relationship between juvenile hormone and …opmental state. furthermore, the effect of juvenile...

J. Insect Physiol. Vol. 32, No. IO, pp. 835-844, 1986 Printed in Great Britain

0022-1910/86 $3.00 +O.OO Pergamon Journals Ltd

RELATIONSHIP BETWEEN JUVENILE HORMONE AND ECDYSTEROIDS IN LARVAL-PUPAL DEVELOPMENT OF

TRZCHOPLUSIA NZ (LEPIDOPTERA: NOCTUIIDAE)

R. A. NEWITT and B. D. HAMMOCK* Department of Entomology, University of California, Davis, CA 95616, U.S.A.

(Received 21 August 1985; revised 19 December 1985)

Abstract-The haemolymph ecdysteroid titre during the late last stadium of the cabbage looper moth, Trichoplusia ni, was monitored in the presence of experimentally altered juvenile hormone levels to test the hypothesis that the appearance of a prepupal burst of juvenile hormone enhances prothoracic gland activity. Two peaks of ecdysteroid activity were detected in the haemolymph of last-stadium larvae (55ng/ml and 950ng/ml) with 20-hydroxyecdysone the most prevalent ecdysteroid. Treatment of post-feeding late last-stadium larvae with juvenile hormone I or the juvenoids, methoprene or epofenonane, had no significant effect on the timing or character of pupation. Application of juvenile hormone I to neck-ligated late last-stadium larvae failed to restore peak ecdysteroid levels to those of unligated controls. Application of the anti-juvenile hormone compounds, fluoromevalonolactone or 3,3-dichloro-2-propenyl hexanoate, disrupted metamorphosis causing delays in tanning and the formation of abnormal pupae. Yet, corresponding deviations in the timing or magnitude of ecdysteroid levels or in the conversion of ecdysone to 20-hydroxyeciiysone were not observed. When early last-stadium larvae were treated with the juvenile hormone esterase inhibitor, 3-octylthio-l,l, I-trifluoro-2-propanone, shifts in the timing of peak ecdysteroid levels, coincident with delayed pupation, were observed. These results suggest that the prepupal prothoracic glands of T. ni may be considerably less sensitive to changes in tile endogenous juvenile hormone titre by the time larvae have begun to wander, even though treatment at this time may still produce toxic effects that influence the success of ecdysis and/or resultant pupal morphology.

Key Word Index: Insect metamorphosis, ecdysteroids, juvenile hormone

INTRODUmION

The initiation of larval-pupal metamorphosis in most Lepidoptera depends upon a decline in the high titre of juvenile hormone present during the last larval-larval ecdysis to undetectable levels. This drop permits a biphasic secretion of ecdysone in the last stadium, which completes the transformation (Lafont et al., 1977; Maroy and Tarnoy, 1978; Dean et af., 1980; Bollenbacher et al., 1981). The first ecdysteroid peak is responsible for a change in the commitment of the epidermal cells and coincides with the onset of wandering, while the second peak promotes apolysis and pupal cuticle formation (Rid- diford, 1980). Juvenile hormone levels continue to remain low except for a short increase in the prepupa (Vargas et al., 1976; Yagi and Kuranochi, 1976; Hsiao and Hsiao, 1977; Sieber and Benz, 1977; Riddiford and Truman, 1978; Bean et al., 1982). This reappearance of juvenile hormone late in the last stadium is critical for successful larval-pupal metamorphosis, since its elimination influences not only the timing of pupation but also the formation of normal pupal morphology (Kiguchi and Riddiford, 1978; Cymborowski and Stolarz, 1979; Sparks and Hammock, 1979; Hiruma, 1980; Safranek et al., 1980; Sieber and Benz, 1980; Gruetzmacher et al., 1984; Jones and Hammock, 1985).

*TO whom correspondence should be addressed.

Considerable speculation has arisen over the specific role of this rise in juvenile hormone levels just prior to pupation (Hiruma, 1980). Of current interest is an observed “prothoracicotrophic effect” of juvenile hormone whereby juvenile hormone is thought to mimic the action of the prothoracicotrophic hormone in modifying the activity of the prepupai prothoracic gland (Hiruma et al., 1978; Cymborowski and Stolarz, 1979; Safranek et al., 1980; Hiruma and Agui, 1982). Yet, the experimental manipulation of endogenous levels of juvenile hormone in the last stadium has had different effects on pupation de- pending on the species examined and its developmental state. Furthermore, the effect of juvenile hormone may be rather involved. For instance, the prepupal prothoracic glands of Munduca sexta are not activated directly by juvenile hormone but indirectly through a fat-body factor whose synthesis or release is somehow triggered by juvenile hormone (Gruetzmacher et al., 1984). However, little data are available from other lepidopteran species to corrobo- rate a universal role for juvenile hormone in specifically influencing ecdysone secretion, whether its action be independent or synergistic with the prothoracicotrophic hormone.

The noctuiid, Trichoplusiu ni, is an excellent model with which to investigate the potential inter- relationships among juvenile hormone, prothoracicotrophic hormone and ecdysteroids since an increase in juvenile hormone titre in the prepupa is

835

836 R. A. NEWITT and B. D. HAMMOCK

absolutely necessary for successful pupation as demonstrated by allatectomy (Jones and Hammock, 1985), neck ligation (Jones et al., 1981) and by the application of compounds with anti-juvenile hormone activity (Sparks, 1984). However, the functional basis for the observed failure to pupate in the absence of juvenile hormone was not disclosed.

