Juvenile hormone esterase activity and ecdysteroid titer in Heliothis virescens larvae injected with Microplitis croceipes teratocytes.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 20:231-242 (1992) JuvenileHormone Esterase Activity and Ecdysteroid Titer in Heliothis virescens Larvae Injected With Microplitis croceipes Teratocytes Deqing Zhang, Douglas L. Dahlman, and Dale B. Gelman Department of Entomology, University of Kentucky, Lexington (D.Z., D.L.D.); Insect Neurobiology and Hormone Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, Maryland (D.B.G.) Juvenile hormone esterase (JHE)activity in the hemolymph of 5th-instar Heliothis virescens larvae injected with Microplitis croceipes teratocytes was inversely related to the number of teratocytes injected. JHE activity in the hemolymph of larvae injected with 750 3-day-old teratocytes (the approximate number from one parasitoid embryo) was depressed to less than 5% of those levels found in control larvae. During the latter portion of the digging stage and in the burrowing-digging (BD) stage JHEactivity in larvae treated with 350 teratocytes was approximately 40% of control values. However, injection of 180 teratocytes did not significantly affect JHEtiters. Two-day-old teratocytes caused the greatest reduction in JHEtiter with decreasing effects observed with injections of 3- to 6-day-oldteratocytes. Nevertheless, because 2-day-old teratocytes were difficult to separate from host hemocytes, 3-day-old teratocytes were used in most of these studies. Injections of nonparasitired H. virescens hemolymph plasma, Micrococcus lureus bacterial cell walls, washed M. croceipes eggs, or teratocytes from Coresia congregata did not depress JHEtiters. Teratocyte injections also significantly reduced growth of host fat body. Ecdysteroid titers in cell formation, day 2 (CF2) larvae injected as new 5th instars with 350 3-day-old teratocytes failed to increase, as compared to noninjected and saline-injected controls. An injection of 1 pg/larvaof 20-hydroxyecdysone at the BD stage permitted normal pupation in 50% of the teratocyte-treated larvae as compared to 0% pupation for teratocyte-treated control larvae not treated with 20-hydroxyecdysone. Teratocytes seem to be responsible for the inhibition of JHE release and thus indirectly impact on ecdysteroid titers. o 1992 ~ i ~ e y - L i s Inc. s, Key words: tobacco budworm, parasitoid, ecdysteroids, Braconidae, Noctuidae Acknowledgments: The authors express appreciation for the technical assistance of T.J. Neary and Becky Wilson for help with insect rearing. The authors gratefully acknowledge support from USDA Competitive Research Grant 89-37250-4705and a grant from R.J. Reynolds. This is paper 91-07-189of the Kentucky Agriculture Experiment Station, Lexington, KY 40546-0091. Received October 31,1991; accepted April 9,1992. Address reprint requeststo Dr. Douglas L. Dahlman, Department of Entomology, 5-225 Agricultural Sciences North, Universityof Kentucky, Lexington, KY 40546-0091. 0 1992 Wiley-Liss, Inc. 232 Zhang et al. INTRODUCTION High titers of JH+are responsible for maintaining the larval stages in holometabolous insects [11. In Lepidoptera, JH titer is high immediately following the last-larval ecdysis but then declines to a very low or even undetectable level [l]. A short burst of ecdysteroid then permits the reprogramming of the larval cells to express their pupal form. This is followedby a second large burst of ecdysteroid which is responsible for the larval-pupal molt. In this sequence of events the degradation of JH by ester hydrolysis is the major pathway of JH catabolism. Indeed, THE has been suggested as the primary agent responsible for the reduction in JH titers, which in turn is necessary for normal insect development . Thus, the decrease in levels of JH is correlated with the level of JHE which degrades JH in the presence of carrier proteins . The change in tissue commitment from larvae to pupa is