Days Of Adult Development Research Articles

Molecular profiling of behavioural development: differential expression of mRNAs for inositol 1,4,5-trisphosphate 3-kinase isoforms in naive and experienced honeybees ( Apis mellifera)

In seeking genetic factors that may control the extended behavioural maturation of adult honeybees we found that inositol 1,4,5-trisphosphate (IP 3) 3-kinase, a key enzyme in the IP 3-mediated signalling cascade, is differentially expressed in brains of naive, newly emerged bees and experienced foragers. DNA sequencing yielded a contig of 21.5 kb spanning the honeybee IP 3K locus and a 3′ flanking gene similar to a transcription factor NFR-κ-B. The IP 3K locus gives rise to three differentially expressed major transcripts produced by alternative splicing that encode proteins with identical, highly conserved C-termini and distinct, non-conserved N-terminal domains. The type A transcript is dominant in the adult brain and its level of expression increases threefold during the first 4 days of adult development. The type B message is expressed in brains of naive bees, but is also found in the thorax and abdomen, whereas transcript C is expressed largely in non-neural tissues and in the antenna. In contrast to type A message, the brain levels of transcript B decrease during the first 4 days of adult life. Our data are evaluated in the context of the contrasting behavioural phenotypes of immature and experienced worker honeybees.

In vitrosynthesis of ecdysteroid by the male accessory reproductive glands, testis and abdominal integument of the adult migratory grasshopper,Melanoplus sanguinipes

Summary Male accessory reproductive glands (MARG), testis (including its sheath) and abdominal integument along with adhering fat body were cultured in vitro to determine their ability to synthesize ecdysteroid during the first 10 days of adult development. Ecdysteroid synthesis by MARG was highest on day 2 (0.180 ng/mg) while the testis showed a peak on day 5 (0.436 ng/mg). In the testis, the sheath is mainly responsible for ecdysteroid synthesis. Ecdysteroid is also synthesized by the abdominal integument with adhering fat body, and peak activity is observed on day 8 (0.50 ng/mg). A time-course study showed that ecdysteroid synthesis increased linearly in all tissues over a 24-h incubation period. However, little storage of ecdysteroid occurred in the tissues.

Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster.

The larval fat body of newly eclosed adults of Drosophila melanogaster was found to contain a single major binding protein specific for juvenile hormone (JH). Binding to this protein was saturable, of high affinity, and specific for JH III. The protein has a subunit molecular weight (Mr) of 85,000, as determined by photoaffinity labeling. The same or similar JH-binding protein was found in larval fat body and cuticle of third instar larvae and in male accessory glands and heads of newly eclosed adults. It was not found in several other tissues in adults. Male accessory gland cytosol from wild-type flies was found to contain a single binder with a dissociation constant (KD) of 6.7 nM for JH III; a binder in similar preparations from the methoprene-tolerant (Met) mutant had a KD value 6-fold higher. JH III stimulated protein synthesis in glands cultured in vitro, but this effect was reduced in Met flies as compared to wild-type flies, establishing a correlation between JH binding and biological activity of the hormone. In addition, glandular protein accumulation during the first 2 days of adult development was less in Met flies than in wild-type flies. These results strongly suggest that the binding protein we have identified mediates this JH effect in male accessory glands and thus is acting as a JH receptor.

Respecification of larval proleg motoneurons during metamorphosis of the tobacco hornworm, Manduca sexta: segmental dependence and hormonal regulation.

The principal locomotory appendages of the Manduca sexta caterpillar, the prolegs, are present on the third through sixth abdominal segments (anal prolegs located on the terminal segment were not included in this study). Previous studies have characterized some of the proleg retractor muscles and their motoneurons. In the present study we identified additional proleg motoneurons and their putative homologs in the non-proleg-bearing segments. One of the motoneurons present in the proleg-bearing segments is absent in the non-proleg-bearing segments. At pupation the prolegs are lost, their muscles degenerate, and some of their motoneurons regress structurally. Subsequently, subsets of the proleg motoneurons and their homologs in other segments die in a segment-specific pattern. This is the first report of segment-specific motoneurons, and of segment-specific death of identified motoneurons, in Manduca. During adult development the surviving proleg motoneurons innervate the tergosternal muscle (TSM) and grow bilateral dendritic arbors. Dendritic growth is completed by about the 12th of the 18 days of adult development. Following adult emergence all but one of the respecified proleg motoneurons dies. The hormonal dependence of dendritic outgrowth was tested by isolating abdomens to eliminate the ecdysteroid-secreting glands in the thorax. Between the second and fifth days after pupation the motoneurons became progressively more competent to undergo dendritic outgrowth following abdomen isolation. The extent of dendritic outgrowth paralleled the degree of morphological development attained by isolated abdomens. It is concluded that ecdysteroids are required for motoneuron outgrowth, but our findings suggest that, unless an abdominal source of ecdysteroids exists in pupae, a relatively small exposure may be sufficient.

