BIOLOGICAL AND BIOTECHNOLOGICAL CONTROL OF INSECT PESTS - CHAPTER 5 ppt

Beckage CONTENTS 5.1 Overview 5.2 Ecdysteroid Agonists 5.3 Juvenile Hormone Agonists 5.4 Azadirachtin References 5.1 OVERVIEW Insect growth regulators IGRs are insecticides that mimic th

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CHAPTER 5 Insect Growth Regulators

Nancy E Beckage

CONTENTS

5.1 Overview 5.2 Ecdysteroid Agonists 5.3 Juvenile Hormone Agonists 5.4 Azadirachtin

References

5.1 OVERVIEW

Insect growth regulators (IGRs) are insecticides that mimic the action of hor-mones on the growth and development of insect pests The advantages of using IGRs instead of classic insecticides in pest control include their reduced toxicity to the environment and their specificity for insects Moreover, because many IGRs act specifically on target pests such as Lepidoptera they show minimal toxicity for beneficial parasites and predators

Williams (1967) was the first to suggest that insect hormones might be utilized

as insect-specific environmentally benign pesticides, and Staal (1982) presented a review of these strategies Mimics of ecdysteroids (molting hormones) and juvenile hormones are two classes of hormone-based pesticides that have been developed for commercial use in insect pest control Azadirachtin, extracted from neem seeds, also appears to disrupt growth and molting in a large number of insect species These three classes of compounds will be discussed in this review

Insect growth and development are regulated by a combination of hormones, including juvenile hormone, ecdysteroids, and several neuropeptides including eclo-sion hormone and ecdysis triggering hormone (see Nijhout, 1994; Riddiford, 1994; Horodyski, 1996; Zitnan et al., 1996) During early larval life, the JH titer is main-tained at a high level, and growth is interrupted by periodic molts that follow the release of 20-hydroxyecdysone by the prothoracic glands The production of ecdysteroids occurs in response to the release of prothoracicotropic hormone from neurosecretory cells localized in the brain and retrocerebral complex Following the production of a new cuticle during molting, eclosion hormone is released, followed

by release of ecdysis-triggering hormone, and the insect sheds the exuvium In Lepidoptera, the JH titer descends to nondetectable levels in the last larval instar,

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providing a signal for release of a small prewandering ecdysteroid peak and a switch from larval to pupal commitment A larger prepupal peak of ecdysteroids, which occurs in the presence of a low titer of JH, then stimulates synthesis of pupal cuticle and the insect makes the transition to the pupal stage Following pupation, ecdys-teroid is again produced to cause formation of adult structures, and the pupal cuticle

is shed at adult eclosion JH then plays a prominent role in the reproduction of many species

Due to the prominence of hormones in the insect life cycle, the administration of hormone agonists and antagonists has disruptive effects on growth and metamorpho-sis One disadvantage of using some hormonally based IGRs, however, is the narrow window of sensitivity during which they must be administered to have any discernable effect on development This can be avoided by the use of compounds with a longer

in vivo half-life, so that the concentration of compound within the animal remains high during the sensitive period Also, some stages of insects may be sensitive to an IGR, whereas other stage(s) are refractory There may be a long lag period between administration of the compound and the observed induction of disruptive effects Thus, it is sometimes difficult to achieve rapid knockdown of an insect population with IGR-based pesticides However, the rapidity with which an insect responds varies with the compound Juvenile hormone agonists may be utilized for control in the long term to disrupt metamorphosis, whereas ecdysone agonists act very quickly (within 24 hours) to trigger an unsuccessful molt Azadirachtin frequently has an immediate antifeedant effect that later disrupts molting or reproduction

Aside from acting as pesticides, hormonally based IGRs are important research tools for the study of hormone action (Oberlander et al., 1995) They offer new insights into how hormones regulate insect growth and development

