Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

The juvenile hormone analog pyriproxyfen affects ecdysteroid-dependent cuticle melanization and shifts the pupal ecdysteroid peak in the honey bee (Apis mellifera)

The control of the pupal melanization in the honey bee by ecdysteroids, and the modulation of these processes by a juvenile hormone analog were investigated by a combination of in vivo and in vitro experiments. Injection of 1-5 microg of 20-hydroxyecdysone (20E) into unpigmented pupae showed a dose- and stage-dependent effect. The higher the dose and the later the injection was performed, the more pronounced was the delay in cuticle pigmentation. This inhibition of cuticular melanization by artificially elevated ecdysteroid titers was corroborated by in vitro experiments, culturing integument from unpigmented, dark-eyed pupae for 1-4 days in the presence of 20E (2 or 5 microg/ml culture medium). Topical application (1 microg) of pyriproxyfen to unpigmented, white-eyed pupae had the opposite effect, leading to precocious and enhanced melanization of the pupal cuticle. In vitro incubation of integuments in the presence of this juvenile hormone analog (1 microg/ml) confirmed these results, showing that pyriproxyfen is apparently capable of triggering melanization. The in vivo mode of action of pyriproxyfen was further investigated by quantifying hemolymph ecdysteroids by radioimmunoassays. Topical application leads to a delay of the pupal ecdysteroid peak by 4 days. The pyriproxyfen-induced low ecdysteroid titers during early pupal development could account for precocious pigmentation by removing an inhibition on prophenoloxidase activation normally imposed by the elevated ecdysteroid titer during this phase.     

Hormone-dependent protein patterns in integument and cuticular pigmentation in Apis mellifera during pharate adult development

The epidermal proteins from staged Apis mellifera pupae and pharate adults and the progress of cuticular pigmentation until adult eclosion were used as parameters to study integument differentiation under hormonal treatment. Groups of bees were treated at the beginning of the pupal stage with the juvenile hormone analog pyriproxyfen (PPN) or as pharate adults with 20-hydroxyecdysone (20E). Another group was treated with both hormones applied successively at these same developmental periods. Controls were maintained without treatment. The epidermal proteins, separated by SDS-PAGE and identified by silver staining, were studied at seven intervals during the pupal and pharate adult stages. The initiation and progress of cuticular pigmentation was also monitored and compared to controls. The results showed that PPN reduced the interval of expression of some epidermal proteins, whereas 20E had an antagonistic effect, promoting a prolongation in the time of expression of the same proteins. In PPN-treated bees, cuticular pigmentation started precociously, whereas in 20E-treated individuals this developmental event was postponed. The double hormonal treatment restored the normal progress of cuticular pigmentation and, to a large extent, the temporal epidermal protein pattern. These results are discussed in relation to the 20E titer modulation and morphogenetic hormone interaction.     

Can larval epidermis omit the secretion of a pupal cuticle in a saturniid moth, Samia cynthia ricini?

When the larval tissue is exposed to the hormonal milieu lacking juvenile hormone, adult characters appear directly omitting the pupal stage in some insects but not in others. In Samia cynthia ricini, a species belonging to the latter group, a possible omission of pupal characters was tested by previously untried experiments. Firstly, the possibility that the larval epidermis of only some stages is capable of responding so as to omit to secrete the pupal cuticle was tested. Pieces of larval integument taken from various developmental stages were implanted into developing (pharate) adults. None of these failed to secrete the pupal cuticle. Secondly, pieces of larval integument were first implanted into brainless pupae and left there for a month to eliminate the effect of a trace of juvenile hormone which might have been carried over by the implants. They were then caused to develop, and they again secreted pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle in this species.     

