arrows) and extended abdomen with transparent posterior end (upper arrow) and no anal gills. E. Pupa with apparent fungal growth (arrow head). Adapted form Khater (2003).

most CSI compouns are very potent against a variety of different pests, especially lepidopterous insects and whiteflies. They are harmless or exert little adverse effect on bees, predators, or parasitoids (Tomlin, 2000) which renders them acceptable for inclusion in IPM programs.

#### **2.2.2 Substances interfering with the action of insect hormones**

Growth and development of insects are regulated by hormones: prothoracicotrophic hormones (PTTH) (brain hormone), ecdysteroids, and juvenile hormones (JH). PTTH controls the secretion of the molting hormone (ecdysone) from the prothoracic gland. Ecdysone is responsible for cellular programming and, together with JH, initiating for the molting process. High secrection of JH from the corpora allata allows the epidermis to be programmed for a larval molt, or else it is programmed for metamorphosis. JH is virtually absent in the pupae, but is present in adults to serve some functions in reproduction. JH suppresses pupation and induces vitellogenesis during the reproductive stage of the insect (Eto, 1990).

#### **<IP_ADDRESS> Ecdysteroid agonist**

Bisacylhydrazine (BSH), a newer class of IGRs, is ecdysone agonists or disruptors with molting hormone activity. BSHs include chromafenozide (Virtu ®), Tebufenozide (Mimic ®, Confirm ®), Halofenozide (Mach-2 ®), and Methoxyfenozide (Intrepid ®). They have a greater metabolic stability than the insect steroid molting hormone 20-hydroxyecdysone (20E) *In vivo* (Retnakaran et al., 1995). They are toxic after ingestion or exposure to higher doses of topical application. Ingestion of BSH creates hyperecdysonism in susceptible insect, including molting events. The effect starts with feeding inhibition within 3-14 h (Retnakaran et al., 1997), which is very important for preventing further crop damage. In the mean while, larvae become moribund, slip their head capsule, and the hind gut may be extruded in extreme cases. Synthesis of new cuticle begins and apolysis of the new cuticle form the old one takes place. The new cuticle is not sclerotized or tanned. Consequently, the food intake by larva is prevented as the mouthparts become soft and mushy. Larval death is due to incomplete molting, starvation, and desiccation due to hemorrhage. Both Tebufenozide and methoxyfenozide, selectively toxic to Lepidopteran pests, have been classified by US EPA as reduced risk pesticides. It worth to mention that azadirachtin in neem tree acts as ecdysone blocker and disrupt insect growth, see botanical insecticides above. In general, BSHs have insect selectivity, reduced risk ecotoxicology, and mammalian profile as they have new mode of action, therefore, they are ideally integrated in IPM programs.

#### **<IP_ADDRESS> Juvenile hormone analogues**

30 Insecticides – Advances in Integrated Pest Management

Fig. 5. Pupal abnormalities of mosquitoes: A. Normal pupa. B. Albino pupa. C. Elephantoid pupa, with enlarged cephalic region and extended abdomen. D. Black cephalothorax (lower arrows) and extended abdomen with transparent posterior end (upper arrow) and no anal gills. E. Pupa with apparent fungal growth (arrow head). Adapted form Khater (2003).

most CSI compouns are very potent against a variety of different pests, especially lepidopterous insects and whiteflies. They are harmless or exert little adverse effect on bees, predators, or parasitoids (Tomlin, 2000) which renders them acceptable for inclusion in IPM

Growth and development of insects are regulated by hormones: prothoracicotrophic hormones (PTTH) (brain hormone), ecdysteroids, and juvenile hormones (JH). PTTH controls the secretion of the molting hormone (ecdysone) from the prothoracic gland. Ecdysone is responsible for cellular programming and, together with JH, initiating for the molting process. High secrection of JH from the corpora allata allows the epidermis to be programmed for a larval molt, or else it is programmed for metamorphosis. JH is virtually absent in the pupae, but is present in adults to serve some functions in reproduction. JH suppresses pupation and induces vitellogenesis during the reproductive stage of the insect

Bisacylhydrazine (BSH), a newer class of IGRs, is ecdysone agonists or disruptors with molting hormone activity. BSHs include chromafenozide (Virtu ®), Tebufenozide (Mimic ®, Confirm ®), Halofenozide (Mach-2 ®), and Methoxyfenozide (Intrepid ®). They have a greater metabolic stability than the insect steroid molting hormone 20-hydroxyecdysone (20E) *In vivo* (Retnakaran et al., 1995). They are toxic after ingestion or exposure to higher doses of topical application. Ingestion of BSH creates hyperecdysonism in susceptible insect, including molting events. The effect starts with feeding inhibition within 3-14 h (Retnakaran et al., 1997), which is very important for preventing further crop damage. In the mean while,

**2.2.2 Substances interfering with the action of insect hormones**

programs.

(Eto, 1990).

**<IP_ADDRESS> Ecdysteroid agonist**

The juvenile hormone analogues (JHA) mimic the activity of naturally occurring JHs and prevent metamorphosis to the adult stage. JH is one of the most pleiotropic hormones known and functions in various aspects such as embryogenesis, molting and metamorphosis, reproduction, diapause, communication, migration/ dispersal, caste differentiation, pigmentation, silk production, and phase transformation. The major function of JH is the maintenance of larval status or the so- called juvenilizing effect, See Tunaz (2004). JHAs are highly effective at the beginning stage of metamorphosis and embryogenesis, for instance, freshly ecdysed last larval instars, freshly ecdysed pupal instars, and deposited eggs. Thus embryogenesis is disrupted when young eggs are treated with JHAs. Application to early last instar larvae would result in the development of supernumerary instars, whereas treatment at the later stage would result in abnormal pupation and development of larval-pupal mosaics or intermediates (Khater, 2003). Treatment of of *M. domestica* and *C. pipiens* with pyriproxyfen (Sumilarv ® Somitomo Co.) interferes with normal metamorphosis and results in various developmental abnormalities including larval pupal intermediates that do not survive (Fig. 1-5); Lower concentrations significantly prolonged larval and papal durations, 10.5 and 11 days (at conc. 0.1 ppm) and 9.9 and 15.1 days (at conc. 4 ppm), respectively, than those of the control (3, 5.7 days and 3.9, 1.1 days, respectively) (Khater, 2003). Methoprene (Altosid ®, Apex ®, Pharorid ®, Precor ®), the first compound introduced into the market, is a terpenoid compound with very low mammalian toxicity and is regularly used for flea control. It is sensitive to light. Thus, It has been used extensively and successfully in indoor environments and on pets in the form of collars, shampoos, sprays, and dips and also as a feed through larvicide for hornfly, *Haematobia,* control on cattle (Graf, 1993). Other IGRs available for use against household and agricultural pests are pyriproxyfen and fenoxycarb. Fenoxycarb (Logic®, Award®, Comply®, Torus®) is a carbamate stomach insecticide that has also JH-type effects when contacted or ingested by various arthropod pests, e.g., ants, roaches, ticks, chiggers and many others. Some JHAs are of plant origin, such as Juvenoids, isolated form plants, the "paper factor" form the balsam fir (*Abies balsamea*) and Juvocimenes from the sweet basil plant (*Ocimum basilicum*) (Bowers & Nishida, 1980). Morphogenetic control is effective for controlling insects that are pests as adults, like most insects of medical and veterinary importance, ex. mosquitoes, nuisance flies, and fire ants. They are not effective against lepidopteran agricultural pests because the larval stage is responsible for plant destruction. Many JH analogs (or mimics) (JHAs) are attractive candidates for pest control because of the ease of their synthesization and their pest selectivity than those of other peptide and steroid hormones (Eto, 1990).

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 33

pheromone is dispersed into the field and the false odor plumes attract males away from females that are waiting to mate, thus reduce the population density of the pests. In contrast to the previous benefits, the high degree of selectivity may be a barrier to large-scale implementation where secondary pests become a problem as insecticides are eliminated (Walker & Welter, 2001). Other obstacles include the following: the lack of an identified pheromone for some pest species, high development and production costs, requirements for specialized application techniques or equipment, and the need to supplement the pheromone program in high pest-pressure situations (Welter et al., 2005). Large-scale implementation projects have yielded significant reductions in pesticide use while maintaining acceptably low crop-damage levels. Pheromones manipulate the behavior of insect pests. With these non-toxic and biodegradable chemicals, insects can be lured into traps or foiled into wasting energy that they normally need for locating food and mates. Pheromones are species-specific chemicals that affect insect behavior and they are not toxic to insects or other non- target organisms, creating opportunities for the biological control of other pest species. They are attractive in extremely low doses and used to bait traps or confuse a mating population of insects. Other advantages include negligible health risks, no accumulation in wildlife or groundwater, limited impacts on other management practices; manage insecticide resistance, and a more rapid registration process. Pheromones can play an important role in integrated pest management for medical or veterinary, structural, agricultural, landscape, and forest pest problems. Pheromone programs are most effective with low to moderate population densities. Because of some difficulties with high populations of pests, these programs should not be viewed as stand-alone strategies but rather as one tactic within a suite of integrated pest management options (Welter et al., 2005). For more information about pheromones, see; Cooping & Menn (2000), Witzgall

(2001), Leal et al. (2003), Bray et al. (2009), and van Emden & Service (2011).

Development of new, ecologically safe technologies to control insect pest populations is of great importance. Photosensitizers are activated by illumination with sunlight or artificial light sources, have been shown to be accumulated in significant amounts by a variety of insects when they are administered in association with suitable baits. The subsequent exposure of such insects to UV/visible light leads induces lethal photochemical reactions and death. The most famous photosensitizers are xanthenes (e.g. phloxin B) and porphyrins (e.g. haematoporphyrin) which appear to be endowed with the highest photoinsecticidal activity. In particular, porphyrins absorb essentially all the UV/visible light wavelengths in the emission spectrum of the sun; hence they are active at very low doses. Photoactive compounds usually used for photosensitization might be effective as pesticide agents, with low impact on the environment, being non-toxic and not mutagenic, see Ben Amor & Jori (2000), Mangan & Moreno (2001), Ragaei & Khater (2004), Lukšienè etal.(2007), and Awad

Several inorganic substances are well know with their insecticidal effect, such as potassium silicate, diatomaceous earth (DE, diatomite or kieselgur), mineral oils, sulfur, boric acid, sodium borate, silica gels, kaolin clay, and soap spray. For more details about inorganic and their uses, see Ware & Whitacare (2004), and Collins (2006). It worth to mention that peracetic

**2.4 Photosenitizers**

et al. (2008) for more fine points.

