Development of the wing discs of Zophobas atratus under natural and experimental conditions: occurrence of a gradual larval-pupal commitment in the epidermis of tenebrionid beetles

Using light and electron microscopy, we studied the development of the wing discs in the large beetle Zophobas atratus, under natural and experimental conditions. A reversible differentiation of the wing discs is usually observed during supernumerary instars of crowded larvae. Juvenile hormone analog (JHA) application during the wandering period or compelled experimental crowding during the larval-pupal switchover - or commitment - inhibits the onset of metamorphosis. Isolation, followed by recrowding, also induces the disc cells to secrete unusual cuticular material. Recrowding is able to trigger the reversal of metamorphosis during the 4-day period when larval-pupal commitment is taking place. Likewise, feeding behaviour which normally stops at commitment often recovers. Ecdysis of intermediate instar animals (prothetelic larvae) corroborates the occurrence of a temporal and spatial variation to commitment, unique to each organ. All these data lead us to consider this 4-day period, which we have called the C period or commitment period, extending from the wandering stage (the previous T period) to the crooked posture stage (i.e. from eyestage 4 to 7) as the physiological time during which the larval organs are gradually committed to differentiate into pupal organs.     

The onset of metamorphosis in Tenebrio molitor L.: Effects of a juvenile hormone analogue and of 20-hydroxyecdysone

Sensitivity to juvenile hormone and to 20-hydroxyecdysone has been investigated during the last-larval stages of Tenebrio molitor. Topical applications of a juvenile hormone analogue (K-421d) showed that the sensitive period, occurring before apolysis, is relatively short (less than 4 days in a 3-week instar) and divided into two phases. Treatment during the first and longest phase induced a delay in development and then an increase in larval moult percentage. Treatment during the second phase induced several abnormal moults (prothetelic larvae and larval-pupal intermediates).Injections of massive doses of 20-hydroxyecdysone (10 μg per animal) also evidenced a period of disturbance of the morphogenetic programme, beginning before pupal apolysis but continuing several days after.Comparison of the sensitive periods to both hormones suggests that a very important and rapid step of the larval-pupal programme change is controlled hormonally just before pupal apolysis.     

Control of juvenile hormone esterase activity in Galleria mellonella larvae

The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae.JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.     

Morphogenesis of the wing Anlagen in the mealworm beetle tenebrio molitor during the last larval instar

The wing Anlagen of Tenebrio develop from epidermal cells located on the lateral margins of meso- and metathoraces. Three to four days after larval ecdysis, these cells start to proliferate slowly, continuing to do so until day 13 which corresponds to the period of the pupal commitment of the remaining epidermis. The wing Anlagen cells then proliferate rapidly until day 18.5. Three days before pupal ecdysis, the mitotic index falls suddenly while 40% of the Anlagen cells disappear owing to cell degeneration. The sudden changes observed in the mitotic index are correlated with two hemolymphatic peaks in ecdysteroid levels. Anlagen of the forewings and hindwings show similar development except for cuticular secretion at the end of the instar which is greater on the upper face of the forewings. A comparison is made with imaginal discs and histoblasts described in other holometabolous insects.     

Ommochrome synthesis and kynurenic acid excretion in relation to metamorphosis and allatectomy in the stick insect, Carausius morosus Br.

End products of tryptophan metabolism in Carausius morosus are the ommochromes ommin and xanthommatin in the epidermis, and kynurenic acid in the faeces. During larval and adult life ommochromes and mainly kynurenic acid are formed. The concentration of kynurenic acid in the faeces of adult females is 2.5 times lower than in the larvae and in adult males. Allatectomy on the first day after a larval moult induces a much longer instar (10 days) than normal. After the following moult, the allatectomized animals are transformed into adultoids. The allatectomized and normal larvae produce similar amounts of kynurenic acid and ommochrome during the larval instar. Twenty days after last ecdysis, the ommochrome content in adult and adultoids is increased. In the faeces of adultoids, however, the concentration of kynurenic acid is higher than in normal female adults, but lower than in males and larvae.     