5.2 ECDYSTEROID AGONISTS

In recent years several nonsteroidal bisacylhydrazine ecdysone agonists have been synthesized by Rohm and Haas (Figure 5.1, compounds 1–5) These chemicals are much more potent than the native hormone 20-hydroxyecdysone in inducing molting They also reduce feeding and weight gain In lepidopteran insects, a lethal molt is induced following administration of the ecdysone agonist, and the animal dies trapped within the exuvial cuticle Feeding stops 4–16 hours after ingestion of toxic doses of the agonist, and molting is initiated in the absence of an ecdysteroid increase, as Wing et al (1988) demonstrated using isolated abdomens that lacked prothoracic glands Usually the animal dies in the slipped head capsule stage fol-lowing onset of apolysis (Dhadialla et al., 1998) However, as described by Oberlander et al (1995), supernumerary larval molts may also occur (Gadenne et al., 1990; Musynska-Pytel et al., 1992) when the JH titer is high

Ecdysteriod agonists have been shown to act in many lepidopterans including

Manduca sexta (Wing et al., 1988), Plodia interpunctella (Silhacek et al., 1990),

Spodoptera littoralis (Smagghe and Degheele, 1992), Spodoptera litura (Tateishi

et al., 1993), Spodoptera exempta (Smagghe and Degheele, 1994a), Spodoptera

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exigua (Smagghe and Degheele, 1994b), and Pieris brassicae (Darvis et al., 1992) RH-5992 (tebufenozide) is more toxic to lepidopteran larvae than RH-5849 (Dhadialla et al., 1998) The compound RH-0345 has a different activity profile and acts on scarabid beetle larvae, cutworms, and webworms (Dhadialla et al., 1998) RH-2485 shows promise because it is more potent than tebufenozide against lepi-dopteran pests of corn (Trisyono and Chippendale, 1997), and cotton (Ishaayaet al., 1995) (see Dhadialla et al., 1998 for review)

Lepidopteran, dipteran, and coleopteran larvae are the primary insects affected

by these ecdysone agonists All larval stages are affected, but the effect induced depends on when during an instar the insect ingests or is treated with the compound

An immediate lethal molt is induced if treatment occurs early in an instar, but if the insect is treated toward the end of an instar, first a normal molt will occur, which

is then followed by the lethal molt (Dhadialla et al., 1998) In adult stage insects, egg production and spermatogenesis may be deleteriously affected by exposure to bisacylhydrazines (Smagghe and Degheele, 1994a; Carpenter and Chandler, 1994) These compounds appear to interact with the ecdysteroid receptor complex (Wing, 1988) and thereby induce their effects Tebufenozide competes with tritiated ponasterone A for binding to ecdysteroid receptors, which are EcR/ultraspiracle

Figure 5.1 Structures of 20-hydroxyecdysone and the bisacylhydrazine ecdysteroid agonists.

Reprinted with permission from Dhadialla et al (1998).

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heterodimers, indicating that the compound acts as an ecdysteroid mimic at the molecular level Experiments using cells (e.g., Drosphila Kc cells — see Wing, 1988; or Chironomus cells — see Spindler-Barth et al., 1991) or tissues cultured

in vitro indicate that the bisacylhydrazines have the same mode of action as 20-hydroxyecdysone, but the effects are much longer lasting compared to 20-HE (Retnakaran et al., 1995) Even though these ecdysone agonists are not steroids, they act similar to 20-HE in causing wing disc eversion (Silhacek et al., 1990; Smagghe

et al., 1996) and other physiological responses, such as initiation of spermatogenesis (Friedlander and Brown, 1995) or adult development in diapausing pupae (Sielezniew and Cymborowski, 1997) that are normally induced by 20-HE The high binding affinity of tebufenozide and RH-2485 to proteins in nuclear extracts of lepidopteran cells is correlated with their selective action on lepidopteran insects (Dhadialla et al., 1998) By contrast, the ecdysteroid receptors of coleopteran insects bind RH 5992 with low affinity (Dhadialla and Tzertzinis, 1997) This difference thereby explains the specificity of this compound for lepidopteran insects The tomato moth Lacanobia oleacea rapidly metabolizes ingested 20-hydroxy-ecdysone, but is susceptible to the ecdysteroid agonists RH-5849 and RH-5992 and undergoes a lethal larval molt (Blackford and Dinan, 1997) This insect feeds on a variety of weeds containing high concentrations of phytoecdysteroids in addition to tomato Blackford and Dinan (1997) propose that the phytoecdysteroids may be rapidly detoxified by conjugation with long-chain fatty acids and excreted in an inactive form, while the ecdysteroid agonists exhibit enhanced in vivo stability

