Juvenile Hormone Esterases of Lepidoptera

Transcription

1 J Comp Physiol (1982) 148:1-10 Journal of Comparative Physiology, B 9 Springer-Verlag 1982 Juvenile Hormone sterases of Lepidoptera. Ativity in the Hemolymph During the Last Larval nstar of 1! Speies Davy Jones, Grae Jones, Keith D. Wing, Maria Rudnika*, and Brue D. Hammok Departments of ntomology and nvironmental Toxiology, University of California, Davis, California 95616, USA Aepted May 1, 1982 Summary. The juvenile hormone esterase (JH) titer was measured during the last larval instar of 11 speies of Lepidoptera (Pier& rapae, Junonia oenia, Danaus plexippus, Hernileua nevadensis, Petinophora gossypiella, Spodoptera exigua, Orgyia vetusta, phestia elutella, Galleria mellonella, Mandua sexta and stigmene area). All speies had a peak of JH at or near the time of wandering. The peak ativity at this time ranged from 0.8 to 388 nmoles JH leaved/min-ml. All speies exept J. oenia had a seond peak of JH during the late prepupal stage. The height of the seond peak ranged from 0.4 to 98.4 nmoles/min. ml. However, there was no apparent orrelation between size of the first and seond JH ativity peaks for the lepidopteran speies examined. There was an apparent relationship between the height of the first and seond JH peaks and reports on titer of JH just prior to these peaks. These data support, with some qualifiations, the extension of developmental information obtained on several well studied speies to a variety of Lepidoptera. ntrodution n holometabolous insets high titers of the juvenile hormones OH's) are thought responsible for maintaining the larval stages. Low JH titers ause a shift from isometri to anisometri development leading to the pupal and adult stages. Degradation of JH by ester hydrolysis is the major initial pathway of JH metabolism in the Lepidoptera exam- * Present address: nstitute of Organi and Physial Chemistry, Tehnial University of Wrotaw, Wybrze~e Wyspifinskie, 27, Wrotaw, Poland Abbreviations: JH juvenile hormone; JH juvenile hormone esterase; PTTHprothoraotropi hormone; Ro (4'- ethylphenoxy)-6,7-epoxy-3-ethyl-7-methylnonane ined and has been suggested as a mode of JH titer regulation neessary for normal development (Gilbert et al. 1978). n fat, reent data are onsistent with a model in whih JH biosynthesis by the orpora allata is redued but not halted, and the low JH titers neessary for prothoraiotropi hormone effets result from inreased JH hydrolysis (Sparks and Hammok 1980). The first lepidopteran titered during the last larval instar for ihemolymph JH esterase (JH) ativity (Mandua sexta L.) possessed two peaks of JH, eah orrelated with an abrupt deline in hemolymph JH (Weirih et al. 1973; Nijhout and Williams 1974; Vine and Gilbert 1977; K. Judy, ited by Sridhara etal. 1978), although Nijhout (1975) noted an initial JH deline in last instar Mandua sexta may not be due to inreased JH ativity. Other lepidopteran speies were subsequently found to possess a last larval instar JH profile similar to Mandua sexta (initially high, falling very low and a prepupal rise; Varjas et al. 1976; Yagi 1976; Hsiao and Hsiao 1977; Mauhamp et al. 1979). The orrelation of JH ativity with the JH delines in Mandua sexta prompted many researhers to generate hypotheses about JH titer regulation in Lepidoptera in general, based on the Mandua sexta model (Riddiford 1980). The appliability of this model to Lepidoptera was supported by the report of two similarly plaed peaks of JH ativity in last larval instar Trihoplusia ni (Hfibner) (Sparks et al. 1979) and by the demonstration of the importane of these peaks for normal development to our (Sparks and Hammok 1980). However, Galeria mellonella (L.) was found to lak the prepupal peak of JH ativity (Hwang-Hsu et al. 1979), although it possessed the prepupal burst of JH (Hsiao and Hsiao 1977). The Galleria mellonella JH profile alls into question the appliability of the Mandua sexta model to all Lepidoptera /82/0148/0001/$ 02.00

2 2 D. Jones et a. : Juvenile Hormone sterase Ativity in Last nstar Lepidoptera The relative heights of the two last instar peaks of JH ativity in Mandua sexta also may not be an appropriate model for all Lepidoptera. n Mandua sexta the first peak in JH ativity is muh greater than the seond peak, while in Trihoplusia ni the heights of these two peaks are about the same. f the amount of JH ativity is an indiator of the importane of JH ester hydrolysis in regulation of the JH titer, then JH may be more important for eliminating prepupal JH in Trihoplusia ni than in Mandua sexta. t also appears that the first peak of JH ativity in the final instar of Mandua sexta, Trihoplusia ni and Galeria mellonella is under neuroseretory ontrol (Vine and Gilbert 1977; MCaleb and Kumaran 1979; G. Jones etal. 1981; Kumaran et al. 1981) while the seond, prepupal peak of JH in the first two speies is indued by a burst of JH from the orpora allata (Hammok et al. 1981). f the pattern of JH ativity during the last instar of other