inhibited when the JH titer is maintained at an artificially high level [4-61. Available evidence suggests a very close relationship between JH titer and JHE level during the last larval stadium [7-91. Based on the measurement of JHE in 11 species of Lepidoptera plus two other species for which data were available, Jones et al. [lo] concluded that most Lepidoptera exhibit two peaks of JHE activity during the last larval instar, the relative size of which varies from species to species. The first peak occurs near the time of maximum weight and the second peak late in the pharate pupal phase. Studies on parasitoid regulation of host development emphasized three independent but interactive sources of regulatory substances which affect the host. They are 1)the adult female parasitoid, which not only deposits eggs but may also inject glandular secretions and polydnavirus, 2) the developing parasitoid larva within the host, and 3) the extra-embryonic tissues, usually called teratocytes. Teratocytes originate from the serosal membrane surrounding the developing parasitoid embryo. These cells are liberated into the host hemolymph when the parasitoid larva hatches [ l l ] and have been suggested to play trophic, immunosuppressive, and secretory roles [12,13]. In a previous study , we found that pupation was prevented when nonparasitized Heliothis virescens larvae were injected with Microplitis croceipes teratocytes. These injected larvae showed behavioral and morphological characteristics similar to larvae parasitized by a wasp and containing the parasitoid larva. We initiated this study to determine if the injected teratocytes were responsible for the reduced levels of ecdysteroids and JHE that were observed previously in parasitized larvae . This experimental design allows a distinction between the effects of teratocytes and those of the parasitoid larva and/or adult wasp products such as poison gland secretions or calyx fluids. *Abbreviations used: BD = burrowing-digging, 5th stadium; CFI = cell formation, day 1; CF2 = cell formation, day 2 ; D1 = digging, day I ; D2 = digging, day 2; D3 = digging, day 3; DLM = delayed larval mortality; 20-OH-ecdysone = 20-hydroxyecdysone; ILPE = incomplete larval pupal ecdysis; JH = juvenile hormone; ]HE = juvenile hormone esterase; N5 = new 5th stadium; P = normal pupa; PP = pharate pupa; PlTH = prothoracicotropic hormone; S = slender, 5th stadium. Teratocyte Effects on JHEand Ecdysteroids 233 MATERIALS AND METHODS Insects Methods of rearing host larvae and parasitoids, collection and preparation of teratocytes, and procedures for injection of teratocytes into nonparasitized hosts have been described previously . Briefly, hemolymph was collected from appropriate age parasitized larvae, diluted with saline, and the teratocytes were separated from hemocytes via several low speed centrifugations. The concentration of teratocytes was determined and the volume was adjusted in order to deliver a specified number of teratocytes in a fixed volume of saline. All nonparasitized larvae used in this study were staged according to the criteria of Webb and Dahlman , which consist of size and behavioral characteristics during the 5th instar. The new and slender stages each last approximately 1 day. The digging stage, usually 2 days long, is followed by a 1-day long burrowing-digging stage. The larva does not feed during the cell formation stage, which lasts 2 days. A l-day long pharate pupal stage precedes pupation, N5 nonparasitized larvae were injected with 10 pl of various doses of teratocytes suspended in Pringle’s saline  using either a Hamilton syringe or a 0.5-cc U-100 insulin syringe (Becton Dickinson, Rochelle Park, NJ). JHE Assay Hemolymph samples for the JHE assay were obtained by clipping a proleg from a chilled (OOC), flaccid larva. The hemolymph from each individual larva was collected into an ice-chilled microcentrifuge tube containing a few crystals of phenylthiourea (to inhibit tyrosinase