Effects of a juvenile hormone analogue on the duration of the fifth instar in the milkweed bug Oncopeltus fasciatus

0022-1910’X1:‘030169-05%0’.00~0 ‘4‘ 19x I Per,@nlorl Pi?,.\\ L/d.

EFFECTS OF A JUVENILE HORMONE ON THE DURATION OF THE FIFTH THE MILKWEED BUG ONCOPELTUS

ANALOGUE INSTAR IN FASCZATUS

WENDY A. SMITH and H. FREDERIK NIJHOUT Department of Zoology,

Duke University.

Durham.

NC 27706. U.S.A.

(Received’ 14 July 1980; revised 10 October 1980) Abstract--Topical application of JHA to fifth instar nymphs of Oncopeltus,fasciutus, immediately following ecdysis from the fourth instar. decreases the duration of the tifth instar by approximately 36 hr in addition to inducing a supernumerary larval moult. JHA appears to accelerate the time of subsequent ecdysis in two ways: first. the onset of ecdysone secretion is accelerated, and is accompanied by a similarly premature initiation of mitotic activity in epidermal cells. This is the classical prothoracicotropic action of JH. Second. the period between the onset of mitotic activity and the time of ecdysis itself is shortened. That is, once cellular activities associated with the moulting cycle are triggered by ecdysone, such activities are completed more rapidly in the presence of JHA. It appears that the larval-larval moult induced by JHA requires intrinsically less time to accomplish than a normal metamorphic moult. Kc,~, Word 1nde.r: Juvenile hormone analogue. On~opelfua fu.uciatu.~. prothoracicotropic effect of JH. ecdysteriod titres

INTRODUCTION THE MOULTING cycle

of insects is provoked when prothoracicotropic hormone (PTTH) from the brain stimulates the prothoracic glands to secrete the moulting hormone, ecdysone. Under certain circumstances, however, juvenile hormone can mimic the ecdysiotropic action of PTTH. This effect was first noted by WILLIAMS (1959) who observed that implanted corpora allata of adult cecropia moths caused brainless pupae to initiate development. GILBERT and SCHNEIDERMAN (1959) subsequently found that partially purified extracts of male cecropia abdomens. containing large amounts of juvenile hormone. could stimulate Ihc development ot brainless saturniid pupae. KRISHNAKUMARAN and SCHNEIDERMAN(1965) later discovered that numerous mimics of juvenile hormone could also activate the prothoracic glands of brainless Cynthia. cecropia and polyphemus pupae. Recently, HIRUMA rt al. (1978) reported that a juvenile hormone mimic could activate the prothoracic pland~ of larval Mun~e.stru hra.s.sic~nr late in the last instar, as well as the prothoracic glands of pupae. The ability of juvenile hormone to activate the prothoracic glands of mature larvae was further contirmcd by Cl MI~OKO~SI
preparing for a supernumerary larval moult. the period between the onset of mitosis and the beginning of new cuticle deposition was considerably shorter than in nymphs undergoing metamorphosis (WI(;C;LESWOKT~I. 1940). that. when triggered in

Wigglesworth

concluded

the prcscnce of _i,uvenile hormone. epidermal changes associated wllh the moulting cycle were completed more rapidly. While Wigglesworth’s experiments did not rule out a possible prothoracicotropic effect of juvenile hormone in hemipteran insects, his alternate explanation for the moult-accelerating effect of juvenile hormone does raise the following question: does juvenile hormone act solely as a prothoracicotropic agent, or, once ecdysone is secreted. can the hormone actually influence the duration of ensuing developmental events? In the present paper we examine this question using the hemipteran insect. Oncopeltus ,fasciatus.

.sesta.