**2.5 Inorganics and organic acid**

#### **<IP_ADDRESS> Antijuvenile hormones**

Antijuvenile hormones, anti- JHs, are very effective pest control agents as they prevent JH production, facilitate JH degradation, or destroy corpus allatum. Intoxicated newly emerged larvae with anti- JH would create miniature pupae, thus abbreviating the distructive part of insect life cylce. Some of these plant-derived substances actually serve to inhibit the development of insects feeding and protecting the host plant. These are referred to broadly as antijuvenile hormones, more accurately, antiallatotropins, or precocenes. Although the mode of action of the precocenes is still unclear, it is known that they depress the level of juvenile hormone below that normally found in immature insects. As anti-JHs is competing with JH in binding to the JH receptors or to the JH carrier proteins, injuring the corpora allata cells, or interfering with JH biosynthesis (Leighton et al., 1981), other JHAs may also function as anti-JHs, such as ETB [ethyl 4-(2- pivaloyloxybutyloxy)-benzoate], which showed JH agonist and antagonist activities in *Manduca sexta* larvae as it compete with JH at the receptor site and become feedback inhibitors of JH biosynthesis (Staal, 1986).

#### **2.2.3 Safety**

Several IGRs are registered by the EPA, such as methoprene (Altosid ®) which is used as a grain protectant, as mosquito growth regulator; Precor ® for indoor control of dog and cat fleas; Hydroprene (Gentrol ® , Mator ®) for use against cockroaches, and stored grain pests; and kinoprene (Enstar II ® ), which is effective against aphids, whiteflies, mealybugs, and scales (both soft and armored) on ornamental plants and vegetable seed crops grown in greenhouses and shadehouses. Although some insects acquired resistance against some IGR- based products, IGRs are typically "safer" to use around humans, pets, and natural enemies than conventional insecticides and acaricides. IGRs are effective when applied in very minute quantities and generally have few or no effects on humans and wildlife. They are, however, nonspecific, since they affect not only the target species, but other arthropods as well. For more information, see Taylor (2001), Tunaz (2004), Ware & Whitacare (2004), Collins (2006), and Gilbert & Gill (2010).

#### **2.3 Pheromones**

Pheromones are a class of semiochemicals that insects and other animals release to communicate with other individuals of the same species. Such behavioral chemicals, range from small hydrophobic molecules to water-soluble peptides, leave the body of the first organism, pass through the air (or water) and reach the second organism, where they are detected by the receiver. Signalling chemicals play an essential role in arthropod life cycles. They provide the means whereby mates, host and oviposition sites are located and recognized (Mordue Luntz, 2003). Pheromones may signal various information. Longlasting pheromones allow marking of food sources or territorial boundaries. Other signals are very short-lived and provide an immediate message, such as a brief period of reproductive readiness or short-term warning of danger.

#### **2.3.1 Uses of pheromones**

There are five principal uses for sex pheromones: population monitoring, mass trapping of insects, movement studies, detection of exotic pests and, mating disruption. Such disturbance is very important in reducing the population density of pests as synthetic pheromone is dispersed into the field and the false odor plumes attract males away from females that are waiting to mate, thus reduce the population density of the pests. In contrast to the previous benefits, the high degree of selectivity may be a barrier to large-scale implementation where secondary pests become a problem as insecticides are eliminated (Walker & Welter, 2001). Other obstacles include the following: the lack of an identified pheromone for some pest species, high development and production costs, requirements for specialized application techniques or equipment, and the need to supplement the pheromone program in high pest-pressure situations (Welter et al., 2005). Large-scale implementation projects have yielded significant reductions in pesticide use while maintaining acceptably low crop-damage levels. Pheromones manipulate the behavior of insect pests. With these non-toxic and biodegradable chemicals, insects can be lured into traps or foiled into wasting energy that they normally need for locating food and mates. Pheromones are species-specific chemicals that affect insect behavior and they are not toxic to insects or other non- target organisms, creating opportunities for the biological control of other pest species. They are attractive in extremely low doses and used to bait traps or confuse a mating population of insects. Other advantages include negligible health risks, no accumulation in wildlife or groundwater, limited impacts on other management practices; manage insecticide resistance, and a more rapid registration process. Pheromones can play an important role in integrated pest management for medical or veterinary, structural, agricultural, landscape, and forest pest problems. Pheromone programs are most effective with low to moderate population densities. Because of some difficulties with high populations of pests, these programs should not be viewed as stand-alone strategies but rather as one tactic within a suite of integrated pest management options (Welter et al., 2005). For more information about pheromones, see; Cooping & Menn (2000), Witzgall (2001), Leal et al. (2003), Bray et al. (2009), and van Emden & Service (2011).

#### **2.4 Photosenitizers**

32 Insecticides – Advances in Integrated Pest Management

Antijuvenile hormones, anti- JHs, are very effective pest control agents as they prevent JH production, facilitate JH degradation, or destroy corpus allatum. Intoxicated newly emerged larvae with anti- JH would create miniature pupae, thus abbreviating the distructive part of insect life cylce. Some of these plant-derived substances actually serve to inhibit the development of insects feeding and protecting the host plant. These are referred to broadly as antijuvenile hormones, more accurately, antiallatotropins, or precocenes. Although the mode of action of the precocenes is still unclear, it is known that they depress the level of juvenile hormone below that normally found in immature insects. As anti-JHs is competing with JH in binding to the JH receptors or to the JH carrier proteins, injuring the corpora allata cells, or interfering with JH biosynthesis (Leighton et al., 1981), other JHAs may also function as anti-JHs, such as ETB [ethyl 4-(2- pivaloyloxybutyloxy)-benzoate], which showed JH agonist and antagonist activities in *Manduca sexta* larvae as it compete with JH at

the receptor site and become feedback inhibitors of JH biosynthesis (Staal, 1986).

Several IGRs are registered by the EPA, such as methoprene (Altosid ®) which is used as a grain protectant, as mosquito growth regulator; Precor ® for indoor control of dog and cat fleas; Hydroprene (Gentrol ® , Mator ®) for use against cockroaches, and stored grain pests; and kinoprene (Enstar II ® ), which is effective against aphids, whiteflies, mealybugs, and scales (both soft and armored) on ornamental plants and vegetable seed crops grown in greenhouses and shadehouses. Although some insects acquired resistance against some IGR- based products, IGRs are typically "safer" to use around humans, pets, and natural enemies than conventional insecticides and acaricides. IGRs are effective when applied in very minute quantities and generally have few or no effects on humans and wildlife. They are, however, nonspecific, since they affect not only the target species, but other arthropods as well. For more information, see Taylor (2001), Tunaz (2004), Ware & Whitacare (2004),

Pheromones are a class of semiochemicals that insects and other animals release to communicate with other individuals of the same species. Such behavioral chemicals, range from small hydrophobic molecules to water-soluble peptides, leave the body of the first organism, pass through the air (or water) and reach the second organism, where they are detected by the receiver. Signalling chemicals play an essential role in arthropod life cycles. They provide the means whereby mates, host and oviposition sites are located and recognized (Mordue Luntz, 2003). Pheromones may signal various information. Longlasting pheromones allow marking of food sources or territorial boundaries. Other signals are very short-lived and provide an immediate message, such as a brief period of

There are five principal uses for sex pheromones: population monitoring, mass trapping of insects, movement studies, detection of exotic pests and, mating disruption. Such disturbance is very important in reducing the population density of pests as synthetic

**<IP_ADDRESS> Antijuvenile hormones**

Collins (2006), and Gilbert & Gill (2010).

reproductive readiness or short-term warning of danger.

**2.2.3 Safety**

**2.3 Pheromones**

**2.3.1 Uses of pheromones**

Development of new, ecologically safe technologies to control insect pest populations is of great importance. Photosensitizers are activated by illumination with sunlight or artificial light sources, have been shown to be accumulated in significant amounts by a variety of insects when they are administered in association with suitable baits. The subsequent exposure of such insects to UV/visible light leads induces lethal photochemical reactions and death. The most famous photosensitizers are xanthenes (e.g. phloxin B) and porphyrins (e.g. haematoporphyrin) which appear to be endowed with the highest photoinsecticidal activity. In particular, porphyrins absorb essentially all the UV/visible light wavelengths in the emission spectrum of the sun; hence they are active at very low doses. Photoactive compounds usually used for photosensitization might be effective as pesticide agents, with low impact on the environment, being non-toxic and not mutagenic, see Ben Amor & Jori (2000), Mangan & Moreno (2001), Ragaei & Khater (2004), Lukšienè etal.(2007), and Awad et al. (2008) for more fine points.

