Cytoskeletal F-actin patterns in whole-mounted insect Malpighian tubules.

The distribution of actin filaments in Malpighian tubules of the fleshfly Sarcophaga bullata (Parker) was investigated before and after metamorphosis by means of the rhodamine phalloidin staining method. The numerous primary cells show a pattern of thick basal actin bundles resembling stress fibres of cultured cells, while the apical microvillar zone shows a bright and homogeneous labelling. The less abundant stellate cells contain no such basal actin bundles and their apical microvillar zone gets only faintly stained. Late larval stages display fingerlike infoldings and an increased actin filament concentration at the apical membrane of the stellate cells. During metamorphosis the Malpighian tubules dedifferentiate and eventually redifferentiate to give rise to adult tubules resembling larval ones. The different types of actin filament organisation in the primary and stellate cells of the Malpighian tubules are discussed.     

The timing of drosophila salivary gland apoptosis displays an l(2)gl-dose response

During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures forming at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae (l(2)gl) gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands which become fully histolysed 12 - 13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue we used transgenic lines expressing reduced ( approximately 0.1) or increased levels of p127 (3.0). Here we show that the timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands.     

Influence of thyroid hormones on neuronal death and differentiation in larval Rana pipiens.

The sphingid moth, Manduca sexta, typically passes through five larval instars, a pupal, and an adult stage. The larval labial glands secrete silk in the first instar and a viscous lubricant in the fifth. During metamorphosis the glands develop into salivary organs which produce an invertase-rich secretion. In normal development, the uniform population of cells in the duct of the larval gland transforms into the four sequentially arranged regions of secretory and conductive cells of the adult gland. In order to determine when competence to form the adult gland is established, fragments of labial gland ducts from first through fifth instar larvae were implanted into pupae. These gland fragments underwent metamorphosis with their hosts, passing through the same developmental phases. Glands from as early as the first instar were competent to form histologically and functionally normal adult regions. In later instars, transplants of measured fragments demonstrated that larval cells were programmed in situ to develop into the four adult cell types.     

Mechanisms of programmed cell death in the midgut and salivary glands from Bradysia hygida (Diptera: Sciaridae) during pupal–adult metamorphosis

Programmed cell death is involved with the degeneration/remodeling of larval tissues and organs during holometabolous development. The midgut is a model to study the types of programmed cell death associated with metamorphosis because its structure while degenerating is a substrate for the formation of the adult organ. Another model is the salivary glands from dipteran because their elimination involves different cell death modes. This study aimed to investigate the models of programmed cell death operating during midgut replacement and salivary gland histolysis in Bradysia hygida. We carried out experiments of real‐time observations, morphological analysis, glycogen detection, filamentous‐actin localization, and nuclear acridine orange staining. Our findings allow us to establish that an intact actin cytoskeleton is required for midgut replacement in B. hygida and nuclear condensation and acridine orange staining precede the death of the larval cells. Salivary glands in histolysis present cytoplasmic blebbing, nuclear retraction, and acridine orange staining. This process can be partially reproduced in vitro. We propose that the larval midgut death involves autophagic and apoptotic features and apoptosis is a mechanism involved with salivary gland histolysis.     

Changes in cytoskeletal actin patterns in the Malpighian tubules of the fleshfly,Sarcophaga bullata (Parker) (Diptera : Calliphoridae), during metamorphosis

Using the rhodamine-labelled phalloidin staining method in combination with detergent extraction, metamorphic changes in actin filament patterns were investigated in the Malpighian tubules of the fleshfly, Sarcophaga bullata (Parker) (Diptera : Calliphoridae). Metamorphosis in this organ implies a process of dedifferentiation, followed by a process of redifferentiation. During dedifferentiation, the large basal actin bundles of the primary cells disappear and the microvillar membrane surface of these cells decreases. Concomitantly, several vesicles are pinched off from infoldings of the brush border. In older pupae, the Malpighian tubules redifferentiate to give rise to adult tubules with actin patterns similar to those of larvae. During redifferentiation of the tubules, the secondary cells display a marked increase in the number of actin filaments in their protrusions. The primary cells in the distal part of the anterior Malpighian tubules of late pupae display a well-developed basal pattern of thick parallel actin bundles. In most cases, major changes in actin filament patterns are found simultaneously with major changes in cell shape, indicating a close relationship between these actin filaments and the process of cellular remodelling.     

Cell differentiation during the ontogeny of larval salivary glands of the fly, Telmatoscopus albipunctatus

Using the larvae, pharate pupa, and pharate adults of the moth fly, Telmatoscopus albipunctatus, histological and ultrastructural features of the salivary glands were investigated. The gland lumen contains a milky secretion from the first instar. This secretion continues to ccur at all subsequent developmental stages; with the onset of the pharate pupal stage, however, the secretion becomes transparent and rather viscous. Histochemical tests revealed that it is mainly proteinaceous. Glands from the same developmental stage may respond differently to PAS-reaction.Various cell organelles were compared at consecutive stages of larval development and of secretory activity of the salivary glands. In first and second instar larvae autophagic vacuoles are virtually absent in the salivary gland cells. They were occasionally found in the third instar, when they appear to be engaged in the process of organelle turnover. Histolysis of the larval glands is initiated towards the close of the fourth instar when the number of autophagic vacuoles starts to increase. Simultaneously, the cytoplasm, previously full of ribosomes and endoplasmic reticulum, starts losing these structures. At the beginning of the pharate adult stage, the cytoplasm becomes practically devoid of all structures other than those engaged in autophagy.Polyteny of the chromosomes during ontogeny of the larval salivary glands is also discussed.     