activity). For each treatment period, eight samples were taken, each from an individual larva; all experiments were repeated at least three times for a total of 24 samples per treatment period. Hemolymph samples were centrifuged for 4 min in a microcentrifugeat 10,OOOg. The supernatant was diluted (usually 1:1,000) in 0.2 M phosphate buffer (pH 7.4) and used as the enzyme source for the JHE assay. Substrates were prepared according to Hammock and Roe . [3H]-JuveniIehormone 111 (17.4pCi/mmol) was purchased from New England Research Products, Boston, MA. Unlabeled JH was obtained from Fluka Chemical Corp., Ronkonkoma, NY,The assay  was based on the fact that JHE hydrolyzes JH to JH acid. The reaction was terminated after a specified time and the JH and JH acid were separated by their different solubilities in immiscible organic and aqueous solvents. The amount of hydrolyzed JH is determined by the amount of radioactivity associated with each fraction when compared to appropriate controls, The assay is designed so that the amount of diluted enzyme used produced a linear increase in product (JH acid). Under most conditions, 100 pl of a 1:1,000 dilution of hemolymph ( = 0.1 pl hemolymph) is incubated with the substrate for 15 rnin. JHE activity is expressed as nmol JH hydrolyzed per min per ml hemolymph. To test the effects of other biological preparations on JHE titers, N5 larvae were injected with either 10 PI of the biological preparations or with 10 p1 of a suspension of the material made up in Pringle’s saline. The test materials included: hemolymph plasma from N5 H. virescens (prepared by a 2-min centrifugation at 10,OOOg); 20 Fg of finely ground lyophilized cells of Micrococcus 234 Zhang et al. luteus; three or four M. croceipes eggs dissected from the ovary of a wasp and then washed in Pringle’s saline four times before injection; and 500-800 teratocytes from Cotesia congvegufa (collected from the hemolymph of parasitized Munducu sextu larvae in a manner similar to the collection of M . croceipes teratocytes). Noninjected and Pringle’s saline-injected larvae served as controls. Hemolymph for the JHE assay was drawn at D2. For each treatment six to eight larvae were injected. For the JHE assay hemolymph samples from individual larvae were collected as described above. Ecdysteroid Titers To determine the effect of teratocytes on hemolymph ecdysteroid levels, N5 larvae were injected with 350 3-day-old teratocytes. Samples were taken at 24-h intervals between BD, CF1 and CF2 and then at two 12-h intervals subsequent to CF2. Because teratocyte-injected larvae seldom demonstrate CF2 characteristics, sample collection was based on chronological age rather than on morphological characters. Each sample contained 50 ~1.1 of hemolymph (10 pl from each of five larvae) placed in 250 pl of ice-cold 70% methanol. The samples were stored at - 75°C. At least three replicates were taken at each time period. Ecdysteroid titers were determined by RIA following the methods of Borst and OConnor  and Bollenbacher et al.  as described by Gelman and Woods  and Gelman et al. . The antiecdysone, a gift from W.W. Bollenbacher, was prepared from a hemisuccinate derivative of ecdysone (at the C-22 hydroxyl group) that had been coupled to thyrogloblin. The antibody had a high affinity for ecdysone and 20-OH-ecdysone . Rescue Studies For the rescue study, N5 N.vivescens larvae were injected with 350 3-day-old teratocytes. When these teratocyte-treated larvae reached BD, they were injected with 1 p1 of various concentrations of 20-OH-ecdysone (Simes, Milano, Italy) in 95% ethanol. Larvae injected with 1 ~1.1of 95% ethanol served as solvent controls. Teratocyte-injected larvae that did not receive the 20-OH-ecdysone injection and larvae that received neither teratocytes nor 20-OH-ecdysone also served as controls. In each experiment six or eight larvae were used for each treatment, and the entire experiment was repeated three times for a