Ecdysteroid levels and integument changes in post-embryonic stages of Anastrepha suspensa

Ecdysteroid levels in larvae and pupae of Anastrepha suspensa (Diptera: Tephritidae) were measured by radioimmunoassay. These levels were correlated with histological changes throughout the development of the post-embryonic stages. Ecdysteroid levels increase rapidly throughout the last-larval instar and on the last day of this stage are 283 pg equivalents of 20-hydroxyecdysone per insect (14.5 ng/g) when wandering behaviour is initiated. At the end of this period the puparium is formed and within 24 h, the ecdysteroid rises to its highest peak (625 pg equivalents of 20-hydroxyecdysone/insect). Larval-pupal apolysis is initiated within 24 h later and the pupal cuticle is then secreted. Two days later, the ecdysteroids reach their lowest levels (75 pg equivalents of 20-hydroxyecdysone/insect or 0.6 ng/g) and most of the larval fat body and other tissues have been histolysed. In 5- to 10-day old pupae ecdysteroid levels again increase and remain relatively high throughout. During this period the larval epidermis is replaced by imaginal epidermis, imaginal discs begin to proliferate and the adult cuticle is secreted. Ecdysteroid levels finally fall 2 days prior to adult emergence. HPLC determinations indicate that 20-hydroxyecdysone is the predominant free ecdysteroid, and along with ecdysone, is readily detectable in all postembryonic stages of this species. An unusually high and unexplained peak of 20-hydroxyecdysone occurs in the pharate adult. This peak probably consists of ecdysone metabolites with retentions similar to that of 20-hydroxyecdysone and to which the antiserum is sensitive.     

Disturbance of adult eclosion by fenoxycarb in the silkworm,Bombyx mori

Fenoxycarb treatment before and after pupal ecdysis of Bombyx mori disturbed adult eclosion and the animals were unable to escape from the pupal exuviae. This effect of fenoxycarb was dose and time dependent with the highest sensitivity around the pupal ecdysis. The sensitivity rapidly diminished within 20 hours of pupal ecdysis. Twenty-hydroxyecdysone (20E) produced similar effects. Fenoxycarb injection at the pupal ecdysis induced higher ecdysteroid production by the prothoracic glands and higher PTTH-secretory activity in the brain-corpora cardiaca-corpora allata complexes. As a result, the fenoxycarb treated pupae contained higher ecdysteroid titres in the haemolymph. Both the fenoxycarb and the 20E treatments resulted in the lack of development of the rectum in pharate adults. This was the main cause of high ecdysteroid titres in the pharate adult stage. This effect was mimicked by either removal of the rectum early in the pharate adult stage or a surgical extirpation of the hindgut at the time of pupal ecdysis. These results suggest that the disturbance of adult eclosion by fenoxycarb is due in part to the inability of the formation of the rectum in the pharate adult stage.     

Hormonal basis of adult eclosion in the gypsy moth (Lepidoptera: Lymantriidae)

Eclosion hormone was found to control the stereotypic adult eclosion behaviour of Lymantria dispar, the gypsy moth. A bioassay for hormonal activity was developed utilizing pharate adult females, and comparisons were made with the Manduca wing assay. The distribution of eclosion hormone activity was confined to the central nervous system tissues including the protocerebrum, corpora allata/corpora cardiaca complex, thoracic and the last abdominal ganglion. Haemolymph ecdysteroid titres were determined daily throughout pupal-adult development, and the peak activity period was found in 3–4 day pupae. Eclosion hormone activity in the brain and corpora allata/corpora cardiaca complex started to increase when the ecdysteroid titre dropped to background levels. Eclosion hormone in the brain peaked in the pharate adult stage, was released in the haemolymph 1 h prior to eclosion, which coincides with the depletion of activity in the retrocerebral complex, and fell to undetectable levels after the adult emerged.     

