Juvenile hormone and moulting hormone titres in diapause- and non-diapause destined flesh flies

.I.InsectPhysiol.. Vol.26,pp. 661 to 664. 'c;Prrgumon Press Ltd. 1980. Printed in Great Britain.

0022-1910180j10014661 $02.00/O

JUVENILE HORMONE AND MOULTING HORMONE TITRES IN DIAPAUSE- AND NON-DIAPAUSE DESTINED FLESH FLIES GREGORY P. WALKERand DAVID L. DENLINGER

Department of Entomology, Ohio State University. Columbus, OH 43210, U.S.A. (Received 27 February 1980; revised 28 April 1980) Abstract-Failure of the brain to stimulate the prothoracic gland to release ecdysone has been widely regarded as the basis for diapause in insect pupae. In diapause-destined flesh flies, the absence of a peak of moulting hormone around the time of pupal head eversion supports this contention, but in addition, major pulses of juvenile hormone (JH) activity with a rhythmicity of 24 hr are unique to flesh flies destined for pupal diapause. JH activity persists during diapause, and a pulse of JH precedes the rise of moulting hormone that initiates adult development. Key Word Index: Juvenile hormone, moulting hormone, ecdysone, diapause. Sarcophaga crassipalpis

INTRODUCTION

MANY insects overwinter in pupal diapause, thus delaying the onset of adult development for several months. WILLIAMS’ (1946, 1947, 1952) classic experiments with silkmoth pupae clearly demonstrated the central role of the brain in presiding over the developmental decision for diapause. By failing to stimulate the prothoracic gland to secrete the moulting hormone ecdysone, the brain was shown to be responsible for the suspension of development. Experiments with flesh flies support this contention: pupal diapause can readily be broken with injections of ecdysteroids (FRAENKELand HSIAO, 1968b; GIBBS, 1976; %AREK and DENLINGER,1975), and the peak of moulting hormone (MH) activity that initiates adult development (SHAAYAand KARLSON,1965) is absent in flies entering pupal diapause (OHTAKI and TAKAHASI,1972). In several species that diapause either as larvae (CHIPPENDALE,1977; YAGI and FUKAYA, 1974) or adults (CASE et al., 1977; DE WILDE et al., 1968; SCH~ONEVELD et al., 1977) juvenile hormone (JH) also plays an important regulatory role, but JH is not known to be involved in pupal diapause. Yet, several interesting observations suggest a potential role for

shortens the duration of diapause (DENLINGER.1980, %AREK and DENLINGER,1975) and synergistically enhances the effect of 20-hydroxyecdysone in terminating diapause @UREK and DENLINGER,1975; DENLINGER,1979). Such effects may be experimental artifacts having no basis in the normal regulatory scheme (BRADHELDand DENLINGER, 1980), but a viable role for JH in pupal diapause of flesh flies cannot be discounted. To explore this possibility, we have monitored JH and’MH titres in diapause and non-diapause destined flesh flies approaching the pupal stage, during diapause, and at the onset of adult development.

MATERIALS AND METHODS Experimental animals

Sarcophaga crassipalpis Macquart was reared as described by DENLINGER(1972) To obtain a high incidence of pupal diapause, adults were reared at 25 rt_1°C 12L:12D (1ight:dark) and larvae and pupae were reared at 20 + 0.5”C, 12L:12D. Non-diapausing flies were obtained by rearing all stages at 25 _t 1°C 15L:9D. JH. The titre of JH in larvae of Mamestra brassicae Newly formed puparia were collected at 2 hr (YAGI. 1976) and Man&-a sexta (DENLINGER, intervals, aged for various periods, frozen and stored BRADFIELD,SAFRANEKand WILLIAMS,unpublished at -20°C until extracted. To stimulate large numbers observations) destined for pupal diapause is higher of flies to break diapause synchronously, pupae in than in their counterparts not entering diapause. Some diapause 20 + 3 days received a topical application of histological evidence suggests that the corpora allata 6 ~1 hexane (DENLINGERet al., 1980). Hexane-treated are active during pupal diapause (HIGHNAM, 1958). pupae were likewise aged for various intervals before When applied to very young third instar flesh fly being frozen and stored at -20°C. larvae, high doses of JH can prevent induction of pupal diapause (DENLINGER,1976). Pupae of several Extraction Lepidoptera break diapause in response to JH The procedure of HSIAOand HSIAO(1977) was used (BRADFIELDand DENLINGER, 1980; GILBERT and SCHNEIDERMAN. 1959; HIRUMA et al., 1978; to extract JH and MH simultaneously from whole KRISHNAKUMARAN and SCHNEIDERMAN, 1965; body homogenates. Each extraction was based on a WILLIAMS,1959) and in diapausing flesh fly pupae, JH sample weighing 21 g (179 k 5 pupae, mean + S.D.). 661