In Spodoptera littoralis, application of RH-5849 delays the onset of wandering

in a dose-dependent manner (Pszczolkowski and Kuszczak, 1996) These results suggest that not only an increase, but also a decrease in ecdysteroid levels appears

to be vital for wandering to be initiated at the normal time

Due to the selectivity of tebufenoxide and RH-2485 for Lepidoptera, the chem-icals can be used without the risk of direct deleterious effects on beneficial species, although there may be indirect effects on parasitoids due to the death of the host Fifth instar Manduca sexta larvae that are injected with 10 µg RH-5849 in DMSO slip their head capsules and later have few or no emerging Cotesia congregata

parasitoids (N.E Beckage and F.F Tan, unpubl data) Brown (1994) analyzed the effects of tebufenoxide on two parasitoids, Ascogaster quadridentata, an endopar-asitoid, and Hyssopus sp., an ectoparasitoid, and assessed its effect on the host–par-asitoid interaction In general, ectoparhost–par-asitoids are less affected by hormonally active IGRs compared to endoparasitoids, which are constantly exposed in vivo to the IGR Both tebufenoxide and RH-2485 are highly active in the field against a variety

of vegetable, fruit, and ornamental pests (Dhadialla et al., 1998), indicating that the compounds have the potential for wide application Importantly, the compounds exhibit minimal toxicity to vertebrates; thus they can be safely utilized in the field without adverse effects on human and animal health

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5.3 JUVENILE HORMONE AGONISTS

The agonists of juvenile hormone include compounds such as methoprene and hydroprene, which are terpenoids, and nonterpenoids such as fenoxycarb and pyriproxyfen (Figure 5.2) In order for metamorphosis to occur in holometabolous insects, JH must descend to nondetectable levels so that ecdysteroid is released in the absence of JH and the switch from larval to pupal commitment occurs (Riddiford, 1994) In hemimetabolous insects, the JH titer must decrease to permit molting to the adult form If the JH titer is maintained at too high a level, due to administration

of a JH agonist in the larval or nymphal stage, then molting to a supernumerary instar or an intermediate (larval–pupal, nymphal–adult, or pupal–adult) is induced Such intermediates are nonviable The supernumerary instars that form may have morphological anomalies, which likewise prevent the animal from completing a normal metamorphosis

JH agonists are highly effective insect growth regulators that cause a wide range

of developmental derangements in susceptible species, affecting embryogenesis, larval development, metamorphosis, and reproduction The JH agonists have low acute toxicity for fish, birds, and mammals (Grenier and Grenier, 1993; Dhadialla

et al., 1998), indicating their use is safe for the environment compared to conven-tional neurotoxic pesticides Indeed, one problem associated with their use is their relative lability, and sustained release formulations are often required to ensure that

Figure 5.2 Structures of the juvenile hormones and juvenile hormone agonists Reprinted with

permission from Dhadialla et al (1998).

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treatment occurs during the window of sensitivity of the pest (e.g., mosquitoes) to

be controlled

Methoprene disrupts embryogenesis and egg hatch as well as larval development

in fleas, and kills mosquitoes as larvae and as pupae prior to adult eclosion Meth-oprene administered to mosquito pupae prevents rotation of the male genitalia (O’Donnell and Klowden, 1997) In houseflies and other Diptera, adult emergence may be prevented by earlier treatment with methoprene, and methoprene adminis-tered as a feed additive to cattle controls horn flies and other veterinary pests that breed in dung Methoprene acts to terminate adult reproductive diapause in houseflies (Kim and Krafsur, 1995) and induce reproductive development It also acts on Lepidoptera to cause molting to supernumerary instars and formation of intermedi-ates Kinoprene is active on whiteflies and other species of Homoptera

