Parasitol Res (1992) 78 : 89-95

Parasitology Research 9 Springer-Verlag1992

Review article

Endocrine strategies for the control of ectoparasites and insect pests Margarethe Spindler-Barth Institut fiir Zoologie,Heinrich-Heine-Universit/itDiisseldorf,Universitfitsstrasse1, W-4000 Dfisseldorf1, Federal Republic of Germany Accepted September 1, 1991

Abstract. The increasing knowledge about endocrine mechanisms in arthropods facilitates the biorational search for drugs against insect pests and parasites that interfere with arthropod hormone action. Juvenile hormone mimics have been successfully applied for about 20 years; however, resistance to juvenile hormone analogues has developed. The introduction of moulting hormone agonists, which compete for binding to the ecdysteroid receptor, is expected in the near future. Despite the considerable progress that has been achieved in peptide hormone research during the last few years, no successful insecticide is currently available, although comparisons of drugs for medical use demonstrate that in principle, successful interference with peptide hormone action is possible. The search for new drugs has been facilitated by advances in cell-culture techniques, which improve the development of suitable screening systems, and by progress in genetic engineering, which could be an important tool in the creation of new strategies for insect pest control.

With the exception of schistosomiasis, all important human parasitic diseases are transmitted by insects that serve either as intermediate or final hosts, as true vectors of pathogens, or as mechanical vehicles of parasites. In addition, insects and mites are important ectoparasites in domestic animals and can also cause endoparasitic diseases such as myasis. The increase in parasitic diseases of humans and animals as well as in human health problems and the need for effective plant protection have advanced efforts in the "struggle against parasites" as outlined by Mehlhorn (1988). New strategies for insect vector and pest control (Casida 1990) are necessary for the following reasons: 1. Resistance to the main insecticides in current use poses a problem even for the use of compounds that have recently been introduced, e.g. pyrethroids (Hutson and Roberts 1985).

2. Concern about environmental problems demands ecologically acceptable approaches. 3. Lower toxicity against vertebrates is mandatory. 4. Empirical screening programs have become considerably less efficient. Several strategies using biorational approaches are presently being applied, including integrated pest management, the use of pheromones, sterilization of male insects (SIT), and the use of predators or insect pathogens such as Bacillus thuringiensis (Kerkut and Gilbert 1985; Casida 1990). Inhibition of insect-specific metabolic pathways such as the synthesis of chitin (Spindler et al. 1990) has been accomplished using benzoylphenylureas (Wright and Retnakaran 1987). Chitin degradation may be an additional target, as may cuticle formation in general. Interference with the endocrine system of arthropods is another promising approach, since most invertebrate hormones differ from those of vertebrates. Indeed, as early as in 1967, Williams introduced this idea of using insect growth regulators as "third-generation insecticides." This strategy has also been considered to be useful against parasitic helminths (Spindler 1988) due to the low vertebrate toxicity of ecdysteroids and juvenile hormones. In the last few years, advanced understanding of endocrine mechanisms of invertebrates, the availability of better test systems, and progress in molecular biology have led to the development of endocrine strategies for insect-vector and pest control. Since possible strategies involving the use of peptide hormones have recently been reviewed elsewhere (Kelly et al. 1990; Harrow et al. 1991 ; O'Shea 1991 ; Shaw and Johnston 1991), the present paper focuses mainly on juvenile hormones and ecdysteroids, which are important for the development of arthropods, and emphasizes the advantage of using insect cell cultures as test and screening systems in a biorational search for insect growth regulators.

90

external cues (e.g.photoperiod) internal cues (e.g. nutritional state)

I%jC

J

HOC

synthesis of neuropeptide hormones

[

Allatotropin (+) AIIostatin (-)

I corpora allata I

~

HO/J'~

R3

~

O

"CHs

CHR1 = OH, R2 = OH, R3 = H 20-OH-Ecdysone RI= H, R2 = OH, R3 = H Ecdysone

PTTH

1

[

prothoracic gland

0

l ecdysteroid synthesis

JH synthesis I

~

~

OH

I

I

juvenile hormones

ecdyaone, 3_dehyrro_ecdysone

transport

Juvmilehormone J~-I-m

transport (free or protein bound)

(JH carrier)

~'~

I"13C N / \ x o

inactivation

(JH esterase) l

11

0

uptake

RH 5849

I

I

H\C:=C ~

] target tissue ] Iintracellular receptors ]

I I I

H3CO O=C

uptake

\

~

/

Oxi/OH

,-c-~ / 1

H..H

~_",? ' - ~ - - A . / ~

/\convers,onto

/

inactivation

active ecdysteroids

--

o L J. l " C" > / " " /i" " o , .,c-c-o/,,

I

1

H$CO--C

excretion

0

Fig. 1. Generalized scheme of the regulation of juvenile (JH) and moulting hormone synthesis as part of a hierarchical system. PTTH, Prothoracicotropic hormone

