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In vivo and in vitro-tissue specific metabolism of juvenile hormone during the last stadium of the cabbage looper, Trichoplusia ni
J. InsectPhysiol.Vol. 42, No. 2, pp. 181-190, 1996
0022-1910(95)00057-7
Pergamon
Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910196 $15.00 + 0.00
In Vivo and In Vitro-tissue Specific Metabolism of Juvenile Hormone During the Last Stadium of the Cabbage Looper, Trichoplusia ni VASANT L. KALLAPUR,*t
CHANDANA MAJUMDER,* R. MICHAEL ROE*1
Received 4 November 1994; revised 28 April 1995
In vitro metabolism of racemic JH III (5 PM) was measured in fat body, midgut and integument homogenate during the last stadium of the cabbage looper, Trichoplusia ni. The JH esterase activity per mg protein peaked in these tissues in prewandering larvae on day 2 and was elevated in the fat body only in prepupae (on day 4). The JH epoxide hydrolase activity peaked on day 2 in midgut and on day 3 (in wandering larvae) in fat body and integument. No differences were noted in the overall JH III metabolism in vitro per insect between wandering (day 3) larvae and prepupae (day 4) and in the in vivo metabolism of lOR,llS-JH II when injected in oil and buffer or when topically applied at physiological concentrations into these same stages. Regardless of the method of introduction of JH II, JH diol was produced in both wandering larvae and prepupae. JH acid and JH acid, diol were also produced but the predominant product was a water soluble metabolite. The preferred substrate for JH epoxide hydrolase at physiological concentrations was JH II as opposed to JH II acid. Approximately half of the JH II after injection into wandering larvae was associated with the alimentary canal and carcass. The metabolites of JH II were evenly distributed. The importance of JH epoxide hydrolase in JH metabolism in last stadium Lepidoptera is discussed. Cabbage looper Munduca sextu
Juvenile hormone Juvenile hormone esterase Epoxide hydrolase
INTRODUCTION
development in several Lepidoptera suggests that JH esterase is an important modulator of JH titer, especially in the regulation of metamorphosis (Weirich et al., 1973; Baker et al., 1987; Jones et al., 1990; Roe and Venkatesh, 1990). The disruption of normal development by specific metabolic inhibitors of hemolymph JH esterase has provided more direct evidence of the functional significance of JH esterase in lepidopteran metamorphosis (Roe and Venkatesh, 1990; Roe et al., 199 1, 1993). In comparison to the studies on JH metabolism in insect hemolymph, much less is known about JH metabolism in other insect tissues. JH esterase and JH epoxide hydrolase activity is found in integument, fat body, midgut, brain, Malpighian tubules and silk glands (Slade and Wilkinson, 1974; Wisniewski et al., 1986; Jesudason et al., 1992). Elevated JH epoxide hydrolase activity in integument, fat body and brain of prewandering, last stadium tobacco hornworms, Manduca sex&, was correlated with increased levels of JH esterase activity in integument, fat body, brain, midgut and hemolymph (Jesudason et al., 1992). Elevated JH epoxide hydrolase and JH esterase activities were also found in prepupal integument and midgut of the same species but not in fat body.
Juvenile hormone (JH) is an important factor in regulating insect growth and development (Gilbert and King, 1973; Hammock, 1985; Roe and Venkatesh, 1990; Riddiford, 1994). The JH titers that control these morphological and physiological changes are regulated by changes in biosynthesis and degradation of JH (Weirich et al., 1973; Hammock, 1985; Roe and Venkatesh, 1990). Two possible, primary routes of JH metabolism are hydrolysis of the methyl ester by JH specific esterase and hydration of the 10,ll -epoxide by epoxide hydrolase producing the carboxylic acid and dial, respectively (Slade and Zibitt, 1972; Ajami and Riddiford, 1973). The only route of JH metabolism in insect hemolymph is ester hydrolysis. The correlation of high levels of hemolymph JH esterase activity with low levels of JH during
*Department of Entomology, Box 7613, North Carolina State University, Raleigh, NC 27695-7613, U.S.A. ton sabbatical from Department of Zoology, Kamataka University, Dharwad 580 003, India. fTo whom all correspondence should be addressed. 181
VASANT
182
L. KALLAPUR
Wing et al. (198 1) measuring the metabolism of R-20458
in last stadium Trichoplusia ni, found a peak in epoxide hydrolase activity in the fat body of wandering larvae when hemolymph and other tissue JH esterase activity was minimal and the reverse was true in prepupae. The JH epoxide hydrolase activity in T. ni was never measured. Based on the relatively low level of R-20458 epoxide hydrolase activity in wandering larvae compared to the peak JH esterase activity in prepupae, Wing et al. (198 1) concluded that JH epoxide hydrolase had a secondary role in JH turnover. It is generally assumed that increased JH metabolism in Lepidoptera is responsible for the initiation of wandering behavior and the regulation of the second JH peak in postwandering larvae. However, most of these studies are based on increased hemolymph metabolism and have not considered the role of other tissues, tissue interactions, and the distribution of JH during development. Considering that the JH metabolite, JH acid, can be a precursor for JH (Sparagana et al., 1985), other routes of JH metabolism other than ester hydrolysis may be more important than originally thought. In this paper, in vitro studies of JH metabolism in specific tissues are correlated with in vivo metabolism, the tissue distribution of JH and substrate preference of epoxide hydrolase, in order to better evaluate the importance of JH esterase and JH epoxide hydrolase in JH titer regulation in last stadium larvae of T. ni. MATERIALS AND METHODS
Insects T. ni larvae (20 per 8 oz wax-coated, cardboard cup) were reared from egg hatch on an artificial diet (diet No. 2 of Roe et al., 1982) at 30 rf: 1°C with a relative humidity of 40% and with a 14:lO h 1ight:dark cycle (lights on at 6 a.m.). Fourth instars that migrated to the sides or top of the rearing containers and that cleared their gut and demonstrated head capsule slippage 6 h prior to lights off, were transferred to 1 oz plastic rearing cups without diet (1 larva per cup). The next morning (2 h after lights on), those larvae that molted (indicated by a caste head capsule) were designated as last stadium day 0 (L5DO) larvae and were provided adequate food to complete the fifth stadium. Any fourth instars remaining at this time were discarded. Nearly 75% of the larvae (gate 1 larvae) wandered on day 3 (10 h after lights on) and molted to the pupa on day 4 at approximately the same time of the day. The remainder molted on day 5 (gate 2). The earliest absolute distinction between gate 1 and gate 2 larvae was at the time of wandering. At earlier ages, a mixture of gate 1 and gate 2 larvae were used. Tissue collection and preparation for enzyme and protein assays Hemolymph was collected from the cut end of the abdominal prolegs into 10 by 75 mm test tubes held in
et al.
ice and containing a few crystals of phenylthiourea (PTU). Hemolymph was pooled from 2 to 3 larvae and centrifuged for 5 min at 1000 g. The supematant was diluted in standard sodium phosphate buffer (ionic strength = 0.2 M, pH 7.4, 0.0 1% PTU) and used immediately for analysis. After hemolymph collection, larvae were immobilized by placing them on crushed ice, and the body cavity was opened and washed with ice-cold buffer (standard phosphate buffer described above with 0.4 mM phenylmethyl sulfonylfluoride, PMSF) to remove residual hemolymph. PMSF is not an inhibitor of JH esterase or JH epoxide hydrolase. The midgut (excluding the gut content), fat body and integument (without fat body and muscles) were removed, washed and temporarily stored on ice. Each tissue was pooled from three larvae and homogenized in 1 ml of standard buffer containing PMSF in a glass homogenizer (with Teflon pestle) held on ice. The homogenate was then centrifuged at 10,000 g for 15 min at 4°C and the supernatant assayed immediately as described later.
Enzyme and protein assays Hemolymph JH esterase activity was determined according to the partition assay described by Hammock and Roe (1985). Hemolymph diluted in standard buffer was incubated at 30°C with racemic 3H-JH III (12 Ci/mmol, tritiated at C-10, New England Nuclear, Boston, MA) mixed with unlabeled racemic JH III (Calbiochem, San Diego, CA). The final JH III concentration was 5 pm, and hemolymph dilutions and incubation times were chosen to produce a linear increase in metabolism with time. Midgut, fat body and integument JH esterase and JH epoxide hydrolase activities were determined by the partition assay of Share and Roe (1988). The 10,000 g supernatant was diluted in standard buffer and 100 ~1 preincubated at 30°C for 10 min with and without 0.1 mM (final concentration) 3 -octylthio- 1, 1,l -trifluoropropan-2-one (OTFP) to inhibit the JH esterase activity. OTFP was synthesized from the n-octyl sulfide and bromotrifluoroacetone in chloroform and purified by fractional distillation and column chromatography to >98% purity as determined by gas chromatography. Studies with 10,000 g supematant from T. ni indicated that 0.1 mM OTFP inhibited 100% of the JH esterase activity and had no effect on the JH epoxide hydrolase activity. JH III substrate (previously described) was then added and incubated at 30°C. Enzyme concentrations and incubation times were chosen which produced linear hydrolysis rates with time. JH esterase and JH epoxide hydrolase activities in midgut, fat body and integument during the last stadium were monitored at 8 h intervals. Protein concentration was determined by the Bio-Rad protein assay (Rockville Centre, NY). The standard was bovine serum albumin, fraction V (Sigma, St. Louis, MO).
