Age-dependent changes of fat body stores and the regulation of fat body lipid synthesis and mobilisation by adipokinetic hormone in the last larval instar of the cricket, Gryllus bimaculatus

Data on the hormonal regulation of the formation and mobilisation of fat body stores are presented and discussed in relation to general parameters of last instar larval development such as growth, food intake, and moulting. Crickets feed voraciously during the first half of the last larval stage. With the onset of feeding, fat body lipid synthesis increases, leading to increasing lipid stores in the fat body with a maximum reached on day 5. Lipid (42% of fat body fresh mass) is the main constituent of the fat body stores, followed by protein (6%) and glycogen (2%). During the second half of the last larval stage, feeding activity dramatically decreases, the glycogen reserves are depleted but lipid and protein reserves in the fat body remain at a high level except for the last day of the last larval stage when lipid and protein in the fat body are also largely depleted. The process of moulting consumes almost three quarters of the caloric equivalents that were acquired during the last larval stage. Adipokinetic hormone (AKH) inhibits effectively the synthesis of lipids in the larval fat body. Furthermore, AKH stimulates lipid mobilisation by activating fat body triacylglycerol lipase (TGL) in last larval and adult crickets. Both effects of AKH are weaker in larvae than in adults. This is the first report on the age-dependent basal activity of TGL in larval and adult insects. In addition, for the first time, an activation of TGL by AKH in a larval insect is shown.     

Prothoracic gland activity and blood titres of ecdysone and ecdysterone during the last larval instar of Locusta migratoria L.

The kinetics of secretion of ecdysone by the prothoracic glands of Locusta migratoria were studied during the last larval instar. Three stages of intense production of ecdysone (α-ecdysone) were monitored during this developmental period: they correspond to three peaks of moulting hormone concentration in the blood, which indicates that the main regulation of the moulting hormone titre is achieved through variations in prothoracic gland activity. In the haemolymph the ratio of ecdysone to ecdysterone (20-hydroxy-ecdysone) is in favour of ecdysone during the two first moulting hormone peaks ecdysterone being by far predominant over ecdysone at the time of the third (major) peak; these results support previous studies on the metabolic fate of injected labelled ecdysone during the same developmental period in Locusta migratoria.     

Influence of leg regeneration on ecdysteroid titres in Acheta larvae

The effects of regeneration on ecdysteroid levels and duration of the 10th larval instar of female house crickets were studied. Three experimental groups were used. The first consisted of larvae regenerating two legs cut off on the first day of this stage. The second group was made up of insects that underwent either an epidermal incision (sham-operated group I) or bleeding (sham-operated group II) at the same time as amputation in the first group. The last group was composed of normal insects.Larval stage length: in the first two groups, the duration of the 10th larval stage was significantly modified. It increased for regenerating animals as well as for sham-operated group I, but decreased for sham-operated group II.Ecdysteroid production: controls showed two periods of intense ecdysteroid activity. The first, which took place between days 4 and 6, was assumed to induce apolysis of the tegument and to render regeneration impossible (it coincided with the so-called critical period for regeneration). The second, which occurred between days 7 and 8 initiated cuticle synthesis. For sham-operated insects neither epidermal injury nor a loss of blood appear to change this hormonal profile significantly. Regenerating crickets however exhibited drastically reduced ecdysteroid peaks: the total apparent production of ecdysteriods was 50% below normal. Moreover, relative concentration of ecdysone to 20-hydroxy-ecdysone was greatly increased. Thus, regeneration obviously has a great and specific influence on ecdysteroid release in insect larvae. This effect may be related to changes observed in the prothoracic gland cycle which suggest that it plays a complex role in the regulation of the regenerative process.     

Moulting hormone level during the last instar of Galleria mellonella larva

Using radioimmunoassay the moulting hormone titres of the greater wax moth were determined during the last larval instar. Two peaks were observed, one when the larvae start to spin and another just before the pupation. The second peak exhibits the higher MH level, equivalent to 3600 ng/g ecdysterone. By TLC-RIA analysis three compounds were detected: ecdysone, ecdysterone and a very polar metabolite (VPM). The pattern of MHs during the last larval instar is described and the possible changes in the activity of enzymes of MH metabolism and ecdysone-ecdysterone conversion is discussed.     