#### **2.5 Inorganics and organic acid**

Several inorganic substances are well know with their insecticidal effect, such as potassium silicate, diatomaceous earth (DE, diatomite or kieselgur), mineral oils, sulfur, boric acid, sodium borate, silica gels, kaolin clay, and soap spray. For more details about inorganic and their uses, see Ware & Whitacare (2004), and Collins (2006). It worth to mention that peracetic

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 35

Virual infection begins in the insect's digestive system after consumption of plant material with viral particles. The virus spread though out the body but the digestive system is among the last part to be destroyed, so the insects usually continue to feed until they die leading to economic loss. See Lacey & Kaya (2007) and McNiel (2010) for more information about biology and ecology of viruses. The virus manipulates the behavior of infected larvae for its own dissemination. For example, larvae of the cabbage moth, *Mamestra brassicae* infected with NPV moved up to five times more than their healthy counterparts during the middle stages of infection (Vasconcelos et al., 1996b). Adult insects can also disperse viruses via vertical and horizontal transmission. Understanding the biology and ecology of viruses is crucial for optimizing pest control strategies of exotic and genetically modified organisms, see Lacy & Kaya (2007) and Szewczyk et al. (2009, 2011) for more details. UV protectants are very important for stability of the viral product, stilbene fluorescent brighteners (e.g. Phorwite AR ®, Blankophor ®, and others) induce the best results. Plant metabolites as peroxidases generate free radicals (Hoover et al., 1998) inactivate BVs. As a result, addition of free radical scavengers such as mannitol or enzyme superoxide dismutase to BV preparations can reduce such inactivation (Zhou et al., 2004). Tillage buries virus particles in the soil, thus good agricultural practices can reduce viral persistence between seasons.

Some BVs are produced as commercial products, mainly for caterpillars, such as Gemstar LC (NPV of *Heliothis*/*Helicoverpa* spp. e.g., corn earworm, tobacco budworm, cotton bollworm); Spod-X LC (NPV of *Spodoptera* spp. e.g., beet armyworm); CYD-X and Virosoft CP4 (GV of *Cydia pomonella*, the codling moth); and CLV LC (NPV of *Anagrapha falcipera*, the celery looper). Successful infections can perpetuate the disease outbreak making repeat

Members of BVs are regarded as safe to vertebrates. Their specificity is usually very narrow, often limited to single insect species. Regarding beneficial arthropods, immature larvae of parasitoids in infected hosts may die not due to virus infection, but relatively to premature loss of the host or to variation in quality of the host. Virus can provide safe, effective and sustainable control of a variety of insect pests as a part of a varied IPM programs, see El-Husseini (2006) and Szewczyk et al. (2009). Entomopathogenic viral agents are ideal for pest management purposes due to their specificity, dispersal capacity, and self propagation. Viruses are the most environmentally acceptable components of direct and integrated management regimes. Accordingly, there is global interest in use of viral agents. Valuable reviews on entomopathogenic viruses are those of Szewczyk et al. (2009, 2011), Lacey &

The insecticidal bacterium, *Bacillus thuringiensis* (Bt) is a widely occurring gram-positive, spore-forming soil bacterium that produces parasporal, proteinaceous, crystal inclusionbodies during sporulation. Bt has been the most successful commercial microbial insecticide, and also has been the subject of the overwhelming majority of genetic engineering studies to improve efficacy (Federici, 2010; Lacey & Kaya, 2010; Mehlhorn, 2011). Bt is actually a

Kaya (2007), McNiel (2010), Ahemed et al. (2011), and Singh et al. (2011).

**3.1.1 Life cycle and viral stability**

**3.1.4 Products**

**3.1.5 Safety**

**3.2 Bacteria**

applications within a season unnecessary.

acid, an organic acid, C2H4O3, has strong acaricidal effect which was discovered for the first time (Khater & Ramadan, 2007). PPA had a great potential as acaricide against the cattle tick, *Boophilus annulatus*, and the fowl tick, *Argas persicus, In vitro.* Two minutes after treatment with 0.5%, PAA induced 100% mortality of both tick species and LC50 values for cattle and fowl ticks, after treatment for 30 min, were 0.06 and 0.05%, respectively. Following treatment with 0.25%, the LT50 values were 0.02 and 3.12 min, respectively. Furthermore, the detrimental effect of PAA against cattle tick extended beyond the adult stage, it significantly prolonged the preoviposition period, shortened the oviposition period, and decreased the mean number of the layed eggs, and such parameters were 14.75 and 6.57 days, and 457.50, respectively, after treatment with 0.25% of PAA. Therefore, PAA is highly effective when used at lower doses and short exposure time. The high speed of killing ticks is very important for avoidance of the hazard ensued by pathogen transmission in the course of delayed mortality caused by the currently used acaricides (Khater & Ramadan, 2007). PAA is highly effective against lice, *In vitro* and soft tick (*In vivo*)*,* (Khater, H.F., Unpublished data).

#### **3. Biological control**

Biological control is the reduction or protection of pest populations by natural enemies which include four categories: microbes or pathogens (such as viruses, bacteria, protozoa, and fungi); entomopathogenic nematodes; predators (such as lady beetles and lacewings); and parasitoids (wasps and some flies). Protozoa, predators, and parasitoids are out of the scope of this chapter. Natural enemies are responsible for natural suppression of pest population. The first successful large scale microbial control application using coidiospores of the fungus *Metarhizium anisopliae* was carried out in the Russian Ukraine against the beet weevil, *Bothynoderes punctiventris* (Metchnikoff, 1879).

#### **3.1 Virus**

Entomopathogenic viruses are obligate disease-causing organisms that can only reproduce within a host insect. Among the fifteen or more families of viruses of invertebrates, it is mainly those having virus particles (virions) occluded within a proteinaceous matrix, an occlusion body (OB), have been used successfully in controlling pest populations. Such families are Entomopoxviridae (Entomopoxviruses, EPVs), Reoviridae (cypoviruses, CPVs), and Baculoviridae (Baculoviruses, BVs) (Lacy & Kaya, 2007). Only BVs have been used as pesticides (Szewczyk et al., 2009, 2011). In the past, the classification of the family Baculoviridae was based on virus morphology. It was divided into two genera: the Nucleopolyhedrovirus (NPVs) and the Granulovirus (GVs). A new division on the basis of comparison of genomic sequences indicate that virus phylogeny followed more closely the classification of the hosts than the virion morphological traits. Accordingly, family Baculoviridae contains four genera: Alphabaculovirus (lepidopteranspecific NPVs), Betabaculovirus (lepidopteran-specific GVs), Gammabaculovirus (hymenopteran-specific NPVs), and Deltabaculovirus (dipteran-specific NPVs) (Jehle et al., 2006). Although wider use of BVs as commercial insecticides was restricted because of their slow killing action and difficulties in large scale production, a very successful project was carried out in Brazil (Moscardi, 1999); over 2.0 million hectare of soybean had been already controlled annually by velvetbean caterpillar BV. Consequently, many countries have increased the area of fields and forests protected by BV pesticides.

#### **3.1.1 Life cycle and viral stability**

34 Insecticides – Advances in Integrated Pest Management

acid, an organic acid, C2H4O3, has strong acaricidal effect which was discovered for the first time (Khater & Ramadan, 2007). PPA had a great potential as acaricide against the cattle tick, *Boophilus annulatus*, and the fowl tick, *Argas persicus, In vitro.* Two minutes after treatment with 0.5%, PAA induced 100% mortality of both tick species and LC50 values for cattle and fowl ticks, after treatment for 30 min, were 0.06 and 0.05%, respectively. Following treatment with 0.25%, the LT50 values were 0.02 and 3.12 min, respectively. Furthermore, the detrimental effect of PAA against cattle tick extended beyond the adult stage, it significantly prolonged the preoviposition period, shortened the oviposition period, and decreased the mean number of the layed eggs, and such parameters were 14.75 and 6.57 days, and 457.50, respectively, after treatment with 0.25% of PAA. Therefore, PAA is highly effective when used at lower doses and short exposure time. The high speed of killing ticks is very important for avoidance of the hazard ensued by pathogen transmission in the course of delayed mortality caused by the currently used acaricides (Khater & Ramadan, 2007). PAA is highly effective against lice, *In*

Biological control is the reduction or protection of pest populations by natural enemies which include four categories: microbes or pathogens (such as viruses, bacteria, protozoa, and fungi); entomopathogenic nematodes; predators (such as lady beetles and lacewings); and parasitoids (wasps and some flies). Protozoa, predators, and parasitoids are out of the scope of this chapter. Natural enemies are responsible for natural suppression of pest population. The first successful large scale microbial control application using coidiospores of the fungus *Metarhizium anisopliae* was carried out in the Russian Ukraine against the beet

Entomopathogenic viruses are obligate disease-causing organisms that can only reproduce within a host insect. Among the fifteen or more families of viruses of invertebrates, it is mainly those having virus particles (virions) occluded within a proteinaceous matrix, an occlusion body (OB), have been used successfully in controlling pest populations. Such families are Entomopoxviridae (Entomopoxviruses, EPVs), Reoviridae (cypoviruses, CPVs), and Baculoviridae (Baculoviruses, BVs) (Lacy & Kaya, 2007). Only BVs have been used as pesticides (Szewczyk et al., 2009, 2011). In the past, the classification of the family Baculoviridae was based on virus morphology. It was divided into two genera: the Nucleopolyhedrovirus (NPVs) and the Granulovirus (GVs). A new division on the basis of comparison of genomic sequences indicate that virus phylogeny followed more closely the classification of the hosts than the virion morphological traits. Accordingly, family Baculoviridae contains four genera: Alphabaculovirus (lepidopteranspecific NPVs), Betabaculovirus (lepidopteran-specific GVs), Gammabaculovirus (hymenopteran-specific NPVs), and Deltabaculovirus (dipteran-specific NPVs) (Jehle et al., 2006). Although wider use of BVs as commercial insecticides was restricted because of their slow killing action and difficulties in large scale production, a very successful project was carried out in Brazil (Moscardi, 1999); over 2.0 million hectare of soybean had been already controlled annually by velvetbean caterpillar BV. Consequently, many countries have increased the area of fields

*vitro* and soft tick (*In vivo*)*,* (Khater, H.F., Unpublished data).

weevil, *Bothynoderes punctiventris* (Metchnikoff, 1879).

and forests protected by BV pesticides.

