Changes in the morphogenetic response of Tenebrio molitor pupae to juvenile hormone in relation to age

Morphogenetic effect of juvenile hormone (JH) and its analogues, dodecyl methyl ether, ethyl trimethyl dodecadienoate and methylenedioxyphenoxy-6-epoxy-3-ethyl-7-methyl-2-nonene, on carefully timed Tenebrio pupae was determined. These results show that the response of pupal epidermal cells to JH varied with age during the first 48 hr after larval-pupal ecdysis. The pupae showed low morphogenetic response soon after pupal ecdysis but their response increased gradually until 18 hr. The response to JH decreased in pupae older than about 32 hr; and 48 hr old pupae were unresponsive to low doses of JH employed in this study. Age-related differences in the pattern of response of the individual body regions to JH were also observed.The synergistic effect of 1 μg of ecdysterone with these JH compounds was also tested in relation to the age of Tenebrio pupa. The results show that the synergistic effect of ecdysterone was generally limited to >18 hr old pupae. This suggests that the physiological basis of the synergistic effect of ecdysterone may be the latter's ability to synchronize epidermal cells.The significance of these observations in the analysis of time of action of juvenile hormone is discussed.     

20-hydroxyecdysone stimulation of juvenile hormone biosynthesis by the mosquito corpora allata

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. JH titer decreases in the last instar larvae allowing pupation and metamorphosis to progress. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which plays an essential role orchestrating reproductive maturation. 20-hydroxyecdysone (20E) prepares the pupae for ecdysis, and would be an ideal candidate to direct a developmental program in the CA of the pharate adult mosquito. In this study, we provide evidence that 20E acts as an age-linked hormonal signal, directing CA activation in the mosquito pupae. Stimulation of the inactive brain-corpora allata-corpora cardiaca complex (Br-CA-CC) of the early pupa (24 h before adult eclosion or −24 h) in vitro with 20E resulted in a remarkable increase in JH biosynthesis, as well as increase in the activity of juvenile hormone acid methyltransferase (JHAMT). Addition of methyl farnesoate but not farnesoic acid also stimulated JH synthesis by the Br-CA-CC of the −24 h pupae, proving that epoxidase activity is present, but not JHAMT activity. Separation of the CA-CC complex from the brain (denervation) in the −24 h pupae also activated JH synthesis. Our results suggest that an increase in 20E titer might override an inhibitory effect of the brain on JH synthesis, phenocopying denervation. All together these findings provide compelling evidence that 20E acts as a developmental signal that ensures proper reactivation of JH synthesis in the mosquito pupae.     

Morphogenetic effects of juvenile hormone and juvenile hormone mimics on adult development of Drosophila

The morphogenetic effects of t,t-farnesol, Law-Williams juvenile hormone analogue, dichlorofarnesenic acid ethyl ester (DFAEE), and a syntetic racemic or isomeric mixture of C₁₈ juvenile hormone (JH), when applied topically to pharate pupae and adults of D. melanogaster have been studied. Of these various agents tested, only DFAEE and JH affected adult development and eclosion and the pharate pupae were the most sensitive to these agents. The racemic mixture of JH induced the secretion, in the abdomen, of a supernumerary cuticle indistinguishable from that of the pupa; it, in addition, retarded the synthesis of brown eye pigments, general body pigmentation, and affected the differentiation of various internal organs and cuticular structures of the abdomen. By comparing the effects of JH with those of Minute (M) and bobbed (bb) mutations on the adult development, it is suggested that JH, by retarding genetic translation mimics M or bb.     