JUVENILE
HORMONE
In vivo JH metabolism
The predominant JH homolog during the last stadium of T. ni is JH II and concentrations ranged from 10 to 400 pg/g insect (Jones et al., 1990). The concentration in wandering larvae was 320 pglg but decreased in prepupae to 10 pg/g insect. In the current study, [ 12, 13-3H,](lOR,l IS)-JH II (58 Ci/mmol, Glenn D. Prestwich, Stony Brook, NY) at the rate of 140 pg/g insect (42 pg/300 to 320 mg wandering larva) was topically applied in light mineral oil to the dorsum of the abdomen or injected in mineral oil or buffer (without PMSF and PTU) in the pleural region of the first abdominal segment. Treatments were made with a 10 ~1 Hamilton syringe (treatment volume 1 ~1) to L5D3 (9 h after lights on) wandering larvae and L5D4 (2 h after lights on) prepupae. After injections, the syringe was held in place for 10 set to insure dispersal of the JH and minimize bleeding. Any insects that bled after the removal of the syringe were discarded. Larvae were held at 30 _+1°C for 0 to 240 min before being analyzed. After incubation, T. ni were homogenized in ethyl acetate:water (1: 1, v:v; 2.5 ml total volume) using a model 10-35 polytron (Brinkmann homogenizer, NY) fitted with a PT 1OST probe generator. The homogenate was centrifuged at 1000 g for 5 min to separate the phases. The top ethyl acetate was removed and the aqueous phase re-extracted twice with 1 ml of ethyl acetate each time. The radioactivity in the aqueous phase and pooled organic extracts were analyzed by liquid scintillation. Further separation of JH II and its metabolites in the organic phase was conducted by thin layer chromatography (TLC). Twenty ~1 of the organic layer were chromatographed in separate lanes with [12,13-3H,]-(10R,1 ls)JH II and its metabolites, i.e. JH II acid, diol and acid, diol on Brinkmann polygram SILG, 0.25 mm thick plastic TLC plates (activated for 30 min at 1OO’C).The plates were developed in hexane : ethyl acetate : glacial acetic acid (66:33:1, v:v:v), each lane was cut into 4 mm fractions and the fractions assayed separately by liquid scintillation. Activity recovered was ~90% of that applied to the plate. The JH acid and JH acid, diol standards were synthesized as described by Share and Roe (1988) and JH diol by the method of Mumby and Hammock (1979). The Rrs of the substrate and metabolites were consistent with that previously published by Share and Roe (1988). To determine the distribution of JH II and its metabolites in different tissues of T. ni, the hormone was injected in mineral oil into L5D3 (wandering) larvae as previously described. The larvae were held at 30°C until analyzed. At different times after injection through 240 min, the larvae were immobilized by placing them on crushed ice, and the body cavity opened in a dissection tray. The hemolymph was collected by flushing the body cavity three times with ice cold phosphate buffer (1 ml each), being careful not to dislodge any fat body. The entire alimentary canal was then removed, leaving fat body, muscles, trachea, nervous system and body wall
METABOLISM
183
IN T. NI
intact (referred to thereafter as carcass). JH II and its metabolites in hemolymph, alimentary canal and carcass were analyzed by extraction with ethyl acetate:water and TLC as previously described. In vitro JH II and JH acid metabolism lase
by epoxide hydro-
Two L5D3 (wandering) T. ni were homogenized in 2.5 ml of standard phosphate buffer and 10,000 g supernatant prepared as before. Supematant was preincubated for 10 min at 30°C with 0.1 mM OTFP to inhibit 100% of the JH esterase activity. Aliquots of 100 ~1 were then incubated with either [12,13-3H,]-(10R,1 IS)-JH II or its acid (prepared as previously described) at a final concentration of 140 pg/ml and incubated at 30°C for 0 to 45 min. After incubation, JH II and the metabolites of JH II were extracted with ethyl acetate and analyzed by TLC. RESULTS In vitro metabolism
of JH during the last stadium
The developmental profile of JH esterase and JH epoxide hydrolase activity in fat body, midgut and integument of last stadium T. ni is presented in Fig. 1. JH metabolic activity in these studies was monitored in vitro in tissue homogenates using racemic JH III at 5 PM (final concentration). A peak in JH esterase activity per mg protein was found in prewandering (L5D2) larval fat body, midgut and integument, and elevated esterase activity was found in fat body but not midgut or integument in prepupae (L5D4). Sparks et al. (1979) found a peak in the hemolymph JH esterase activity in both L5D2 and L5D4 larvae which was later validated in several other investigations (reviewed by Roe and Venkatesh, 1990). Hemolymph is devoid of JH epoxide hydrolase. However, a peak in JH epoxide hydrolase activity was found in the midgut of prewandering L5D2 larvae (Fig. 1) which correlated with peaks in JH esterase activity in fat body, midgut, integument and hemolymph. JH epoxide hydrolase activity peaked in fat body and integument on day 3 of the last stadium. For each tissue examined (fat body, midgut and integument) there was no significant difference (t test, (Y= 0.05) between the highest JH esterase activity detected and the highest JH epoxide hydrolase activity. The JH metabolic activity per mg protein was highest in fat body, followed in order by midgut and integument. Overall in vitro JH III metabolic activity (both JH esterase and JH expoxide hydrolase activity) per insect was approximately equivalent between wandering larvae (L5D3) and prepupae (L5D4) of T. ni (4650 versus 4950 pmol per min per larval equivalent, respectively; Table 1). However in wandering larvae, 75% of JH metabolism was by JH epoxide hydrolase while in prepupae, 85% of the JH metabolic activity was due to JH esterase. The JH epoxide hydrolase activity was found mostly in the fat body in both wandering larvae (80% of the total JH epox-
184
VASANT
L. KALLAPUR
et al.
EPOXIDE HYDROLASE
JR ESTERASE 1.20 - Fat body
1.40 1.20 1.00 0.80
0.60 0.60 0.40
0.20 0.00 ’
I
1
0.20 ’
Integument
0.00’ LS DO
L5 Dl
L5 D2
L5 D3 AGE
PP (DAYS)
Integument
’
L5 DO
W Dl
L5 D2
LS D3
PP
IN LAST INSTAR
FIGURE 1, JH esterase and JH epoxide hydrolase activity in homogenates of fat body, midgut and integument during the last stadium of the cabbage looper, Trichoplusia ni. Each point is the mean of three samples; each sample consisted of tissue pooled from three insects. Vertical lines are fl SEM. L5DO = Day 0 in the last (fifth) stadium; PP = prepupa.
ide hydrolase activity) and prepupae (46%). The JH esterase activity was found mostly in the fat body in wandering larvae (52%) as opposed to the hemolymph in prepupae (68%). In prepupae the JH esterase activity in midgut and integument was minimal as compared to other tissues (3% each). Although there was a 14-fold increase in the hemolymph JH esterase activity from LSD3 to LSD4, the increase in JH esterase activity per insect was only 4-fold and 25% of the JH metabolic activity in wandering larvae was JH esterase.
In viva metabolism prepupae
of JH
in wandering
larvae
and
Physiological concentrations of 3H- 1OR, 1 lS-JH II were injected in light mineral oil into the hemocoel of wandering larvae and prepupae of the cabbage looper. At 5 min after the introduction of JH, overall metabolism was higher in prepupae (38%) as compared to 8% in wandering (LSD3) larvae (t-test CY= 0.05) (Fig. 2). There was also significantly lower metabolism in L5D3 larvae at 15 min but no differences were found in metabolism
JUVENILE TABLE
1. In vitro metabolism
of lOR,SJH
HORMONE
III in different
METABOLISM
tissues
of wandering
pmol JH III min-’ JH esterase Tissue
Wandering
Hemolymph Fat body Midgut Integument Whole
200 600 180 170
insect
2850 1120 130 110
(LSD4)
4210
*JH metabolic activity is the mean of five replicates fl SEM. Figure in paranthesis Hemolymph JH easterase activity per insect was calculated from the hemolymph by Wing (1981).
at 60 and 120 min. The only JH II product that appeared to increase consistently with time in both wandering larvae and prepupae was a polar metabolite that was water soluble and could not be extracted by ethyl acetate (Fig. 3). In our TLC system (silica gel and 66:33:1 of hexane:ethyl acetate:glacial acetic acid), the polar metabolite did not move from the origin. This polar metabolite was also by far the predominant product from the metabolism of JH II at most time points examined in both L5D3 and L5D4 larvae but the rate of production was greater in the former (55% versus 35%, respectively, at 120 min). JH diol, JH acid and JH acid, diol were produced in both developmental stages of T. ni examined.
loopers,
Trichoplusiu ni
(%)*
Wandering
f 280.0 (68) + 54.0 (27) + 10.0 (3) f 6.0 (3)
1150
Tissue distribution
larval-equivalent-’
cabbage
JH epoxide
Prepupal
(17) (52) (16) (15)
185
and prepupal
activity
(L5D3)
* 10.0 k 30.0 rf: 3.0 + 4.0
IN T. NI
(L5D3)
hydrolase
activity Prepupal
(L5D4)
2800 _+ 23.0 (80) 300 * 30.0 (9) 400 + 8.0 (11)
340 + 33.0 (46) 100 + 7.0 (34) 150 f 8.0 (20)
3500
740
indicates the percentage of overall activity in each tissue. JH esterase activity per ml x the blood volume determined
Five min after injection, 9 1.6% ized and of this unmetabolized was found in the hemolymph associated with the alimentary
of the JH was unmetabolJH, approximately 50% and the other 50% was canal and carcass (insect
ACID - DIOL
of JH and its metabolites
The tissue distribution of 3H-10R,1 lS-JH II and its metabolites at different times after injection in light mineral oil into wandering T. ni is shown in Figs 4 and 5.
-
0’
WANDERING
I
0
30
I
60
I
90
,
320
5
15 45 120 60 TIME AFTER INJECTION (MINUTES)
TIME AFTER INJECTION (MINUTES) FIGURE 2. Percent metabolism at different times after injection of [12,13-3H,]-(10R,1 IS)-JH II in oil (140 pg JH II/g insect) into wandering (fifth stadium, day 3) larvae and prepupae (fifth stadium, day 4) of the cabbage looper, Trichoplusia ni. After injection, larvae were incubated at 30°C. Unmetabolized JH II was identified and quantitated by thin layer chromatography. Each point is the mean of three samples; each sample consisted of three insects. Vertical lines are +l SEM.
FIGURE 3. Percent metabolism of [12,13-3H,J-(10R,1 Is)-JH II to JH II acid, dial, acid-dial and polar metabolite at different times after injection in oil (140 pg JH II/g insect) into wandering (fifth stadium, day 3) larvae (a) and prepupae (fifth instar, day 4) (b) of the cabbage looper, Trichoplusia ni. After injection, larvae were incubated at 30°C. Metabolites were identified and quantitated by thin layer chromatography. Each point is the mean of three samples; each sample consisted of three insects. Vertical lines are +l SEM.
VASANT L. KALLAPUR
186
&m
( 91.6
)
( 64.2 )
( 30.5
)
et al.
