Exuvial Space Research Articles

Molting-related proteases in the brown planthopper, Nilaparvata lugens

Digestion and absorption of old cuticles during insect molting are necessary for new cuticle formation, during which complicated enzyme catalysis is essential. To date, a few carboxypeptidases, aminopeptidases and serine proteases (mostly trypsins) connected with cuticle digestion, zymogen activation and histological differentiation during the ecdysis of lepidopteran, dipteran and hymenopteran insects have been identified. However, little is known about these proteins in hemimetabolous insects. In this study, we identified 33 candidate trypsin and trypsin-like homologs, 14 metallocarboxypeptidase and 32 aminopeptidase genes in the brown planthopper Nilaparvata lugens, a hemipteran rice pest. Among the proteins encoded by these genes, 9 trypsin-like proteases, 3 metallocarboxypeptidases and 1 aminopeptidase were selected as potential procuticle hydrolases by bioinformatics analysis and in vivo validation. RNA interference targeting these genes demonstrated that 3 trypsin-like proteases (NlTrypsin-8, NlTrypsin-29 and NlTrypsin-32) genes and 1 metallocarboxypeptidase (NlCpB) gene were found to be essential for ecdysis in N. lugens; specifically, gene silencing led to incomplete cuticle degradation and arrested ecdysis, causing lethal morphological phenotype acquisition. Spatiotemporal expression profiling by quantitative PCR and western blotting revealed their specific expression in the integument and their periodic expression during each stadium, with a peak before ecdysis and eclosion. Transmission electron microscopy demonstrated corresponding ultrastructural defects after RNAi targeting, with NlCpB-silenced specimens having the most undigested old procuticles. Immunohistochemical staining revealed that NlTrypsin-8, NlTrypsin-29 and NlCpB were predominantly located in the exuvial space. This research further adds to our understanding of proteases and its potential role in insect ecdysis.

Mechano-chemical enforcement of tendon apical ECM into nano-filaments during Drosophila flight muscle development

Contractile tension is critical for musculoskeletal system development and maintenance. In insects, the muscular force is transmitted to the exoskeleton through the tendon cells and tendon apical extracellular matrix (ECM). In Drosophila, we found tendon cells secrete Dumpy (Dpy), a zona pellucida domain (ZPD) protein, to form the force-resistant filaments in the exuvial space, anchoring the tendon cells to the pupal cuticle. We showed that Dpy undergoes filamentous conversion in response to the tension increment during indirect flight muscle development. We also found another ZPD protein Quasimodo (Qsm) protects the notum epidermis from collapsing under the muscle tension by enhancing the tensile strength of Dpy filaments. Qsm is co-transported with Dpy in the intracellular vesicles and diffuses into the exuvial space after secretion. Tissue-specific qsm expression rescued the qsm mutant phenotypes in distant tissues, suggesting Qsm can function in a long-range, non-cell-autonomous manner. In the cell culture assay, Qsm interacts with Dpy-ZPD and promotes secretion and polymerization of Dpy-ZPD. The roles of Qsm underlies the positive feedback mechanism of force-dependent organization of Dpy filaments, providing new insights into apical ECM remodeling through the unconventional interaction of ZPD proteins.

Mechano-Chemical Enforcement of Tendon Apical ECM into Nano-Filaments During Drosophila Flight Muscle Development

Contractile tension is critical for musculoskeletal system development and maintenance. In insects, the force transmission from the muscle to the exoskeleton depends on the tendon cells and tendon apical extracellular matrix (aECM). In Drosophila, tendon cells secrete Dumpy (Dpy), a Zona pellucida domain (ZPD) protein, to form the force-resistant filaments in the exuvial space, anchoring the tendon cells to the pupal cuticle. By live imaging, we showed that Dpy filaments undergo remodeling in response to the tension increase during indirect flight muscle development. Additionally, we found another ZPD protein, Quasimodo (Qsm), which plays a long-range, non-cell-autonomous role in Dpy filaments enforcement. Inside the cells, Qsm promotes secretion and polymerization of Dpy-ZPD. The dual role of Qsm underlies the positive feedback mechanism of force-dependent remodeling of Dpy filaments, providing new insights into aECM remodeling through the unconventional interaction of ZPD proteins.

