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J. Insect fhysiol. Vol. 40, No. 2, pp. 129-136, 1994 Copyright c 1994 Elsevier Science Ltd

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Regulation of Vitellogenin Production in Armyworm Moths, Pseudaletia unipuncta MICHEL CUSSON,*,t C. G. YU,* KERRY CARRUTHERS,$ GERARD R. WYATT,1 STEPHEN S. TOBE,* JEREMY N. McNEILg

Received 30 April 1993; revised 29 July 1993

The vitellogenin (Vg) of Pseuduktiu unipnctu was identified by SDS-PAGE of egg extracts and male and female hemolymph. It dissociated into two apoproteins of 164 and 40 kDa, the larger of which reacted specifically with a polyclonal antibody raised against the native Vg. An enzyme-linked immunosorbant assay (ELBA) was used to examine Vg production in virgin and mated P. unipuncta females, as well as in neck-ligated individuals to which juvenile hormone (JH) homologs or analogs were applied. During the 2 days following emergence, Vg titers were low (ca 0.2 mg/ml) in virgin females, but they increased steadily thereafter, reaching values of cu 2.4 mg/ml on day 7. Twenty-four h after mating, Vg titers were significantly higher in mated females than in virgins of the same age, but at 48 h, Vg titers of mated individuals were significantly lower than those of virgins, suggesting that Vg was taken up by the developing oiicytes. Females neck-ligated immediately after emergence did not produce Vg, but production was restored in a dose-dependent manner by topical applications of JH homologs or analogs. JH I and JH II displayed approx. 10 times more biological activity than JH III, and two JH analogs were still more active (ED,, pg/moth: JH III, 13.0; JH II, 1.8; JH I, 1.0; methoprene, 0.04, pyriproxyfen, 0.003). In a time-course analysis, pyriproxyfen-activated Vg accumulation began between day 1 and day 2 and reached a plateau on day 3 after treatment.

Juvenile hormone Methoprene Pyriproxyfen Vitellogenin True armyworm Pseudaletia unipuncta Mating

INTRODUCTION

Important differences exist in the timing and hormonal regulation of vitellogenesis among the Lepidoptera. Whereas vitellogenin (Vg) production begins during the last larval stadium in the gypsy moth, Lymantria &spar (Davis et al., 1990; Lamison et al., 1991), it is initiated in the prepupal stage of the silkmoth, Hyalophora cecropia (Pan, 1971, 1977), during early pupal develop- ment in Bombyx mori (Tsuchida et al., 1987), and in the late pharate adult stages of the Indian meal moth, Plodia interpunctella (Shirk et al., 1990) and the tobacco hawkmoth, Manduca sexta (Imboden and Law, 1983; Kawooya et al., 1987; Nijhout and Riddiford 1974, 1979). In other species such as the Monarch butterfly, Danaus plexippus (Pan and Wyatt, 1971,1976) and the corn earworm, Helicoverpa (Heliothis) zea

*Department of Zoology, University of Toronto, Toronto, Ontario,

Canada M5S 1Al.

TPresent address: Natural Resources Canada, Canadian Forest

Service-Quebec Region, 1055 du P.E.P.S., P.O. Box 3800, Sainte-

Foy, Qubbec, Canada GlV 4C7.

SDepartment of Biology, Queen’s University, Kingston, Ontario,

Canada K7L 3N6.

§DCpartement de biologic, Universitk Laval, Sainte-Foy, Qutbec, Canada GlK 7P4.

(Satyanarayana et al., 1992), Vg synthesis does not begin until after adult emergence.

Although juvenile hormone (JH) is responsible for the induction of Vg synthesis in the majority of insect species examined (Engelmann, 1983), stimulation by JH during late larval and pupal development would be in conflict with the regulation of metamorphosis, which requires low titers of JH. Thus, when vitellogenesis is initiated before adult eclosion, the regulatory signals appear to be provided by the common hormonal events of meta- morphosis (see Shirk et al., 1990). In L. dispar, a low or declining JH titer in the last larval instar is necessary for Vg accumulation, as shown by the suppression of Vg production in larvae treated with the JH analog, metho- prene (Davis et al., 1990; Hiremath and Jones, 1992). In B. mori, Vg synthesis begins a few days after pupal ecdysis, coincident with a peak in the ecdysteroid titer, and injection of 20-hydroxyecdysone in debrained B. mori pupae stimulated Vg production (Tsuchida et al., 1987). Conversely, treatment with 20-hydroxyecdysone inhibited Vg synthesis in P. interpunctella, a species in which vitellogenesis coincides with the decline in the ecdysteroid titer of the pharate adult (Shirk et al., 1990). The tobacco hawkmoth, M. sexta, presents a special case in which vitellogenesis begins 3-4 days before moth

