Microplitis croceipes teratocytes cause developmental arrest of Heliothis virescens larvae.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 12:51-61 (1 989) Microplitis croceipes Teratocytes Cause Developmental Arrest of Hekothis virescens Larvae Deqing Zhang and D.L. Dahlman Department of Entomology, University of Kentucky, Lexington, Kentucky Microplitis croceipes teratocytes placed into nonparasitized Heliothis virescens larvae survived in the absence of a parasitoid larva and caused developmental changes in the host. Expressions of these changes included delayed larval mortality, incomplete larval-pupal ecdysis, or delayed pupation. Two day old 4th stadium H. virescens larvae were more sensitive t o injected teratocytes than were 5th stadium larvae. Three day old teratocytes were more effective than were 6 day old teratocytes. The degree of response was related to the number of injected teratocytes. For example, 750 three day old teratocytes (the approximate number from a single parasitoid egg) caused delayed larval mortality in 96% of the treated larvae whereas 175 three day old teratocytes caused delayed larval mortality in only 33% of the treated larvae. Even a dose of 80 teratocytes resulted i n 15% incomplete larval-pupal ecdysis compared to 0% for controls. Treatment with hernocyte- and teratocyte-free hernolymph from parasitized larvae, hemocytes from nonparasitized H. virescens, unfertilized M. croceipes eggs, Cotesia congregata teratocytes, or Micrococcus lysodeikticus cells all had very little effect either on larval growth or development time. Key words: tobacco budworm, Braconidae, trophoserosa cells, juvenile hormone esterase, polydnavirus INTRODUCTION Many previous studies have described changes in host development, behavior, physiology and morphology caused by its parasitoid [l-41. These changes were considered to result from either direct feeding and secretions by the young Acknowledgments: The authors express appreciation for the technical assistance of T.J. Neary and Becky Wilson and Deidre Jacobson for help with insect rearing. The authors gratefully acknowledge support of the U.S. Department of Agriculture (85-CRCR-1-1764)and a grant from R.J. Reynolds. This is paper 89-7-56 of the Kentucky Agriculture Experiment Station, Lexington, KY 40546-0091. Received March 20,1989; accepted August 10,1989. Address reprint request to Dr. D.L. Dahlman, Dept. Entomology, University of Kentucky, Lexington, KY 40546-0091. 0 1989 Alan R. Liss, Inc. 52 Zhang and Dahlman parasitoid larvae within the host andior some factors injected by the adult female during oviposition. Numerous changes in the host have been observed during the development of the parasitoid. The simultaneous expression of interdependent factors associated with host regulation makes it difficult to determine the specific role of individual components. These parasitoid-derived components  include polydnavirus , secretions from the venom and other accessory glands , and teratocytes [8-111. The teratocytes are cells derived from the serosal membrane of the egg of parasitic braconids , and although these cells undoubtedly play an important role in the parasitoid-host interaction, they have not been as extensively studied as the other factors. Vinson and Iwantsch [ 121 thoroughly reviewed the teratocyte literature through 1979. Several studies have examined the transfer of teratocytes to nonparasitized hosts. For example, Cardiochiles nigriceps Viereck teratocytes injected into nonparasitized Heliothis virescens (Fab.) larvae lived and grew in the absence of the parasitoid, and the injected larvae all eventually pupated [lo]. Several years later it was shown that teratocytes from this same species exhibited juvenile hormone activity . Buhler et al.  suggested that teratocytes from a Chelonus species may be responsible for altered development of Trichopluszu ni (Hubner), but they could show no conclusive evidence to support their suggestion. Subsequently, Jones  demonstrated that teratocytes were not involved in this system. Teratocytes have been shown to improve in vitro growth of some parasitoids [16,17] but not others [MI. None of the investigations to date has examined the host response to different numbers of teratocytes, teratocytes of different ages, and hosts of various