Changes in ecdysone titre during pupal-adult development in the silkworm, Bombyx mori

Changes in the ecdysone titre of the silkworm, Bombyx mori, during pupal-adult development were estimated. The average value of the maximum titre, which was observed on the second day after pupal ecdysis, was about 0·8 μg equivalent of β-ecdysone/g of live weight in both sexes.

There is a distinct sexual dimorphism in the pattern of the ecdysone titre. The male exhibited a single sharp peak on the second day whereas the female showed the second peak on the fifth day. When the female was ovariectomized, the ‘female type’ ecdysone pattern was converted to the ‘male type’. In the female pharate adult 7 days after pupal ecdysis, ecdysone activity accumulated in the ovaries.

The relationship between the ecdysone titre and adult differentiation, especially during ovarian development, is discussed.     

Stem cells of the beetle midgut epithelium

At the completion of metamorphosis, adult insect cells have traditionally been assumed to halt cell divisions and terminally differentiate. While this model of differentiation holds for adult ectodermal epithelia that secrete cuticular specializations of exoskeletons, adult endodermal epithelia are populated by discrete three-dimensional aggregates of stem cells that continue to divide and differentiate after adult emergence. Aggregates of these presumptive adult stem cells are scattered throughout larval and pupal midgut monolayers. At the beginning of adult development (pupal-adult apolysis), the number of cells within each aggregate begins to increase rapidly. Dividing cells form three-dimensional, coherent populations that project as regenerative pouches of stem cells into the hemocoel surrounding the midgut. Stem cell pouches are regularly spaced throughout endodermal monolayers, having adopted a spacing pattern suggesting that each incipient pouch inhibits the formation of a similar pouch within a certain radius of itself—a process referred to as lateral inhibition. At completion of adult development (pupal-adult ecdysis), a distinct basal-luminal polarity has been established within each regenerative pouch. Dividing stem cells occupying the basal region are arranged in three-dimensional aggregates. As these are displaced toward the lumen, they transform into two-dimensional monolayers of differentiated epithelial cells whose apical surfaces are covered by microvilli. This organization of stem cell pouches in insect midguts closely parallels that of regenerative crypts in mammalian intestines.     

Simultaneous determination of molting and juvenile hormone titers of the greater wax moth

A simple and rapid extraction procedure was developed to determine simultaneously the molting hormone (MH) and juvenile hormone (JH) activity in a single insect tissue sample. From the onset of the last larval stage to adult eclosion of the greater wax moth, Galleria mellonella, three JH peaks were noted: at the time of the sixth larval ecdysis, 1 day before the seventh larval ecdysis, and at the time of adult eclosion. Three MH peaks were recorded for the male: at 1 day before the sixth larval ecdysis, 1 day before the seventh larval ecdysis, and 2 days after pupation. In the female, a fourth peak was shown at the time of adult eclosion. This fourth peak exhibits the highest molting hormone activity of all samples, 1600 Musca units/g of fresh tissue or an equivalent of 5.6 μg/g of ecdysterone. Eighty per cent of this MH accumulated in the ovary. The significance of MH and JH titers as related to the endocrine regulation of development is discussed in the light of this finding.     

Effects of methoprene and juvenile hormone on larval ecdysis,emergence, and metamorphosis of the endoparasitic wasp, Apanteles congregatus

Topical application of juvenile hormone I and III or the hormone analogue methoprene to parasitized Manduca sexta larvae inhibited subsequent emergence of the endoparasitic wasp Apanteles congregatus. Methoprene treatment inhibited wasp emergence in a dose-dependent manner, causing either a delay or total inhibition of emergence. These results were interpreted as reflecting inhibitory effects of juvenile hormone on the second-larval ecdysis of the parasitoid that normally occurs during emergence from the host larva. Parasitoid ecdysis was disrupted even when methoprene was applied to host larvae a few hours prior to the normal expected time of emergence. A correlation between the number of emerging parasitoids and the timing of emergence was seen in methoprene-treated hosts, and few parasitoids emerged after day 9 of the host's fifth-instar. Our findings suggest that the suppression of emergence by juvenile hormone analogues noted in previous studies may be due to a similar inhibitory effect on parasitoid ecdysis. We also observed that parasitoids emerging from hosts treated with a low dose of methoprene (1 μg) later pupated normally but then formed nonviable pupal-adult intermediates. Thus use of this insect growth regulator must be undertaken carefully to prevent possible adverse effects on natural parasitoid populations.     