129

130 MICHEL CUSSON et al.

emergence (Nijhout and Riddiford, 1974; Imboden and Law, 1983) and proceeds normally in the absence of the pupal corpora allata (Nijhout and Riddiford, 1974), whereas completion of oiicyte maturation in the young female moth is JH-dependent, although the exact action of JH is unclear since it does not appear to be essential for Vg synthesis (Nijhout and Riddiford, 1974). At the other end of the spectrum are several noctuid moths [Pseuduletia unipuncta (Cusson and McNeil, 1989a); He/i&his virescens (Ramaswamy and Cohen, 1991); H. zeu (Satyanarayana et al., 1991, 1992); Leucaniu separata (Wu and Quo, 1963)] and butterflies [Nymph&s antiopa (Herman and Bennett, 1975); D. plexippus (Pan and Wyatt, 1971, 1976)] in which oiicyte growth is strictly a post-eclosion, JH-dependent phenomenon. Among these insects, induction of Vg synthesis by JH or JH analogs has been demonstrated for H. zea, D. plexippus and N. antiopa (references given above). In summary, the above observations are in agreement with the generalization (Satyanarayana et al., 1992; Sroka and Gilbert, 1971; Wu and Quo, 1963) that JH-dependent, post-emergence egg maturation is found only in long-lived, feeding adults.

In the context of post-emergence vitellogenesis, an important question is the relative abilities of the three main lepidopteran JHs (JH I, II and III) to promote ovarian development. In the species examined, D. plex- ippus (Lessman et al., 1982), M. sexta (Nijhout and Riddiford, 1974), and H. virescens (Ramaswamy and Cohen, 1991), JH I and JH II were much more effective than JH III in inducing oiicyte maturation. Whether this pattern reflects a greater stimulation of Vg production by the higher homologs than by JH III has not been determined.

In the true armyworm moth, Pseuduletiu unipuncta (Noctuidae), oiicyte maturation takes place after adult ecdysis, is dependent on the presence of JH (Cusson and McNeil, 1989a), and is considerably slowed or delayed when female moths are maintained under low-tempera- ture and short-day conditions (Cusson et al., 1990). Furthermore, a correlation has been observed between ovarian growth and the in vitro biosynthetic activity of the corpora allata (Cusson et al., 1990, 1993). In this paper we report on Vg titers in virgin and mated P. unipuncta females and on the effects of topically applied JH homologs and analogs on the accumulation of Vg in neck-ligated females.

MATERIAJS AND METHODS

Rearing

Insects came from a laboratory colony stocked with P. unipuncta adult moths collected in the field in 1989 and 1990. Larvae were reared individually on an arti- ficial diet (Shorey and Hale 1965) under a 16 h light : 8 h dark (16L : 8D) regime at 25°C and the sexes were separated at the pupal stage (Breeland, 1958). Pupae and adults were held under these same environ- mental conditions, except for individuals employed in

the dose-response experiments and in the time-course analysis, which were maintained under a 12L : 12D photoperiod. Following emergence, adults were held in individual cages and fed with an 8% sucrose solution.

Protein identljkation and isolation

Hemolymph was collected from punctures made along the abdominal dorsal vessel. To identify the polypeptide components of Vg, an egg extract as well as hemolymph samples from males, and non-vitellogenic (newly- emerged) and vitellogenic (45 days old) females were analyzed by SDS-PAGE (Laemmli, 1970) on homo- geneous 12.5% gels and 415% linear gradient gels. Before analysis, hemolymph was diluted in PBS (0.14 M NaCl, 8.0 mM Na,HPO,, 1.4 mM KHrPO,, 2.7 mM KCl, pH 7.2-7.4) saturated with phenylthiourea (PTU), and centrifuged at 10,000 g for 10 min at 4°C to remove hemocytes.