',

~-

0 Azadirachtin

O

Basic features of the insect endocrine system Retinoic acid

In general, the same principles of endocrine regulation that are known in vertebrates also apply to invertebrates. As exemplified by ecdysteroids and juvenile hormones, the endocrine system exhibits a hierarchical organization that includes multiple feedback regulation and neurosecretory control of peripheral endocrine glands (Fig. 1). The occurrence of the two main classes of hormones that are involved in the regulation of development, juvenile hormones and ecdysteroids (Fig. 2), is restricted to invertebrates. Juvenile hormones are found only in insects and crustaceans (Cusson et al. 1991), whereas ecdysteroids have also been detected in helminths (Franke and K/iuser 1989). Some steroid hormones of the "vertebrate type" have also been observed in invertebrates, but their biological function is unknown (De Loof 1987). Due to the high-sequence homology of peptide hormones in invertebrates with those in vertebrates, a clear distinction of the two types is not possible (De Loof and Schoofs 1990; De Loof et al. 1990; Keller 1990). Although a considerable number of peptide hormones and of trophic and releasing factors have been characterized, new peptides with hormonal properties are continuously being detected due to the recent progress made in separation techniques. Arthropods exhibit hormone signal-transducing systems similar to those displayed by vertebrates. For example, the regulation of ecdysteroid

Fig. 2. Structures of some representative compounds discussed in

this review

synthesis by neurosecretory peptides is mediated via cyclic adenosine 5'-monophosphate (cAMP) and involves Ca 2§ and protein kinase C (Watson et al. 1989; Smith and Sedlmeier 1990); this is also the case for the endocrine regulation of diuresis in insects (Spring 1990). The hormonal signal of ecdysteroids and juvenile hormones is transduced by intracellular receptors (SpindlerBarth and Spindler 1987; Bidmon and Sliter 1990; Palli et al. 1991). These receptors belong to the large superfamily of ligand-activated transcription factors (Beato 1991), including all steroid hormone receptors in vertebrates, vitamin D3, retinoic acid, and thyroxin (Evans 1988). Juvenile hormones exhibit a dihomosequiterpenoid skeleton that shows some similarity to the nonsteroidal structure of retinoic acid (Fig. 2). Indeed, some retinoids exhibit weak juvenile-hormone-mimicking activity (Palli etal. 1991). For juvenile hormones, additional membrane recognition and effector sites have been described (Ilenchuk and Davey 1985, 1987; Yamamoto et al. 1988), whereas the existence of corresponding sites for ecdysteroids remains a matter of debate (Koolman and Spindler 1983; Spindler and Spindler-Barth 1989).

91 Due to the rigid exoskeleton of insects, their development from egg to adult involves several moults. The number of moults, the accompanying morphogenetic processes, and, to a certain degree, the hormonal regulation of moulting are typical for a given systematic group of insects, with the most complex and advanced regulation being found in holometabolic insects (Fig. 1). This essential feature of insect life, the moulting process, occurs under a rather complex process of regulation by several hormones and is also influenced by other cues, e.g., environmental ones (Ohnishi and Ishizaki 1990). As illustrated in Fig. 1, it is obvious that either interference with one of the steps in this cascade or a disturbance of the delicate balance between juvenile hormone and ecdysteroids that is typical for a certain developmental stage might be a suitable approach for insect pest control. In general, signal-transducing mechanisms at the postreceptor level seem to be similar in vertebrates and invertebrates. However, as to little is known about these mechanisms, a determination cannot be made as to whether they exhibit differences that would enable successful interference at this point of the endocrine system in arthropods. To date, efforts have been concentrated on the hormone receptor or prereceptor level, i.e., interference with the synthesis, degradation, and inactivation of hormones as well as with agonistic and antagonistic effects at the receptor level. Manipulation of hormone levels can also be achieved by the integration of genes coding for insect hormones or for hormone-metabolizing enzymes, among other substances, into an appropriate vector. In addition, the interruption of hormone action seems to be feasible through the development of antihormones that bind selectively to hormones and thus inactivate their action. Furthermore, the use of antisense DNA, which binds to m R N A coding for peptide hormones, is being considered. Peptide hormones In principle, there are various approaches for interfering with the action of peptide hormones. Although drugs for medical use have demonstrated the effectiveness of interference with peptide hormone synthesis, as has been shown for captopril (Ondetti et al. 1977), or with hormone action, as has been reported for a cholecystokinin mimic (Evans et al. 1986), and the efficacy of inhibition of hormone-degrading enzymes, e.g., that of the enkephalin-degrading enzyme by thiorphan (Roques et al. 1980), comparative compounds for use in arthropod pest control or as antiparasitic agents are not yet available. The structure of a considerable number of peptide hormones is known, but no peptide hormone receptor has thus far been isolated and characterized. The construction of synthetic binding molecules or antihormones that prevent the biological action of hormones requires extensive studies on structure-activity relationships. Most of such research has been conducted on the adipokinetic (AKH) and myotropic hormones of insects and has enabled receptor modelling (Golds-