More recently, nonterpenoidal JH mimics such as fenoxycarb and pyriproxyfen have appeared on the market These usually have greater stability in the environment, and are more active in vivo compared to the terpenoid IGRs

Fenoxycarb is a non-neurotoxic carbamate with a high level of JH-like activity

in a wide range of insects, including Heteroptera, Lepidoptera, Hymenoptera, Coleoptera, Diptera, Dictyoptera, Isoptera, and Homoptera (see Grenier and Grenier,

1993 for review) The administration of fenoxycarb has been reported to cause sterility, kill insect eggs, interfere with metamorphosis, induce permanent larvae, cause formation of nonviable intermediates, and affect caste differentiation Ultralow doses of fenoxycarb (100 picograms) induce permanent larvae in the silkworm

Bombyx mori (Monconduit and Mauchamp, 1998), which have inactive prothoracic glands Incubation of prothoracic glands in the presence of fenoxycarb reduces production of ecdysteroid by the glands (Dedos and Fugo, 1996) Larval growth and food consumption is reduced following the administration of fenoxycarb in B mori

(Leonardi et al., 1996) Fenoxycarb appears to qualitatively affect the biosynthesis

of fatty acids by the fat body in the Eastern spruce budworm Choristoneura fumifer-ana (Mulye and Gordon, 1993)

Moreno et al (1993a,b) investigated the effects of fenoxycarb on the parasitoid

Phanerotoma ocularis and found signficant adverse effects on parasitoid develop-ment whether the compound was applied via the host (Moreno et al., 1993a) or directly to the parasitoid (Moreno et al., 1993b) Development of the tachinid

Pseudoperichaeta nigrolineata is also disrupted by fenoxycarb applied to its host (Grenier and Plantevin, 1990) Other JH agonists such as methoprene may also deleteriously affect the emergence and metamorphosis of parasitoids (Beckage and Riddiford, 1982), so hormonally active IGRs may have some adverse effects on parasitoid populations but overall the effects seem less detrimental compared to the effects of conventional neurotoxic pesticides

Pyriproxyfen is another potent JH agonist that is active in a wide range of arthropods, including ants (Vail and Williams, 1995; Vail et al., 1996), fleas (Palma

et al., 1993), mole crickets (Parkman and Frank, 1998), mosquitoes (Kono et al., 1997), flies (Hargrove and Langley, 1993; Bull and Meola, 1993), whiteflies (Ishaaya

et al., 1994; Ishaaya and Horowitz, 1995), scales (Peleg, 1988), cockroaches (Koehler and Patterson, 1991; Lim and Yap, 1996), and ticks (Teal et al., 1996; Donahueet al., 1997), as well as lepidopterans (Smagghe and Degheele, 1994b) As

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seen with other JH agonists, multiple effects are induced in a single species The compound interferes with embryogenesis, disrupts the metamorphic molt, and causes morphological deformities in the desert locust, Schistocerca gregaria (Vennard et al., 1998) In the tobacco cutworm, Spodoptera litura, topical application of 0.3 ng pyroproxyfen to day 0 female pupae reduces the number of eggs oviposited, because the treated females lack an oviposition-stimulating factor, which is necessary for the deposition of eggs (Hatakoshi, 1992) Thus, reproduction may be inhibited through oviposition inhibition as well toxic effects on developing oocytes following treatment with pyriproxyfen

Similar to methoprene, pyriproxyfen fed to cattle and other animals has an insecticidal effect on the subsequent emergence of horn flies, face flies, and house-flies from manure (Miller, 1989; Miller and Miller, 1994) Pyriproxyfen also disrupts development of horn flies following direct application to the fly (Bull and Meola, 1993)