Control of neurosecretion in the moth Manduca sexta: physiological regulation of the eclosion hormone cells.

Metamorphosis in the moth Manduca sexta culminates with the secretion of the peptide eclosion hormone (EH), which triggers the stereotyped behavior of adult emergence (eclosion) from the pupal cuticle. In restrained but spontaneously behaving animals, the release of EH occurred shortly before the onset of subjective night (Fig. 3) and coincided with a depletion of EH from the neurohemal organs of the brain, the corpora cardiaca-corpora allata complex (CC-CA; Fig. 4). EH is produced by neurons within a bilaterally paired group of brain neurosecretory cells (Group Ia) which project to the CC-CA via the nervi corporis cardiaci- 1 + 2 (NCC-1 + 2; Fig. 1). Electrical stimulation of the NCC-1 + 2 caused a marked increase in the levels of EH secreted from isolated CC-CA (Fig. 2), while stimulation of the other nerves innervating the neurohemal organs did not. Electrical activity in the NCC-1 + 2 paralleled that of the cerebral neurosecretory cells (Fig. 1). Chronic extracellular recordings revealed a sudden increase in the tonic firing of several units within this nerve approximately 2 to 3 h before normal eclosion (Fig. 5), coincident with the release of EH bioactivity from the CC-CA (Fig. 6). The Group Ia neurons were electrically inactive on the day before eclosion (Day-1), but on the day of eclosion (Day 0) a subgroup of these cells exhibited both enhanced synaptic input and elevated rates of tonic firing during the normal time of EH release (Fig. 7). No significant differences in resting membrane potential or spike waveform characteristics were detected among the various subsets of Group Ia cells on either Day-1 or Day 0, while a significant increase in the resting input resistance was seen in the active subgroup on Day 0 (Fig. 8). This increase may be due to the regulatory effects of the steroid 20-hydroxyecdysone, which inhibits the release of EH and may act by preventing the synaptic activation of the EH neurons until the final day of adult development.

Endocrine regulation of terminal muscle differentiation: Atrophy and degeneration of the intersegmental muscles of Lepidoptera

The intersegmental muscles of the Lepidoptera pass through three separate, sequential differentiated states during pharate adult development: status quo; atrophy; degeneration. Each of these developmental programs is characterized by a distinct morphology, physiology and endocrine responsiveness. The factors responsible for regulating these differentiative changes are ecdysteroids. In Manduca sexta, the haemolymph ecdysteroid titre declines in a circadian-modified fashion during the last three days of adult development, which parallels the maturation of the intersegmental muscles. Abdomen-ligation, which causes a precipitous decline in the ecdysteroid titre, causes the precocious atrophy and degeneration of these muscles, whereas injection of, or infusion with, 20-hydroxyecdysone greatly delays such changes. While the terminal differentiation of the epidermis and nervous system is also regulated by ecdysteroids, endocrine manipulations have suggested that the development of the intersegmental muscles is independent of these tissues. In the silkmoth Antheraea polyphemus, ecdysteroids are also responsible for regulating intersegmental muscle differentiation, but eclosion hormone (a peptide) acts as the proximal trigger for the activation of the degeneration program. The declining ecdysteroid titre initiates the atrophy program and subsequently determines the timing of both release of eclosion hormone and intersegmental muscle sensitivity to the peptide. Eclosion hormone then acts directly on the muscles, via cGMP, to activate the degeneration program. Ecdysteroids appear to prevent premature muscle degeneration by regulating a biochemical step distal to both the eclosion hormone receptor and the rise in cGMP.