A partial purification of the Vg-related protein, vitellin (Vn), was achieved by gel filtration on a Seph- acryl S-300 (Pharmacia) column. Mature eggs (500 mg) were placed in 5 ml of PTU-saturated PBS and ground in a glass homogenizer. One ml of the homogenate was applied to the column and the resulting fractions were analyzed by SDS-PAGE to determine the distribution of Vn and other proteins. For a quantitative determination, the gel was scanned using an Ultroscan XL Enhanced Laser and analyzed with Gelscan@ software. This par- tially purified Vn was then used as a standard in ELISA assays to determine the amount of Vg in hemolymph samples (see below).

Production of polyclonal antibody

Antibodies specific for the female-limited protein, Vg, were produced by using female hemolymph as an anti- gen for polyclonal ascites fluid induction in BALB/c mice, followed by exhaustive absorption with male hemolymph. One injection of 0.3 ml of 2,6,10,14- tetramethylpentadecane (pristane, Sigma) was per- formed prior to the immunization schedule to enhance the production of immune ascites fluid (Lacy and Voss, 1986). The immunogen (hemolymph from mated female moths) was diluted in 0.1 M PBS, emulsified (1 : 1, v/v) with Freund’s complete adjuvant (Sigma), and injected (1OOpg of protein in 0.4 ml per mouse per injection) intraperitoneally (i.p.). The mice were boosted twice, 7 and 14 days after the first injection, with the antigen (same dose as above) in Freund’s incomplete adjuvant. To induce the production of ascites, Fox-NY cells were injected i.p. at a concentration of lo6 viable cells (0.5 ml) per mouse. The cells were first cultured (Yu et al., 1992) and the viable ones enumerated microscopically using a hemocytometer after staining with 0.5% trypan blue (Aldrich).

Ascites fluid was collected i.p. using a sterile 16-gauge hypodermic needle, clarified by centrifugation at 1OOOg for 5 min to remove debris and clots, and stored frozen. The proteins were precipitated using ammonium sulfate (Harlow and Lane, 1988). The antibody (IgG) was then

VITELLOGENIN REGULATION BY JH IN PSEUDALETIA 131

purified by protein A affinity chromatography (Goding, 1978) using the protocol described by the manufacturer (Bio-Rad, Affi-Gel Protein A MAPS II set). To separ- ate Vg-specific IgG from other antibodies, the protein A-purified antibodies were loaded onto a column containing CNBr-activated Sepharose4B (Bio-Rad) coupled to male hemolymph proteins. The Vg-specific IgG was eluted with PBS and collected, and the column was then washed with glycine buffer (0.1 M, pH 3.0) to remove bound unwanted antibodies. The PBS fractions were run through the column again and this procedure was repeated four times.

Characterization of antibody

IgG. Immunoreactive protein bands were visualized _

The antibody was characterized on Western blots as described by Yu et al. (1992). Female and male hemo- lymph proteins were separated by SDS-PAGE on a 12.5% gel and transferred to nitrocellulose membranes. Binding sites on the membranes were then blocked with 1% BSA in TBS (0.05 M Tris-HCl, 0.15 M NaCl, pH 7.5) and incubated with either purified or unpurified antibodies. After washing, the membranes were incu- bated with alkaline phosphatase-labeled goat anti-mouse Electrophoretic separation of hemolymph proteins

. from P. unipuncta males and vitellogenic females, under

subsequently checked for the presence of a sperma- tophore. Hemolymph samples were taken 0, 24 or 48 h after mating. For controls, hemolymph samples were drawn from virgin females of equivalent ages.

Ligations and hormone treatments

To determine the effects of JH homologs and analogs on Vg titers of female moths, newly-emerged females were neck-ligated and treated with a range of doses of JH I, JH II (Calbiochem), JH III (Sigma), methoprene (gift of Zoecon Corporation, Dallas, TX, U.S.A.), and pyriproxyfen (gift of Sumitomo Chemical Co., Osaka, Japan), prepared in acetone and applied topically to the ventral abdomen. All ligated females were maintained by daily injections of 100 ~1 of 5% sucrose. Hemolymph samples were taken 3 days (for dose-response determi- nations) and from 1 to 6 days (for time-course analysis) after treatment.