worthy et al. 1990; Holman et al. 1990; Nachman et al. 1990). The peptide structure precludes the direct use of these hormones, even if they could be synthesized in sufficient quantities. Severe problems involving their stability, penetration, absorption, and sequence homology with vertebrate peptide hormones must be taken into account. However, drug targeting through the use of vectors (e.g., baculoviruses) in which the genes coding for peptide hormones, antihormones, or hormone-degrading enzymes are integrated seems feasible. Juvenile hormones Juvenile hormones or synthetic compounds exhibiting juvenoid activity have been successfully used for insect pest control for about 20 years (Retnakaran et al. 1985). Their lack of stability under field conditions and their sensitivity to esterolytic cleavage can be circumvented by the development of appropriate synthetic juvenoids. Due to the pleiotropic action of juvenile hormones' ovicidal and larvicidal effects, disturbances of the diapause, metamorphosis, and reproduction of insects as well as effects on their life span have been noted (Retnakaran et al. 1985; Shaaya and Spindler 1990; Riddiford and Ashburner 1991 ; Shaaya et al. 1991). However, the occurrence of giant larvae due to the metamorphosis-inhibiting action ofjuvenoids should be taken into account, whereby damage by larval feeding poses a problem. For this reason, a juvenile hormone antagonist would be preferable. However, the juvenile hormone receptor protein has thus for been only poorly characterized and the corresponding gene has not been identified. If this can be accomplished and the receptor can be expressed in vitro, the establishment of screening systems for intensive binding studies will be facilitated. Interference with the synthesis of juvenile hormones is also possible. Precocenes selectively destroy the corpora allata, the hormone gland in which juvenile hormone synthesis takes place, and thus act as anti-juvenile hormones. Due to the high doses required and to the finding that most holometabolous insects are not attacked by precocenes, these substances can be used as useful experimental probes but not as control agents in the field (Retnakaran et al. 1985). The same holds true for thiosemicarbazones (Barton et al. 1989) and for the fermentation product sinefungin, which competitively inhibits the synthesis of methyl farnesoate the penultimate step in juvenile hormone synthesis (Ferenz and Peter 1987). Attempts to interfere with juvenile hormone synthesis on the basis of endocrine neurosecretory factors currently remain speculative. Allostatins and allotropins that exert inhibitory or stimulatory action on the corpora allata (Fig. 1) have been identified, but it is too early to speculate on their potential in pesticide control. In some insect orders, the juvenile hormone titer is regulated mainly by a specific esterase. Although specific inhibitors of this enzyme occur at least in Lepidoptera, e.g., 3-octylthio-l,l,l,-trifluoropropan-2-one, their practical application as insecticides is not feasible, mainly due

92 to toxicological problems (Hammock et al. 1984). A further possibility would involve the integration of the esterase gene in vectors such as nuclear polyhedrosis viruses, which has successfully been accomplished (Hammock et al. 1990); however, the expression of the juvenile hormone esterase gene to date has not been sufficiently stable to ensure the continued success of this approach.

Ecdysteroids Due to the low vertebrate toxicity of ecdysteroids, the protection of vertebrate hosts seems to be possible. The use of ecdysteroids against parasitic helminths has been proposed (Spindler 1988; Barker et al. 1990). Indeed, following the application of moulting hormones at an inappropriate time and in nonphysiologically high concentrations, deleterious effects have been observed both in vivo and in vitro not only during larval insect development (hyperecdysonism) but also during embryogenesis (Diibendorfer 1989) and even in helminths (Mehlhorn et al. 1986; Spindler etal. 1986; Franke and K/iuser 1988; Barker et al. 1990). A wide spectrum of ecdysteroids occur in various plants, and the possibility that they might provide protection against nonadapted phytophagous insects has been discussed (Lafont and Horn 1989). However, the extremely effective metabolism and excretion of ingested moulting hormones by insects leads to difficulties in maintaining elevated ecdysteroid levels for a sufficient time. Considerable efforts have been made to interfere with ecdysteroid synthesis. Insects cannot synthesize sterols de novo from small molecules such as acetate and are therefore forced to take up cholesterol as a precursor. A number of compounds, especially azasteroids, have been tested for their ability to inhibit the enzymes involved in the biosynthesis of ecdysteroids. Although effective inhibitors were found, no commercial product was developed, possibly due to practical reasons such as high production costs or environmental instability (Retnakaran etal. 1985; Spindler-Barth and Spindler 1987). However, the synthesis of ecdysteroids is inhibited by benzoylphenylureas (Soltani et al. 1984; Londershausen et al. 1989), and this may contribute to the insecticidal action of the latter; however, the main target of these growth regulators, which have been used as insecticides for about 20 years, is the inhibition of chitin synthesis. Azadirachtins (Fig. 2), a group of closely related compounds isolated from the neem tree, cause a variety of developmental disturbances besides their antifeedant effects (Schmutterer 1988). These compounds are used in developing countries mainly as a plant extract. Azadirachtins lower ecdysteroid titers, presumably by depressing the release of prothoracicotropic hormone from the brain; this hormone stimulates ecdysone synthesis in the prothoracic glands (Koolman et al. 1988; Garcia et al. 1990). Although the chemical structure of these compounds is too complicated to enable their chemical synthesis in suitable quantities, they can serve as models,

