Pupal-adult Transformation Research Articles

Effects of X-irradiation on egg hatchability, larval and pupal survival in the tobacco hornworm, Manduca sexta

The effects of X-irradiation upon Manduca sexta egg hatchability and larval survival were studied using LD 50's. Radiosensitivity declined with age in developing eggs (LD 50 of 9.4 kR in 2.5 to 3-days eggs contrasted to 18.2 kR in 4 to 4.5-day eggs) and in larvae. The dose-response patterns obtained agree with those previously reported for other insects. The maximal (ED 0) and minimal (ED 100) X-ray exposures resulting in eclosion successes of 100% and 0%, respectively, were utilized throughout pupal-adult development as measures of radiation sensitivity. A pronounced decrease in radiosensitivity was noted through the day of eclosion (Day 0: ED 0 = 7.3 kR; ED 100 = 18.7 kR; Day 22: ED 0 = 45.0 kR, ED 100 = 105.0 kR). X-irradiation during the pupal-adult transformation was observed to inhibit eclosion without disrupting normal metamorphosis.

The Occurrence of "Extranumerary" Cuticle Deposition in Transplanted Larval Integument of the Sikworm Bombyx mori (Lepidoptera : Bombycidae)

The author indicated in previous reports (KATO, 1973, 1975) that in Bombyx mori pieces of larval integument, if transplanted to a pupa, produced two different layers of cuticle in the process of pupal-adult transformation. In the present experiments, attempts were made to examine the occurrence of the extranumerary cuticle deposition in transplants by preparing the larval donor from different stages of development and by using hosts possessing different hormonal conditions. Pieces of integument taken from larvae of an earlie instar (1st, 2nd or 3rd) and transplanted to a pupa deposited two different layers of cuticle at a higher rate than those from larvae of later instars (4th or 5th) before the host pupa ecdysed to na adult. When placed in a penultimate or last-instar larva, integument pieces from earlier instars always deposited only a single layer of cuticle at the time when the host moulted to the next stage. The extranumerary cuticle deposition in transplants took place even when allatectomized or decapitated pupae were employed as the host. Furthermore, this evidence was obseved in both male and female pupae as the host.

Action of juvenoids on the metamorphosis of cyclorrhaphous Diptera

Administrations of high doses of juvenoids to the last instar larvae of cyclorrhaphous flies cause occasionally lethal defects in puparium formation but mostly affect only the pupal-adult transformation. In pupae, juvenoids impede proliferation and differentiation of the imaginal disks and of abdominal histoblasts: at low doses they cause incomplete rotation of male genitalia and deformations of the ovipositor, at higher doses their effects gradually spread from the tip of abdomen towards the middle of the body. The highest amounts influence the entire abdomen, size and pigmentation of the eyes, and development of hairs and sclerotization of the integument on the head and thorax. Various species slightly differ in the pattern of morphological effects produced, in the ability of affected insects to leave the puparium, and in the sensitivity to juvenoids of different types. A uniform scale for classification of morphological effects in the species examined is described in this paper. The most potent juvenoids are effective at doses around one nanogramme per specimen. Out of 29 selected compounds tested, isopropyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate is the most active juvenoid for Drosophila, Musca, and Sarcophaga; methyl 10,11-epoxy-3,7,11-trimethyl-2,6-tridecadienoate is the most active juvenoid for Ceratilis; and isopropyl 11-chloro-3,7,11-trimethyl-2-dodecenoate possesses the highest activity for Calliphora.

Biochemical correlations during metamorphosis in Pieris brassicae

Various biochemical parameters including DNA, RNA, proteins, uric acid, and pteridines have been examined during the pupal-adult transformation of Pieris brassicae. Special consideration was given to two tissues: the wings and abdominal fat body. They showed characteristic patterns of development with respect to macromolecular syntheses that could be related to histological data and tentatively correlated with ecdysone levels during the same period. All these data were collected in order to know the in situ behaviour of some organs before the in vitro study of ecdysone action.

Effect of a juvenile hormone analogue on phosphatase activity in pupae of the stable fly, Stomoxys calcitrans

The alkaline phosphatase activity of stable fly pharate pupae treated with 10 ng of an insect juvenile hormone analogue (JHA), Stauffer R-20458, or untreated pupae was rhythmic and peaked 48 and 96 hr after larval-pupal apolysis. Those treated with 10 μg were not rhythmic and peaked at 48 and 120 hr. Acid phosphatase activity showed a general increase throughout pupal-adult transformation and was three- to four-fold higher than alkaline phosphatase activity. At 24 and 48 hr after larval-pupal apolysis, acid phosphatase was lower in the treated animals, but at 72 and 120 hr, it was higher. Neither inhibition nor enhancement of acid and alkaline phosphatase activity or by three other JHAs could be demonstrated in vitro.