Proteomic analysis of Bombyx mori molting fluid: Insights into the molting process

Molting is an essential biological process occurring multiple times throughout the life cycle of most Ecdysozoa. Molting fluids accumulate and function in the exuvial space during the molting process. In this study, we used liquid chromatography-tandem mass spectrometry to investigate the molting fluids to analyze the molecular mechanisms of molting in the silkworm, Bombyx mori. In total, 375 proteins were identified in molting fluids from the silkworm at 14–16h before pupation and eclosion, including 12 chitin metabolism-related enzymes, 35 serine proteases, 15 peptidases, and 38 protease inhibitors. Gene ontology analysis indicated that “catalytic” constitutes the most enriched function in the molting fluid. Gene expression patterns and bioinformatic analyses suggested that numerous enzymes are involved in the degradation of cuticle proteins and chitin. Protein-protein interaction network and activity analyses showed that protease inhibitors are involved in the regulation of multiple pathways in molting fluid. Additionally, many immune-related proteins may be involved in the immune defense during molting. These results provide a comprehensive proteomic insight into proteolytic enzymes and protease inhibitors in molting fluid, and will likely improve the current understanding of physiological processes in insect molting. Biological significanceInsect molting constitutes a dynamic physiological process. To better understand this process, we used LC-MS/MS to investigate the proteome of silkworm molting fluids and identified key proteins involved in silkworm molting. The biological processes of the old cuticle degradation pathway and immune defense response were analyzed in the proteome of silkworm molting fluid. We report that protease inhibitors serve as key factors in the regulation of the molting process. The proteomic results provide new insight into biological molting processes in insects.

Modulation of foregut synaptic activity controls resorption of molting fluid during larval molts of the moth Manduca sexta.

We examined the role of the foregut in the resorption of molting fluid (MF) from the exuvial space during the last larval-larval molt of the moth Manduca sexta. In intermolt larvae, the activity of the foregut is characterized by robust peristaltic contractions. With the onset of the molt, MF is secreted into the exuvial space where it digests and weakens the old cuticle. The appearance of MF in the exuvial space is accompanied by a dramatic reduction in the amplitude of the foregut contractions. Foregut peristalsis returned about halfway through the molt, followed shortly by the appearance of MF in the gut. These observations suggested that larvae use their foreguts to remove MF from the exuvial space. Animals whose foreguts were surgically inactivated did not resorb their MF and most failed to successfully shed their old cuticles. The reduction in foregut motility at the onset of the molt was correlated with a sharp decline in the amplitude of the excitatory junctional potentials. With the onset of the molt there was also a decline in the number of presynaptic terminals on the foregut that loaded with the activity-dependent dye FM1-43. In the second half of the molt, the appearance of MF in the foregut and the return of foregut motility was correlated with an increase in FM1-43 loading. These data reveal that during a larval-larval molt, vesicle release and/or recycling of the presynaptic endings on the foregut muscles is modulated to assure the proper timing of MF resorption.

Cellular organization and fine structure of type II microtrich sensilla in gammaridean amphipods (Crustacea)

The cellular organization of type II microtrich sensilla was studied in male Anonyx lilljeborgi Boeck, 1871 (Lysianassoidea) by light and transmission electron microscopy. The sensillum consists of two bipolar sensory neurons and three concentric sheath cells. The sensory cell bodies are subepidermal. In each sensillum both dendrites are enclosed by the thecogen cell process. The inner dendritic segments are filled with mitochondria and lucent vesicles and expand in the epidermis into a spindle-shaped swelling. One of the neurons gives rise to two cilia and the second to a single cilium. These three outer dendritic segments lie in the receptorlymph cavity. The dendritic sheath, secreted by the thecogen cell process, completely ensheaths the outer dendritic segments. The trichogen (middle) cell and the tormogen (outer) cell incompletely enclose the thecogen cell, but their processes form autojunctions around the dendritic sheath in the apical epidermis. In premolt, the trichogen cell processes project into the exuvial space. The cytoplasm of the tormogen cell and the bordering epidermal cells contains coarse osmiophilic inclusions. All the cells of the sensillum are joined by desmosomes. The sensilla structurally resemble chemosensory (gustatory) insect sensilla.