ages. To define the potential role of teratocytes in the parasitoid-host relationship, we transplanted Microplitis croceipes (Cresson) teratocytes into nonparasitized H. virescens hosts of different ages and demonstrated that teratocytes cause retardation of host H. virescens larval development. METHODS H.virescens larvae were obtained from the laboratory colony reared on Vanderzant’s artificial diet . The developmental terms for specific physiological phases of the larvae used in this study were described by Webb and Dahlman . M. croceipes larvae were maintained in the laboratory using H . virescens larvae as hosts. Parasitoid adult wasps were held in plastic boxes (27 x 9 x 19cm) with tap water and 1:l honey:water available on cotton plugs. An equal number male and female wasps were placed in the box and were allowed to mate for at least 24 h before females were used for parasitization. Premolt third-instar H. virescens larvae were parasitized by exposing them to female wasps in a 15 cm glass Petri dish under a high-intensity, IR-filtered lamp for approximately 50 min. The ratio of H. virescens larvae to M . croceipes females was about 8:l. Each parasitized larva was held in a 18.5 ml cup holding 6-9 ml of artificial diet; the temperature was 25 2°C under long-day conditions (16L:8D). Under these conditions, the M . croceipes embryo hatched in approximately 2 days and the host was in the second day of the 4th stadium * Teratocytes Cause Larval Development Arrest 53 (D2-4+).The host larva was a newly molted 5th instar (N-5) 2 days later, and after an additional 4 days, it had reached the burrowing-digging stage (BD-5). In nonparasitized larvae, pupation occurred four days after BD-5. The age of the teratocytes was measured from the estimated time of egg hatch. Therefore, for example, 2 day old teratocytes were obtained from larvae that had been parasitized 4 days earlier. At least 30 larvae of each stage were injected either with 10 p1 of Pringle's saline  or with a suspension of teratocytes in saline obtained from larvae of different ages. Approximately 750 teratocytes are derived from each M . croceipes egg , and their size increases with age [ll]. Teratocytes from 4, 5, 6, 7, or 8 day post-parasitized larvae were collected from the hemolymph by cutting an anterior proleg of the parasitized larva and collecting the teratocyte-containing hemolymph in a 1.5 ml microcentrifuge tube containing 1ml of Pringle's saline. Teratocytes were concentrated by a 30 s spin on a microcentrifuge. Following centrifugation, approximately the upper half of the supernatant was discarded before the soft pellet containing teratocytes and hemocytes was resuspended in the remaining supernatant. This suspension was mixed with Pringle's saline to a final volume of 2.5 ml in a 13 x 100 mm tube held in ice. The teratocytes were allowed to settle for approximately 15 min, and approximately 1.25 ml of the upper portion was removed and replaced with an equal volume of fresh Pringle's saline. The teratocytes were resuspended by gentle swirling of the tube. This washing procedure was repeated at least three times and was estimated to remove at least 90% of the hemocytes. After the final wash, the teratocytes were allowed to settle for approximately 30 min and all but 250 pl of the overlying saline was removed. The teratocyte concentration was then determined and Pringle's saline was gradually added until the desired dilution was obtained. Dilutions were verified by actual counts of the number of teratocytes in a 2 pl suspension placed in a depression slide. Parasitized larval hemolymph was obtained as above from 5 day postparasitized larvae by collecting 1ml of hemolymph into a 1.5ml microcentrifuge tube containing 0.5 ml Pringle's saline. The hemolymph was centrifuged for 2 min to ensure complete removal of cellular material, and the supernatant was used for injection. Control hemolymph was collected from S-5 nonparasitized larvae that were equivalent in age to 5 day post-parasitized larvae. The hemolymph was treated as described above, and the supernatant was used for injection. Hemocytes from nonparasitized S-5 larvae were concentrated from 300 pl of hemolymph diluted with 600 pl of Pringle's saline by centrifugation for 2 min followed by resuspension of the hemocyte pellet