Factors influencing the developmental capacity of Galleria larval epidermis

The nature of the cuticle secreted by integument from a day-1 penultimate instar larval Galleria when cultured in vivo in the abdomen of a last instar larva varied with the age of the host. When placed in a day-5 last instar larva, the implanted integument secreted a pupal cuticle at the time the host metamorphosed and became a pupa. However, when placed in a day-7 last instar larva the implant, from the same stage donor, secreted a larval cuticle at the time the host pupated. Experimental studies involving implantation of the integument for a 24 hr period, into various developmental stages of normal and ligated last instar larvae, pupae, and pharate adults, prior to placing it in a day-7 last instar larva suggest that a non-hormonal factor present in day-4 and -5 last instar larvae is important to initiate pupal syntheses.     

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.     

Ecdysteroid titers in pupae of highly social bees relate to distinct modes of caste development

Modifications in endocrine programs are common mechanisms that generate alternative phenotypes. In order to understand how such changes may have evolved, we analyzed the pupal ecdysteroid titers in two closely related, highly social bees: the honey bee, Apis mellifera, and a stingless bee, Melipona quadrifasciata. In both species, the ecdysteroid titers in queens reached their peak levels earlier than in workers. Titer levels at peak maxima did not differ for the honey bee castes, but in Melipona they were twofold higher in queens than in workers. During the second half of pupal development, when the ecdysteroid titers decrease and the cuticle progressively melanizes, the titer in honey bee queens remained higher than in workers, while the reverse situation was observed in Melipona. Application of the juvenile hormone analog Pyriproxyfen^® to spinning-stage larvae of Melipona induced queen development. Endocrinologically this was manifest in a queen-like profile of the pupal ecdysteroid titer. Comparing these data with previous results on preimaginal hormone titers in another stingless bee, we conclude that the timing and height of the pupal ecdysteroid peak may depend on the nature of the specific stimuli that initially trigger diverging queen/worker development. In contrast, the interspecific differences in the late pupal ecdysteroid titer profiles mainly seem to be related to caste-specific programs in tissue differentiation, including cuticle pigmentation.     

Rectal sac distention is induced by 20-hydroxyecdysone in the pupa of Bombyx mori

Holometabolous insects do not excrete but store metabolic wastes during the pupal period. The waste is called meconium and is purged after adult emergence. Although the contents of meconium are well-studied, the developmental and physiological regulation of meconium accumulation is poorly understood. In Bombyx mori, meconium is accumulated in the rectal sac; thereby, the rectal sac distends at the late pupal stage. Here, we show that rectal sac distention occurs between 4 and 5 days after pupation. The distention is halted by brain-removal just after larval-pupal ecdysis but not by brain-removal 1 day after pupation. In the pupae, brain-removal just after ecdysis kept the hemolymph ecdysteroid titer low during early and mid-pupal stages. An injection of 20-hydroxyecdysone (20E) evoked the distention that was halted by brain-removal in a dose-dependent manner. Therefore, brain-removal caused the lack of ecdysteroid, and rectal sac distention did not appear in the brain-removed pupae because of the lack of ecdysteroid. We conclude that rectal sac distention is one of the developmental events regulated by 20E during the pupal period in B. mori.     

Development of the prepupal Verson's gland of the tobacco hornworm, Manduca sexta, and its hormonal control

The segmentally arranged Verson's glands are epidermal derivatives comprised of three cells: the duct, saccule, and secretory cells. The development of these glands was followed through the 5th instar and larval-pupal transition of Manduca sexta. The glands are relatively small during the feeding stage, begin to grow at wandering, and undergo about a 50-fold increase in size during the prepupal period. The increase in size is due mainly to the hypertrophy of the secretory cell which synthesizes a heterogeneous set of proteinaceous secretory products. Three prominent 11 to 12 kiloDalton (kD) polypeptides are made by the pharate fifth larval gland, whereas the pupal gland produces polypeptides ranging from 14 to 75 kD with a major complex at 30 to 34 kD. The secretory product is poured out onto the surface of the new cuticle at the time of ecdysis and contains all of the major proteins detected in extracts of the whole gland. The accumulation of secretory products by the gland occurs during the prepupal peak of ecdysteroid and is blocked if this rise is prevented by abdominal isolation. Infusion of 30 micrograms 20-hydroxyecdysone (20-HE) into such isolated abdomens caused synthesis of the pupal products. Treatment with the juvenile hormone mimic, methoprene, during the fifth instar showed that the commitment of the glands to produce the pupal proteins is independent of and occurs before the overlying epidermis becomes committed to make pupal cuticle.     