total of 22 larvae per treatment. All larvae were weighed daily after the initial injection of teratocytes and records were kept on developmental progress. The final status attained by treated larvae was either successful pupation, incomplete larval-pupal ecdysis, or an extended larval instar followed by death. These parameters have been more fully described by Zhang and Dahlman . Teratocyte Effect on Host Fat Body Effect of teratocytes on nonparasitized host fat body mass was evaluated by treating N5 larvae with either 10 p1 of Pringle’s saline or 10 pl of saline containing 350 3-day-old teratocytes. Larvae were dissected 3 days later when the controls reached the D2 stage. The larvae were chilled on crushed ice, an incision was made along the dorsal midline, and the digestive tract was removed. In order to reduce variation among samples, only the fat body found between the first and fifth abdominal segments was collected. Extraneous hemolymph Teratocyte Effects on JHEand Ecdysteroids 235 was removed from the fat body by capillary action (using absorbent paper), and the net weight of the fat body from each larva was determined to the nearest 0.1 mg. A total of 27 larvae were sampled for each treatment. RESULTS Teratocyte-injected larvae exhibited abnormally low levels of hemolymph JHE with the degree of change induced by the teratocytes being dose-dependent (Table 1, Fig. 1).For each teratocyte treatment (180,350, 750) there was a separate set of controls, the mean values of which, with a few exceptions, were not significantly different from each other. It should be noted that the trauma associated with injection resulted in an additional day in the active feeding stage (D3) and, thus, a delay of 1day to maximum JHE activity, as compared to noninjected controls. It also resulted in lower JHE values at BD as compared to noninjected controls. There were two JHE peaks for both the noninjected and saline-injected controls. The first peak was large and broad and associated with the last day of the D phase and with the BD phase. The second peak was small and associated with the PP phase. The timing and titer of both peaks was as expected, based on information about JHE titers in other Lepidoptera [lo]. A dose of 750 teratocytes prevented the appearance of significant amounts of JHE at all times during the 5th stadium. Even a dose of 350 teratocytes (approximately one-half the number derived from a single parasitoid embryo) kept the JHE titer from reaching more than 40% of the control level. However, a dose of 180 teratocytes had no apparent effect on JHE titer. JHE activity was inversely correlated with the age of teratocytes injected into the larvae (Table 2). The reduction in JHE activity caused by 2-day-old teratocytes was significantly greater than that caused by 3-day-old teratocytes. JHE activity from larvae treated with either 5- or 6-day-old teratocytes was significantly greater than the JHE levels in larvae treated with either 2-or 3-day-old teratocytes. However, even in larvae treated with 6-day-old teratocytes, JHE levels were significantly reduced, only being 20% of those found in salinetreated controls. Results from experiments designed to test the effects of other biological preparations on JHE levels support the hypothesis that M. croceipes teratocytes are responsible for the low JHE levels observed. No reduction in JHE titer was observed after any of the treatments other than M . croceipes teratocytes (Table 3). The developmental rate of all larvae treated with these other biological preparations was similar and all pupated. In a separate test, larvae that had received washed M. croceipes eggs were dissected but teratocytes were not observed in the host hemolymph. To determine the effect of teratocyte injection on ecdysteroid titers, larval hemolymph was sampled daily from BD to . ' I P Ecdysteroid titers in noninjected controls and Pringle's saline-injected controls both demonstrated the expected large premolt peak at CF2 + 12 h (Table 4).In contrast, the ecdysteroid titers in teratocyte-treated larvae were significantly higher (approximately 150-200 pg/pl greater) than in either control during the earlier stages (BD through CFI), but then remained unchanged during the time when ecdysteroid titers surged in the controls. 