**3. Biological control**

**3.1 Virus**

Virual infection begins in the insect's digestive system after consumption of plant material with viral particles. The virus spread though out the body but the digestive system is among the last part to be destroyed, so the insects usually continue to feed until they die leading to economic loss. See Lacey & Kaya (2007) and McNiel (2010) for more information about biology and ecology of viruses. The virus manipulates the behavior of infected larvae for its own dissemination. For example, larvae of the cabbage moth, *Mamestra brassicae* infected with NPV moved up to five times more than their healthy counterparts during the middle stages of infection (Vasconcelos et al., 1996b). Adult insects can also disperse viruses via vertical and horizontal transmission. Understanding the biology and ecology of viruses is crucial for optimizing pest control strategies of exotic and genetically modified organisms, see Lacy & Kaya (2007) and Szewczyk et al. (2009, 2011) for more details. UV protectants are very important for stability of the viral product, stilbene fluorescent brighteners (e.g. Phorwite AR ®, Blankophor ®, and others) induce the best results. Plant metabolites as peroxidases generate free radicals (Hoover et al., 1998) inactivate BVs. As a result, addition of free radical scavengers such as mannitol or enzyme superoxide dismutase to BV preparations can reduce such inactivation (Zhou et al., 2004). Tillage buries virus particles in the soil, thus good agricultural practices can reduce viral persistence between seasons.

#### **3.1.4 Products**

Some BVs are produced as commercial products, mainly for caterpillars, such as Gemstar LC (NPV of *Heliothis*/*Helicoverpa* spp. e.g., corn earworm, tobacco budworm, cotton bollworm); Spod-X LC (NPV of *Spodoptera* spp. e.g., beet armyworm); CYD-X and Virosoft CP4 (GV of *Cydia pomonella*, the codling moth); and CLV LC (NPV of *Anagrapha falcipera*, the celery looper). Successful infections can perpetuate the disease outbreak making repeat applications within a season unnecessary.

#### **3.1.5 Safety**

Members of BVs are regarded as safe to vertebrates. Their specificity is usually very narrow, often limited to single insect species. Regarding beneficial arthropods, immature larvae of parasitoids in infected hosts may die not due to virus infection, but relatively to premature loss of the host or to variation in quality of the host. Virus can provide safe, effective and sustainable control of a variety of insect pests as a part of a varied IPM programs, see El-Husseini (2006) and Szewczyk et al. (2009). Entomopathogenic viral agents are ideal for pest management purposes due to their specificity, dispersal capacity, and self propagation. Viruses are the most environmentally acceptable components of direct and integrated management regimes. Accordingly, there is global interest in use of viral agents. Valuable reviews on entomopathogenic viruses are those of Szewczyk et al. (2009, 2011), Lacey & Kaya (2007), McNiel (2010), Ahemed et al. (2011), and Singh et al. (2011).

#### **3.2 Bacteria**

The insecticidal bacterium, *Bacillus thuringiensis* (Bt) is a widely occurring gram-positive, spore-forming soil bacterium that produces parasporal, proteinaceous, crystal inclusionbodies during sporulation. Bt has been the most successful commercial microbial insecticide, and also has been the subject of the overwhelming majority of genetic engineering studies to improve efficacy (Federici, 2010; Lacey & Kaya, 2010; Mehlhorn, 2011). Bt is actually a

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 37

are classified according to their insecticidal activity and molecular relationship into four major gorups (Hӧfte and Whiteley, 1989): Cry I toxins, active against larvae of lepidopterans; Cry II toxins, active against larvae of lepidopterans and dipterans; Cry III toxins, active against larvae of coleopterans (Chrysomelidae); and Cry IV toxins, active against larvae of nematoceran flies. Cry toxins bind to glycoprotein or glycolipid receptors. The toxins can also be further categorized into subclasses, ex. CryIA etc. The genes of the toxins are named as the toxins but without capital letters and in italic (e.g., *cry*IV). Cytotoxins (Cyt) found in strains toxic to larvae of nematoceran flies and coleopterans. The CytA protein binds to lipids and does not exhibit the specific binding mechanism which the Cry proteins do (Hӧfte and Whiteley, 1989). Cyt proteins are thought to have a similar mode of action to that of Cry proteins, with the exception that they directly bind to the microvillar lipid bilayer. Similar to Bti, the toxicity of Bs is due to protein endotoxins produced during sporulation and assembled into a parasporal body. The main toxin of Bs is a binary toxin (BinA and BinB) which are proteolytically activated in the mosquito midgut to release peptides (43 and 39 kDa, respectively) to form the binary toxin. The toxins bind to microvilli of the midgut epithelium, trigger hypertrophy and lysis of cells, leading to destruction of

Despite decades of use, there is no reported resistance among mosquito populations probably due to biochemical properties of Cyt protein. Bti produces a parasporal body that contains four major endotoxins, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa. Both Cry and Cyt are must be ingested to yield active toxins in the midget of insect. Cyt protein synergizes the toxicity of the Bti Cry proteins resulting in delayed the phenotypic expression of the evolution of resistance. Such synergistic interactions are also known to occur between the Bti Cry proteins. When Cyt protein combined with the Bs Bin toxin, it can also overcome resistance to Bti. For detailed information, see Wirth et al. (2005) and Federici (2010). As a rule, bacterial control agents are less likely to provoke resistance because their mode of action is more complex (Wirth et al., 2005; Federici, 2010). However, resistance had developed against Btk in some pests, e.x., the stored grain pest, *Plodia interpunctella*, and the diamondback moth, *Plutella xylostella*. In most cases, resistance has been associated with a recessive or partially recessive trait(s) and appears to be linked to a single gene. The combination of protoxins in Bti is very important in reducing the likelihood of resistance. In contrary, when the gene encoding a single toxin protein was cloned into a microorganism and then fed to larval mosquitoes, resistance was induced within a few generations; see transgenic organisms below and Mehlhorn (2011) for more details. For avoidance of resistance, products with different arrays of endotoxins should be alternated (Schuster &

The U.S. Environmental Protection Agency (USEPA) categorizes the risk posed by Bt strains to nontarget organisms as minimal to nonexistent. After using of Bt products for more than 50 years, it can be concluded that Bt belong to the most environmentally safe products as they kill target organisms and usually do not harm nontarget organisms, such as beneficial insects, plants, or humans. The safety record for occupational exposure to Bt based biopesticides with regard to human health is considered good (Barfod, 2010). Some Bt

midgut and death of mosquito larva.

**3.2.3 Resistance**

Stansly, 2006).

**3.2.4 Safety**

complex of bacterial subspecies that occur in soil, leaf litter, leaf surfaces, insect feces, and as a part of the flora in the midguts of many insect species. There are several insecticides based on various sub-species of *Bacillus thuringiensis* Berliner (*Bt*), such as *B thuringiensis israelensis*  (Bti), with activity against mosquito larvae, black fly (simuliid), fungus gnats, and related dipterans species*; B thuringiensis kurstaki* (Btk) and *B thuringiensis aizawai* (Bta)with activity against lepidopteran larval species; *B thuringiensis tenebrionis* (Btt), with activity against coleopteran adults and larvae; and *B thuringiensis japonensis* (Btj) strain *buibui*, with activity against soil-inhabiting beetles.

#### **3.2.1 Mode of action**

The insecticidal properties of Bt are largely a function of the presence of extra-chromosomal plasmids in the cell. These carry genes such as *cry* genes that encode a diverse array of these protein crystalline inclusion bodies which are toxic to insects. Upon ingestion by an insect, the crystal proteins (Cry) are solubilised and the insect gut proteases convert the original protoxin into smaller toxins. These hydrolysed toxins bind to the insect's midgut cells at high-affinity and specific receptor binding sites where they interfere with the potassium ion dependent, active amino acid symport mechanism decreasing absorption of minerals and nutrition from midgut and finally death of the columnar cells. This disruption causes the formation of large cation-selective pores that increase the water permeability of the cell membrane. A large uptake of water causes cell swelling and eventual rupture, disintegrating the midgut lining. Different toxins bind to different receptors with different intensities and this explains the selectivity of different Bt strains in different insect species (Baum et al., 1999; Cooping & Menn, 2000; Lacey & Kaya, 2007; Federici, 2010). Delayed larval mortalities (2–48 h) is caused by the crystal inclusions which have to be ingested and then processed within the insect's gut. Biological control products may contain the endotoxins plus live bacterial cells. The toxin stops feeding, this action hinder further damage caused by the feeding larva, and do not directly kill insects, but young larvae may starve to death and may die from bacterial infection over a longer period. Some commercial products contain Bti crystal proteins and spores, such as Bactimos and VectoBac (Valent BioSciences). VectoBac, Bti, (12 AS, Wady El- Niel for agricultural development Co. Egypt) is highly effective against *C. pipence* than *M. domestica*, LC50 values were 1x10-5 and 3.86 x103 spores/ml, respectively, and LC 90 values were 0.04 and 37.28 x 103 spores/ ml, respectively. Survived mosquito larvae died as pupae (Khater, 2003). Bti is a practical substitute to organophosphate insecticides, but it is unsuitable for application to environmentally sensitive water bodies. *Bacillus sphaericus* control mosquito larvae, particularly *Culex* and anopheline spp., especially those breeding in polluted water. It controls also black fly, *Simulium* sp., the vector of river blindness disease. Bs is effectively controlled *C. pipiens* in Egypt (Ragaei et al., 2004). It is widely used in Europe and Africa. Registered *B. sphaericus* product is Vectolex CG (Valent BioSciences). Recent studies in Kenya have shown that at least in some areas, biting rates by *Anopheles gambiae* can be reduced by more than 90% by using a combination of existing commercial formulations of Bti and Bs (Filinger & Lindsay, 2000).