( 29 )
E90
% OF TOTAL JH II REMAINING / INSECT
B n CARCASS
0 HEMOLYMFH I3 ALiMENTARYCANAL
30
5
TIME
120
AFTER INJECTION
240
(MINUTES)
FIGURE 4. Distribution of [12,13-3H,]-(10R,1 IS)-JH II in carcass, hemolymph injection in oil (140 pg JH II/g insect) into wandering (fifth instar, day 3) larvae injection, larvae were incubated at 30°C. JH II was identified and quantitated by mean of three samples; each sample consisted of three insects.
minus hemolymph and alimentary canal) (Fig. 4). Extensive efforts were made to wash the alimentary canal and carcass with buffer to remove contaminating hemolymph. Whether the JH II associated with the hard tissues was
and alimentary canal at different times after of the cabbage looper, Trichoplusia ni. After thin layer chromatography. Each point is the Vertical lines are +l SEM.
surface bound or intracellular was not determined. percentage JH II in hemolymph, alimentary canal carcass did not significantly change from 5 through min (t-test, (Y = 0.05) even though the percentage
d 70 i3 E 6o 8 40 * 30 20 10 5
120
30
240
e! 300 IIzb aD 1~~
5
30
120
240
b3 1y 90 880 ? 70 fl 60
T
g 70
T
2 60 E 5o 8 40 be30 20 10 a 5
TIME AFTit
240
INJECTI~“(MINUl’RS)
FIGURE 5. Percent metabolism of [12,13-3H2]-( lOR,l Is)-JH II to polar metabolite (a), acid, diol (b), diol (c) and acid (d) in
carcass, hemolymph and alimentary canal at different times after injection in oil (140 pg JH II/g insect) into wandering (fifth stadium, day 3) larvae of the cabbage looper, Trichoplusia ni. After injection, larvae were incubated at 30°C. Metabolites were identified and quantitated by thin layer chromatography. Each point is the mean of three samples; each sample consisted of three insects. Vertical lines are fl SEM.
The and 120 JH
187
JUVENILE HORMONE METABOLISM IN T. NI
remaining declined to 30.5%. At 240 min, there was a significant increase in the percentage JH II in hemolymph over that at 120 min and a significant decrease in carcass. The metabolites of JH II, i.e., polar metabolite, JH acid, diol, JH diol and JH acid, were evenly distributed in hemolymph, alimentary canal and carcass after injection (Fig. 5). In vivo JH metabolism following administration
d@erent routes of
The method of introduction of 3H-10R,1 lS-JH II into wandering (L5D3) larvae and prepupae (L5D4) affected JH metabolism 60 min after treatment (Table 2). Overall JH metabolism was greater when JH was either applied topically in oil or injected in buffer as compared to injection in oil. This was the case for both wandering larvae and prepupae. For example, the remaining JH when injected in oil into L5D3 larvae was 46% while only 8.4% and 2.3% was remaining for topical in oil and buffer injected, respectively (Table 2). Interestingly, the method of introduction also affected the production of JH acid. The topical application of JH consistently produced more JH acid in L5D3 and L5D4 T. ni as compared to injection in either buffer or oil. No JH acid was detected when JH II was injected in buffer even though in prepupae, hemolymph JH esterase activity is at peak levels at this developmental time (Table 1). JH acid, diol was detected. There was no significant difference (t-test, (Y= 0.05) in the in vivo metabolism 60 min after injection between wandering larvae and prepupae topically treated with JH II or injected with oil; prepupae had statistically higher JH metabolism when JH was introduced by injection in buffer but the difference was less than 2% and JH metabolism was almost 100% at 60 min after injection. Irrespective of the method of introduction, JH diol and polar conjugate was produced with the latter consistently being the predominant metabolite of JH II ranging from 33 to 71% in L5D3 larvae to 23 to 79% in prepupae. Greater polar metabolite was produced in wandering larvae (L5D3) than in prepupae for all methods of introduc-
tion although the difference was not statistically significant for buffer injection. Metabolism of JH II versus JH II acid by epoxide hydrolase When epoxide hydrolase from whole body homogenate of wandering T. ni was incubated with 3H-10R,1 ISJH II or its acid at physiological concentrations (140 pg/ml of homogenate or 450 pg/insect equivalent), JH was metabolized at least 3-times faster than JH acid (Fig. 6). At 45 min after injection, 14% of the JH II was metabolized while only 4% of the JH acid was metabolized at the same time.
‘15 *
JHII
.+
JHAdd
12 -
10
0
30
20 TIME
40
50
(MINUTES)
FIGURE 6. Time course of percent metabolism of [12,13-3H,](lOR,llS)-JH II and its acid when incubated in vitro at 30°C in 10,000 g supematant prepared from the homogenization of wandering (fifth instar, day 3) larvae of the cabbage looper, Trichoplusia ni. Supematant was preincubated with octylthio- 1, l , l-trifluoropropan-2one (final concentration 4.1 mM) for 10 min at 30°C prior to the addition of substrate (final concentration 140 pg/ml). JH diol and JH diol, acid were identified and quantitated by thin layer chromatography. Each point is the mean of three samples. Vertical lines are kl SEM.
TABLE 2. In vivo metabolism of lOR,l lS’-JH II introduced by different methods to wandering larvae (and prepupae) of the cabbage looper, Trichoplusia ni % JH or JH metabolite 60 min alter treatment* Application method Topical in oil Injected in buffer Injected in oil
Polar 70.5 (34.8 82.6 (78.7 33.3 (22.6
f + f f + f
4.2 4.9) 5.6 0.8) 0.9 2.0)
Acid-diol 8.1 (15.1 8.6 (12.1 8.1 (6.9
+ f + + + +
2.7 2.1) 0.6 0.8) 2.4 1.5)
Diol 1.9 k (7.1 + 2.7 + (9.1 f 6.7 f (2.2 f
1.6 0.3) 1.5 1.0) 2.4 0.6)
*Values are the mean of three replicates zkl SEM for wandering larvae and prepupae in parenthesis.
Acid
JH
11.8 f 1.6 (26.0 + 2.1)
8.4 k 5.3 (17.0 f 3.7) 2.3 k 0.3 (0) 46.1 + 4.6 (48.1 + 3.6)
(k 6.0 + 1.0 (20.1 f 0.5)
VASANT
188
L. KALLAPUR
DISCUSSION
It is generally accepted that increases in JH metabolism are responsible for the removal of JH in last stadium Lepidoptera and this regulates the timing for the initiation of wandering behavior midway through the last stadium and the proper expression of the pupal stage just prior to the final larval ecdysis (Hammock, 1985; Roe and Venkatesh, 1990). The current view is that in Lepidoptera, JH esterase is responsible for the primary metabolism of JH at these two periods in the last stadium and JH epoxide hydrolase converts JH acid to JH acid, diol. Based on levels of R20458 epoxide hydration compared to JH ester hydrolysis in last stadium T. ni, Wing et al. (198 1) concluded that epoxide hydrolase had a secondary role in JH metabolism. Touhara and Prestwich (1993) more recently provided kinetic evidence that binding protein protected JH from JH epoxide hydrolase but not JH esterase, thus arguing that JH esterase was the primary metabolic route for JH in M. sextu. There is also direct evidence of the importance of JH esterase metabolism in JH regulation and the regulation of metamorphosis. The specific inhibition of JH esterase with polarized alcohols and organophosphorus JH mimics delayed wandering behavior, delayed pupation, and produced what appeared as larval-pupal intermediates in last stadium T. ni (Roe et al., 1991, 1993; Linderman et al., 1991). Jones et al. (1990) showed that this inhibition resulted in an increase in the JH titer in the same insect. JH esterase inhibition in adult virgins of T. ni also caused egg laying, presumably by reducing JH metabolism and elevating the JH titer (Roe et al., 1991). Available evidence in the Dipteran, Culex quinquefasciatus (the yellow fever mosquito), suggests that JH epoxide hydrolase is more important than JH esterase in JH metabolism and suggests a functional role for JH epoxide hydrolase in both metamorphosis and adult reproduction. During the last stadium of C. quinquefasciatus, two peaks in JH epoxide hydrolase activity were reported at the exclusion of any change in the JH esterase activity (Lassiter et al., 1995a). Apparently, epoxide hydrolase and not JH esterase has a dynamic role in JH turnover in last instars of this insect. The in vitro JH III epoxide hydrolase activity exceeded that of JH esterase throughout the last stadium by as much as 6-fold. More JH III diol was found than JH acid in both in vitro and in vivo assays of adult C. quinquefasciatus. Blood feeding increased JH epoxide hydration 36 h later, presumably to reduce JH levels and allow the production of ecdysone necessary for the initiation of vitellogenesis (Lassiter e2 al., 1994, 1995b). Unfortunately, no inhibitors for epoxide hydrolase are available to directly address the importance of epoxide hydrolase. However, the evidence, although not conclusive, does point to JH epoxide hydrolase and not JH esterase as the most important factor in the primary metabolism of JH in C. quinquefasciatus. In last stadium tobacco homworm larvae at the time of the first and second hemolymph JH esterase peak,
et al.
