Increase In Ecdysteroid Titer Research Articles

Ecdysteroid promotes cell cycle progression in the Bombyx wing disc through activation of c-Myc

Developmental switching from growth to metamorphosis in imaginal primordia is an essential process of adult body planning in holometabolous insects. Although it is disciplined by a sequential action of the ecdysteroid, molecular mechanisms linking to cell proliferation are poorly understood. In the present study, we investigated the expression control of cell cycle–related genes by the ecdysteroid using the wing disc of the final-instar larvae of the silkworm, Bombyx mori. We found that the expression level of c-myc was remarkably elevated in the post-feeding cell proliferation phase, which coincided with a small increase in ecdysteroid titer. An in vitro wing disc culture showed that supplementation of the moderate level of the ecdysteroid upregulated c-myc expression within an hour and subsequently increased the expression of cell cycle core regulators, including A-, B-, D-, and E-type cyclin genes, Cdc25 and E2F1. We demonstrated that c-myc upregulation by the ecdysteroid was not inhibited in the presence of a protein synthesis inhibitor, suggesting a possibility that the ecdysteroid directly stimulates c-myc expression. Finally, results from the administration of a c-Myc inhibitor demonstrated that c-Myc plays an essential role in 20E-inducible cell proliferation. These findings suggested a novel pathway for ecdysteroid-inducible cell proliferation in insects, and it is likely to be conserved between insects and mammals in terms of steroid hormone regulation.

Possible roles of Juvenile Hormone and Juvenile Hormone binding protein on changes in the integument during termination of larval diapause in the bamboo borer Omphisa fuscidentalis

AbstractTo understand the role of Juvenile Hormone (JH) on changes in the integument of Omphisa fuscidentalis Hampson (Lepidoptera: Pyralidae) during larval–pupal development, the expression patterns of transcripts of the O. fuscidentalis JH binding protein gene (OfJHBP) are determined in the integument of diapausing larvae. Quantitative real‐time polymerase chain reaction (qRT‐PCR) studies show that the relative levels OfJHBP mRNA in the integument do not differ significantly in different segments of the body. Application of the JH analogue (JHA) methoprene to diapausing larvae results in a relatively low expression of OfJHBP mRNA from days 0 to 8 after JHA application and reaches maximal levels in the pupal stage. Changes in OfJHBP expression after 20‐hydroxyecdysone (20E) injection also show low levels of OfJHBP mRNA from days 0 to 8 and show a peak of expression in the pupal stage. Incubation of the integument in vitro in the presence of JHA (3.22 µm) induces high levels of OfJHBP expression within 120 min, whereas incubation with 20E (2.08 µm) induces gene expression at 150 min. To study the cooperative effect on OfJHBP expression of these two hormones, dose–response experiments are performed. Larval integument is maintained in the presence of 0.32 µm JHA or 2.08 µm 20E or a combination of both hormones in vitro for 120 min. Induction of OfJHBP expression by JHA and 20E in combination is significantly higher than that of either hormone alone. The results impact not only on our understanding of how methoprene (and hence JH) terminates larval diapause through an increase in ecdysteroid titre in the haemolymph, but also on how JH acts directly on the integument to stimulate the expression of a JH binding protein gene.

The role of 20-hydroxyecdysone and juvenile hormone in pupal commitment of the epidermis of the silkworm, Bombyx mori

During the pupal metamorphosis in insects, cellular commitment for pupal differentiation must precede before its differentiation. The pupal commitment of Bombyx mori epidermis occurred from day 3 to day 6 last (5th) instar larvae in response to the gradual increase in ecdysteroid titer in the presence of a small amount of juvenile hormone (JH). Yet the concealed preparatory process of the commitment had begun in the newly synthesized 5th instar larval epidermis (∼6h before the ecdysis) as a competence phase, in which pupal commitment in vitro was induced by 20-hydroxyecdysone (20E) but inhibited by JH. This competence phase continued until day 2 5th instar, and the decrease and increase in cellular sensitivity to JH and 20E, respectively, occurred gradually during this period. In early day 3, autonomous pupal commitment began in vitro and 20E stimulated the commitment, but JH could only partially prevent the commitment in both cases. This apparent reversible to irreversible transition ended in early day 6 by the completion of pupal commitment, when the cells completely lost their sensitivity to JH and no longer expressed the larval cuticle protein gene 30. The expression of the transcription factor, broad, closely followed the commitment, so that we could use this gene expression as a molecular marker for pupal commitment. These results indicate that exposure to 20E and loss of the sensitivity of the epidermal cells to JH are required for the completion of pupal commitment, and suggest that the unusually long process over 3days could be due to the presence of the detectable JH during the commitment.

Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson

During larval diapause lasting 9 months from September to May, trehalase activity in the midgut of the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae) was low from December to April, followed by a fourfold increase in May that remained high during the pupal stage in July. An application of juvenile hormone analog (JHA) produced increases in the ecdysteroid titer, while trehalase activity was increased by both JHA and 20-hydroxyecdysone (20E) injection. The trehalase activity in the midgut of diapausing larvae was doubled by incubating the midgut with 20E for 48h. During diapause as well as after JHA application, expression of two ecdysone receptor isoform genes (EcR-A and EcR-B1) in the midgut increased simultaneously with the increase in hemolymph ecdysteroid titer, followed by an increase in trehalase activity. The hemolymph of diapausing larvae contained a trehalase inhibitor and inhibitory activity was high during diapause. After 20E injection, trehalase inhibition decreased as midgut trehalase activity increased. Taken together, at least two factors may participate in the change in midgut trehalase activity: increase in trehalase activity and decrease in trehalase inhibitor activity, both of which may be induced by 20E.

Host-parasitoid interactions relating to penetration of the whitefly, Eretmocerus mundus

It has been reported that the aphelinid wasp Eertmocerus mundus parasitizes all four nymphal instars of the sweet potato whitefly, Bemisia tabaci (Biotype B), with 3rd instars being the preferred hosts. The parasitoid lays its egg on the leaf underneath the host nymph. First instars hatch and later penetrate the whitefly. Previous studies have shown that the initiation of parasitoid penetration induces the host to form a cellular capsule around the parasitoid. As described here, females never oviposited once the 4th instar whitefly nymph had initiated adult development. First instar E. mundus larvae were observed under 2nd, 3rd and 4th instar whitefly nymphs, however, penetration did not occur until the whitefly had reached the 4th instar. The non-penetrating E. mundus larva almost always induced permanent developmental arrest in its 4th instar whitefly host and also caused a reduction in whole body host ecdysteroid titers. Therefore, unless there is a peak in molting hormone titer in the area local to penetration, it appears that the induction of capsule formation is not due to an increase in ecdysteroid titer. As the capsule formed around the penetrating parasitoid, host epidermal cells multiplied and became cuboidal and columnar, and relatively thick layers of new cuticle were deposited within the developing capsule, particularly near its ventral opening. The newly formed host cuticle was thinner in the dorsal part of the capsule and appeared to be absent at its apex. These results provide new information regarding the timing and dynamics of parasitoid oviposition and egg hatch as related to larval penetration, parasitoid-induced changes in whitefly development, molting hormone titers and the process of capsule formation.

Host-parasitoid interactions relating to penetration of the whitefly, Bemisia tabaci, by the parasitoid wasp, Eretmocerus mundus

It has been reported that the aphelinid wasp Eertmocerus mundus parasitizes all four nymphal instars of the sweet potato whitefly, Bemisia tabaci (Biotype B), with 3rd instars being the preferred hosts. The parasitoid lays its egg on the leaf underneath the host nymph. First instars hatch and later penetrate the whitefly. Previous studies have shown that the initiation of parasitoid penetration induces the host to form a cellular capsule around the parasitoid. As described here, females never oviposited once the 4th instar whitefly nymph had initiated adult development. First instar E. mundus larvae were observed under 2nd, 3rd and 4th instar whitefly nymphs, however, penetration did not occur until the whitefly had reached the 4th instar. The non-penetrating E. mundus larva almost always induced permanent developmental arrest in its 4th instar whitefly host and also caused a reduction in whole body host ecdysteroid titers. Therefore, unless there is a peak in molting hormone titer in the area local to penetration, it appears that the induction of capsule formation is not due to an increase in ecdysteroid titer. As the capsule formed around the penetrating parasitoid, host epidermal cells multiplied and became cuboidal and columnar, and relatively thick layers of new cuticle were deposited within the developing capsule, particularly near its ventral opening. The newly formed host cuticle was thinner in the dorsal part of the capsule and appeared to be absent at its apex. These results provide new information regarding the timing and dynamics of parasitoid oviposition and egg hatch as related to larval penetration, parasitoid-induced changes in whitefly development, molting hormone titers and the process of capsule formation.