The present study explores the possible interaction between the prepupal juvenile hormone level in T. ni and the programming of ecdysone secretion. Ecdy- steroid levels were monitored by radioimmunoassay (RIA) in haemolymph from late last-stadium T. ni larvae whose endogenous juvenile hormone titre was experimentally altered by topical applications of juvenile hormone I, or the juvenoids epofenonane or methoprene, or the compounds, fluoromevalonolactone (FMEV) or 3,3-dicholoro-2-propenyl hexanoate (DPH) which possess anti-juvenile hormone activity. Finally, the contribution of specific ecdysteroids to the total titre during periods of peak ecdysteroid activity was determined in haemolymph from treated and control late last-stadium larvae by high performance liquid chromatography.

MATERIAL AND METHODS

Insect staging

Fourth stadium moulting to 5th stadium Tricho- plusia ni (bubbleheads) were removed from mass culture between 0500-0900 h after lights on and reared 10 to a container on an artificial diet at 28 + 2°C with a photoperiodic regime of 14 h light: 10 h darkness (Shorey and Hale, 1965). Here- after, all times are reported as after lights on. Fifth- ,stadium larvae usually pupate in 4 days following ecdysis (gate l), but some require 5 days (gate 2). Gate-l early last-stadium larvae were separated from gate-2 larvae according to the weight and time data of Sparks et al. (1979). For intervening time points, only larvae with weights within a specified range were collected to increase the probability of obtaining gate-l larvae (Table 1). Gate-l larvae were collected on days 3 and 4 within predefined time limits derived from the morphological markers of Jones et al., (1981) with modifications depicted in Table 1. Hae- molymph was collected from the appropriate stage and stored at -20°C with I-phenyl-2-thiourea until radioimmunoassay (RIA).

Extraction and assay of ecdysteroids

Haemolymph was centrifuged (lOOOg, 3 min) and the supernatant was extracted twice in 70% methanol (1:4, v/v), then centrifuged again (8000 g, 10 min). The pooled supernatant was partitioned countercurrently against 3 equal volumes of a 1: 1 mixture of petroleum ether-ethyl ether or hexane. The pooled hypophase was evaporated under nitrogen to dryness, then dis- solved in 70% methanol. Duplicate aliquots from at least four different concentrations of a sample were analyzed for ecdysteroids by RIA. In later experiments where high ecdysteroid titres were expected, haemolymph was diluted with borate buffer pH 8.4 and analyzed directly by RIA.

The ecdysteroid antiserum used preferentially bound ecdysone over 20-hydroxyecdysone 2.3: 1 at a final incubation concentration of l/8000, a dilution

that bound 40-50% of the total radiolabel added. Assays were performed according to the procedure of Chang and O’Connor (1979) with a total sera concentration of 6%. Data were expressed as nanogram equivalents of 20-hydroxyecdysone generated from a logit transformation of bound/lOO% bound versus log dose, where 100% bound represents that bound when no unlabelled ecdysteroid is present. Radiolabelled [23,24-3H]ecdysone (65 Ci/mmol) was supplied by New England Nuclear. Purity and concentration of unlabelled ecdysone and 20-hydroxyecdysone were periodically checked by ultraviolet spectrophotometry at 254 nm in methanol (E-12400; Horn, 1971).

Application of compounds to late last-stadium larvae to assess effects on pupation

Topical applications of juvenile hormone I, epofenonane, methoprene and fluoromevalonolactone (FMEV) and 3,3-dichloro-2-propenyl hexanoate (DPH) were performed to test their effects on the character and timing of pupation in T. ni. Epofenonane (I+‘-ethylphenoxyl-3-ethyl-7-methyl- 6,7-epoxynonane) and methoprene (isopropyl [2E,4E] - 3,7,11- trimethyl - 1 1 - methoxy - 2,4 - dodeca - dienoate) are potent juvenile hormone mimics (Zurflueh, 1976) whereas fluoromevalonolactone (tetrahydro-4-fluoromethyl-4-hydroxy-2H-pyran-2- one) (Kramer and Staal, 1981; Quistad et al., 1981; Staal et al., 1981) and 3,3-dichloro-2-propenyl hexanoate (Quistad et al., 1985) presumably function by directly inhibiting two different enzymes responsible for juvenile hormone biosynthesis. Groups of 3B-40 wandering 5th~stadium larvae were either neck- ligated at 0400 h or left unligated then treated topically on the dorsum of the thorax with a single dose of either juvenile hormone I (100 or 200 nmol), the juvenoids epofenonane (100, 200 or 400 nmol) or methoprene (200 nmol) or the anti-juvenile hormone compounds FMev (100, 265 or 530 nmol) or DPH (890, 1340, or 1785 nmol) in 2 ~1 of ethanol at various times on day 3. Ligated and unligated controls received ethanol only. As the normal time to pupation approached, larvae were examined for visible developmental abnormalities and the percentage of larvae that tanned was recorded as a measure of the success of pupation. Tanning served as an adequate measure of pupation since it occurs rapidly following ecdysis (Sparks, 1984). The time required for 50% of the larvae to tan on the dorsum of the thorax (TM”) was determined by probit analysis of the cumulative percentage response versus log time. Means from treated and control groups were compared by a two tailed Student’s t-test (a = 0.05).

Application of compounds to late last-stadium larvae to assess effects on ecdysteroid titre

Wandering Sth-stadium larvae were ligated either at 0400, 0700 or 0900 h on day 3 and then immediately treated topically on the dorsum of the thorax with a single dose of juvenile hormone I (200 nmol) in 2 ~1 of ethanol. Alternatively, unligated wandering larvae were treated with a single dose of either FMEV (530nmol) at 0400 h or at 0700 h or DPH (1340 nmol) at 0500 h on day 3. Unligated and ligated controls received only ethanol. Haemolymph was

Ecdysteroid activity 837

collected from the same number of larvae (usually 15-25) within each treatment group between lOO@-1200h,1400-1600h,l800-2000honday3and between 0600-0800 h, 100&1200 h and 160&1800 h on day 4, for each time of treatment on day 3 and assayed for ecdysteroids.