Lepidoptera is similar to that of Mandua sexta and Trihoplusia ni, then the JH regulatory mehanisms found in Mandua sexta and Trihoplusia ni may apply to the other speies as well. f the interation between JH and JH titers during the last instar in Mandua sexta is to serve as a omprehensive model for the Lepidoptera, with Galleria mellonella being an exeptional speies, the JH titer in many more lepidopteran speies must be examined and ompared to that in Mandua sexta. Thus a projet to monitor hemolymph JH ativity during the last instar of 1 speies of Lepidoptera representing 10 families was undertaken, and the results are reported here. The biohemial properties of the hemolymph binding proteins and JH esterases from some of these speies are disussed in a ompanion manusript (Wing et al., in preparation). Materials and Methods Speies used in the study, their soure and rearing onditions are given in Table 1. All weighings and bleedings were made between 14:00 and 17:00 h (day begins at lights on, 5:00 h a.m.), exept where otherwise noted. ah larva was bled only one. Developmental markers were used whenever possible to stage and preisely time subjet animals in an effort to insure that points of highest hemolymph JH ativity would not be missed. D. Jones et al. (1981) previously found that use of devel- Table i. Speies, soure of speies, and rearing onditions of larvae titered for JH esterase during the last larval instar ~ Family Speies Soure Diet medium Pieridae Nymphalidae b Pieris rapae (abbage butterfly) Junonia oenia (bukeye butterfly) F~ progeny of field adults Laboratory ulture Brassia Passiflora or Althaea Danaidae Danaus plexippus (monarh butterfly) Field olleted larvae Aslepias Saturniidae Gelehiidae Notuidae Hemileua nevadensis (Nevada buk-moth) Petinophora gossypiella (pink bollworm) Spodoptera exigua (beet armyworm) Field olleted larvae Laboratory ulture Laboratory ulture Salix Wheat germ base Pinto bean base Liparidae Orgyia vetusta (western tussok moth) Field olleted larvae rigonum Pyralidae Pyralidae Artiidae Sphingidae u phestia elutella (tobao moth) Galleria mellonella (greater wax moth) stigmene area (salt marsh aterpillar) Mandua sexta (tobao hornworm) Laboratory ulture Laboratory ulture F1 progeny of field larvae Laboratory ulture Bran-wheat germ base Cereal ~ Pinto bean base Corn grit base a All larvae reared under 14 light: 10 dark, 28 ~ onditions, exept for G. mellonella whih were kept in total darkness u Diet and larvae purhased from Carolina Biologial Supplies Bhaskaran, personal ommuniation

3 D. Jones et al. : Juvenile Hormone sterase Ativity in Last [nstar Lepidoptera 3 opmental markers was helpful in staging Trihophtsia ni larvae used in determining levels of last instar JH ativity. t is reognized that rearing onditions, photoperiod, temperature, et. an affet the observed JH ativity. Thus, although hemolymph JH ativity in Galleria mellonella and Mandua sexta has been reported by other laboratories, these two speies were monitored again here using animals reared in this laboratory and our assay proedure to failitate omparisons. JH ativity was monitored using 3H-C10 labeled JH (New ngland Nulear, 11 Ci/mmole or 470 GBq/nmole) as a substrate as previously desribed (Hammok and Sparks 1977). Hemolymph was diluted with sodium phosphate buffer (ph = 7.4, [ = 0.2 M % phenylthiourea) and held on ie at 4 ~ until assayed (usually within 48 tl). Several authors have found that in Mandua sexta there is a broad ph optimum for JH ativity over several ph units (Weirih et al. 1973; Vine and Gilbert 1977), and this is also true for JH ativity in Trihoplusia ni (Wing et al. 1981), Heliothis viresens, Spodoptera exigua and Galleria mellonella (unp. data). Therefore, observed differenes in JH ativities among speies probably reflet true differenes and are not artifats of hypothetially different ph optima. The hemolymph was diluted with buffer suh that the appearane of JH aid was linear with time during the ourse of the assay. n eah ase, the appearane of JH aid was dependent on time and hemolymph onentration. Assays were performed at 30 ~ with 5 x 10-6 M substrate added in ethanol. ah larva served as a single repliation for the time point at whih it was bled. The number of repliations was variable, but the points of highest and lowest ativity reeived the most repliations (see figure legends for more details). Statistial multiple omparisons were made to determine the number of peaks of hemolymph JH ativity during the last larval instar, to ompare the amounts of ativity of the two peaks within a speies (if there was more than one peak), and to ompare peaks from different speies. For the purposes of this study a peak is defined as a statistially signifiant rise in ativity as ompared with ativities at adjaent (though not neessarily immediately adjaent) time points. However, the skewed nature of the data, the unequal varianes and unequal sample sizes meant that the assumptions for parametri proedures were usually not satisfied. Therefore, the