Patterns of biosynthesis and accumulation of hydrocarbons and contact sex pheromone in the female german cockroach,Blattella germanica

De novo synthesis of contact female sex pheromone and hydrocarbons in Blattella germanica was examined using short in vivo incubations. Accumulation of pheromone on the epicuticular surface and the internal pheromone titer were related to age-specific changes in hydrocarbon synthesis and accumulation in normal and allatectomized females. The incorporation of radiolabel from [1-¹⁴C]propionate into the cuticular methyl ketone pheromone fraction was positively related to corpora allata activity during two gonotrophic cycles. During peak pheromone production the total internal lipid fraction contained greater titers of pheromone than the cuticular surface, and it too exhibited a cycle internally, preceding the rise in external pheromone. This suggests that synthesis and accumulation of pheromone internally are followed by transport of pheromone to the epicuticular surface where it accumulates. Radiolabel was incorporated efficiently into both cuticular and internal hydrocarbons after the imaginal molt and until the peak of pheromone synthesis, but it declined to lower levels before ovulation and throughout pregnancy. The internal hydrocarbon titer decreased 58% after oviposition, suggesting deposition in the egg case. It remained relatively unchanged during pregnancy and increased again during the second gonotrophic cycle. In allatectomized females, hydrocarbon synthesis was reduced relative to control females until oviposition in the latter. However, subsequent rates of hydrocarbon synthesis in allatectomized females (without oothecae) exceeded the rates in sham-operated females (with oothecae). In the absence of ovarian uptake of hydrocarbons, the internal titer increased without the decline found in control females at oviposition. As internal hydrocarbons increased, so did cuticular hydrocarbons and both internal and cuticular methyl ketone pheromones. These patterns corresponded well with feeding patterns in sham-operated and allatectomized females, suggesting that pheromone production is normally regulated by stage-specific feeding-induced hydrocarbon synthesis (precursor accumulation internally) and juvenile hormoneinduced conversion of hydrocarbon to pheromone. They also suggest that both the cuticle and the ovaries might be target sites for hydrocarbon and possibly methyl ketone deposition. © 1994 Wiley-Liss, Inc.     

Molting hormone titer changes and their significance during development of the colorado beetle, Leptinotarsa decemlineata

Molting hormone (MH) titer in whole animal extracts of Leptinotarsa decemlineata was determined by chemical extraction and the Musca test (1 MU = 3.5 ng ecdysterone) during the developmental span from newly-ecdysed fourth instar larva to an adult 3 days after eclosion. Within the 17-day period, 21 age groups were chosen to estimate the MH titer. Two peaks of MH titer were detected, one in the post-feeding larval stage and the other during the pupal and pharate adult stage. MH activity was first detected in 2-day-old post-feeding larvae, and reached a maximum of 23.5 MU/g tissue on the third day. It began to decline on 3.5 days, and fell to 5.5 MU/g tissue on 4.5 days, the time of larval-pupal ecdysis. In the pupal and pharate adult stage MH rose after the first day and increased to a maximum of 91.5 MU/g tissue on the third day. The titer again declined on the fourth day, and became undetectable one day before adult emergence and in adults 3 days after emergence. MH was demonstrated to be produced by isolated larval abdomens. A peak of 11.5 MU/g tissue was detected in 7-day post-ligation preparations. The titer decreased to 6.9 MU/g tissue in 10-day post-ligation preparations, which was the time of the ecdysis. The finding raises questions concerning the rôle of MH synthesis by other tissues in relation to the function of the prothoracic glands during insect development.     

Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.     

Juvenile Hormone Binding Protein Titers During Adult Metamorphosis in Manduca sexta

ABSTRACT ABSTRACT During the pupal and adult stages, the JHBP levels displayed a sex-related difference, with females showing higher levels than males. A sharp increase in JHBP levels was observed at day 2 of the pupal stage. After day 2 the JHBP titer declined precipitously, and then remained unchanged until day 12 in males. JHBP titers in females decreased slightly after day 2 and then remained relatively constant until day 12. In both sexes, the JHBP levels showed a steep increase and peaked around day 15. During the previtellogenic period, the JHBP titers declined dramatically until adult ecdysis. During early adult stage, JHBP titiers in the female remained constant at preecdysis level. This information could broaden our understanding of pest physiology during adult metamorphosis, and could have extensive implications for developing insect growth regulators to control agricultural pests.     

Activities of hormone metabolizing enzymes in house flies treated with some substituted urea growth regulators.

The insect growth regulators (IGR) TH 6038 and TH 6040 affect larvae of various species by interfering with cuticle development. In a biochemical study of their effects, larvae of the house fly, $\text{Musca domestica}$ L. were reared for 2 days on diets containing 1.7 to 166.7 ppm of these compounds, then assayed for activities of the microsomal oxidases and the enzyme(s) which metabolize β-ecdysone. The activities of these enzymes were compared with the percentage of treated larvae completing pupal-adult ecdysis. The two compounds reduced the activity of the β-ecdysone metabolizing enzyme(s) by as much as 57%, reduced pupal-adult ecdysis by 43% to 100%, and stimulated microsomal oxidase activity 4- to 12-fold. Supplementation of the diet of the treated insects with the Cecropia juvenile hormone, JH I, partially restored pupal-adult ecdysis but supplementation with β-ecdysone had no effect. The mode of action indicated by these results is that the IGRs cause an accumulation of β-ecdysone in the treated larvae. This stimulates the enzyme, chitinase, which degrades chitin in preparation for formation of the new cuticle. The hormone may also cause a JH deficiency and the stimulation of DOPA decarboxylase and phenol oxidase which would further disrupt the normal molting process.     

Testicular maturation in the sheep bot fly Oestrus ovis

The process of testicular maturation in relation to intrapuparial development was studied in the sheep nasal bot fly, Oestrus ovis L. (Diptera: Oestridae). After formation of the puparium during larval-pupal apolysis and the cryptocephalic pupal stage (approximately 24-72 h), spermatogonia had undergone mitotic divisions and sperm cysts had been formed. Five days after pupariation, spermatogonia transformed into primary spermatocytes during the phanerocephalic pupal stage, and secondary spermatocytes first appeared during the pupal-adult apolysis. Secondary spermatocytes began undergoing the second meiotic division by day 8 (transparent-eye pharate adult stage). By days 9 and 10, round spermatids were present and began to elongate by day 11. By day 12, the first bundles of tailed spermatozoa had appeared. By day 15 (the yellow-orange eye pharate adult stage), round, elongated, tailed and bundled spermatids were predominant and by day 17 differentiating spermatids occupied nearly 35% of the testicular cavity, and 60% was occupied by free sperm. By day 21 (the red-brown eye pharate adult stage), spermatozoa colonized the seminal vesicle. At emergence (approximately day 22), a complement of free sperm occupied the testis and the seminal vesicle, but groups of developing cells frequently remained in certain zones. Spermatogenesis was carried out after pupariation and spermiogenesis occurred during the pharate adult stage. After emergence, males possessed fully formed spermatozoa ready for ejaculation.     