Programmed cell death in the larval salivary glands of <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera,Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.     

麦蛾柔茧蜂幼虫唾液腺的显微与超微形态观察

本文采用解剖学观察、显微摄影、透射电镜等方法对麦蛾柔茧蜂Habrobracon hebetor幼虫唾液腺的显微形态、超微结构以及发育特性进行了观察和分析。麦蛾柔茧蜂幼虫唾液腺为一对无色透明至乳白色的管状腺体,自口腔沿中肠两侧向后延伸,单侧腺体在中部先分支、后合并成一不规则环状,端部呈单盲管状。唾液腺管道长度随幼虫龄期增加而呈线性增长。对唾液腺切片进行超微结构观察,发现腺管由两类差异明显的单层细胞组成,I型细胞微绒毛层较厚,胞内除有丰富的内质网和线粒体之外,还含有大量囊泡,并观察到囊泡运输分泌颗粒的现象;II型细胞微绒毛短,胞内的内质网和线粒体数量丰富。本文研究为深入探究寄生蜂幼虫的消化生理以及寄生蜂-寄主互作机制奠定了基础。     

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.     

Rearrangement of the tubulin and actin cytoskeleton during programmed cell death in Drosophila salivary glands

During larva-to-pupa metamorphosis Drosophila salivary glands undergo programmed cell death by autophagocytosis. Although ultrastructure of Drosophila salivary glands has been extensively studied in the past, little is known about mechanism of programmed cell death, especially the role of the cytoskeleton. In this paper we describe changes in microtubule and actin filament network compared to the progress of DNA fragmentation and redistribution of acid phosphatase. In feeding and wandering larvae microtubules and actin filaments form regular networks localized mostly along the plasma membrane. The first major rearrangement of microtubules and actin filaments occurred when larvae everted spiracles and the glands shifted their secretion from saliva to mucoprotein glue (stage L1). Microtubule cytoskeleton became denser and actin filaments concentrated along cell boundaries. At the same time nuclei flattened and migrated into the microtubule-rich layer near the basal membrane. In late prepupae (8-10 h after P1) the microtubule network became fainter, and actin filaments appeared frequently deeper in cytoplasm, gradually concentrating around nuclei. Simultaneously large patches of acid phosphatase activity surrounded nuclei and shortly thereafter chromosomal DNA began to fragment. During the final collapse of the gland (early pupae, 13.5 h after formation of white puparium) cellular fragments and autophagic vacuoles contained a continuous F-actin lining and the microtubule network displayed signs of extensive degradation. The results are consistent with the hypothesis that, in Drosophila salivary glands, extensive autophagic activities target nuclei for degradation; that this process occurs late in the course of programmed cell death; and that it directly involves cytoskeletal structures which are altered far earlier during the course of cell death.     

The humoral immune response of sheep to antigens from larvae of the sheep blowfly (Lucilia cuprina)

The sheep blowfly, Lucilia cuprina, is a myiasis-causing insect whose larvae evoke an immune response in sheep. By means of an immuno-dot blot and Western immuno-blot assays it has been demonstrated that sheep experimentally infected with larvae produce antibodies against a wide array of components from all three larval instars, with each instar displaying a differing set of antigens. The electrophoretic profiles of the proteins in various larval extracts and the patterns of antibody reactivity were very different. Of the extracts tested (1st, 2nd and 3rd instar larval excretions/secretions and visceral homogenates, extracts of 3rd instar salivary glands, mid guts, haemolymph and cuticle) the most intense antibody reaction was detected against the salivary gland extract: preparations of larval excretions/secretions and from the larval mid gut also reacted strongly. In contrast a cuticle extract reacted minimally with infected sheep sera.     

Cellular metamorphosis. II. The larval labial gland duct and its prospective adult fates in the tobacco hornworm.

The larval labial gland of the sphingid moth, Manduca sexta, produces a viscous secretion, presumably a lubricant, facilitating the burrowing which precedes pupation. During metamorphosis, the gland transforms into a salivary organ, producing an invertase-rich digestive secretion. The single-cell type found in the duct of the larval gland transforms into the four structurally and functionally distinct cell types found in the four sequentially arranged secretory and conductive regions of the adult salivary gland. Surgical experiments were performed to study the prospective fates of different parts of the larval gland. The glands were bisected and one or both fragments were left in situ to undergo metamorphosis. In addition, fragments of the larval gland were implanted in pupal hosts and went through metamorphosis free of their prior attachments. The four linearly arrayed adult regions originate from correspondingly positioned areas in the larval duct.     