Development of the Stomach in Euphausia Superba Dana (Euphausiacea)

ABSTRACT Development of the stomach, with emphasis on the filter system, was investigated in the metanauplius, calyptopis, and furcilia stages of the Antarctic krill Euphausia superba. The metanauplius lacks an elaborated stomach as well as a connection between the esophagus and the midgut. In calyptopis I, the stomach becomes functional. The armature of the cardiac stomach consists of basic components such as lateral and ventral projections and stout spines, but not a primary filter. The latter develops before molt beneath the cuticle and is seen in the exuvial space of calyptopis II. With molting to calyptopis III, the primary filter is elaborated and becomes capable of sorting food particles and of separating a dorsal food from a ventral filtration channel. Although in furcilia IV the spacing of spines in the primary filter is nearly identical to that in the adult specimens, the filter spines in calyptopis III are more widely separated. In all larval stages examined (except furcilia IV), the development of the pyloric stomach is delayed with respect to that of the cardiac stomach. Residues of a pyloric secondary filter system were not found in developing stages of Euphausia superba.

Ultrastructure des phaneres distaux (a1) des appendices du premier sternite abdominal chez Campodea sensillifera (Conde et Mathieu) (Insecta : Diplura)

The fine structure of the sensilla situated at the end of appendages of the first abdominal sternite in both sexes of C. sensillifera (Diplura) reveals that they are mechanoreceptors and taste receptors. Each sensory unit is made of 3 dendrites ensheathed by 3 cells (thecogen, trichogen and tormogen). The thecogen cell is voluminous and secretes a large quantity of a dense secretory material accumulated in a reservoir. At the bottom of this reservoir, the original inner lymph cavity appears as a clear space, surrounding the basal part of the cilia. In the lateral part of the tormogen cell, there is a subspherical cavity in which a lightly electron-dense substance forms a deposit on the wall. This cavity takes form after ecdysis, then the deposit occurs and reaches its maximum thickness during the feeding phase. After apolysis, it is eliminated in exuvial space. It may strengthen the upper part of the cell and improve the reception of mechanical stimuli during the feeding phase.

Ultrastructure of the integument of a pelagic Crustacean: moult cycle related studies on the Antarctic krill, Euphausia superba

The ultrastructure of euphausiid integument was examined in relation to the moult cycle and supplemented by investigations of chitinase activity in the integument and content of N-acetyl-β-D-glucosamine in the hemolymph. The Antarctic krill, Euphausia superba was collected in 1983 in Admiralty Bay, King George Island, Antarctica. Some specimens of the Northern krill, Meganyctiphanes norvegica, from the Danish Kattegat served for comparison. As a major aim of the study, the moult staging system developed for living tissue could be verified by ultrastructural findings. Under experimental high production conditions of the Antarctic summer, no period of rest or “intermoult” between post- and premoult was observed in subadult E. superba. Neither was a resting phase seen at the cellular level, the epidermis remained active. The epidermal gland cells did not show any cyclical changes, and the organelles of protein synthesis were generally well developed in all moult stages. In order to follow the physiological course of events, structural and biochemical methods were combined and showed as a result that the last moult stage before ecdysis is characterized by massive cuticular resorption. The epicuticle remained ultrastructurally unchanged before and after ecdysis, even though its permeability should alter at ecdysis. The existence of muscle insertions which connect the old and the new cuticle across the exuvial space suggests an answer to the question why E. superba is hardly impaired in swimming almost up to the time of ecdysis.