RESULTS

IdentiJication of Vg

using 5-bromo-4-chloro-3-indolyl phosphate and nitro- blue tetrazolium.

ELISA

Vitellogenin was quantified using an enzyme-linked immunosorbant assay (ELISA). Hemolymph samples (5 ~1 in 50 ~1 PTU-saturated PBS, frozen at -20°C until analysed) were diluted (1 : 27,000) in TBS, transferred to ELISA plates (Falcon 39 12, MicroTest III) (100 p l/well), and incubated for 2 h. Partially-purified Vn and TBS were used as standard and blank, respectively. Binding sites on the plate were blocked with 2% BSA in TBS (45 min incubation). After washing with TBS, 100 ~1 of the purified antibody (1 : 32 dilution in TBS con- taining 0.1% BSA) were added to each well and the plate was incubated overnight at 4°C. After washing the plate three times with TBS, 100~1 of biotinylated sheep anti-rabbit IgG were added to each well (1 : 500 dilution in TBS containing 0.1% BSA) and incubated for 1.5 h at room temperature. After three washes with TBS, 100 p 1 of strepavidin-labelled peroxidase were added to each well (1 : 2000 dilution in TBS containing 1% BSA) and incubated for 40 min. After five washes with TBS, the substrate (ABTS) was added. After 30 min incubation, the reaction was stopped with 0.02 M PBS containing 0.02% NaN,, pH 7.4, and A405nm was read.

bands (Fig. 1). Polypeptides with identical mobilities denaturing conditions, revealed two female-specific

were also present in an egg extract, but not in the hemolymph of pre-vitellogenic females (data not shown). The molecular weights of the two female-specific polypeptides were estimated to be 164 and 40 kDa on 12.5% SDS-PAGE. These values are similar to those of the large [apoVg I, sensu Imboden and Law, (1983)] and small (apoVg II) components of Vg reported for other species of moths (see Discussion). We conclude that they are the two apoproteins of Vg (hemolymph) and Vn (egg extract) in P. unipuncta.

Vg titers in virgin and mated females

Hemolymph samples were collected from virgin fe- males 0, 1,2, 3, 5 or 7 days after emergence. To examine the effect of mating, 20 5-day-old virgin females were each paired with a virgin male of the same age and allowed to mate during the scotophase; each female was

Western blotting of hemolymph proteins demon- strated the specificity of the purified antibody which recognized only one polypeptide in female hemolymph [Fig. 2(A)], whereas the unpurified one yielded many immunoreactive bands [Fig. 2(B)]. This polypeptide, which was not detected by the antibody in male hemo- lymph, had the same mobility as apoVg I, as determined by staining of a portion of the same gel used for blotting, indicating that the purified antibody was specific for apoVg I. Figure 3 shows the dilution-dependent immunoreactivity of the antibody using female hemo- lymph (native Vg protein), as well as the lack of immunoreactivity in male hemolymph and TBS blanks.

Fractionation of an egg extract by gel filtration chro- matography yielded a preparation of Vn estimated as 70% pure by SDS-PAGE and densitometric analysis of the stained polypeptide bands. This partially purified protein was used as a standard for the ELISA assays.

Vg titers in virgin and mated females

Vitellogenin titers, determined by ELISA, were low (0.1-0.3 mg/ml) in hemolymph of virgin females O-2 days following emergence, but then underwent a steady

132 MICHEL CUSSON et al.

1 2

FIGURE 1. Electrophoretic separation of hemolymph proteins from male (1) and female (2) P. unipuncra moths on a 12.5% SDS-PAGE gel. Numbers on the left show positions of molecular weight markers (kDa). Arrows on the right point to the two female-specific Vg

apoprotein bands: upper arrow, 164 kDa; lower arrow, 40 kDa.

increase, reaching values of about 2.4 mg/ml by day 7 (Fig. 4). In another experiment, the effect of mating on hemolymph Vg titers was examined (Fig. 5). After copulation, Vg titers were significantly higher at 24 h (Student’s t = 2.68, P = 0.031) and lower at 48 h (t = 4.67, P = 0.0012), than in similarly aged virgins.