81.5 f 9.3 29.4 f 7.7 21.0 f 4.5 77.8 i 8.0 22.8 i 7.9 1.5 +- 1.3 35.2 2 10.5 19.9 2 8.9 3.8 +- 1.0 28.6 % 8.9 15.9 2 3.8 1.4 +- 0.9 21.0 f 3.9 18.8 2 3.5 1.9 2 1.0 18.5 f 8.5 13.8 +- 3.5 0.9 f 0.7 Control Saline Teratocytes Control Saline Teratocytes D3 Phases BD CFl * -a 0.3 2 0.2 0.4 +- 0.3 1.0 i7 0.9 0.1 % 0.1 1.5 % 0.9 0.6 +- 0.3 1.9 & 0.8 0.1 ? 0.1 1.9 ? 1.0 CF2 * f 0.7 f 3.0 f 4.2 4.5 1.9 1.9 i 1.4 1.5 2 1.4 6.8 9.3 1.9 7.8 +- 0.9 4.4 2 2.9 8.0f 2.9 PP S.E.in Heliothis virescens Larvae Injected at N-5With Various Numbers 180 Teratocytes per larva 41.22 4.4 18.5 f 9.6 47.0 f 7.7 20.1 f 5.1 18.0 f 5.9 40.7 f 8.2 19.0 i: 6.4 16.4 f 6.6 350 Teratocytes per larva 65.5 f 6.7 21.2 i 8.8 44.4 f 8.7 18.8 ? 8.6 76.6 +- 9.8 30.1 f 4.0 19.2 f 2.8 2.1 i 1.4 750 Teratocytes per larva 76.4 f 8.5 19.6 f 4.2 10.8 f 4.9 56.8 10.8 49.4 f 9.4 2.5 f 1.4 5.0 f 3.3 1.9 +- 1.1 2 *n = 24 for all samples. "Stress from injection causes an extra day in the digging state that is not present in the noninjected control larvae. 74.0 f 8.3 44.6 f 9.8 40.1 f 3.9 29.6 i- 3.8 21.5 2 8.7 18.1 2 4.6 D2 23.3 f 3.5 20.8 f 4.5 15.1i 4.4 D1 Control Saline Teratocytes S TABLE 1. JHE Activity (nmol JH hydrolyzedmidml hernolymph) of 3-DayOld Microplitis croceipes Teratocytes* Teratocyte Effects on jHE and Ecdysteroids - 237 0control <.- 80 2.- 60 EE3 Pringle's saline = K E teratocytes T N e I -0 2 40 S D1 D2 03 ED CF1 CF2 PP STAGE Fig. 1. JHEactivity (nmol JHhydrolyzed/min/ml hernolymph) ? S.E. in Hehothis virescens larvae injected at N-5 with 750 3-day-old Microplitis croceipes teratocytes. (n = 24 for each sample time and treatment.) One-half of the N5 H . virescens larvae treated with 350 teratocytes died as larvae (DLM) approximately 5 days after the BD stage, well beyond the 4 days typically needed for pupation (Table 5 ) . The remaining treated larvae expired as the result of unsuccessful larval-pupalecdyses (ILPE). Treatments with ethanol or 20-OH-ecdysone (0.1 @larva) did not significantly alter this response. However, a 0.25 pg/larva dose of 20-OH-ecdysone at the BD stage resulted in 13.6% successful pupation. At doses of 1 and 2.5 pg/larvae, pupation rates TABLE 2. JHE Activity (nmol JH hydrolyzedmidml hemolymph) 2 S.E. in D2 Heliathis virescens Larvae Injected at N5 With 350 Teratocytes of Various Ages Obtained From Heliothis virescens Hosts Parasitizedby Microplitis croceipes Treatment (Teratocyte age) 2 Days 3 Days 4 Days 5 Days 6 Days Saline Noninjected Parasitizedb n JHE Activity" 6 6 6 3.0 1.7 6.7 f 0.8 9.0 f 9.6 12.8 f 6.5 14.5 2 7.7 76.5 f 2.7 83.0 f 3.3 2.9 f 0.3 6 6 4 6 - * a b bc C C d d - 'Means followed by the same letter are not significantly different (a = 0.05; new Duncan's multigle range test). From Dahlman et al. . 238 Zhang et al. TABLE 3. Effect of Various Biological Preparations on JHE Activity in Heliothis virescens Lame* * Treatment n JHE Activity S.E. (nmoles JH hydrolyzed/min/ml) N.virescens plasma (10 p1) M. luteus cell wall (20 pg) M. croceipes eggs (3 or 4) C. congreguta teratocytes (500-800) M . croceipes teratocytes (350) 7 6 6 8 8 6 8 103.0 -+ 42.0 172.7 t 61.3 1078 2 39.1 114.9 19.8 21.0 2 4.5 128.5 26.4 149.6 t 10.1 Saline control (10 pl) Noninjected control * *Larvaewere injected at N5 and JHE was measured at D2. were increased to 50% or more and delayed larval mortality was either significantly reduced or absent. There was a 2-day delay in pupation of 20-OH-ecdysone-treated larvae as compared to that observed for noninjected controls. The time required to express ILPE ranged from 5.2 to 7.9 days beyond BD. A dose of 350 3-day-old teratocytes significantly reduced the mass of fat body