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Role of juvenile hormone-esterase in mating-stimulated egg
development in the moth Heliothis virescens
Sonny B. Ramaswamy
a,*, Shengqiang Shu
a, George N. Mbata
1,a, Anna Rachinsky
a,
Yong Ihl Park
2,a, Lakisha Crigler
b, Shalana Donald
b, Asok Srinivasan
baDepartment of Entomology, Kansas State University, 123 West Waters Hall, Manhattan, KS 66506, USA bDepartment of Biology, Tougaloo College, Tougaloo, MS 39714, USA
Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000
Abstract
Juvenile hormone (JH) titer in virgin females of Heliothis virescens is significantly lower than that in mated females of the same age. The JH titer in virgin females follows a diel pattern in which it begins to increase towards the end of photophase, remains high around the onset of scotophase, and declines during scotophase. The titer reaches its lowest levels at the onset of photophase, and remains low during the first half of photophase. In mated females, the diel pattern of JH titers is not as pronounced. JH-esterase (JHE) activity in mated females is significantly lower than that of virgin females during photophase; JHE levels in the former are similar to levels seen in newly emerged females. JHE activity in mated females also exhibits a diel pattern, in which activity is low during photophase and high at the onset of scotophase. Evidence for the indirect involvement of JHE in the mating-stimulated egg development is provided by the effect of selected JHE inhibitors in inhibiting JHE activity and stimulating egg production in virgin females.2000 Elsevier Science Ltd. All rights reserved.
Keywords: JH titer; JH-esterase; Inhibitors; Trifluoropropanones; OTFP; BTFP; Reproduction; Lepidoptera; Noctuidae
1. Introduction
Juvenile hormone (JH) produced in corpora allata (CA) regulates reproduction in adult females of various species of insects (Nijhout, 1994). In many Lepidoptera, JH is crucial for egg production (Ramaswamy et al., 1997). In females of some species, mating causes a multi-fold increase in JH titer (e.g. Lacanobia oleracea, Edwards et al., 1995; Heliothis virescens, Shu et al., 1998; Choristoneura spp., Cusson et al., 1999; Cydia
pomonella, Webb et al., 1999).
Three hypotheses have been postulated for this mat-ing-induced increase in JH titer (Ramaswamy et al.,
* Corresponding author. Tel.:+1-785-532-6154; fax:+ 1-785-532-6232.
E-mail address: [email protected] (S.B. Ramaswamy).
1 Present address: Department of Entomology and Plant Pathology,
127/110 Noble Research Centre, Oklahoma State University, Stillwater, OK 74078, USA.
2 Pressent address: Department of Entomology, North Carolina
State University, Raleigh, NC 27695, USA.
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 5 0 - 3
1997): (1) transfer of JH by the male to the female; (2) inhibition of JH degradation in mated females; and (3) mating-triggered allatotropic effect in females. Male moths of some species are known to transfer JH to females during mating (Park et al., 1998), but not in others (Cusson et al., 1999). Mating reduces JH-esterase (JHE) activity by more than 95% in Trichoplusia ni (Venkatesh et al., 1988), whereas it causes a significant increase in JHE activity in both Choristoneura
fumifer-ana and C. rosacefumifer-ana (Cusson and Delisle, 1996).
2. Materials and methods
2.1. Insects
Heliothis virescens larvae reared on a wheat germ diet
were obtained from the USDA-ARS Gast Rearing Facility, Mississippi State, Mississippi. Pupae were sexed, and males and females were held in separate jars at 27±2°C in an incubator with a 14:10 light:dark cycle. Under our conditions, peak moth emergence occurs approximately 2 h after the onset of scotophase. Newly emerged females were collected every 15 min from 2 h to 4 h after the onset of scotophase, designated as time 0, and aged to the required length of time. Females were allowed to couple with virgin males when they were 24 h old, and uncoupling occurred when they were ca. 30 h old. During scotophase, insects were handled under red light.