Moulting and ecdysone release in response to electrical stimulation of protocerebral neurosecretory cells in Locusta migratoria

Locusta migratoria larvae were submitted to electrical stimulation of the protocerebral neurosecretory cells (median neurosecretory cells of the pars intercerebralis and lateral neurosecretory cells), during the last larval instar. The effects of the treatment were observed both on the duration of the stage and on the variations in haemolymph ecdysone levels. In untreated larvae, there was an initial ecdysone peak at the beginning of day 5, which was followed by 4 larger peaks between days 6 and 8. Stimulation of the median neurosecretory cells at the beginning of the instar resulted in the formation of one very large hormonal peak at the end of day 3: a day and a half earlier than in the control groups. Moulting was likewise accelerated. Stimulation also increased the size of the peaks, as compared with the controls. Stimulation of the lateral neurosecretory cells had a weaker ecdysiotropic effect; neither the number nor the size of the peaks were changed, though, like ecdysis, they occurred earlier. Stimulation of the deutocerebrum had no effect on either ecdysone titres or moulting. Electrical stimulation of the median neurosecretory cells at the end of day 5, that is after the occurrence of the first ecdysone peak, shortened the larval stage while having no significant effect on ecdysone levels in the haemolymph. The neuroendocrine control of ecdysis in Locusta is discussed.     

Hormonal regulation of pupation in the codling moth, Laspeyresia pomonella

ABSTRACT. According to the different reactions to the juvenoid Altosid®, the last larval instar (L₅) of Laspeyresia pomonella (L.) (Tortricidae) reared under 'long day' conditions (constant light) was subdivided into three sensitive phases: an additional larval instar, a larval–pupal intermediate, or a pupa. Under short day conditions, the prothoracotropic effect of juvenile hormone (JH) in L₅, which have a continuous high titre of JH during the whole instar, indicated that it is not a particular titre of JH but a rise in the titre that can induce the production of moulting hormone. Neck-ligation experiments showed that JH acts not directly on the prothoracic glands but via the head, probably via the neurosecretory system. The meaning of the JH-peak in mature L₅ reared under long days was determined either by injections with the anti-JH, precocene II, in combination with applications of Altosid, or by forcing precocene-treated larvae to a precocious moult by injecting them with ecdysterone. Precocene delayed, and JH accelerated pupation if administered 4.5 days after the L₅ -moult. JH was also found to stimulate the growth and differentiation of the imaginal discs. Moulting hormone in long-days reared insects was detected one day after the larvae had spun their cocoon, with a maximum on the second day after spinning. The hormone was also present in freshly moulted pupae. Neck-ligation of mature larvae indicated that the delay between activation of the prothoracic glands and the production of an effective amount of moulting hormone is less than one day.     

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.     

The metabolism of 3H-moulting hormone in Calliphora erythrocephala during larval development

The activity in whole insects for converting ³H-α-ecdysone to ³H-β-ecdysone after injection is low (half-maximal) in young last instar larvae, maximal in mature larvae, and minimal (fourth-maximal) at the white puparial stage. Because moulting hormone titre is low throughout the last larval instar and increases at the formation of the puparium it appears that hydroxylation at C-20 is not a key step in regulating β-ecdysone biosynthesis during larval development.The activity for catabolizing ³H-β-ecdysone is maximal in second instar larvae, about thirdmaximal throughout most of the third instar, and minimal at pupariation (thirtieth-maximal). Thus inactivation may play a rôle in regulating moulting hormone titre during larval development.     

Temporal organization of endocrine events underlying larval-larval ecdysis in the silkworm, Bombyx mori

The timing of ecdysis in the penultimate instar of Bombyx mori was demonstrated to be under the control of a circadian clock. The temporal organization of secretion of prothoracicotropic hormone (PTTH), ecdysone and juvenile hormone was studied with particular attention to the circadian control of the timing of hormone release. PTTH release occurs, at least, in the second and third night. The latter is responsible for evoking the larval ecdysis. Prothoracic gland initiates ecdysone secretion abruptly with a very short span after the second PTTH release and secrete enough amount of ecdysone for larval moulting, which takes place 11 h later. Juvenile hormone titer is relatively high before the second PTTH release and corpus allatum becomes dispensable for ensuring the larval moulting in 1.5 h. Based on these findings, interpretations for the endocrine system underlying precocious pupation and formation of intermediates, which are produced by neck ligation, are presented.     