The hormonal regulation of the larval diapause in the codling moth, Laspeyresia pomonella (Lep. Tortricidae)

The hormonal control of the facultative diapause of the codling moth has been investigated. The diapause can be divided into 4 phases or periods: (1) diapause induction by short-day conditions (SD) in young larvae, (2) initiation of the diapause in the early last larval instar by a high titre of juvenile hormone, (3) onset and maintenance of diapause with inactivity of the neuroendocrine system, as evidenced by the results of neck-ligation experiments, (4)termination of diapause by the production of ecdysteroid.Diapause-induced larvae pupated after spinning the cocoon, if the state of induction was changed by injection with the anti-juvenile hormone precocene II at the beginning of the last larval instar and subsequent results of neck-ligation experiments, (4) termination of diapause by the production of ecdysteroid. treated with juvenile hormone during the first 1.5 days after the last larval moult and subsequently reared under SD. Under LD, continuous application of juvenile hormone during the last larval instar and after spinning did not prevent the insects from moulting to either a supernumerary larva, a pupa or a larval-pupal intermediate. Termination of diapause, i.e. pupation, was achieved by injecting diapausing larvae with 20-hydroxyecdysone. Although juvenile hormone was found to have a prothoractropic effect in diapausing larvae, no pupal moult could be induced by the application of the hormone. Contrary to the hormonal situation before pupation of nondiapausing larvae, no juvenile hormone could be detected before or during the pupation of larvae after diapause.     

Lepidopteran anti-juvenile hormones: Effects on development of Apanteles congregatus in Manduca sexta

Parasitism by the braconid wasp Apanteles congregatus decreases the effectiveness of the anti-juvenile hormone agents ETB (ethyl 4-[2-{ittert-butyl carbonyloxy}bytoxy]benzoate) and fluoromevalonolactone (FMev) in inducing precocious metamorphosis of Manduca sexta larvae. Topical application of 1–200 μg ETB to parasitized third-instar larvae had no effect on either host or parasite development, whereas doses of 50μg or more ETB applied to unparasitized third-instar larvae caused formation of larval-pupal intermediates after the fourth instar. Parasitism also decreased the effectiveness of 100–200 μg FMev in causing metamorphosis at the moult following its application. In contrast to ETB, FMev disrupted development of the parasitoids. No wasps emerged when preterminal stage hosts were treated with FMev and the hosts formed larval-pupal intermediates. After treatment of terminal stage hosts with FMev, the number of emerging parasitoids was reduced by one-third. Precocene II (100 μg per larvae) had no effect on development of either M. sexta or A. congregatus.     

Progress of developmental programme during the last larval instar of Bombyx mori: Relationships with food intake,ecdysteroids and juvenile hormone

The progress of developmental programme in the epidermal cells of last instar larvae of Bombyx mori was determined by ecdysteroid injections in normal and in JH-treated larvae. To clarify the importance of food intake in the control of development, starved animals were also used.The instar begins with a period during which the larval programme is expressed: this occurs in the presence of 20-hydroxyecdysone. Epidermal cells can thereafter secrete pupal cuticle after ecdysteroid injection although the larval programme is normally still present. During the last period only pupal characters can be expressed either in normal or in 20-hydroxyecdysone-injected larvae.These different developmental phases are not correlated with obligatory and facultative feeding periods.Transition from the first to the second phases is correlated with the absence of JH effects on pupal genes. JH applications during the second period, however, prevent the expression of pupal characters after 20-hydroxyecdysone injection. Thus, during this period, the pupal programme is not stabilized. Cellular reprogramming itself occurs at the onset of the last developmental period and is probably under the control of ecdysteroids.     

Effects of previous diet and frequency of feeding on development of larval damselflies

SUMMARY. 1. Food availability during instar F-3 affected duration of instar F-3 of larval Ischnura vertiealis (Say) maintained in the laboratory but did not affect the increase in head width at the moult to instar F-2.
2. Food availability during instar F-2 altered both duration in instar F-2 and weight per unit head width of newly moulted F-l larvae, but not the increase in head width at the moult to instar F-1.
3. Food availability during instar F-3 had no effect on instar duration, weight per unit head width, or increase in head width of F-2 larvae at the moult to instar F-l.
4. Survival of larval Coenagrion resolution (Hagen) reared in the laboratory was dependent on mean number of Daphnia offered per day. In addition, larvae fed less frequently, but on the same mean number of Daphnia per day. had lower survival rates than larvae fed more frequently.     

