Changes in lipophorin composition during the fourth to fifth instar molt of Manduca sexta

During the feeding stage of the fourth instar of the tobacco hornworm, Manduca sexta, lipophorin has a density of 1.144 g/ml and is comprised of 55% protein and 45% lipid, mainly diacylglycerol and phospholipid. These values are similar to those found during the corresponding period of the fifth instar. However, during the intermolt period the lipid composition of lipophorin changes to 45% protein and 55% lipid and the density decreases to 1.112 g/ml. The onset of these changes in lipid content correspond to the increase in ecdysteroid titer which signals the beginning of molting. At the end of molting both the lipid content and density return to those characteristic of the fifth instar. These data further confirm the complex nature of lipophorin metabolism in M. sexta larva and suggest that ecdysone may be the trigger for lipid mobilization during the intermolt period.     

The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust

The presence of juvenile hormone in the haemolymph of larvae of Locusta has been detected by a modified Galleria bioassay and these results are compared with indirect methods of estimating corpus allatum activity. Juvenile hormone is present in the haemolymph during the fourth larval instar except on the last day of the instar, and is absent from the haemolymph of the fifth and final larval instar except on the last day of the instar. Changes in the volumes of the corpora allata simply reflect changes in the growth of the whole insect and are of no value in predicting endocrine activity. Changes in the size of the cells of the corpora allata can be correlated with the presence of juvenile hormone in the haemolymph in the fourth larval instar, but similar changes in cell size occur in the fifth larval instar when no juvenile hormone is present in the haemolymph. The effects of the implantation of corpora allata are unreliable as estimates of corpus allatum activity as isolated corpora allata from fifth instar larvae release juvenile hormone. Indirect methods of measuring corpus allatum activity are thus shown to be unreliable. The R_f value of Locusta juvenile hormone as determined by thin-layer chromatography differs from that of Roeller's juvenile hormone, suggesting that the two hormones might be chemically distinct.     

Synchrony of juvenile hormone-sensitive periods for internal and external development in last-instar larvae of Oncopeltus fasciatus

Breakdown of the moulting glands in Oncopeltus can be completely inhibited by topical application of a juvenile hormone analogue prior to day 2 of the fifth instar, and partially inhibited by application prior to day 4. The analogue-sensitive period for the inhibition of external metamorphosis is very similar to that for the inhibition of cell death in the moulting glands. A decline in response to the analogue between days 2 and 4 is correlated with rising ecdysteroid levels in the haemolymph. That this rise in ecdysteroids may be responsible for termination of the juvenile hormone-sensitive period is suggested by premature loss of sensitivity to the analogue by the moulting glands following premature exposure to 20-hydroxyecdysone.     

Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta

The titer of ecdysone in whole animal extracts of Manduca sexta was determined by radioimmunoassay during the fifth (last) larval instar, pharate pupal development and pupation. A subtle peak in ecdysone concentration was noted at day 4 (just prior to the onset of the wandering stage) and a second and greater peak at day 8.5 (coincident with pharate pupal development). The titer fluctuations during development were a result of changes in tissue ecdysone and not of alterations in the ecdysone content of the gut. When prothoracic gland secretory activity was analyzed in vitro at the same stages, the most rapid rate of α-ecdysone secretion was shown to occur on day 7 (one day prior to the peak in whole-animal ecdysone concentration). An earlier peak in prothoracic gland activity may occur at day 4–5. Thin layer and gas-liquid chromatographic analyses revealed developmental changes in the ratio of β:α-ecdysone in hemolymph and whole-animal extracts. It is suggested that the steroid-hydroxylating capacity of the insect increases during the instar.     

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

The temporal organization of endocrine events underlying larval-pupal moult was examined. After the phagoperiod of 6–7 days, the last instar larvae of Bombyx mori purged the gut contents and then pupated 4–5 days thereafter. Juvenile hormone titer was considerably high on the first day of the fifth instar, but declined to an undetectable level on day 3. Release of prothoracicotropic hormone (PTTH) began after the decline in the junvile hormone titer and completed during the night of day 4. Prothoracic glands began to secrete ecdysone on day 5. Larvae could pupate on schedule if their brains were removed after the PTTH release. One time of PTTH release during the feeding stage could satisfy the requirement for pupation occuring on schedule. The phagoperiod could be shortened by allatectomy early in the fifth instar and prolonged by the injection of a certain dose of juvenile hormone analogue before the gated PTTH release, accounting for the role of juvenile hormone in the timing of PTTH release.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