nonparametri test whih is least sensitive to skewness in distributions and unequal varianes of sampled populations (median test) was used to determine if there was a differene among the entral loations (medians) of the sampled populations. The median test was provided with an experimentwise error rate similar to that of Dunan's new multiple range test, a ommon parametri proedure by use of the Bonferroni nequality. The number of statistially signifiant JH ativity peaks delared for eah speies with the median test was the same as when heked with parametri proedures. Variation among repliations in eah time point is reported as standard deviation. Results Pieris rapae (L.). The duration of and weight gain during the last instar are given in Fig. 1 a. After essation of feeding, weight was not used to stage larvae but instead the following developmental markers were used: migration to top of rearing ontainers, bak girdle formation (Smith and Smilowitz 1976), loss of ability to walk, loss of ability to url, newly molted soft pupa, soft pupa with slerotized horns, 24 h old pupa. -g Z~- T 1-- "3 "5 O- r "-& 160- v U= 120- o) 80- HCS 90rng 185mg -r BGCN H D P t ' T BGCN NMP HCS /-, Day of fast instor Fig. 1. a Weight gain and physiologial markers observed during the iast larval instar of Pieris rapae (Tmigration to top of ontainer; BG bak girdle formation; C an url, not walk; N not able to url; NMP newly molted pupa; H soft pupa, horns slerotized; D1P day 1 pupa), b Hemolymph JH esterase ativity during the last instar (n = 4-14 repliates per time point) The profile of JH ativity during the last instar and early pupa is given in Fig. 1 b. Two statistially signifiant peaks were found, one ourring near the time of maximum larval weight, and the seond peak, more diffiult to detet, ourring lose to pupal edysis. The first peak was signifiantly higher than the seond peak. Junonia oenia (Htibner). The duration of and weight gain during the last instar are given in Fig. 2a. There appeared to be a bimodal distribution in maximum weight ahieved, perhaps due to sexual dimorphism, but sine it was not severe it was not further investigated. After essation of feeding, developmental markers were used to stage the larvae and these inluded: migration to top of rearing ontainer, first hanging from ontainer top, first hanging +6 h, first hanging +12 h, newly molted wet pupa, semi-slerotized pupa (~ 12 h old), and day pupa. The profile of JH ativity during the last instar and early pupa is given in Fig. 2b. A slow rise to a single statistially signifiant peak was found whih reahed a maximum 6 h after first hanging from the top of the rearing ontainer. The titer delined bak to a low point (12 h pupa) and appeared to rise again through day 3 pupal stage. This titer profile was repliated on 4 different generations of larvae.

4 4 D. Jones et al. : Juvenile Hormone sterase Ativity in Last nstar Lepidoptera r- "o -d H "T -'~ ,' 500- ~ & H N NNPD P mg 399 &99rag 599rng / 8- b) 6- ~ P HCS Doy of last instar Fig. 2. a Weight gain and physiologial markers observed during the last larval instar of Junonia eoenia (T migration to top of ontainer; H hanging by tip of abdomen; NMP newly molted pupa; D1P day 1 pupa), b Hemolymph JH esterase ativity during the last larval instar and early pupa (H+ 6 hanging for 6 h, et.) (n = 5 to 18) >* o._r u H O HCS /v W H C NC NHP S Weight (g) Physiologial marker Fig. 3. Hemolymph JH ativity during the last instar of Danaus plexippus (HCS head apsule slippage; W wandering stage; H hanging from top of ontainer; C able to url, but not walk; NC not able to url up; NMP newly molted pupa; S pigmented but still soft pupa) (n = 3 to 9) Danaus plexippus (L.). During the feeding stage, larvae were synhronized by weight while postfeeding larvae were synhronized by the following developmental markers: wandering, migration to top of rearing hamber, hanging by terminal prolegs but still able to walk, loss of ability to walk, loss of ability to url, newly molted pupa, soft pupa with some pupal oloration. The profile of JH ativity during the last instar is given in Fig. 3. Ativity was very low, yet two statistially signifiant peaks were found. The "{D 20- u 10-- T O 0- r- ~7-5- Weight (rag) b) Day of wonder g-l& 2 S SQ PPNMP Most S wondering ome ~ t.,ooon wondering --! /spinning 1_4]_31 21_ Days from maximum weight Fig. 4. a Weight gain and physiologial markers observed during the last larval instar of phestia elutella, b Hemolymph JH esterase ativity during the last instar (S ooon spinning; SQ larva an squirm but not rawl; PP larva a stiff prepupa; NMP newly molted untanned pupa) (n = 8 to 26) first peak ourred definitely prior to ahievement of maximum weight and the seond during the late prepupal stage. The heights of the two peaks were not signifiantly different. phestia elutella (Hiibner). The duration of and weight gain during muh of the last larval instar are given in Fig. 4 a. After essation of feeding, larvae were not staged by weight