2.2. JH titers
Hemolymph was obtained from females during the various sampling periods by making an incision in the dorsal blood vessel and allowing the hemolymph to flow into graduated micropipets (Ramaswamy et al., 1995).
Twentyµl of hemolymph were pooled from four moths
each contributing 5µl and mixed with 100 µl
acetonitr-ile, 100 µl 2% NaCl solution, and 200 pg JH III ethyl
ester (internal standard) in pre-cleaned and silanized glass vials with screwcaps lined with Teflon. Samples were processed and JHs (JH I, JH II, and JH III) were quantified by gas chromatography coupled to a mass spectrometer in the selected ion mode (GC–MS–SIM) according to Shu et al. (1997). JH titer (total of JH I, JH II, and JH III) is expressed as ng/ml of hemolymph. Under our conditions the lower limit of detection of the different JH homologs is 10 pg with better than 90% efficiency (Shu et al., 1997).
2.3. Assay for JH-esterase
Hemolymph was collected from virgin and mated females of appropriate ages, dispensed into 0.5 ml microcentrifuge tubes containing a few crystals of phe-nylthiourea (PTU), and centrifuged for 5 min at 6000g
and 4°C. An appropriate volume of the cell-free serum
was added to a 99-fold volume (=1:100 dilution) of
modified PBS buffer (0.2 M PBS with 0.1% PTU, 10% sucrose, pH 7.4), and stored at280°C until it was used for determining JHE activity. Triplicates of enzyme
ali-quots (100 µl each) were incubated at 30°C for 20 min
with 2 µl of 3H-labeled JH III (New England Nuclear,
Boston, MA) solution containing 0.5 nmol of unlabeled JH III and ca. 25 000 DPM. Hemolymph JHE activity was quantified according to Hammock and Sparks
(1977), and is expressed in nmol/min/ml (amount of JH III in nmol hydrolyzed in 1 min per ml of hemolymph).
2.4. JH-esterase inhibitors
JHE inhibitors synthesized and provided by C. Devor-shak and M. Roe (North Carolina State University) were used to determine their effectiveness at stimulating egg production and inhibiting JHE in virgin H. virescens
females. Inhibitors included selected
trifluoropro-panones: butyl-1,1,1-trifluoro-2-propanone (BTFP);
dodecyl-1,1,1-trifluoro-2-propanone (DTFP);
octyl-1,1,1-trifluoro-2-propanone (OTFP); and phenylthio-1,1,1-trifluoro-2-propanone (PTFP). The inhibitors were dissolved at the appropriate concentrations in ethanol,
and 5µl were applied topically to the abdominal venter
of virgin females within 2 h of emergence. Females were dissected 24 h later to allow counting of chorionated oocytes contained in ovarioles. Control females received
5 µl ethanol only or remained untreated.
In a second experiment, virgin females were treated within 2 h of emergence during scotophase with BTFP
or OTFP at a dose of 1 µmol in 5 µl ethanol. Controls
included either untreated moths or ethanol-treated moths.
Twelve or 24 h later, 5–10 µl hemolymph from each
female were collected. The hemolymph was dispensed into microcentrifuge tubes containing a few crystals of phenylthiourea (PTU) and processed as above for determining JHE activity. Protein concentrations in ali-quots were measured by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as standard. JHE activity is expressed as JH III nmol hydrolyzed in 1 min per mg protein.
2.5. Data analysis
Data were subjected to analysis of variance (ANOVA) followed by Fisher’s protected least significant differ-ence test (LSD) or Student–Newman–Keul’s multiple range test or t-tests.