Development of terminal filaments and ovariole envelopes in Thermobia domestica (Insecta, Zygentoma) larvae

The 3rd instar female larvae of Thermobia domestica have five pairs of gonad primordia, each enclosed within a basal lamina (tunica propria). At the end of the 3rd instar some somatic cells scattered on the outer surface of the lamina are seen. During the 4th larval instar the gonad primordia start to form the ovarioles. Each ovariole is elongated and polarized, having anterior and posterior ends. The anterior group of outer somatic cells proliferate to form the terminal filament. At the 6th larval stage the ovarioles are already formed. The terminal filament is separated from the germarium by a thick basal lamina (transverse septum). There are three types of cell building the terminal filament. 1/Basal cells with numerous fingerlike projections; 2/Cells with electron lucent cytoplasm and large nuclei, and 3/Cells with darker cytoplasm containing bundles of fibers and more compact nuclei. The outer surface of the filament is covered by a thick, fibrous basal lamina. The somatic cells that in the previous stages were scattered on the tunica propria as distinct cells, in the 6th larval stage form a cellular envelope (tunica externa). This envelope is formed by a layer of flat cells, and contains numerous tracheae.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

Relationship of developmental stage to initiation of replication in polytene nuclei

The various types of autoradiographic patterns occurring in salivary gland nuclei ofD. melanogaster following a short pulse of tritiated thymidine have been described and the probable order of their sequence within a polytenic replication cycle assigned. On the basis of that assignment, the distribution of those patterns in the nuclei of salivary glands of 160 staged larvae and prepupae has been assessed and the following interpretations made: Throughout the third larval instar and prepupal period there is a continuing decrease in frequency of labeled nuclei. Within the larval period that decrease is reflected in a proportional decrease in label patterns characteristic of both initiation and propagation phases of the replication cycle. At the beginning of the prepupal period there is a rise in the proportion of labeled cells displaying initiation type patterns followed by an abrupt cessation of such patterns. It is proposed that termination of initiation of polytenic replication is amongst the physiologic manifestations of the induction of pupation.     

Decreased JH biosynthesis is related to precocious metamorphosis in recessive trimolter (rt) mutants of the silkworm, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of ecdysteroidogenic activity of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The cellular mechanism underlying ecdysteroidogenesis throughout the last larval instar of the silkworm, Bombyx mori, was analyzed by determining the in vitro ecdysteroid secretory activity of the prothoracic glands and cAMP accumulation of gland cells, as well as changes in responsiveness to stimulation by prothoracicotropic hormone (PTTH) and 1-methyl-3-isobutylxanthine (MIX). It was found that the prothoracic glands during the first 3 days of the last instar cannot produce detectable ecdysteroid and showed no response to stimulation by PTTH or 1-methyl-3-isobutylxanthine (MIX). However, artificial elevation of cellular cAMP levels by in vitro dibutyryl cAMP treatment stimulated the glands to secrete detectable ecdysteroid, implying the presence of a cAMP-dependent ecdysteroidogenic apparatus during this stage. From days 3 to 8, basal gland activities fluctuated, but the glands showed activation responses to PTTH and to the chemicals that increase cellular cAMP levels. After the occurrence of the peak in basal gland activity on day 9, glands on day 10 showed no response to PTTH, implying a refractory state of the glands to PTTH stimulation. For cAMP accumulation, it was found that glands on day 2 began to show increased cAMP accumulation to PTTH, implying that the acquisition of gland competency for elevation of cAMP levels after stimulation by PTTH precedes that of ecdysteroid production. Moreover, during most parts of the last larval instar (between days 3 and 8) and at the pupation stage, greatly increased cAMP accumulation upon stimulation by PTTH was observed only in the presence of MIX, indicating that cAMP phosphodiesterase levels may be high during these stages. From these results, we concluded that development-specific PTTH signal transduction during the last larval instar, which shows a different pattern from that of the penultimate larval instar, may play an important role in regulating changes in prothoracic gland activity and in leading to larval-pupal metamorphosis.     

Actin-mediated retraction of the larval epidermis during metamorphosis of the sand dollar,Dendraster excentricus

Summary During the first 15 to 20 min of metamorphosis the larval arms are retracted and resorbed into the aboral surface of the juvenile. Arms excised from metamorphosing larvae will undergo a sequence of contraction and histolysis that is identical to that occurring in intact larvae. Prior to and during metamorphosis, epidermal cells contain bundles of 5 to 7-nm microfilaments in arrays radiating apically from the base of the cells. Sparse microfilaments also occur near the plasmalemma of epidermal cells and some mesenchymal cells in larvae fixed during metamorphosis. Contraction of excised arms is reversibly inhibited by treatment with cytochalasin B, and microfilaments bind myosin subfragment-l. Indirect immunofluorescence of larval arms using an antibody against chicken-muscle actin and staining with the F-actin specific probe, NDB phallacidin indicate that the arms contain actin distributed in a manner consistent with ultrastructural findings. It is proposed that retraction of the larval arms during metamorphosis is produced by an actin-mediated change in shape of the epidermal cells.