Effects of juvenile hormone on the secretion of prothoracicotropic hormone in the last- and penultimate-instar larvae of the silkworm Bombyx mori

The effect of juvenile hormone (JH) on the secretion of the prothoracicotropic hormone (PTTH) was investigated, by examining the changes in hemolymph PTTH titer after the topical application of JH-I on the larvae of the silkworm, Bombyx mori. The titer of PTTH was determined by the time-resolved fluoroimmunoassay. JH-I application at very early stages of development in the fifth (last) instar resulted in a significant increase in the PTTH titer, but this effect became less evident thereafter. After the onset of wandering (day 6 of the fifth instar), JH-I did not affect the hemolymph PTTH titer. JH-I application on day 5 resulted in the delay of spinneret pigmentation on day 6, which is induced by an increase in the ecdysteroid titer on day 5 and is the first visible indication of larval–pupal transformation. However, the JH-I application did not suppress the increase in either PTTH or ecdysteroid titer on day 5, suggesting that JH-I acts on the spinneret to inhibit the response of the tissue to ecdysteroids. JH-I also exhibited a PTTH titer-elevating effect in the fourth instar. These results suggest that JH has a role as a potent stimulator of PTTH secretion in both the penultimate and last instar of the silkworm.

Effect of nonsteroidal ecdysteroid agonists on ecdysteroid titer in Spodoptera exigua and Leptinotarsa decemlineata

In control last-instar larvae of Spodoptera exigua, and Leptinotarsa decemlineata, major hemolymph ecdysteroid peaks appeared around day 3–4, and 8–9, respectively, which is correlated with normal apolysis. Later on, hormone titers declined to a basal level. In Colorado potato beetle last-instars, a small rise was noted around day 4, which represents the time of digging. In last-instar larvae of S. exigua treated with either RH-5849 or tebufenozide, the ecdysteroid titer remained at a basal level without any elevation. Such larvae showed signs of untimely apolysis within 12–24 h after treatment followed by cessation of fresh weight gain. Likewise, hemolymph ecdysteroid levels and larval weight gain were reduced in last-instar larvae of L. decemlineata treated with RH-5849 as compared with controls. Otherwise, treatment of Colorado potato beetle larvae with tebufenozide caused no hormonal and physiological abnormalities as compared with controls. It is suggested that both nonsteroidal ecdysteroid agonists act via ecdysteroid receptor binding without an increase in ecdysteroid titer.

Developmental changes and acetylcholinesterase activity in the metamorphosing brain of Tenebrio molitor: Correlation to ecdysteroid titers

AbstractThe brain of Tenebrio molitor exhibited marked fluctuations in acetylcholinesterase (AChE) activity throughout metamorphosis. This was true AChE activity, since it was inhibited by high substrate concentrations and by 10 μM of the specific AChE inhibitor BW284C51 [(1,5‐bis'4‐allyldimethylammoniumphenyl)‐pentan‐3‐one dibromide] but not by iso‐OMPA (tetraisopropylpyrophosphoramide), a cholinesterase (but not AChE) inhibitor. The histochemical AChE activity was localized in the neuropile and the nuclear envelope of neurons and glial cells. The enzyme extracted from brains with 1% Triton X‐100 and 1 M NaCl sedimented as a single peak in a sucrose density gradient, with a sedimentation coefficient of 5.4S. This single AChE sedimentation peak was mainly due to an amphiphilic dimeric form. AChE activity per brain increased in newly ecdysed pupa. AChE activity per milligram of protein exhibited a peak in the mid‐pupa which could be correlated to the increase in ecdysteroid titers. © 1994 Wiley‐Liss, Inc.

Correlation of ecdysteroids with ovarian development and yolk protein synthesis in the adult stable fly, Stomoxys calcitrans

1. 1. The ecdysteroids in the adult stable fly were analysed through the first oogenie cycle. 2. 2. In females, both hemolymph and whole bodies showed a dramatic increase in ecdysteroid titer on days 4–5 when the ovaries began to accumulate yolk. 3. 3. The ecdysteroid titer decreased after the first clutch of eggs was deposited. 4. 4. The change in ecdysteroid titer was not observed in male and ovariectomized female flies. 5. 5. HPLC/RIA analysis indicated that ecdysone and 20-hydroxyecdysone were the major ecdysteroids. 6. 6. The correlation between the sex-specific change in the ecdysteroid titer and ovarian development suggests a role for ecdysteroids in female reproduction of the stable fly.