which might lead to the development of more simple molecules whose biological activity would be retained. A promising tool for the reduction of ecdysteroid titers, an ecdysteroid-uridine diphosphate-glycosyltransferase occurring naturally in certain insect pathogenic baculoviruses, has recently been detected (O'Reilley and Miller 1989). The conjugation of ecdysteroids with a glycoside moiety or the formation of sulfate or phosphate esters is a well-known mechanism for the inactivation of ecdysteroids (Lafont and Connat 1989). This mechanism is used physiologically by arthropods to store considerable amounts of biologically inactive ecdysteroids in the egg; these are subsequently activated by hydrolytic cleavage during embryonic development. Infection with viruses that constitutively express this enzyme leads to the inactivation of ecdysteroids in these insects at an inappropriate time. Since numerous baculoviruses exhibit limited host specificity, selective pest control by viral infection seems possible. A great number of different ecdysteroids that were mainly isolated from plants have been tested for their agonistic or antagonistic ecdysteroid action (Bergamasco and Horn 1980; Cherbas et al. 1980; Hetru et al. 1986; Spindler-Barth and Spindler 1987; Lehmann et al. 1988; Dinan 1989; Lafont and Horn 1989; Spindler et al., in preparation). Although a considerable understanding of the structure-activity relationship between ecdysteroid ligands and their corresponding hormone receptors has been gained from such studies, the ecdysteroid agonist R H 5849 (Fig. 2) was detected only recently (Wing 1988; Wing et al. 1988). The detection of R H 5849 represented the first time that an insecticide had been found by means of a screening system especially designed for the detection of hormone-interfering substances using a cell line from Drosophila. The test is based on the induction of acetylcholinesterase by ecdysteroids in insect cell cultures (Spindler-Barth et al. 1988). The increase in acetylcholinesterase activity evoked by ecdysteroids can amount to as much as 30 times the control value, which enables the detection of small differences in moulting hormone potency. Moreover, the detection of hormone antagonists is possible. Simultaneous controls of cell density and viability allows the exclusion of cytotoxic effects that can be misinterpreted as antagonistic hormone effects when in vivo tests are used (Spindler et al., in preparation). If the compound of interest is applied simultaneously with a concentration of 20-OH-ecdysone that is sufficient to induce half-maximal levels of acetylcholinesterase activity, both agonistic and antagonistic as well as cytotoxic effects can be determined using the same assay. As acetylcholinesterase activity is easy to detect and the test can be automated, this assay is well suited for screening tests, especially when cell lines growing in suspension are used. Since the increase in acetylcholinesterase activity is a late hormone response that occurs in the presence of a permanently high ecdysteroid level (Spindler-Barth 1991), care must be taken that hormone metabolism as well as synthesis by the cells is excluded (Spindler and Spindler-Barth 1991). If this is the case, as has been

93 Table 1. Characteristics of the nonsteroidal ecdysteroid agonist RH

5849 and its effects on insects I.

Binds to intracellular ecdysteroid receptors from Drosophila melanogaster a, Plodia interpunctella a, Chironomus tentans c and Astacus leptodactylus a with high affinity (ICso, between 1 and

3 gM) II. Does not interfere with mammalian glucocorticoid and estradiol receptor c III. Evokes the same biological effects that are induced by ecdysteroids: 1. Induces acetylcholinesterase in hormone-sensitive cell lines from Drosophila ~ and Chironomus ~ 2. Induces evagination of imaginal discs and chitin synthesis in P. interpunctella ~ 3. Inhibits chitin synthesis ~ and dopa decarboxylase activity f in a C. tentans cell line 4. Inhibits cell proliferation in cell lines from D. melanogaster b, C. tentans c and P. interpunctella e 5. Breaks larval diapause in Ostrinia nobilis g IV. Exerts insectic~cdaleffects in a variety of insect species.... g' ~ V. Low acute mammalian toxicity (rat LDso : 435 mg/kg; dermal, > 5,000 mg/kg)" VI. Essential nontoxicity to fish and birds and only low toxicity to Daphnia (acute 48-h LCso, 7 mg/1)a a Wing and Aller 1990 b Wing 1988 c Spindler-Barth et al. 1991 a Quack and Turberg, personal communication Silhacek et al. 1990 f Baumeister, personal communication g Gadenne et al. 1990 h Wing et al. 1988