The preparatory behavior rhythm of the mothManduca sexta: an ecdysteroid-triggered circadian rhythm that is independent of the brain

1. On the last day of adult development, individuals of the mothManduca sexta showed a 3 to 4 h bout of abdominal movements termed the preparatory behavior which was then followed a few hours later by the emergence (eclosion) of the moth from the old pupal cuticle. When the developing insects were exposed to daily cycles of light and temperature (2° C amplitude), both behaviors occurred at characteristic times of day. 2. When insects were exposed to light and temperature cycles at various phases to one another, the time of the preparatory behavior was phase-locked to the temperature step-up, whereas the timing of ecdysis was a function of both the light and the temperature cycles (Fig. 3). 3. Under conditions of constant light and temperature the preparatory behavior showed a clear free-running rhythm (Fig. 4) indicating that its onset is controlled by a circadian clock. 4. Removal of the brain from developing moths did not interfere with the entrainment of the preparatory behavior rhythm by day-night cycles of light and temperature (Fig. 5). 5. When the abdomen of the moth was separated from the head and thorax late in adult development, the abdomen was still capable of performing the preparatory behavior and in many cases the behavioral onset occurred earlier than in controls (Fig. 6). It was concluded that some factor from the anterior end of the insect normally inhibited the behavior. 6. This factor proved to be the ecdysteroids, the steroid molting hormones, which undergo a normal decline late in adult development. Injections of the ecdysteroid, 20-hydroxyecdysone (20-HE), delayed the onset of the preparatory behavior in both isolated abdomens and intact animals (Fig. 7). In the latter the delay was dosage dependent and caused by physiological concentrations of steroid. 7. It was concluded that the preparatory behavior is triggered by the declining titer of ecdysteroids late in adult development. The synchronization of the behavior with the day-night cycle results from the action of a temperature-sensitive, circadian clock that resides outside of the insect's brain. This clock presumably controls the phase of the ecdysteroid decay which, in turn, determines the time of the preparatory behavior.

Eclosion Hormone and Bursicon Titres and the Onset of Hormonal Responsiveness During the Last Day of Adult Development in Manduca Sexta (L)

ABSTRACT The blood titres of eclosion hormone in Manduca sexta indicate that the hormone is liberated into the blood during a span of about 20 min and then disappears with a half-life of about 45 min. Eclosion normally follows 2·5 h after the appearance of the hormone in the blood. Bursicon appears in the blood within 2 min after the newly emerged moth comes to rest at a wing spreading site. Hormone is apparently released during the following 10 min and then disappears with a of 40–50 min. The target tissues for the two hormones become responsive during the last day of development. The wing epidermis and nervous system appear to become sensitive to the eclosion hormone at about the same time, approximately 4 h before the hormone’s release. Wings become responsive to bursicon about 2 h earlier. During the last day of adult development, Manduca show a precise onset of responsiveness to bursicon and eclosion hormone followed a few hours later by the gated release of the two hormones. This appearance of hormone sensitivity is likely to be due to a photoperiodically gated event which occurs late in adult development but prior to eclosion hormone release. The nature of this event is unknown.

Alanine aminotransferase: properties and distribution in the desert locust, Schistocerca gregaria

Developmental kinetics of l-alanine:2-oxoglutarate aminotransferase, followed from the 3rd instar to the 10th day of adult development of the desert locust, Schistocerca gregaria, showed that the absolute enzyme activity of both the flight muscle and the gut tissue was the function of the age of the insect as well as protein synthesis. Specific activity was found to follow Ω-pattern. pH optima of partially purified flight muscle mitochondrial enzyme were 7.4 and 8.3 in phosphate and borate buffer, respectively. The enzyme was highly sensitive to sulfhydryl reagent, p-chloromercuribenzoate and also to carbonyl reagents hydroxylamine and phenylhydrazine. Kinetics of transamination was studied in the direction of pyruvate formation. The absolute K m values for alanine and α-ketoglutarate were 19.05 and 1.60 mM respectively and absolute maximal velocity of 0.048 μmol pyruvate formed per minute.

