Effects of a nonsteroidal ecdysone agonist tebufenozide on hostparasitoid interactions.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 26:235-248 (1994) Effects of a Nonsteroidal Ecdysone Agonist, Tebufenozide, on Host/Parasitoid Interactions JohnJ. Brown Department of Entomology, WashingtonState University,Pullman Tebufenozide (RH-5992), a nonsteroidal ecdysone agonist, stimulated significant ( P 2 0.05) growth in both testes of post-diapausing codling moth larvae and a dormant Ascogaster larva in its overwintering host's hemocoel. Tebufenozide elicited the same responses in post-diapausingtestes and Ascogaster larvae as were reported earlier in insects treated with 20-hydroxyecdysone (20-OHE) [Friedlander, J Insect Physiol 35:29 (1989); Brown et al., Endocrinological Frontiers in Physiological Insect Ecology. Wroclaw: Wroclaw Technical University Press, pp 443-447 (1988)]. Only a trace (I1%) of ''C-tebufenozide was recovered from gonads and exuviae of healthy larvae, or from Ascogaster larvae removed from parasitized hosts; however, the renewed growth of testes and Ascogaster larvae and apolysis of codling moth integument were an obvious response to the hormone agonist. Most of the injected 14C-tebufenozidewas recovered from host fat body, while the alimentary canal retained approximately 40% of the 14C-tebufenozide fed in an artificial diet. Host exposure to tebufenozide did not cause apolysis in endo- or ectoparasitic hymenopteransfeeding on treated codling moth larvae; however, the endoparasitoid trapped in the host's hemocoel died as its host's tissue deteriorated. Different results were observed on ectoparasitoidsdeveloping on treated hosts. Ectoparasitic Hyssopus sp. (Eulophidae) larvae feeding on tebufenozide treated hosts pupated in the normal length of time. Hyssopus adults which developed from larvae fed tebufenozide treated hosts were fertile and produced as many progeny as adults reared from solvent fed controls. There was no evidence of secondary poisoning to Hyssopus sp. and codling moth exposure to tebufenozide may actually benefit the rearing of this eulophid by maintaining the host in the susceptible larval stage and preventing the host larva from spinning a cocoon. Q 1994 WiIey-Liss, Inc. Key words: Ascogaster quadridentata, Cydia pornonella, testes, tebufenozide, codling moth Acknowledgments: The author appreciates the gift of ''C-tebufenozidefrom Drs.A.C.T. H5uandC.R. Carlson of Rhom & Haas, Spring House, PA, and thetechnicalhelpof D. Reed Larsen.This research was supported in part by a grant to J.J.B. from the Washington State Tree Fruit Research Commission 6405. Received February 4, 1993; accepted March 5, 1993. Address reprint requests to Dr. John J. Brown, Department of Entomology, Washington State University, Pullman, WA 991644382, 0 1994 Wiley-Liss, Inc. 236 Brown INTRODUCTION The testes of diapausing codling moth, Cydia pornonella L. (Tortricidae)larvae are diminutive, darkly melanized, kidney-shaped tissues, with arrested spermatogenesis. Dormancy is maintained by the absence of molting hormone ill. Dormant testes apparently respond to hemolymph ecdysteroids occurring during diapause termination, because the addition of ZO-OHE*to in vitro cultures of dormant testes initiates growth and a renewal of spermatogenesis [21. In parasitized codling moths, an Ascogaster quadridentata Wesmael (Braconidae) larva diapauses as a small (V = 10.150 mm3) first instar parasitoid in the hemocoel of its overwinteringhost. Dormancy of both insects is assured because the host’s endocrine system is inactive, and host ptg activity is necessary for post-diapause development of the parasitoid larva [31. Some Li parasitoid larvae may not have a functional ptg and they may rely upon ecdysteroids in their h o d s hemolymph . This would be a logical explanation for dormancy in the Cydia/Ascogaster system. Ligation of the host, caudal to the ptg, isolates the dormant parasitoid larva in the abdomen and prevents the renewal of the parasitoid’s growth, even when the host is transferred from dormancy-maintaining (OP:24S, 4°C) to DTC (16P:8S, 25°C). However, previous experiments have shown that an injection of 20-OHE into the ligated host abdomen will cause renewed parasitoid growth [51. In summary, both testes and A. quadridentata larvae respond to a postdiapausing pulse of hemolymph ecdysteroids by renewing their growth and development. However, parasitized codling moth