Incorporation of 14C from glucose into CO2, chitin, lipid, protein and soluble carbohydrate during metamorphosis of the blowfly, Phormia regina

The metabolism of [14C]glucose was ascertained throughout metamorphosis of the blowfly, Phormia regina. Following injection of labelled substrate, individual insects were fractionated at 24-hour intervals during metamorphosis to obtain carbohydrate, protein, lipid, and chitin fractions as well as CO2. Each fraction was assayed for 14C activity. Maximum metabloic activity occurred at two periods which correspond to the larval-pupal and pupal-adult transformations. By the end of metamorphosis, label from injected glucose was present in each fraction in the following proportions: 41.5 per cent in CO2; 8.9 per cent in protein; 8.3 per cent in chitin; 3.6 per cent in glycogen; 3.0 per cent in lipid; and 4.0 per cent in non-glycogen carbohydrate. The remainder was contained in non-protein amino-acid.

CHANGES IN THE LIPID AND NITROGEN CONTENT DURING POSTEMBRYONIC DEVELOPMENT OF PSEUDALETIA UNIPUNCTA (LEPIDOPTER A: NOCTUIDAE)

AbstractChanges were recorded in dry matter content and in the metabolism of lipids and nitrogen during the postembryonic development of Pseudaletia unipuncta (Haw.) on corn. The study revealed that 22 to 24% of the lipids were used during moulting and pupal–adult transformation and that nitrogen was not lost during such periods. The coefficient of apparent utilization of nitrogen remained more or less constant; however, that of lipids decreased with larval development. No significant difference was evident in the relationship between lipid consumption and weight gain, and nitrogen consumption and weight gain during larval development. A direct linear relationship was obtained between nitrogen and lipid utilization during all instars. In terms of heterauxesis, the accumulation of lipids during larval development was opposed to that of nitrogen.

Effects of ecdysterone in vitro on hindgut and hemocytes of Manduca sexta (Lepidoptera)

Tissues from the tobacco hornworm, Manduca sexla (Johannson), were maintained in vitro for up to 10 months. When treated with the molt-stimulating hormone ecdysterone (β-ecdysone), these tissues underwent morphogenetic changes that closely resembled those normally occurring in vivo. However, when treated with the inactive isomer 22-isoecdysone, no such changes occurred. When tissues from lastinstar larvae were treated, the changes were characteristic of those occurring during the larval-pupal transformation; and when tissues from diapausing pupae were treated, the changes were characteristic of those that occur during the pupal-adult transformation. Hemocytes also responded to treatment with an increase in migratory activity.

DNase as a component of ‘multi-enzyme complexes’ in the blood of saturniid moths

Five distinct bands of DNase activity can be demonstrated by zone electrophoresis of the blood of diapausing Cecropia pupae. This enzyme activity, which resembles vertebrate DNase II, and other hydrolytic enzymes were found to be closely associated with blood pigment. These pigment-enzyme complexes resist dissociation by mild means, although partial separation of different bands was achieved by varying the salt concentration of the blood. Preliminary gradient density studies of one of the complexes revealed a sedimentation constant of approximately 23 S, which is consistent with the presence of several enzyme molecules bound to a lipid molecule. The demonstration of DNase activity in the blood by histochemical means was confirmed by studies of in vitro and in vivo degradation of 14C-labelled exogenous DNA. The patterns of appearance and disappearance of different DNase bands in the blood and tissues during the pupal-adult transformation are also reported. The possible significance of these multi-enzyme complexes and particularly DNase is discussed.