but by the following developmental markers: Days i and 2 of wandering, on day 3 of wandering the larva spins a ooon but retains the ability to walk, the larva an squirm but not rawl (Day prepupa), larva is a stiff, motionless prepupa unable to url (Day 2 prepupa), newly molted pupa. The profile of JH ativity during the last larval instar is given in Fig. 4b. Two statistially signifiant peaks were found. The first peak ourred on the first day of wandering. The seond peak, whih had signifiantly greater ativity than the first, ourred during the late prepupal stage. The deline of the seond peak ontinued into the early pupal stage. stigmene area (Drury). The duration of and weight gain during the last larval instar are given in Fig. 5 a. There was a signifiant sexual dimorphism between the maximum weights of females

5 D. Jones et al. : Juvenile Hormone sterase Ativity in Last nstar Lepidoptera 5 --v._ 7 % z- Physiologial weight or weight at bleeding HCS S C NCNMP ] ~ O6 b) _ j" j/[ \ 0.5 HCS Day of tast instor tt S CNCNMP t Fig. 5. a Weight gain and physiologial markers observed during the last larval instar of stigmene area (HCS head apsule slippage to the last instar; S ooon spinning; C larva an url but not walk; NC loss of ability to url; NMP newly molted pupa), b Hemolymph JH esterase ativity during the last instar (n=4 to 10) ") > 0 _.r u -r 7-6 G r~ \ S WUPR NC NMP 6 7 B DiP Day of last instar Fig. 6. JH esterase ativity during the last larval instar and early pupa of Galleria mellonella (S first ooon spinning; W dense ooon built but larva an still walk; UPR larva annot walk but an upright itself; NC no longer able to url; NM,P newly molted pupa; DP day 1 pupa) (n = 5 to 12) 0~ -6 Days from max wt Day of blaken. Day of PP & D.P 10 1 l 4 x,a (1188 rag) and males (864 rag) (median test, P< 0.05). Only females were bled for assay. After reahing maximum weight, larvae were synhronized with developmental markers. The markers used were: ooon spinning, loss of ability to walk, loss of ability to url, and newly molted pupa. The profile of JH ativity during the last larval instar is given in Fig. 5 b. Two statistially signifiant peaks were found. The first peak ourred at the time of maximum weight and the seond peak at the loss of ability to walk. The heights of the two peaks were not signifiantly different. Galleria mellonella (L.). This speies was not monitored for weight gain during the last larval instar. Staging was done by day during the feeding stage and by physiologial markers after feeding. Developmental markers used were: first ooon spinning, dense ooon with ability to walk, loss of ability to walk, loss of ability to url, newly molted pupa (untanned), and tanning but still soft pupa (early day 1 pupa). The profile of JH ativity during the last instar is given in Fig. 6. The first statistially signifiant peak found ourred on day 5 and ativity then slowly delined bak down as the pupal molt approahed. The peak ourred over 24 h before the usual time of ooon spinning. A seond tran- Color PP NMP ;, -- blakening 2,75- o; ~ ~ _51_&1_3t_21_ *L Days from maximum weight Fig. 7. a Weight gain and physiologial markers observed during the last larval instar of male and female Hemileua nevadensis (PP prepupa; NMP newly molted pupa), b Hemolymph JH esterase ativity during the last instar (DP day 1 pupa) (n = 3 to 23)9 JH esterase ativity remained low through day 3 of the pupal stage sient and more diffiult to detet small JH peak ourred at the pupal molt. Data used to define this peak were derived from bleedings from 3 separate generations. The duration of the peak is apparently muh less than 24 h. The ativity of the first peak was signifiantly greater than that of the seond peak. Hemileua nevadensis (Streth)9 The duration of and weight gain during the last larval instar are given in Fig. 7 a. There was a high degree of sexual dimorphism in size of the last instar, the females

6 6 D. Jones et al. : Juvenile Hormone sterase Ativity in Last nstar Lepidoptera reahing a signifiantly greater maximum weight (2.8 g) than males (2.1 g) (median test P<0.05). To stage feeding larvae, only several gl of hemolymph were drawn for assay. The larvae were then reared to pupation to determine the time from bleeding until maximum weight. Larvae bled and reared in this manner usually had weight gains similar to unbled ontrols. After essation of feeding, the following developmental markers were used to synhronize test larvae: olor hanges from bright yellow to dull yellow with inreasing blak (days 1, 2, 3, and 5 of this ondition), days 1, 2, 3 of prepupa (prepupa defined as a postwandering larva whih ould not walk or url so that the head touhed the tail), newly molted soft pupa, slerotized pupa (data only shown for day 1). No parasites emerged from any of these field olleted larvae. The profile of JH ativity during the last instar and early pupal stage is given in Fig. 7b. Two statistially signifiant peaks were observed. The first peak ourred at the time of