Development and hormonal release of adult behavior patterns in silkmoths

1. When the pupal cuticle was peeled from aroundAntheraea pernyi andHyalophora cecropia moths on the last day of adult development, the animals showed reduced levels of adult behavior. Clawing and extension movements of the legs as well as wing flapping were seen, but complex behaviors such as the righting response and walking were absent (A. pernyi) or only occasionally observed (H. cecropia). 2. Injection of the eclosion hormone into “peeled” moths was followed 2 to 3 hours later by the onset of the eclosion movements. Walking behavior and the righting response appeared abruptly at the time of eclosion. 3. The development of certain adult behaviors was examined by peelingA. pernyi moths that were in the 14th through 19th (last) day of adult development. Animals peeled on days 16 or 17 showed distinct eclosion movements which started within a few minutes after peeling. But on day 18, this behavior became suppressed and could no longer be elicited by peeling. 4. Sensitivity to the eclosion hormone did not appear until day 18 and became fully developed on day 19. 5. Injection of picrotoxin into peeled day 19A. pernyi resulted in the abrupt onset of walking behavior. 6. It was concluded that certain adult behaviors such as eclosion and walking become complete and functional during adult development. But as development progresses, these then become repressed—presumably through neural inhibition. The eclosion hormone apparently acts by turning off this inhibition.

Ultrastructural studies on neuromuscular contacts and the formation of junctions in the flight muscle of Antheraea polyphemus (Lep.) I. Normal adult development.

The ultrastructure of neuromuscular connections on developing dorsolongitudinal flight muscles were studied in the moth Antheraea polyphemus. Undifferentiated membrane contacts between axon terminals and muscle-fiber anlagen are present in the diapause pupa. They persist during the period of nerve outgrowth, which probably provides a pathway of contact guidance. By the 4th day of adult development some of these contact areas have differentiated into structures similar to neuromuscular junctions although differentiation of muscle structure does not start earlier than the eighth day. Dense-cored vesicles are abundant in many axon terminals at the beginning of development. They later decrease in number quite rapidly. The significance of the above-mentioned early junctions, their possible mode of action and the role of the dense-cored vesicles are discussed. It is proposed that they exercise a stimulating (trophic) influence on the growth of the undifferentiated muscular tissue. The imaginal neuromuscular junctions are formed during the second half of adult development. Clusters of vesicles and electron-dense depositions along the inner face of the axo- and lemma seem to initiate junction formation. Glial processes then grow between the axo- and sarcolemma and divide the large contact area into several small segments. Mutual invaginations and protrusions of the sarcolemma and the glial cell membrane subsequently form an extensive "rete synapticum." Six days before eclosion the glial and sarcoplasmic parts of the rete synapticum are similar in size. Up to eclosion, all glial processes shrink and increase in electron density. Most of the observations are discussed also in relation to findings in vertebrates.

Morphological and physiological studies of the silkmoth ovary I. The general histology and some histochemical properties of the ovary of Samia Cynthia (Drury)

Ovaries of the giant silkmoth Samia cynthia Drury have been studied histologically and histochemically during diapause, adult development, and after injury to the diapause animal. In addition to cellular changes involved in follicular growth and vitellogenesis, changes in the intermediate layer cells are very striking, showing a distinct pattern during ovarian development and after injury. Two types of granules, one periodic acid-Schiff (PAS) positive, the other paraldehyde-fuchsin (PF) positive, change in their number and distribution in the intermediate cells during development and after injury. The PAS-positive granules appear during the first one-third of development, increase in number and size until the tenth day, then gradually dwindle in number and size, and disappear in the adult cells. The PF-positive granules, present in moderate numbers in the diapause animal, decrease in number until the tenth day of adult development, then reappear, and are present again in adult intermediate cells. After injury to a diapause pupa, intermediate cells show a greatly increased number of PF-positive granules, but none that are PAS-positive. The PAS-positive granules are a neutral or mildly acidic mucosubstance, and may correspond to lysosomal activity, while the PF-positive granules may be a neurosecretory-like substance or the product of hormone-dependent protein synthesis, since the patterns of granule distribution in the intermediate cells seem to reflect both general metabolic and hormone-related events.

THE ORIGIN, DISTRIBUTION AND FATE OF THE MOLTING FLUID PROTEINS OF THE CECROPIA SILKWORM.