In the Colorado potato beetle, pyriproxyfen inhibits expression of diapause protein 1, and induces expression of vitellogenin following application to short-day adults destined to diapause (De Kort et al., 1997) In last instar nymphs, the com-pound prevents metamorphosis at low doses Pyriproxyfen also induces synthesis

of vitellogenin in Locusta migratoria (Edwards et al., 1993) and inhibits the syn-thesis of larva-specific hemolymph proteins (De Kort and Koopmanschap, 1991) Pyriproxyfen resistance has been generated in houseflies selected for 17 gener-ations for resistance (Zhang and Shono, 1997; Zhang et al., 1997; Zhang et al., 1998)

In these flies, cytochrome P450 monooxygenase enzymes in the fat body and gut play critical roles in the metabolism of pyriproxyfen (Zhang et al., 1998) Resistance

to pyriproxyfen has also been reported in the homopteran Bamesia tabaci (Horowitz and Ishaaya, 1994) This is not surprising, given that resistance to other JH agonists such as methoprene can also be induced (Turner and Wilson, 1995)

5.4 AZADIRACHTIN

Azadirachtin is a tetranortriterpenoid present in seeds from the Indian neem tree,

Azadirachta indica This compound was originally isolated by Butterworth and Morgan (1968) and its structure was subsequently described (Zanno et al., 1975; Kraus et al., 1985) Azadirachtin has strong antifeedant and repellant activity (Ascher, 1993; Simmonds and Blaney, 1996) and has pleiotropic effects on growth, develop-ment, and reproduction (Schmutterer, 1990; Mordue (Luntz) and Blackwell, 1993)

In contrast to the wealth of information we have about its effects on development, its biochemical effects at the cellular and molecular levels are still barely known

A wide range (>200 species; Ascher, 1993) of both chewing and sucking phy-tophagous insects, and stored product pests have been shown to be affected by azadirachtin Insects that are susceptible include aphids, lepidopterans, hemipterans, cockroches, beetles, and orthopterans Azadirachtin is taken up systemically and translocated into the tissues of treated plants (Arpaia and Van Loon, 1993), in addition to affecting insects via the leaf surface or by direct contact with the target pest Aquatic species such as mosquitoes are also affected (Mordue (Luntz) and

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Blackwell, 1993) While neem derivatives have been reported to provide broad-spectrum control of pest species, they appear less toxic to natural enemies of insect pests than to the pests themselves (Schmutterer, 1990; Banken and Stark, 1997) The activity of azadirachtin is due to a complex combination of antifeedant and toxic properties that affect growth, molting, and reproduction (see Schmutterer, 1990, Ascher, 1993, and Mordue (Luntz) and Blackwell, 1993 for reviews) Aside from its insecticidal properties, it also exhibits nematocidal, antiviral, and antifungal properties (Mordue (Luntz) and Blackwell, 1993) Protozoa (e.g., malaria parasites) are also deleteriously affected (Jones et al., 1994) by azadirachtin

Azadirachtin’s antifeedant properties to some extent reflect its action on gustatory chemoreceptors and its activity in suppressing food consumption has been reported

in numerous species (Mordue (Luntz) and Blackwell, 1993) However, topical appli-cation of azadiractin also has potent effects, indicating a nongustatory pathway also

is involved in its mechanism of action Second, azadirachtin affects ecdysteroid and juvenile hormone titers, resulting in severe growth and molting aberrations The neuropeptides regulating ecdysteroid and JH production may be affected Disruption

of molting leads to formation of larval–pupal, nymphal–pupal, nymphal–adult, and pupal–adult intermediates Formation of permanent larvae is also observed Third,

it has direct detrimental effects on many insect tissues such as endocrine glands, muscles, fat body, and gut epithelial cells Effects on reproduction include effects

on spermatogenesis in males, and fecundity and oviposition in females Also, lon-gevity of adult males or females may be reduced

Azadirachtin is a growth retardant in Periplaneta americana and reduced inges-tion of food in the immature stages results in smaller adults, which exhibit a decrease

in the rate of reproduction relative to untreated control insects (Richter et al., 1997) Effects on egg viability have been reported in many species