proven for the epithelial cell line from C h i r o n o m u s tent a n s , the capability to induce acetylcholinesterase activity is quantitatively correlated with the affinity to the ecdysteroid receptor (Table 1). This contrasts with chitin synthesis, which is also modulated by ecdysteroids; since chitin synthesis is a multistep process, only a qualitative correlation with the binding affinity seems possible (Silhacek et al. 1990; Spindler-Barth et al. 1991). At the first glance, the structure of R H 5849 as an ecdysteroid agonist seems surprising. However, computer modeling revealed that the molecule can in fact be folded in such a way that it fits satisfactorily with the steroid-binding domain of the ecdysteroid receptor. This is reminiscent o f the nonsteroidal stilbenes, which exhibit estrogen activity. The simple chemical structure of the phenylhydrazine derivative R H 5849 is a prerequisite for its use in pest control, since the complex chemical structure of all biologically active ecdysteroids thus far tested has been a major drawback to their use. The binding of R H 5849 (Table 1) is restricted to ecdysteroid receptors, and this compound does not compete with vertebrate steroids for their corresponding receptors (Spindler-Barth et al. 1991). The low toxicity (Wing and Aller 1990) of this compound may even enable its use against human parasites. Although receptor-binding studies have revealed a

lower affinity to the ecdysteroid receptor for R H 5849 as compared with 20-OH-ecdysone (Wing 1988; Spindler-Barth etal. 1991), severe deleterious effects have been observed in vivo following treatment with R H 5849 (Wing et al. 1988; Silhacek et al. 1990; Wing and Aller 1990), in contrast to the application of ecdysteroids. This is attributable to the enhanced metabolic stability of R H 5849. Despite the interaction with a basic mechanism, which is c o m m o n to all arthropods, no adverse effects have been observed in vivo in D a p h n i a or crayfish (Wing and Aller 1990), although R H 5849 also competes for the ecdysteroid receptor in crayfish (Quack and Turberg, personal communication). Field studies on R H 5849 that yielded positive results have been reported (Wing and Aller 1990). Outlook

The search for new compounds for use in pest control is dependent on effective test systems. Using gene technology, sufficient amounts of peptide hormones can be synthesized to enable binding studies to be carried out for the development of antihormones, which "inactiv a t e " endogenous hormones by binding to their receptor. In addition, investigations o f metabolism, inactivation, and processing of peptide hormones are facilitated. The isolation of hormone receptors by classic proteinseparation techniques is a difficult task due to the minute amounts that are present and to the lability of these molecules. In contrast to the case in vertebrates, only recently was a hormone (ecdysteroid) receptor purified from a D r o s o p h i l a cell line (Luo et al. 1991). Identification of receptor genes (Seagraves and Hogness 1990) and their subsequent expression in a suitable system can provide enough material for receptor characterization and for the performance of binding studies to investigate structure-activity relationships. Tests for hormone function using organs or tissues of insects are time-consuming. The use of hormone-sensitive insect cells facilitates this task considerably (Oberlander and Miller 1987; Dinan et al. 1990) as demonstrated by the detection of R H 5849 (Wing 1988). Presently, only hormone mimics are used in insect pest control. To date, no hormone antagonist has been made available. The use of enzymes that modify hormone levels seems feasible in the near future. All other strategies mentioned herein require additional basic research before their practical application can be expected. The initial hope of Williams (1967) to circumvent the resistance of insects to compounds that are applied for pest control by using physiologically occurring substances has not been fulfilled (Shemshedini and Wilson 1990). A better understanding of insect physiology, which is exemplified in this review by endocrine regulation and its underlying mechanisms of arthropod metabolism and development, opens new possibilities for insect pest control. These new strategies can help to cope with the increasing demand for effective and environmentally safe pest control. The need for new drugs is underscored by the fact that despite the considerable effort expended

94 tO date, n o effective i m m u n i z a t i o n a g a i n s t a n y o f the m a i n p a r a s i t o s e s t r a n s m i t t e d b y a r t h r o p o d s is available. H o w e v e r , resistance r e m a i n s a p r o b l e m t h a t c a n at b e s t be d e l a y e d b y the d e l i b e r a t e use o f pesticides a n d the c o n s t a n t effort e x e r t e d in the search for new c o m p o u n d s .