1. Molting in insects is always accompanied by the production of a molting fluid which fills the exuvial space between the new and the old cuticle and digests the inner layers of the old cuticle. In Hyalophora cecropia, molting fluid is secreted at the outset of adult development and persists until two days before eclosion, whereupon it is absorbed. 2. The present report examines the protein composition of the molting fluid of Cecropia, the origin of the molting fluid proteins, the relation of these proteins to blood proteins and the exchange of macromolecules between the molting fluid and the blood. It also examines the sites of absorption of molting fluid. 3. Disc electrophoresis on acrylamide gels reveals that the molting fluid of Cecropia contains about fifteen protein bands which can be resolved at pH 8.6. Some of these protein bands are detected in the molting fluid at all stages, whereas others appear only at specific times. About ten of the bands are peculiar to molting fluid and are not detected in the blood. About five bands are detectable in both blood and molting fluid, but none of these common bands appears to be a major component of the molting fluid, and only one is a major blood protein. In contrast, the epidermis contains most of the major protein bands found in molting fluid but lacks all but one of the major protein bands present in the blood. 4. Immunological analysis reveals that blood and molting fluid share five antigens. At least four of these common antigens also occur in the epidermis which appears to secrete these antigens into both the molting fluid and the blood. 5. Native and foreign proteins do not penetrate from the exuvial space into the blood or vice versa. Apparently the epidermis and cuticle act as a barrier to the exchange of most macromolecules between the blood and molting fluid. The exuvial space is clearly a separate fluid compartment. 6. In addition the exuvial space itself is compartmentalized and the fluids in the compartments do not admix several days before eclosion. 7. Absorption of molting fluid during the final two days of adult development occurs most readily through particular regions of the integument. In the abdomen the principal sites of absorption are pits which represent the points through which tonofibrils make attachment to the old cuticle. Two days before ecdysis, the attachments between the tonofibrils and the pupal cuticle rupture, exposing the points of attachment on the new cuticle. It is through these exposed surfaces that much of the molting fluid is absorbed. Molting fluid is also absorbed in the head and thorax through various flexible membranes at the bases of the appendages.

Programmed cell death—V. Cytolytic enzymes in relation to the breakdown of the intersegmental muscles of silkmoths

During the 3 week period of adult development cytolytic enzymes are synthesized and sequestered in lysosome-like particles in the intersegmental muscles of the silkmoth abdomen. A cathepsin begins to increase in titre during the first few days of adult development and continues to increase slowly during the first 2 weeks of this period. Then, during the final week of adult development, it increases at a logarithmic rate of about 30 per cent per day. In centrifuged homogenates prepared in sucrose, most of the enzymatic activity accompanies the mitochondrial ‘fraction’, from which a substantial proportion can be relased by osmotic shock or by detergents. In sucrose homogenates of degenerating muscle, a lesser fraction of the enzyme is sedimentable, and a greater percentage of the total enzymatic activity is ‘free’ prior to the addition of detergent. An acid phosphatase follows a similar pattern but is more readily solubilized by the homogenization procedure. These enzymes are considered to be directly responsible for the destruction of the intersegmental muscles.

Programmed cell death—III. Neural control of the breakdown of the intersegmental muscles of silkmoths

The breakdown of the intersegmental muscles of the silkmoth abdomen is potentiated by exposure to ecdysone during the first few days of adult development. Three weeks later, after the completion of adult development, the actual breakdown is triggered by a neural mechanism. The latter consists of a sudden curtailing or cessation of the outflow of impulses in the motor nerves which innervate the abdominal muscles. The muscles attain the capacity to respond to this signal during the final 3 days prior to the moth's ecdysis. By chronic electrical stimulation of the nerves, the breakdown of the muscles can be opposed or prevented.

The nucleic acid content of tissues of cecropia silkmoth pupae relations to body size and development

1. 1. The levels of DNA and total nucleic acid, giving RNA by difference, have been determined in the wing tissue and fat body of a series of pupae of the cecropia silkmoth in diapause and at an early stage of imaginal development. Exhaustive dialysis of the fat body tissue residue was essential to remove uric acid and facilitate extraction of nucleic acids. 2. 2. In the fat body, the ratio of tissue-residue weight to DNA varies in proportion to the fresh weight of the individual pupae. This is interpreted on the basis of the correlation of cell size with animal size. The wing tissue does not show such a relationship. 3. 3. In the wing tissue, the level of DNA approximately doubles, and that of RNA rises some four-fold, during the transition from diapause to the second day of adult development. In the fat body, there is no significant change in the level of either type of nucleic acid.