The role of juvenile hormone in the larval diapause of the European corn borer,Ostrinia nubilalis

The possible role of juvenile hormone (JH) in the induction and termination of larval diapause in the European corn borer, Ostrinia nubilalis, was investigated using topical applications of both JH I and a JH mimic as well as by monitoring JH titers with the Galleria bioassay. Neither JH nor the JH mimic ZR515 was capable of influencing diapause termination when administered topically. The Galleria bioassay revealed little or no JH in the hemolymph of mid diapause (>30 days) insects, indicating no demonstrable role for JH in diapause maintenance. When ZR515 was administered to nondiapause, newly ecdysed fifth instar larvae the pupal molting cycle was delayed. By use of photoperiodic regimes we were able to show that the molting delay was not equivalent to diapause induction. The Galleria bioassay showed differences in JH titer profiles between diapause and nondiapause animals during the final larval stadium. The nondiapause insects showed titers that decline rapidly to trace amounts following the molt to fifth instar then rose prior to pupation. The diapause insects had generally higher titers and exhibited a more gradual decline after the molt. No evidence was obtained to support the hypothesis that JH plays a key role in the induction, maintenance, or termination of larval diapause.     

Purification,characterization and developmental changes in the titer of a new larval serum protein of the silkworm,Bombyx mori

A new protein was found as a major component of hemolymph proteins up to day 1 of the last larval instar of Bombyx mori, and was named Bombyx mori larval serum protein (BmLSP). The BmLSP was purified to homogeneity by ammonium sulfate precipitation, CM-cellulose column chromatography and gel permeation chromatography. The molecular weight of BmLSP was estimated to be 30,000 by SDS-PAGE and 25,000 by gel permeation chromatography. The amino acid composition of BmLSP was similar to that of 30 kDa proteins which are the major serum proteins in the older last (fifth) instar larvae. The 20 NH₂-terminal amino acids were sequenced and found to be quite different from those of the 30 kDa proteins. Developmental changes in BmLSP titer were followed throughout post-embryonic life by Western blotting using a specific antiserum against BmLSP. Within 1 day after larval hatching, BmLSP appeared in the hemolymph and remained at an almost constant level until day 1 of the last instar. On day 2 of the last instar, the BmLSP level suddenly fell and then gradually decreased toward larval-pupal metamorphosis. Thus, BmLSP is a true larval serum protein and is different from proteins stored for metamorphosis.     

Activity of the prothoracic gland and its sensitivity to prothoracicotropic hormone in the penultimate and last-larval instar of Bombyx mori

The time course of secretion of ecdysone in vitro by the prothoracic glands of Bombyx mori was studied through the penultimate and last-larval instars. Ecdysone was produced by the glands in high amounts by the penultimate instar at 72 and 84 h while the glands in the last instar exhibited a high activity over 4 days around the time of gut purge and thereafter. The glands in the penultimate instar produced ecdysone at a low level throughout the instar before the sharp peak of activity, when they became inactive and remained so for the first 3 days of the last instar after when they regained secretory activity. Sensitivity of the glands to prothoracicotropic hormone varied in accord with the changes in their secretory activity. Inactive glands were not stimulated by 22K-prothoracicotropic hormone. In addition, glands with maximal activity in the penultimate instar were insensitive to 22K-prothoracicotropic hormone. These results suggest that the prothoracic glands in the penultimate and last-instar larvae are physiologically different.     

亚洲玉米螟末龄幼虫促前胸腺激素与蜕皮甾类激素滴度的变化

用放射免疫分析法(Radioimmunoassay,RIA)以12小时间隔测定了亚洲玉米螟Ostrinia tfurnacalis末龄非滞育幼虫血淋巴中蜕皮甾类激素滴度.通过前胸腺体外培养,以12小时间隔测定了前胸腺体外分泌活性的变化.发现二者的变化在相同发育阶段是一致的.在亚洲玉米螟上建立了促前胸腺激素(PTTH)体外测定法,并用此法以24小时间隔测定了末龄幼虫脑和血淋巴中PTTH滴度.发现血淋巴中PTTH滴度在末龄第5和7天各有一高峰,脑中只在第5天有一高峰.     