GREGORY P. WALKER AND

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Bioassays

MH activity was determined using the Musca bioassay procedure described by KAPLANIS et al. (1966). Most MH extracts were diluted with 0.4 ml distilled water, but highly viscous extracts weighing over 70 mg (about 10% of samples) were diluted with 0.5 ml. For each extract, 20 test abdomens were injected with 3 ~1 sample using an electronic microapplicator. The response was converted to microgram 20-hydroxyecdysone equivalents by comparison to a 20-hydroxyecdysone (Simes, Milan) standard. The detection limit of this bioassay is 0.001 pg 20-hydroxyecdysone equivalents. The Galleria wax wound bioassay (DE WILDEet al., 1968, DE LOOFand VANDE VEIRE, 1972) was used to determine JH activity. The greater wax moth, Galleria mellonella, was reared at 31 “C using a larval diet composed of 8 oz Gerbers baby cereal, 200 ml honey, and 100 ml glycerine. Peanut oil, by itself, showed no JH activity and was used for preparing serial dilutions of the JH extract. Each bioassay score was based on the response of 10 pupae and expressed in Galleria units (GU) per gram fresh weight. One GU corresponds to 5 pg JHI. RESULTS Titre of moulting hormone

Following puparium formation the cyclorrhaphous flies progress rapidly through several developmental stages: larval-pupal apolysis leads to the stage of the cryptocephalic pupa, a stage in which the pupal head remains inverted. Muscular contractions then force the pupal head to evert and mark the beginning of the phanerocephalic stage. As shown by FRAENKELand HSIAO(1968a), the onset of pupal diapause coincides with eversion of the pupal head (indicated by arrow in Fig. 1). The MH titre begins to rise shortly before pupariation (SHAAYAand KARLSON, 1965; OHTAKI and TAKAHASHI,1972; HODGETTSet al., 1977) and reaches a maximum of 0.1 pg 20-hydroxyecdysone equivalents/g both in flies destined for continuous

DAVID L. DENLINGER

development (Fig. 1A) and flies destined for pupal diapause (Fig. 1B). In both groups of flies, the MH titre then drops, but in flies developing immediately into adults a second peak of MH appears around the time of head eversion. In diapause destined flies, the second peak is missing and the MH level remains at an undetectable level throughout diapause. Using hexane as a tool for breaking diapause, we find the MH level rises within 9 hr after hexane application and remains elevated for several days (Fig. 1B). Titre of juvenile hormone

In both non-diapausing (Fig. 1A) and diapausing (Fig. 1B) flies, the peak of MH responsible for initiating adult development is directly preceded by a brief pulse of JH. But, there is a striking difference observed immediately after pupariation in the JH titre of diapause and non-diapause destined flies. In flies programmed for continuous development, JH activity remains consistently low except for the pulse of activity preceding the second peak of MH. By contrast, flies programmed for pupal diapause show regular pulses of JH activity at 24 hr intervals after pupariation. Between peaks of the cycle JH activity is undetectable, in contrast to the low, but consistently detectable activity observed in non-diapause destined flies. Since each extraction group consisted of individuals pupariating at different times during the sidereal day, the observed rhythm is in phase with ‘post pupariation time’ rather than absolute sidereal time. The cycles persist for at least 4 days after pupariation (2 days after onset of diapause). JH activity is detectable in groups of flies 10 and 20 days in diapause, but whether JH pulses continue throughout diapause still remains unknown since the extractions of 10 and 20 day pupae are based on asynchronous groups of flies potentially representing different phases of a JH cycle.