maximum weight and the seond peak during the late prepupa, shortly before edysis. The first peak was signifiantly greater than the seond. Mandua sexta (L.). During the feeding phase of the last larval instar, weight was primarily used to stage larvae. Toward the end of the feeding phase, the appearane of a white, halky material on the fees (' frosted frass ', Nijhout and Williams 1974) was used. After frosted frass, larvae were staged by exposure of the dorsal vessel, wandering, the day of the prepupal phase, metathorai bars (Vine and Gilbert 1977) and newly molted pupa. The profile of JH ativity during the last larval instar is given in Fig. 8. The two statistially signifiant peaks found ourred at the appearane of frosted frass and metathorai bars, respetively. The ativity during the first peak was signifiantly geater than during the seond. Orgyia vetusta (Boisduval). The weight gain and developmental markers observed during the last larval instar are given in Fig. 9 a. There was a high degree of sexual dimorphism in maximum weight, the females weighing signifiantly more (525 mg) than the males (225 mg) at maximum weight (median test, P<0.05). Therefore, several gl of blood were taken from eah larva, and these larvae were then reared through to determine time of bleeding prior to maximum weight or pupation. n most ases, development of these larvae followed the general profile of ontrol larvae. All larvae were also observed to see whih, if any, of these field q- --) L 3g 9g FF DV W PP1 PP4 NNP Physiologiol st(re ot time of bleeding Fig. 8. Hemolymph JH esterase ativity during the last larval instar of Mandua sexta (g gram weight; FF frosted frass ; D V exposure of dorsal vessel; W wandering; PP prepupa; MB metathorai bars; NMP newly molted pupa) (n = 5 to 12) & 300- %] u 200- noo- O- bl j- o) ~ r_ S PP NN D 1 P L -L *3 Doys from moximum weight Fig. 9. a Weight gain and physiologial markers observed during the last larval instar of male and female Orgyia vetusta (S ooon spinning; PP prepupa; NMP newly molted pupa; DP day 1 pupa), b Hemolymph JH esterase ativity during the last instar and early pupa (n = 2 to 9). sterase data from male and female larvae were ombined. The seond peak ativity was 28.7 nmoles JH leaved/min vs 7.2 and 12,9 for adjaent time points olleted larvae were parasitized. Toward the end of the instar, larvae were not staged by distane from maximum weight but by distane from pupation. The profile of JH ativity during the last instar is given in Fig. 9 b. Two statistially signifiant

7 D. Jones et al. : Juvenile Hormone sterase Ativity in Last nstar Lepidoptera 7 Weight {rag) or physiologial marker at Needing Naxirnum weight ~-lday NW PPNMPD P ~u 30 -d-~ 20- ~.~ lo- 0- ~ NNPD, -~ HCS & Day of last instar Fig. 10. a Weight gain and physiologial markers observed during the last larval instar of Petinophora gossypiella (PP larva a stiff prepupa; NW not able to walk; NMP newly molted pupa; D~P day i pupa), b Hemolymph JH esterase ativity during the last instar and early pupa (n = L,O- -7-.g 0- ~ 0.2- ~z (33 ~ 0.1- Physiologial marker or weight at bleeding 9%121-15%189- HCS 10913&lL "~ "hi / ( " b] J P&tioo ell pp 0 HCS Day of last instar DiP Fig. 11. a Weight gain and physiologial markers observed during the last larval instar of Spodoptera exigua (HCS head apsule slippage of penultimate instar; PP larva is stiff prepupa; D1P day pupa. b Hemolymph JH esterase during the last instar (n = 3 to 25). Weights are mg at top of b peaks were found. The first and signifiantly greater peak ourred at the time of maximum weight and the seond peak one day prior to pupation. The delining seond peak ontinued on into the early pupal stage. Tahinid parasites emerged from 3 larvae bled at the time of host spinning and from 2 hosts bled as prepupae. The mean JH ativity in these hosts was almost exatly that of unparasitized hosts. Petinophora gossypiella (Saunders). The duration of and weight gain during the last larval instar are given in Fig. 10a. Post-feeding markers used to stage larvae inluded: 1 and 2 days post-maximum weight, loss of ability to walk, prepupa (defined as an immobile, stiff larva), newly molted pupa, slerotized day 1 pupa. The profile of JH ativity during the last larval instar and early pupal stage is given in Fig. 10 b. Two statistially signifiant peaks of ativity were found. The first peak ourred one day prior to maximum weight and the seond peak ourred at the time of loss of the ability to walk. The seond peak was signifiantly higher than the first. Spodoptera exigua (Htihner). The duration of and weight gain during the last larval instar are given in Fig. 1 la. After essation of feeding, larvae were not staged by day but by several developmental markers. These markers were: digging of a pupation ell, prepupa (defined as loss of the ability to walk or url), slerotized day 1 pupa. The profile of JH ativity during the last instar is given in Fig. 11 b. Two statistially signifiant peaks were found. The first peak ourred