Haemolymph ecdysteroid titres during larval-adult development in Rhodnius prolixus: Correlations with moulting hormone action and brain neurosecretory cell activity

A haemolymph ecdysteroid titre of the fifth (last)-larval instar of the hemipteran, Rhodnius prolixus has been determined by radioimmunoassay. During the last-larval stadium the ecdysteroid titre increases from a negligible level in the unfed insect to a detectable level within minutes following a blood meal. The titre reaches a plateau of ～50–70 ng/ml at 3–4 hr and this level is maintained until day 5–6, the time of the head-critical period in Rhodnius. At the head-critical period the titre begins to increase again, this time dramatically, reaching a peak of ～ 3500 ng/ml at day 13. From day 14 to ecdysis (day 21) the titre declines to a low level, ～ 30 ng/ml. Basal levels of ecdysteroids, ～ 15 ng/ml, were detectable in young adult males and females. A survey of haemolymph volumes during the last-larval instar indicates that the changes in the ecdysteroid titre reflect changes in the rates of ecdysteroid synthesis, and not changes in haemolymph volume. Excretion of ecdysteroids varies systematically during the instar, suggesting that control of ecdysteroid excretion may be important in regulation of the haemolymph titre. Qualitative analysis of the haemolymph ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxy-ecdysone. For the large peak preceding larval-adult ecdysis, 20-hydroxy-ecdysone was the predominant hormone. These results indicate that there may be two periods of release of prothoracicotropic hormone (PTTH) from the brain in Rhodnius, one immediately following the blood meal and the second on day 5 or 6. The significance of these times of PTTH release is discussed in relation to classical evidence of the timing of moulting hormone action, the response of target tissues, and with more recent findings on the timing of release of neurosecretory material from the brain of Rhodnius during moulting.     

Action in vivo d'ecdysones sur la morphogénèse imaginale d'Aeshna cyanea (Odonata)

Single amounts of α or β ecdysone were injected during the last larval instar of Aeshna cyanea at various times after ecdysis. In these experimental conditions, α and β ecdysone had similar effects. Very large amounts of brown or black cuticle appeared on the tarsal claws soon after hormone injection, so that the cuticular synthesis of the larvae which were injected at the beginning of the last stage appears about two or three times more quickly than in controls. Nearly all the larval characters were exhibited by animals injected on the day of or the day after the last larval ecdysis. If the hormonal injection was further delayed, only adultoid forms were obtained. No perfect adults appeared. The effects evoked by α or β ecdysone may be different from one organ to another.On the other hand, some results were different according to the type of ecdysone. Darkening of the tarsal claws (and perhaps sclerotization) appears sooner when β ecdysone is supplied. The morphology of the external organs which degenerate during metamorphosis is not always the same after injection of equal amounts of α or β ecdysone at the same time. The regression of the larval organs seems to be more explicit and appears sooner when β ecdysone was administrated. The morphogenesis of the organs which grow during metamorphosis was either weaker or non-existent with β ecdysone.These results are discussed with regard to previous work.     

Spiny lobster development: mechanisms inducing metamorphosis to the puerulus: a review