Application of 3-octylthio-l,l, 1-tripuoro-2-propanone to early last-stadium larvae

Newly ecdysed day-1 Sth-stadium larvae were treated topically on the dorsum of the thorax at 0500 h with 1 ~1 of either ethanol or a 0.4 M ethanolic solution of 3-octyl:.hio-l,l,l-trifluoro-2-propanone (OTFP) and the procedure repeated at 0830, 1200 and 1600 h thereafter and again on day 2. As a potent juvenile hormone esterase inhibitor, 3-octylthio- 1,1, I-trifluoro-2-propanone (OTFP) selectively cur- tails juvenile hormone degradation and can prolong the duration of high juvenile hormone titre (Abdel- Aal and Hammock, 1985). On the morning of the 3rd day at 0800 h and on consecutive mornings thereafter, the number of larvae displaying wandering behaviour in both control and treated groups were recorded. Those treated larvae that failed to wander by the morning of the 3rd day were considered to be affected by OTFP. Haemolymph was collected separately from the same number of gate-1 controls and OTFP-affected larvae (15-25) for RIA and then again at 1200 and 1800 h on days 3 and 4. Unaffected OTFP-treated larvae were also bled at 1200 and 1800 h on day 3 to determine if the ecdysteroid levels in these larvae were similar to those of gate-l controls. The OTFP-treated group surviving beyond day 3 includes both affected and unaffected larvae, since some control larvae normally wander on day 4 (gate-2 controls). OTFP-affected larvae that delay pupation by 1 day are indistinguishable from gate-2 controls on the morning of day 4. OTFP-treated larvae with pupation delayed past day 4 were bled at either or both of the later times on the day they first exhibited wandering behaviour, when ecdysteroid levels were expected to be high. In addition, those control larvae pupating on day 5 (gate 2) were also bled at 1200 and 1800 h on day 4.

HPLC of ecdysteroiris

Ecdysteroids were separated by HPLC and quantified by RIA to confirm the expected prevalence of 20-hydroxyecdysone as the major contributor to high RIA activity and to record changes in their respective levels in response to topical application of compounds affecting pupation. Haemolymph (3 ml), obtained separately from control and FMEV-treated larvae between 1500-1700 h on day 3, was handled initially as previously described. FMEV-treated larvae received a single dose of 265 nmol at 0400 h on day 3, while controls received only ethanol. The pooled, delipidated aqueous hypophase was evaporated under reduced pressure at 40°C to remove methanol, then extracted countercurrently against three equal volumes of water saturated I-butanol. The I-butanol phases were washed twice with an equal volume of I-butanol-saturated water, pooled and then evaporated to dryness under reduced pressure. The aqueous phases were also saved and analyzed for ecdysteroid activity by RIA. The residue of

the I-butanol phases was redissolved in 200 ~1 of 30% methanol and loaded onto a Sep-pak C,8 cartridge (Waters Associates). Highly polar ecdysteroids were eluted with 4 ml of 30% methanol (fraction no. 1) while free ecdysteroids and less polar ecdysteroids were eluted with 4 ml of absolute methanol [fraction no. 21 (Gibson et al., 1984). Both fractions nos 1 and 2 were passed through a 0.45 pm membrane filter (Millipore), evaporated to dryness under nitrogen and redissolved in 400~1 of 20% acetonitrile in water. Aliquots of the pooled I-butanol phases, fraction no. 1, and fraction no. 2 were removed for RIA. Another aliquot of fraction no. 2 (20~1) was subjected to HPLC. Fractionation of ecdysteroids was carried out on a C,, column (15 cm x 4.6 mm; Supelco) in a linear gradient of IWO% acetonitrile in water at 1.5 ml/min over 25 min with an ultraviolet detector set at 242 nm. Authentic 20-hydroxyecdysone and ecdysone standards eluted at 7 and 10 min, respectively. Fractions (1 ml) were collected, evaporated to dryness and analyzed by RIA.

RESULTS

Ecdysteroid titre profile

During the last stadium of T. ni, a small peak of RIA activity (80 ng/ml) was found at 1800 h on day 2, which dropped 34 h later [55 ng/ml] (Fig. I). A much larger peak began around 1000 h on day 3 after

Doy I Doy 2 Doy 3 Dcy 4

Hours AL0

Fig. 1. Haemolymph ecdysteroid titre during the last-larval stadium of T. ni. Haemolymph from 40-60 larvae was removed at designated day and time, partially purified, and quantified by radioimmunoassay (RIA). Duplicate aliquots from at least 5 different dilutions of a given sample were assayed. The numbers in parenthesis indicate the number of times that the procedure was independently replicated, while bars represent standard deviation. Abbreviations include: W-initiation of wandering behavior; PP-prepupal stage; E-ecdysis to pupa. Hours ALO-hours after lights on.