elevated JH esterase and epoxide hydrolase activity occurred in integument, midgut, fat body and brain, with the exception of the fat body at the time of the second peak (Jesudason et al., 1992). This synchrony between increases in JH esterase activity and JH epoxide hydrolase activity was not found in T. ni (Fig. 1). The JH esterase activity peaked in fat body, midgut and integument in last stadium cabbage loopers at day 2 and elevated JH esterase activity was found in the fat body in prepupae (day 4), while the midgut JH epoxide hydrolase activity peaked on day 2 and the fat body and integument peaked on day 3. Wing et al. (198 1) reported an order of magnitude lower amount of R20458 epoxide hydrolase activity compared to JH esterase activity in last stadium T. ni. In a similar analysis in our experiments using racemic JH III, no significant differences were noted at peak levels between in vitro JH esterase and JH epoxide hydrolase activity in fat body, midgut and integument (Fig. 1, activity expressed per mg protein). Similar results where the in vitro JH epoxide hydrolase activity was equivalent or in some cases exceeded the JH esterase in specific tissues in prewandering and prepupal last stadium M. sexta was previously reported by Jesudason et al. (1992) and in last stadium Galleria mellonella by Wisniewski et al. (1986). So, based simply on the concentration of activity in specific tissues (excluding the hemolymph), JH esterase and JH epoxide hydrolase appear to have similar importance in JH metabolism in these Lepidoptera. It was interesting to note that JH epoxide hydrolase activity per mg protein (measured in vitro with racemic JH III) peaked in fat body and integument on day 3 in T. ni and JH esterase activity was elevated in fat body and hemolymph but not the other tissues examined on day 4 (Fig. 1, Sparks et al. 1979, Roe and Venkatesh 1990). Overall JH III metabolism in vitro was essentially the same at these two time points (Table 1) and when 1OR,11S-JH II at physiological concentrations were injected in oil into wandering larvae and prepupae, the level of metabolism was also similar. In fact, injection in oil or buffer or the topical application of lOR,l lS-JH II produced the same results; the rate of overall metabolism was the same in wandering larvae and prepupae (Table 2). However, on L5D3 the JH epoxide hydrolase activity per insect was 3 times that of the JH esterase while in prepupae essentially the reverse occurred, i.e. the JH esterase activity was 5.7-times that of the JH epoxide hyrolase. Jones et al. (1990) found that the JH titer peaked on L5D3 at 350 pglg tissue and decreased to 10 pg/g by L5D4 in T. ni. It was concluded that the increase in JH esterase activity in prepupae noted in hemolymph and other tissues by Wing et al. (1981) was responsible for this reduction along with decreased biosynthesis. Considering that the overall levels of metabolism are apparently the same between wandering larvae and prepupae based on the in vitro and in vivo experiments presented here, there is obviously a mechanism for the maintenance
JUVENILE
HORMONE
of high JH titers in wandering larvae in the presence of high tissue JH epoxide hydrolase activity. Note that approximately half of the injected JH II in wandering T. ni was associated with gut and carcass (fat body and integument) (Fig. 4), also the sites for high JH epoxide hydrolase activity. This was true at most of the time points after injection. Thus, differences between the tissue distribution of JH epoxide hydrolase and that of JH does not appear to explain why high JH titers exist in the presence of elevated JH metabolic activity. Touhara and Prestwich (1993) found that JH binding protein prevented the metabolism of JH by soluble JH epoxide hydrolase but not JH esterase in A4. sextu and hypothesized that the binding protein directed the metabolism of JH by JH esterase first. Because of the low affinity of the binding protein for JH acid, the acid would be released and subsequently metabolized by JH epoxide hydrolase. If this model is applicable to last stadium cabbage loopers and considering that Wing et al. (1981) demonstrated that the hemolymph JH binding protein concentration did not change between wandering larvae and prepupae of T. ni, competition with high affinity binding protein might explain the existence of high JH titers in day 3 wandering larvae in the presence of high JH epoxide hydrolase and low JH esterase activity. However, at different times after injection of lOR, 1IS-JH II into wandering and prepupal T. ni, one of the primary metabolites of JH that was found was JH II diol (Fig. 3). Despite the method of introduction, JH diol was always one of the primary metabolites (Table 2), although usually at a lower concentration than that of JH acid. The major product formed from the injection of JH II was a polar metabolite, which formed faster in wandering larvae than in prepupae and which was formed irrespective of the method of introduction of JH (Fig. 3 and Table 2). The polar metabolite was by far the predominant product of JH metabolism in both wandering and prepupal T. ni. Halarnkar et al. (1993) identified the polar metabolite in A4. sexta as a phosphate conjugate of JH diol, which was the principle end product of JH I metabolism in newly molted last stadium tobacco hornworm larvae. No JH acid, diol-phosphate conjugate has been found in A4. sextu (personal communication with D. Schooley, Department of Biochemistry, University of Nevada, Reno). These results with M. sextu argue that the major metabolite in both wandering larval and prepupal T. ni either injected or topically treated with JH might be JH diol and not JH acid (Fig. 3 and Table 2); also note that only small amounts of JH acid, diol were found especially as compared to the polar metabolite. The importance of JH epoxide hydrolase in the primary metabolism of JH is further argued by the discovery that at physiological concentrations of 1OR, 1 lS-JH II in vitro, the preferred substrate for JH epoxide hydrolase was JH II rather than JH II acid (Fig. 6). The role of JH epoxide hydrolase in the regulation of JH titer and metamorphosis is not yet resolved. In the model proposed by Touhara and Prestwich (1993) for A4.
METABOLISM
IN T. NI
189
sex&, the effect of specific and non-specific JH binding
by various tissues and the distribution of JH and JH metabolic enzymes in these tissues during development on JH metabolism was not included in their analysis. In the work presented here on T. ni and that of Halarnkar et al. (1993) working with A4. sex&, an argument can also be made that a single injection of JH in vivo, even when introduced at a final concentration after injection which is comparable to normal physiological levels of JH, does not mimic the natural introduction of JH into the hemolymph by the corpora allata. On the other hand, Hidayat and Goodman (1994) found in M. sexta larvae that < 1% of the JH-specific binding protein was hormone-loaded at any one time during the fourth stadium. Considering the high affinity of JH binding protein for JH and binding protein abundance, the only limiting factor in single injection experiments into hemolymph which is poorly circulated is compartmentalization and limited mixing. Finally, note that the distribution of each JH metabolite was approximately the same between tissues (hemolymph, carcass, and alimentary canal; Fig. 5) at different times after injection, indicating no tissue specific storage of a particular metabolite. It was also interesting that even though the JH esterase activity is low in the hemolymph of wandering T. ni (0.07-times that of prepupae on a per insect basis), the JH esterase activity in the other tissues were quite similar (950 pmol JH III mini larvalequivalent-’ for wandering larvae versus 1360 pmol min-’ larval-equivalent- ’ for prepupae). Recall, that approximately 50% of the injected lOR,llS-JH II was found in tissues other than hemolymph in wandering larvae of T. ni. The significance of this rather substantial gut and carcass JH esterase activity associated with JH in wandering larvae is not clear. It does indicate a similar clearance rate for tissue JH in wandering larvae and prepupae. In terms of JH esterase metabolism of JH, the major change that occurs between day 3 and day 4 in the last stadium of T. ni, is an increase in the clearance rate in the hemolymph, not the other tissues. Finally, it should be noted that the method of introduction of JH can affect the route of metabolism (Table 2). Therefore, care should be taken in the interpretation of experiments involving the in vivo metabolism of JH. In summary, a peak in JH III esterase activity per mg protein measured in vitro was found in fat body, midgut, integument and hemolymph in prewandering (day 2) last stadium T. ni and in fat body and hemolymph of prepupae (on day 4). Midgut JH epoxide hydrolase activity peaked on day 2 and in fat body and integument on day 3. No significant differences were noted in the overall metabolism of JH III in vitro between wandering larvae and prepupae and in vivo when lOR,l l,S-JH II was introduced at physiological concentrations by different methods. JH II diol and a predominant JH II polar metabolite was found, the latter which in M. sexta was a phosphate conjugate of JH diol. JH II epoxide hydrolase metabolized JH II at a faster rate than JH acid. These studies in toto suggest a more important role for JH epoxide
190
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hydrolase in the metabolism of JH than was originally thought. REFERENCES Ajami A. M. and Riddiford L. M. (1973) Comparative metabolism of the cecropia juvenile hormone. .Z. Insect Physiol. 19, 6355645. Baker F. C., Tsai L. W., Reuter C. C. and Schooley D. A. (1987) In viva fluctuation of JH, JH acid, and ecdysteroid titer, and JH esterase activity, during development of fifth stadium Manduca sexta. Insect Biochem. 17, 989-996. Gilbert L. I. and King D.S. (1973) Physiology of growth and development. Endocrine aspects. In The Physiology of Znsecta, 2nd Edition (Ed. Rockstein M.), pp. 249-370. Academic Press, New York. Halarnkar P. P., Jackson G. P., Straub K. M. and Schooley D. A. ( 1993) Juvenile hormone catabolism in Manduca sexta: homologue selectivity of catabolism and identification of a diol-phosphate conjugate as a major end product. Experientia 49, 988-994. Hammock B. D. (1985) Regulation of juvenile hormone titer: degradation. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 7 (Eds Kerkut G. A. and Gilbert L. I.), pp. 43 l-472. Pergamon Press, Oxford. Hammock B. D. and Roe R. M. (1985) Analysis of juvenile hormone esterase activity. Methods Enzymol. 111, 487494. Hidayat P. and Goodman W. G. (1994) Juvenile hormone and hemolymph juvenile hormone binding protein titers and their interaction in the hemolymph of fourth stadium Manduca sexta. Insect Biochem. Molec. Biol. 24, 709-715. Jesudason P., Anspaugh D. D. and Roe R. M. (1992) Juvenile hormone metabolism in the plasma, integument, midgut, fat body, and brain during the last instar of the tobacco hornwom, Manduca sexta (L.). Arch. Znsect Biochem. Physiol. 20, 87-105. Jones G., Hanzlik T., Hammock B. D., Schooley D. A., Miller C. A., Tsai L. W. and Baker F. C. (1990) The juvenile hormone titre during the penultimate and ultimate larval stadia of Trichoplusia ni. J. Insect Physiol. 36, 77-83. Lassiter M. T., Apperson C. S. and Roe R. M. (1995a) Juvenile hormone metabolism during the fourth stadium and pupal stage of the southern house mosquito, Culex quinquefasciatus Say. J. Insect Physiol. 41, 869-876. Lassiter M. T., Apperson C. S. and Roe R. M. (1995b) Juvenile hormone metabolism in the ovary, gut, head and carcass after blood feeding in the southern house mosquito, Culex quinquefasciatus. Comp. Biochem. Physiol. In press. Lassiter M. T., Apperson C. S., Crawford C. L. and Roe R. M. (1994) Juvenile hormone metabolism during adult development of Cufex quinquefasciatus (Diptera:Culicidae). J. Med. Entomol. 31, 586 593. Linderman R. J., Tshering T., Venkatesh K., Goodlett D. R., Dauterman W. C. and Roe R. M. (1991) Organophosphorus inhibitors of insect juvenile hormone esterase. Pestic. Biochem. Physiol. 39, 57-73. Mumby S. M. and Hammock B. D. (1979) A partition assay for epoxide hydrases acting on insect juvenile hormone and an epoxidecontaining juvenoid. Anal. Biochem. 92, 162 1. Riddiford L. M. (1994) Cellular and molecular actions of juvenile hormone I. General considerations and premetamotphic actions. Adv. Insect Physiol. 24, 2 13-274.