#### **3.2.2 Bacterial toxins**

The use of insect-specific toxins from Bt and Bs is forming an increasingly component of biological control strategies. The protein crystals (protoxins) contain several toxins which

are classified according to their insecticidal activity and molecular relationship into four major gorups (Hӧfte and Whiteley, 1989): Cry I toxins, active against larvae of lepidopterans; Cry II toxins, active against larvae of lepidopterans and dipterans; Cry III toxins, active against larvae of coleopterans (Chrysomelidae); and Cry IV toxins, active against larvae of nematoceran flies. Cry toxins bind to glycoprotein or glycolipid receptors. The toxins can also be further categorized into subclasses, ex. CryIA etc. The genes of the toxins are named as the toxins but without capital letters and in italic (e.g., *cry*IV). Cytotoxins (Cyt) found in strains toxic to larvae of nematoceran flies and coleopterans. The CytA protein binds to lipids and does not exhibit the specific binding mechanism which the Cry proteins do (Hӧfte and Whiteley, 1989). Cyt proteins are thought to have a similar mode of action to that of Cry proteins, with the exception that they directly bind to the microvillar lipid bilayer. Similar to Bti, the toxicity of Bs is due to protein endotoxins produced during sporulation and assembled into a parasporal body. The main toxin of Bs is a binary toxin (BinA and BinB) which are proteolytically activated in the mosquito midgut to release peptides (43 and 39 kDa, respectively) to form the binary toxin. The toxins bind to microvilli of the midgut epithelium, trigger hypertrophy and lysis of cells, leading to destruction of midgut and death of mosquito larva.

#### **3.2.3 Resistance**

36 Insecticides – Advances in Integrated Pest Management

complex of bacterial subspecies that occur in soil, leaf litter, leaf surfaces, insect feces, and as a part of the flora in the midguts of many insect species. There are several insecticides based on various sub-species of *Bacillus thuringiensis* Berliner (*Bt*), such as *B thuringiensis israelensis*  (Bti), with activity against mosquito larvae, black fly (simuliid), fungus gnats, and related dipterans species*; B thuringiensis kurstaki* (Btk) and *B thuringiensis aizawai* (Bta)with activity against lepidopteran larval species; *B thuringiensis tenebrionis* (Btt), with activity against coleopteran adults and larvae; and *B thuringiensis japonensis* (Btj) strain *buibui*, with activity

The insecticidal properties of Bt are largely a function of the presence of extra-chromosomal plasmids in the cell. These carry genes such as *cry* genes that encode a diverse array of these protein crystalline inclusion bodies which are toxic to insects. Upon ingestion by an insect, the crystal proteins (Cry) are solubilised and the insect gut proteases convert the original protoxin into smaller toxins. These hydrolysed toxins bind to the insect's midgut cells at high-affinity and specific receptor binding sites where they interfere with the potassium ion dependent, active amino acid symport mechanism decreasing absorption of minerals and nutrition from midgut and finally death of the columnar cells. This disruption causes the formation of large cation-selective pores that increase the water permeability of the cell membrane. A large uptake of water causes cell swelling and eventual rupture, disintegrating the midgut lining. Different toxins bind to different receptors with different intensities and this explains the selectivity of different Bt strains in different insect species (Baum et al., 1999; Cooping & Menn, 2000; Lacey & Kaya, 2007; Federici, 2010). Delayed larval mortalities (2–48 h) is caused by the crystal inclusions which have to be ingested and then processed within the insect's gut. Biological control products may contain the endotoxins plus live bacterial cells. The toxin stops feeding, this action hinder further damage caused by the feeding larva, and do not directly kill insects, but young larvae may starve to death and may die from bacterial infection over a longer period. Some commercial products contain Bti crystal proteins and spores, such as Bactimos and VectoBac (Valent BioSciences). VectoBac, Bti, (12 AS, Wady El- Niel for agricultural development Co. Egypt) is highly effective against *C. pipence* than *M. domestica*, LC50 values were 1x10-5 and 3.86 x103 spores/ml, respectively, and LC 90 values were 0.04 and 37.28 x 103 spores/ ml, respectively. Survived mosquito larvae died as pupae (Khater, 2003). Bti is a practical substitute to organophosphate insecticides, but it is unsuitable for application to environmentally sensitive water bodies. *Bacillus sphaericus* control mosquito larvae, particularly *Culex* and anopheline spp., especially those breeding in polluted water. It controls also black fly, *Simulium* sp., the vector of river blindness disease. Bs is effectively controlled *C. pipiens* in Egypt (Ragaei et al., 2004). It is widely used in Europe and Africa. Registered *B. sphaericus* product is Vectolex CG (Valent BioSciences). Recent studies in Kenya have shown that at least in some areas, biting rates by *Anopheles gambiae* can be reduced by more than 90% by using a combination of existing commercial formulations of

The use of insect-specific toxins from Bt and Bs is forming an increasingly component of biological control strategies. The protein crystals (protoxins) contain several toxins which

against soil-inhabiting beetles.

Bti and Bs (Filinger & Lindsay, 2000).

**3.2.2 Bacterial toxins**

**3.2.1 Mode of action**

Despite decades of use, there is no reported resistance among mosquito populations probably due to biochemical properties of Cyt protein. Bti produces a parasporal body that contains four major endotoxins, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa. Both Cry and Cyt are must be ingested to yield active toxins in the midget of insect. Cyt protein synergizes the toxicity of the Bti Cry proteins resulting in delayed the phenotypic expression of the evolution of resistance. Such synergistic interactions are also known to occur between the Bti Cry proteins. When Cyt protein combined with the Bs Bin toxin, it can also overcome resistance to Bti. For detailed information, see Wirth et al. (2005) and Federici (2010). As a rule, bacterial control agents are less likely to provoke resistance because their mode of action is more complex (Wirth et al., 2005; Federici, 2010). However, resistance had developed against Btk in some pests, e.x., the stored grain pest, *Plodia interpunctella*, and the diamondback moth, *Plutella xylostella*. In most cases, resistance has been associated with a recessive or partially recessive trait(s) and appears to be linked to a single gene. The combination of protoxins in Bti is very important in reducing the likelihood of resistance. In contrary, when the gene encoding a single toxin protein was cloned into a microorganism and then fed to larval mosquitoes, resistance was induced within a few generations; see transgenic organisms below and Mehlhorn (2011) for more details. For avoidance of resistance, products with different arrays of endotoxins should be alternated (Schuster & Stansly, 2006).

#### **3.2.4 Safety**

The U.S. Environmental Protection Agency (USEPA) categorizes the risk posed by Bt strains to nontarget organisms as minimal to nonexistent. After using of Bt products for more than 50 years, it can be concluded that Bt belong to the most environmentally safe products as they kill target organisms and usually do not harm nontarget organisms, such as beneficial insects, plants, or humans. The safety record for occupational exposure to Bt based biopesticides with regard to human health is considered good (Barfod, 2010). Some Bt

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 39

effectiveness is limited by high temperatures. The fungus is not effective for mosquitoes in brackish or organically rich aquatic habitats (Merriam and Axtell, 1982). *L. giganteum* has a wide geographical distribution: North America, Europe, Africa, Asia, Antarctica (Federici, 1981). Fungal reproduction is both asexual (zoospores) and sexual (oospores) (Federici, 1981). In order to infect mosquito larvae, biflagellate zoospores must be formed. Motile zoospores are the asexual stage of the fungus. Oospores, the sexual stage of *L.giganteum*, can also be used as inoculum. They are dormant propagules, resistant to desiccation and mechanical abrasion and stable for at least seven years, which allows multivoltine

*Lagenidium* spp. was isolated form Egypt for the first time form *Culex pipiens* larvae infesting a polluted creak in Miet El- Attar, Benha, Egypt, by Khater (2003). Such fungus was propagated on SDA medium and Peptone yeast glucose (PYG) for sporulation. Five concentrations (5 x105- 1.6 x107) were used to infect *C. pipiens* late 3rd larval stage *In vitro* and the biological parameters had been followed up till emergence of adults. LC50 and LC90 values were 2.79 x106 and 3.94 x108 spores/ ml, respectively. At the lowest concentration, larval duration reached 8.3 days (3.6 days in the control group) and morphological changes (symptoms of mycosis) was 66.7%, (Fig. 4,5). All survived larvae died as pupae; consequently, the isolated fungus inhibited adult emergence. As this fungus has the ability to be self- propagated, it could be an effective control agent for the vector of Bancroftian filariasis and Rift valley fever virus in Egypt (Khater, 2003). The fungus has caused high mortalities in mosquito populations, see Schotle et al. (2004) for a review. Results from a small scale field trial in North Carolina indicated that *L. giganteum*  recycled for an entire season despite periodic scarcity of hosts and short-term drought with infections ranging from 0-100% (Jaronski & Axtell, 1983). A large-scale field trial in Californian rice fields, using mycelium from either 20 or 30 liters of fermentation beer per hectare resulted in 40%-90% infection of *Culex tarsalis* and *Anopheles freeborni* sentinel larvae (Kerwin &

The relative potency indicated that Vectobac (Bti), and *Lagenidium* spp. were 12x1011 and 4.3 times, respectively, more effective than the fungal product Biosect®, *Beauveria bassiana*, on the basis of LC50 values (Khater, 2003). *L. giganteum* is compatible with the bacterial agents Bti and Bs Meyer and Neide when used against *Culex quinquefasciatus* (Orduz & Axtell, 1991), with the fungus having the distinct advantage over Bti in that it is able to recycle in stagnant water, infecting multiple and overlapping generations of mosquitoes (Legner, 1995). In field trials in which Laginex 25 was compared with Vectobac-12AS (Bti), Laginex reduced *C. quinquefasciatus* larvae by 100% for 22 days whereas Vectobac-12AS required

Unfortunately, mass production yields of oospores remain orders of magnitude below that of the less stable mycelial (asexual, presporangial) stage, and continued problems with spore activation have prevented large-scale field tests (Kerwin and Petersen, 1997). For solving this problem, improving oospore yields would be much more useful than zoospores in large-scale operational mosquito control programs. In contrast to the previous obstacles, Laginex® is a *L.giganteum*-based product by AgraQuest (California, USA) until 1999. It is effective against larvae of most pest mosquito species; remains infective in the environment through dry periods but it is unable to survive high summertime temperatures; the kind of

persistence of the fungus in some habitats (Kerwin et al., 1994).

Washino, 1987).