Table I. Staging criteria for gate-l last-stadium T. ni larvae

Day Time of last (b after Weight

stadium lights on) (mg) Morphological markers

I 2400&00 140-190 07w1200 I S-220 130&1800 190-240 1900 24C-280

2 24004x00 260-290 070% I 200 280-3 IO 130&1900 290-330 larvae approaching maximal weight, ending feeding stage; 2000 3oc-350 maximal weight attained, larvae exhibit faded dorsal white

stripes, gut purging underway; 3 240&0300 28&310 white dorsal stripes barely visible, gut purging nearly

terminated, some larvae turning pale green; 030~700 25&290 gut purging completed, larvae develop uniform pale-green

colour and migrate to top of container (wanderers); 0700-0900 24&280 larvae start to spin cocoon; 100&1800 23&260 cocoon a thick bubble of silk; 1900 22&260 larvae terminated cocoon spinning, retract abdominal prolegs;

4 24OIXO700 22&260 larvae exhibit pronounced body segmentation and cream-coloured posterior, ventral side is found facing away from surface

080&l 100 22&250 contact; larvae have enlarged anterior preceding ecdysis, following ecdysis wings become distinct, dorsum of abdomen light brown while remainder of body green;

1200 210-250 pupae become fully tanned and sclerotized

the initiation of cocoon spinning and later reached a maximum between 16OG2000 h on day 3 (950 ng/ml). These maximal ecdysteroid levels were present approx 9-13 h after the onset of wandering (uniform pale-green color) and 13-l 7 h before ecdysis. A grad- ual decline to baseline levels (S 50 ng/ml) was completed by about 1200 h on day 4.

Effect of applications of compounds on pupation

Juvenile hormone and juvenoids. Pupal ecdysis and tanning in T. ni usually began between 0700-1000 h on day 4, approx 27-30 h following the onset of wandering. When a single dose of either juvenile hormone I, epofenonane, or methoprene was topically applied to unligated wandering larvae around 0400 or 0700 h on day 3, no significant change in the Tso” value for tanning from that of controls was observed (Table 2). Applications of a higher dose of

epofenonane (400 nmol) to larvae early on day 3 also failed to significantly alter the time to pupation. Furthermore, no effect was observed when epofenonane was applied as early as 1900 h on day 2 (data not shown). Other morphological changes lead- ing to pupation such as cocoon spinning, crochet retraction, ecdysis and wing formation appeared normal in these treated larvae. Thus, an elevation of juvenile hormone above existing levels in larvae during the prepupal stage failed to elicit observable changes in the character or timing of pupation. However, the incidence of adult emergence was not assessed in these treated larvae.

Attempts to determine if applications of juvenile hormone I or epofenonane could restore pupation in ligated T. ni proved experimentally difficult because ligation resulted in high mortality prior to tanning. Only a limited number of ligated larvae survived long

Table 2. Effect of topical application of juvenile hormone 1. epofenonane, methoprene, and Ruoromevalonolactone on time to pupation in unligated late last stadium T. ni larvae

Compound

Juvenile hormone I

Epofenonane

Methoprene

Fluoromevalonolactone

Dose (nmol)

200 200 100 100 400 200 200 100 100 200 200 530 530 265 265

Time of treatment on day 3 of last

stadium (hours ALO)

033w530 07O(M830 04ow530 0730-0830 040&0430 0400-0530 0700-0730 o3ow3o 0700-0730 043w5Oa 07004l730 0400430 0630-0830 0400-0530 0630-0800

Change in T”” for tanning relative to controls*t

(hours)

+0.38 k 0.97 (lO)t +0.52 k 0.42 (3) +0.68*0.97(13) +0.08 +_ 0.88 (7) +O.l5 f 0.08 (2) f0.23 + 1.00 (5) -0.38 + I .40 (4) f0.47 f 0.38 (5) +0.03 5 0.90 (5) -0.82 f 0.82 (7) -0.75 * l.l5(5)

- 13.75 f 2.73 (3) - 5.00 + 3.38 (8) -6.87 f 1.62(4) -5.07 f 0.78 (2)

100 0400-0500 -3.88 + 0.68 (3j

‘Tanning advanced (+) or delayed (-) with respect to controls. tEach treatment group contained 40-60 wandering (day 3) larvae. $Number of independent replicates in parentheses. AL0 = after lights on.


enough to tan, usually after a minimum of 50 h following ligation. Some of these survivors had ecdysed as evidenced by tracheal withdrawal. Never- theless, no clear effect of juvenile hormone I or epofenonane on the time of tanning was observed.

FMEV and DPH. The treatment of unligated wandering larvae on day 3 before 0700 h with a single dose of FMEV (100, 265 or 530 nmol) led to the formation of fatal “larval-pupal intermediates”. At lower doses (50 nmol) fewer larvae developed into larval-pupal intermediates. Development in treated larvae paralleled that of controls until the morning of day 4, when abnormalities appeared and they failed to either initiate or successfully complete ecdysis. An enlarged head and exuviae from tracheal cuticle found along the lateral posterior portion of the body suggested an attempt to develop pupal structures and ecdyse. Yet, larval morphology still predominated. Thoracic legs remained intact and wings failed to develop underneath the larval cuticle. Although these larval-pupal intermediates were unable to ecdyse, they did tan. Tanning occurred in discrete dorsal bands or stripes which remained confined, instead of spreading uniformly over the body as in normal tanning.

A closer examination of the effect of FMEV re- vealed an associated dose-dependent delay in the time of tanning. When unligated wandering larvae were treated topically with a single dose (530nmol) between 0400-0430 h on day 3, there was a 13.75 + 2.73 h delay in tanning when compared to controls (Table 2). If the dose was decreased, the retarding effect of FMEV on the time to tanning was correspondingly reduced. Postponement of treatment to between 0630-0800 h on day 3 significantly low- ered the potency of FMEV. This effect was more dramatic at the higher dose (53Omnol).