shortly before maximum weight and the seond peak ourred during the short prepupal stage. The heights of the first and seond peaks were not statistially different. Comparison of JH Ativity in Different Speies Table 2 gives a statistial omparison of the median JH ativities of the first and seond peaks among the 11 speies, plus 2 other speies for whih data was available. n general, the speies ould be plaed in disrete groups by the amount of JH ativity. The first peak in Orgyia vetusta (388 nmoles/min'ml) was signifiantly higher than that in any other speies. Spodoptera exigua and Pseudoplusia inludens (Walker), with a first peak of 95.8 and 71.5, respetively, were not signifiantly different, although their ativity was higher than in the remaining speies. The next group was Trihoplusia ni and Galleria mellonea (38.1 and 20.7 nmoles/min.ml), while Hemileua nevadensis, Mandua sexta, phestia elutella, Junonia oenia, and Petinophora gossypiella had lower but very similar ativities ( nmoles/min.ml). The next group stigmene area and Pieris rapae had

8 8 D. Jones et al. : Juvenile Hormone sterase Ativity in Last nstar Lepidoptera Table 2. Comparison of last larval instar hemolymph JH esterase ativity at the first and seond JH peaks in 13 speies of Lepidoptera a Speies (Mean) median first peak JH titer 1st peak (Mean) median seond peak JH titer 2nd peak nmoles/min-ml ng/ml (or g) nmoles/min.ml ng/ml (or g) Orgyia vetusta (350.1) 388.0a (28.1) 23.7d Spodoptera exigua (95.8) 95.8b 85 e (94.5) 98.4b 50 e Pseudoplusia inludens b (71.5) 71.5b (126.0) 126.0a Trihoplusia ni ~ (40.0) 38,1 (3:t.4) 27,5 Galleria mellonella (24.9) 20.7d 1.9 e (8.2) 8.5d 0,044 e Hemileua nevadensis (8.9) 8.3de (3.1) 2.6e Mandua sexta (10.3) 8.2de 1.3& (3.2) 3.2e 0.72 e phestia elutella (8.1) 8.1de (27.7) 29.1 Junonia oenia d' f (6.9) 6.4e (3.2) 3.8e Petinophora gossypiella (6.6) 5.9e (22.2) 21.6d Pieris rapae ~ (4.5) 4.4ef 0.03 e (2.6) 2.2e 0.02 e stigmene area (4.2) 3.2f (7.6) 6.6d Danaus plexippus r (0.8) 0.8g (0.5) 0.4f a Speies medians followed by a different letter have signifiantly different peak JH ativity, nonparametri multiple omparison test b Data on Pseudoplusia inludens (soybean looper) from Sparks, personal ommuniation using partition assay, JH substrate Data for first peak obtained from study of Wing et al. (unpublished); for seond peak from Jones et al. (unpublished), using partition assay, JH substrate d This speies had no seond peak. JH ativity is from prepupa shortly before edysis orresponding to the time of the seond peak in the other speies e Spodoptera exigua from Yagi (1976) using data for Spodoptera littura; Mandua sexta from Fain and Riddiford (1975), Sridhara et al. (1978); Pieris rapae from Varjas et al. (1976) using data for Pieris brassieae; Galleria mellonella from Hsiao and Hsiao (1977) Butterflies. The remaining speies are moths o (_1 C Do~ R (ug) Fig. 12. Hemolymph JH ativity of day 1 pupae of P. rapae topially applied with various doses of the juvenoid Ro and bled 24 h later (n = 10 individuals per data point) very little ativity ( nmoles/min-ml) and the final speies, Danaus plexippus, had extremely low ativity (0.8 nmoles/min-ml). The speies groups listed above for the ativity during the first peak were not the same groups as those for the seond peak of ativity. There was no relationship between the heights of the first and seond peak (rs not signifiant at 0.05 level). ndution of Pupal JH Ativity in Pieris brassiae t has been shown that JH or JH ative juvenoids will indue JH ativity in the pupae of several speies of moths. Sine JH ativity in the butterflies was generally lower than that in moths, it was desirable to test whether juvenoid appliations would indue JH ativity in butterfly pupae. Fig. 12 shows that JH ativity ould be indued in a statistially signifiant dose-dependent manner by topial appliations of the juvenoid Ro (Hoffman-La Rohe). The absolute level of indued ativity was muh lower than that in juvenoid treated Trihoplusia ni pupae (Wing et al. 1981), as well as the fold indution (3 fold, vs 6 fold in Trihoplusia ni). Disussion There are several results of this study whih enable onlusions to be drawn about JH's in Lepidoptera. First, the ourrene of two signifiant peaks of JH ativity in the last larval instar is by far the most ommon situation. n many of these speies the JH esterase ativity is due to a single enzyme or to a group of very similar enzymes (Wing et al., in preparation). Only one speies, Junonia oenia, was found to have a single peak of JH ativity during the last larval instar. Seond, there is great variability in the relative ativities of the first and seond peaks. n some speies (Mandua sexta, Hemileua nevadensis, Galleria mellonella, Pieris rapae, Orgyia vetusta),

