Defence by plant toxins in milkweed bugs (Heteroptera: Lygaeinae) through the evolution of a sophisticated storage compartment

Species of the heteropteran subfamily Lygaeinae possess special subcuticular compartments to store cardiac glycosides, plant‐derived defensive compounds, which they release upon predator attack. In adults of the large milkweed bug, Oncopeltus fasciatus, these storage compartments have previously been described as a modified integument, forming a fluid‐filled dorsolateral space. Here we use three‐dimensional imaging of serial histological sections and synchrotron radiation‐based micro‐computed tomography data to reveal the morphology of these storage compartments and the mechanisms used for the release of a cardiac glycoside‐rich fluid upon attack. Our comparative analysis revealed that the morphology and release mechanism vary among the species investigated. By reconstructing these traits on a recent molecular phylogeny of the Lygaeinae, we demonstrate that the adaptations for the storage and release of cardiac glycosides have evolved in a stepwise manner.     

Ultrastructure of the nymphal integument in the camel tick Hyalomma (Hyalomma) dromedarii (Ixodoidea: Ixodidae)

The ultrastructure of the nymphal integument in the ixodid tick Hyalomma (Hyalomma) dromedarii is compared for stages of development during and after feeding, and up to the first step of molting, apolysis. The integument comprises a cuticular layer and underlying epidermal cells. The body cuticle, which consists of both sclerotized and non-sclerotized parts, is divided into an outer, thin epicuticle, and an inner, thick, fibrillar procuticle. Pore canals in the procuticle are continuous with wax canals which traverse the epicuticle. As feeding progresses, the parallel, extensible epicuticular folds disappear due to the gut filling with ingested blood. The procuticular zone, however, becomes subdivided into an exocuticle, similar to the previously seen procuticle, and a lamellate endocuticle. Pore canals lose their parallel pattern and appear to have become deformed by stretching of the cuticle. The flat epidermal cells grow asynchronously during feeding; their cytoplasm becomes packed with well-developed rough endoplasmic reticulum (RER), while the cell apices project long microvilli extending deep into the procuticle. The RER undergoes ultrastructural changes indicating synthetic activity. Dense material released through the microvilli may serve to lyse the endocuticle and thus cause separation of the cuticle from the epidermis during apolysis. The lysed area, the exuvial cavity, is filled with lysed components which are probably withdrawn by endocytosis into the apical coated vesicles which appear in epidermal cells. Two types of integumental glands, which may participate in wax production, are observed in this study. The ultrastructure of their previously undescribed cuticular ducts is described, in addition to other hypodermal structures including epidermis-muscle attachments and sensory receptors.     

Ultrastructure of an integumental organ with probable sensory function in Paragordius varius (nematomorpha)

The cuticle of late parasitic stages of Paragordius varius (Leidy, 1851) is composed of a layer with large fibres and a second layer (often named the areolar layer) distal from it. In this paper, organs are described that start at the basal side of the epidermis, pass the epidermis and the fibrous layer of the cuticle and merge with large, cushion‐like structures in the distal layer of the cuticle. The epidermal part of the organs is composed of darkly stained cells, which are probably in contact with the basi‐epidermal nervous system. Up to four processes of this cell traverse the cuticle. These processes might include cilia, because they contain microtubule‐like structures. The probable connection to nerve cells and the connection to the cushion‐like structures in the outer cuticular layer make it likely that the organs described here are sensory in function.     

Ultrastructure of the contact surfaces of Passalurus ambiguus (Rudolphi, 1819) (Nematoda)

The ultrastructure of the contact surfaces (integument and intestinal wall) of the nematode Passalurus ambiguus has been studied. The integument is composed according to the scheme common for all nematodes and includes a cuticle, hypodermis and a muscular layer. The specificity is with regard to the epicuticle, the different number of the cuticular sublayers in the anterior, central and the posterior parts of the worm body and the absence of a basal cuticular membrane. The intestinal wall consists of epithelial cells with microvilli. The ultrastructural characteristics of both contact surfaces indicate their main functions--absorption, secretion, transport, protection, movement, etc.     

