Inactive proteinases in silkmoth moulting gel

The moulting gel of silkmoths lacks proteolytic activity but contains an inactive form of the proteinases which are later found in the moulting fluid. These inactive enzymes are activatable in vitro by dilution, activation proceeding most rapidly at low ionic strength. Activation proceeds as a first-order process and is not autocatalytic. Approximately the full amount of proteinases ultimately found in moulting fluid are already present in the gel. Moulting gel does not inhibit the active proteinases of moulting fluid; moreover, the proteolytic activity elicited by dilution of the moulting gel does not disappear upon reconcentration. These observations suggest that the proteinases in moulting gel are not inhibited by a stable, dissociable inhibitor; they may be present either as compartmentalized active enzymes or as proenzymes. Several possible mechanisms for the in vivo activation at the time of gel to fluid transformation are discussed.     

Seasonal production of moulting hormone in the barnacle Semibalanus balanoides

An analytical procedure for the quantification of ecdysteroids (crustacean moulting hormone) in barnacles was devised so that minimum sample size could be used. A combination of solvent partitions, Sephadex chromatography, silylation and gas chromatography with electron capture detection was devised, enabling ecdysteroids to be determined down to 20 pg. This was used to determine the amount of moulting hormone in a population of barnacles over a 30 month period. Levels varied from barely detectable in winter months to a maximum value of 1.5 μg kg^− 1 of wet weight of barnacles in September. Polar conjugates of 20-hydroxyecdysone were detected only during the winter months. The number of barnacles moulting at any time corresponded roughly to the titre of hormone present at that time.     

Prothoracic gland involution related to moulting hormone levels during the metamorphosis of Tenebrio molitor L.

Prothoracic gland (PG) of Tenebrio shows ultrastructural changes which can be correlated with ecdysteroid levels (measured by radioimmunoassay) during larval-pupal development. However, the gland cells begin to degenerate before pupal-adult ecdysis: the PG involution is completed before the moulting hormone peak which triggers pupal-adult development. These facts strongly suggest that another endocrine organ produces moulting hormone needed for adult development.     

Effects of moulting hormone on RNA and cuticle synthesis in the epidermis of cockroach embryos cultured in vitro

In epidermal cells of embryos of Blabera cultured in a medium without exogenous moulting hormone, 2 peaks in RNA synthesis occur respectively on days 7 and 11, followed by two peaks in cuticular products synthesis: a peak in protein synthesis on day 16 and a peak in chitin synthesis on day 17. Addition of exogenous moulting hormone to the culture medium hastens the two peaks in RNA synthesis and brings them closer together on days 1 and 4. The peaks in protein and chitin synthesis are similarly brought forward, and both appear on day 5.Localisation of the synthesised cuticular products is a function of the stage of differentiation reached in culture by the epidermal cells. Initially, there are little of the synthesised products and they are distributed uniformly throughout the cells. Progressively, the rate of synthesis increases and the products appear concentrated at the tips of the cells and then pass into the developing cuticle. At the beginning, the whole thickness of the deposited cuticle is labelled, then only the basal regions. Finally, the intensity of the label decreases.The results of incorporation rates of uridine, proline and glucosamine, the observation of the localisation of the label, and the secretion of the cuticle show that RNA synthesis occur before protein and chitin syntheses which are tied to the secretion of the procuticle.The hormone acts directly on the epidermal cells by triggering, in a general manner, the events which usually occur under the endogenous moulting hormone control.     

Neutral metal chelator-sensitive protease in insect moulting fluid

Proteolytic activity in moulting fluid from the sphingid Manduca sexta has at least two pH optima; these occur at pH 7 and at pH 7·7. The latter activity is shown to be trypsin-like in that it is susceptible to inhibition by diisopropylfluorophosphate. By contrast, the peak at neutral pH consists of proteolytic activity not hitherto described in invertebrates. This activity shows little or no inhibition with diisopropylfluorophosphate or p-hydroxymercuribenzoate but is strongly inhibited by chelators such as 1,10-phenanthroline, 8-hydroxyquinoline, and EDTA. The neutral metal chelator-sensitive activity requires calcium but the inhibitor data permit the conclusion that the metal ion inhibited by the chelators belongs to the first transition series and thus cannot be calcium. The neutral protease appears to be similar to proteases previously characterized from bacteria and snake venom. In moulting fluid from Manduca, proteolytic activity in vitro is very low in the presence of 1,10-phenanthroline at every pH studied except pH 7·7; in vivo, ecdysis is inhibited in Manduca larvae fed on diet containing a sufficient level (0·02 per cent or higher) of 1,10-phenanthroline. The metal chelator-sensitive proteolytic activity appears to be an essential moulting protease in Manduca.     