THE JUVENILE HORMONE. II. ITS ROLE IN THE ENDOCRINE CONTROL OF MOLTING, PUPATION, AND ADULT DEVELOPMENT IN THE CECROPIA SILKWORM

1. By means of a standardized "pupal assay" for juvenile hormone, the endocrine activity of the corpora allata was found to undergo large and systematic changes during the postembryonic development of the Cecropia silkworm.2. In each of the larval instars that were studied, the glands are least active just prior to the larval molt and most active shortly after the molt. Larval molting therefore appears to take place in the presence of a declining titer of juvenile hormone—an endocrine situation which apparently permits the striking changes in morphology and pigmentation which occur in successive larval stages of the Cecropia silkworm.3. The corpora allata show low but definite activity at the time of pupation. If the glands are extirpated so that pupation occurs in the absence of juvenile hormone many larval tissues overleap the pupal stage and undergo precocious adult differentiation. Therefore, the low concentration of juvenile hormone in the mature larva plays a definite role in the endocrine control of pupation.4. The corpora allata are inactive throughout the entire pupal stage. This inactivity persists during the first two-thirds of adult development. During the final week of adult development, the glands recover their endocrine function and are maximally active by the time of emergence of the adult moth.5. The absence of juvenile hormone proves to be an obligatory feature of the initial phase of adult differentiation. If active corpora allata are implanted into a pupa so that adult development is initiated in the presence of juvenile hormone, the pupa develops and molts into a creature which is a mixture of pupa and adult. The higher the titer of juvenile hormone, the more extensive is the preservation of pupal characters.6. Juvenile hormone is effective in blocking adult differentiation only when it is present during the first five days of adult development. Consequently, the target of juvenile hormone appears to be certain early events which are the normal reactions to ecdyson at the outset of adult development. If unopposed by juvenile hormone, these events commit the cells to developmental reactions accompanied by metamorphosis.7. A comprehensive theory is presented for the action of juvenile hormone in the Cecropia silkworm. According to this theory, juvenile hormone modifies the cellular reactions to ecdyson by opposing the de-repression, de-coding or acting-out of fresh genetic information prerequisite for progressive differentiation but not prerequisite for growth in an unchanging state.

The Moulting Fluid of the Cecropia Silkworm

ABSTRACT The initiation of adult development in the pupa of the Cecropia silkworm is accompanied by a retraction of the hypodermis from the pupal cuticle; the exuvial space is simultaneously flooded with the moulting fluid. During the first two-thirds of adult development the moulting fluid is a dilute, aqueous, proteinaceous gel resembling egg albumen ; at this time it is without effects on the pupal cuticle. On approximately the fourteenth day of adult development the gel is converted into a sol and shows a considerable increase in chitinase activity and the first demonstrable proteolytic activity. The late, active moulting fluid then begins to hydrolyse the protein and chitin in the overlying pupal endocuticle. By the twentieth day the old endocuticle has disappeared. The tanned proteinchitin complex of the sclerotized exocuticle is not attacked. Finally, the moulting fluid is completely resorbed into the underlying insect. The residual pupal exocuticle forms the bulk of the exuviae. Moulting fluid and blood are compared in respect to the following properties : consistency, cellular content, solubility, refractive index, water content, chemical composition, total nitrogen, protein nitrogen, pH, redox potential, and enzymatic content. The moulting fluid shows numerous quantitative and qualitative differences from the blood. The fluid is, evidently, a distinctive secretory product of the underlying integument—presumably of the hypodermis itself. A dynamic state exists between the moulting fluid and the underlying insect. Radioactive glycine, injected into the moulting fluid, was promptly resorbed and incorporated into the protein of the adult moth. Several lines of evidence suggest that the secretion or activation of the moulting fluid enzymes are synchronized with the sclerotization of the underlying new cuticle. This synchronization, it is suggested, may serve to protect the new cuticle from the hydrolytic action of the moulting fluid.