Cyanoglucoside storing cuticle of Zygaena Larvae (Insecta,Lepidoptera)

Summary As a deterrent against predators, larvae of Zygaena trifolii release droplets of fluid containing cyanoglucosides from segmentally arranged cuticular cavities. Histological examinations show that during the moulting period, the old cuticle, including the cavities and the secretion within them, is degraded, with the exception of a thin mesocuticular layer forming the exuviae. When the endocuticular layer of the new cuticle is deposited, the cuticle detaches from the underlying epidermis in specific areas, which leads to the formation of the cuticular cavities. During a moult-intermoult sequence the concentration of cyanoglucosides in both the haemolymph and the defensive secretion shows specific changes. These changes seem to be related to the formation and degradation of the cavities. We suggest that during the moult the cyanoglucosides are transported through the epidermis into the haemolymph to prevent them from being wasted with the exuviae and, after ecdysis, are retranslocated into the newly formed cavities.     

The permeability of the midgut of three insects to cardiac glycosides

The uptake of a polar and nonpolar cardiac glycoside by three insects, Oncopeltus fasciatus, Schistocerca gregaria and Periplaneta americana was investigated. Of these insects, the midgut of only O. fasciatus was found to be permeable to cardiac glycosides. Ouabain was not metabolized by this insect and crossed the midgut slowly and passively. It was sequestered from the haemolymph into the dorsolateral spaces against a concentration gradient and at relatively fast rates suggesting that uptake from the gut is rate limiting. Digitoxin was metabolized at the level of the midgut but not in the isolated haemolymph or dorsolateral space fluids. Twenty-four hours after feeding O. fasciatus labelled digioxin, digitoxin metabolites but no unchanged digitoxin could be detected in the haemolymph while both metabolites and a small amount of unchanged digitoxin could be detected in the dorsolateral space fluids.     

Comparative morphology of echinoderm calcified tissues: Histology and ultrastructure of ophiuroid scales (Echinodermata,Ophiuroida)

Summary The calcified body wall of an ophiuroid was investigated by a new method and compared with that of other echinoderms. The previous opinion that the epidermis of ophiuroid arm shields consists of a reduced syncytium continuous with the underlying dermis is incorrect. The epidermis is distinctly separated from the dermis by a basal layer and consists of (1) supporting cells which bear the cuticle, (2) ciliated cells (hitherto unknown and probably sensory), (3) gland cells, and (4) nerve cells with the basal nerve plexus. The overall structure of the epidermis is a three-dimensional tube system (marked by the basal lamina) which penetrates the dermal tissue of the scale's pore space and continues with nerve cords situated below the scale. This arrangement is unique in echinoderms.The dermal sclerocytes largely conform with those of the echinoid Eucidaris. The mineral skeleton is produced intracellularly or intrasyncytially. Moreover, dermal sclerocytes were found to release extracellular microfibrils which have nothing to do with calcite deposition. The attachment of the cuticle to the dermis is achieved by means of epidermal coupling areas. Collagen fibers fasten the scale to the underlying connective tissue sheath. The supposed fibrocytes within this sheath resemble sclerocytes. Each collagen bundle is provided with a strand of nerve fibers which, in contrast to the basal nerve plexus, are naked. They are said to infuence the mechanical properties of the connective tissue.Structures associated with cilia occur in cell types which normally lack a cilium. This finding suggests that most echinoderm cells are potentially monociliate.Abbreviations A apical shield - asp secretory products - B bacteria - bb basal body - bl basal lamina - C ciliated cell - ca coupling area - ci cilium, - cf collagen fibrils - cs cell surface - CTS connective tissue sheath - cu _i inner cuticular layer - cu _m middle cuticular layer - dp distal processes (Sc) - EC epineural canal - G Golgi complex - gv granular vesicle - H haemal vessel - hb homogeneous body - hl horizontal lamina (Su) - j cell junction - L lateral shield - le boundary layer (Sc) - lo distal lobe (Su) - M intervertebral muscle or its attachment - m mitochondrium - mf microfibrils - mu mucus - mv microvilli - mvb multivesicular body - N nerve cell - n nucleus - nf neurofibrils - ng neurogranules - nn naked neurofibrils - O oral shield - P tube foot - Pc phagocyte - pg pigment granules - rl rootlet - RN radial nerve - RV radial vessel - Sc sclerocyte - sh cytoplasmic sheath (Sc) - sj septate junction - Su supporting cell - sv secretory vesicle - T calcite trabeculum - V vertebral ossicle - v vesicle (Su)     

Detection of haemocyte proteins in the integument of the developing Mediterranean fruit flyCeratitis capitata