Effects of hormones and injury on RNA synthesis in Saturniid moths

The present report examines the rate of RNA synthesis during the post-embryonic life of the Cecropia silkworm and other Saturniid moths. Autoradiographic determinations were made of the rate of incorporation of tritiated uridine in various tissues during normal development and in insects subjected to endocrinological and surgical manœuvres. The effects of inhibitors of RNA and protein synthesis during various stages of the life cycle were also analysed. The rate of RNA synthesis in many tissues roughly parallelled the rate of respiration during various stages of the life cycle, but there were significant differences in temporal patterns of RNA synthesis not only between tissues but within a tissue. During larval moult cycles chitogenous epithelia such as epidermis, tracheae, and foregut showed temporal patterns of RNA synthesis which correlated directly with the process of moulting. In other tissues the temporal patterns of RNA synthesis did not parallel the events of the moult cycle. The rate of RNA synthesis was higher during larval life than in any other stage. After pupation RNA synthesis decreases in most tissues and is extremely low during diapause at which time only a few tissues such as haemocytes, brain, oenocytes, Malpighian tubules, and perigonadal fat body continue to synthesize RNA at significant rates. During the first half of the pupal-adult transformation, the rate of RNA synthesis increased initially, but thereafter it decreased in many tissues. Integumentary injury to diapausing pupae stimulated RNA synthesis. A local stimulation occurred within 2 hr of injury and was characterized by intense RNA synthesis in tissues near the injury. A systemic stimulation appeared later in all of the tissues examined except the gonads. This systemic stimulation of RNA synthesis roughly paralleled the increased respiration stimulated by injury. A pupal cuticle was synthesized by the regenerating epidermis in the absence of juvenile hormone. When RNA synthesis was blocked in diapausing pupae with actinomycin D, pupae survived and continued to synthesize proteins for 24 days, suggesting that messenger RNAs survive for many days. When protein synthesis was blocked with puromycin, diapausing pupae died within 17 days. During periods of rapid RNA synthesis such as larval life, early stages of adult development and after injury, the insects were promptly killed by low doses of actinomycin. When RNA synthesis was low, such as during diapause, in late states of adult development, and during adult life, they were insensitive to large doses of actinomycin. Experiments on developing adults indicated that the mRNA for many specific developmental events was made many days prior to the event.

Fatty acid composition of the silkworm, Bombyx mori L.

The fatty acid compositions of lipids of Bombyx mori L. from egg to adult, of larval organs, and of mulberry leaves were determined by gas-liquid chromatography. Fourteen fatty acids, including minor or trace elements, were identified with the extracts from the silkworm and seventeen acids from mulberry leaves. Linoleic acid predominated throughout development, usually constituting one-third of the total acids, and it was also in most of the larval organs. The fatty acid composition varied greatly during the larval period; young larvae contained much more stearic and linoleic acids and much less palmitic and oleic acids than older larvae. From the mid-fifth instar to the adult stage a gradual change in composition was observed. The distinct divergence of the composition of the larva from that of the mulberry leaves suggested some aspects of fatty acid synthesis. The fatty acid pattern showed large variations among larval organs. Male pupae contained more fatty acids throughout than female pupae. The female utilized fatty acids during the pupal-adult transformation, whereas fatty acids were reserved in male pupae. The unsaturated fatty acids predominated over the saturated in both sexes, but a higher proportion of unsaturated acids was observed in female pupae. Furthermore, it was shown that the fatty acid pattern was different in the sexes.

Cellular membrane formation by plasmatocytes of diapausing Cecropia pupae

The early phase of wound healing in diapausing Hyalophora cecropia pupae is dominated by the formation of an acellular membrane by plasmatocytes. Phase contrast microscopic observations of blood from unwounded pupae reveal few circulating haemocytes, approximately half of which are plasmatocytes of varying morphology. In the days following injury both the absolute and relative number of plasmatocytes increase. The initial increase in cell number is not attributable to mitosis because no mitotic figures are seen until 18–24 hr after wounding, whereas substantial increases in cell number are observed by this time. A relative increase in large fusiform plasmatocytes suggests instead that the initial cellular response to injury is the release of haemocytes from depots within the insect. Subsequently, mitoses of haemocytes, particularly plasmatocytes, become rather frequent, and further increases in cell number appear to result from these mitoses. Time lapse sequences of wound healing were photographed with phase optics through the optically flat surfaces of a plastic chamber which connected the haemocoeles of two pupae in telobiosis. As plasmatocytes touch the plastic surface of the chamber, they lose much of their heteromorphy. On these surfaces a cell typically flattens, produces a ruffled membrane, and migrates randomly over the plastic. As the population of attached haemocytes increases, movement of the individual cells is progressively restricted. A complete cellular sheet eventually covers the plastic surfaces. This cellular membrane is subsequently thickened by the deposition of additional plasmatocytes on the blood side. The cellular membrane then degenerates and tears free from the plastic. The broken membrane fragments are pulled into the wound areas by circulatory movements of the haemolymph and partially seal the pupae from the blood chambers. Wound closure is aided by the tendency of hyaline plasmatocytes to adhere to each other and to other cells and thereby to form large clumps of cells. These clumps attach to membrane fragments and form plugs that stop the flow of haemolymph into the blood chamber. Tracheae, accompanied by epithelial sheets, grow from the margins of the wound. The extending tracheal system aids in wound closure and is the means whereby a continuous epithelium is established across the injured area. Since hypodermal cells apparently do not undergo mitosis in the absence of ecdysone, and since only occasional hypodermal cells and a few tracheoblasts synthesize DNA in the absence of hormone, the ability of the cells of the tracheal system to migrate and produce an epithelial sheet assumes great importance for successful wound healing in diapausing pupae. Evidently the replacement of thinned-out epidermal cells as well as the secretion of a cuticle in the wound area is delayed until the hormonally mediated pupal-adult transformation.