3. Results
3.1. JH titer and JHE activity
JH titer in virgin females begins to increase towards the end of photophase, remains high around the onset of scotophase, and declines during scotophase. The titer reaches its lowest level at the onset of photophase (Fig. 1A). At emergence, hemolymph JH titers in virgin H.
virescens is undetectable and increases to almost 75
Fig. 1. (A) Juvenile hormone titers in the hemolymph of virgin and mated Heliothis virescens females from emergence through 78 h of age. Females were allowed to couple with virgin males when they were 24 h old, and uncoupling occurred when they were ca. 30 h old. Vertical dotted bars denote the 10 h scotophase, except in the first bar which shows only 8 h as moths typically emerge 2 h into scotophase. (B) Hemolymph juvenile hormone esterase activity in virgin and mated
Heliothis virescens females from emergence through 84 h of age.
Females were allowed to couple with virgin males when they were 24 h old, and uncoupling occurred when they were ca. 30 h old. Vertical dotted bars denote scotophase as above. JH and JHE titers were obtained from four replicates of four moths per replicate.
is around 125 ng/ml, while that in comparably aged vir-gin females is undetectable. The titer in mated females is significantly higher than that of virgin females at all sampling periods, and continues to increase significantly with age of mated females, reaching over 200 ng/ml when mated females are 72 h old (Fig. 1A). Interest-ingly, at 6 h after uncoupling (Fig. 1A), there is a pre-cipitous decline in JH titers in the mated female; JH in comparably aged virgin females is undetectable at our level of detection.
Virgin females exhibit a slow increase in JHE activity with age, increasing from just over 2 nmol/min/ml of JHE activity in the hemolymph of newly emerged females to ca. 10 nmol by about 30 h after emergence (Fig. 1B). After this age, JHE activity remains high. JHE activity in virgin females also exhibits a diel pattern,
especially beginning after the second scotophase (Fig. 1B), similar to the pattern seen in JH titers. JHE activity in mated females is significantly lower than that of virgin females during photophase (Fig. 1B). JHE activity in mated females shows a more pronounced periodicity, with activity increasing during the latter half of photo-phase and peaking at the onset of scotophoto-phase. Mating does not result in immediate inhibition of JHE, since JHE activity 0 h after uncoupling is similarly high as in comparably aged virgin females. JHE activity in mated females during photophase is significantly depressed, reaching only around 2 nmol or less JHE activity per min per ml of hemolymph, which is comparable to that seen in newly emerged females (Fig. 1B). The mating effect on increasing JH titer and on decreasing JHE activity during photophase is not transient. JHE activity
in mated females is further depressed (,2 nmol JHE
activity) as they grow older, but in similarly aged virgin
females, JHE activity continues to remain high (.10
nmol JHE activity) (Fig. 1B), greater than a five-fold dif-ference.
3.2. JHE inhibitors
Because of the inhibition of JHE in mated females, we hypothesized that JHE was involved in the mating-induced gonadotropic effect. This hypothesis was tested using JHE inhibitors.
All four JHE inhibitors caused varying levels of gona-dotropic activity; BTFP elicited the most pronounced dose response (Fig. 2). The JHE inhibitors caused a two-fold to almost a four-two-fold stimulation of egg production in treated virgin females by 24 h after treatment, which is similar to that seen in mated females, 24 h after mating. When BTFP and OTFP were tested for their effective-ness at inhibiting JHE activity, both caused inhibition, but OTFP was the more effective JHE inhibitor, com-pared with the solvent-treated controls. BTFP appeared to exhibit significant inhibition of JHE activity 26 h after treatment (Fig. 3). With OTFP, inhibition also was more pronounced at 26 h after treatment, where JHE activity was depressed by greater than four-fold. Moths treated with OTFP exhibited general malaise with elongated and distended abdomens at 24 h after treatment.
4. Discussion
As had been demonstrated earlier (Ramaswamy et al., 1997; Zeng et al., 1997; Shu et al., 1998), mated H.
vire-scens females exhibited significantly higher titers of JH
Fig. 2. Gonadotropic effect of selected juvenile hormone esterase inhibitors in virgin Heliothis virescens females. Controls included untreated and solvent treated females. Twelve to 13 females were used for each treatment. See text for inhibitors used and treatment protocols.