A single application of DPH (1340 nmol) to wandering Sth-stadium larvae early on day 3 also resulted in the formation of tanned abnormal pupae and/or larval-pupal intermediates similar to the effects of FMEV but was less potent. About 90% of DPH- treated larvae attempted ecdysis, yet successful pu-

pation was minimal (18%) due to morphological abnormalities which resulted in eventual death. Many larvae acquired pupal characters but failed to com- pletely shed the exoskeleton and thus were trapped, while others remained less developed and formed larval-pupal intermediates. Nevertheless, both eventually tanned. Treatment with DPH also led to delays in the time to pupation even in those larvae that appeared normal. By 0900 h on day 4 when control larvae had completed pupation, only 15% of DPH- treated larvae had initiated ecdysis, while tanning was delayed by about 16 h. Results from a limited number of experiments also indicated that at higher doses of DPH (1785 nmol) there is increased mortality before ecdysis (26%) and a higher incidence of abnormally formed pupae (93%), while at lower doses (890 nmol) 40% of the treated larvae appear as normal pupae.

Effects of compounds on the ecdysteroid titre

Juvenile hormone I. The ecdysteroid titre of both unligated and ligated late last-stadium larvae was highest between 1400-2000 h on day 3 (Table 3). Yet, the peak titres of ligated larvae only approached the magnitude of those from the unligated controls when ligation on day 3 was postponed to 0900 h. By 0600-0800 h on day 4, the ecdysteroid titre had dropped to nearly normal baseline levels for both unligated and ligated larvae.

The application of juvenile hormone I (200 nmol) to ligated larvae failed to restore peak RIA activity to levels found in unligated larvae, nor were the levels significantly different from those in the ligated controls (Table 3). Furthermore, the application of juvenile hormone I to neck-ligated larvae did not shift or prolong the relative duration of the ecdysteroid peak. However, small differences in the peak ecdysteroid titre in response to juvenile hormone I may have been overlooked due to the frequency with which haemolymph samples were taken (4 h intervals) or due to the time-course of the experiment (32 h) following treatment.

FMEV and DPH. A single dose of FMEV (530nmol) applied topically to unligated late last-

Table 3. Haemolymph ecdysteroid titre of late last-stadium T. ni larvae following treatment with juvenile hormone I*

Treatment at 0400 h Treatment at 0700 h Treatment at 0900 h AL0 on day 3 AL0 on day 3 AL0 on day 3 -- -

Unligated Ligated Ligated Ligated Ligated Ligated Ligated Day of last Time bled5 controls + JH-I + JH-I JH-I

stadium (hours ALO) WWt (w/ml) (w&W hwiml) 3 100~1200 107 i 35 63 +55 83 k 24

(3)X (3) (2) (4: h’, 14OISl600 775 Ifr 450 101 + 52 75 k 58 87k11 64 + 29 462 ? 155 588 f 79

(7) (3) (3) (2) (2) (2) (2) 1800-2000 593 f 291 140 * 74 215 f 122 215 f 100 168 +45 266 f 197 294 + 172

(5) (4) (4) (2) (2) (2) (2) 4 060&0800 48F 17 59 ? 26 54+_8 ItOk 133k24 23 i 6 49* IO

(7) (5) (31 (2) (2) (3) (3) 1000-1200 59 _+ 32 49 -+ 9 60 f 42 69+43 81 +77 48 f 35 46 + 26

(6) (3) (4) (2) (2) (2) (3) 160&1800 46 * 20 35s 13 49 f 27

(2) (4) (3)

*Wandering larvae were neck-ligated and then immediately treated with either ethanol (ligated controls) or 200 nmol of juvenile hormone I in ethanol at the specified times on day 3. Unligated controls received only ethanol similar to the ligated controls.

tData are expressed as nanogram 20-hydroxyecdysone equivalents/ml haemolymph. tNumber of independent replicates in parentheses. @From each treatment group. IO-20 larvae were bled within designated time. AL0 = after lights on.


Table 4. Haemolymph ecdysteroid titre of late last-stadium T. rri larvae following treatment with fluoromevalonolactone (FMEV)*

Day of last stadium

Time bled? (h ALO)

3 100~1200

i40&-1600

180&2000

4 0600+00

1000-1200

1500

Controls (“g/ml)1

Treated at 0400 h AL0 on day 3

(“g/ml)

487 f 173 (7)§

840 k 214 (11)

731 & 284 (9)

79 * 54 (11)

635 I7 (12)

I58 *41 (2)

574 f 201 (5)

741 f 267 (6)

914i310 (5)

121 +88 (7)

3529 (5)

Treated at 0700 h AL0 on day 3

(“g/ml)

562 + 172 (3)

978-70 (4)

71 I + 214 (4)

82 * 35 (4)

93 * 39 (4)

I31 & I4 12)

*Wandering larvae were treated with either ethanol (controls) or 530 nmol of FMEV in ethanol at the specified time. tFrom each treatment group, IO-20 larvae were bled wlthin the designated time. fData expressed as nanogram ZO-hydroxyecdysone equivalents/ml of haemolymph. BNumber of independent reolicates in oarentheses. ALO = after lighis on. .

Table 5. Haemolymph ecdysteroid titre of late last-stadium T. n; larvae following treatment with 3,3-dichloro-2-propenyl hexanoate

(DPH)*

Day of last Time bled? Co”trols Treated stadium (h ALO) (“g/ml)1 (“g/ml)

3 0630 453 0900 781 f 615 353 i 127 II30 777 * 133 735 i_ 33 1530 1544 f 475 1434 k 176 1830 1264 +_ 329 1267 + 307

4 0530 86 380 0830 I31 685 II30 317

*Wandering larvae were treated with ethanol or 1340 nmol of DPH in ethanol at 0500 h AL0 on day 3.

tFrom each treatment group, 20-25 larvae were bled for each time point.

$Data expressed as nanogram 20-hydroxyecdysone equivalents/ml haemolymph.