Fluctuations in ecdysteroid titre and development during the early stage of the last larval instar of trimoulter strains of the silkworm, Bombyx mori

The correlation between slight fluctuations in haemolymph ecdysteroid titre and development of the larvae were explored in recessive and dominant trimoulter strains of the silkworm, Bombyx mori. In recessive trimoulters, the decision for precocious metamorphosis is reversible, and perfect supernumerary larval moults can be induced by the addition of 20-hydroxyecdysone to food sources during the early stage of the last (fourth) instar. Changes in haemolymph ecdysteroid titre show that a small peak occurs at 24 h. On the contrary, for dominant trimoulters, as well as for recessive trimoulters to which an imidazole compound (KK-42) is applied, or which are exposed to high temperature and moisture during the early stage of the penultimate (third) larval instar, the decision for precocious metamorphosis becomes irreversible in some tissues, and a perfect supernumerary larval moult can never be induced through 20-hydroxyecdysone feeding after the third ecdysis. Ecdysteroid titre levels remained low during the early stage of the last larval instar. We propose that the observed decrease after small increase in ecdysteroid titre in recessive trimoulters is related to changes in the developmental programme from larva to pupa. The mechanism by which the controlled fluctuations in ecdysteroid titres are regulated is discussed.

Ecdysteroid synthesis and molting by the tobacco hornworm, Manduca sexta, in the absence of prothoracic glands

When a pair of prothoracic glands (PGs) were removed from Manduca sexta pupae on the day of pupation, the hemolymph ecdysteroid titer remained at a low level. When a portion of the gland pair was extirpated from pupae after the critical period for prothoracicotropic hormone release, the maximum hemolymph ecdysteroid titer was reduced in proportion to the mass of the PGs removed. These findings clearly showed that the PGs in intact pupae are responsible for the elevated ecdysteroid titer required to elicit adult development on schedule. When brains were removed on the day of pupation, the initiation of adult development was delayed for weeks or months. In contrast, pupae whose PGs were removed on the day of pupation initiated development only 7 days late, indicating the existence of an additional source of pupal ecdysteroids. Further, abdomens of male M. sexta that were isolated on the day of pupation initiated adult development spontaneously within 70 days. The implantation of day 0 pupal brains into these isolated abdomens accelerated the initiation of adult development and elicited synchronous adult development. The hemolymph ecdysteroid titer of those isolated abdomens receiving implants of brains increased within 5 days and reached a maximum level of 1.5 micrograms/ml. The analysis of hemolymph ecdysteroids by reverse-phase HPLC revealed that ecdysone was the major moiety and that the ecdysteroid composition was similar to that of normal, intact pupae that had just initiated adult development. These results demonstrate that the PGs are not requisite for adult development. An increased hemolymph ecdysteroid titer was also observed in isolated abdomens from which the testes were removed and in abdomens devoid of their digestive tract. Indeed, in the latter case, the ecdysteroid titer attained much higher levels than those observed for abdomens with intact guts. Despite numerous attempts to identify the tissue(s) in the isolated abdomens responsible for the increase in ecdysteroid titer, its identity remains unknown.

Interactions between limb regeneration and ecdysteroid titres in last larval instar Rhodnius prolixus (Hemiptera)

Leg-amputation experiments performed on populations of carefully staged last-instar Rhodnius prolixus indicate that the regeneration critical period in this species occurs 6–7 days following feeding. Decapitation experiments indicate that the regeneration critical period is also the time of the head critical period for moulting. The head critical period is associated with a rapid increase in ecdysteroid titre in response to a release of prothoracicotropic hormone by the brain. Leg amputations performed prior to the head critical period result in a temporary decrease in ecdysteroid titre lasting 24–36 h, while the same operation performed after the head critical period has no effect on ecdysteroid titre. When the period of temporary depression in titre encompasses the head critical period, the rapid increase in ecdysteroid titre associated with this time is delayed. Thus, the ability to regenerate a leg after initiation of the moult is dependent upon when the injury occurs in relation to the release of prothoracicotropic hormone at the head critical period. The mechanism responsible for the decline in ecdysteroid titre following leg amputation is unknown but does not appear to involve prothoracicotropic hormone or the prothoracic glands. Injection of tetrodotoxin at the time of a leg amputation prolongs the period for which the titre is depressed. In these animals the titre rises in conjunction with resumption of electrical activity in the corpus cardiacum, indicating that prothoracicotropic hormone release may be necessary for recovery of the titre following leg amputation. Results are discussed in relation to existing evidence concerning possible interactions between regeneration, prothoracicotropic hormone release and moulting.