References

Barker GC, Chitwood DJ, Rees HH (1990) Ecdysteroids in helminths and annelids. Invertebr Reprod Dev 18:1-11 Barton AE, Wing KD, Le DP, Slawecki RA, Feyereisen R (1989) Arylpyridyl-thiosemicarbazones: a new class of anti-juvenile hormones active against Lepidoptera. Experientia 45 : 580-583 Beato M (1991) Transcriptional control by nuclear receptors. FASEB J 7:2044-2051 Bergamasco R, Horn DHS (1980) The biological activities of ecdysteroids and ecdysteroid analogues. In: Hoffmann JA (ed) Progress in ecdysone research. Elsevier/North-Holland, Amsterdam, pp 299-324 Bidmon HJ, Sliter TJ (1990) The ecdysteroid receptor. Invertebr Reprod Dev 18:13-27 Casida JE (1990) Pesticides and alternatives: innovative chemical and biological approaches to pest control. Elsevier, Amsterdam, p 586 Cherbas L, Yonge CP, Cherbas P, Williams CM (1980) The morphological response of Kc-H cells to ecdysteroids: hormonal specificity. Wilhelm Roux's Arch Entwicklungsmech Org 189:1-15 Cusson M, Yagi K J, Ding Q, Duve H, Thorpe A, McNeil JN, Tobe SS (1991) Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: the search for a unifying arthropod endocrinology. Insect Biochem 21 : 1 6 De Loof A (1987) The impact of the discovery of vertebrate-type steroids and peptide hormone-like substances in insects. Entomol Exp App145 : 105-113 De Loof A, Schoofs L (1990) Homologies between the amino acid sequences of some vertebrate peptide hormones and peptides isolated from invertebrate sources. Comp Biochem Physiol [B] 95:459-468 De Loof A, Schoofs L, Broeck JV (1990) Molecular structures of some vertebrate-tpe messenger peptides in invertebrates. In: Epple A, Scannes CG, Stetson MH (eds) Progress in comparative endocrinology. Wiley-Liss, New York, pp 16-21 Dinah L (1989) Ecdysteroid structure and hormonal activity. In: Koolman J (ed) Ecdysone. From chemistry to mode of action. Georg Thieme, Stuttgart, pp 345-353 Dinan L, Spindler-Barth M, Spindler KD (1990) Insect cell lines as tools for studying ecdysteroid action. Invertebr Reprod Dev 18 : 43-53 Diibendorfer A (1989) Ecdysteroid action in embryonic systems. In: Koolman J (ed) Ecdysone. From chemistry to mode of action. Georg Thieme, Stuttgart, pp 421-425 Evans BE, Bock MG, Rittle KE, DiPardo RM, Whitter WL, Veber DF, Anderson PS, Freidinger RM (1986) Design of potent, orally effective, nonpeptidal antagonists of the peptide hormone cholecystokinin. Proc Natl Acad Sci USA 83:4918-4922 Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240:889-895 Ferenz H J, Peter MG (1987) The inhibitory effect of sinefungin on juvenile hormone biosynthesis and development in locusts. Insect Biochem 17:1119-1122 Franke S, Kfiuser G (1989) Occurrence and hormonal role of ecdysteroids in non-arthropods. In: Koolman J (ed) Ecdysone. From chemistry to mode of action. Georg Thieme, Stuttgart, pp 296-307 Gadenne C, Varjas L, Mauchamp B (1990) Effects of the nonsteroidal ecdysone mimic, RH-5849, on diapause and non-dia-