Roe R. M. and Venkatesh K. (1990) Metabolism of juvenile hormones: degradation and titer regulation. In Morphogenetic Hormones of Arthropods, Vol. 1 (Ed. Gupta A. P.), pp. 126179. Rutgers Univ. Press, New Brunswick. Roe R. M., Hammond A. M. Jr and Sparks T. C. (1982) Growth of larval Diutraea saccharulis (Lepidoptera: Pyralidae) on an artificial diet and synchronization of the last larval stadium. An. Entomol. Sot. Amer. 75, 421429. Roe R. M., Jest&son P., Venkatesh K., Kallapur V. L., Anspaugh D. D., Majumder C., Linderman R. J. and Graves D. M. (1993) Developmental role of juvenile hormone metabolism in Lepidoptera. Amer. Zool. 33, 375-383. Roe R. M., Venkatesh K., Anspaugh D. D., Linderman R. J. and Graves D. M. (1991) Chemical and biological approaches to juvenile hormone esterase based insecticides. In Reviews in Pesticide Toxicology, Vol. I (Eds Hodgson E., Roe R. M. and Motoyama N.), pp. 249-259. North Carolina State Univ., Raleigh. Share M. R. and Roe R. M. (1988) A partition assay for the simultaneous determination of insect juvenile hormone esterase and epoxide hydrolase activity. Anal. Biochem. 169, 81-88. Slade M. and Wilkinson C. F. (1974) Degradation and conjugation of cecropia juvenile hormone by the southern armyworm (Prodenia eridania). Comp. Biochem. Physiol. 49B, 99-103. Slade M. and Zibitt C. H. (1972) Metabolism of cecropia juvenile hormone in insects and in mammals. In Insect Juvenile Hormones: Chemistry and Action (Eds Menn J. J. and Beroza M.), pp. 155176. Academic Press, Orlando, Florida. Sparagana S. P., Bhaskaran G. and Barrera P. (1985) Juvenile hormone acid methyltransferase activity in imaginal discs of Manducu sexta prepupae. Arch. Insect Biochem. Physiol. 2, 19 l-202. Sparks T. C., Willis W. S., Shorey H. H. and Hammock B. D. (1979) Haemolymph juvenile hormone esterase activity in synchronous last instar larvae of the cabbage looper, Trichoplusia ni. J. Insect Physiol. 25, 125-132. Touhara K. and Prestwich G. D. (1993) Juvenile hormone epoxide hydrolase: photoaffinity labeling, purification and characterization from tobacco homworm eggs. J. Biol. Chem. 268, 19,60619,609. Weirich G., Wren J. and Siddal J. B. (1973) Developmental changes of the juvenile hormone esterase in hemolymph of the tobacco homworm, Manduca sextu. Insect Biochem. 3, 397407. Wing K. D. (198 1) Juvenile Hormone Esteruses of Trichoplusiu ni and Other Lepidoptera: Characteristics, Site of Synthesis, Interaction with Other Proteins and Inhibition. Ph.D. dissertation, University of California, Riverside. 157 pp. Wing K. D., Sparks T. C., Love11V. M., Levinson S. 0. and Hammock B. D. (1981) The distribution of juvenile hormone esterase and its interrelationship with other proteins influencing juvenile hormone metabolism in the cabbage looper, Trichoplusia ni. Insect Biochem. 11, 473-485. Wisniewski J. R., Rudnicka M. and Kochman M. (1986) Tissue specific juvenile hormone degradation in Gulleria mellonellu. Insect Biochem. 16, 843-849.
Acknowledgements-This research was supported by the North Carolina Agricultural Research Service and the USDA Competitive Grants Program (90-37263 and 93-01607).
0022-1910(95)00057-7
Pergamon
Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910196 $15.00 + 0.00
In Vivo and In Vitro-tissue Specific Metabolism of Juvenile Hormone During the Last Stadium of the Cabbage Looper, Trichoplusia ni VASANT L. KALLAPUR,*t
CHANDANA MAJUMDER,* R. MICHAEL ROE*1
Received 4 November 1994; revised 28 April 1995
In vitro metabolism of racemic JH III (5 PM) was measured in fat body, midgut and integument homogenate during the last stadium of the cabbage looper, Trichoplusia ni. The JH esterase activity per mg protein peaked in these tissues in prewandering larvae on day 2 and was elevated in the fat body only in prepupae (on day 4). The JH epoxide hydrolase activity peaked on day 2 in midgut and on day 3 (in wandering larvae) in fat body and integument. No differences were noted in the overall JH III metabolism in vitro per insect between wandering (day 3) larvae and prepupae (day 4) and in the in vivo metabolism of lOR,llS-JH II when injected in oil and buffer or when topically applied at physiological concentrations into these same stages. Regardless of the method of introduction of JH II, JH diol was produced in both wandering larvae and prepupae. JH acid and JH acid, diol were also produced but the predominant product was a water soluble metabolite. The preferred substrate for JH epoxide hydrolase at physiological concentrations was JH II as opposed to JH II acid. Approximately half of the JH II after injection into wandering larvae was associated with the alimentary canal and carcass. The metabolites of JH II were evenly distributed. The importance of JH epoxide hydrolase in JH metabolism in last stadium Lepidoptera is discussed. Cabbage looper Munduca sextu
Juvenile hormone Juvenile hormone esterase Epoxide hydrolase
INTRODUCTION
development in several Lepidoptera suggests that JH esterase is an important modulator of JH titer, especially in the regulation of metamorphosis (Weirich et al., 1973; Baker et al., 1987; Jones et al., 1990; Roe and Venkatesh, 1990). The disruption of normal development by specific metabolic inhibitors of hemolymph JH esterase has provided more direct evidence of the functional significance of JH esterase in lepidopteran metamorphosis (Roe and Venkatesh, 1990; Roe et al., 199 1, 1993). In comparison to the studies on JH metabolism in insect hemolymph, much less is known about JH metabolism in other insect tissues. JH esterase and JH epoxide hydrolase activity is found in integument, fat body, midgut, brain, Malpighian tubules and silk glands (Slade and Wilkinson, 1974; Wisniewski et al., 1986; Jesudason et al., 1992). Elevated JH epoxide hydrolase activity in integument, fat body and brain of prewandering, last stadium tobacco hornworms, Manduca sex&, was correlated with increased levels of JH esterase activity in integument, fat body, brain, midgut and hemolymph (Jesudason et al., 1992). Elevated JH epoxide hydrolase and JH esterase activities were also found in prepupal integument and midgut of the same species but not in fat body.
Juvenile hormone (JH) is an important factor in regulating insect growth and development (Gilbert and King, 1973; Hammock, 1985; Roe and Venkatesh, 1990; Riddiford, 1994). The JH titers that control these morphological and physiological changes are regulated by changes in biosynthesis and degradation of JH (Weirich et al., 1973; Hammock, 1985; Roe and Venkatesh, 1990). Two possible, primary routes of JH metabolism are hydrolysis of the methyl ester by JH specific esterase and hydration of the 10,ll -epoxide by epoxide hydrolase producing the carboxylic acid and dial, respectively (Slade and Zibitt, 1972; Ajami and Riddiford, 1973). The only route of JH metabolism in insect hemolymph is ester hydrolysis. The correlation of high levels of hemolymph JH esterase activity with low levels of JH during
*Department of Entomology, Box 7613, North Carolina State University, Raleigh, NC 27695-7613, U.S.A. ton sabbatical from Department of Zoology, Kamataka University, Dharwad 580 003, India. fTo whom all correspondence should be addressed. 181
VASANT
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L. KALLAPUR
Wing et al. (198 1) measuring the metabolism of R-20458
in last stadium Trichoplusia ni, found a peak in epoxide hydrolase activity in the fat body of wandering larvae when hemolymph and other tissue JH esterase activity was minimal and the reverse was true in prepupae. The JH epoxide hydrolase activity in T. ni was never measured. Based on the relatively low level of R-20458 epoxide hydrolase activity in wandering larvae compared to the peak JH esterase activity in prepupae, Wing et al. (198 1) concluded that JH epoxide hydrolase had a secondary role in JH turnover. It is generally assumed that increased JH metabolism in Lepidoptera is responsible for the initiation of wandering behavior and the regulation of the second JH peak in postwandering larvae. However, most of these studies are based on increased hemolymph metabolism and have not considered the role of other tissues, tissue interactions, and the distribution of JH during development. Considering that the JH metabolite, JH acid, can be a precursor for JH (Sparagana et al., 1985), other routes of JH metabolism other than ester hydrolysis may be more important than originally thought. In this paper, in vitro studies of JH metabolism in specific tissues are correlated with in vivo metabolism, the tissue distribution of JH and substrate preference of epoxide hydrolase, in order to better evaluate the importance of JH esterase and JH epoxide hydrolase in JH titer regulation in last stadium larvae of T. ni. MATERIALS AND METHODS
Insects T. ni larvae (20 per 8 oz wax-coated, cardboard cup) were reared from egg hatch on an artificial diet (diet No. 2 of Roe et al., 1982) at 30 rf: 1°C with a relative humidity of 40% and with a 14:lO h 1ight:dark cycle (lights on at 6 a.m.). Fourth instars that migrated to the sides or top of the rearing containers and that cleared their gut and demonstrated head capsule slippage 6 h prior to lights off, were transferred to 1 oz plastic rearing cups without diet (1 larva per cup). The next morning (2 h after lights on), those larvae that molted (indicated by a caste head capsule) were designated as last stadium day 0 (L5DO) larvae and were provided adequate food to complete the fifth stadium. Any fourth instars remaining at this time were discarded. Nearly 75% of the larvae (gate 1 larvae) wandered on day 3 (10 h after lights on) and molted to the pupa on day 4 at approximately the same time of the day. The remainder molted on day 5 (gate 2). The earliest absolute distinction between gate 1 and gate 2 larvae was at the time of wandering. At earlier ages, a mixture of gate 1 and gate 2 larvae were used. Tissue collection and preparation for enzyme and protein assays Hemolymph was collected from the cut end of the abdominal prolegs into 10 by 75 mm test tubes held in
et al.
ice and containing a few crystals of phenylthiourea (PTU). Hemolymph was pooled from 2 to 3 larvae and centrifuged for 5 min at 1000 g. The supematant was diluted in standard sodium phosphate buffer (ionic strength = 0.2 M, pH 7.4, 0.0 1% PTU) and used immediately for analysis. After hemolymph collection, larvae were immobilized by placing them on crushed ice, and the body cavity was opened and washed with ice-cold buffer (standard phosphate buffer described above with 0.4 mM phenylmethyl sulfonylfluoride, PMSF) to remove residual hemolymph. PMSF is not an inhibitor of JH esterase or JH epoxide hydrolase. The midgut (excluding the gut content), fat body and integument (without fat body and muscles) were removed, washed and temporarily stored on ice. Each tissue was pooled from three larvae and homogenized in 1 ml of standard buffer containing PMSF in a glass homogenizer (with Teflon pestle) held on ice. The homogenate was then centrifuged at 10,000 g for 15 min at 4°C and the supernatant assayed immediately as described later.