in 200 pl of Pringle's saline. Cotesiu congregutu (Say)teratocytes obtained fromManducu sextu (L.) hemolymph were prepared similar to the Microplitis teratocytes. Unfertilized M . croceipes eggs were dissected from the ovaries. Two to four eggs were injectedlHe2iothis larva. Lyophilized Micrococcus lysodeikticus cells (Sigma Chemical Co., St. Louis, MO) (200 pg/ml) were suspended in Pringle's saline. Larvae in stage D2-4, N-5, or BD-5 were injected with 10 pl volumes of the different preparations. *Abbreviations used: BD-5 = burrowing-digging, 5th stadium; CFI-5 = cell formation, day 1; CF2-5 = cell formation, day2; D2-4 = day 2,4th stadium; D1-5 = digging, day 1,5th stadium; D2-5 = digging, day 2, 5th stadium; D3-5 = digging, day 3, 5th stadium; DLM = delayed larval mortality; DP = delayed pupation; ILPE = incomplete larval pupal ecdysis; N-5 = new 5th stadium; P = normal pupae at expected time; S-5 = slender 5th stadium. 54 Zhang and Dahlman All the larvae were rinsed in 75% ethanol solution for 2-3 s to retard subsequent fungal and bacterial growth on the larvae and then anesthetized with C 0 2before injection. Injections were performed using a 50 pl Hamilton syringe. The needle was directed posteriorly and entered dorsally through either the left or right side behind the head capsule. Each larva received a total volume of 10 pl- Larvae that bled profusely following injection were discarded. Care was taken to prevent puncturing of the gut wall, which would result in early (1-2 days post-injection) death of the larvae. Data from these larvae were not included in the study. After injection, larvae were removed and placed in individual fresh diet cups to insure an optimum growth condition. Larvae were weighed daily, and the behavioral and morphological markers were observed. RESULTS Preliminary studies with teratocyte injections of H . virescens larvae suggest that teratocytes interfered with the larval-pupal molt. Four categories of host response were established as follows: DLM (usually occurred near the expected time of CF2 when larvae were injected at D2-4 or N-5 or occurred 1to several days later than the expected pupation date when injected at BD-5); ILPE (evidence of an attempt to pupate included formation and tanning of pupal cuticle underneath the unshed larval cuticle, anterior projection of the larval head capsule, and/or partial shedding of the larval cuticle accompanied with partial removal of the tracheal cuticle); DP (normal pupae but occurred 1-4 days later than expected); and P (normal pupae at expected time). DLM was considered to be the maximum response because of termination of growth at an earlier point in the developmental process. Three day old teratocytes were more effectivethan were 6 day old teratocytes (Table 1)with greater than 70% of the mortality observed as DLM compared to less than 20% with 6 day old teratocytes. However, 6 day old teratocytes were active enough to produce approximately 70% ILPEs when injected into either D2-4 or N-5 larvae. When compared to injections with saline, both 3 and 6 day old teratocytes significantly reduced subsequent weight gain when injected into D2-4 larvae (Table 1).The only combination that produced a minimum response was when 6 day old teratocytes were injected into BD-5 larvae. However, even then, 36% of the larvae failed to pupate as compared to 100% pupation of the controls. The only apparent effect of the saline injection was a 1 day delay in pupation compared to noninjected controls. The actual delay in development occurred just after injection because most saline-treated larvae spent an extra day in the active feeding period of the ultimate larval stadium. Teratocytes were checked for viability using absorption of 0.1% solutions of neutral red and exclusion of trypan blue in Pringle's saline as criteria. Teratocytes harvested from all parasitized larvae 3-10 days post-parasitization turned red when placed in a solution of neutral red but retained their original pale-yellow color for at least 20 min when placed in a solution of trypan blue. Both tests confirmed the teratocytes were living cells. Damage to teratocytes resulting from collection, centrifugation, and washing procedures ranged between 0.9 and 2.7%. In order to determine viability of transplanted teratocytes, approx- Teratocytes Cause larval Development Arrest 55 TABLE 1. Heliothis virescens Host Response as a Function of DifferentAge Microplitis croceipes Teratocytes and Host Age* Response Host age DLM (%) Days to larval mortality ( 2SD) ILPE (%) Days to ILPE ( tSD) Delayed pupation” (days) ( 2 SD) (%) 0 - 0 0 - 0 + 161.4B ( f 45.7) + 150.7B 0 - 0 ( f 39.6) - 3.8A ( f 0.1) 3.3AB (20.8) 2.9 A ( 20.3) 0 DP (%) P Weightb gain (mg) 3 day old teratocytes D2-4 N-5 BD-5 72.7 83.3 84.3 9.2A‘ ( + 1.7) 7.8B ( 21.0) 7.7B ( 20.6) 27.3 7.1 A ( 2 0.4) 16.7 7.0A ( t0.8) 15.7 6.8A (r 0.4) 6 day old teratocytes D24 16.7 8.0B 66.7 ( ? 