Diflubenzuron-Induced alterations during in vitro development of Tenebrio molitor pupal integument

The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20-hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (? 10 μg/ml) resulted in epicuticle deposition. These observations confirm in vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells. In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after in vivo DFB treatment in Tenebrio pupae.     

Monoclonal antibodies recognizing larval- and pupal-specific cuticular proteins of Tenebrio molitor (Insecta,Coleoptera)

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type 2, a 26 kDa polypeptide, and type 3, three polypeptides of 18.5, 19.5 and 21.5 kDa. They did not immunoreact with any protein of fat bodies or haemolymph from pharate pupae, suggesting that the antigens originate from the epidermis. The stage-specificity was confirmed by electron microscopic immunogold labelling. Type 1 and 3 MABs recognized antigens characterizing larval and pupal preecdysial sclerotized cuticles, while the antigens recognized by type 2 were localized in the first few lamellae of unsclerotized postecdysial cuticle. When the expression of the adult programme was inhibited by application of a juvenile hormone analogue, the larval-/pupal-specific CPs were detected in the supernumerary pupal cuticle. These results suggest that the genes encoding these proteins are juvenile hormone dependent. These MABs should be useful tools to isolate pupal-specific genes whose regulation sems to be different from that of the adult-specific ones.     

Haemolymph ecdysteroid titres in diapause- and non-diapause-destined larvae and pupae of Sarcophaga argyrostoma

No differences were observed between the rates of development of larvae and pupae from diapause- and non-diapause-destined lines of Sarcophaga argyrostoma except that those destined for diapause have a longer post-feeding, wandering, larval phase associated with a lower haemolymph ecdysteroid titre, as measured by radioimmunoassay. Following pupariation, both cultures show a high haemolymph titre associated with larval/pupal apolysis. The developing culture displays an ecdysteroid peak at 72 h after pupariation which may be involved with pupal/adult apolysis and the initiation of pharate-adult development. This peak is reduced in the diapause-destined culture. Following the initiation of pharate adult development, there is a very large peak at 85–90 h. Those pupae entering diapause display very low titres as a result of the failure of the brain/prothoracic gland axis to release ecdysone. There are no quantitative or qualitative differences between the titres of specific ecdysteroids in the prepupae of the two lines as determined by reverse-phase high-performance liquid chromatography. A preliminary examination of the levels of free and conjugated ecdysteroids has provided the basis for proposing a mechanism of ecdysone metabolism in this insect.     

棉铃虫蛹期血淋巴的蜕皮甾类

目前为止仅在少数几种昆虫中研究过蛹期的蜕皮激素。关于蜕皮甾类的性质分析,结果也颇不一致。本文采用放射免疫分析、薄层层析、高压液相色谱及质谱对棉铃虫Heliothis armigera蛹血淋巴内的蜕皮激素进行了研究。结果如下:1.物理-化学方法证明蛹血淋巴内存在二种蜕皮甾类:蜕皮酮和20-羟基蜕皮酮。2.蛹期蜕皮甾类滴度呈一宽峰,高峰出现在化蛹后的第5天(3435ng/ml)。3.在高峰时,蜕皮酮与20-羟基蜕皮酮的比例为1:3.57,说明20-羟基蜕皮酮是主要的蜕皮甾类。4.比较雌雄两性蛹的蜕皮甾类滴度,未见明显差异。研究表明在棉铃虫中影响成虫发育的主要激素是20-羟基蜕皮酮而不是蜕皮酮。     

Induction of dauer pupae by fenoxycarb in the silkworm,Bombyx mori

Topical application of fenoxycarb (1 μg per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.