produced in a host. At the D2 stage there was only about 60% the amount of fat body tissue in the insects that received the teratocytes (35.6 1.6 mg fresh weight) as compared to 56.5 k 1.7 mg for the saline controls and 56.8 & 1.4mg for noninjected controls. Injection of saline had no effect on fat body development. * DISCUSSION Teratocytes reduced hemolymph JHEina dose-dependent manner. JHE activity remained at very low levels in D2-BD larvae injected with the embryoequivalent of 750 3-day-old teratocytes, in contrast to high levels observed in normal larvae. Larvae treated with 350 teratocytes had somewhat higher JHE activity, although levels remained significantlybelow normal. The specific mode of action of teratocyte inhibition of JHE remains unknown. It is possible that the teratocytes release an inhibitor which blocks JHE synthesis and/or release. Since fat body development was retarded by teratocyte injections and fat body synthesizes JHE , inhibition of JHE synthesis, either directly via some prod- * TABLE 4. EcdysteroidTiter (pg 20-OH-Ecdysoneequivalentdpl hemolymph S.E.)in Heliothis virescens Larvae Injected With 350 3-Day-Old Microplitis croceipes Teratocytes at N5 Larvae Control Saline Teratocytes BD CFl 47 c 12 (5)a 77 f 18 (6) 363 f 35 (6) 130 f 26 (6) 45 k 18 (6) 300 k 28 (6) aNumbers in parentheses = n. Phases CF2 or CF1 $. 24h 469 k 114 (3) 405 -t 96 (5) 173 35 * (6) CF2 + 12 h or CFl + 36h CF2 + 24 h or CF1 + 48 h 1,426 5 463 (6) 2,534 577 (2) 314 f 61 (2) 4,293 905 (3) 4,235 2 682 (3) 254 f 81 (3) * * Teratocyte Effects on JHEand Ecdysteroids 239 TABLE 5. Effects of Rescue With 20-OH-Ecdysone on Heliothis virescens Larvae Treated With 350 3-Day-Old Microplitis croceipes Teratocytes at N5* Treatment (CLg per larva) 2.5 DLM (”/.I 0 1.0 5.5 0.25 40.9 0.10 63.6 Days to larval mortality” ( 2 S.D.) - ILPE (“/.I 45.5 6.0 45.5 6.3 ( + 1.5) 6.9 45.5 (-1 36.4 ( 2 1.4) Solvent 59.1 Nonrescued 50.0 5.9 ( + 1.2) 40.9 5.2 50.0 (f0.4) Noninjected control 0 - 0 Days to ILPE ( 2 S.D.) 5.2 ( * 0.9) 6.0 ( * 1.2) 7.9 ( 2 1.7) 7.6 ( k 0.5) 6.7 ( 2 1.7) 6.0 (i0.9) - P (%) 54.5 Days to P ( k S.D.) 0 5.6 0.9) 6.1 ( * 1.1) 6.0 (f1.0) - 0 - 0 - 100 3.8 ( + 0.4) (k 50.0 13.6 *20-OH-Ecdysone treatment applied at BD, 22 larvae per treatment. aDaysmeasured from time of 20-OH-ecdysone treatment at BD. uct released by the teratocytes or indirectly via general inhibition of fat body growth, may be responsible for the observed results. Alternatively, Hayakawa [24,25] reported purifying a 4,500-dalton peptide JHE repressor factor from the hemolymph of Pseuduletia sepuruta parasitized by Apunteles karzyui. Perhaps this peptide was released by the teratocytes from A . kariyui and teratocytes from M. croceipes release a similar factor. It is important to recognize that ail ages of teratocyte tested were effective, even though the same number of 6-day-old teratocytes were less efficient than a similar number of 2-day-old teratocytes. Although the teratocytes do not undergo cellular division, the ploidy number increases  and they increase in size [ll];thus, the older teratocytes are larger and perhaps have a greater capacity to secrete substances that inhibit JHE production. Because we observed a slight reduction of potency in older teratocytes, their effectiveness per unit weight may truly be reduced. Under our experimental conditions, the teratocytes in parasitized larvae would be approximately 6 days old at the time of JHE release (D2-BD) in nonparasitized larvae. However, they were present in the host as free floating cells all during the 5th stadium and could have been affecting fat body development during this entire time. The minimum time between teratocyte injection and JHE inhibition has not been determined but will be the subject of future studies. We did not conduct an exhaustive test for other biological preparations that may cause reduced levels of JHE, but we did show that several foreign materials, including M. croceipes