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.     

Lipid metabolism of the female crab Pachygrapsus marmoratus during the moulting cycle

The moult induces important variations in the concentration and fatty acids composition of lipid classes during the moulting cycle of the female crab Pachygrapsus marmoratus regardless of the maturational degree of ovaries. Sexual maturity is characterized by a rise in lipids. Juveniles contain high levels of polyunsaturated fatty acids except in the ovary, whereas adults are composed mainly of monoethylenic acids. The moulting decrease of saturated fatty acids shows their importance during this crucial period, owing to their utilization as energy sources at the time of ecdysis. The late premoult fall of hepatopancreatic lipids results at once from inanition during the period before the exuviation and transfer of lipids through the hemolymph to the periphery in order to realize numerous changes occurring during the moulting process. The relative stability of lipid composition of ovaries in sexual pause during the moulting cycle agrees with a reduced metabolism of ovarian cells.     

Stretch-induced moulting in Oncopeltus fasciatus

Fifth instar nymphs of Oncopeltus fasciatus initiate a moulting cycle when they reach a critical weight of 27.9±1.1 (±S.D.) mg for females or 23.7±1.2 mg for males. This critical weight is not absolute. Its precise value depends on the somatic size of the individual. Large-bodied individuals have a higher critical weight than small-bodied ones. Sexual size-dimorphism accounts largely for sex differences in critical weights. Animals of a subcritical weight can be induced to moult by means of a saline injection, in the absence of further food intake or growth. Since a saline injection only stretches the abdominal wall it is suggested that moulting in Oncopeltus is triggered by stimulation of abdominal stretch receptors, as in bloodsucking Reduviidae. The critical weight is thus an index of the size at which a critical degree of stretch is achieved.     

Larval moulting hormone of trichophoran Hemiptera-Heteroptera: Makisterone A,not 20-hydroxyecdysone

The haemolymph ecdysteroids were examined in fifth-stage larvae of Nezara viridula, Podisus maculiventris and Dysdercus cingulatus (Hemiptera-Heteroptera) using high-pressure liquid chromatography to separate the ecdysteroids and a radioimmunoassay to detect the fractionated ecdysteroids. The length of the fifth stage ranged from 5 to 8 days, and a peak in ecdysteroid titre (1700–2650 ng/ml) occurred 2–3 days prior to ecdysis to the adult. An ecdysteroid matching the retention time of makisterone A (24-methyl-20-hydroxyecdysone) was clearly present in haemolymph taken at the time of peak titre in all 3 of these true bugs, whereas little, if any, ecdysone or 20-hydroxyecdysone was detected. These data, along with previously reported data for the milkweed bug Oncopeltus fasciatus, are persuasive evidence that makisterone A is the larval moulting hormone of a group of closely related Heteroptera called the Trichophora (Lygaeoida, Pentatomoidea, Pyrrhocoroidea and Coreoidea).     

Towards a mechanistic understanding of insect life history evolution: oxygen‐dependent induction of moulting explains moulting sizes

Moults characterise insect growth trajectories, typically following a consistent pattern known as Dyar's rule; proportional size increments remain constant across inter‐instar moults. Empirical work suggests that oxygen limitation triggers moulting. The insect respiratory system, and its oxygen supply capacity, grows primarily at moults. It is hypothesized that the oxygen demand increases with increasing body mass, eventually meeting the oxygen supply capacity at an instar‐specific critical mass where moulting is triggered. Deriving from this hypothesis, we develop a novel mathematical model for moulting and growth in insect larvae. Our mechanistic model has great success in predicting moulting sizes in four butterfly species, indirectly supporting a size‐dependent mechanism underlying moulting. The results demonstrate that an oxygen‐dependent induction of moulting mechanism would be sufficient to explain moulting sizes in the study species. Model predictions deviated slightly from Dyar's rule, the deviations being typically negligible within the present data range. The developmental decisions (e.g. moulting) made by growing larvae significantly affect age and size at maturity, which has important life history implications. The successful modelling of moulting presented here provides a novel framework for the development of realistic insect growth models, which are required for a better understanding of life history evolution.     

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.     

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.     