§Mean f range for 2 replicates. AL0 = after lights on.

stadium larvae at either 0400 h or 0700 h on day 3 did not significantly change the ecdysteroid profile from that found in control larvae during the time-course of these experiments (Table 4).

Except for a few time points, the single dose of DPH (1340 nmol) was also unable to appreciably reduce peak ecdysteroid levels below those found in controls when applied early on day 3 (Table 5). During periods of peak ecdysteroid activity (1130, 1530 and 1830 h) there was no significant difference between the levels in treated and control larvae.

However, at 0900 h on day 3, treated larvae possessed lower levels than the controls while on day 4, treated T. ni larvae possessed elevated ecdysteroid levels above those of controls which had already declined to near baseline levels. A shift in the timing of ecdysteroid secretion seemed unlikely since peak titres of about equal magnitude were found on day 3 for treated and control larvae.

Effects of 3-octylthio-I,l,l-trifluoro-2-propanone on pupation and on peak ecdysteroid activity

When the juvenile hormone esterase inhibitor, OTFP, was applied repeatedly at a concentration of 4 x IO-’ M to newly ecdysed Sth-stadium larvae over the course of the first 2 days, a significantly lower proportion of OTFP-treated larvae (25%) pupated within the normal 4 days following the last larval-larval ecdysis as compared to controls (87%) (Table 6). By the 6th day most of the OTFP-treated larvae had pupated (92%), yet some still did not pupate until the 7th day. In contrast, all control farvae completed pupation by the 5th day.

This temporal delay in wandering was associated with coincident changes in the ecdysteroid titre and may account for the inhibition of metamorphosis. Larvae affected by OTFP exhibited low ecdysteroid levels _(< 100 ng/ml) on day 3, whereas those of controls were correspondingly high [2000 ng/ml] (Fig. 2). Larvae that were unaffected by OTFP (25%) act like gate-l controls with elevated ecdysteroid

Table 6. Effect of topical application of 3-octylthio-l,l,l-trifluoro-2-propanone (OTFP) on time to pupation in last stadium T. ni larvae*

Time expired following Percentage of total population completing pupation? the last larval-larval

__~.

ecdysis Controls Treated _~___. Four days 87 f 7t 25 f IO Five days or less 98 f 2 69 k I I Six days or less 92 + 6 Seven days or less 98 k 2

*Larvae treated topically 4 times a day with either ethanol (controls) or 0.4 pmol of OTFP in ethanol on the first 2 days of the last stadium.

*Mean f standard deviation from 3 replicates. $Each treatment group started with 200 larvae.

841 Ecdysteroid activity

HRS AL0

Fig. 2. Protile of haemolymph ecdysteroid titre in OTFP-treated and untreated last-stadium larvae. Larvae were treated with either ethanol or 0.4 ymol of OTFP in ethanol 4 times a day for the first 2 days of the last stadium at 4 h intervals. On the morning of the 3rd day, wandering larvae were separated from nonwanderers in both control and OTFP-treated groups. Over 85% of controls had displayed wandering behaviour indicative of gate-l larvae (N). In a well synchronized population, 100% wandering should occur on day 3. Control larvae that failed to wander on day 3 were subsequently sampled on day 4, accounting for gate 11s (0). Those larvae that had been treated with OTPP, yet exhibited wandering behaviour or day 3 were considered unaffected and as such acted like gate 1s ( q ). Those that had been treated and showed no sign of wandering behaviour on day 3 were assumed at&ted (a). These larve were sampled on subsequent days as they exhibited wandering behaviour. Haemolymph from 15-25 larvae was removed at the designated times on each day. The bars represent standard deviation of data from

3 experiments. Where absent, data are from fewer replicates.

levels on day 3. The ecdysteroid titre of OTFP affected larvae on day 3 also showed no indication of a rise over background levels during the time of expected peak ecdysteroid activity. On day 4, when 25% of OTFP-treated larvae initiated wandering, the ecdysteroid titre increased in a manner similar to normal untreated larvae on the previous day. Except for the temporary delay, once they began wandering OTFP-treated larvae developed morphologically and endocrinologically like the controls. A proportion of OTFP-treated larvae on day 4 were undoubtedly unaffected gate-2 larvae (11%) and could account for some of the high ecdysteroid levels found later that night in these larvae as they approached ecdysis. However, the further delays in ecdysone secretion to day 5 and 6 are more likely a result of OTFP treatment, since controls have completed pupation by day 5.

HPLC of ecdysteroids

When haemolymph obtained from gate-l day-2 larvae containing low levels of ecdysteroids (80-90ng/ml) was spiked with either ecdysone or 20-hydroxyecdysone, recovery was about 59%. RIA activity was found almost exclusively in the butanol phase, while the aqueous phase retained an insignificant amount (4%). Both ecdysone and 20.hydroxyecdysone were recovered about equally. Free ecdysteroids like ecdysone and 20-hydroxyecdysone were further separated from highly polar materials on the C,* Sep-pak cartridge, since almost

all of the RIA activity was associated with the fraction eluted in methanol (fraction no. 2) as op- posed to the fraction eluted with 30% methanol (fraction no. 1). Recovery of samples from fraction no. 2 injected into the HPLC was greater than 70% with clear separation of ecdysone from 20-hydroxyecdysone. The ratio of 20-hydroxyecdysone to ecdysone in a single sample of haemolymph from day-2 larvae was 2.4: 1. Authentic 20-hydroxyecdysone (100 ng) and ecdysone (100 ng) were routinely used to verify retention times, 7 and 10min respectively. Blank solvent runs following the injection of these standards determined that contamination from previous runs was less than 1 ng for either ecdysteroid. The amount of ecdysone present in the corresponding HPLC fractions was determined by extrapolation from an ecdysone standard curve prepared over a wide range of concentrations, similar to the procedure used for 20-hydroxyecdysone.