eggs and teratocytes from another braconid species, did not prevent synthesis and release of JHE in H. virescens. Only M. croceipes teratocytes caused a marked reduction in hemolymph JHE levels. Hayakawa  reported that the peptide he isolated had limited in vivo activity in P. sepurutu. However, he showed that multiple injections (five injections 240 Zhang et al. maximum) performed at daily intervals kept the JHE titer depressed for as long as the injection regimen was maintained. Presumably, when the inhibitory effect of the peptide diminished, JHE was released, the JH titer dropped, and the larval-pupal molt was initiated. We have not determined JH titers in parasitized or teratocyte-injected larvae but they might be expected to remain at a level similar to those found in the larval stage prior to the release of JHE. The actual mechanisms by which JHE activity is suppressed and larval developmental events are mediated are not known. Prior to pupal commitment, high JH titers inhibit ecdysone production [27,28] by preventing the release of PTTH from the brain, as well as by influencing the competency of the prothoracic glands to respond to MTH and a hemolymph ecdysiotropic factor [29,30]. In teratocyte-injected larvae, high JH titers may continue to prevent the release of PTTH and/or may keep the prothoracic gland in an incompetent state. While JH titers prior to JHE release are primarily controlled by synthesis , the timely release of JHE is responsible for the rapid degradation of existing JH in the hemolymph , thus setting the stage for subsequent developmental events. It has been reported that treatment of young larvae with JH decreases the JHE activity or inhibits its production at the appropriate time [30,31]. Without a drop in JH titer and the resulting return to compe tency of the prothoracic gland, ecdysteroid production should be minimal. However, there are other reports that JH production initiates JHE synthesis and/or release . Thus, teratocytes may cause JH titers to be low and the JH-induced JHE synthesis is never initiated. During the earlier part of the 5th stadium, ecdysteroid titers in N.virescens parasitized by M. croceipes were higher (150-200 pg/Fl) than in controls . In this report we observed the same pattern in larvae treated with teratocytes. Presumably this titer is adequate to program larval tissues for pupal commitment, but larvae do not advance in development beyond CF1 because the second large peak of ecdysteroid is absent. Parasitized larvae showed tissue response to injections of 20-OH-ecdysone by forming pupal cuticle and attempting to ecdyse , and we have shown here that 20-OHecdysone rescued a significant number of larvae previously injected with teratocytes. We suggest that the action of teratocytes, while not specifically known, impacts the complex interplay of hormone titers and feedback mechanisms to prevent the synthesis, release, and/or catabolism of adequate amounts of ecdysteroid. In the case of M . croceipes, the action is independent of any effect from parasitoid venom or calyx fluid. In contrast, Wani et al.  showed that teratocytes from A . kariyui prevent larval-pupal ecdysis only in the presence of both venom and calyx fluid. As an alternative, the teratocytes could be synthesizing and releasing JH  which could keep ecdysteroid titers depressed via feedback mechanisms . However, if JH levels were high, the 20-OHecdysone rescue experiments should have caused larval-larval molts rather than larval-pupal molts unless JH levels dropped for a short time (to allow tissue commitment) and then increased again. In either case, the parasitized and teratocyte-treated larvae remain in the CF1 stage because of the inadequate titers of ecdysteroid necessary to drive the cells to the larval-pupal transformation. Teratocyte Effects on JHEand Ecdysteroids 241 LITERATURE CITED 1. Riddiford LM: Hormone action at the cellular level. 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