An ecdysone receptor from the pentatomomorphan, Nezara viridula, shows similar affinities for moulting hormones makisterone A and 20-hydroxyecdysone

It has been suggested that Pentatomomorpha utilise the C₂₈ ecdysteroid, makisterone A (MakA), as the major moulting hormone rather than the more common C₂₇ hormone, 20-hydroxyecdsyone (20E). The present study is the first to examine this postulate at the level of the ecdysone receptor protein, a heterodimer of nuclear receptors EcR and USP. cDNAs encoding two alternatively spliced isoforms of EcR and a single USP were isolated from a high-quality cDNA library prepared from a representative pentatomomorphan, Nezara viridula (Nv). NvEcR and NvUSP were found to group phylogenetically with heteropteran and other insect EcRs and USP/RXRs, respectively. Sequence comparison and phylogenetic analysis of these proteins found them to be distinct from those belonging to other hemipteran ecdysone receptors characterised to date. Co-expression of the His₆-tagged ligand binding regions (LBRs) of the two NvEcR variants with the FLAG-tagged LBR of NvUSP was achieved in insect cells employing appropriately constructed baculoviruses. The corresponding heterodimers, designated NvE10 and NvE11, were purified by affinity chromatography utilising the His₆ tags on their NvEcR subunits. The heterodimers displayed nanomolar affinity for [³H]ponasterone A (K_d = 6.8-7.5 nM), characteristic of ecdysone receptors. MakA has a similar affinity to 20E for both NvE10 and NvE11, consistent with MakA being a major moulting hormone in N. viridula.     

Degeneration of moulting glands in male crickets

The degeneration of the prothoracic glands of the male cricket, Gryllus bimaculatus, was analyzed by using an in vitro assay for ecdysteroid release from the moulting glands in last instar nymphs as well as in adult animals, and correlated with light and transmission electron microscopy. Apoptosis was examined by the TUNEL-reaction. The ability to synthesize ecdysteroids reached a peak at the 8th day of the last larval instar, identified as the moulting peak. After adult ecdysis it decreased to barely measurable values. Prothoracic gland degeneration was initiated at the time of the moulting peak, characterized by TUNEL positive reactions, nuclear and cytoplasmatic condensation, a striking abundance of residual basal laminae; besides a great amount of autophagic vacuoles are observed. The results reveal that apoptosis and autophagy are the basic mechanisms for programmed cell death in the prothoracic gland of Gryllus bimaculatus.     

Effect of turbulence on the feeding and moulting of the cirripede Balanus Balanoides (L.) given an algal diet

The shell growth, moulting, and faecal pellet production of Balanus balanoides (L.) have been investigated experimentally in turbulent and quiet conditions. In turbulent conditions an algal food level of 50% was sufficient to maintain the same shell growth as at the 100% level, whereas at this level in quiet conditions growth was less than at 50% with turbulence. At the 10% food level growth was markedly reduced in quiet conditions. On the assumption that production of faeces is a measure of feeding activity, the latter is stimulated by turbulence as is the moulting rate. The relation between production of faeces and moulting is, however, independent of water movement. Faecal pellet production continues at higher algal food levels after the growth rate has become constant indicating that when assimilation is maximal there is no feed-back mechanism regulating food intake.     

Exclosure study on the exploitation of macrophytes by summering and moulting waterbirds at Lower Lake Constance

Owing to synchronous moult, most waterbird species are constrained by flightlessness and limited mobility for several weeks. As new feather production is energy demanding, these birds need to choose a safe moulting site with appropriate food supply. Up to 20,000 waterbirds carry out moult at Lake Constance, gathering at sites where they find food close to safe hiding places from predators and human-caused disturbance. In this study, we focused on the food supply at one prominent moulting site, Mettnau Südbucht, at Lower Lake Constance. We aimed to determine the food items and quantity as well as their utilization by summering and moulting waterbirds. We conducted experiments with exclosure cages which protected macrophytes from bird grazing and compared these sites with unprotected grazed sites. In these experiments, we found that the summering and moulting waterbird community, dominated by Eurasian Coots (Fulica atra Linnaeus), caused a significant decline of the macrophyte biomass at 1.5-m depth (MWL), where they were responsible for a loss of over 40% of the total charophyte biomass. No grazing effect was found at a greater depth (2-m MWL). The available food consisted mostly of Chara spp. with a biomass density of about 350 g m^?2. Animal food items were negligible: Macroinvertebrates, mainly Asellus aquaticus Linnaeus, that were associated with the macrophytes, made up only 2% of total biomass, and were very unevenly distributed.     