**<IP_ADDRESS> Production**

**<IP_ADDRESS> The relative potency**

retreatment by the 10th day (Hallmon et al., 2000).

subspecies produce other types of toxins besides Cry proteins as ß-exotoxin and α-toxin. Both toxins are toxic to vertebrates and they are banned in Bt products, see Lacey & Kaya (2007) and Makonde et al. (2010) for more particulars about bacterial toxins. Bt and BS are the most used biocontrol agents against nuisance, pest or vector species. **T**hey are strongly incorporated in IPM programs because they are highly efficient, easily be mass-produced, easy to handle, stable when stored, cost-effective, pest specific, and safe to people and the environment. For more information about bacteria as a biological control agent, see Lacey & Kaya (2007), Gilbert & Gill (2010), Ahmed et al. (20 11), Mehlhorn (2011), Singh et al. (2011), and van Emden & Service (2011).

#### **3.3 Entomophathogenic fungi**

Entomophathogenic fungi are important natural control agents that limit insect populations. Survival of fungi requires a delicat balance of interactions between fungi, host, and the environment. Most fungi cause insect diseases spread by means of asexual spores called conidia which vary greatly in their ability to survive adverse environmental conditions, desiccation, and ultraviolet radiation.

#### **3.3.1 Life cycle**

In general, insects get infected when they come into contact with spores on the bodies of dead insects, on the surface of plants, in the soil, or in the air as windborne particles. High humidity is usually required for germination of conidia on the insect cuticle and production of germ tube that allow the fungus to penetrate the cuticle often at joints or creases where the insect's protective covering is thinner. In contrast to virus and bacteria, fungi do not have to be ingested to cause infections. The fungus multiplies within the host insect and kill it. Many fungi produce toxins to increase the speed of kill or prevent competition from other microbes. Death is due to toxin produced by the fungus or fungal multiplication. Under favorable conditions, the fungus grows out of the cadavers, usually at thinner areas and form conidiophores or analogous structures and sporulates. Some species go into a resting stage which survive periods of adverse conditions before forming or releasing spores. Soil incorporating fungi usually avoid the adverse effects of ultraviolet radiation and desiccation, but other microorganisms that act as competitors or antagonists often alter pathogen effectiveness. Fungal pathogens differ in the range of hosts. Many important fungi attack eggs, immature, and adults of a variety of insect species. Others are more specific to immature stages or to a narrow range of insect species. Several fungi are used to control insects such as *Leptolegnia spp., Coelomomyces spp., Hirsutella thompsonii, Nomuraea rileyi,* and *Vericillium lecanii*. The most famous entomophathogenic fungi will be disused below.

#### **3.3.2 Oomycetes (water molds)**

*Lagenidium giganteum* was first described by Couch (1935) from a combined collection of copepods and mosquito larvae, *Culex* and *Anopheles,* in North Carolina, USA. It has a wide geographical distribution: North America, Europe, Africa, Asia, Antarctica (Federici, 1981). The only species of the genus *Lagenidium* is known to be pathogenic to mosquito larvae is *L. giganteum (*formerly*: L. culicidum)*. As a facultative parasite, *L.giganteum* can grow as pathogen on mosquito larvae, or as a saprophyte in aquatic environments (Sur et al., 2001). This aquatic fungus is highly infectious to larvae of several mosquito genera. It cycles effectively in the aquatic environment even when mosquito density is low, but its effectiveness is limited by high temperatures. The fungus is not effective for mosquitoes in brackish or organically rich aquatic habitats (Merriam and Axtell, 1982). *L. giganteum* has a wide geographical distribution: North America, Europe, Africa, Asia, Antarctica (Federici, 1981). Fungal reproduction is both asexual (zoospores) and sexual (oospores) (Federici, 1981). In order to infect mosquito larvae, biflagellate zoospores must be formed. Motile zoospores are the asexual stage of the fungus. Oospores, the sexual stage of *L.giganteum*, can also be used as inoculum. They are dormant propagules, resistant to desiccation and mechanical abrasion and stable for at least seven years, which allows multivoltine persistence of the fungus in some habitats (Kerwin et al., 1994).

*Lagenidium* spp. was isolated form Egypt for the first time form *Culex pipiens* larvae infesting a polluted creak in Miet El- Attar, Benha, Egypt, by Khater (2003). Such fungus was propagated on SDA medium and Peptone yeast glucose (PYG) for sporulation. Five concentrations (5 x105- 1.6 x107) were used to infect *C. pipiens* late 3rd larval stage *In vitro* and the biological parameters had been followed up till emergence of adults. LC50 and LC90 values were 2.79 x106 and 3.94 x108 spores/ ml, respectively. At the lowest concentration, larval duration reached 8.3 days (3.6 days in the control group) and morphological changes (symptoms of mycosis) was 66.7%, (Fig. 4,5). All survived larvae died as pupae; consequently, the isolated fungus inhibited adult emergence. As this fungus has the ability to be self- propagated, it could be an effective control agent for the vector of Bancroftian filariasis and Rift valley fever virus in Egypt (Khater, 2003). The fungus has caused high mortalities in mosquito populations, see Schotle et al. (2004) for a review. Results from a small scale field trial in North Carolina indicated that *L. giganteum*  recycled for an entire season despite periodic scarcity of hosts and short-term drought with infections ranging from 0-100% (Jaronski & Axtell, 1983). A large-scale field trial in Californian rice fields, using mycelium from either 20 or 30 liters of fermentation beer per hectare resulted in 40%-90% infection of *Culex tarsalis* and *Anopheles freeborni* sentinel larvae (Kerwin & Washino, 1987).

#### **<IP_ADDRESS> The relative potency**

38 Insecticides – Advances in Integrated Pest Management

subspecies produce other types of toxins besides Cry proteins as ß-exotoxin and α-toxin. Both toxins are toxic to vertebrates and they are banned in Bt products, see Lacey & Kaya (2007) and Makonde et al. (2010) for more particulars about bacterial toxins. Bt and BS are the most used biocontrol agents against nuisance, pest or vector species. **T**hey are strongly incorporated in IPM programs because they are highly efficient, easily be mass-produced, easy to handle, stable when stored, cost-effective, pest specific, and safe to people and the environment. For more information about bacteria as a biological control agent, see Lacey & Kaya (2007), Gilbert & Gill (2010), Ahmed et al. (20 11), Mehlhorn (2011), Singh et al. (2011),

Entomophathogenic fungi are important natural control agents that limit insect populations. Survival of fungi requires a delicat balance of interactions between fungi, host, and the environment. Most fungi cause insect diseases spread by means of asexual spores called conidia which vary greatly in their ability to survive adverse environmental conditions,

In general, insects get infected when they come into contact with spores on the bodies of dead insects, on the surface of plants, in the soil, or in the air as windborne particles. High humidity is usually required for germination of conidia on the insect cuticle and production of germ tube that allow the fungus to penetrate the cuticle often at joints or creases where the insect's protective covering is thinner. In contrast to virus and bacteria, fungi do not have to be ingested to cause infections. The fungus multiplies within the host insect and kill it. Many fungi produce toxins to increase the speed of kill or prevent competition from other microbes. Death is due to toxin produced by the fungus or fungal multiplication. Under favorable conditions, the fungus grows out of the cadavers, usually at thinner areas and form conidiophores or analogous structures and sporulates. Some species go into a resting stage which survive periods of adverse conditions before forming or releasing spores. Soil incorporating fungi usually avoid the adverse effects of ultraviolet radiation and desiccation, but other microorganisms that act as competitors or antagonists often alter pathogen effectiveness. Fungal pathogens differ in the range of hosts. Many important fungi attack eggs, immature, and adults of a variety of insect species. Others are more specific to immature stages or to a narrow range of insect species. Several fungi are used to control insects such as *Leptolegnia spp., Coelomomyces spp., Hirsutella thompsonii, Nomuraea rileyi,* and *Vericillium lecanii*. The most famous entomophathogenic fungi will be disused below.

*Lagenidium giganteum* was first described by Couch (1935) from a combined collection of copepods and mosquito larvae, *Culex* and *Anopheles,* in North Carolina, USA. It has a wide geographical distribution: North America, Europe, Africa, Asia, Antarctica (Federici, 1981). The only species of the genus *Lagenidium* is known to be pathogenic to mosquito larvae is *L. giganteum (*formerly*: L. culicidum)*. As a facultative parasite, *L.giganteum* can grow as pathogen on mosquito larvae, or as a saprophyte in aquatic environments (Sur et al., 2001). This aquatic fungus is highly infectious to larvae of several mosquito genera. It cycles effectively in the aquatic environment even when mosquito density is low, but its

and van Emden & Service (2011).

**3.3 Entomophathogenic fungi**

desiccation, and ultraviolet radiation.

**3.3.2 Oomycetes (water molds)**

**3.3.1 Life cycle**

The relative potency indicated that Vectobac (Bti), and *Lagenidium* spp. were 12x1011 and 4.3 times, respectively, more effective than the fungal product Biosect®, *Beauveria bassiana*, on the basis of LC50 values (Khater, 2003). *L. giganteum* is compatible with the bacterial agents Bti and Bs Meyer and Neide when used against *Culex quinquefasciatus* (Orduz & Axtell, 1991), with the fungus having the distinct advantage over Bti in that it is able to recycle in stagnant water, infecting multiple and overlapping generations of mosquitoes (Legner, 1995). In field trials in which Laginex 25 was compared with Vectobac-12AS (Bti), Laginex reduced *C. quinquefasciatus* larvae by 100% for 22 days whereas Vectobac-12AS required retreatment by the 10th day (Hallmon et al., 2000).