The prothoracicotropic effect of juvenile hormone has been clearly demonstrated only in lepidopteran insects. Juvenile hormone can also accelerate the moulting process in Hemiptera, an effect first noted by WIGGLESWORTH (1934, 1936) who observed that tifth instar nymphs of Rhodnius prolixus took less lime 11)undergo a supernumerary larval moult in the prehence ofjuvenile hormone than they did to moult to an ;idul[. He later found that in tifth instar nymphs 169

MATERIALS

AND METHODS

Insects Oncopeltus fasciatus were reared at 27°C under a 16~:80 photoperiod. Water and field-collected milkweed seeds (Asclepias q’riaca) were supplied ad libitum. For experimental work. freshly-ecdysed (pale) fifth instar nymphs were collected at approx. 2 hr intervals throughout the photoperiod and maintained in glass jars in groups of 5-10. Fresh water and seeds were supplied in each jar. Instar lengths were measured by examining treated nymphs at 12-hr intervals and noting the number of animals that had ecdysed during each interval.

WENDV A. SMITH AND H. FREDER~K NIJHOUT

170

Application

mixture of 2% antiecdysone, 24% control rabbit serum and 74% borate buffer, and (2) the incubation period was reduced to 20 min at 37°C. followed by 1 hr at 4°C. All other aspects of the assay were identical to those reported by BORST and O’CONNOR (1972).

of juvenile hormone analogue (JHA)

The juvenile hormone analogue ZR-515 (Zoecon Corp., Palo Alto, CA, U.S.A.) was used throughout this study. JHA was dissolved in acetone and diluted to yield a range of concentrations between 0.1 and 10.0 pg/pl. One ~1 aliquots of the appropriate dose of JHA were then applied to the prothorax of newlyecdysed fifth instar nymphs. Control animals received 1 ~1 of acetone alone, or no treatment, as specified below.

Mitotic

indices

Samples of dorsal epidermis from the first 3 abdominal segments were fixed in Carnoy’s for l-3 hr and stained in haematoxylin and nuclear fast red. The tissues were then dehydrated in ethanol, cleared in xylene and mounted on microscope slides with Permount (Fisher Scientific, Pittsburgh, PA, U.S.A.). Mitotic bodies were counted in 4 separate regions of each epidermal sample, including one intersegmental region per sample. Each region comprised approx. 1500 cells. Five replicate samples were assayed for each reported time period.

Radioimmunoassay Haemolymph ecdysteroid levels were determined with the use of radioimmunoassay. Samples were collected by removing the legs and antennae from nymphs and gently aspirating the exuded haemolymph. We found that a minimum of 10 ~1 of haemolymph were required for an accurate determination of ecdysteroid concentrations which necessitated the pooling of haemolymph from 5 to 10 animals. Each 10 ~1 sample was extracted with 200 ~1 methanol, and the extract was dried under nitrogen. Dried samples were stored at -20°C until the time of assay. Ecdysone antiserum was a generous gift of Dr. John D. O’Connor (University of California, Los Angeles, U.S.A.). Tritiated 20hydroxyecdysone (2.2 Ci/mmol) was obtained from the New England Nuclear Corporation (Boston, MA, U.S.A). Our assay procedure was based on that described by BORST and O’CONNOR (1972), with the following modifications suggested by KUNKEL (1977): (1) the concentration of antiserum in the incubation medium was increased. resulting in a final incubation

RESULTS Effects of JHA on duration of the fifth

instar

The median duration of the fifth instar in control nymphs treated with acetone immediately upon ecdysis from fourth to fifth nymph was 174 hr. range = 156-192 hr (Fig. 1). Such animals emerged as normal adults, with well-developed wings and genitalia. Because acetone had no effect upon the normal duration of the fifth instar (median duration at 27C = 175 hr range = 156-192), nor upon the morphology of emergent adults, control animals in subsequent experiments received no acetone

IOC

I2 2 e

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LEGEND A lO.OJlg A 6.Oug 0 3.olq 0 i.oAJg n 0.1 JJg cl control --

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r

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1

108

120

132

144

156

168

180

192

TIME

IN HOURS

Fig. 1. Effects of JHA on duration of the fifth instar in Oncopelcus fasciatus under our laboratory conditions. JHA was topically applied to fifth instar nymphs immediately following ecdysis from the fourth instar.A~~l0.0~g,n=9;~~~6.0~g.n=32:o--o3.0~g.~~=l8:0-o1.O~g,n=16:~--H 0.1 pg. n= 17: U--U control (acetone), n= 33. All groups were examined at 12 hr intervals following treatment (abscissa), and the cumulative percentage of nymphs that had moulted during each interval was noted (ordinate). A Mann-Whitney U-test (SOKALand ROHLF,1969) was used to analyze differences in the duration of the fifth instar between groups. The following groups differed significantly: 1.O-10.0 pg JHA vs control, p < 0.001: 1.0-10.0 pg JHA vs 0.1 pg JHA, p < 0.01; 0. I pg JHA vs control. p < 0.01.