Factors influencing juvenile hormone esterase activity in the wax moth, Galleria mellonella

The effects of juvenile hormone, antiallatotropins, selected surgical procedures and starvation on the juvenile hormone esterase levels in Galleria larvae and pupae were investigated. JH reduced JH esterase activity in larvae but induced the enzyme in 1-day-old pupae. In vitro studies confirmed that the peak of synthesis and/or release of JH esterase from the fat body of last instar larvae occurred 4 days after ecdysis. These studies also showed that fat body from JH-treated larvae released much less enzyme than controls. Antiallatotropins, precocene 2 and ZR 2646 also reduced JH esterase levels in larvae, but ZR 2646 induced JH esterase in pupae. In starved larvae, JH esterase did not increase during the first five days. A minimum of 36 hr of feeding was necessary for the larval esterase activity to increase on schedule on day 4 of the last larval stadium. When day-l larvae were ligated behind the head or the prothorax, they had lower JH esterase levels and yet showed a slight increase in the enzyme when the larvae reached the age of 4 days. The significance of these results is discussed in relation to the possible control of esterase activity during metamorphosis.     

Juvenile hormone: Its assay and effects on pupae of Manduca sexta

Non-diapausing pupae of Manduca sexta were used to develop a bioassay for juvenile hormone (JH). The period of maximal sensitivity to Cecropia C17-JH injected in olive oil was found to be 24 to 30 hr after pupal ecdysis at the time of the beginning of epidermal retraction. The dose-response curves for C16-, C17-, and C18-JH at 29±1 hr after pupal ecdysis were determined and those for the latter two compounds were found to be similar and to be linear between 10^?2 and 10 μg/g body weight. C16-JH was 300 times less active than C17- and C18-JH in this bioassay. The effectiveness of 0·5 μg/g C18-JH at 29±1 hr was determined by the carrier media in which it was injected. The highest scores were obtained when the carrier was light mineral oil or loive oil whereas the lowest scores were obtained using 10% BSA or Tween 80. These scores are consistent with the kinetics of equilibration of the injected JH with the haemolymph. Thus, the injected hormone is more effective when it slowly leaks into the blood, presumably because it is metabolized much more slowly.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Disturbance of Adult Eclosion by Fenoxycarb,a Juvenile Hormone Mimic,in the Beet Armyworm,Spodoptera exigua

Effect of exogenous juvenile hormones (JHs) on pupal development was assayed in the beet armyworm, Spodoptera exigua. Fenoxycarb, a potent JH mimic, was applied topically to different ages of the pupae, and showed significant inhibition of normal adult eclosion even at 0.1 μg dose when it was applied at the early pupal stage (day 0). As the pupal development underwent, the susceptibility of the pupae fenoxycarb decreased. RH5992, a potent ecdysteroid mimic, did not, however, any similar inhibitory effect on the pupae. Natural JH types (JH I, JH II, and JH III) were applied on day 0 pupae to compare their inhibitory effects on adult eclosion. Both JH I and JH II significantly inhibited adult eclosion at 1.0 μg dose, but JH III did not even at 10.0 μg dose. It was noted that fenoxycarb-treated pupae showed little rectum development. Fenoxycarb did not, however, show any negative effect on the development of compound eye and wing imaginal discs, and on the pupal hemolymph protein pattern. These results suggest that there should be a commitment period requiring an absence of JH for a normal adult metamorphosis during early pupal development and that the endogenous type of JH in S. exigua is JH I or JH II or both JHs like other lepidopteran species.     

Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm, Heliothis zea

In Heliothis zea, pupal diapause is not due to a deficiency of the prothoracicotropic hormone (PTTH), as it is in many other insects. However, PTTH is essential for diapause termination and adult development. Removal of the pupal brain 4 hr after larval-pupal ecdysis blocks the insect's ability to initiate adult development. Transplantation of brain neurosecretory cells restores this ability, whereas other tissues such as corpora allata have no effect. In the diapausing pupa, PTTH is released from the brain within 24 hr after larval-pupal ecdysis. Subsequent removal of the brain fails to block the ability for diapause termination, because PTTH potentiates the ability for adult development. Since diapause termination is suppressed in a temperature of 21°C, the bollworm retains the ability to initiate development in 27°C whereas it remains in diapause in 21°C. Diapause continues even though pupae are supplied with additional PTTH via neurosecretory cell transplantation.Ecdysone injection and prothoracic gland-ablation experiments indicate that the prothoracic glands are the source of the prohormone α-ecdysone, and that diapause is maintained by an α-ecdysone deficiency. This evidence, in conjunction with the above results, suggests that PTTH release potentiates prothoracic gland function in the diapausing pupa which is then regulated by a temperature dependent process.     