Reproductive effects have been noted in Orthoptera, Heteroptera, Homoptera, Hymenoptera, Coleoptera, Lepidoptera, and Diptera (Schmutterer, 1990) In females, azadirachtin treatment may result in total sterility Degenerative changes are seen in the follicle cells as well as the oocytes themselves Observations include a separation

of the follicle from the oocyte, and lack of vitellogenin in both fat body and hemolymph as well as in developing oocytes The effects of azadirachtin may be rescuable by treatment with juvenile hormone (Sayah et al., 1996) Azadirachtin inhibits the growth of oocytes of Rhodnius prolixus when administered in a blood meal, and phospholipid transfer from lipophorin to the developing oocytes is inhib-ited (Moreira et al., 1994)

In males, effects are seen on testes development and spermatogenesis, resulting

in a decrease in fertility (Dorn, 1986; Shimizu, 1988) Injection of azadirachtin inhibits the growth of testes in the desert locust, Schistocerca gregaria, and sperm meiosis is halted at metaphase I (Linton et al., 1997) Dihydrozadirachtin binds sperm in the testes and reduces sperm motility in the locust Schistocerca gregaria

(Nisbet et al., 1996)

In the African armyworm Spodoptera exempta, a highly damaging pest of cereal crops in Africa, azadirachtin treatment lowers food intake, efficiency of conversion

of ingested food, and efficiency of conversion of digested food to body mass (Tanzubil, 1995) At 10 µg per insect, growth is drastically curtailed In this

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iment, azadirachtin was applied topically, confirming that the compound can interfere with feeding via a nonsensory mechanism

Aside from affecting feeding, azadirachtin deleteriously affects the gut itself Azadirachtin disrupts the ultrastructural organization of the epithelial cells lining the midgut of Rhodnius prolixus (Nogueira et al., 1997) Changes include clustering

of the microvilli, disorganization of the extracellular membrane layers, and alter-ations in the basal portion of the epithelial cells The midgut is also disrupted in the locusts Schistocerca gregaria and Locusta migratoria, and the effects are distinctly different from those induced by starvation (Nasiruddin and Mordue, 1993) Salannin, which is also extracted from neem seeds, deters feeding, delays molting

by increasing larval duration, causes larval and pupal mortalities, and decreases pupal weights of Spodoptera litura (Govinachari et al., 1996) Smaller pupae yield smaller adults with reduced fecundity

Azadirachtin affects the ultrastructure of the ring gland of Lucilia cuprina

(Meurant et al., 1994) The ring gland is comprised of the prothoracic gland, corpus cardiacum, and corpus allatum Changes seen include crenulation of nuclear shape, clumping of the heterochromatin, and pyknosis The observed effects likely contrib-ute to a generalized disturbance of neuroendocrine function Rembold et al.(1989) reported that azadirachtin targets the corpus cardiacum and causes disruption of its function Ecdysis may be inhibited (Kubo and Klocke, 1982; Garcia and Rembold, 1984; Arpaia and Van Loon, 1993), suggesting that the production of eclosion hormone (Horodyscki, 1996) or ecdysis triggering hormone (Zitnan et al., 1996) may be affected Levels of ecdysteroids and prothoracicotropic hormone are altered following treatment with azadirachtin (Barnby and Klocke, 1990) Thus, the observed developmental disturbances likely reflect changes caused in the endocrine glands of treated insects, and consequently hormone titers

Azadirachtin, along with three other neem seed compounds, inhibits ecdysone 20-monooxygenase in a dose-dependent manner, indicating ecdysteroid metabolism

is slowed (Mitchell et al., 1997) by these agents Metabolism of hormones as well

as their synthesis may be disrupted, contributing to alterations in the endocrine milieu

Azadirachtin binds to the nuclei of Sf9 cells in culture, and binding is irreversible and saturable (Nisbet et al., 1997) Isolated cells in culture can therefore be used to study the mechanism of action of azadirachtin