As expected, the predominant ecdysteroid in the haemolymph of gate-l untreated Sth-stadium larvae during the second peak of RIA activity (between 1500-1700 h on day 3) was 20-hydroxyecdysone, with ratios of 20-hydroxyecdysone to ecdysone around 9.6 + 5.4:1. Although RIA activity was found in fractions more polar than those associated with ecdysone or 20-hydroxyecdysone, the amounts were neg- ligible in comparison. Prior exposure to FMEV in doses causing the formation of larval-pupal intermediates also failed to significantly change the ratio of 20-hydroxyecdysone to ecdysone (12.4 f 8.7: 1)


from that seen in controls. The amount of 20-hydroxyecdysone detected in the haemolymph of both treated and control larvae was more consistent between samples than the levels of ecdysone. Some of the variability in the ratios of 20-hydroxyecdysone to ecdysone may be attributable to the lower accuracy with which the minute levels of ecdysone were mea- sured.

DISCUSSION

The ecdysteroid profile in the haemolymph of the noctuiid, Trichoplusiu ni, during the last stadium fits the general pattern found in other lepidopterans, where at least two major peaks of ecdysteroid activity with functional importance are found with 20-hydroxyecdysone being the most prevalent ecdysteroid (Bollenbacher et al., 1981; Dean et al., 1980; Maroy and Taroy, 1978; Lafont et al., 1977; Calvez et al., 1976). The timing of the ecdysteroid peaks

H enerated from RIA is also compatible with the data

rom previous ligation studies in T. ni (Jones et al., 1981) and by radioimmunoassay (Jones, 1985).

Previous findings demonstrating changes in the time to pupation following topical application of juvenile hormone or juvenoids were not confirmed in the present study with T. ni. Sparks (1984) had reported an observed advance in the time to tanning in unligated larvae of 4.4 h in response to a single dose of juvenile hormone I (200 nmol), while a delay of 1.5 h was found following similar treatment with epofenonane. However, no dose response or time dependence was reported for these compounds. In the present study, no significant effect of juvenile hormone I, epofenonane, or methoprene on time to tanning was observed in numerous attempts at various doses, whether application was performed early or late on day 3.

The inability of juvenile hormone I, epofenonane, and methoprene to promote precocious metamorphosis in late last-stadium T. ni larvae, similar to Ephestiu cauteflu (Lazarovici et al., 1984), is in marked contrast to the high sensitivity found in similarly aged larvae of M. sextn (Gruetzmacher et al., 1984; Safranek et al., 1980), Spodoptera littoralis (Cymborowski and Stolarz, 1979) and Mamestra brassicue (Hiruma et al., 1978). However, the doses used in the present experiments with T. ni were within the linear range of response reported from the previous studies and should have been physiologically effective. Furthermore, there is no reason to suspect that juvenile hormone I was rapidly degraded, since it was previously reported by Sparks (1984) that following topical application of 200 nmol to late last-stadium T. ni larvae, a high titre remains in the haemolymph for 6.5 h. Possibly the effects of juvenile hormone 1 may be more evident in those lepidopteran species where the prepupal stage has a longer duration than the 1.5 days following gut purge as in T. ni.

In the present study, the anti-juvenile hormone compound FMEV produced the most marked effects on the time of tanning and pupal morphology and these data are in close agreement with those of Sparks (1984). Several lines of evidence suggest that the extensive delays in time to tanning and resultant developmental aberrations observed for concen-

trations of FMEV above 50nmol are a direct consequence of reduce juvenile hormone levels (Quistad et al.. 1985; Sparks, 1984; Edwards et al., 1983; Quistad et al., 1981). However, FMEV can cause moderate toxicity at high doses for lepidopterans which are less sensitive to the compound (Quistad et al., 1981). It is noteworthy that DPH was far less active when applied to last-stadium T. ni larvae than FMEV. For instance, there was little delay in tanning with an application of 895 nmol of DPH early on day 3, while a corresponding 5 h delay was observed for 530 nmol of FMEV. Quistad et al. (1985) found DPH to be much more active than FMEV in Heliothis uirescens larvae of an earlier age, whereas the reverse was observed with similarly aged M. sexta. Several explanations to account for the differences in response to FMEV and DPH seem plausible. First, the response could be species dependent. Second, age may play a significant role since the higher metabolic capability present in the last larval stadium of many lepidopterans could preferentially degrade the more lipophilic DPH relative to FMEV. Finally, it is conceivable that DPH could be a suicide substrate requiring activation by the corpora allata. Since the prepupal peak of juvenile hormone is so brief, there may not be sufficient time for DPH to appreciably inactivate juvenile hormone biosynthesis.

Ecdysone secretion from the prepupal prothoracic gland in T. ni in the presence of prothoracicotrophic hormone seems either independent or extremely sensitive to juvenile hormone, since no change in the magnitude or timing of the second peak of ecdysteroid activity following juvenile hormone titre manipulation was observed. It is possible that the corpora allata from last-stadium larvae are less sensitive to FMEV than those from earlier aged larvae, such that the circulating titre of juvenile hormone was not reduced sufficiently to offset normal ecdysteroid secretion in the presence of normal levels of prothoracicotrophic hormone. Instead, FMEV produced toxic effects that led to the formation of larval-pupal intermediates. As a consequence, tanning was delayed. Compounds that alter juvenile hormone titre may only have an impact on ecdysteroid secretory activity in T. ni when applied earlier in the last stadium when they can indirectly effect the release of prothoracicotrophic hormone even though abnormalities still arise in pupal morphology when application is done in the prepupal stage.