NEUROANATOMICAL STUDIES OF PROTHORACIC GLANDS OF COTTON BOLLWORM HELZCOVERPA ARMZCERA (LEPIDOPTERA: NOCTUIDAE)*

Abstract Gross anatomy, ultrastructure, innervation and ultrastructural alterations of the prothoracic gland (PTG) of cotton bollworm, Helicover pa armigera (Lepidoptera: Noctuidae) are illustrated for the last larval and early pupal stages as observed by light and electron microscopy. The T-shaped, paired (PTGs) consist each of 76–116 cells which are classified morphologically as large and small gland cells. In addition, another kind of small (about 6μ in diameter) gland cell was found in the PTGs of last instar larvae. The PTGs are innervated by the branches of 3 nerves! and tracheae and tracheoles are abundantly distributed to these glands. PTGs disappeared completely by the third day after ecdysis to the pupal stage (at temperature 28 C with a photoperiod L15^:D9). An intercellular channel system (ICS) is formed by numerous, deep invaginations of the plasma membrane of gland cells. This ICS gradually increases in depth and width and reaches maximum development around the time of the major ecdysteroid secretion peak during the last larval instar. Numerous multivesicular sacs (MVS) with their remnants and an extensive rough endoplasmic reticulum were observed within ICS and cytoplasm, respectively, on the fourth day of the last larval instar. At that time the matrix of mitochondria became much more electron lucent. Freeze-fracture replicas of the glandular epithelium were made from last instar (4th day larvae. Dynamics of structure are related to data from others concerning secretory states of the prothoracic glands of this species.     

Requirements for molting of the crochet epidermis of the tobacco hornworm larva in vivo and in vitro

Summary In the tobacco hornworm,Manduca sexta, the epidermis which underlies the larval crochets is the first tissue to become independent of the prothoracic glands (PG) in a larval molt. In each successive larval molt, crochet forming cells increase in size, form hooks at their distal ends and, finally, secrete cuticle. This paper examines the endocrine requirements for competence to molt and describes parallel cultures in vivo and in vitro to define the hormonal control of crochet molting. When implanted into a fourth instar host larva prior to initiation of the last larval molt, competent crochet epidermis molted, forming crochets synchronously with its host. In the fourth instar, competence to form crochets is attained slowly during the first two days following ecdysis from the third instar. During the feeding phase of the fifth (last) instar, the crochet epidermis remains competent to molt (to form an extra sixth instar set of crochets) until the larva attains a weight of about 4.5 gm. Then, concurrent with the decline in the titer of juvenile hormone (JH) in the hemolymph, competence to form crochets declines. A similar loss of competence did not occur when fourth instar crochet epidermis was exposed to a declining JH titer by culture in either fourth instar isolated abdomens for 72 h or in fifth instar host larvae between 4 and 7 gm. Responses of crochet epidermis cultured in vitro also were examined. Competent fourth instar crochet epidermis formed crochets following 3–6 h exposure to ecdysone in vitro. Six ×10^–7M -ecdysone was required for 50% response, whereas a 10–50-fold higher concentration of -ecdysone was necessary. Although formation of morphologically complete crochets in vitro proceeded with similar time course to that in situ, no molt-induced growth occurred in vitro. When crochet epidermis was exposed to ecdysone in vitro immediately after explantation, exogenous JH was not required for molting. But when tissue was first cultured for 72 h without hormones, subsequent molting in vitro could not be elicited, although molting still could occur when the tissue subsequently was implanted into a fourth instar host. Exposure to corpora allata or to JH during the 72 h of culture in vivo partially prevented the loss in capacity to respond to ecdysone in vitro, suggesting that JH may be one factor involved directly or indirectly in maintenance of tissue responsiveness.Preliminary presentation of some of this work given at the December, 1973 Meeting of the American Society of Zoologists (Fain and Riddiford, 1973)     

Developmental response of the brown-winged green bug, Plautia stali (Heteroptera: Pentatomidae), to food shortage