JH effects in Oncoprltu.,

1

0

r

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I

24

48

72

96

TIME

IN

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1

1

120

144

168

HOURS

Fig. 2. Effects of JHA on ecdysteroid titres (expressed in 20-hydroxyecdysone) during Onto~~~/tu.rfa.v~~~~1~,~.~~~6.O~~~JHA~~~~control(notreatme~~~.Eachpointrepresen~sthemeanofl0 samples; bars indicate standard deviations.

treatment. Animals that received 0.1 hlg JHA emerged significantly earlier than control nymphs. the median duration of the instar being 160 hr. range = 132-168 (Fig. 1). Such animals developed into nymphal-adult intermediates, with foreshortened wings and underdeveloped genitalia. Following application of 1.0 pg JHA. the duration of the instar ( I35 hr. range = 120-168) was significantly shorter than that noted for the 0.1 p(g JHA and acetonetreated groups. Of the animals that received a dose of 1.O pg JHA, 88”,/, emerged as supernumerary nymphs. retaining thick wing pads and nymphal genitalia. The remaining 12% were predominantly nymphal in character. Applications of doses larger than 1.0 pg JHA had no further significant effect upon the median time of ecdysis, nor upon the morphology of the nymphs at emergence. However, the 6.0 and 10.0 pg doses of JHA appeared to induce a more uniform response, with fewer late-emerging animals (Fig. 1) and a higher percentage of supernumerary nymphs ( >95”/,). To reduce variability among JHA-treated animals in subsequent experiments, we chose to use a 6.0 pg dose of JHA.

Accerated ecdysis following treatment with JHA may be due to a premature stimulation of ecdysone a more rapid completion of cellular secretion. activities provoked by ecdysone, or by an acceleration of both processes. We next examined the effects of JHA on the appearance of ecdysteroids in the haemolymph during the fifth instar. In untreated nymphs maintained at 27°C ecdysteroid levels began to rise gradually between 48 and 72 hr of the fifth instar, and then rose sharply until 108 hr, reaching near-basal levels just prior to the time of ecdysis to an adult (168 hr). By contrast, in nymphs exposed to

the tifth instar

oi

6.0 bg JHA immediately after ecdysis to the fifth instar. ecdysteroid levels began to rise approx. 24 hr earlier and reached peak levels at 84 hr (Fig. 2). Ecdysteroid titres in JHA-treated animals also declined earlier, such that by 120 hr, haemolymph concentrations were similar to those in 168 hr control nymphs. Effects ofJHA on epidermat mirosis and the duration of the moulting process The foregoing results demonstrated that treatment with JHA during the first day of the fifth instar accelerated the onset of ecdysteroid secretion. This would imply that the initiation of cellular changes associated with the moulting process was also accelerated. We chose initiation of mitotic activity in the abdominal epidermis as an objective measure of the onset of moulting in fifth instar nymphs. In untreated nymphs, a rapid increase in mitotic activity began during the second day of the fifth instar (Fig. 3). This was correlated with the initial increase in ecdysteroid levels in the haemolymph (Fig. 2). Mitotic activity in untreated nymphs remained elevated for about 36 hr, and then declined to undetectable levels by 108 hr. In nymphs treated with 6.0 pg JHA, the onset of mitosis was accelerated by about 12 hr as compared to untreated control animals, corresponding to an early increase in haemolymph ecdysteroids. In contrast to control nymphs, mitotic activity remained elevated for only 24 hr declining to undetectable levels by 84 hr. Finally, the period between the onset of mitosis and emergence was reduced in JHA-treated nymphs. In control animals this period lasted from 60 to 174 hr, or a total of 114 hr. By contrast, in JHA-treated nymphs, this period lasted from 48 to 135 hr, or only 87 hr. Thus an acceleration in the onset of ecdysone

WENDYA.