Developmental endocrinology of larval moulting in the tobacco hornworm, Manduca sexta.

A larval moult in the tobacco hornworm, Manduca sexta, involves an endocrine cascade that begins with the release of a cerebral peptide hormone, the prothoracicotropic hormone (PTTH). The release of PTTH is gated, occurs during the scotophase and appears to be developmentally cued. In fourth instar Manduca larvae, PTTH release into the haemolymph occurs as a single burst over a few hours during the head critical period, i.e. the time during which the head (brain) is needed for the initiation of the moult to the fifth (last) instar. Released PTTH activates the prothoracic glands (PGs), and within a few hours the cumulative effect of this event results in a dramatic increase in the haemolymph ecdysteroid titre, which then elicits the moult. An assessment of the capacity of the corpora allata (CA) to synthesize juvenile hormone (JH) in vitro indicates that the above sequence of endocrine events begins only when JH synthesis has reached a nadir for the instar. Since CA activity is an indirect measure of the haemolymph titre of the hormone, it is conceivable that the developmentally cued release of PTTH is permissively controlled by a decreasing haemolymph titre of JH. With the increase in the ecdysteroid titre which marks the end of this endocrine cascade, the CA again become active, presumably to cause the increase in the JH haemolymph titre which directs the larval moult. This investigation has thus established the temporal and quantitative dynamics of the PTTH-PG axis that drive larval moulting and provides insight into the interendocrine regulatory relationships that may exist between the ecdysteroids and JHs. These possible relationships and the role of the brain in their regulation are discussed.

Effects of decapitation at the head critical period for moulting on haemolymph ecdysteroid titres in final-instar male and female Rhodnius prolixus (Hemiptera)

Decapitation experiments performed on large populations of carefully staged last-instar Rhodnius prolixus indicate that there is a 24 h sex difference in the time of the head critical period. Males pass the head critical period over a 36 h period beginning at 1200 A.Z.T. (Arbitrary Zeitgeber Time) day 6 after feeding, while females pass the head critical period over a 36 h period beginning at 1200 A.Z.T. day 5 after feeding. Radioimmunoassay determinations of haemolymph ecdysteroid titres show that there is also a 24 h sex difference in the time of the major increase in ecdysteroid titre that commences at the head critical period. These results imply that there is a sex difference in the time at which the release of prothoracicotropic hormone (PTTH) associated with the head critical period occurs. Decapitation prior to the head critical period results in a rapid, permanent decrease in ecdysteroid titre, while decapitation after the head critical period does not prevent normal increases in ecdysteroid levels. We infer that the presence of the head is essential until just prior to the head critical period and its associated increase in at the head critical period also indicates that the head critical period represents a change in the activity of the prothoracic glands indicative of a response to a significant release of PTTH at this time. The significance of these results is discussed in relation to both classical and recent evidence concerning the timing of PTTH release and changes in ecdysteroid titres in Rhodnius prolixus.

Changes in lipophorin composition during the fourth to fifth instar molt of Manduca sexta

During the feeding stage of the fourth instar of the tobacco hornworm, Manduca sexta, lipophorin has a density of 1.144 g/ml and is comprised of 55% protein and 45% lipid, mainly diacylglycerol and phospholipid. These values are similar to those found during the corresponding period of the fifth instar. However, during the intermolt period the lipid composition of lipophorin changes to 45% protein and 55% lipid and the density decreases to 1.112 g/ml. The onset of these changes in lipid content correspond to the increase in ecdysteroid titer which signals the beginning of molting. At the end of molting both the lipid content and density return to those characteristic of the fifth instar. These data further confirm the complex nature of lipophorin metabolism in M. sexta larva and suggest that ecdysone may be the trigger for lipid mobilization during the intermolt period.

Ecdysteroid influence on the development of the host Heliothis virescens and its endoparasite Microplitis croceipes

Ecdysteroid injections into H. virescens larvae parasitized by M. croceipes resulted in an array of effects dependent on the developmental phase of injected larvae. Injections into early 5th instars had little or no effect on either the parasite or the host. Injections into older pre-wandering 5th instars initiated both premature parasite emergence and premature entry of the host into the wandering phase. Injections of ecdysteroid into post-wandering, pre-parasite emergence larvae resulted in both a dose-dependent inhibition of parasite emergence and formation and tanning of host pupal cuticle. Injections into post-wandering, post-parasite-emergence larvae resulted in tanning of the host cuticle. A temperature effect was observed in which effects of the ecdysteroid injections administered at wandering and post-wandering phases were enhanced at the higher rearing temperature of 32°C. RIA of haemolymph from unparasitized larvae showed an early 8-fold increase in ecdysteroid titre 2 days before pupation. This increase was not observed in parasitized larvae of the same age.