larvae generally do not have developing testes 1161; therefore, the parasitoid larva is the recipient of ecdysteroids in the host hemocoel. In the absence of host testes, the parasitoid can monitor and interpret the host’s endocrine message signalling the end of dormancy. The parasitoid resumes growth in response to the host’s release of a post-diapausing pulse of ecdysteroids [3,71. Ecdysteroids play a major role in host/parasitoid interactions and until recently researchers had to depend upon commercially available 20-OHE to study these endocrine interactions [81. This strategy would be comparable to using JH rather than methoprene to study endocrine interactions. Besides being prohibitively expensive, the action of in vivo degradative enzymes requires the researcher to use dosages higher than the physiological titer of the natural hormone. Tebufenozide (ring substituted 1,2 dibenzoyl-1 tert-butyl hydrazide; RH-59921, is an ecdysteroid agonist structurally similar to the initial hydrazide compound (RH-5849) discovered by Hsu and Aller [91. Tebufenozide may be used to further our understanding of the role of ecdysteroids in host/parasitoid endocrine relationships, just as methoprene has aided our understanding of JH involvement. *Abbreviations used: DMSO = dimethyl sulioxide; DTC = diapause terminating conditions; EC50 =effective concentration needed to cause head capsuleslippage in 50% of the treated population; IGR= insectgrowth rcgu1ator;JH=juvenilehormone;JHA=juvenile hormoneanalog;LD= longday; L, =defines the instar or an age-day within a stadium (i.e., L,, L2, L1);20-OHE=2O-hydroxyecdysone; P = photophase; ptg = prothoracic gland/s; RH = Rohm and Haas; S = scotophase; SD = short day. Host Specific Ecdysone Agonist 237 Here, I report the response of testes in healthy codling moth larvae, or an Li Ascogaster larva in the abdomen of its host, to an injection of tebufenozide. Presumably if tebufenozide is acting as an ecdysone agonist, both testes in the healthy larva and the L1 parasitoid larva should initiate post-dormancy growth and development in response to the hormone mimic, as these processes were demonstrated earlier to be under the control of ecdysteroids [2,51. This manuscript also examines the possible indirect toxic effects of the growth regulator on hymenopteran endo- and ectoparasitoids feeding on hosts fed or injected with tebufenozide. MATERIALS AND METHODS Insects Nonparasitized codling moth larvae and those parasitized by A. quadridentutu were reared individually in 35 cc plastic cups containing an agar-wheat germ-casein diet . Long-day (LD = 16P8S) conditions promoted continuous growth, while exposure to short-day (SD = 8P:16S) photoregimes at 25°C induced dormancy. Dormancy of codling moth larvae was maintained in a walk-in cooler (OP24S, 4°C) for 3 months. Dormancy was rapidly terminated in these larvae by transferring healthy or parasitized codling moth larvae to DTC (LD, 25°C). Tebufenozide was incorporated into artificial diet and presented to nondiapausing codling moth larvae or dissolved in acetone and topically applied to nondiapausing codling moths or injected with DMSO directly into the hemocoel of diapausing larvae, to observe the effect of the ecdysone agonist on host testicular development or endoparasitic A. quadridentutu larval growth and survival. Testes and A. quadridentutu larvae were measured by an ocular micrometer, 5 days following treatment of codling moth larvae with enough tebufenozide to initiate integumental apolysis within a 24 h period following treatment. Some hosts, which had previously been exposed to various concentrations of tebufenozide (four hosts/concentration/treatment, repeated three times) by either interhemocoelic injection or via diet, were presented (one host/one parasitoid adult) to Hyssops (Eluchertus) sp. (Eulophidae)adults after the host’s head capsule had slipped forward, and ovipositional behavior and reproductive success of these ectoparasitoids was noted. Chemicals Tebufenozide was weighed and dissolved in DMSO for addition to an artificial diet or direct injection into the larva’s hemocoel. Rohm and Haas (Spring House, PA) provided U 14C-A-ringlabeled tebufenozide (25.52mCi/mmol). Aliquots of ‘q-tebufenozide stored in methanol were evaporated to dryness under N2 and redissolved in an equal volume of either DMSO, for incorporation into artificial diet and injection into the host’s hemocoel, or acetone for topical