0.4) N-5 18.1 7.6 B 72.7 ( 20.2) BD-5 18.1 6.7C ( + 0.3) 7.5A 1.1) 7.5A ( 20.2) 7.3 A ( 20.4) 16.7 (i- 18.1 0.9 63.8 0 + 158.2B ( *40.1) + 133.1B (k 0 49.6) - Pringle’s saline D2-4 0 0 - 100.0 1.2 c 0.4) 1.1 c ( k 0.2) 0.9C ( 20.1) 0 (k N-5 0 BD-5 0 - 0 - 100.0 0 - 100.0 0 + 358.3 A (+ 40.9) + 145.78 ( 253.6) 0 - *Each treatment consisted of between 30 and 45 larvae injected either with Pringle‘s saline or with 750 teratocytes in saline. ”Daysafter expected pupation day. bDifferencebetween initial and maximum weight. ‘NOS. in the same column followed by the same letter are not significantly different (P < .05) by Duncan’s multiple range test. imately 350 2 day old teratocytes were injected into a group of N-5 larvae and subsampled on successive days after treatment (5-5 to pharate pupa). Nearly all the teratocytes were viable for the first 5 days, but on the sixth day all had lost their viability. The effect of different numbers of teratocytes on the development of host larvae was also examined. Based on the results previously described, 3 day old teratocytes were used with the BD-5 stage host because this stage was larger and responded as well as the smaller sized, younger N-5 stage. We found a dose-dependent effect and as few as 175 three day old teratocytes were able to prevent pupation of 80% of the injected larvae (Table 2). A shift in mortality from DLM to ILPE was observed with decreasing teratocyte numbers. Even a dose of 80 teratocytes resulted in 15%ILPEs. An injection of 750 teratocytes of different ages produced an inverse relationship between teratocyte age and Heliothis larval response (Table 3). Two 56 Zhang and Dahlman TABLE 2. Host Response of BD-5 Heliothis virescms Larvae to Injections of Various Numbers of 3 day old Microplitis croceipes Teratocytes* Response No. injected teratocytes DLM 1,400 86.7 1%) Days to larval mortality (? SD) 8.0 A“ ILPE (%) 13.3 Days to ILPE (-+ SD) 175 33.3 7.4 A ( * 1.2) 46.7 80 0 - 15.4 Saline 0 - 0 8.0 A ( + 0.7) 7.7 AB ( * 1.2) 6.8 B ( * 1.0) 5.3 c ( * 0.4) 5.0 C (-+ 0.4) - Control 0 - 0 - ( ? 1.3) 750 76.2 8.1 A 23.8 ( ? 1.2) 350 46.7 7.5 A 40.0 (t0.5) DP (days) ( * SD) (%) 0 - 0 0 - 0 2.8A 0 DP (%) 13.3 P ( * 0.2) 20.0 84.6 97.6 0 1.9B ( * 1.0) 1.6B ( ? 1.3) 1.OB ( ? 0.1) - 0 0 2.4 100.0 “Each treatment consisted 30-45 BD-5 larvae. a N ~in~the . same column followed by the same letter are not significantly different (P > .05) by Duncan’s multiple test. and 3 day old teratocytes prevented pupation in all treated larvae, whereas 4 and 5 day old teratocytes successfully prevented pupation in approximately one-half of the treated larvae (Table 3). Although 6 day old teratocytes were even less effective, they still prevented pupation in 36%of those treated. Delayed pupation associated with injected teratocytes was significantly longer than control individuals (Table 3, compare teratocyte treatments to hemolymph plasma and saline treatments). The effect of an injection of the equivalent of lop1 of either hemocyte- and teratocyte-free hemolymph from parasitized larvae or hemocyte-free hemolymph from control larvae were evaluated and the results are included in Table 3. The 13.3% ILPE obtained from the parasitized hemolymph treatment may have been in response to unidentified cell-free materials associated with the parasitized condition of the host from which the hemolymph plasma was derived. Hemolymph from nonparasitized HeZiothis larvae and Pringle’s saline did not produce either DLM or ILPEs even though approximately one-half of the larvae experienced a delay in pupation, presumably as the result of trauma related to the injection (see also saline controls in Tables 1 and 2). Injections of H. virescens