Enzyme and protein assays Hemolymph JH esterase activity was determined according to the partition assay described by Hammock and Roe (1985). Hemolymph diluted in standard buffer was incubated at 30°C with racemic 3H-JH III (12 Ci/mmol, tritiated at C-10, New England Nuclear, Boston, MA) mixed with unlabeled racemic JH III (Calbiochem, San Diego, CA). The final JH III concentration was 5 pm, and hemolymph dilutions and incubation times were chosen to produce a linear increase in metabolism with time. Midgut, fat body and integument JH esterase and JH epoxide hydrolase activities were determined by the partition assay of Share and Roe (1988). The 10,000 g supernatant was diluted in standard buffer and 100 ~1 preincubated at 30°C for 10 min with and without 0.1 mM (final concentration) 3 -octylthio- 1, 1,l -trifluoropropan-2-one (OTFP) to inhibit the JH esterase activity. OTFP was synthesized from the n-octyl sulfide and bromotrifluoroacetone in chloroform and purified by fractional distillation and column chromatography to >98% purity as determined by gas chromatography. Studies with 10,000 g supematant from T. ni indicated that 0.1 mM OTFP inhibited 100% of the JH esterase activity and had no effect on the JH epoxide hydrolase activity. JH III substrate (previously described) was then added and incubated at 30°C. Enzyme concentrations and incubation times were chosen which produced linear hydrolysis rates with time. JH esterase and JH epoxide hydrolase activities in midgut, fat body and integument during the last stadium were monitored at 8 h intervals. Protein concentration was determined by the Bio-Rad protein assay (Rockville Centre, NY). The standard was bovine serum albumin, fraction V (Sigma, St. Louis, MO).
JUVENILE
HORMONE
In vivo JH metabolism
The predominant JH homolog during the last stadium of T. ni is JH II and concentrations ranged from 10 to 400 pg/g insect (Jones et al., 1990). The concentration in wandering larvae was 320 pglg but decreased in prepupae to 10 pg/g insect. In the current study, [ 12, 13-3H,](lOR,l IS)-JH II (58 Ci/mmol, Glenn D. Prestwich, Stony Brook, NY) at the rate of 140 pg/g insect (42 pg/300 to 320 mg wandering larva) was topically applied in light mineral oil to the dorsum of the abdomen or injected in mineral oil or buffer (without PMSF and PTU) in the pleural region of the first abdominal segment. Treatments were made with a 10 ~1 Hamilton syringe (treatment volume 1 ~1) to L5D3 (9 h after lights on) wandering larvae and L5D4 (2 h after lights on) prepupae. After injections, the syringe was held in place for 10 set to insure dispersal of the JH and minimize bleeding. Any insects that bled after the removal of the syringe were discarded. Larvae were held at 30 _+1°C for 0 to 240 min before being analyzed. After incubation, T. ni were homogenized in ethyl acetate:water (1: 1, v:v; 2.5 ml total volume) using a model 10-35 polytron (Brinkmann homogenizer, NY) fitted with a PT 1OST probe generator. The homogenate was centrifuged at 1000 g for 5 min to separate the phases. The top ethyl acetate was removed and the aqueous phase re-extracted twice with 1 ml of ethyl acetate each time. The radioactivity in the aqueous phase and pooled organic extracts were analyzed by liquid scintillation. Further separation of JH II and its metabolites in the organic phase was conducted by thin layer chromatography (TLC). Twenty ~1 of the organic layer were chromatographed in separate lanes with [12,13-3H,]-(10R,1 ls)JH II and its metabolites, i.e. JH II acid, diol and acid, diol on Brinkmann polygram SILG, 0.25 mm thick plastic TLC plates (activated for 30 min at 1OO’C).The plates were developed in hexane : ethyl acetate : glacial acetic acid (66:33:1, v:v:v), each lane was cut into 4 mm fractions and the fractions assayed separately by liquid scintillation. Activity recovered was ~90% of that applied to the plate. The JH acid and JH acid, diol standards were synthesized as described by Share and Roe (1988) and JH diol by the method of Mumby and Hammock (1979). The Rrs of the substrate and metabolites were consistent with that previously published by Share and Roe (1988). To determine the distribution of JH II and its metabolites in different tissues of T. ni, the hormone was injected in mineral oil into L5D3 (wandering) larvae as previously described. The larvae were held at 30°C until analyzed. At different times after injection through 240 min, the larvae were immobilized by placing them on crushed ice, and the body cavity opened in a dissection tray. The hemolymph was collected by flushing the body cavity three times with ice cold phosphate buffer (1 ml each), being careful not to dislodge any fat body. The entire alimentary canal was then removed, leaving fat body, muscles, trachea, nervous system and body wall
METABOLISM
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IN T. NI
intact (referred to thereafter as carcass). JH II and its metabolites in hemolymph, alimentary canal and carcass were analyzed by extraction with ethyl acetate:water and TLC as previously described. In vitro JH II and JH acid metabolism lase
by epoxide hydro-
Two L5D3 (wandering) T. ni were homogenized in 2.5 ml of standard phosphate buffer and 10,000 g supernatant prepared as before. Supematant was preincubated for 10 min at 30°C with 0.1 mM OTFP to inhibit 100% of the JH esterase activity. Aliquots of 100 ~1 were then incubated with either [12,13-3H,]-(10R,1 IS)-JH II or its acid (prepared as previously described) at a final concentration of 140 pg/ml and incubated at 30°C for 0 to 45 min. After incubation, JH II and the metabolites of JH II were extracted with ethyl acetate and analyzed by TLC. RESULTS In vitro metabolism
of JH during the last stadium
The developmental profile of JH esterase and JH epoxide hydrolase activity in fat body, midgut and integument of last stadium T. ni is presented in Fig. 1. JH metabolic activity in these studies was monitored in vitro in tissue homogenates using racemic JH III at 5 PM (final concentration). A peak in JH esterase activity per mg protein was found in prewandering (L5D2) larval fat body, midgut and integument, and elevated esterase activity was found in fat body but not midgut or integument in prepupae (L5D4). Sparks et al. (1979) found a peak in the hemolymph JH esterase activity in both L5D2 and L5D4 larvae which was later validated in several other investigations (reviewed by Roe and Venkatesh, 1990). Hemolymph is devoid of JH epoxide hydrolase. However, a peak in JH epoxide hydrolase activity was found in the midgut of prewandering L5D2 larvae (Fig. 1) which correlated with peaks in JH esterase activity in fat body, midgut, integument and hemolymph. JH epoxide hydrolase activity peaked in fat body and integument on day 3 of the last stadium. For each tissue examined (fat body, midgut and integument) there was no significant difference (t test, (Y= 0.05) between the highest JH esterase activity detected and the highest JH epoxide hydrolase activity. The JH metabolic activity per mg protein was highest in fat body, followed in order by midgut and integument. Overall in vitro JH III metabolic activity (both JH esterase and JH expoxide hydrolase activity) per insect was approximately equivalent between wandering larvae (L5D3) and prepupae (L5D4) of T. ni (4650 versus 4950 pmol per min per larval equivalent, respectively; Table 1). However in wandering larvae, 75% of JH metabolism was by JH epoxide hydrolase while in prepupae, 85% of the JH metabolic activity was due to JH esterase. The JH epoxide hydrolase activity was found mostly in the fat body in both wandering larvae (80% of the total JH epox-
184
VASANT
L. KALLAPUR
et al.
EPOXIDE HYDROLASE
JR ESTERASE 1.20 - Fat body
1.40 1.20 1.00 0.80
0.60 0.60 0.40
0.20 0.00 ’
I
1
0.20 ’
Integument
0.00’ LS DO
L5 Dl
L5 D2
L5 D3 AGE
PP (DAYS)
Integument
’
L5 DO
W Dl
L5 D2
LS D3
PP
IN LAST INSTAR
FIGURE 1, JH esterase and JH epoxide hydrolase activity in homogenates of fat body, midgut and integument during the last stadium of the cabbage looper, Trichoplusia ni. Each point is the mean of three samples; each sample consisted of tissue pooled from three insects. Vertical lines are fl SEM. L5DO = Day 0 in the last (fifth) stadium; PP = prepupa.
ide hydrolase activity) and prepupae (46%). The JH esterase activity was found mostly in the fat body in wandering larvae (52%) as opposed to the hemolymph in prepupae (68%). In prepupae the JH esterase activity in midgut and integument was minimal as compared to other tissues (3% each). Although there was a 14-fold increase in the hemolymph JH esterase activity from LSD3 to LSD4, the increase in JH esterase activity per insect was only 4-fold and 25% of the JH metabolic activity in wandering larvae was JH esterase.