Programmed cell death—II. Endocrine potentiation of the breakdown of the intersegmental muscles of silkmoths

The abdominal intersegmental muscles of saturniid pupae are well preserved during the entire pupal period and throughout the three weeks required for the development of the adult moth. Then, within 48 hr after the emergence of the moth, the muscles break down and disappear. The breakdown is a result of developmental reactions within the muscles themselves; their dissolution, in this sense, is a carefully timed, final episode in their own metamorphosis. Thus, the endocrine conditions obtaining at the onset of adult development (the presence of ecdysone and the absence of juvenile hormone) dictate both the formation of all adult structures, including muscles, and the eventual death of the intersegmental muscles. If juvenile hormone is injected, the breakdown does not occur in the intersegmental muscles or in any of the other tissues which are normally destroyed during the pupal-adult transformation. The final signal for the initiation of breakdown does not appear to be endocrine in character. Thus, when pairs of pupae were joined in parabiosis so that they shared the same blood, the breakdown of muscle in one individual did not hasten or retard the breakdown in the other individual. This finding points to some further signalling system which triggers the initiation of cell death in the endocrinologically potentiated muscles.

The content of juvenile hormone and lipid in lepidoptera: Sexual differences and developmental changes

Juvenile hormone, lipid and water content were measured in H. cecropia at representative stages during its life cycle. Alterations in juvenile hormone titer were consistent with our present understanding of the endocrine control of insect metamorphosis. It is present in the larval stages but absent prior to the molt from pupa to adult. The finding of hormone in the egg and embryo can be explained in part by the concentration of juvenile hormone in the adult female but a measurable amount is produced during embryonic development. The adult moths have the highest hormone concentration and the male adult usually has a greater quantity of hormone than the female. The sexual dimorphism in hormone content is due to greater secretion by the large male corpora allata and could not be equalized by the surgical techniques conducted. Of the many species assayed, the male Cecropia moth yielded the most hormone. The studies on total lipid changes revealed that the male Cecropia utilizes little if any net lipid during the pupal-adult transformation, but uses lipid rapidly as a flight fuel during adult life. During adult life the total lipid of both males and females is rapidly depleted and may be one renson that these moths die in about 10 days.

Immunological studies of insect metamorphosis. II. The role of a sex-limited blood protein in egg formation by the Cecropia silkworm.

1. In the pupal stage of the cecropia silkworm, antigen 7, a protein with the solubility characteristics of an albumin, is present in female blood in approximately a thousand times higher concentration than in the blood of males. Antigen 7 is undetectable in the blood of larvae of either sex. It first appears in the blood after the larva has spun its cocoon, and is present throughout all subsequent stages of metamorphosis. Late in the pupal-adult transformation, when the eggs are produced, the concentration of antigen 7 in female blood decreases significantly. 2. An antigen which could not be distinguished from antigen 7 immunologically is present in solution in the yolk of unfertilized eggs. 3. In females which, by ovariectomy, were prevented from forming eggs, the concentration of antigen 7 in the blood increased during the usual period of egg formation rather than undergoing the normal decrease. Ovaries transferred to the hemocoel of males produced eggs but were unable to incorporate antigen 7 in the yolk unless a detectable amount of the protein was present in the blood. The ovaries of polyphemus females which had been transfused with cecropia blood incorporated cecropia antigen 7 into the eggs they produced. These lines of evidence indicate that antigen 7 is secreted into the blood by some tissue other than the ovaries, and that it is subsequently drawn from the blood and deposited in the yolk. 4. The concentration of antigen 7 in the clear, liquid fraction of the yolk is four times higher than the maximum concentration attained in the blood during metamorphosis, and twenty times higher than that of the blood at the conclusion of egg formation. The protein thus appears to be transferred from blood to yolk against a concentration gradient.