This review outlines current knowledge of mechanisms effecting metamorphosis in decapod crustaceans and insects. The comparative approach demonstrates some of the complexities that need resolving to find an answer to the question raised frequently by ecologists: “What triggers metamorphosis in spiny lobsters?” It is evident that crustacean moulting and metamorphosis are genetically controlled through endocrine systems that mediate gene expression. The molecular mechanisms underlying these developmental processes have been studied intensively in insects, particularly in the fruitfly, Drosophila melanogaster (Diptera), and some lepidopteran species. Comparatively, there is minimal information available for a few decapod crustacean species, but none for spiny lobsters (Palinuridae). Nothing was known of hormone signalling transduction pathways, via nuclear receptors (NRs) and gene activation during larval moults in palinurids—until a recent, ground-breaking study of early phyllosomal development of Panulirus ornatus by Wilson et al. (Rock Lobster Enhancement and Aquaculture Subprogram. FRDC Project 2000/263, Australian Govt, Fisheries Research and Development Corporation and Australian Institute of Marine Science, Nov 2005). Their study not only identified homologues of five hormone NRs of D. melanogaster, but also patterns of gene regulation showing strong similarities to those of gene expression found in insect larval development. Their results indicated that control of moulting and metamorphosis in palinurids closely parallels that in insects, suggesting that insects can serve as model systems for elucidating molecular mechanisms in larval decapods. In insects and crustaceans, the steroid hormone, ecdysone, (20E) initiates moulting. In insects, juvenile hormone (JH) mediates the type of larval moult that occurs, either anamorphic or metamorphic. The latter results when the level of JH in the haemolymph drops in the final larval instar. High levels of JH inhibit the metamorphic moult during insect larval development. The interaction of 20E and JH is not fully understood, and the operative molecular mechanisms are still being elucidated. No nuclear receptor for JH has been identified, and alternative JH signalling pathways await identification. In decapod crustaceans, methyl farnesoate (MF), a precursor of JH, replaces the latter in other functions mediated by JH in insects; but there is little evidence indicating that MF plays a similar ‘antimetamorphic’ role in decapod larval moults.     

Ecdysteroid levels and developmental events during larval moulting in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, apolysis of the spiracle is the first visible sign of the initiation of a larval moult. After spiracular apolysis, the characteristic sequence of new spiracle formation can be recognized easily through the cuticle around the old spiracle. This sequence, called the spiracle index, was used as the basis for a precise developmental chronology of the larval moulting period. At certain spiracle-index stages, histological changes in the larval ventral abdominal integument were also examined. By allatectomy and abdominal ligations, the critical periods for the secretion of juvenile hormone and ecdysone respectively were found to be at the initiation of spiracular apolysis and at the time when general apolysis was complete. The haemolymph-ecdysteroid level as measured by radioimmunoassay was about 30–50 ng/ml during the first 2 days of the 4th instar, 60–70 ng/ml at the time of initiation of spiracular apolysis, and 200 ng/ml at the time of completion of apolysis of the general body surface. The maximal level of the hormone was about 290 ng/ml at the mid-moulting period. The relationship between the ecdysteroid titre and morphological events occurring during the larval moult was discussed.     

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.     

Reduced titres of ecdysone following juvenile hormone treatment in the German cockroach, Blattella germanica

Changes in ecdysone titre of the larvae of the German cockroach, Blattella germanica, exposed continuously to the juvenile hormone (JH), or to the insect growth regulator (IGR) with JH activity, can be correlated with the nature of the substance applied, its dose, and the time of application. The younger larvae exposed to the high dose of the IGR die in the next ecdysis, whereas the same treatment induces a diapause-like stage of developmental arrest in the last larval stage. The affected larvae have very little or no ecdysone, the synthesis of which takes place in the second part of the instar. The same treatment after this period has a lesser effect. The extent of the effect is correlated to the amount of ecdysone synthesized before the application of IGR. Last instar larvae exposed to the lower dose of the IGR or JH lack the peak of ecdysone normally found in the controls at the end of the second third of the instar when metamorphosis takes place. In these insects the first rise of the ecdysone titre begins towards the end of the instar, and ecdysis into the supernumerary larval stage is initiated when the ecdysone titre reached a level permitting ecdysis.A direct or indirect antagonism between these hormones, both fundamental to insect development, can explain the morphogenetic, inhibitory, and lethal effects observed in insects treated with JH or IGR with JH activity.     