Summary Studies of the synthesis of integumental proteins during the feeding and non-feeding stages ofCeratitis capitata demonstrated stage specificity. The synthetic profile changed dramatically, showing a maximum of protein synthesis just before the larval wandering stage, followed by an abrupt decline. The comparison between synthetic and accumulation profiles indicated that some polypeptides must be internalized into the integument from the haemolymph. The major haemolymph proteins or arylphorins have already been documented to be incorporated into the integument. In the present work, we demonstrated the interalization of some haemocyte proteins into the integument. For that purpose, polyclonal antibodies were raised against total haemocyte proteins. Immunoblot analysis of haemocyte salt extractable proteins revealed that the protein bands at 36, 54, 58, 84, 110 and 130 kDa were immunoreactive with the total haemocyte antibodies. Cell-free protein synthesis, organ culture experiments and immunoblot analysis indicated that the 36-, 54- and 58-kDa polypeptides were synthesized only in the haemocytes and were probably internalized into the integument from the serum. The 36-kDa polypeptide was also demonstrated to be internalized into the fat body of white puparia. The immunofluorescence experiments suggested that the internalization of haemocyte proteins first occurs into the epidermal cells and then into the cuticle. The presence of haemocyte proteins in the integument was also demonstrated by immunofluorescence experiments in twoC. capitata mutants. These mutations affect the darkening and stiffening of the cuticle. The demonstration of 36-, 54- and 58-kDa haemocyte polypeptides in the integument reveals a hitherto unknown function of this cell type. Moreover, the demonstration of tyrosine binding to the 54- and 58-kDa polypeptides points to their potential involvement in the sclerotization process in the cuticle.     

An ultrastructural study of the larval integument of the midge,Chironomus riparius meigen (Diptera: Chironomidae)

Summary The larval integument of the midge, Chironomus riparius Mg., is unusually thin although it conforms with the normal insect pattern. The cuticle of the post-cephalic segments is about 3 m thick and overlies an epidermis which has an irregular basal plasma membrane resulting in spaces occurring between it and the basement membrane. The ventral tubuli have a similar epidermis but the cuticle is somewhat thinner. The anal papillae have the thinnest cuticular covering with a uniquely folded epicuticle of variable thickness, and their epidermis has the characteristics of a transporting epithelium. No evidence of pore canals could be found in the cuticle of any part except the head capsule which has a remarkably smooth epicuticle and a distinct layer which may represent the exocuticle. There are no spaces between the basement membrane and basal plasma membrane of the epidermis in the head. Ultrastructural evidence would suggest that gaseous exchange can occur across most of the post-cephalic integument.The author is indebted to Mrs. L. Rolph and Mr. R.L. Jones for their technical assistance     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

The integumental ultrastructure of Parathalestris harpactoides (Claus, 1863) (copepoda,harpacticoida)

The integument of Parathalestris harpactoides (Claus, 1863) is studied by scanning and transmission electron microscopy. The general structure of the integument conforms to the common pattern known from Copepoda. Emphasis is given to the structural variation of the cuticle in different regions of the body. The cuticle measures about 6 µm in most parts of the body, and shows a laminate appearance. The epicuticle is about 60 nm thick. Numerous pore canals containing muscular tonofilaments penetrate the procuticular layer of the integument. A peculiar feature is the presence of a honeycombed layer in the outermost zone of the cuticle of some parts of the body. The epidermal layer, muscle insertions and integumental pores are of common type. The cuticle of some specimens, both males and females, is covered with microorganisms.     

Development of the crystalliferous cuticle of Chamaecyparis lawsoniana (A. Murr.) Pari. (Gupressaceae)

A developmental study of the cuticle has shown that it consists of a homogeneous cuticle proper apposed on the wall and a heterogeneous cuticular layer generated by intussusception of cutin into the wall. At an early stage, the adcrusted cuticle proper is underlain by a ruthenium red-positive layer in which the cuticular layer originates. The origin of the anticlinal flange is referable to an electron-dense, ruthenium red-positive ridge which arises above the anticlinal wall and which also becomes cutinized. At leaf maturity, the inner surface of the cuticular layer, including that of the flange, forms interdigitating protuberances with the cell wall.
Development of the cuticle coincides with deposition of crystals of calcium oxalate in the epidermal cell wall. Initiation of large, early-formed crystals is associated with electron-opaque membranous structures formed close and parallel to the plasmalemma in the young cell wall. Crystals undergo periclinal and anticlinal growth and subsequently become engulfed within the cuticle by development of the cuticular layer. Cutin/polysaccharide interaction during development and the significance of crystal deposition are discussed.     