172

SMITH AND

H. FREDERIKNIJHOUT

I

36

48

60

72

84

96

108

TIME IN HOURS Fig. 3. Effects of JHA on mitotic activity during the fifth instar of Oncopeltusfusciurus. O---O 6.0 pg JHA; -0 control (no treatment). Each point represents the mean mitotic index ofdorsal abdominal epidermis from 5 individuals:

bars indicate

secretion, and the closely associated initiation of epidermal mitosis, was not sufficient to account for the entire shortening of the moult cycle in JHA-treated nymphs. Rather, both the onset and duration of the moult cycle were modified by treatment with JHA. DISCUSSION When the juvenile hormone analogue ZR-5 15 was applied to Oncopeltus fusciatus nymphs immediately following ecdysis from fourth to fifth instar, the remainder of the instar was reduced by approx. 36 hr (21%). Our results indicate that JHA accelerated the development of fifth instar nymphs in two ways. First, the hormone accelerated the initiation of moultrelated changes in the epidermis by approx. 12 hr (7%) as judged by the onset of mitotic activity. Second. the period between the onset of mitosis and ecdysis itself was shortened by about 1 day (14%). That is, cellular activities associated with the moult cycle were initiated sooner, and, once initiated, were completed more rapidly as a result of exposure to JHA. The overall pattern of changes in haemolymph ecdysteroids was similar in JHA-treated and control nymphs-a gradual rise in ecdysteroid titres, followed by a rapid increase to a single peak concentration, and a subsequent decline to near-basal levels at the time of ecdysis. This pattern of ecdysteroid secretion was similar to that normally observed in the last nymphal instar of Schistocerca gregaria (WILSON and MORGAN, 1978) and Blatta orientalis (KUNKEL, 1977). No evidence was found for multiple peaks of ecdysteroids, such as those seen in the final larval instar of holometabolous insects, e.g. Man&z sexta (BOLLENBACHER et al., 1975), or in some hemimetabolous insects, e.g. Locusta migratoria

standard

deviations.

(HIRN et al., 1979) and Panstrongylus megistus (FURTADO et al.. 1976). Samples of blood in our assays were, of necessity, obtained from groups of 5-10 animals. This fact, combined with the asynchronous nature of development in Oncopeltus nymphs would make small, transient peaks of ecdysteroids extremely difficult to detect. Ecdysteroid levels in JHA-treated nymphs did rise approx. 24 hr earlier than those in control nymphs. The accelerated onset of mitotic activity in JHAtreated animals reflected this early increase in haemolymph ecdysteroids. It is of interest to note that in both JHA and untreated animals, mitotic activity was stimulated by very low levels of ecdysteroids, thus, the onset of mitotic activity appears to be a very sensitive correlate of ecdysone secretion. This study was not designed to test the site of action of JHA in accelerating the onset of ecdysone secretion of fifth instar nymphs of Oncopeltus. However, in light of previous studies (GILBERT and SCHNEIDERMAN,1959: KRISHNAKUMARANand SCHNEIDERMAN,1965; HIRUMA et al., 1978; CYMBOROWSKI and STOLARZ, 1979; SAFRANEKet al., 1980), it is likely that the hormone in some manner activates the prothoracic glands, as opposed to stimulating the secretion of PTTH from the brain. The second effect of JHA, acceleration of the developmental sequence itself, remains completely unexplained at this time. WIGGLESWORTH (1936) suggested that JH accelerates the onset of cuticule deposition in Rhodnius. He further hypothesized that an early initiation of cuticule deposition could play a causative role in the inhibition of metamorphosis by fixing the morphology of the cuticule before the expression of adult genes could take place. However, as demonstrated by LAWRENCE