DISCUSSION At puparium formation, the MH titre in S. crassipalpis

-10000 A Pqromm.d Continuous 8. Programmed

for D.velop,ment for

Pupal

Diapoura

(solid circles) and moulting hormone (open circles) titres in Sarcophaga for (A) continuous development without interruption of pupal diapause (25”C, 15:9 L:D) and (B) pupal diapause (2O”C, 12:12 L:D). Time scales are adjusted to align comparable developmental stages; arrow indicates time of pupal head eversion.

Fig.

1. Juvenile

hormone

crassipalpisprogrammed

Hormone

titres in diapause

663

is high (around 0.1 pg 20-hydroxyecdysone induce diapause in long day flies by mimicking the JH equivalents/g) in both diapause and non-diapause pulses with exogenous JH have failed. Diapause in destined flies and is well within the range of flesh flies is characterized by regular cycles of oxygen physiological levels reported in other Diptera (SHAAYA consumption (DENLINGER et al., 1972) that may and KARLSON,1965; GALBRAITHet al., 1969, OHMORI correlate with the JH cycles. A regular release of JH and OHTAKI, 1973; FRISTROMet al., 1973; MILNERand could also be involved in measuring passage of time. SANG, 1974; YUND and FRISTROM,1975; HODGETTSet An effect elicited by JH could gradually accumulate al., 1977). In Calliphoru, SHAAYAand KARLSON(1965) until a threshold is reached and thus play a critical role reported a second peak of MH activity coinciding with in determining the duration of diapause. If such is the initiation of adult development. The absence of this case, it should be possible to mimic the passage of time second peak of MH in flies destined for pupal diapause with exogenous JH. JH analogue applied to was predicted by FRAENKELand HSIAO (1968b) and diapausing pupae does indeed shorten diapause has now been confirmed for both S. peregrina (OHTAKI (%%REK and DENLINGER.1975) and application of JH and TAKAHASHI,1972) and S. crassipalpis. In diapause, analogue to third instar larvae on the day before development is arrested shortly after pupal head pupariation can reduce the duration of pupal diapause eversion, thus suggesting that the first peak of MH from 80 to 40 days (DENLINGER.1980). triggers, not only puparium formation, but also Aclino~frdgett,entsWe thank our colleagues ANN L. pupation and pupal head eversion. GNAGEY, JAMES Y. BRADFIELD. VINCEU C. HENKIC’H. and By failing to immediately release a second peak of MICHAEL P. MURTAUGH for their comments. This research MH, short day flies are locked into a period of was supported in part by the Science and Education developmental arrest. Low levels of MH have been Administration of the U.S. Department of Agriculture under detected in diapausing pupae of Hyalophora cecropia Grant No. 7800595 from the Competitive Research Grants Office using a radioimmune assay (MCDANIEL, 1979), but with the less sensitive Musca bioassay we have detected no MH activity in flesh flies during diapause. REFERENCES When the brain of the diapausing fly pupa receives an appropriate environmental cue (high temperature, BRADFIELDJ. Y., IV and DENLINGER D. L. (1980) Diapause DENLINGER, 1972) or chemical shock (hexane, development in the tobacco hornworm: a role for ecdysone DENLINGER et al., 1980) the prothoracic gland or juvenile hormone’? G‘rn. conzp. Eudocrinol. 41, component of the ring gland is again stimulated to 101-107. CARMEL P. W.. ARAU S. and FERIN M. (1976) Pituitary stalk release ecdysone. 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destined

flies is unclear.

Thus

far, attempts

to

synthesis

in the mosquito:

the role ofjuvenile

hormone

in

664

GREGORY P. WALKER AND DAVID

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