When tested in combination with gypsy moth NPV, azadirachtin plus NPV was significantly more effective in killing gypsy moth larvae compared to NPV alone or azadirachtin only (Cook et al., 1996) The combination of azadirachtin and virus was predicted to result in good foliage protection if used against gypsy moth larvae However, the addition of azadirachtin to viral formulations might also result in less virus being produced within the larval cadaver and released into the environment because the treated larvae are smaller (Cook et al., 1996)

Aside from the IGRs discussed here, several other insect growth regulators are presently being utilized in pest control, including chitin synthesis inhibitors (e.g., benzoylphenyl ureas; Cohen, 1993) and other compounds such as cyromazine, which act on cuticle deposition (Venuela and Budia, 1994), Chitinases also offer

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nities for development of new biopesticides (Kramer and Muthukrishnan, 1997) Insect chitinase genes can be expressed in plants, thereby disrupting the feeding and growth of the insect feeding upon the plant, or expressed in insect baculoviruses, enhancing the insecticidal activity and speed of kill of the viruses (Gopalakrishnan

et al., 1995) Insect neuropeptides also offer potential for development of new insec-ticides similar to the hormonally based IGRs that have already been discovered and utilized in pest control (Masler et al., 1993)

REFERENCES

Arpaia, S and J.J.A Van Loon Effects of azadirachtin after systemic uptake into Brassica oleracea on larvae of Pieris brassicae Entomologia Experimentalis et Applicata 66, 39–45, 1993.

Ascher, K.R.S Nonconventional insecticidal effects of pesticides available from the neem tree, Azadirachta indica Archives of Insect Biochemistry and Physiology 22, 433–449, 1993.

Banken, J.A.O and J.D Stark Stage and age influence on the susceptibility of Coiccinella septempunctata (Coleoptera: Coccinellidae) after direct exposure to Neemix, a neem insecticide. Journal of Economic Entomology 90, 1102–1105, 1997.

Barnby, M.A and J Klocke Effects of azadirachtin on levels of ecdysteroids and prothorac-icotropic hormone-like activity in Heliothis virescens (Fabr.) larvae Journal of Insect Physiology 36, 125–131, 1990.

Beckage, N.E and L.M Riddiford Effects of methoprene and juvenile hormone on larval ecdysis, emergence, and metamorphosis of the endoparasitic wasp, Apanteles congregatus Journal of Insect Physiology 28, 329–334, 1982.

Blackford, M and L Dinan The tomato moth Lacaobia oleracea (Lepidoptera: Noctuidae) detoxifies ingested 20-hydroxyecdysoine, but is susceptible to the ecdysteroid agonists RH-5849 and RH-5992 Insect Biochemistry and Molecular Biology 27, 291–309, 1997 Brown, J.J Effects of a nonsteroidal ecdysone agonist, tebufenozide, on host/parasitoid interactions Archives of Insect Biochemistry and Physiology, 235–248, 1994.

Bull, D.L and R.W Meola Effect and fate of the insect growth regulator pyriproxyfen after application to the horn fly (Diptera: Muscidae) Journal of Economic Entomology 86, 1754–1760, 1993.

Butterworth, J.H and E.D Morgan Isolation of a substance that suppresses feeding in locusts.

J Chem Soc., Chem Commun., 23–24, 1968.

Carpenter, J.E and L.D Chandler Effects of sublethal doses of two insect growth regulators

on Heliocoverpa zea (Lepidoptera: Noctuidae) reproduction Journal of Entomological Science 29, 425–435, 1994.

Cohen, E Chitin synthesis and degradation as targets for pesticide action Archives of Insect Biochemistry and Physiology 22, 245–261, 1993.

Cook, S.P., R.E Webb, and K.E Thorpe Potential enhancement of the gypsy moth (Lepi-doptera: Lymantriidae) nuclear polyhedrosis virus with the triterpene azadirachtin.

Environmental Entomology 25, 1209–1214, 1966.

Darvis, B., L Polgar, M.H Tag El-Din, E Katakin, and K.D Wing Developmental distur-bances in different insect orders caused by an ecdysteroid agonist, RH 5849 Journal of Economic Entomology 85, 2107–2112, 1992.

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