Previous work had clearly shown a cumulative delayed pupation in T. ni resulting from the topical application of several juvenile hormone esterase inhibitors during the early days of the last stadium (Hammock et al., 1984; Sparks and Hammock, 1980), whereas later applications during the prepupal stage had only a limited effect (Sparks, 1984) unless very high concentrations were used (Jones and Ham- mock, unpublished). The repeated treatment of early last-stadium larvae with OTFP clearly demonstrated that a disruption of pupation via manipulation of juvenile hormone levels can be correlated with de- tectable disturbances in the timing of normal peak ecdysteroid levels in the prepupal stage. This result was important because it showed that the timing of application is crucial. OTFP may have indirectly blocked the first release of prothoracicotrophic hor-


mone thereby preventing the change from larval to pupal programming promoted by the release of ecdysone. Once OTFP was metabolized and juvenile hormone esterase activity returned to normal, juvenile hormone levels presumably declined enabling prothoracicotrophic hormone secretion. Devel- opment then proceeded and normal ecdysteroid levels were restored when sufficient prothoracicotrophic hormone had been released. A similar change in the pattern of ecdysteroid secretion was observed in Epftestia cautella in response to early applications of methoprene (Lazarovici et al., 1984). The parallel in the effects of elevating juvenile hormone titre before the wandering stage upon the timing and magnitude of peak ecdysteroid activity between T. ni and E. cautella are striking, although OTFP causes delayed pupation while methoprene promotes the production of giant larvae.

If the diminished levels of juvenile hormone in the presence of anti-juvenile hormone compounds were still sufficient to stimulate the activity of the prothoracic gland, then neck ligation or allatectomy should have been more efficient in preventing both the prothoracicotrophic hormone and juvenile hormone from reaching the prothoracic glands. Al- latectomy would have been the preferred meth- odology, however, the procedure in T. ni requires extreme precision and our attempts were met with poor success. Alternatively, neck ligation of wandering T. ni at successively later times on day 3 should have led to a progressively longer exposure of the prothoracic glands to normal circulating titres of juvenile hormone and prothoracicotrophic hormone since juvenile hormone levels in T. ni gradually rise from mid-day 2 and continue during cocoon spinning following 1000 h on day 3 and then peak at mid-day 3 (Jones et al., 1981; Jones et al., unpublished). Presumably, the levels of prothoracicotrophic hormone rise during the early hours of day 3 and remain elevated through 0930 h on day 3, since the first peak of ecdysteroid activity (1800 h on day 2) dropped during the early hours of day 3 then rose again at about 1000 h. This corresponds to a time just prior to or during the attainment of the uniform pale-green colour, but before the initiation of cocoon spinning. Neck-ligation of last stadium M. sexta larvae prior to the pale-green colour marker prevents both tanning and successful pupation, both events associated with a second period of prothoracicotrophic hormone release (Truman and Riddiford, 1974). Higher ecdysteroid levels were observed in T. ni larvae ligated later on day 3 when compared to those levels found in larvae ligated earlier. However, the ecdysteroid titre of juvenile hormone I-treated ligated larvae never approached the levels of unligated controls or exceeded levels in ligated controls, contrary to what might be expected for there to be a prothoracicotrophic effect of juvenile hormone on the prepupal prothoracic glands. The low ecdysteroid titre observed in ligated larvae may just reflect the diminished levels of prothoracicotrophic hormone.

It is possible that the effect of juvenile hormone on the function of prepupal prothoracic glands can only be observed in ligated larvae after an extended time. Gruetzmacher et al. (1984) demonstrated that a near normal, yet delayed, haemolymph ecdysteroid titre

occurs 24 or 36 h following the application of either hydroprene or juvenile hormone I respectively to neck ligated M. sexta larvae. However, in neck- ligated T. ni, a significant change in the profile of peak ecdysteroid activity in response to juvenile hormone treatment was not observed 25-32 h following treatment.

The success of larvae to withstand neck ligation may be a limiting factor in the present experimental design for T. ni. The duration of metamorphosis and responsiveness to hormonal or physical manipulation may also be quite different between species. Success- ful pupation eventually occurs in untreated, neck ligated M. sexta (10 days following ligation) if ligation is performed after the first release of prothoracicotrophic hormone due to leakage of ecdysone, whereas in untreated neck ligated T. ni successful pupation is blocked (Truman, 1972).

Thus, the present study failed to identify a prothoracicotrophic effect of juvenile hormone on the prepupal prothoracic gland of T. ni. The only significant change observed in the peak ecdysteroid activity was in response to manipulation of the juvenile hormone titre at the beginning of the last stadium, which presumably prevents the first release of prothoracicotrophic hormone. Why topical application of FMEV or DPH to T. ni prepupae led to such dramatic changes in morphology and rate of tanning remains an enigma. Further work on this species should focus on other experimental designs that can be used to test how prepupal juvenile hormone acts to contribute to successful pupation.

Acknowledgements-This work was supported in part by United States Public Health Services Grant 7-ROI- ES027 1 O-06 and a grant from the Herman Frasch Founda- tion. R.A.N. received partial support from a Jastro-Shields Fellowship and B.D.H. from a NIEHS Research Career Development Award ES-00107-05. The authors wish to thank Dr D. A. Schooley and Dr G. B. Staal of Zoecon Corp. for generously providing the compounds FMEV and DPH as well as Dr T. Kingan from Columbia University for the ecdysteroid antiserum.

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relationship between juvenile hormone and …opmental state. furthermore, the effect of juvenile...

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