application. Recovery of 14C-Tebufenozide Samples of whole insects, specific tissues, or artificial diet were homogenized in 200 p1 saturated NaCl in 1%aqueous HC1 and extracted 3 times with 400 pl of ethyl acetate:acetonitrile (2:1),with vigorous vortexing and centrifugation at 2,940g for 10 min between washes [lo]. The pooled organic layers were evapo- 238 Brown rated to dryness, resolubilized in 100pl of ethyl acetate:acetonitrile(2:l). Recovery rates ranged from 50 to 70%. Eighty microliters were used for liquid scintillation counting, while 10 pl of the remaining volume was spotted onto 250 pm normal-phase silica gel TLC plates. One lane on each TLC plate used to analyze each sample was devoted to the parent U-*'k-A-ring labeled tebufenozide. Radioactivity was analyzed by scraping 1 cm portions of each lane into separate scintillation vials, adding Scinti Verse I1 (Fisher Scientific, Fairlawn, NJ), and counting in a liquid scintillation counter. When the TLC plates were developed in dich1oromethane:acetonitrile:aceticacid (67:33:1), tebufenozide had an Rf of 0.70. Recovery of 'k-tebufenozide from pooled samples of various host tissue or the parasitoids body was reported as a percentage of cpm detected in each fraction. This allowed comparisonsto be made between pooled samples without regard to the actual wet weight of each tissue. The amount of ' k -tebufenozide recovered from the host, Hyssopus pupae, or cpm present in frass voided by Hyssopus larvae prior to pupation, was reported as cpm/mg wet weight of host or parasitoid frass. Statistical Analysis The size of testes were measured by an optic-micrometer and volume was calculated for each testis [V = 7c/6(L x W)21or fused testes [V = 4/3n x radius3] [111.Similar measurements were made of parasitoid larvae [V = 7c/4 x L x W2] [121. Mean volumes of each group were analyzed by one-way analysis of variance (Fisher's LSD, P < 0.05). RESULTS Effect of Tebufenozide on Post-Diapause Development of Ascogaster Larvae or Host Testes Since diapausing codling moth larvae do not feed, tebufenozide was injected into their hemocoel with a 33 gauge needle. All larvae were ligated posterior to the site of injection, so that testes in healthy larvae or Ascogaster larvae in parasitized hosts were incubated in vivo with tebufenozide, but isolated in their host's abdomen, without exchange of hemolymph that bathes the ptg of the codling moth larvae. An injection of either 2 or 4 pg tebufenozide/pl DMSO caused both nonparasitized and larvae parasitized by Ascogas ter to initiate apolysis in the abdominal integument within 24 h following treatment. All injected and ligated larvae were held individually in LD, 25"C, 70% RH for 5 days, then testes from healthy larvae or Ascogaster larvae from hosts were measured and compared to those from codling moth larvae injected with the solvent carrier DMSO. Testes in healthy larvae responded to an injection of tebufenozide by initiating rapid growth (Fig. 1).The testes exposed to 4 pg of tebufenozide were significantly (P I0.05) larger than those excised from larvae injected with DMSO alone. The injection of tebufenozide caused the abdominal integument to initiate apolysis in all codling moth larvae and a large percentage of gonads fused in response to the ecdysone agonist. Testes generally fuse in the pharate pupal stage. Ascogaster larvae removed from hosts injected with either 2 or 4 pg tebufenozide were significantly (P I 0.05) larger than those excised from hosts injected h E E ! Host Specific Ecdysone Agonist 239 A 0.2 0.0 0.1 T DMSO Only 2119 4Kl Tebufenozide Fig. 1. Testicular volume 5 days after post-diapausing male codling moth larvae were treated with either 2 or 4 pg tebufenozide in DMSO or DMSO alone. The percentage of testes fused during the time of the experiment i s shown within the column representingthat population. Mean (* S.D.) lengths of parasitoid larvae, followed by the same letter, are not significantly different at P S 0.5. with only DMSO. All hosts injected with tebufenozide initiated integumental apolysis and a significant (P I0.05) number of Ascogaster larvae excised from treated hosts had molted to their second instar (Fig. 2). None of the hosts injected with DMSO alone initiated apolysis and all Ascogaster larvae dissected from these solvent treated hosts were in their first stadium. Recovery of l4 C-Tebufenozide From Injected Larvae Both healthy and parasitized diapausing