JH effects in Oncop~/rus

(1969), maintenance of larval characters is not dependent upon an accelerated deposition of cuticle. Lawrence administered small doses of JH to fifth instar Oncopeltus and found that while these did not alter the duration of the instar, they did result in a mosaic cuticle in which patches of juvenile cuticle were retained. Conversely, WILLIS and HOLLOWELL ( 1976) found that the moult cycle of Oncopeltus could be accelerated significantly by ecdysone with minimal effects on the morphology of the resultant adult. We think that the most reasonable explanation for the observed acceleration of the developmental sequence is that the ‘larval programme’ triggered by ecdysone in the presence of JH is completed more rapidly than a programme involving the more extensive changes resulting in metamorphosis from nymph to adult. According to this hypothesis, by determining the type of programme to be completed during a given instar, JH would influence both the ~BILII’C and duration ofensuing developmental events.

Acknowledgements-We thank Dr. J. H. WILLIS for her generous contribution of A. syriaca seeds; and Dr. M. M. NIJHOU~ for a critical reading of the manuscript. This work was supported, in part, by NSF Grants Nos. I%M76-80518 and PCM79-11779 to HFN, and by a Cocos Foundation Training Grant in Morphology to WAS.

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(Heteroptera:Reduviidae). Cr. hehd. SPanc. Awd. Sri.. Paris 283, 1077-1080. GILBERT L. 1. and SCHNEIDERMANH. A. (1959)Prothoracic gland stimulation by juvenile hormone extracts of insects. Nature, Lond. 184, 171-173. HIRN M., HETRU C., LAGUEUX M. and HOFFMAN J. A. (1979) Prothoracic gland activity and blood titres of ecdysone and ecdysterone during the last larval instar of Locu.yta migratoria L. J. Insect Physiol. 25, 255-261. HIRUMA K., SHIMADA H. and YAGI S. (1978) Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mumestra bra.rsicar. J. Insect Physiol. 24. 215-220. KRISHNAKUMARAN A. and SCHNEIDERMAN H. A. (1965) Prothoracotrophic activity of compounds that mimic juvenile hormone. J. Insect Phvsio(. 11, 1517-1532. KUNKEL J. G. (1977) Cockroach moulting: II. The nature of regeneration-induced delay of moulting hormone secretion. Biol. Bull., Mar. hiol. Lah. Woody Hole 153, 145-162. LAWRENCE P. A. (1969) Cellular differentation and pattern formation during metamorphosis of the milkweed bug Oncopeltus. Devel. Biol. 19, 12-40. SAFRANEK L., CYMBOROWSKI B. and WILLIAMS C. M. (1980) Effects ofjuvenile hormone on ecdysone-dependent development in the tobacco hornworm. Munduca .se.yfu. Biol. Bull. Mar. biol. Lab., Woods Hole 158, 248-256. SOKAL R. R. and ROHLF F. J. (1969) Biomerry. W. H. Freeman, San Francisco, U.S.A. WIGGLESWORTH V. B. (1934) The physiology of ecdysis in Rhodnius prolixus (Hemiptera) 11. Factors controlling moulting and metamorphosis. JI. micros<. ‘Ci. 77, 191-222. WIGGLESWORTH V. B. (1936) The function of the corpus allatum in the growth and reproduction of Rhodnius prolixus (Hemiptera). Q. JI. micros<,. Sci. 79, 9 l-l 21. WIGGLESWORTH V. B. (1940) The determination of characters at metamorphosis in Rhodniw proliuu., (Hemiptera). J. exp. Biol. 17, 201-222. WILLIAMS C. M. (1959) The juvenile hormone. I. Endocrine activity of the corpora allata of the adult Cecropia silkworm. Biol. Bull. Mar. hiol. Lab.. Wood,v Hole J 16, 323 -338. WILLIS J. H. and HOLLOWELL M. P. (1976) The interaction of juvenile hormone and ecdysone: Antagonistic, synergistic, or permissive? In: The Juvenile Hormones (Ed. by GILBERT L. I.) pp. 270-287. Plenum Press. New York. WILSON 1. D. and MORGAN E. D. (1978) Variations in ecdysteroid levels in 5th instar larvae of Schislocrrca gregario in gregarious and solitary phases. J. In.sect Physiol. 24, 751-756.