hemocytes, C. congregutu teratocytes, unfertilized M. croceipes eggs or a suspension of lyophylized M. lysodeikticus cells all failed to produce anything other than a 1day delay in pupation time. The behavioral and morphological changes in 5th-instar Heliothis larvae injected at the N-5 stage with 750 teratocytes included changes in pigmentation, heart beat, ocellar pigmentation, ocellar bar, feeding behavior, and tactile response (Table 4). These characters were first described nonparasitized and parasitized larvae by Webb and Dahlman . Teratocytes Cause Larval Development Arrest 57 TABLE 3. Host Response of BD-5 Heliofhis virescens Larvae to 750 Microplitis croceipes Teratocytes of Different Ages* Response Teratocyte age (days) DLM 2 85.0 (%) Days to larval mortality ( 2SD) ILPE (%) 15.0 4 - 0 0 - 0 3.9A 1.8) 3.5AB ( k 1.0) 3.0B ( k 1.5) 1.5C ( t1.2) 1.4C ( t0.2) 1.OC ( * 0.1) 0 0 - 0 0 - 0 - 63.3 0 - 0 - 0 40.0 6 18.1 Control 0 0 43.3 5 Parasitized hemolymph' Control hemolymph' Saline (%) 5.5 AB ( + 0.7) 5.2 AB (+ 0.3) 5.0 B ( t1.1) - 33.3 6.4 A (a) DP (days) ( tSD) DP 9.2 A" 0.7) 7.8 B ( t0.6) 7.7 B ( k 1.1) 6.5 C ( t1.0) 6.2 C ( k 1.5) - P ( & 1.3) (k 3 Days to ILPE ( + SD) 66.7 6.1 AB ( & 0.7) 16.7 5.7 AB 40.0 ( ? 1.4) 6.7 18.1 13.3 (k 53.3 63.8 46.7 50.0 - 0 0 40.0 50.0 36.7 100.0 *Each treatment consisted of between 30-45 BD-5 larvae. a N ~in~the . same column followed by the same letter are not significantly different ( P > .05) by Duncan's multiple range test. 'Hemocyte- and teratocyte-free. 'Hemocyte-free. The nonparasitized larva takes 9 days to complete 5th-instar development and pupate. As the insect aged, the integument pigmentation changed from very dark to light, the dorsal heart became very apparent on BD-5, the ocellar pigments were retracted to the maximum on PP, and the ocellar bar was completely gone from CF2-5 . Injections of N-5 larvae with 10 p1 of Pringle's saline modified this scheme to some degree. The primary effect was to add an additional day, digging 3 (D3-5) to the normal development time. This resulted in slight modification of the schedule of morphological and behavioral changes that occurred in noninjected controls , including slight differences in pigmentation, an extension of the time the dorsal vessel was apparent, and a delay in the complete disappearance of the ocellar bar until early CF2-5 (Table4). Pupation of Pringle's saline-injected larvae was normal, but usually 1day later than untreated larvae. In contrast, teratocyte-treated larvae appeared to stop development at CF1-5. These individuals did not pupate and only gradually, over a period of days, attained some of characteristicsassociated with CF2-5 (Table 4). Pigmentation did not change as much as saline-injected larvae, heart beat was observed on D3-5 and BD-5, but was not as pronounced, ocelli began to retract 3 days following cell formation (a delay of 2 days), and the ocellar bar was still recognizable at the time of larval death. After treatment, larval feeding was restricted to the diet surface for 4-5 days in contrast to only 3 days for saline-injected S N 7 - ++ + CF C ++ CF C W CF - + ++++ - - + PP 8 ++ ++ CF1 CF2 6 D1 S N - - - - - + + - + - - - - - R SU SU E SU R E SU E SU CF CF CF - CF ++ + CF + ++ - E S L S L S L S L S L D ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ ++ - - + + + + + + + + + + + + + + + + +++ +++ +++ +++ +++ +++ ++ 2 1 0 Days from treatment 3 4 5 6 7 8 9 1 0... Phases D2 D3 BD CF1 CF CF CF . . .b +lDa +2D +3D 750 Microplitis croceipes teratocytes ”Daysafter CFl . bAfterCF + 3D, ILPE, DLM or Larvae were observed. “Intensityof response indicated by the number of + ’s. dFeedingbehavior: SU = surface; D = digging; CF = cell formation; W = wandering. ‘Tactile response: R = rearing, mandibles extended; E = active escape; C = curling; W = Wriggling; SL = sluggish. Pigment++++‘++++++++ +++ ++ ++ ation Heartbeat + ++++ visible Ocellar pigment retracted Ocellarbar + + + + + + + + + + + + + + + + + + + + + + + W SU SU SU D D Feeding behaviord E R R R E E Tactile responsee 1 0 Pringle‘s saline Days from treatment 2 3 4 5 Phases D2 D3 BD D1 TABLE 4. Behavioral and Morphological Changes in 5th-Instar Heliothis virescens Larvae Subjected to Injection Teratocytes Cause Larval Development Arrest 59 controls. On the 6th day, treated