codling moth larvae were injected with 'k-tebufenozide and 5 days later various tissues were collected and extracted to recover the radiochemical. Eighty percent of the 'k-tebufenozide recovered from post-diapausing codling moth larvae was isolated from perivisceral fat body and peripheral fat body associated with the new integument (Table 1).One percent or less of the 'k-tebufenozide was recovered from testes of healthy larvae or from Ascogaster larvae in parasitized individuals. The head capsule and alimentary canal of both healthy and parasitized insects contained approximately 4 and 11% of the recovered 'k-tebufenozide, respectively. Reasons for the consistent 3-fold increase in "k-tebufenozide recovered from the new integument of parasitized individuals compared to that recovered from healthy larvae is unclear, and histological studies are planned to interpret these findings. Recovery of 14C-TebufenozideFrom Orally Fed Larvae 24 H Post-Treatment The alimentary canal of treated larvae stored tebufenozide. The gut always contained approximately 40% of the recovered I4C labeled hormone agonist, regardless of the concentration of tebufenozide in the artificial diet. Larvae fed 240 Brown h E E v 3.5 3.0 B 1 B - I 'c1 .-0 I m 2.5 c 0 2.0 CI v) Q 5 m ~. C 1.5 DMSO 4P9 Te buf e nozi de 2P9 Only Fig. 2. Length of parasitoid's body 5 days after post-diapausing parasitized codling moth larvae were treated with either 2 or 4 pg tebufenozide in DMSO or DMSO alone. The percentage of Ascogaster larvae that molted into their second instar during the time of the experiment is shown within the column representingthat population; none of the parasitoids removed from solvent treated control hosts molted. Mean (kS.D.) lengths of parasitoid larvaefollowed by the same letter are not significantly different at P 4 0.5. a diet containing 25 ppm had a greater percentage of labeled tebufenozide in the perivisceral fat body and carcass, and a lower percentage recovered from the alimentary canal and hemolymph, when compared to the distribution of IT-tebufenozide recovered from individuals fed a diet containing 6.25 ppm (Table 2). Once excessive amounts of tebufenozide penetrate the hemocoel, either by absorption across the gut wall or direct injection, then increasing amounts of "k-tebufenozide will be recovered from the fat body. This partitioning of the compound, which is first taken up by the gut and then enters into fat body tissue, should be kept in mind in the following discussion of secondary poisoning and indirect effects of tebufenozide on developing endo- and ectoparasitoid Hymenoptera. TABLE 1. Percent Recovery of Tebufenozide From Pooled Samples of Various Tissues From Healthy Male or Ascogaster Parasitized Post-Diapausing Codling Moth Larvae (n = 15) 5 Days After Injection of 1 pg 'k-Tebufenozide in 1 pl DMSO Healthy larvae Tissue Alimentary canal Perivisceral fat body Testes or Ascogusfu larva Body wall (including fat body) Exuvium Head capsule - Ascogaster parasitized larvae x% S.D. X% S.D. 11.3 f 2.4 40.0 5.9 0.8 f 0.3 40.7 f 1.7 4.0 f 0.8 3.3 f 1.2 11.3k 4.5 38.0 f 11.4 1 . o i 0.2 31.0+ 9.4 14.7f 2.1 4.7f 1.2 + Host Specific Ecdysone Agonist 241 TABLE 2. Percent Recovery of Tebufenozide From Pooled Samples of Various Tissues From Healthy Nondiapausing Codling Moth Larvae, 24 H After Being Offered Diet Containing 'k-Tebuf enozide Diet with 6.25 pprn tebufenozide Tissue Alimentary canal Perivisceral fat body Hemolymph Remaining carcass X% Diet with 25 ppm tebufenozide - x% S.D. S.D. 43.2i6.6 13.4 2.6 10.2 2.6 33.0 3.0 36.6 f 3.9 14.6 & 1.6 9.1 i3.7 40.0 f 1.8 * * * Effect of Tebufenozide on Parasitoid Survival Reared on Orally or Topically Treated Nondiapausing Hosts Codling moth larvae parasitized by A. quadridenfafawere isolated from diet and held as pharate individuals until each had ecdysed into their 4th stadium, then they were offered diet containing various concentrations of tebufenozide for 24 h. All A. quadridenfafalarvae are in their first stadium at this time 1131. The EC50 was 2.19 ppm and all host larvae fed diet containing 5 or more ppm tebufenozide initiated apolysis within 24 h. Other hosts that had exited the diet and had initiated the wandering stage were isolated from the general colony and topically treated with either 1 pg tebufenozide dissolved in acetone or acetone alone. The occurrence of host apolysis, in response to tebufenozide in the diet or after topical