pause larvae of the European corn borer, Ostrinia nubilalis Hbn. J Insect Physiol 36 : 555-559 Garcia ES, Luz N, Azambuja P, Rembold H (1990) Azadirachtin depresses the release of prothoracicotropic hormone in Rhodnius prolixus larvae: evidence from head transplantations. J Insect Physiol 36 : 679 682 Goldsworthy GJ, Wheeler CH, Cusinato O, Wilmot CM (1990) Adipokinetic hormones: structures and functions. In: Epple A, Scannes CG, Stetson MH (eds) Progress in comparative endocrinology, vol 342. Wiley-Liss, New York, pp 28-34 Hammock BD, Abdel-Aal YAI, Mullin CA, Hanzlik TN, Roe RM (1984) Substituted thiotrifluoropropanones as potent selective inhibitors of juvenile hormone esterase. Pest Biochem Physiol 22: 209-223 Hammock BD, Bonning BC, Possee RD, Hanzlik TN, Maeda S (1990) Expression and effects of the juvenile hormone esterase in a baculovirus vector. Nature 344:458-461 Harrow ID, Gration KAF, Evans NA (1991) Neurobiology of arthropod parasites. Parasitology 102:$59-$69 Hetru C, Roussel JP, Mori K, Nakatani Y (1986) Activit~ antiecdyst6roide de brassinost6roides. C R Acad Sci [II] 302:417-420 Holman GM, Nachman RJ, Wright MS (1990) Comparative aspects of insect myotropic peptides. In : Epple A, Scannes CG, Stetson MH (eds) Progress in comparative endocrinology, vol 342. Wiley-Liss, New York, pp 35-39 Hutson DH, Roberts TR (eds) (1985) Insecticides, vol 5. John Wiley & Sons, Chichester, p 385 Ilenchuk TT, Davey KG (1985) The binding of juvenile hormone to membranes of follicle cells in the insect Rhodnius prolixus. Can J Biochem Cell Biol 63:102-106 Ilenchuk TT, Davey KG (1987) Effects of various compounds on Na/K-ATPase activity, JH I binding capacity and patency response in follicles of Rhodnius prolixus. Insect Biochem 17:1085-1088 Keller R (1990) Neurosekretion und Neuropeptide im Nervensystern von dekapoden Crustaceen. Verh Dtsch Zool Ges 83:313327 Kelly TJ, Masler EP, Menn JJ (1990) Insect neuropeptides: new strategies for insect control. In: Casida JE (ed) Pesticides and alternatives: innovative chemical and biological approaches to pest control. Elsevier, Amsterdam, pp 283-297 Kerkut GA, Gilbert LI (eds) (1985) Comprehensive insect physiology, biochemistry and pharmacology, vol 12. Insect control. Pergamon Press, Oxford, p 849 Koolman J, Spindler KD (1983) Mechanism of action of ecdysteroids. In: Downer HRGH, Laufer H (eds) Endocrinology of insects. A. Liss, New York, pp 179-201 Koolman J, Bidmon HJ, Lehmann M, K/iuser G (1988) On the mode of action of azadirachtin in blowfly larvae and pupae. In: Sehnal F, Zabza A, Denlinger DL (eds) Endocrinological Frontiers in Physiological Insect Ecology. Wrodrov Technical University Press, Wodrov, pp 55 67 Lafont R, Connat JL (1989) Pathways of ecdysone metabolism. In: Kootman J (ed) Ecdysone. From chemistry to mode of action. Georg Thieme, Stuttgart, pp 167-173 Lafont R, Horn DHS (1989) Phytoecdysteroids: structure and occurrence. In: Koolman J (ed) Ecdysone. From chemistry to mode of action. Georg Thieme, Stuttgart, pp 39-64 Lehmann M, Vorbrodt HM, Adam G, Koolman J (1988) Antiecdysteroid activity of brassinosteroids. Experientia 44:355 356 Londershausen M, Spindler-Barth M, Spindler KD (1989) Influence of the insect growth regulator SIR 8514 on chitin synthesis, chitin degradation and ecdysteroid titer. In: Skjak-Baerk G, Anthonson T, Sandford P (eds) Chitin and chitosan, Elsevier Applied Science, London, pp 233-242 Luo Y, Amin J, Voellmy R (1991) Ecdysterone receptor is a sequence-specific transcription factor involved in the developmental regulation of heat shock genes. Mol Cell Biol 11:36603675 Mehlhorn H (ed) (1988) Parasitology in focus. Facts and trends. Springer, Berlin, p 924

95 Mehlhorn H, Spindler KD, Spindler-Barth M, Walldorf V, Andrews P, Thomas H (1986) The effect of precocene II and 20OH-ecdysone on Litosomoides carinii and Dipetalonema viteae in vivo. Z Parasitenkd 72 : 843 845 Nachman RJ, Roberts VA, Holman GM, Trainer JA (1990) Consensus chemistry and conformation of an insect neuropeptide family analogous to tachykinins. In: Epple A, Scannes CG, Stetson MH (eds) Progress in comparative endocrinology, vol 342. Wiley-Liss, New York, pp 60-66 Oberlander H, Miller S (1987) Lepidopteran cell lines: tools for research in physiology, development, and genetics. In: Maramorosch K (ed) Advances in cell culture, vol 5. Academic Press, New York, pp 187-207 Ondetti MA, Rubin B, Cushman DW (1977) Design of specific inhibitors of angiotensin-convertingenzyme: new class of orally active antihypertensive agents. Science 196:441-444 Ohnishi E, Ishizaki H (1990) (eds) Molting and metamorphosis. Japan Scientific Societies Press, Tokyo, p 270 O'Reilley DR, Miller LK (1989) A bacutovirus blocks insect molting by producing ecdysteroid UDP-glycosyl transferase. Science 245:1110-1112 O'Shea M (1991) Introduction to neuropeptides: perspectives for the parasitologist. Parasitology 102:$71-$75 Palli RS, Riddiford LM, Hiruma K (1991) Juvenile hormone and "retinoic acid" receptors in Manduea epidermis. Insect Biochem 21:7-15 Retnakaran A, Granett J, Ennis T (1985) Insect growth regulators. In: Kerkut GA, Gilbert LI (eds) Comprehensive insect physiology, biochemistry and pharmacology, vol 12. Pergamon Press, Oxford, pp 529 601 Riddiford LM, Ashburner M (1991) Effects of juvenile hormone mimics on larval development and metamorphosis of Drosophila melanogaster. Gen Comp Endocrinol 2:172-183 Roques BP, Fourni6-Zaluski MC, Soroca E, Lecomte JM, Malfroy B, Llorens C, Schwartz JC (1980) The enkephalinase inhibitor thiorphan shows antinociceptive activity in mice. Nature 288 : 286-288 Schmutterer H (1988) Potential of azadirachtin-containing pesticides for integrated pest control in developing and industrialized countries. J Insect Physiol 34:713-719 Seagraves WA, Hogness DS (1990) The E75 ecdysone-inducible gene responsible for the 95B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes Dev 4 :204-219 Shaaya E, Spindler KD (1990) The effect of methoprene on RNA and chitinolytic enzyme synthesis and on ecdysteroid titer in some insects. In: Casida JE (ed) Pesticides and alternatives: innovative chemical and biological approaches to pest control. Elsevier, Amsterdam, pp 271-282 Shaaya E, Calderon M, Pisarev V, Spindler KD (1991) The effect of juvenile hormone on life span, egg production and ecdysterold titer in Ephestia cautella females. Arch Insect Biochem Physioi (in press) Shaw C, Johnston CF (1991) Role of regulatory peptides in parasitic platyhelminths and their vertebrate hosts : possible novel factors in host-parasite interactions. Parasitology 102:$93-S105 Shemshedini L, Wilson TG (1990) Resistance to juvenile hormone and an insect growth regulator in Drosophila is associated with an altered cytosolic juvenile hormone-binding protein. Proc Natl Acad Sci USA 87:2072 2076 Silhacek DL, Oberlander H, Porcheron P (1990) Action of RH 5849, a non-steroidal ecdysteroid mimic, on Plodia interpunctel-