The composition of larval-pupal moulting fluid in the tobacco hornworm, Manduca sexta

The concentrations of sodium, potassium, chloride, bicarbonate, total phosphate, labile phosphate, inorganic phosphate, protein, polypeptides, amino acids, trehalose, and glucose, as well as pH and osmotic pressure of larval-pupal moulting fluid and haemolymph were measured in the tobacco hornworm, Manduca sexta, during the secretion and resorption of moulting fluid. Moulting fluid is a mildly alkaline (pH 7.2), iso-osmotic (330 mOsm) potassium bicarbonate salt solution containing within it the sol form of moulting gel. Bicarbonate is the principal anion in moulting fluid. It is only a minor component of haemolymph. Significant differences in the concentrations of potassium, chloride, bicarbonate, non-labile phosphate, labile phosphate, inorganic phosphate, protein, polypeptide, amino acids, trehalose, and glucose indicate that the integumentary epithelium does not act as a semi-permeable membrane, and that free diffusion between moulting fluid and haemolymph can account for only a small fraction of the molecular species movement between these two fluids.     

Structure of the toad epidermis during the moulting cycle

Summary Skin samples were taken from 17 toads (Bufo bufo) in various phases of the moulting cycle. The phase was determined by recording a number of moulting intervals prior to sacrifice and by study of the macroscopic appearance of the skin and the moulting behaviour at sacrifice. Eight of the toads were in the moult when the samples were taken.Characteristic morphological changes were found to be restricted to a period immediately prior to and after shedding of the slough. Chemical changes of the membranes of stratum corneum (or material adhered to them) were observed already prior to adoption of the moulting posture. The separation from the underlying epidermis prior to shedding was accompanied by a swelling of the stratum corneum cells. After final detachment of the slough the replacement layer was differentiated into a new stratum corneum within 24 hours. Significant changes in the morphology of the flask cells were not observed.The findings are discussed with emphasis on the processes of separation and differentiation of the stratum corneum. Based on the morphology of the epidermis the following terminology is proposed for the phases of the moulting cycle: Intermoult phase, preparation phase, early shedding phase, late shedding phase, and differentiation phase.The authors wish to thank professor C.B. Jørgensen, Dr. D. P. Knight and Dr. E.H. Larsen for valuable discussions. The technical assistance of Miss Susanne Binzer and Mrs. Grete Budtz is gratefully acknowledged. G.B. kept records of the moults. Without her patient and careful observations during several weeks it had not been possible to obtain the present material.     

Chilled Galleria mellonella larvae: mechanism of supernumerary moulting

ABSTRACT. Supernumerary larval instars were produced when Galleria mellonella L. (Lepidoptera) larvae were chilled at 0°C. Although sensitivity to cooling stress of the last instar and younger larvae were generally the same, only penultimate and the last instar larvae showed a significant correlation between their age and the number of additional larval moults. Chilling stress induced a rapid and persistent increase in the JH titre of the last instar larvae. Severing the ventral nerve cord resulted in a predictable loss of the ability to produce supernumerary moults in chilled last instar larvae. The data suggest that sensory input stimulates allatotropic hormone secretion by the brain of chilled larvae. The possible mechanism controlling supernumerary moulting is discussed.     

Hormone,vitamin and contaminant status during the moulting/fasting period in ringed seals (Pusa [Phoca] hispida) from Svalbard

This study investigates the potential effects of moulting, and the concomitant period of fasting undertaken by ringed seals, on hormone, vitamin and contaminant status in adult animals in a population from Svalbard, Norway, which has relatively low contaminant levels. Concentrations of circulating total and free thyroxine and triiodothyronine, circulating and hepatic vitamin A, hepatic persistent organic pollutants and their circulating hydroxyl metabolites were higher in moulting seals compared to pre-moulting seals. The opposite trend was observed for body condition, circulating calcitriol levels and hepatic mRNA expression of thyroid hormone receptor β. No differences were observed for circulating or hepatic vitamin E levels or hepatic mRNA expressions for deioidinase 1 or 2, or retinoic acid receptor α between the two seal groups. The observed differences are likely the result of increased metabolic rates required during moulting to maintain thermal balance and replace the pelage, in combination with mobilization of lipid soluble compounds from blubber stores during the fasting period that is associated with moulting. The present study shows that contaminant levels and their relationships with physiological or endogenous variables can be highly confounded by moulting/fasting status. Thus, moulting status and body condition should be taken into consideration when using variables related to thyroid, calcium or vitamin A homeostasis as biomarkers for contaminant effects.