application, determined the fate of the Ascogaster endoparasitoid. Ectoparasitic L3 Ascogaster larvae egressed from hosts that did not initiate apolysis, but no L3 parasitoid larvae emerged from hosts that had initiated apolysis in response to the hormone agonist. First instar Ascogaster larvae, of various lengths, were found in hosts dissected for destructive sampling. Generally, the endoparasitoid (L1) larva remained alive within the host hemocoel, as long as the host tissue did not become discolored and start to decompose. Hyssopus sp. females stung and oviposited on tebufenozide fed hosts. The number of Hyssopus larvae surviving on a tebufenozide treated host was not significantly ( P 2 0.05) different from the number expected to develop on a nontreated host. When Hyssopus females were given a choice of a treated or healthy host, they attacked and oviposited freely on both. Tebufenozide treated hosts did not cause acute secondary effects or head capsule slippage in ectoparasitoid larvae; however, endoparasitic Ascogasfev larvae eventually died because of the onset of host tissue deterioration. Post-diapausing codling moth larvae injected with 'k-tebufenozide directly into the hemocoel also supported Hyssopus larval development, but adult eclosion of the wasps was reduced. These Hyssopus pupae, after feeding as larvae on hosts injected with 0.1,0.5, or 1 pg 'v-tebufenozide, accumulated and tebufenozide recovered from retained the hormone agonist in their bodies. these Hyssopus pupae had the same relative mobility as the parent compound, when separated on TLC plates. Nondiapausing codling moth larvae fed various concentrations (up to 25 ppm) "k-tebufenozide in an artificial diet for 24 h were removed from the diet and caged along with 3 Hyssopus females for 7 days. Hyssopus pupae reared from larvae that had fed on these orally tebufenozide 'v- 242 Brown treated hosts failed to accumulate or at least retain 'k-tebufenozide; however, the frass collected from larvae voiding their gut prior to pupation contained increasing amounts of tebufenozide proportionate to the concentration in the diet fed to their hosts (Table3). The number of progeny/host produced by these Hyssopus adults, reared from larvae fed tebufenozide treated hosts, did not differ significantly (P 50.05) from the averagenumber expected from a Hyssopus attack on a nontreated host. DISCUSSION Ecdysteroid Agonistic Effects of Tebufenozide For two decades [141 researchers have used synthetic JHA as tools to understand physiological responses to JH. Hormone analogs are not as susceptible to degradative enzymes, which rapidly metabolize exogenously applied natural hormone, so synthetic analogs are persistent in vivo. Physiologists and endocrinologists studying host /parasitoid endocrine interactionshave often applied a JHA, such as methoprene, to the host and recorded its effect on parasitoid development 181. Now researchers have a family of ecdysteroid agonists, including RH-5849 [15-181 and tebufenozide (RH-5992),that mimics the action of 20-OHE. These ecdysteroid agonists are highly selective for lepidopterans and coleopterans 1191 and are reported to have low acute toxicity to bees 1201. Therefore, tebufenozide should be of particular importance to researchers investigating the endocrine interactionsbetween a lepidopteran host, susceptible to the actions of this hormone mimic, and hymenopteran parasitoids, which presumably would be predicted to tolerate the hormone agonist. Nondiapausing hosts fed tebufenozide during the first day of their 4th stadium initiated apolysis within 24 h; however, all endoparasitic Ascogaster larvae, removed from these pharate hosts, were still in their first stadium. This was an expected result, because an A. quadridentutu larva remains in the first stadium while its host undergoes 3 larval-larval molts, and it is only after the host ceases feeding, wanders, and spins a cocoon, that the parasitoid larva molts into its second instar [131. Grossniklaus-Burgin and Lanzrein  surveyed the JHand ecdysteroid titers of Trichoplusia ni attacked by another chelonine species, Chefonus sp. The life cycles of Ascogaster and Chelonus are nearly identical. The Chelonus species molts to its second instar, when its host JH titer has declined to an undetectable level .Lepidopteran larvae, including the codling moth ,generally cease feeding when the hemolymph titer of JH declines to an undetectable level [231. Therefore, if tebufenozide is administered orally, the host JH titer should be expected to be high, thus preventing an Ascogaster molt. The sudden exposure to an ecdysteroid agonist along with a high endogenous hemolymph titer of JH would not differ from conditions the endoparasitoid had experienced in earlier host larval- larval molts. Wandering codling moth larvae would be expected to have a low titer of hemolymph JH . A topical application of 1 pg tebufenozide administered to these larvae still did not initiate a molt in Ascogaster larvae in their host's hemocoel, but it did elicit an integumental apolysis in all hosts. Diapause in both the codling moth larva and its endoparasiticAscogaster larva is maintained by the inactivity of the host neuroendocrine system and dor- 0.1 0.5 1.0 Amount injected into each host (kg) Hyssopus pupae fed injected host 2.8 37.4 165.3 Injected hosts 18.7 121.4 294.2 Amount (cpm/mg) recovered from: 1.25 6.25 25.00 Concentration in diet (pg/g) 3.3 6.1 46.5 Tebufenozide fed hosts 1.7 3.2 1.5 3.9 8.5 11.4 Hyssopus pupae Frass from Hyssopus reared from hosts larvae fed orally fed tebufenozide poisoned hosts Amount (cpm/mg) recovered from: TABLE 3. Comparison of the cpmlmg Wet Weight Recovered From Hosts, Ectoparasitoid, and Frass of Ectoparasitoid Larvae Fed a Coding Moth Larva Either Fed or Injected With 14C-Tebufenozide 244 Brown mancy is terminated in both by a pulse of 20-OHE in the absence of JH 13,241. Diapausing larvae do not feed, so the tebufenozide was injected directly into the hemocoel and the hosts were immediately ligated, isolating the dormant endoparasitoid in the host’s abdomen away from the host’s prothoracic gland. A significant ( P 20.05) number of these endoparasitoid larvae molted to their second instar in hosts treated with either 2 or 4 pg of tebufenozide, compared to hosts injected only with DMSO (Fig. 2). Apparently RH-5849, another ecdysteroid agonist, can terminate diapause in Ostrinia nubilalis after a topical application of 1 pg dissolved in acetone 1161. A transdermal route would avoid the insult of puncturing the hemocoel, although ligation should still be used to block the influence of ecdysteroids produced by host ptg on testicular growth in healthy larvae or parasitoid development in parasitized individuals. Friedlander and Reynolds [251 have reviewed examples, including C. POrnonellu 1261,where testes in diapausing lepidopterans respond to 20-OHE with resumption of spermatogenicevents. Loeb 1271 emphasized renewed testicular growth in response to 20-OHE. Our studies show that testes in diapausing codling moth larvae are an excellent bioassay tissue for an ecdysteroid agonist such as tebufenozide. Indeed, testes excised from larvae injected 5 days earlier with 4 pg tebufenozide were significantly (P I0.05) larger in volume and more of these testes were fused, as compared to testes removed from solvent treated larvae (Fig. 1).However, lepidopteran testes are themselves a source of ecdysteroids 1281and they may synthesize enough ecdysteroids to control their own internal milieu 1291.In this case, tebufenozide, an ecdysteroid mimic, may serve as a hemolymph message, the same signal represented by 20-OHE in untreated hosts, and is interpeted by the Ascogaster larva in a castrated host to mean that the host’s diapause is terminating. Secondary Effects of Tebufenozide on Endo- and Ectoparasitoidsof the Codling Moth Beneficial parasitoids are extremely susceptible to conventional neurotoxic insecticides because of their searching activities, and if they are not killed outright, their exposure to these pesticides often has sublethal effects [301. Host exposure to IGRs can sometimes benefit the parasitoid population [311, but more often host exposure to any pesticide is detrimental to parasitoids, and especially jeopardizes endoparasitoid survival in treated hosts. Stark et al. I311 lists several reviews of the effect of pesticides on nontarget invertebrates and Beckage et al.  summarized the effects of IGRs on insect endoparasitoids as being either direct or indirect. Tebufenozide action must be considered an indirect effect that causes the host to stop feeding prematurely due to precocious stimulation of molting. The anti-JH fluoromevalonolactone causes Munduca sexta to cease feeding and benzyl-l,3-benzodioxole apparently causes morphological deformities in the mandibles of the host, preventing feeding . Tebufenozide