la (Huebner) in vivo and in vitro. Arch Insect Biochem Physiol 15:201-212 Smith W, Sedlmeier D (1990) Neurohormonal control of ecdysone production: comparison of insects and crustaceans. Invertebr Reprod Dev 18 : 77-89 Soltani N, Delbeque JP, Delachambre J, Mauchamp B (1984) Inhibition of ecdysteroid increase by diflubenzuron in Tenebrio molitor pupae and compensation of diflubenzuron effect on cuticle secretion by 20-hydroxyecdysone. Int J Invertebr Reprod Dev 7 : 323-332 Spindler KD (1988) Parasites and hormones. In: Mehlhorn H (ed) Parasitology in focus. Facts and trends. Springer, Berlin, pp 465-476 Spindler KD, Spindler-Barth M (1989) Uptake of ecdysteroids. In: Koolman J (ed) Ecdysone. From chemistry to mode of action. Georg Thieme, Stuttgart, pp 245-249 Spindler KD, Spindler-Barth M (1991) Ecdysteroid production and metabolism by an epithelial cell line from Chironornus tentans. Naturwissenschaften 78 : 78 79 Spindler KD, Spindler-Barth M, Mehlhorn H (1986) Effects of the juvenile hormone antagonist precocene II and the moulting hormone 20-OH-ecdysone on Litosomoides carinii and Dipetalonema viteae in vitro. Z Parasitenkd 72:837-841 Spindler KD, Spindler-Barth M, Londershausen M (1990) Chitin metabolism: a target for drugs against parasites. Parasitol Res 76:283-288 Spindler-Barth M (1991) Hormonal regulation of acetylcholinesterase in an epithelial cell line from Chironomus tentans. Z Naturforsch (in press) Spindler-Barth M, Spindler KD (1987) Antiecdysteroids and receptors. In: Agarwal MK (ed) Receptor mediated antisteroid action. Walter de Gruyter, Berlin, pp 497-511 Spindler-Barth M, Schmidt H, Drews U, Spindler KD (1988) Increase in activity of acetylcholinesterase by 20-OH-ecdysone in a Chironomus tentans cell line. Roux's Arch Dev Biol 197:366-369 Spindler-Barth M, Turberg A, Spindler KD (1991) On the action of RH 5849, a nonsteroidal ecdysteroid agonist, on a cell line from Chironomus tentans. Arch Insect Biochem Physiol 16:1118 Spring JH (1990) Endocrine regulation of diuresis in insects. J Insect Physiol 36:13-22 Watson RD, Spaziani E, Bollenbacher WE (1989) Regulation of ecdysone biosynthesis in insects and crustaceans : a comparison. In: Koolman J (ed) Ecdysone. From chemistry to mode of action. Georg Thieme, Stuttgart, pp 188-203 Williams CM (1967) Third generation pesticides. Sci Am 217:13 17 Wing KD (1988) RH 5849, a nonsteroidal ecdysone agonist: effects on a Drosophila cell line. Science 241:467-469 Wing KD, Aller HE (1990) Ecdysteroid agonists as novel insect growth regulators. In: Casida JE (ed) Pesticides and alternatives: innovative chemical and biological approaches to pest control. Elsevier, Amsterdam, pp 251-257 Wing KD, Slawecki RA, Carlson GR (1988) RH 5849, a nonsteroidal ecdysone agonist: effects on larval Lepidoptera. Science 241 : 470-472 Wright JE, Retnakaran A (eds) (1987) Chitin and benzoylphenyl ureas. Dr. W. Junk, Dordrecht, p 309 Yamamoto K, Chadarevian A, Pellegrini M (1988) Juvenile hormone action mediated in male accessory glands of Drosophila by calcium and kinase C. Science 239:916-919