larvae formed a cell and remained there until they died. Tactile response was very sluggish during the latter portion of development. Death ultimately occurred, either as DLM or ILPE. DISCUSSION It should be recognized that, although not free of host hemocytes, the method used to prepare the teratocytes reduced the number of hemocytes by at least 90%. The fact that injections of concentrated preparations of hemocytes from nonparasitized larvae did not alter the growth parameters of the larvae further supports our contention that the hemocytes are not a critical component in causing the observed phenomena. An alternative approach for collection of teratocytes would be to use in vitro rearing techniques similar to those described by Greany . Although such a procedure would provide teratocytes free from host hemocytes, at this time we have not developed expertise sufficient to provide the quantity of teratocytes needed to conduct the work. A confounding issue that must be considered in future studies is the fact that polydnavirus from Cumpoletis sonorensis (Cameron) is found in the nuclei of hemocytes, tracheal epithelium, muscle, and fat body cells of its H . virescens host even though replication of the virus was not observed . In addition, both male and female wasps contain DNA sequences homologous to the polydnavirus DNA and there may be at least some integration of the viral sequences into the wasp genome . This suggests that the virus is transmitted vertically through the germline. Although C. sonorensis is an Ichneumonidae and does not have teratocytes, both Ichneumonidae and Braconidae have polydnavirus. It is likely that all tissue associated with the egg, including the teratocytes, contain the polydnavirus genome. Therefore, our conclusions that teratocytes cause the described retardation of host development may actually be the result of the expression of viral genome via the teratocytes. Nevertheless, our results clearly demonstrate an important role of the teratocyte in the parasitoid-host interaction. The growth and development of teratocyte-injected larvae were very similar to those of larvae following parasitism except for those that developed into ILPE. Our findings were similar to those of Vinson [lo] who demonstrated that H. virescens larvae injected with approximately 100 C. nigriceps teratocytes (50% of the normal number) formed abnormal pupae that became sclerotized and appeared normal in the head and abdominal regions, whereas the ventral thoracic region did not become sclerotized because of the failure of the wing pads to develop. Our results suggest that the injected teratocytes inhibit the process of larval-pupal transformation. According to our investigations, the inhibitor(s) are more active in younger teratocytes and younger hosts are more sensitive than are older hosts. Even though the older teratocytes are less effective in producing altered growth in H . virescens larvae, it should be emphasized that it is only a case of degree and even teratocytes that were collected from naturally parasitized hosts 6 days after the estimated eclosion time of the parasitoid egg still had major impact (Tables 1 and 3). There does seem to be some trauma to the teratocytes caused by the collection process because the transplanted cells were only via- 60 Zhang and Dahlman ble for 5 days, whereas teratocytes from natural parasitization were still viable 10 days post-parasitization. It is also possible that a synergisticreaction occurs between the teratocytes and other factors associated with a truly parasitized host that enhances teratocyte survival. In at least one case, transplanted teratocytes attained even larger sizes when introduced into nonparasitized larvae 1101. The action of the teratocytes appears to be directed toward the latter part of the last stadium because the response of both N-5 and BD-5 larvae to 3 day old teratocytes were similar (Table 1). These data support the conclusion that the action occurs quickly, but they also support the possibility that the larvae may be conditioned several days prior to the time of response. The specific nature of the inhibitors remain undetermined. Joiner et al.  reported that extracts of teratocytes of C. nigriceps exert juvenile hormone activity. If this also is the case with M . croceipes, it is likely that the juvenile hormone titer is higher in younger teratocytes. Alternatively, juvenile hormone titers in the host may be higher as the result of a failure of juvenile hormone esterase to clear the hemolymph. Dahlman et al.  have reported reduced levels of juvenile hormone esterase in H. virescens parasitized by M. croceipes and Zhang and Dahlman (unpublished data) have shown that transplants of M . croceipes teratocytes inhibit juvenile hormone esterase production in H. virescens larvae. Further studies will undoubtedly enable us to develop an essential understanding of the importance that teratocytes play in this parasitoidhost interaction. This is the first report that has demonstrated quantitative differences in hosts treated with various numbers and ages of teratocytes in the absence of a parasitoid larva. LITERATURE CITED 1. Beckage NE, Riddiford LM: Developmental interactions between the tobacco hornworm, Ivlunducu sexfu and its parasite Apunteles congregutus. Entomol Exp Appl23, 139 (1978). 2. Johnson B: Effect of parasitization by Aphidus plutensis Brethes on the developmental physiology of its host, Aphis cruccivoru Koch. Entomol Exp Appl2,82 (1959). 3. Vinson SB, Barras DJ: Effects of the parasitoid Curdiochiles nigriceps on the growth, development and tissues of Heliothis virescens. J Insect Physioll6,1329 (1970). 4. Webb BC, Dahlman DL: Developmental pathology of Heliothis virescens parasite-mediated host developmental arrest. Arch Insect Biochem Physiol2,131(1985). 5. Stoltz DB Interactions between parasitoid-derived products and host insects: An overview. J Insect Physiol32,347 (1986). 6. Stoltz DB, Vinson SB, MacKinnon, EA: Baculovirus-like particles in the reproductive tracts of female parasitoid wasps. Can J Microbiol22,1013 (1976). 7. Weseloh RM: Dufour’s gland: Source of sex pheromones in a hymenopterous parasitoid. Science 193, 695 (1976). 8. Jackson DJ: Giant cells in insects parasitized by hymenopterous larvae. Nature 235,1040 (1935). 9. Salt G: The resistance of insect parasitoids to the defense reactions of their hosts. Biol Rev 43,200 (1968). 10. Vinson SB: Development and possible function of teratocytes in host-parasite association. J Invert PatholZ6,93 (1970). 11. Vinson SB, Lewis WJ: Teratocytes: Growth and numbers in the hemocoel of Heliuthis virescens attacked by Microplitis croceipes. J Invert Pathol22,351(1973). 12. Vinson SB, Iwantsch GF: Host regulation by insect parasitoids. Q Rev Biol55,143 (1980). 13. Joiner RL, Vinson SB, Benskin JB: Teratocytes as source of juvenile hormone activity in parasitoid-host relationship. Nature New Biol246, 120 (1973). Teratocytes Cause Larval Development Arrest 61 14. Biihler A, Hanzlik TN, Hammock B D Effect of parasitization of Trichoplusiu ni by Chelonus sp. Physiol Entomol 20,383 (1985). 15. Jones D Material from adult female Chelonus sp. directs expression of altered developmental programme of host Lepidoptera. J Insect Physiol22,129 (1987). 16. Greany P: In vitro culture of hymenopterous larval endoparasitoids. J Insect Physiol32, 409 (1986). 17. Strand MR, Vinson SB, Nettles WC, Jr, Xie ZN: In vitro culture of the egg parasitoid Telenornus heliofhidis: The role of teratocytes and medium consumption in development. Entomol Exp Appl46,71(1988). 18. Rotundo G, CavalloroR, Tremblay E: In vitro rearing of Qsiphlebusfabururn[Hym:Braconidae]. Entomophaga 33,261 (1988). 19. Vanderzant ES, Richardson CD, Fort SW, Jr: Rearing the bollworm on artificial diet. J Econ Entomol55,140 (1962). 20. Pringle JWS: Proprioception in insects. J Exp Biol25,101(1938). 21. Vinson SB: Teratocytes: Growth and numbers in the hemocoel of Heliothis virescens attacked by Microplitis croceipes. J Invert Pathol22,351(1973). 22. Stoitz DB, Vinson SB: Penetration into caterpillar cells of virus-iike particles injected during oviposition by parasitoid ichneumonid wasps. Can J MicrobiolZ5,207 (1979). 23. Fleming JGW, Summers MD: Curnpoletis sonorensis endoparasitic wasps contain forms of C. sonorensis virus DNA suggestive of integrated and extrachromosomal polydnavirus DNAs. J Virol57,552 (1986). 24. Dahlman DL, Coar DL, Koller CN, Neary TJ: Contributing factors to reduced ecdysteroid titers in Heliothis virescens parasitized by Microplitis croceipes. Arch Insect Biochem Physiol (in press).