To study developmental response of the brown-winged green bug, Plautia stali Scott, to food shortage we reared nymphs under restricted feeding conditions produced by shortening the feeding period after molt or withholding food from second to fifth instars. For second instars, molting rates were significantly reduced as the feeding period was shortened. Shortening the feeding periods for third to fifth instars also reduced molting rates, but less so; some nymphs were able to complete their developments even if no food was given to any of the instars. Compared with controls for which feeding was not restricted, nymphs that successfully reached the next instar had reduced postmolt body size as a result of restricted feeding for all instars except the fifth (2-day feeding for second instar, and 1-day feeding for third and fourth instars), whereas instar duration was unchanged or only slightly prolonged for all instars. These results suggest that only nymphs with nutritional accumulation over a specific threshold in each instar can progress to the next instar, and that, particularly for second to fourth instars, nymphs develop on schedule without prolonging instar durations to compensate for reduced growth under conditions of food shortage.     

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta. There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O,O-diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.     

In vitro stimulation of cell death in the moulting glands of Oncopeltus fasciatus by 20-hydroxyecdysone

The moulting glands of the milkweed bug, Oncopeltus fasciatus, normally degenerate just before the time of ecdysis to an adult (day 7 of the fifth instar). Morphologically normal cell death can be prematurely stimulated in vitro by 20-hydroxyecdysone. Breakdown is triggered by a 24-hr period of exposure to 20-hydroxyecdysone, but an additional incubation period is required before clear signs of degeneration are manifested. Glands removed after the onset of endogenous ecdysteroid secretion degenerate in vitro in the absence of added hormones. Thus, in the moulting glands of Oncopeltus, ecdysteroids appear to act as an important trigger for metamorphic cell death.     

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

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

In vitro rearing of Toxoneuron nigriceps (Hymenoptera: Braconidae), a larval endoparasitoid of Heliothis virescens (Lepidoptera: Noctuidae) from early second instar to third instar larvae

The early second instar larvae of Toxoneuron nigriceps, a larval endoparastioid of Heliothis virescens, were incubated in artificial rearing media, supplemented with hemolymph of the unparasitized and parasitized fifth instar larvae of the host, H. virescens. The parasitoid larvae were incubated in both a semisolid and liquid form of the artificial rearing medium, and their growth and development were evaluated. The growth in size (increase in length and width), development (molting), and survival of the incubated larvae were observed for 10 days. The incubated larvae exhibited some level of growth in all nine types of media tested, including the control (without host hemolymph). However, ingesting the semisolid rearing media supplemented with the hemolymph from the late fifth instar (day 5, 7 and 9) parasitized host resulted in 100% of the larvae molting to third instars. Some of the in vitro reared third instar larvae demonstrated behavioral changes that could be interpreted as the preparation for cocoon formation or pupation i.e. oral secretion of a whitish material and lots of twisting and turning; however, none produced a cocoon nor pupa.     

Growth and silk formation of silkworm larvae influenced by phytoecdysones

The fourth and fifth instar larvae of the silkworm were reared on artificial diets containing ponasterone A, ecdysterone, and inokosterone. The growth of the larvae and their silk glands, fibroin-synthesizing activity, and silk formation have been investigated. With a diet containing ponasterone A, the fourth instar larvae grew slowly and only a few larvae could ecdyse, while the growth of the fifth instar larvae was disturbed and they died with a darkening of the skin. Ponasterone A also inhibited the growth of the silk glands during the fifth instar. In contrast, the other two phytoecdysones did not greatly influence larval growth. The fourth instar larvae grew rapidly and their ecdysis was advanced with a diet which contained 10 μg of inokosterone/1 g of dry diet. The diet which contained 5 μg of ecdysterone or 10 μg of inokosterone/1 g of dry diet accelerated maturation, while that containing 10 or 20 μg of ecdysterone, or 40 μg of inokosterone, delayed maturation of the fifth instar larvae.Only phytoecdysones caused a decrease in growth of the silk glands in the early half of the instar, and a large amount of phytoecdysones accelerated their growth during the last part of the fifth instar. The fibroin-synthesizing activity was levelled up by feeding ecdysterone and inokosterone, and inokosterone appreciably stimulated activity. Assay of in vitro fibroin synthesis showed that ponasterone A competed with ecdysterone in a stimulative action. Silk formation was much lower in larvae fed the diet containing 5 μg of ecdysterone or 10 μg of inokosterone/1 g of dry diet and was far greater in larvae fed the diet containing 40 μg of inokosterone than in the controls.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.