9 D. Jones et al. : Juvenile Hormone sterase Ativity in Last nstar Lepidoptera 9 the first peak is signifiantly higher than the seond. n other speies (Trihoplusia hi, Spodoptera exigua, Danaus plexippus, stigmene area), the peaks demonstrated similar levels of JH ativity. Finally, in still other speies (Petinophora gossypiella and phestia elutella), the seond peak is signifiantly higher than the first. t also appears that the butterflies, in general, have muh lower hemolymph JH ativity than the moths, although JH ativity an be inreased in the hemolymph pool by JH or JH analog appliations in speies from both groups. Another possible generalization onerns the time of ourrene of the two peaks. No speies possess a peak of JH ativity early in the last larval instar. nstead, there is a rise, variable in steepness, usually ulminating in a first peak at or near the time of maximum weight. The timing of the first peak in Samia ynthia Drury, Saturniidae (Weirih and Wren 1976) also appears to be similarly plaed. The JH ativity usually then delines bak down to, or lose to, the level ourring early in the instar. The seond peak then ours in the prepupal stage, shortly before the pupal molt. The seond peak seems most losely orrelated with the physiologial marker of loss of the ability to walk or url, whih ours shortly before pupation. The speies with just one peak, Junonia oenia, also begins the last larval instar with a low JH ativity and also shows a subsequent rise near the time of maximum weight. The time of peak ativity, however, ours midway through the prepupal stage, whih is in between the time of the 2 peaks found in the other speies. The ativity then gradually delines bak to the level of the early instar. Apparently, in this speies one of the two last instar peaks that is missing. t should be noted that the JH ativity of Galleria melonella published by Hwang-Hsu et al. (1977) differs from that found here in that the ativity observed in their study did not deline after wandering and then show a prepupal rise. nstead, it delined to an intermediate level at whih it hovered until pupation. n their study, larvae were bled at 24 h intervals and thus a small, transient rise in JH ativity at pupation may have gone undeteted. Also, the maximum ativity observed in this study for Mandua sexta and Galleria mellonella is lower than that previously published. Although this ould be due to different assay proedures, or strain differenes, we have found seasonal differenes in Trihoplusia ni ativity as did Vine and Gilbert (1977) for Mandua sexta. n both Mandua sexta and Gatteria mellonella the juvenile hormone titer delines to an extremely low level (Nijhout and Williams 1974; Hsiao and Hsiao 1977) onomitant with the first peak of JH ativity (Vine and Gilbert 1977; Hwang-Hsu et al. 1979). This JH peak has been hypothesized to be neessary for eliminating residual hemolymph JH one the orpora allata have been inativated. The great differenes among the 13 speies in Table 2 in the heights of the first JH ativity peak indiate that degradation may be a more important mode of JH learane during the mid-instar in some speies than in others. The differenes in magnitude of peaks of the ativity prompt hypotheses onerning the endorine events involved in lepidopteran metamorphosis. The galleria wax test has been used to measure the JH titer in Mandua sexta, Galleria rnellonella, Pieris brassiae and Spodoptera littura (F.). The use of the same assay proedure failitates omparison of the JH titers in eah of these speies. The titers in Pieris brassiae and Spodoptera littura are onsidered here as representative of the titers in Pieris rapae and Spodoptera exigua. The titer in Galleria mellonella was determined on a per gram tissue basis, while in the others the unit was per ml hemolymph. However, the JH onentration per gram of tissue in Mandua sexta is essentially the same as the onentration per ml hemolymph (Shooley, personal ommuniation). Sine the prepupal peak of Trihoplusia ni JH ativity appears to be indued by the prepupal burst of JH and an also be indued in moths and butterflies in a dose-dependent manner by topial appliations of JH and juvenoids, it would be of interest to see whether the heights of prepupal peaks of JH and JH in different speies are related. The regression of JH ativity on JH onentration using the data in Table 2 (Manduea sexta, Galleria mellonella, Pieris rapae and Spodoptera littura) yields a statistially (P= 0.05) signifiant and provoative rank orrelation value (r s = 0.80). The brain-subesophageal ganglion-ontrolled first peak of JH ativity is modulated but apparently not totally regulated by JH sine JH or juvenoid appliation only slightly inreases the first peak of esterase ativity in Mandua sexta and Trihoplusia ni while allatetomy redues but does not eliminate the peak (Sparks and Hammok 1979; Bhaskaran et al., Riddiford and Hammok and G. Jones, unpublished information). Nevertheless, this peak has been hypothesized to be important in learane of JH so that PTTH-edysone release may proeed and metamorphosis begin. The regression of JH ativity on JH onentration (first peaks) using the data in Table 2 again yields a signifiant (P < 0.05) and provoative rank orrelation of r S = 1.00.