100 ng/ml of JH, while JH was almost undetectable in hemolymph of comparably aged virgin females. This JH in the mated female can be accounted for almost com-pletely by the JH transferred by males during copulation (Park et al., 1998), which is reduced drastically by 6 h after uncoupling. JH titers continue to increase signifi-cantly in mated females, while the titers in virgin females decrease (Fig. 1A). This latter continued increase in JH titers is the result of an allatotropic effect of mating in H. virescens (Park et al., 1998), a phenom-enon observed in cockroaches (e.g. Diploptera punctata; Stay and Tobe, 1977), but not in the tortricids C.
fumifer-ana and C. rosacefumifer-ana (Delisle and Cusson, 1999). In the
latter tortricid species, males did not transfer JH to females during copulation (Cusson et al., 1999); the spe-cific physiological basis for the increased JH titer in mated females in these species remains to be determined. In an earlier study, we had observed that JH pro-duction and release in virgin H. virescens females may exhibit diel periodicity, but hemolymph samples were not taken beyond 48 h of age (Shu et al., 1998). The
pronounced pattern of changes in JH titers in virgin females, and the less pronounced pattern in mated females (Fig. 1A), demonstrated in the current study, has hitherto not been described in other species of insects. We propose that such a diel pattern, i.e. inclining JH titer during the latter part of photophase, may have a role in regulating egg development. During scotophase, under declining JH titer, the female is likely to be physiologically geared to dispersing to find potential host plants, calling and sex pheromone emission in vir-gin females, and to laying eggs in mated females (Ramaswamy, 1990). Whether such a periodicity is observed in JH-regulated egg development remains to be determined in H. virescens and other lepidopterans.
In addition to the pronounced allatotropic effect of mating and the transfer of JH during copulation in H.
virescens (Park et al., 1998), we had speculated on an
Fig. 3. Inhibition of juvenile hormone esterase in virgin Heliothis
virescens females by selected juvenile hormone esterase inhibitors.
Controls included untreated and solvent treated females. Eleven to 25 females were used for each treatment. See text for inhibitors used and treatment protocols.
when JHE activity is similar to that seen immediately after moth emergence (Fig. 1B). Interestingly, JHE activity also exhibits a diel periodicity similar to that seen in JH titers, and the two may be regulating each other. Mating-induced suppression of JHE has been observed in noctuid species only thus far (this study; Venkatesh et al., 1988), because in the tortricids, C.
fum-iferana and C. rosaceana, there is no difference in JHE
levels of mated and virgin females (Cusson and Deli-sle, 1996).
JHE activity in H. virescens is only around 2 nmol/min/ml hemolymph in females at emergence, which increases to about 10 nmol by 30 h after emerg-ence. But in T. ni, JHE activity at emergence is ca. 25 nmol/min/ml hemolymph; the level remains relatively constant in virgins but begins to decrease in mated females to less than 2 nmol by 6 days after emergence (Venkatesh et al., 1988). The reason for the approxi-mately one order of magnitude difference in JHE activity in virgin females of the two noctuids is unknown, and may be related to levels of JH production and release in the two species. However, details of the dynamics of JH titers in the hemolymph of T. ni remain to be determined. The potential for JHE involvement in indirectly affect-ing egg development is corroborated by results from the experiments on the JHE inhibitors. All four JHE inhibi-tors, OTFP, BTFP, PTFP, and DTFP, stimulated egg development, with BTFP causing the best dose-depen-dent increase in egg development in treated virgin females (Fig. 2). The JHE inhibitors caused a two-fold to almost a four-fold stimulation of egg production in
treated virgin females by 24 h after treatment, which is similar to that seen in mated females, 24 h after mating. When two of these compounds, BTFP and OTFP, were tested for their effectiveness, at 12 h post-treatment there was a significant inhibition by OTFP of JHE activity in treated females (Fig. 3); at 24 h post-treatment, JHE activity was lowered in OTFP- and BTFP-treated females, especially those treated with OTFP. However, females treated with the higher doses of OTFP exhibited general malaise, which may indeed be responsible for the reduced JHE activity. This latter malaise effect of such compounds needs to be studied, to determine their physiological effects. The reciprocal effects of BTFP and OTFP on JHE inhibition and stimulation of egg develop-ment, i.e. higher JHE inhibition but lower stimulation of egg development or vice versa (Figs. 2 and 3), also require additional study.