Cellular events in the prothoracic glands and ecdysteroid titres during the last-larval instar of Spodoptera littoralis

Changes in prothoracic gland morphology were correlated to developmental events and ecdysteroid titres (20-hydroxyecdysone equivalents) during the last-larval instar in Spodoptera littoralis. After ecdysis to the last-larval instar the haemolymph ecdysteroid titre remained at about 45 ng/ml, when the prothoracic glands appeared quiescent. The first signs of distinct gland activity, indicated by increased cell size and radial channel formation, were observed at about 12 h prior to the cessation of feeding (36 h after the last-larval moult), accompanied by a gradual increase in ecdysteroid titre to 110 ng/ml haemolymph, at the onset of metamorphosis. During this phase ecdysteroid titres remained at a constant level (140–210 ng/ml haemolymph) and prothoracic gland cellular activity was absent for a short period. The construction of pupation cells occurred when haemolymph ecdysteroids titres increased to 700 ng/ml. A rapid increase in ecdysteroids began on the fourth night (1600 ng/ml haemolymph) reaching a maximal level (4000 ng/ml haemolymph) at the beginning of the fourth day. In freshly moulted pupae a relatively high ecdysteroid titre (1100 ng/ml haemolymph) was still observed, although during a decrease to almost negligible levels. The increase in ecdysteroid level during the third and the fourth nights of the last-larval instar was correlated with the period when almost all the prothoracic gland cells showed signs of high activity. Neck-ligation experiments indicated the necessity of head factors for normal metamorphosis up to the second to third day of the instar. The possibility that the prothoracic glands are under prothoracicotropic hormone regulation at these times is discussed.

Analysis of metamorphosis in Drosophila melanogaster: Characterization of giant, an ecdysteroid-deficient mutant

The mutant allele giant of Drosophila melanogaster affects the timing and the level of increase in ecdysteroid titer normally occurring at puparium formation. The third larval instar is extended by 4 days in phenotypically “giant” individuals during which the imaginal discs mature slower than normal and finally take on the folding pattern characteristic of maturity at a time when normal individuals have already formed puparia. After puparium formation, development occurs at the same rate in giant and wild-type animals. Feeding 20-hydroxyecdysone at 94 hr after oviposition allows giant larvae to develop at the same rate as wild-type larvae and to produce normal-sized adults (although at 94 hr the imaginal discs of giant lack much of the folding pattern of mature discs). Radioimmunological determination of ecdysteroid titers in giant and normal individuals indicates that the peak of ecdysteroid activity associated with puparium formation is lower in giant and occurs 4 days later than normal. These results indicate that giant is an ecdysteroid-deficient mutant with major effects on metamorphosis. Unlike previously reported ecdysteroid-deficient mutants, however, giant larvae eventually develop into adults and may be induced to undergo complete metamorphosis at the same time as wild type by feeding 20-hydroxyecdysone.

Cytophysiological correlations between prothoracic gland activity and hemolymph ecdysteroid concentrations in Rhodnius prolixus during the fifth larval instar: Further studies in normal and decapitated larvae

Hemolymph ecdysteroid titers in fifth instar larvae of Rhodnius prolixus were determined by radioimmunoassay, and their prothoracic glands were excised and examined by electron microscopy. During the last larval instar, the titer of ecdysteroid increased between the head-critical period until Day 13, at which time the peak titer was 3100 pg 20-hydroxyecdysone equivalents/μl. The activation of secretory cells at the time of the second period of prothoracicotropic hormone release was correlated with the development of major cellular organelles. The smooth endoplasmic reticulum first appeared at the head-critical period and the proliferated in close relation to the increase in ecdysteroid titer until Day 13, after which time it disappeared. Mitochondria expand and develop tubular cristae. They are closely associated with the smooth endoplasmic reticulum. When insects were decapitated, hemolymph ecdysteroid titer remained below 10 pg/μl and the prothoracic gland cells failed to develop smooth endoplasmic reticulum. We conclude that in the prothoracic gland cells as well as other steroidogenic tissues the smooth endoplasmic reticulum in association with mitochondria is involved in ecdysone biosynthesis.