10 10 D. Jones et al. : Juvenile Hormone sterase Ativity in Last nstar Lepidoptera Another fator whih ould impinge on the indution of JH by JH is the fration of irulating JH whih is bound to a arrier protein. Using the data of Wing et al. in preparation of the binding protein parameters in Trihoplusia ni, Heliothis zea, Mandua sexta, Galleria mellonella and stigmene area, and a heuristi JH titre of 10.8 M, the fration of JH in bound form ranged from 83-99%. A weak trend for JH ativity to be negatively orrelated with fration of JH bound was observed, but no onlusions ould be drawn. n summary, then, it appears that most Lepidoptera have 2 last instar peaks of JH ativity, whih vary in relative size from speies to speies. The peaks our near the time of maximum weight and late in the prepupal stage. Finally, JH an indue JH ativity in at least pupae and probably prepupae of both moths and butterflies. Aknowledgements. This researh was supported in part by Grant No. 5-R01-S from the National nstitutes of Health. B.D. Hammok was supported by NHS Researh Career Development Award K04 S The authors thank Drs. Thomas Baker and Mihael Rust for aess to ultures of some of the speies used in this study. Referenes Gilbert L, Goodman W, Granger N (1978) Regulation of juvenile hormone titer in Lepidoptera. n: Gaillard P J, Boer HH (eds) Comparative endorinology. lsevier/north-holland Biomedial Press, Amsterdam Fain M, Riddiford LM (1975) Juvenile hormone titers in the hemolymph during late larval development of the tobao hornworm, Mandua sexta (L.). Biol Bull 149: Hammok BD, Sparks TC (1977) A rapid assay for inset juvenile hormone esterase ativity. Anal Biohem 82 : Hammok BD, Jones D, Jones G, Rudnika M, Sparks TC, Wing K (1981) Regulation of juvenile hormone esterase in the abbage looper, Trihoplusia ni. n: Kloza M (ed) Regulation of inset development and behaviour. nt Conf, Karpaz, Poland, June 23-28, 1980, part, pp Hsiao TH, Hsiao C (1977) Simultaneous determination of molting and juvenile hormone titers of the greater wax moth. J nset Physiol 23 : Hwang-Hsu K, Reddy G, Kumaran KA, Botlenbaher W, Gilbert L (1979) Correlations between juvenile hormone esterase ativity, edysone titer and ellular programming in Galleria mellonella. J nset Physiol 25: Jones D, Jones G, Hammok BD (1981) Growth parameters assoiated with endorine events in larval Trihoplusia ni (Hfibner) and timing of these events with developmental markers. J nset Physiol 27 : Jones G, Wing KD, Jones D, Hammok B (1981) Soure and ation of head fators regulating juvenile hormone esterase in larvae of the abbage looper, Trihoplusia ni. J nset Physiol 27:85-91 Kumaran AK, Brook MM, Khipple S (1981) Juvenile hormone esterase ativity in Galleria: effets of JH, ligation and brain implantation, n: Pratt G, Brooks GT (eds) Juvenile hormone biohemistry. lsevier, Amsterdam, pp 17%184 Mauhamp B, LaFont R, Jourdain D (1979) Mass fragmentographi analysis of juvenile hormone level during the last instar of Pieris brassiae. J nset Physiol 25 : MCaleb DC, Kumaran AK (1979) Control of juvenile hormone esterase ativity in Galleria mellonella. J nset Physiol 25 : Nijhout HF (1975) Dynamis of juvenile hormone ation in larvae of the tobao hornworm, Mandua sexta (L.). Biol Bull 149: Nijhout HF, Williams CM (1974) Control of molting and metamorphosis in the tobao hornworm, Mandua sexta (L.): essation of juvenile hormone seretion as a trigger for pupation. J xp Biol 61: Riddiford LM (1980) nteration of edysteroids and juvenile hormone in regulation of larval growth and metamorphosis of the tobao hornworm. n: Hoffman JA (ed) Progress in edysone researh. lsevier, Amsterdam, pp Smith CL, Smilowitz Z (1976) Growth and development of Pieris rapae larvae parasitized by Apanteles glomeratus. ntomol xp Appl 19: Sparks TC, Hammok BD (1979) ndution and regulation of juvenile hormone esterases during the last larval instar of the abbage looper, Trihoplusia hi. J nset Physiol 25: Sparks TC, Hammok BD (1980) Comparative inhibition of the juvenile hormone esterases from Triehoplusia ni, Tenebrio molitor, and Musa domestiea. Pesti Biohem Physiol 14: Sparks TC, Willis WS, Shorey HH, Hammok BD (1979) Haemolymph juvenile hormone esterase ativity in synhronous last instar larvae of the abbage looper, Trihoplusia ni. J nset Physiol 25: Sridhara S, Nowok J, Gilbert L (1978) Biohemial endorinology of inset growth and development. n: Rikenberg HV (ed) Biohemistry and mode of ation of hormones, vol. University Park Press, Baltimore, pp Varjas L, Paguia P, Dewilde J (1978) Juvenile hormone titers of penultimate and last instar larvae of Pieris brassieae and Barathra brassiae in relation to the effet ofjuvenoid appliation. xperientia 32: Vine RK, Gilbert L (1977) Juvenile hormone esterase ativity in preisely timed last instar larvae and pharate pupae of Mandua sexta. nset Biohem 7 : Weirih G, Wren J (1976) Juvenile hormone esterase in inset development: A omparative study. Physiol Zool 49: Weirih G, Wren J, Siddall JB (1973) Developmental hanges of the juvenile hormone esterase ativity in haemolymph of the tobao hornworm, Manduea sexta. nset Biohem 3 : Wing KD, Sparks TC, Lovell VM, Levinson SO, Hammok BD (1981) The distribution of juvenile hormone esterase and its interrelationships with other proteins influening juvenile hormone in the abbage looper, Trihoplusia ni. nset Biohem 11: Yagi S (1976) The role of juvenile hormone in diapause and phase variation in some lepidopterous inset. n: Gilbert L (ed) The juvenile hormones. Plenum Press, New York, pp