The role of juvenile hormone in endocrine control of pigmentation in Manduca sexta

Spatial and temporal distribution of insecticyanin was studied in the fourth and fifth larval instars of Manduca sexta. The protein was distributed between the epidermis (62%), the hemolymph (37%) and the pericardial cells (0.5%). Hemolymph insecticyanin (HINS) was highest (0.6 mg/ml) in the very early fourth instar, gradually declining to 0.3 mg/ml. Levels in the fifth instar decreased after ecdysis (0.15 mg/ml), began to rise at wandering, and nearly doubled by the time of pupation. Titers of epidermal insecticyanin (EINS) followed the general growth patterns during the fourth and early fifth instar. At 76 hr after fifth instar ecdysis, titers of EINS dropped precipitously and then rose again to peak just after the wandering stage. Levels of EINS again rapidly declined and could not be detected after 180 hr. Ecdysteroids appear to shut off synthesis of EINS but this response is quantitatively modified in the presence of JH. Endocrine manipulation of the last larval-larval molt indicated that juvenile hormone (JH) acts quantitatively on EINS to induce a dose-dependent increase. The JH-induced increase can be as much as 4-fold, depending upon the body region.     

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP₃R-operated mobilization of endoplasmic reticulum Ca²⁺ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.     

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta. There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O,O-diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.     

The effect of juvenile hormone on pupal pigmentation of Pieris brassicae L.

The role of juvenile hormone (JH) in the morphological colour adaptation of pupae of Pieris brassicae controlled by environmental factor was analyzed. First the effects of JH I and its analogue, Farnesyl-Methyl-Ether (FME) were tested. Secondly the JH-titres of the last instar larvae were measured under various light conditions which influence the future pigmentation of the pupae.During the sensitive period, which occurs before pupation, blue light (410 nm) produces the strongest, darkness medium, and yellow light (570 nm) the lightest pigmentation of the pupae.JH I as well as FME has an inhibiting effect on the formation of the black spots in the cuticle. However, this effect only becomes apparent (a) if the insects are kept under blue light during the sensitive period (which normally leads to a strong black pigmentation) and (b) only when these animals were treated with JH I or FME either at the beginning, or 10 hr after the beginning of the sensitive period. In the last larvae instar, JH could be found only during the sensitive period. Fourteen hours after the beginning of the sensitive period the JH concentration reaches a maximum of 30–100 pg JH per insect. The JH-titre resulting from blue light conditions is significantly different from those of the larvae kept under white or yellow light. An additional maximum of 60 pg JH per animal was found 8 hr/after the beginning of the sensitive period. Obviously, JH affects the process of pigmentation of the pupae, but the described results are not sufficient to explain thoroughly the regulation of pigmentation modified by environmental factors. The effects of further factors are discussed.     

In vivo fluctuation of JH,JH acid,and ecdysteroid titer,and JH esterase activity,during development of fifth stadium Manduca sexta