Dose-response experiments

Neck-ligated females did not produce Vg unless treated with JH or JH analogs. Sigmoidal dose-response curves were obtained when hemolymph Vg was assayed 3 days after treatment of ligated females with the JH homologs I, II and III (Fig. 6), or the JH analogs methoprene (Fig. 7) and pyriproxyfen (Fig. 8). From

FIGURE 2. Western blot transfer from an SDS-PAGE gel following separation of proteins from female hemolymph. Strip A was incubated with the purified (1 : 10 dilution) Vg-specific antibody and strip B was incubated with the unpurified (1 : 5000 dilution) antibody. Arrow on

the left shows position of apoVg I.

these curves, the maximal response to the treatments and the ED,, were estimated (Table 1). The JH analogs evoked 50% of the maximal response at much lower doses than the natural JH, with pyriproxyfen being the most effective, followed by methoprene, JH I, JH II and JH III. Juvenile hormones I and II had similar levels of effectiveness and were approx. 10 times more active than JH III. However, the order of effectiveness for maximal level of response was more or less reversed, with

VITELLOGENIN REGULATION BY JH IN PSEUDALETIA 133

0.6

2 E 0.5

s 0.4

d d 0.3

0.2

10,000 100,000 1,000,000

Dilution of hemolymph

FIGURE 3. Sensitivity of Vg-specific antibody (as determined by

ELISA) to P. unipuncta female and male hemolymph. Blank is TBS only.

3r

2

l +.

\ l Female hemolymph

o Male hemolymph

‘\ A Blank

l \

‘\

%

1.

\ ‘\

0 1 2 3 4 5 6 7 8

Age (days)

FIGURE 4. Vitellogenin titer (mg/ml) in hemolymph of virgin P. unipuncta females held at 2S”C, 16 h light : 8 h dark, as a function of age.

Titers were determined by ELISA using partially purified P. unipunctu vitellin as a standard. Error bars: S.E.M., N = 5 for each age.

l

0

p virgins T

I I I

0 24 48

Time after mating (h)

FIGURE 5. Effect of mating on vitellogenin titers (mg/ml) in P. unipuncra females. On the day of mating (time 0) females were 5 days

old. Solid circles: virgin females; open circles: mated females. Titers

were determined as explained in Fig. 4. Error bars: S.E.M., N > 5 for

each time and each group.

20 -

z E

2 15- . JHI

. JH II .E z

b JH III

z = 0 10 - .Z >

c

2 x 5-

z E

:

0 L l/T/ 11 1 I I

-3 -2 -1 0 1 2 3

Juvenile hormone applied (log,, vg)

FIGURE 6. Effect of increasing doses of JH I, JH II, and JH III on

vitellogenin titers of neck-ligated P. unipuncta females. Experimental

moths were ligated and treated topically with the hormones immedi-

ately after emergence. Vitellogenin titer determinations were made 3

days after treatment. Titers were determined as explained in Fig. 4.

Error bars: S.E.M., N = 6 for each dose.

15 r

0 I I I I I I -4 -3 -2 -1 0 1 2

Methoprene applied (log,,, pg)

FIGURE 7. Effect of increasing doses of methoprene on vitellogenin

titers of neck-ligated P. unipuncta females. See Fig. 6 for details.

OL -6 -5 -4 -3 -2 -1 0 1 2

Pyriproxyfen applied (log,, pg)

FIGURE 8. Effect of increasing doses of pyriproxyfen on vitellogenin

titers of neck-ligated P. unipuncta females. See Fig. 6 for details.

134 MICHEL CUSSON et al.

TABLE 1 Doses of JH, methoprene, and pyriproxyfen necessary to

invoke 50% of the Vg level at maximal response (ED,) 72 h after

topical application to neck-ligated P. unipunctu females

Hormone or analog ED,, Maximal response

oig) Cvg, mg/ml hemolymph)

JH III 13.0 16.0

JH II 1.8 12.0

JH I 1.0 11.6

Methoprene 0.04 12.0

Pyriproxyfen 0.003 6.5

pyriproxyfen giving a hemolymph Vg level less than half that of JH III.