Etude ultrastructurale de l'évolution du tégument de la Sangsue Hirudo medicinalis L. (Annélide,Hirudinée) au cours d'un cycle de mue / Developmental changes in the integument of the leech Hirudo medicinalis L. (Annelida,Hirudinea) during a molting cycle. An ultrastructural study

Summary

The integument of the leech Hirudo medicinalis is mainly composed of a single layer of cuticle-secreting epidermal cells. The cuticle is made up of collagen fibers which support a layer of membrane-bound epicuticular projections.

Shedding of the old cuticle is preceded by the formation of a new cuticle. The epicuticular projections are the first to develop: they originate from the tips of numerous microvilli of the epidermal cells. As soon as it appears, the newly-formed collagen layer is firmly attached to the epidermal cells by numerous hemidesmo-somes, whereas the old cuticle is no longer connected with the epidermal surface. The epidermal cells exhibit marked characteristics of secretory activity during the laying down of the new cuticle.

The observations are discussed in connexion with recent findings of high ecdysteroid levels in leeches at the beginning of the molting cycle.     

A new glandular sensory organ inCatanema sp. (Nematoda,Stilbonematinae)

Summary A new multicellular glandular sensory organ is described forCatanema sp. (Nematoda, Stilbonematinae). The organs terminate in setae and are distributed in six longitudinal rows along the body. Two types of glandular cells (type A and type B), one monociliary sensory cell and one undifferentiated epidermal cell are combined in the basiepidermal organ. A comparison of epidermal glands as well as sensory organs in Nematoda is made. A causal relationship between the development of such complex, large and numerous glandular sensory organs and the occurrence of species-specific, symbiotic epibacteria inCatanema sp. seems probable, although there is no simple correlation between the distribution of these organs and epibacteria. A mucous cover over the bacterial layer, released by the glandular sensory organs, may create a microenvironment for the interaction between epibionts and host.Abbreviations (used in figures) a amphid - A1–A4 type 1–4 granules of type A gland cell - an annuli - b bacteria - B1–B3 type 1–3 granules of type B gland cell - bl basal lamina - bp basal part of seta - bz basal zone of cuticle - c cuticle - ca canal - cg caudal gland - ci cilium - cz cortical zone of cuticle - d dictyosomes - e epidermis - e co extracellular coat - em extracellular matrix - ep epicuticle - f filaments - gcA type 1 gland cell - gcB type 2 gland cell - i lp inner labial papillae - m mitochondrion - me membranes of type 2 gland cell - mo mouth opening - mz median zone of cuticle - n nucleus - nu nucleolus - p process - pv primary vesicle of type A gland cell - r ribosomes - s seta - sc sensory cell - sp secretory product - tj tight junction - tp terminal part of seta - uc undifferentiated epidermal cell - va vacuoles or vesicles of epidermal cells - ve vesicles of sensory cell     

The fine structure of wound repair in an insect (Rhodnius prolixus)

The cells surrounding a wound in the integument of Rhodnius adults show an increase in RNA content, cytochrome oxidase and esterase activity. An excision in the integument is filled by blood which coagulates and is tanned into an insoluble membrane. The basement membrane of the adjoining epidermis acts as a self-sealing membrane and contracts to cover the excision. The epidermis is attached to the cuticle by the subcuticular layer which it resorbs and by pore canal filaments which are left behind as it migrates. The epidermis migrates as a sheet in contact with the cuticle then with the coagulated blood and basement membrane which cover the excision. Blood cells migrate individually into an excision and do not adhere to a surface in the process. Microtubules cannot be identified with movement. Both epidermal and blood cells remove the cells killed by wounding as evidenced by the appearance of coated vesicles and phagocytic bodies in both cell types. The reconstituted integument consists of a surface membrane in which the layers of the epicuticle are not distinguishable, a nonlamellate cuticle secreted by an epidermis which also appears to secrete the new basement membrane.     

Ultrastructure and cytochemistry of the integument of Modiolicola gracilis,parasitic copepod in mussel gills (Mytilus galloprovincialis and Mytilus edulis)

Of mussels taken from the Ebro Delta River (E. Spain), 3% have a nonmodified copepod, Modiolicola gracilis, in the gill tissues. The cuticle of different segments of the body has an epicuticle with two layers, which show external microvilli-like projections. Weakly positive reactivity to the PTA technique has been detected in the external region. The procuticle has the helicoidal architecture of the chitinous tegument in arthropods, whereas the cuticle shows discontinuities in the regions of ducts in tegumental glands. The integument is comprised of three types of cells. Epidermal cells are flat with numerous mitochondria. Muscle cells show well-developed mitochondria with several longitudinally distributed cristae. A third and secretory cell shows a well-developed rough endoplasmic reticulum and Golgi complex in the basal zone. Its apical portion is full of secretory granules. Through the cuticle, these integumental glands open directly to the cuticular surface via a short duct coated by epicuticle. The composition and specializations of this complex cuticular architecture differ markedly from those shown by an endoparasitic copepod detected in the digestive gland of the mussel. It does not appear that the specializations detected in the cuticle of M. gracilis lead to any histopathological alteration in host tissues. © 1994 Wiley-Liss, Inc.     