causes the host’s head capsule to slip forward within 7 to 24 h; as a result the host stops feeding. Tebufenozide also may cause apolysis in the ectodermally derived fore- and hindgut of the host, although we have no histological evidence for this action. Regardless of the mode of action of tebufenozide on the host, the endoparasitic Ascogaster larva dies in its first stadium. Host Specific Ecdysone Agonist 245 The ectoparasitic Hyssopus attacked and parasitized treated hosts, regardless of the concentration or manner of delivery of tebufenozide to the hosts. However, tebufenozide injected hosts caused some mortality in eulophid larvae and pupae, and few adult wasps eclosed. Injection directly into the host’s hemocoel probably delivered more tebufenozide to the hemolymph than what would enter across the midgut of a host fed tebufenozide in an artificial diet. The hemolymph RH-5849 peak in treated larvae and the 20-OHE peak in solvent treated larvae of M. sexta were measured by Wing et al. [lo] and both decayed in an identical pattern. The fat body is the tar et tissue for both ecdysteroids 1351 and RH-5849 [361. Forty percent of the 9-tebufenozide injected into codling moth larvae accumulated in the perivisceral fat body, while the bulk of orally administered 14C-tebufenozideremained in the alimentary canal. If fat body uptake was measured concurrent to hemolymph loss we might be able to determine the fate of these IGRs in an insect. Fat body uptake of tebufenozide might explain the deIayed growth regulatory effects observed several instars after exposure to these IGRs 1371. Premetamorphic energy demands that utilize fat body reserves may cause the accumulated IGR to reenter the hemolymph at a crucial endocrine-dependent time just prior to pupation. The fate of ingested tebufenozide includes retention in the alimentary canal, slow absorption into the hemocoel and uptake by the fat body. Hosts fed 25 ppm tebufenozide in an artificial diet, a 10-foldgreater concentration than the EC50, still supported an average number of progeny, and adult Hyssops resulting from these larvae reared on treated hosts were fertile. Lower concentrations of tebuenozide may even be beneficial in parasitoid rearing programs in that host susceptibility is prolonged. Hosts exposed to the hormone agonist can not spin a cocoon which can be an effectivephysical barrier against Hyssopus attack; it also prevents complete pupation, thus maintaining the host in a susceptible stage for Hyssopus attack. Endoparasitoids are inherently more susceptible than an ectoparasitoid feeding on an IGR treated host because of their physiology and life cycle. An endoparasitoid inhabiting the hemocoel of an IGR treated host is in constant contact with the growth regulator and some of the ecydsone agonist may penetrate the parasitoid’s integument. The tebufenozide penetrating an endoparasitoid larva and metabolic waste products are retained until a meconium can be deposited prior to pupation. We did not observe symptoms indicative of an ecdysone agonist in A . quadridentutu larvae excised from tebufenozide hosts; however Ascogaster larvae always died in hosts which had slipped their head capsules in response to the IGR. An earlier study of Myzus persicae (Sulz) parasitized by Aphidius matricariae (Hal.)simply observed death of endoparasitoids encased in an unshed old and new (double) cuticle, where apolysis had occurred in the host integument but ecdysis was incomplete [381. Alternatively, an ectoparasitoid may regurgitate enzymes into the hemocoel of their host and then the imbibed predigested body fluids containing tebufenozide must still cross the midgut of the ectoparasitoid larva or the growth regulator will be eliminated in the parasitoid’s frass (Table 3). Refuge from dermal contact, preingestive enzymatic action on host tissues, selective absorption across the midgut cells, and continuous discharge of frass pellets collectively give a measure of protection to ectoparasitoids, not found in endoparasitoids. !? 246 Brown LITERATURE CITED 1. Sieber R, Benz G (1980): Hormonal regulation of pupation in the codling moth, Luspeyresiu pornonella. Physiol Entomol5:283. 2. Friedlander M (1989): 20-Hydroxyecdysone induces glycogen accumulation within the testicular sheath during in vitro spermatogenesis renewal in diapausing codling moth (Cydiu pornonella). J Insect Physiol35:29. 3. 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