Epidermal cell development during the pupal-adult metamorphosis of Hyalophora cecropia

To establish a base for studying the hormonal control of insect epidermal cell activity, the ultrastructure of abdominal epidermis was analyzed during the normal pupal-adult development of Hyalophora cecropia. Adjacent epidermal cells could be distinguished on the basis of organelle content and staining intensity, suggesting that this monolayer is not composed of a homogenous cell population. At the onset of adult development the form of the epidermal cell is transformed from that typical of a quiescent cell with free ribosomes and few mitochondria to one which is metabolically active and possesses numerous apical membrane microvilli, rough endoplasmic reticulum and numerous mitochondria. On about day 5 of pharate adult development the apical plasma membrane is no longer folded but becomes folded again several days later when cuticulin and endocuticle are deposited. On about day 7, giant autophagic vacuoles are discerned that may be important in cellular reprogramming. After adult ecdysis, the epidermal cells continue to deposit endocuticle.     

Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary

We previously reported purification of the cysteine protease from Bombyx eggs (BCP) and the occurrence of the enzyme in various tissues of this insect. In the present paper, we present a detailed analysis of stage-specific changes in activity of BCP between the fourth larval instar and pupal-adult development. A synthetic fluorescent peptide, carbobenzoxy-L-Phenylalanyl-L-Arginine4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA), was used to assay proteolytic activity. When tissue extracts were treated with anti-BCP serum before assay of enzyme activity, most activity towards Z-Phe-Arg-MCA was removed from the extracts. Therefore proteolytic activity in the present experiments is due mainly to BCP. We used Western blot and Northern blot analyses to determine tissue and stage specific expression of the enzyme. In the 5th larval fat body and hemolymph, BCP activity dramatically increased at the time of spinning, returning to the basal level before ecdysis. Northern blot analysis showed that a 1.5 kilobase mRNA which hybridizes to BCPcDNA suddenly appears during this period. Similar results were obtained in 4th instar fat body. In pupal hemolymph and fat body, low basal activity of BCP was detected early (day 0 to day 3 after pupal ecdysis), followed by a pronounced increase to a maximum six days after ecdysis, before returning to the basal level. In ovariectomized female pupae, a significant amount of proteolytic activity accumulated in hemolymph, suggesting that the enzyme is synthesized in the fat body and transferred into the ovary along with vitellogenin. BCP activity increased three days after injection of 20-hydroxyecdysone into ligated pupae. Furthermore, putative BCPmRNA appeared in the fat body within 24 hours after injection. This increase was completely blocked by the administration of cycloheximide. The results suggest that, BCP is synthesized in extraovarian tissues such as fat body and ovarian follicle cells and accumulates in the ovary, thus representing a new class of yolk protein.     

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.     

Regulation of 20-hydroxyecdysone on the larval pigmentation and the expression of melanin synthesis enzymes and yellow gene of the swallowtail butterfly, Papilio xuthus

The swallowtail butterfly, Papilio xuthus, changes its larval body pattern dramatically during the fourth ecdysis. Cuticular pigmentation occurs with precise timing just before ecdysis. We previously found that the cuticular pigmentation was regulated by three melanin synthesis genes, tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. We discovered that yellow is strongly expressed in the presumptive black markings earlier than TH and DDC. Because the ecdysis is triggered by 20-hydroxyecdysone (20E), the effects of 20E on the pigmentation and expression of the melanin synthesis genes were examined. Here, we established a method for the topical application of 20E to molting specimens, so that 20E has only a partial effect, resulting in successful ecdysis. When we applied 20E during the mid-phase of the molting period, when the 20E titer is declining, cuticular pigmentation was completely inhibited. The cessation of hormonal treatments caused delayed pigmentation. yellow expression was promoted by a high titer of 20E, whereas the expression of TH, DDC, and ebony was suppressed, suggesting that a decline in the 20E concentration is necessary for the induction of the expression of the latter three genes. These results indicate that cuticular pigmentation is controlled by the exposure to 20E and its removal.     