The formation of the epicuticle and associated structures inOniscus asellus (Crustacea,Isopoda)

Summary The integument of the woodlouse,Oniscus asellus, consists of a two-layered epicuticle, a largely lamellate procuticle — itself divided into two regions (pre-and postecdysial cuticles), and the epidermis. At the initiation of new cuticle production the epidermal cells become vacuolated and retract away from the cuticle. Apolysis occurs immediately after the cessation of postecdysial cuticle production. The formation of the epicuticle is unique among the arthropods since material aggregates along the distal epidermal membrane. By indenting, doubling back on itself, and incorporating septa, the epicuticle forms surface structures such as plaques and tricorns.The innervation, and so the receptive function of the tricorns is confirmed, but since there is no connection between the old and new receptors during premoult, sensory information from these exoreceptors must be severely curtailed. This may explain the biphasic moult in all isopods since it ensures that only half the body experiences this sensory deprivation at any one time. In terrestrial species there is the additional advantage of restricting the area of permeable new cuticle. The frequency of moulting may be due to the need to renew disrupted receptor surfaces.Tricorns do not appear to be the mechanoreceptors involved in the marked thigmotactic response of woodlice since they do not have the typical internal structure of such receptors; rather, the dendrite —which extends into the lumen of the tricorn —is protected from deformation by the previously unreported combination of a dendritic sheath and a cuticular tube. The modality of tricorns is possibly one of hygro-perception. One of the behavioural responses of woodlice to desiccation is aggregation. The numerical distribution of tricorns over the body surface is admirably suited to assist in the formation and maintenance of such aggregates during desiccation and to their observed dispersal when the relative humidity rises.     

The comparative morphology of epidermal glands in Pentatomoidea (Heteroptera)

The Heteroptera show a diversity of glands associated with the epidermis. They have multiple roles including the production of noxious scents. Here, we examine the cellular arrangement and cytoskeletal components of the scent glands of pentatomoid Heteroptera in three families, Pentatomidae (stink bugs), Tessaratomidae, and Scutelleridae (shield-backed bugs or jewel bugs). The glands are; (1) the dorsal abdominal glands, (2) the tubular glands of the composite metathoracic gland, and (3) the accessory gland component of the composite metathoracic gland. The dorsal abdominal glands are at their largest in nymphs and decrease in size in adults. The metathoracic gland is an adult-specific gland unit with a reservoir and multiple types of gland cells. The accessory gland is composed of many unicellular glands concentrated in a sinuous line across the reservoir wall. The lateral tubular gland is composed of two-cell units. The dorsal abdominal glands of nymphs are composed of three-cell units with a prominent cuticular component derived from the saccule cell sitting between the duct and receiving canal. The cuticular components that channel secretion from the microvilli of the secretory cell to the exterior differ in the three gland types. The significance of the numbers of cells comprising gland units is related to the role of cells in regenerating the cuticular components of the glands at moulting in nymphs.     

Cell polarity during wound healing in an insect epidermis

The insect integument displays uniform posterior orientation of cuticular denticles or bristles formed by the epidermal cells. We want to understand how cell polarities become uniformly oriented in the plane of the epidermal sheet. Here we test whether directed cell migration disturbs the orientation of denticles. Burning a circular area of epidermal cells beneath the cuticle causes cells to migrate into the resulting wound and the cuticle pattern observed after the subsequent moult depends on the time interval between burning and ecdysis. After a short wound-healing period cuticular protrusions tend to point away from the wound. With increasing would healing periods they tend to point more and more towards the wound centre. These results suggest that the migrating cells tend to orient cuticular protrusions in the direction of cell movement while continued cell movement will bend nascent cuticular protrusions outwards. Cell shape may also determine denticle orientation. I propose that the asymmetric localization of cell components known to determine the orientation of cell migration may also determine denticle orientation in insect epidermal cells.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.