In viva metabolism prepupae
of JH
in wandering
larvae
and
Physiological concentrations of 3H- 1OR, 1 lS-JH II were injected in light mineral oil into the hemocoel of wandering larvae and prepupae of the cabbage looper. At 5 min after the introduction of JH, overall metabolism was higher in prepupae (38%) as compared to 8% in wandering (LSD3) larvae (t-test CY= 0.05) (Fig. 2). There was also significantly lower metabolism in L5D3 larvae at 15 min but no differences were found in metabolism
JUVENILE TABLE
1. In vitro metabolism
of lOR,SJH
HORMONE
III in different
METABOLISM
tissues
of wandering
pmol JH III min-’ JH esterase Tissue
Wandering
Hemolymph Fat body Midgut Integument Whole
200 600 180 170
insect
2850 1120 130 110
(LSD4)
4210
*JH metabolic activity is the mean of five replicates fl SEM. Figure in paranthesis Hemolymph JH easterase activity per insect was calculated from the hemolymph by Wing (1981).
at 60 and 120 min. The only JH II product that appeared to increase consistently with time in both wandering larvae and prepupae was a polar metabolite that was water soluble and could not be extracted by ethyl acetate (Fig. 3). In our TLC system (silica gel and 66:33:1 of hexane:ethyl acetate:glacial acetic acid), the polar metabolite did not move from the origin. This polar metabolite was also by far the predominant product from the metabolism of JH II at most time points examined in both L5D3 and L5D4 larvae but the rate of production was greater in the former (55% versus 35%, respectively, at 120 min). JH diol, JH acid and JH acid, diol were produced in both developmental stages of T. ni examined.
loopers,
Trichoplusiu ni
(%)*
Wandering
f 280.0 (68) + 54.0 (27) + 10.0 (3) f 6.0 (3)
1150
Tissue distribution
larval-equivalent-’
cabbage
JH epoxide
Prepupal
(17) (52) (16) (15)
185
and prepupal
activity
(L5D3)
* 10.0 k 30.0 rf: 3.0 + 4.0
IN T. NI
(L5D3)
hydrolase
activity Prepupal
(L5D4)
2800 _+ 23.0 (80) 300 * 30.0 (9) 400 + 8.0 (11)
340 + 33.0 (46) 100 + 7.0 (34) 150 f 8.0 (20)
3500
740
indicates the percentage of overall activity in each tissue. JH esterase activity per ml x the blood volume determined
Five min after injection, 9 1.6% ized and of this unmetabolized was found in the hemolymph associated with the alimentary
of the JH was unmetabolJH, approximately 50% and the other 50% was canal and carcass (insect
ACID - DIOL
of JH and its metabolites
The tissue distribution of 3H-10R,1 lS-JH II and its metabolites at different times after injection in light mineral oil into wandering T. ni is shown in Figs 4 and 5.
-
0’
WANDERING
I
0
30
I
60
I
90
,
320
5
15 45 120 60 TIME AFTER INJECTION (MINUTES)
TIME AFTER INJECTION (MINUTES) FIGURE 2. Percent metabolism at different times after injection of [12,13-3H,]-(10R,1 IS)-JH II in oil (140 pg JH II/g insect) into wandering (fifth stadium, day 3) larvae and prepupae (fifth stadium, day 4) of the cabbage looper, Trichoplusia ni. After injection, larvae were incubated at 30°C. Unmetabolized JH II was identified and quantitated by thin layer chromatography. Each point is the mean of three samples; each sample consisted of three insects. Vertical lines are +l SEM.
FIGURE 3. Percent metabolism of [12,13-3H,J-(10R,1 Is)-JH II to JH II acid, dial, acid-dial and polar metabolite at different times after injection in oil (140 pg JH II/g insect) into wandering (fifth stadium, day 3) larvae (a) and prepupae (fifth instar, day 4) (b) of the cabbage looper, Trichoplusia ni. After injection, larvae were incubated at 30°C. Metabolites were identified and quantitated by thin layer chromatography. Each point is the mean of three samples; each sample consisted of three insects. Vertical lines are +l SEM.
VASANT L. KALLAPUR
186
&m
( 91.6
)
( 64.2 )
( 30.5
)
et al.
( 29 )
E90
% OF TOTAL JH II REMAINING / INSECT
B n CARCASS
0 HEMOLYMFH I3 ALiMENTARYCANAL
30
5
TIME
120
AFTER INJECTION
240
(MINUTES)
FIGURE 4. Distribution of [12,13-3H,]-(10R,1 IS)-JH II in carcass, hemolymph injection in oil (140 pg JH II/g insect) into wandering (fifth instar, day 3) larvae injection, larvae were incubated at 30°C. JH II was identified and quantitated by mean of three samples; each sample consisted of three insects.
minus hemolymph and alimentary canal) (Fig. 4). Extensive efforts were made to wash the alimentary canal and carcass with buffer to remove contaminating hemolymph. Whether the JH II associated with the hard tissues was
and alimentary canal at different times after of the cabbage looper, Trichoplusia ni. After thin layer chromatography. Each point is the Vertical lines are +l SEM.
surface bound or intracellular was not determined. percentage JH II in hemolymph, alimentary canal carcass did not significantly change from 5 through min (t-test, (Y = 0.05) even though the percentage
d 70 i3 E 6o 8 40 * 30 20 10 5
120
30
240
e! 300 IIzb aD 1~~
5
30
120
240
b3 1y 90 880 ? 70 fl 60
T
g 70
T
2 60 E 5o 8 40 be30 20 10 a 5
TIME AFTit
240
INJECTI~“(MINUl’RS)
FIGURE 5. Percent metabolism of [12,13-3H2]-( lOR,l Is)-JH II to polar metabolite (a), acid, diol (b), diol (c) and acid (d) in
carcass, hemolymph and alimentary canal at different times after injection in oil (140 pg JH II/g insect) into wandering (fifth stadium, day 3) larvae of the cabbage looper, Trichoplusia ni. After injection, larvae were incubated at 30°C. Metabolites were identified and quantitated by thin layer chromatography. Each point is the mean of three samples; each sample consisted of three insects. Vertical lines are fl SEM.
The and 120 JH
187
JUVENILE HORMONE METABOLISM IN T. NI
remaining declined to 30.5%. At 240 min, there was a significant increase in the percentage JH II in hemolymph over that at 120 min and a significant decrease in carcass. The metabolites of JH II, i.e., polar metabolite, JH acid, diol, JH diol and JH acid, were evenly distributed in hemolymph, alimentary canal and carcass after injection (Fig. 5). In vivo JH metabolism following administration
d@erent routes of
The method of introduction of 3H-10R,1 lS-JH II into wandering (L5D3) larvae and prepupae (L5D4) affected JH metabolism 60 min after treatment (Table 2). Overall JH metabolism was greater when JH was either applied topically in oil or injected in buffer as compared to injection in oil. This was the case for both wandering larvae and prepupae. For example, the remaining JH when injected in oil into L5D3 larvae was 46% while only 8.4% and 2.3% was remaining for topical in oil and buffer injected, respectively (Table 2). Interestingly, the method of introduction also affected the production of JH acid. The topical application of JH consistently produced more JH acid in L5D3 and L5D4 T. ni as compared to injection in either buffer or oil. No JH acid was detected when JH II was injected in buffer even though in prepupae, hemolymph JH esterase activity is at peak levels at this developmental time (Table 1). JH acid, diol was detected. There was no significant difference (t-test, (Y= 0.05) in the in vivo metabolism 60 min after injection between wandering larvae and prepupae topically treated with JH II or injected with oil; prepupae had statistically higher JH metabolism when JH was introduced by injection in buffer but the difference was less than 2% and JH metabolism was almost 100% at 60 min after injection. Irrespective of the method of introduction, JH diol and polar conjugate was produced with the latter consistently being the predominant metabolite of JH II ranging from 33 to 71% in L5D3 larvae to 23 to 79% in prepupae. Greater polar metabolite was produced in wandering larvae (L5D3) than in prepupae for all methods of introduc-
tion although the difference was not statistically significant for buffer injection. Metabolism of JH II versus JH II acid by epoxide hydrolase When epoxide hydrolase from whole body homogenate of wandering T. ni was incubated with 3H-10R,1 ISJH II or its acid at physiological concentrations (140 pg/ml of homogenate or 450 pg/insect equivalent), JH was metabolized at least 3-times faster than JH acid (Fig. 6). At 45 min after injection, 14% of the JH II was metabolized while only 4% of the JH acid was metabolized at the same time.
‘15 *
JHII
.+
JHAdd
12 -
10
0
30
20 TIME
40
50
(MINUTES)
FIGURE 6. Time course of percent metabolism of [12,13-3H,](lOR,llS)-JH II and its acid when incubated in vitro at 30°C in 10,000 g supematant prepared from the homogenization of wandering (fifth instar, day 3) larvae of the cabbage looper, Trichoplusia ni. Supematant was preincubated with octylthio- 1, l , l-trifluoropropan-2one (final concentration 4.1 mM) for 10 min at 30°C prior to the addition of substrate (final concentration 140 pg/ml). JH diol and JH diol, acid were identified and quantitated by thin layer chromatography. Each point is the mean of three samples. Vertical lines are kl SEM.
TABLE 2. In vivo metabolism of lOR,l lS’-JH II introduced by different methods to wandering larvae (and prepupae) of the cabbage looper, Trichoplusia ni % JH or JH metabolite 60 min alter treatment* Application method Topical in oil Injected in buffer Injected in oil
Polar 70.5 (34.8 82.6 (78.7 33.3 (22.6
f + f f + f
4.2 4.9) 5.6 0.8) 0.9 2.0)
Acid-diol 8.1 (15.1 8.6 (12.1 8.1 (6.9
+ f + + + +
2.7 2.1) 0.6 0.8) 2.4 1.5)
Diol 1.9 k (7.1 + 2.7 + (9.1 f 6.7 f (2.2 f
1.6 0.3) 1.5 1.0) 2.4 0.6)
*Values are the mean of three replicates zkl SEM for wandering larvae and prepupae in parenthesis.