#### **<IP_ADDRESS> Production**

Unfortunately, mass production yields of oospores remain orders of magnitude below that of the less stable mycelial (asexual, presporangial) stage, and continued problems with spore activation have prevented large-scale field tests (Kerwin and Petersen, 1997). For solving this problem, improving oospore yields would be much more useful than zoospores in large-scale operational mosquito control programs. In contrast to the previous obstacles, Laginex® is a *L.giganteum*-based product by AgraQuest (California, USA) until 1999. It is effective against larvae of most pest mosquito species; remains infective in the environment through dry periods but it is unable to survive high summertime temperatures; the kind of

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 41

pupal intermediates. Mortality and morphological changes of pupae (symptoms of mycosis) reached 100% at 8 x 102 spores /ml. Adult mortalities, deformity, and emergence reduction rates increased as the concentrations increased (79.3%, 60%, 98.2%, respectively, at 4x102 spores/ ml). Adult abnormalities were failure of adult eclosion, small adults, and deformed wings and legs (Fig. 1-3). In addition, this pathogen manipulates the behavior of infected host for his own dissemination as the apparently normal adults exhibited the characteristic position of *Entomophthora* spp. (Fig. 6) (Krasnoff et al., 1995; and Khater, 2003)

Fig. 6. Characters of adult house fly infected with *Entomophthora* spp.: A. Adult exhibited elevated abdomen, wings, and extended legs whereas the mouth parts were stoked

(white color), D. Hyphal body (zoosporangia) right arrow, spore (left arrow) of

*Entomophthora* spp. Adapted form Khater (2003).

downwards (anterior view), B. Adult with extended legs (ventral view), C. Conidial shower

First, adults died in elevated positions, with the proboscis extended and attached to the substrate, legs spread, the abdomen angled away form the substrate, and the wings rose above the thorax. The conidial discharge reached 3-9 cm, depending on the size of the cadavers (Fig. 6). Conidia are forcibly ejected from conidiophores that emerge most profusely from the intersegmental membranes on the insect's abdomen, such posture enhance dispersal of conidia (Krasnoff et al., 1995; Khater, 2003). Similarly, an onion maggot fly, *Delia antiqua* (Meigen), infected by *E.muscae* Cohn (Fresenius) and dying at the top of a grass stem with its proboscis fastened to the substrate and its abdomen well raised by an atypical disposition of the legs appears to be an ideal launch pad for maximizing the effective area covered by the conidial shower (see Krasnoff et al., 1995 for more details). Second, the pathogen also benefits indirectly from the sexual attractiveness of female cadavers. Males attempting necrophilous copulations invariably become infected with the

as follows:

spore used was not mentioned. Shathele (2009) made an excellent potential for investigations on the use of newly developed isolate (Saudi-1 and Saudi-2) media for an isolated strain of *L. giganteum* form Louisiana for its maintenance and zoospore release.

#### **<IP_ADDRESS>. Safety**

Zoospores of the fungus appear harmless to vertebrates (Kerwin et al., 1990), most aquatic invertebrates (Nestrud & Anderson 1994) except *Daphnia* spp. and copepods (Couch, 1935), three cladoceran species and a chironomid species (Nestrud & Anderson, 1994).

#### **3.3.3 Hyphomycetes**

Most soil fungi used for the control of insect pests belong to the group hyphomycetes, such as *Beauveria bassiana* and *Metarhizium anisopliae,* formerly known as *Entomophthora anisopliae* . Some species have been developed as commercial products because of their ability to be mass produced. Most fungi in this group can cause natural outbreaks, to a wide range of insect hosts, on their own when environmental conditions are favorable. Soil provides protection against UV along with optimal conditions of temperature and moisture. Furthermore, the fungi may survive in the soil through recycling in insects or roots (Leger, 2008). Thus, they provide a long-term strategy for larvae and puparia control (Quesada-Moraga et al., 2006). *B. bassiana* infects both larvae and adults of a broad host range. Understanding the interactions between *B. bassiana* and other soil microorganisms is very important for the success of using of this fungus. Commertially available products based on *B. bassiana* are Mycotrol O (Emerald BioAgriculture), Naturalis Home and Garden (H&G), Naturalis L (Troy BioSciences, Inc.), and Biosect® (Kafr El Zayat - KZ Chemicals, Egypt). Khater (2003) used Biosect® to control larvae of both *M. domestica* and *C.pepiens In vitro*. LC50 an LC90 values were 29.2 x107 and 9.97 x108 spores/ ml for house flies and 1.2x107 and 4.17x109 spores/ ml, for mosquitoes, respectively. The total larval mortalities of mosquitoes were almost 100%, the few survived larvae died as pupae. At the lowest concentrations, Larval and pupal malformations were 30% and 45%, See fig (1,2,4,5) for symptoms of mycosis.

#### **3.3.4 Entomophthorales**

Entomophthorales is a group of fungi that tend to be much more host specific and can cause natural outbreaks in insect populations. Several different *Entomophthora muscae sensu stricto* genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily **(**Jensen et al., 2001)**.** Currently, no commercially-based product is available because of difficulties in mass production. All available literature deal with *E. muscae* as a pathogenic fungi of adult *M. domestica,* but Khater (2003) isolated it for the first time in Egypt, form Moshtohor, Toukh, Qlubia governorate, such strain has a unique ability to infect larvae of house flies for the first time also. The isolated fungus was replicated on Sabouraud Dextrose Agar (SDA) medium for hyphenation and on liquid medium for laboratory – scale Entomophthora' hyphal production for enhancement of zoosporogenesis. Khater (2003) also made a bioassay using early 3rd larval stages of house flies which were infected *In vitro* by adding different concentrations of *E. muscae* (from 25 to 16 x 102 spores/ml) to the breeding medium of larvae and followrd up till adult emergence. LC50 and LC90 values for larval mortalities were 2.19 x 102 and 20 x 102 spores/ ml, respectively. At lower concentrations, larval durations were elongated (7.2 days) than that of the control group (2.6 days). Larval abnormalities were larval pigmentation, black, red- pin point pigmentation, ulceration, maceration and larval

spore used was not mentioned. Shathele (2009) made an excellent potential for investigations on the use of newly developed isolate (Saudi-1 and Saudi-2) media for an isolated strain of *L. giganteum* form Louisiana for its maintenance and zoospore release.

Zoospores of the fungus appear harmless to vertebrates (Kerwin et al., 1990), most aquatic invertebrates (Nestrud & Anderson 1994) except *Daphnia* spp. and copepods (Couch, 1935),

Most soil fungi used for the control of insect pests belong to the group hyphomycetes, such as *Beauveria bassiana* and *Metarhizium anisopliae,* formerly known as *Entomophthora anisopliae* . Some species have been developed as commercial products because of their ability to be mass produced. Most fungi in this group can cause natural outbreaks, to a wide range of insect hosts, on their own when environmental conditions are favorable. Soil provides protection against UV along with optimal conditions of temperature and moisture. Furthermore, the fungi may survive in the soil through recycling in insects or roots (Leger, 2008). Thus, they provide a long-term strategy for larvae and puparia control (Quesada-Moraga et al., 2006). *B. bassiana* infects both larvae and adults of a broad host range. Understanding the interactions between *B. bassiana* and other soil microorganisms is very important for the success of using of this fungus. Commertially available products based on *B. bassiana* are Mycotrol O (Emerald BioAgriculture), Naturalis Home and Garden (H&G), Naturalis L (Troy BioSciences, Inc.), and Biosect® (Kafr El Zayat - KZ Chemicals, Egypt). Khater (2003) used Biosect® to control larvae of both *M. domestica* and *C.pepiens In vitro*. LC50 an LC90 values were 29.2 x107 and 9.97 x108 spores/ ml for house flies and 1.2x107 and 4.17x109 spores/ ml, for mosquitoes, respectively. The total larval mortalities of mosquitoes were almost 100%, the few survived larvae died as pupae. At the lowest concentrations, Larval and pupal malformations were 30% and 45%, See

Entomophthorales is a group of fungi that tend to be much more host specific and can cause natural outbreaks in insect populations. Several different *Entomophthora muscae sensu stricto* genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily **(**Jensen et al., 2001)**.** Currently, no commercially-based product is available because of difficulties in mass production. All available literature deal with *E. muscae* as a pathogenic fungi of adult *M. domestica,* but Khater (2003) isolated it for the first time in Egypt, form Moshtohor, Toukh, Qlubia governorate, such strain has a unique ability to infect larvae of house flies for the first time also. The isolated fungus was replicated on Sabouraud Dextrose Agar (SDA) medium for hyphenation and on liquid medium for laboratory – scale Entomophthora' hyphal production for enhancement of zoosporogenesis. Khater (2003) also made a bioassay using early 3rd larval stages of house flies which were infected *In vitro* by adding different concentrations of *E. muscae* (from 25 to 16 x 102 spores/ml) to the breeding medium of larvae and followrd up till adult emergence. LC50 and LC90 values for larval mortalities were 2.19 x 102 and 20 x 102 spores/ ml, respectively. At lower concentrations, larval durations were elongated (7.2 days) than that of the control group (2.6 days). Larval abnormalities were larval pigmentation, black, red- pin point pigmentation, ulceration, maceration and larval

three cladoceran species and a chironomid species (Nestrud & Anderson, 1994).

**<IP_ADDRESS>. Safety**

**3.3.3 Hyphomycetes**

fig (1,2,4,5) for symptoms of mycosis.

**3.3.4 Entomophthorales**

pupal intermediates. Mortality and morphological changes of pupae (symptoms of mycosis) reached 100% at 8 x 102 spores /ml. Adult mortalities, deformity, and emergence reduction rates increased as the concentrations increased (79.3%, 60%, 98.2%, respectively, at 4x102 spores/ ml). Adult abnormalities were failure of adult eclosion, small adults, and deformed wings and legs (Fig. 1-3). In addition, this pathogen manipulates the behavior of infected host for his own dissemination as the apparently normal adults exhibited the characteristic position of *Entomophthora* spp. (Fig. 6) (Krasnoff et al., 1995; and Khater, 2003) as follows:

Fig. 6. Characters of adult house fly infected with *Entomophthora* spp.: A. Adult exhibited elevated abdomen, wings, and extended legs whereas the mouth parts were stoked downwards (anterior view), B. Adult with extended legs (ventral view), C. Conidial shower (white color), D. Hyphal body (zoosporangia) right arrow, spore (left arrow) of *Entomophthora* spp. Adapted form Khater (2003).