Juvenile hormone (JH), JH acid, and ecdysteroid titer, and JH esterase activity, were measured in hemolymph from synchronous last stadium larvae of Manduca sexta. JH and JH acids were identified and quantified by GC-MS: JH I and II (and the corresponding acids) were the predominant JH homologs detected in males or females. Maximum levels of JHs and JH acids were observed just following ecdysis to the fifth (last) stadium (day 0, 0 hr) and at the prepupal stage (day 6–day 7). JH titer (≥ 1 ng JH I or II/ml) was higher than JH acid titer (∼0.7 ng JH I acid or JH II acid/ml) in very early fifth stadium larvae. However, this was reversed at the prepupal stage when higher titers of JH acids than JH were observed. JH acid titer began to rise prior to JH titer at the prepupal stage. JH esterase activity rose significantly only after JH or JH acid titers had begun to decline; maximum JH esterase activity was observed at day 3 and day 8. Ecdysteroid titer (measured by RIA) decreased during the last larval molt to a low level by day 0 (0 hr) and to undetectable levels at day 0 (12 hr) of the fifth stadium, by which time JH and JH acid levels had also declined substantially. Just prior to wandering, a small ecdysteroid peak was noted and a slightly elevated level of ecdysteroid was maintained for a further 2 days before a surge in ecdysteroid titer occurred at the prepupal stage, in synchrony with JH and JH acid titer maxima. There was no sexual dimorphism in timing or magnitude of JH, JH acid, and ecdysteroid titer or JH esterase activity.     

Hormonal regulation of JP29 in the epidermis during larval development and metamorphosis in the tobacco hornworm,Manduca sexta

Previous studies have shown that the larval epidermis of the tobacco hornworm, Manduca sexta, contains a 29 kDa nuclear protein (JP29) that binds pothoaffinity analogs of juvenile hormone (JH), but does not bind JH I with high affinity. We now find that JP29 is also associated with the insecticyanin granules, and we show that JP29 mRNA is regulated in a complex fashion by both 20-hydroxyecdysone (20E) and JH. Studies with day 2 fourth instar larval epidermis in vitro showed that a molting concentration 12 μg/ml) of 20E caused the disappearance of JP29 mRNA, irrespective of the presence or absence of JH; this effect was dependent on the concentration of 20E (ED₅₀=200 ng/ml). The reappearance of JP29 mRNA around the time of ecdysis required the presence of JH at head capsule slippage (HCS), since little appeared in larvae allatectomized about 6 h before HCS unless JH I was applied at the time of HCS. Maintenance of JP29 mRNA in fifth instar epidermis also required the continued presence of JH in both isolated abdomens and in vitro. Culture of either day 1 or day 2 fifth instar epidermis without hormones for 24 h caused decline of JP29 mRNA, which was accelerated by 20E in a concentration-dependent manner (ED₅₀ = 30 and 10 ng/ml 20E respectively). When day 2 epidermis was exposed to 500 ng/ml 20E for 24 h to cause pupal commitment, JP29 mRNA disappeared. Neither methoprene nor JH I (in either the presence or the absence of the esterase inhibitor O-ethyl, S-phenyl phosphamidethiolate [EPPAT]) was able to prevent this loss, although both slowed its rate. The mRNA for the larval cuticle protein LCP14 was found to be regulated similarly to that for JP29 by 20E, but differently by JH. The JP29 protein was relatively long-live, persisting after the disappearance of its mRNA for at least 19 h during the larval molt and for more than 24 h in vitro. Although trace amounts of JP29 are found for the first 12 h after pupal ecdysis, injection of 5 μg JH II into pupae during the critical period to cause the synthesis of a second pupal cuticle had no effect on the amount of JP29 present. Thus, although the presence of JP29 in larval epidermis is associated with and dependent on JH, high amounts are not associated with the “status quo” action of JH on the pupa. The role of this protein consequently remains obscure. Arch. Insect Biochem. Physiol. 34:409–428, 1997. © 1997 Wiley-Liss, Inc.     

Cuticle laccase of the silkworm,Bombyx mori: Purification,gene identification and presence of its inactive precursor in the cuticle.

Laccase is a multi-copper enzyme found in variety of organisms including plants, fungi and bacteria. In insects, laccase is thought to play an important role in cuticle sclerotization with its ability to catalyze the oxidation of phenolic compounds to their corresponding quinones. From the newly ecdysed pupae of the silkworm, Bombyx mori, we purified a dimer form of cuticular laccase with 70-kDa polypeptides. Mass spectrometric analysis of the tryptic fragments and cDNA sequence analysis revealed that the gene for the purified laccase (BmLaccase2) is an ortholog of laccase2, one of the multiple laccase genes found in insect genomes. BmLaccase2 is highly expressed in the epidermis prior to ecdysis, suggesting that the BmLaccase2 protein accumulates before ecdysis. However, the cuticle of newly ecdysed pupa does not have laccase activity, and the activity only becomes detectable several hours after ecdysis. These data suggest that cuticle laccase is synthesized as an inactive precursor, which is later activated after ecdysis. We also found that urea-solubilized cuticle protein extract contains an inactive form of laccase that can be activated by trypsin treatment.     