Time -course analysis

The accumulation of Vg over time was examined by treating neck-ligated females with pyriproxyfen at a dose of 0.1 ,ug/moth. Vitellogenin became detectable in the hemolymph l-2 days after treatment and attained near-maximal titers by day 3 (Fig. 9).

DISCUSSION

The apoprotein composition of the Vg of P. unipuncta is consistent with that reported for other lepidopteran species, i.e. a large (15&200 kDa) and a small [4&65 kDa (Raikhel and Dhadialla, 1992)] apoprotein. In terms of apoprotein molecular weights, the Vg of P. unipuncta (If3 and 40 kDa) shows greater resem- blance to that of the related noctuid H. zea [165 and 45 kDa (Satyanarayana et al., 1992)] than to those of more distantly related species (M. sexta, a sphingid [177 kDa and 45 kDa (Osir et al., 1986)], and Hyalophora cecropia, a saturniid [180 kDa and 47 kDa (Harnish and White 198211). It would be of value to determine whether these differences in size reflect degrees of immunological similarity and evolutionary distance, as they may provide another means of measuring relat- edness. Certainly, the presence of only two Vg apo- proteins in the Lepidoptera differs from the variable and

9r

Time after treatment (days)

FIGURE 9. Time-course analysis of vitellogenin production in neck-

ligated females in response to topical application of pyriproxyfen

(0.1 pg) on the day of emergence. Titers were determined as explained

in Fig. 4. Error bars: S.E.M., N = 6 for each sampling time.

sometimes complex picture seen in other insect orders (Harnish and White, 1982; Kunkel and Nordin, 1985).

As previously shown for M. sexta (Osir et al., 1986), only the large apoprotein of the Vg of P. unipuncta was recognized by the antibody raised against the native protein. This indicates that the two apoproteins are immunologically distinct and that apoVg II is likely positioned inside the native molecule.

Vitellogenin titers in virgin females (Fig. 4) increased, from the time of emergence, in a fashion similar to that reported for the in vitro rates of JH biosynthesis (see Cusson et al., 1990, 1993). The first major increase in the Vg titer was seen between days 2 and 3, matching the elevation in the biosynthetic activity of the corpora allata during the same period [for females originating in the same colony used for the present experiments (Cusson et al., 1993)]. Thereafter, both Vg titer and corpus allatum activity increased steadily until the last sampling day (day 7). A correlation is also seen between Vg titer and oiicyte growth (see Cusson and McNeil, 1989b; Cusson et al., 1990), although the oiicytes began increasing in size immediately after emergence whereas Vg titers remained low for the first 2 days (Fig. 4) suggesting that the initial oiicyte growth does not involve vitellogenesis, or, possibly, that Vg is taken up at the same rate as it is synthesized. Determinations of follicle patency or oocyte Vn content could clarify this question.

Mating resulted in a significant increase in the Vg titer, 24 h post-copulation, above that seen in virgin females of the same age (Fig. 5). At the time they are ready to mate, females have normally accumulated ca 300 mature eggs in their ovaries (Cusson and McNeil, 1989b), and on the first and second nights following successful mating in our experiments, they laid an average of 4 16 and 409 eggs, respectively. This represents more than 50% of the total number of eggs produced during a l&l5 day oviposition period (McNeil, unpublished). Such rapid maturation of eggs would require a sharp increase in Vg production similar to that measured here in the O-24 h post-mating interval. The fact that by 48 h after mating, Vg titers were significantly lower than in virgin females of the same age is likely the result of Vg uptake into the ca 500 eggs produced during this period. Given the dependency of Vg synthesis on JH in this species, the post-mating increase in Vg production suggests a parallel rise in the JH titer, a possibility that remains to be verified. Mating in another noctuid, Trichloplusia ni, results in a decline in the activity of the JH-specific esterase, thereby favoring an increase in the titer of JH (Venkatesh et al., 1988). Work is currently under way to determine whether mating induces changes in JH biosynthesis and/or degradation in P. unipuncta.