Age-dependent cyclic locomotor activity in the cricket, Gryllus bimaculatus, and the effect of adipokinetic hormone on locomotion and excitability

Excitability and locomotor activity of male and female last instar larvae and adults of the two-spotted cricket are measured under crowded conditions, allowing the animals to interact with conspecifics during observations. Male and female last instar larvae display age-dependent cyclic patterns of activity with maxima during early to mid scotophase and minima during early photophase. A period of low locomotor activity without time of day-dependent cyclic changes starts 1 day before the final moult and lasts until 1 day after the moult. Then, both excitability and locomotor activity increase and become cyclic again within 2 or 3 days. The cyclic changes gradually dampen in adult females older than 6 days and finally cease. When injected into photophase larvae and adults, adipokinetic hormone (AKH) increases excitability and locomotor activity in a dose-dependent manner, whereas it has no such effect when injected into scotophase animals. Other behaviours (jumping, hind wing trembling) that mostly occur in scotophase crickets are also increased by injecting AKH into photophase crickets. We argue that AKH could be responsible for linking the endogenous clock output with the cyclic changes in locomotor activity. Furthermore, AKH may serve to synchronise metabolism and behaviour to optimise larval development and reproduction.     

Insulin/IGF signaling regulates the change in commitment in imaginal discs and primordia by overriding the effect of juvenile hormone

At the beginning of the final larval (fifth) instar of Manduca sexta, imaginal precursors including wing discs and eye primordia initiate metamorphic changes, such as pupal commitment, patterning and cell proliferation. Juvenile hormone (JH) prevents these changes in earlier instars and in starved final instar larvae, but nutrient intake overcomes this effect of JH in the latter. In this study, we show that a molecular marker of pupal commitment, broad, is up-regulated in the wing discs by feeding on sucrose or by bovine insulin or Manduca bombyxin in starved final instar larvae. This effect of insulin could not be prevented by JH. In vitro insulin had no effect on broad expression but relieved the suppression of broad expression by JH. This effect of insulin was directly on the disc as shown by its reduction in the presence of insulin receptor dsRNA. In starved penultimate fourth instar larvae, broad expression in the wing disc was not up-regulated by insulin. The discs became responsive to this action of insulin during the molt to the fifth instar together with the ability to become pupally committed in response to 20-hydroxyecdysone. Thus, the Manduca bombyxin acts as a metamorphosis-initiating factor in the imaginal precursors.     

A reinvestigation of the effects of cortisol on growth in insects

Incorporation of cortisol, or various derivatives and analogues of cortisol, into the diet had no effect on the rate of growth of Tenebrio molitor. Injections of cortisol, or its derivatives, into fourth instar larvae of Schistocerca americana gregaria, had no effect on their rate of growth. The moult to fifth instar was normal. Administration in the diet, or injections of cortisol had no effect on the rate of growth of fourth and fifth instar larvae of Manduca sexta, or on the insect's uptake and utilization of food. The development of larvae to pupae and adults was unaffected. It is concluded, contrary to the findings of two previously published reports, that the vertebrate hormone cortisol does not affect the growth and development of insects.     

The role of eclosion hormone in the larval ecdyses of Manduca sexta

Each larval moult in Manduca sexta consists of an identical series of developmental and behavioural events leading up to ecdysis. Injections of eclosion hormone into staged larvae in any instar resulted in the premature elicitation of the larval pre-ecdysis behaviour, comprising a rhythmic sequence of muscle contractions, followed by the larval ecdysis behaviour.A marked depletion of eclosion hormone stores form the ventral chain of ganglia coincided with each larval ecdysis and in the moult to the fifth instar, eclosion hormone activity appeared in the blood at the onset of the pre-ecdysis behaviour.Responsiveness to eclosion hormone for pre-ecdysis and ecdysis behaviour developed about 12 and 6 hr before normal ecdysis, respectively. Elicitation of ecdysis behaviour by exogenous hormone inhibited both subsequent behavioural responses to eclosion hormone and endogenous hormonal release.In conclusion, the behavioural programme involved in each larval ecdysis appears to be controlled by the eclosion hormone.     