Effect of topical application of a juvenile hormone mimic on the duration of the moulting cycle in fifth instar Rhodnius

Fifth instar Rhodnius prolixus given a single topical application of synthetic juvenile hormone (JH) on one of the first 12 days after feeding showed a sigmoid relationship between day of treatment and duration of the moulting cycle. A similar relationship was observed between day of treatment and mean ‘juvenile score’ in subsequently emerging insects. The linear relationship between mean ‘juvenile score’ and duration of the moulting cycle suggests that the increased time from feeding to ecdysis for late-treated insects is a result of the morphogenetic effects of the JH treatment. Some of the partially juvenilized insects obtained by treatment after day 5 produced an odour characteristically detected only from adult insects. The possible significance of this observation is discussed.     

Developmental profiles of the mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta

When fat body mRNA from the tobacco hornworm larva, Manduca sexta, was translated in a rabbit reticulocyte lysate system, three major polypeptides were found, each having a different developmental profile. One mRNA coded for a 74 kilodalton (K) polypeptide doublet precipitated by an antibody to the arylphorin (manducin). This mRNA was present only during the intermolt feeding phase of the penultimate and the final larval instars. Its appearance 16–24 hr after larval ecdysis was dependent upon the incoming nutrient supply and independent of the juvenile hormone (JH) level. Immunoblots of proteins of the fat body, epidermis, and cuticle revealed the presence of arylphorin in all three tissues. Additionally, several small polypeptides that cross-reacted with the arylphorin antibody were found in the fat body during and up to 24 hr after the last larval molt and in the tanning pupal cuticle. The larval epidermis was also found to contain a small amount of arylphorin mRNA. At the time of the JH decline prior to the onset of metamorphosis, a female-specific mRNA coding for a 79 K translation product appeared. In allatectomized larvae this mRNA was detectable earlier, and its appearance in intact larvae was prevented by application of methoprene, indicating that JH regulates its appearance. At wandering a new mRNA that also codes for a 79 K polypeptide appeared in both sexes and was the major messenger present during the prepupal stage. Neither it nor the female-specific mRNA were translatable after pupal ecdysis.     

Rôle des organes perisympathiques et dynamique de la secretion de la bursicon chez Tenebrio molitor

Light and electron microscope observations have been made on the perisympathetic organs (P.O.) situated on the dorsal somatic abdominal nerves in pupae of Tenebrio molitor.Tests carried out with extracts of P.O. using a bioassay method on pupae of Tenebrio at various developmental stages indicate that these organs have an important role in the storage and release of the tanning hormone, bursicon. The P.O. extracts from the abdominal segments (2 to 5) of 5-day-old pupae were found effective but not those from pharate adult (2 days before ecdysis). The storage and release of bursicon occur later from the P.O. of the posterior abdominal segments (6 to 8).The activity of the blood begins to be effective in the pharate adult 2 days before ecdysis, reaches a maximum 1 hr after the adult ecdysis, and decreases thereafter.This study carried out on the various parts of the nervous system shows that all these parts are effective at a certain stage of the pupal and adult development following an antero-posterior gradient. In the cephalic region, this activity is early and long-lasting, but it appears at later stages and only for a short time in the thorax and abdomen. This phenomenon indicates that bursicon is either passed along the ventral nervous chain or it is synthesized in different parts of the nervous system at successive developmental stages. In the present paper, we have tried to give a comprehensive summary of the dynamics of bursicon in insects.