First, adults died in elevated positions, with the proboscis extended and attached to the substrate, legs spread, the abdomen angled away form the substrate, and the wings rose above the thorax. The conidial discharge reached 3-9 cm, depending on the size of the cadavers (Fig. 6). Conidia are forcibly ejected from conidiophores that emerge most profusely from the intersegmental membranes on the insect's abdomen, such posture enhance dispersal of conidia (Krasnoff et al., 1995; Khater, 2003). Similarly, an onion maggot fly, *Delia antiqua* (Meigen), infected by *E.muscae* Cohn (Fresenius) and dying at the top of a grass stem with its proboscis fastened to the substrate and its abdomen well raised by an atypical disposition of the legs appears to be an ideal launch pad for maximizing the effective area covered by the conidial shower (see Krasnoff et al., 1995 for more details). Second, the pathogen also benefits indirectly from the sexual attractiveness of female cadavers. Males attempting necrophilous copulations invariably become infected with the

Ecosmart Biorational Insecticides: Alternative Insect Control Strategies 43

juveniles release bacteria that live symbiotically within the nematode's gut. The nematodebacterium relationship is highly specific: only *Xenorhabdus* spp. bacteria co-exist with steinermatids, and only *Photorhabdus* bacteria co-exist with heterorhabditids. Under optimal conditions, it takes 3–7 days for steinernemadtids and heterorhabditids to complete one life cycle inside a host from egg to egg. Emergence of infective juveniles from the host requires about 6–11days for steinernemadtids and 12–14 days for heterorhabditids (Kaya & Koppenhöfer, 1999). The ability of any biological control nematode to infect a particular insect can be affected by nematode and insect behavior, physical barriers, and immune

Over 30 species of beneficial nematodes have been identified. Seven species have been commercialized worldwide and seven are currently available in the United States: *Steinernema carpocapsae, S. feltiae, S. glaseri, S. riobravis, Heterorhabditis bacteriophora, H. megidis,* and *H. marelatus.* (Kaya & Koppenhöfer, 1999). There were two commercial

The use of the entomopathogenic nematodes *S. feltiae* and *H. megidis* in high numbers was shown to be effective against housefly larvae (Renn, 1995) and effective after formulation into a housefly bait, as effective as the spraying of a carbamate pesticide in pig units (Renn, 1998). Also the nematode *Paraiotonchium muscadomesticae* could infect housefly larvae, but mortality was low except at high nematode concentrations, but *P. muscadomesticae* infect housefly larvae and descendants of the nematodes invade and damage the ovaries of adult female flies and are deposited in the larval habitat when the flies attempt to oviposit. Infected adults lived about half as long as uninfected flies (Geden, 1997; Khater, unbuplished data); consequently, *P. muscadomesticae* reduce fly population indirectly. Nematodes were effective in the laboratory but persisted in manure only for 3-7 days. Moreover, mermithid nematodes which parasitize grasshoppers, cockroaches, and mosquitoes can play a part in natural suppression of pest insects. Nematodes must be raised

Entomopathogenic nematodes are not harmful to humans, animals, plants, or earthworms. They can therefore be used as biological control organisms. Such beneficial nematodes have been released extensively in the field with negligible effects on nontarget insects and are

Finally, microbial control agents provide a more environmentally acceptable and sustainable form of insect pest management than chemical insecticides, but are most effective when underpinned by a detailed knowledge of specific host-pathogen interactions. For more information about successful application of entompathgenic nematodes, see Mill et al. (2000), Gaugler (2002), Crow (2006), Hajek et al. (2007), Lacey & Kaya (2007), and Gilbert &

Genetically modified organisms (GMO) and crops (GMC) could be engineered to augment pest control. Transgenic organisms are genetically altered by artificial introduction of DNA

nematode products available for termite control, Spear ® and Saf T-Shield ®.

in live insects and this is not a very efficient means of production.

regarded as remarkably safe to the environment.

responses.

**3.4.3 Safety**

Gill (2010).

**4. Transgenic pesticides**

**3.4.2 Using beneficial nematodes**

fungus (Moller, 1993). Third, both field observations (Roffey, 1968) and controlled experiments (Bellini et al., 1992; Khater 2003, unpublished data) indicate that host mortality usually occurs in the late afternoon. This may be a temporal manipulation serving to enhance the likelihood that conidia produced from the dead host will be released at a time when the possibility for germination is highest, after dark when dew has set and surface moisture has had a chance to accumulate. The presumed adaptive payoff of elevation and posture for the fungus enhances dispersal of propagules.

#### **3.3.5 Safety**

Concerning the safety of entomopathogenic fungi developed for commercial use in microbial control of insect pests. Most fungi showed no infectivity to man or other vertebrates. Safety tests with *Nomuraea rileyi* , *Hirsutella thompsonii , Verticillium lecnii,* and *L. giganteum* assured negative findings to different mammals and birds. On the other hand, *B. bassiana* has been reported to cause allergies in humans and is at least an opportunistic pathogen to man and other mammals. *B. bassiana* and *M. anisopliae* affect non-target invertebrates with various degrees; see El-Husseini (2006) for more details. Soil treatment with *M. anisopliae* and *B. bassiana* on *Tapinoma nigerrimum* colonies indicated that there were no significant differences in ant, as a non- target host, activity before and after fungal treatment (Garrido-Jurado et al., 2011). For more information about entomophathogenic fungi, see Hakjek et al. (2007), Lacey & Kaya (2007), Gilbert & Gill (2010), Singh et al. (2011), and van Emden & Service (2001). For using of fungi for controlling arthropods of medical and veterinary importance, see Steenberg et al. (2001), Samuels, et al. (2002), Khater (2003), Kirkland et al. (2004), Schotle et al. (2004), Hartelt et al. (2008), Zabalgogeazcoa et al. (2008), Mochi et al. (2010), and Stephen & Kurtböke (2011).

#### **3.4 Nematodes**

Entomopathogenic nematodes cause damage to soilborne insect pests. Nematodes from the families Steinernematidae and Heterorhabditidae have proven to be the most effective as biological control organisms (Lacy & Kaya, 2007) to control a wide range of insect pests including filth flies, German cockroaches, cat fleas, armyworms, carpenter worms, crown borers, cutworms, flea beetles, leaf miners, mole crickets, phorid flies, plume moths, root weevils, sciarid flies, stem borers, webworms, and white grubs (Smart, 1995).

#### **3.4.1 Life cycle**

Generally speaking, the life cycle of most nematodes includes an egg stage, four juvenile stages, and an adult stage. The third juvenile stage, dauer, is the only infective and freeliving stage which is capable of surviving in the soil; its function is to locate, attack, and infect an insect host through its breathing holes, mouth, or anus, but some species are capable of penetrating thin areas of the insect's cuticle. After that, the nematodes release special bacteria into the insect. The toxins produced by the bacteria kill the insect after a few days. The bacteria multiply inside the body of the insect and the nematodes eat the bacteria. The nematodes mature, mate, and multiply inside the insect. Eventually, the insect's body becomes filled with nematodes. Infective stage nematodes then exit the insect body searching of other insects to infect. Once inside the body cavity of the host, the infective juveniles release bacteria that live symbiotically within the nematode's gut. The nematodebacterium relationship is highly specific: only *Xenorhabdus* spp. bacteria co-exist with steinermatids, and only *Photorhabdus* bacteria co-exist with heterorhabditids. Under optimal conditions, it takes 3–7 days for steinernemadtids and heterorhabditids to complete one life cycle inside a host from egg to egg. Emergence of infective juveniles from the host requires about 6–11days for steinernemadtids and 12–14 days for heterorhabditids (Kaya & Koppenhöfer, 1999). The ability of any biological control nematode to infect a particular insect can be affected by nematode and insect behavior, physical barriers, and immune responses.

#### **3.4.2 Using beneficial nematodes**

42 Insecticides – Advances in Integrated Pest Management

fungus (Moller, 1993). Third, both field observations (Roffey, 1968) and controlled experiments (Bellini et al., 1992; Khater 2003, unpublished data) indicate that host mortality usually occurs in the late afternoon. This may be a temporal manipulation serving to enhance the likelihood that conidia produced from the dead host will be released at a time when the possibility for germination is highest, after dark when dew has set and surface moisture has had a chance to accumulate. The presumed adaptive payoff of elevation and

Concerning the safety of entomopathogenic fungi developed for commercial use in microbial control of insect pests. Most fungi showed no infectivity to man or other vertebrates. Safety tests with *Nomuraea rileyi* , *Hirsutella thompsonii , Verticillium lecnii,* and *L. giganteum* assured negative findings to different mammals and birds. On the other hand, *B. bassiana* has been reported to cause allergies in humans and is at least an opportunistic pathogen to man and other mammals. *B. bassiana* and *M. anisopliae* affect non-target invertebrates with various degrees; see El-Husseini (2006) for more details. Soil treatment with *M. anisopliae* and *B. bassiana* on *Tapinoma nigerrimum* colonies indicated that there were no significant differences in ant, as a non- target host, activity before and after fungal treatment (Garrido-Jurado et al., 2011). For more information about entomophathogenic fungi, see Hakjek et al. (2007), Lacey & Kaya (2007), Gilbert & Gill (2010), Singh et al. (2011), and van Emden & Service (2001). For using of fungi for controlling arthropods of medical and veterinary importance, see Steenberg et al. (2001), Samuels, et al. (2002), Khater (2003), Kirkland et al. (2004), Schotle et al. (2004), Hartelt et al. (2008), Zabalgogeazcoa et al. (2008),

Entomopathogenic nematodes cause damage to soilborne insect pests. Nematodes from the families Steinernematidae and Heterorhabditidae have proven to be the most effective as biological control organisms (Lacy & Kaya, 2007) to control a wide range of insect pests including filth flies, German cockroaches, cat fleas, armyworms, carpenter worms, crown borers, cutworms, flea beetles, leaf miners, mole crickets, phorid flies, plume moths, root weevils, sciarid flies, stem borers, webworms, and white grubs