Acid
JH
11.8 f 1.6 (26.0 + 2.1)
8.4 k 5.3 (17.0 f 3.7) 2.3 k 0.3 (0) 46.1 + 4.6 (48.1 + 3.6)
(k 6.0 + 1.0 (20.1 f 0.5)
VASANT
188
L. KALLAPUR
DISCUSSION
It is generally accepted that increases in JH metabolism are responsible for the removal of JH in last stadium Lepidoptera and this regulates the timing for the initiation of wandering behavior midway through the last stadium and the proper expression of the pupal stage just prior to the final larval ecdysis (Hammock, 1985; Roe and Venkatesh, 1990). The current view is that in Lepidoptera, JH esterase is responsible for the primary metabolism of JH at these two periods in the last stadium and JH epoxide hydrolase converts JH acid to JH acid, diol. Based on levels of R20458 epoxide hydration compared to JH ester hydrolysis in last stadium T. ni, Wing et al. (198 1) concluded that epoxide hydrolase had a secondary role in JH metabolism. Touhara and Prestwich (1993) more recently provided kinetic evidence that binding protein protected JH from JH epoxide hydrolase but not JH esterase, thus arguing that JH esterase was the primary metabolic route for JH in M. sextu. There is also direct evidence of the importance of JH esterase metabolism in JH regulation and the regulation of metamorphosis. The specific inhibition of JH esterase with polarized alcohols and organophosphorus JH mimics delayed wandering behavior, delayed pupation, and produced what appeared as larval-pupal intermediates in last stadium T. ni (Roe et al., 1991, 1993; Linderman et al., 1991). Jones et al. (1990) showed that this inhibition resulted in an increase in the JH titer in the same insect. JH esterase inhibition in adult virgins of T. ni also caused egg laying, presumably by reducing JH metabolism and elevating the JH titer (Roe et al., 1991). Available evidence in the Dipteran, Culex quinquefasciatus (the yellow fever mosquito), suggests that JH epoxide hydrolase is more important than JH esterase in JH metabolism and suggests a functional role for JH epoxide hydrolase in both metamorphosis and adult reproduction. During the last stadium of C. quinquefasciatus, two peaks in JH epoxide hydrolase activity were reported at the exclusion of any change in the JH esterase activity (Lassiter et al., 1995a). Apparently, epoxide hydrolase and not JH esterase has a dynamic role in JH turnover in last instars of this insect. The in vitro JH III epoxide hydrolase activity exceeded that of JH esterase throughout the last stadium by as much as 6-fold. More JH III diol was found than JH acid in both in vitro and in vivo assays of adult C. quinquefasciatus. Blood feeding increased JH epoxide hydration 36 h later, presumably to reduce JH levels and allow the production of ecdysone necessary for the initiation of vitellogenesis (Lassiter e2 al., 1994, 1995b). Unfortunately, no inhibitors for epoxide hydrolase are available to directly address the importance of epoxide hydrolase. However, the evidence, although not conclusive, does point to JH epoxide hydrolase and not JH esterase as the most important factor in the primary metabolism of JH in C. quinquefasciatus. In last stadium tobacco homworm larvae at the time of the first and second hemolymph JH esterase peak,
et al.
elevated JH esterase and epoxide hydrolase activity occurred in integument, midgut, fat body and brain, with the exception of the fat body at the time of the second peak (Jesudason et al., 1992). This synchrony between increases in JH esterase activity and JH epoxide hydrolase activity was not found in T. ni (Fig. 1). The JH esterase activity peaked in fat body, midgut and integument in last stadium cabbage loopers at day 2 and elevated JH esterase activity was found in the fat body in prepupae (day 4), while the midgut JH epoxide hydrolase activity peaked on day 2 and the fat body and integument peaked on day 3. Wing et al. (198 1) reported an order of magnitude lower amount of R20458 epoxide hydrolase activity compared to JH esterase activity in last stadium T. ni. In a similar analysis in our experiments using racemic JH III, no significant differences were noted at peak levels between in vitro JH esterase and JH epoxide hydrolase activity in fat body, midgut and integument (Fig. 1, activity expressed per mg protein). Similar results where the in vitro JH epoxide hydrolase activity was equivalent or in some cases exceeded the JH esterase in specific tissues in prewandering and prepupal last stadium M. sexta was previously reported by Jesudason et al. (1992) and in last stadium Galleria mellonella by Wisniewski et al. (1986). So, based simply on the concentration of activity in specific tissues (excluding the hemolymph), JH esterase and JH epoxide hydrolase appear to have similar importance in JH metabolism in these Lepidoptera. It was interesting to note that JH epoxide hydrolase activity per mg protein (measured in vitro with racemic JH III) peaked in fat body and integument on day 3 in T. ni and JH esterase activity was elevated in fat body and hemolymph but not the other tissues examined on day 4 (Fig. 1, Sparks et al. 1979, Roe and Venkatesh 1990). Overall JH III metabolism in vitro was essentially the same at these two time points (Table 1) and when 1OR,11S-JH II at physiological concentrations were injected in oil into wandering larvae and prepupae, the level of metabolism was also similar. In fact, injection in oil or buffer or the topical application of lOR,l lS-JH II produced the same results; the rate of overall metabolism was the same in wandering larvae and prepupae (Table 2). However, on L5D3 the JH epoxide hydrolase activity per insect was 3 times that of the JH esterase while in prepupae essentially the reverse occurred, i.e. the JH esterase activity was 5.7-times that of the JH epoxide hyrolase. Jones et al. (1990) found that the JH titer peaked on L5D3 at 350 pglg tissue and decreased to 10 pg/g by L5D4 in T. ni. It was concluded that the increase in JH esterase activity in prepupae noted in hemolymph and other tissues by Wing et al. (1981) was responsible for this reduction along with decreased biosynthesis. Considering that the overall levels of metabolism are apparently the same between wandering larvae and prepupae based on the in vitro and in vivo experiments presented here, there is obviously a mechanism for the maintenance
JUVENILE
HORMONE
of high JH titers in wandering larvae in the presence of high tissue JH epoxide hydrolase activity. Note that approximately half of the injected JH II in wandering T. ni was associated with gut and carcass (fat body and integument) (Fig. 4), also the sites for high JH epoxide hydrolase activity. This was true at most of the time points after injection. Thus, differences between the tissue distribution of JH epoxide hydrolase and that of JH does not appear to explain why high JH titers exist in the presence of elevated JH metabolic activity. Touhara and Prestwich (1993) found that JH binding protein prevented the metabolism of JH by soluble JH epoxide hydrolase but not JH esterase in A4. sextu and hypothesized that the binding protein directed the metabolism of JH by JH esterase first. Because of the low affinity of the binding protein for JH acid, the acid would be released and subsequently metabolized by JH epoxide hydrolase. If this model is applicable to last stadium cabbage loopers and considering that Wing et al. (1981) demonstrated that the hemolymph JH binding protein concentration did not change between wandering larvae and prepupae of T. ni, competition with high affinity binding protein might explain the existence of high JH titers in day 3 wandering larvae in the presence of high JH epoxide hydrolase and low JH esterase activity. However, at different times after injection of lOR, 1IS-JH II into wandering and prepupal T. ni, one of the primary metabolites of JH that was found was JH II diol (Fig. 3). Despite the method of introduction, JH diol was always one of the primary metabolites (Table 2), although usually at a lower concentration than that of JH acid. The major product formed from the injection of JH II was a polar metabolite, which formed faster in wandering larvae than in prepupae and which was formed irrespective of the method of introduction of JH (Fig. 3 and Table 2). The polar metabolite was by far the predominant product of JH metabolism in both wandering and prepupal T. ni. Halarnkar et al. (1993) identified the polar metabolite in A4. sexta as a phosphate conjugate of JH diol, which was the principle end product of JH I metabolism in newly molted last stadium tobacco hornworm larvae. No JH acid, diol-phosphate conjugate has been found in A4. sextu (personal communication with D. Schooley, Department of Biochemistry, University of Nevada, Reno). These results with M. sextu argue that the major metabolite in both wandering larval and prepupal T. ni either injected or topically treated with JH might be JH diol and not JH acid (Fig. 3 and Table 2); also note that only small amounts of JH acid, diol were found especially as compared to the polar metabolite. The importance of JH epoxide hydrolase in the primary metabolism of JH is further argued by the discovery that at physiological concentrations of 1OR, 1 lS-JH II in vitro, the preferred substrate for JH epoxide hydrolase was JH II rather than JH II acid (Fig. 6). The role of JH epoxide hydrolase in the regulation of JH titer and metamorphosis is not yet resolved. In the model proposed by Touhara and Prestwich (1993) for A4.
METABOLISM
IN T. NI
189
sex&, the effect of specific and non-specific JH binding
by various tissues and the distribution of JH and JH metabolic enzymes in these tissues during development on JH metabolism was not included in their analysis. In the work presented here on T. ni and that of Halarnkar et al. (1993) working with A4. sex&, an argument can also be made that a single injection of JH in vivo, even when introduced at a final concentration after injection which is comparable to normal physiological levels of JH, does not mimic the natural introduction of JH into the hemolymph by the corpora allata. On the other hand, Hidayat and Goodman (1994) found in M. sexta larvae that < 1% of the JH-specific binding protein was hormone-loaded at any one time during the fourth stadium. Considering the high affinity of JH binding protein for JH and binding protein abundance, the only limiting factor in single injection experiments into hemolymph which is poorly circulated is compartmentalization and limited mixing. Finally, note that the distribution of each JH metabolite was approximately the same between tissues (hemolymph, carcass, and alimentary canal; Fig. 5) at different times after injection, indicating no tissue specific storage of a particular metabolite. It was also interesting that even though the JH esterase activity is low in the hemolymph of wandering T. ni (0.07-times that of prepupae on a per insect basis), the JH esterase activity in the other tissues were quite similar (950 pmol JH III mini larvalequivalent-’ for wandering larvae versus 1360 pmol min-’ larval-equivalent- ’ for prepupae). Recall, that approximately 50% of the injected lOR,llS-JH II was found in tissues other than hemolymph in wandering larvae of T. ni. The significance of this rather substantial gut and carcass JH esterase activity associated with JH in wandering larvae is not clear. It does indicate a similar clearance rate for tissue JH in wandering larvae and prepupae. In terms of JH esterase metabolism of JH, the major change that occurs between day 3 and day 4 in the last stadium of T. ni, is an increase in the clearance rate in the hemolymph, not the other tissues. Finally, it should be noted that the method of introduction of JH can affect the route of metabolism (Table 2). Therefore, care should be taken in the interpretation of experiments involving the in vivo metabolism of JH. In summary, a peak in JH III esterase activity per mg protein measured in vitro was found in fat body, midgut, integument and hemolymph in prewandering (day 2) last stadium T. ni and in fat body and hemolymph of prepupae (on day 4). Midgut JH epoxide hydrolase activity peaked on day 2 and in fat body and integument on day 3. No significant differences were noted in the overall metabolism of JH III in vitro between wandering larvae and prepupae and in vivo when lOR,l l,S-JH II was introduced at physiological concentrations by different methods. JH II diol and a predominant JH II polar metabolite was found, the latter which in M. sexta was a phosphate conjugate of JH diol. JH II epoxide hydrolase metabolized JH II at a faster rate than JH acid. These studies in toto suggest a more important role for JH epoxide
190
VASANT L. KALLAPUR et al.
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Acknowledgements-This research was supported by the North Carolina Agricultural Research Service and the USDA Competitive Grants Program (90-37263 and 93-01607).