Inhibitory effects of parasitism by the gregarious endoparasitoid Cotesia congregata on host testicular development

Cotesia congregata is a gregarious larval endoparasitoid of the tobacco hornworm, Manduca sexta. Parasitized larvae exhibit a variety of physiological and developmental aberrations, the most obvious of which is the induction of developmental arrest characterized by the absence of wandering behavior and suppression of pupation. This arrest appears attributable to continued maintenance of an elevated titer of juvenile hormone and reduced levels of hemolymph juvenile hormone esterase activity. Injection of the wasp's polydnavirus into nonparasitized larvae also causes arrest and the larvae eventually form larval-pupal intermediates instead of normal pupae, indicating the virus may be partially responsible. Aside from causing arrested host development, parasitism also inhibits the normal development and differentiation of testes in male host larvae, so that the testes atrophy instead of growing synchronously with other larval tissues. Here we report that parasitism has pronounced disruptive cytological effects on the developing reproductive organs of male hosts, in addition to causing them to atrophy. Parasitism results in a reduction in testicular volume attributable to a reduction in the number of developing germ cells. Microscopy revealed that the structural integrity of the sheaths surrounding the testicular follicles also is disrupted, so that the tissues appear grossly abnormal compared to those of nonparasitized larvae. Intrahemocoelic injection of purified C. congregata polydnavirus in combination with venom into nonparasitized fourth instar larvae, or topical application of 100 μg of methoprene to fourth instar larvae, also alters sheath integrity and reduces the numbers of developing germ cells, but not to the same degree as the pattern observed in truly parasitized hosts. The occurrence of cell death in the male gonad was documented using the vital dyes acridine orange and ethidium bromide. Arch. Insect Biochem. Physiol. 36:95–114, 1997. © 1997 Wiley-Liss, Inc.     

Effects of starvation on moult cycle and hepatopancreas of Stage I lobster(Homarus americanus) larvae

Effects of feeding and starvation on the moult cycle and on the ultrastructure of hepatopancreas cells were studied in Stage I lobster larvae (Homarus americanus Milne-Edwards). The relative significance of yolk and first food was quite different in larvae originating from two females. This difference was evident also in the amounts of stored lipid in the R-cells of the larval hepatopancreas. Most larvae from one hatch were, in principle, able to develop exclusively with yolk reserves (without food) to the second instar. The larvae from the second hatch showed lecithotrophic development only to the transition between late intermoult and early premoult (Stages C/D₀ of Drachs's moult cycle) of the first larval instar. When initial starvation in this group lasted for 3 days or more, the point of no return (PNR) was exceeded. After the PNR, consumption of food was still possible, but development ceased in the transition C/D₀ or in late premoult (D_3–4). It is suggested that these stages of the moult cycle are critical points were cessation of development and increased mortality are particularly likely in early larval lobsters under nutritional stress. Examination of hepatopancreas R-cells suggested that the PNR is caused by an irreversible loss of the ability to restore lipid reserves depleted during initial starvation. Initial periods of starvation ending before the PNR prolonged mainly Stage D₀ of the same instar (I). During this delay, structural changes in the R-cells caused by the preceding period of starvation were reversed: reduced lipid inclusions, swollen mitochondria, an increased number of residual bodies indicating autolysis, and a reduction of the microvillous processes. Continually starved larvae which showed lecithotrophic development throughout the first instar and were then re-fed after moulting successfully, had later a prolonged intermoult (Stage C) period in the second instar. This shows that, despite occasional lecithotrophy, food is an important factor in early larval development of the lobster.     

Treatment of larvae with repeated doses of precocene II induces precocious metamorphic changes in Spodoptera mauritia (Lepidoptera: Noctuidae)

In Spodoptera mauritia repeated daily treatments of larvae with 40 μg of precocene II throughout the fifth and sixth instar larval period had no effect on the larval-larval period but prolonged larval-pupal ecdysis. The resulting pupae showed precocious adult differentiation of mouth parts, wings, eyes, legs, and fat body.     

Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.     

Ecdysteroid titres during larval life and metamorphosis of Galleria mellonella

The ecdysteroid titre in Galleria larvae normally follows either a short larval cycle, which is characterized by a single peak on day 2, or a long metamorphic cycle, which includes a small peak on day 6 and a large one on day 7. Intermediate cycles and moult induction with reduced ecdysteroid concentrations occur under experimental conditions. The cycles and peaks are initiated and, until the brain critical period, driven by prothoracicotropic hormone (PTTH), while juvenile hormone (JH) titre determines which type of cycle will be activated. In larval development JH stimulates PTTH release but the metamorphic programme of neuroendocrine regulations involves blocking PTTH production by JH. This hormone then causes an interruption of the ecdysteroid cycle and extension of the last larval instar.