Induction of female sex pheromone production in male houseflies by ovary implants or 20-hydroxyecdysone

Implanting ovaries or injecting 20-hydroxyecdysone into male houseflies induced sex pheromone production, including (Z)-9-tricosene (muscalure), 9,10-epoxytricosane and (Z)-14-tricosen-10-one, which normally occurs only in vitellogenic females. Control males did not produce detectable amounts of these compounds. Injection of 20-hydroxyecdysone (5 μg/insect per day) for 3 days resulted in the accumulation of 1.81 μg/insect of (Z)-9-tricosene, 0.97 μg/insect of 9,10-epoxytricosane and 0.12 μg/insect (Z)-14-tricosen-10-one. Multiple injections of 20-hydroxyecdysone at doses as low as 50 ng resulted in the accumulation of 23:1, C₂₃ epoxide and C₂₃ ketone; shifted the distribution of label within the alkenes from 27:1 to 23:1 and decreased the amount of label in the hydrocarbon fractions as alkenes. Structures of the C₂₃ alkene and epoxide produced by the males were verified by gas chromatography-mass spectrometry. Radioactivity from [1-¹⁴C] acetate was incorporated into the C₂₃ alkene, epoxide and ketone in male insects after ovaries were implanted or they were injected with 20-hydroxyecdysone. Synthesis of the C₂₃ pheromone components decreased rapidly within several days after the administration of 20-hydroxyecdysone ceased, indicating that the enzymes involved in sex pheromone production were not permanently induced by hormone treatment. Ecdysone was also effective in initianing pheromone production in males, whereas inokosterone and cholesterol were not effective. Data presented demonstrate that male houseflies possess the metabolic capability to produce the sex pheromone components, and this suggests that 20-hydroxyecdysone alters the production of cuticular hydrocarbons such that the C₂₃ sex pheromone components become major products.     

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.     

Diuresis in the cabbage white butterfly, Pieris brassicae: Fluid secretion by the malpighian tubules

The sudden weight loss at the pupal-adult ecdysis of the cabbage white butterfly, Peiris brassicae, is caused by a diuresis which greatly reduces the blood volume after emergence. Study of the Malpighian tubules as isolated preparations shows that only those of the adult insect possess the ability to secrete fluid at extremely fast rates when stimulated. In contrast to the fast-secreting tubules of bloodsucking insects, the mechanism of fluid secretion is not unusual, probably depending on the active transport of potassium ions.     

Patterns of leucine incorporation in the spermathecal accessory glands of the post-ecdysial adult female Tenebrio molitor

The spermathecal accessory glands of female mealworm beetles become fully active by six days after pupal-adult ecdysis. Coincident with the morphological maturation, increases occur in protein and RNA content and in total leucine incorporation. Three classes of differentiation-specific proteins are increasingly prominent and show heightened rates of leucine incorporation by the sixth day.     

Cuticular sclerotization in larval and adult locusts, Schistocerca gregaria

The sclerotization of both larval and adult cuticle from the desert locust, Schistocerca gregaria, has been studied by measuring the incorporation of radioactive dopamine and N-acetyldopamine into the cuticle. The results are compared with the degree of sclerotization of the cuticle and the amount of sclerotizing enzyme present. The various parts of the cuticle differ considerably with respect to the degree of sclerotization: in adult locusts the mandibles and the dorsal mesothoracic cuticle contain about twenty times as much cross-linking material per mg cuticle than is present in the abdominal tergites and sclerites.The degree of sclerotization in the various types of cuticle is apparently not determined by the amounts of sclerotizing enzyme present, and the rate at which radioactive dopamine or N-acetyldopamine is incorporated into the cuticle appears also to be unrelated to the amount of enzyme.The degree of sclerotization of the various parts of the cuticle from fifth instar larvae corresponds with the amounts of labelled dopamine which are incorporated during the first day after ecdysis, whereas there is no correlation between sclerotization and the amounts of labelled dopamine which are incorporated in older larvae. The degree of sclerotization of adult cuticle after 1 day corresponds to the incorporation of dopamine during the first day. When older animals are compared only little correlation is observed. The relative rates of sclerotization in the various parts of the cuticle must therefore change as the adult insect grows older.The changes in the incorporation pattern during the development of the locust are discussed in relation to the physiological control of the sclerotization process.