The Vg titer found in hemolymph 3 days after admin- istration of JH or analogs to neck-ligated females has been used as an assay of their relative activities. In Locusta migratoria, even though both Vg synthesis in the fat body and uptake into the oijcytes are JH-dependent, assay of hemolymph Vg has been found to provide a simple method giving the same ED,, for a JH analog

VITELLOGENIN REGULATION BY JH IN PSEUDALETZA 135

as was obtained for measurements of Vg synthesis in isolated fat body (Edwards et al., 1993). Our results for P. unipuncta show that Vg production is dependent on the presence of JH or an analog (Figs 6-8), as suggested by a previous study on the effects of allatectomy and JH replacement therapy on ovarian growth (Cusson and McNeil 1989a). The greater effectiveness of JH I and JH II, relative to JH III, in promoting Vg accumulation is not surprising given that the higher homologs are much more effective than JH III in inducing ovarian growth in three other lepidopteran species (Lessman et al., 1982; Nijhout and Riddiford, 1974; Ramaswamy and Cohen, 1991). Furthermore, this pattern of effectiveness matches the in vitro production of JH by CA of P. unipuncta, with more JH I and JH II being produced than JH III (Cusson et al., 1993). The lower effectiveness of JH III could result from more rapid enzymatic degradation or lower affinity for the JH receptor. However, JH I and JH III are hydrolyzed at equal rates in M. sexta hemolymph (Peter, 1981) and it appears likely that the observed differences in effectiveness reflect, at least in part, differential action at the receptor level. To our knowledge, our work is the first to demonstrate a difference among the three major JH homologs with respect to their effectiveness in triggering a specific biochemical process,

Treatment with methoprene and pyriproxyfen resulted in the induction of Vg production at much lower doses than with JH itself, probably as a result of their great metabolic stability. Methoprene, although structurally similar to JH, resists degradation by JH esterase through substitution of the methyl ester with an isopropyl ester (Hammock and Quistad, 1976). Pyriproxyfen, on the other hand, shows little chemical similarity to JH and bears no functional ester grouping. The relative affinities of these compounds for the JH receptor, which must also be important, are at present unknown.

The compound with the lowest ED,,, pyriproxyfen, elicited a lower maximal response than methoprene (Table 1). There is evidence from experiments with L. migratoria that pyriproxyfen at high doses may act as an antagonist, as well as an inducer, of Vg synthesis (Edwards et al., 1993), which could result in a reduced maximal response. Pyriproxyfen might also have a rela- tively greater effect on Vg uptake into the obcytes, because of differential affinity for receptors in the fat body and ovarian follicle cells or longer persistence in the hemolymph. The lower Vg titers observed in hemolymph of intact moths, compared with JH/JH analog treated ligated females, may also be due to the sustained JH production by the corpora allata. The relative effects of JH and JH analogs on the distinct processes of fat body Vg synthesis and ovarian Vg uptake need to be studied in a single species,

After topical application of pyriproxyfen, there was a delay of approx. 1 day before the appearance of Vg in the hemolymph was detected (Fig. 9). A similar lag was observed in L. migratoria following application of methoprene or pyriproxyfen (Chinzei and Wyatt, 1985;

Edwards et al., 1993), and has been shown to represent the time needed for Vg mRNA accumulation (Dhadialla et al., 1987). In P. unipuncta, maximal Vg titers were reached 3 days after treatment with pyriproxyfen, indi- cating that the interval chosen was appropriate for our dose-response assays.

In conclusion, hormonal regulation of Vg production in P. unipuncta fits the “short-lived vs long-lived” classification of Lepidoptera, as armyworm moths meet both requirements for JH-regulated ovarian growth: they can live up to 15 days as reproductive individuals and they need to feed as adults. With P. unipuncta, a species in which the physiological and behavioral aspects of reproduction have already been established (Cusson and McNeil, 1989a, b; Cusson et al., 1990, 1993; Delisle and McNeil, 1986, 1987a, b; Dumont and McNeil, 1992; Fitzpatrick and McNeil, 1988, 1989; Fitzpatrick et al., 1988, 1989) we have now characterized the induction of a specific biochemical process (Vg synthesis) by defined compounds, thus providing a basis for studies on the hormonal regulation of reproduction at the molecular level.

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Acknowledgements-We wish to thank D. Trudel for her assistance in the preparation of the figures. Funding for this research has been provided, in part, by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Fonds F.C.A.R., and Insect Biotech Canada, one of the 15 Networks of Centres of Excellence supported by the Government of Canada.