Influence of a juvenile hormone analogue on vitellogenin synthesis and oögenesis in larvae of Nauphoeta cinerea

In experiments on the synthesis of the vitellogenic protein, farnesylmethylester, a juvenile hormone (JH) analogue, was injected into female Nauphoeta cinerea larvae at various stages during their development. Two and 4 days after injection, 2 μl of haemolymph were assayed in a vitellogenin immunodiffusion test. In second last and last instar larvae less than 6 days before adult ecdysis, high doses (100 μg) of farnesylmethylester are necessary to induce vitellogenin synthesis, whereas older last stage larvae and decapitated adults respond to small doses (1 μg) with the synthesis of vitellogenin. It seems that the competence to synthesize the vitellogenic protein changes at the time of induction of the moulting process. If farnesylmethylester is injected into last instar larvae with a supposedly high titre of ecdysone, the vitellogenic protein can be detected in the haemolymph of a small percentage of animals only.Oöcyte maturation can be observed in last instar larvae injected after the fifth to ninth day with farnesylmethylester. The observed volume changes of the corpora allata suggest that an absence of JH for a short time is necessary for the oöcytes to become competent to grow. Last instar larvae treated with farnesylmethylester become larval-adult intermediates with partly developed oöcytes, demonstrating a simultaneous juvenilizing and gonadotropic influence of the JH analogue. In last instar larvae injected with farnesylmethylester a partial degeneration of already maturing oöcytes is induced at the time when the ecdysone titre is supposedly high and the possible reasons for this are discussed.     

Sinusoidal magnetic fields and chawki (silkworm) rearing in sericulture

Effects of sinusoidal magnetic fields on chawki silkworm rearing have been studied. The experiment was conducted using a multi X Bi silkworm hybrid, PM x CSR 2. Disease-free layings were reared from hatching to cocooning and by subjecting first and second instar to three magnetic field frequencies: 0.1, 1.0, and 10 Hz at 1500 nT, pp, for six days at six hours per day. Controls were maintained simultaneously. Larval durations for both young-stage chawki (I and II) and late stage (III, IV, V) were calculated in days and hours, including the feeding and moulting periods for I to IV instars, and the feeding period for V instar up to the time of spinning. The study revealed that the magnetic exposures reduced both feeding and moulting times with no adverse effects on larval growth. The substantial reductions in time, 33 hours in instars I and II, and 64 hours in total larval duration, could be commercially important in chawki rearing, saving time, leaf consumption, and labor expenses.     

Ecdysones and imaginal disc development during the last larval instar of Pieris brassicae

Ecdysone haemolymph levels and in vivo development of imaginal wing discs have been studied during the last larval instar of Pieris brassicae.During this period, β-ecdysone variations show two successive peaks, the first one related to the induction of wandering stage, and the second (main) one to pupal cuticle synthesis. The observed situation is very similar to that of Manduca sexta. Imaginal wing disc growth is composed of several genetically programmed steps that need the presence of ecdysone, but do not appear very closely linked to circulating hormone levels. It seems that ecdysone haemolymph peaks should be considered as periods where ecdysone levels are above a threshold value.     

Humoral inhibition of the corpora allata in larvae of Diploptera punctata: Role of the brain and ecdysteroids

Juvenile hormone synthesis by adult female corpora allata was inhibited following implantation into final-larval-instar males; inhibition was prevented by decapitation of the larval hosts on day 11 (prior to the head critical period for moulting), but not by decapitation on day 13. Implantation of one larval protocerebrum restored inhibition of implanted corpora allata, demonstrating that the brain releases an inhibitory factor. Corpora allata implanted into larvae decapitated on day 11 were inhibited by injections of 20-hydroxyecdysone. Since treatment of corpora allata with 20-hydroxyecdysone in vitro did not inhibit juvenile hormone synthesis, ecdysteroids probably act indirectly on the corpora allata. Juvenile hormone synthesis and haemolymph ecdysteroid concentration were measured following implantation of corpora allata along with two larval brains into larval hosts. Brain implantation did not affect ecdysteroid concentration, but did inhibit juvenile hormone synthesis, even in animals with low haemolymph ecdysteroid concentration. Incubation with farnesoic acid stimulated juvenile hormone synthesis by corpora allata from males early in the final larval stadium, but not after day 8, showing that one of the final two reactions of juvenile hormone synthesis is rate-limiting in larval corpora allata at this stage. Adult female corpora allata which had been humorally inhibited by implantation into larvae were stimulated by farnesoic acid.