Venkatesh et al. (1988) had earlier demonstrated the effectiveness of JHE inhibitors such as OTFP and other compounds at eliciting better than 90% inhibition of JHE activity. These authors, however, did not explicitly test these compounds for gonadotropic activity, although they speculated that JHE was somehow involved in gon-adotropism in mated females (Roe et al., 1993). The effectiveness of substituted trifluoropropanones as potent and selective JHE inhibitors has been known for several years (e.g. Hammock et al., 1984). The current study is the first to demonstrate that such compounds indirectly stimulate egg development by inhibiting JHE activity in virgin females, thus providing support for the nexus between mating, inhibition of JHE activity, and stimu-lation of egg development.
JHE activity is likely to be one of three main factors that regulate JH titer dynamics in females, along with JH biosynthesis in CA and JH transfer by males. We hypothesize that JH biosynthesis activity parallels the JH titer pattern in virgin females and is high over the entire 24 h period in mated females. These two driving forces, JH biosynthesis and JH degradation, acting in concert could explain the diel pattern in JH titer of virgin females and increasing JH titer with age of mated females. JHE activity around the onset of photophase coupled with the likely inactivity of CA in virgin females may result in the reduction in circulating JHs produced during the preceding period. Continuous pro-duction of JHs coupled with low JHE activity during photophase could explain increasing JH titers in mated females.
sco-tophase. But during photophase the levels are signifi-cantly depressed, which is when, interestingly, JH titers are also low.
We have demonstrated that in H. virescens mating results in the transfer of JH from male to female and, further, that mating results in the activation of the mated female’s CA to biosynthesize JH, while concurrently the JH degradative system is inhibited, albeit slowly. We propose that the co-occurrence of these three mech-anisms accounts for the post-mating increase in hemo-lymph JH titers of mated females, resulting in stimu-lation of egg production.
Whether the male’s gonadotropic factor elicits its effect as a humoral or neuronal signal and/or by acting directly on the female’s CA is unknown. Studies with crickets have suggested that mating may trigger an allatostatic/allatotropic signal via the neuronal pathway acting on the female’s CA (Loher et al., 1987). Similarly, in M. sexta, mechanical stimuli from the bursa affect the CA via a neuronal pathway (Sasaki and Riddiford, 1984; Stringer et al., 1985). However, in H. virescens, tran-section of the ventral nerve cord of females does not prevent the mating-induced stimulation of egg pro-duction (Park and Ramaswamy, 1998). Additionally,
decapitation of mated females immediately upon
uncoupling or up to 6 h after uncoupling abolishes an increase in egg production. But decapitation from 6 h to 12 h after uncoupling results in a significant increase in egg production, which increases further if females are not decapitated until 24 h after uncoupling. Taken together, these data suggest that the mating-induced gon-adotropic stimulus in H. virescens appears to entail a humoral route either stimulating the CA directly or by affecting the brain to release allatotropins and/or affect JH-degradation. The finer details of the interaction between the humoral male factor and the brain regulation of the CA of mated H. virescens females remain to be delineated.
Acknowledgements
Drs Kun Yan Zhu, Tracey Webb, and Damon Crook, along with two anonymous reviewers, are thanked for comments on this manuscript. Funded in part by grants USDA/CSREES No. 96-35302-3715 and No. 98-35302-6985 and NIH-NIGMS GM08110. Contribution No. 00-111-J, Kansas State University Agricultural Experi-ment Station.
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