Ultrastructure and ontogeny of the double-walled sensilla on the funicle of Calliphora erythrocephala meigen (diptera : calliphoridae)

The fine structure and ontogenesis of the double-walled hair sensilla on the funicle of Calliphora erythrocephala (Diptera : Calliphoridae) was investigated with transmission and scanning electron microscopes. The small ribbed pegs, situated on the surface of the antenna and the sensilla coeloconica found exclusively in olfactory pits, belong to the group of double-walled sensilla with wall pores. No pore-tubule system is present. An electron-dense substance lying between the wall pores presumably functions as the stimulus-conducting system. Because it can be extracted by fat-dissolving detergents, it is assumed to contain lipids. The double-walled sensilla are enclosed by 4 envelope cells. Ontogenesis is initiated by the sensory cilia enclosed in a dendritic sheath growing into the exuvial space. The hair-forming cell extends as finger-shaped cell processes along the dendritic sheath. Constriction of these cell processes leads to the formation of the longitudinal ridges and the “inner” hair wall. The trichogen cell secretes cuticular material in all directions. In the course of ontogenesis, the dendritic sheath, lying inside the hair shaft, degenerates. It is assumed that the trichogen cell participates in this process. The electron-dense substance, which lies between the pores in the imago, is interpreted as the remnants of the dendritic sheath material.

Chloride transport across the integumentary epithelium of Manduca sexta (Lepidoptera: Sphingidae).

Moulting fluid of Manduca sexta contains high concentrations of potassium and bicarbonate (100 mM) and low concentrations of chloride (5 mM). This fluid begins to disappear from the exuvial space approximately 9-10 h before the actual shedding of the integument. During this time, the integument can be isolated in an Ussing cell and electrical properties measured in vitro. In a normal 32 mM KHCO3 saline, potential difference (PD) is around 10 mV, exuvial side positive, and short-circuit current (SCC) is 15-20 microA cm-2. Substitution of chloride slightly reduces both PD and SCC, although resistance does not change significantly. Measurement of chloride transport in the absence of K+ indicates that 100% of the SCC can be accounted for by the net chloride flux (approximately 2 microA cm-2). The Km and Jmax for transepithelial chloride transport are 14 mM and 0.1 microEq cm-2 h-1. Bilateral potassium addition stimulates chloride transport, doubling net chloride flux as potassium concentration increases from 2 to 5 mM. Chloride net flux is not inhibited by the presence of furosemide (1 mM), nor in HCO3- -free saline by thiocyanate (1 or 10 mM) or acetazolamide (0.1 mM), but is inhibited by 100% N2. The pattern of chloride transport in M. sexta is similar to that previously reported for the rectum of locusts. As chloride is normally at low concentrations in the moulting fluid, it is suggested that this transport system acts to maintain low intracellular concentrations which may be necessary for enzymatic functions in the epidermal cells and has little importance in fluid transport.

Ultrastructure and ontogeny of the hair sensilla on the funicle of Calliphora erythrocephala (Insecta, Diptera)

Four envelope cells are responsible for the formation of the basiconical sensilla of Calliphora. They are the thecogen, trichogen, and tormogen cells, and envelope cell 4. In early stages of development the still subepithelial sensory cilia are completely enclosed by the innermost thecogen cell. The first formation movements are initiated by a growth thrust of the hair-forming cell into the exuvial space. The sensory cilia only begin to grow into the hair anlage when the hair-forming cell has almost reached its final length. As soon as growth is completed the trichogen cell, tormogen cell, and envelope cell 4 start to excrete cuticular material. The trichogen cell forms the perforated part of the hair shaft and the stimulus-conducting system consisting of the pore tubules. The tormogen cell is responsible for the excretion of the basal non-perforated hair shaft and sheath cell 4 forms the proximal part of the socket region. The thecogen cell only begin to produce dendritic sheath material when the sensory hair is almost complete. Approximately 7–8 days after pupation the tormogen cell degenerates, having, by this time, produced about two-thirds of the sensilla cuticle. The surrounding envelope cells incorporate cell fragments of the tormogen cell. The trichogen cell continues the secretion where the tormogen cell left off. When the secretion of cuticle is finished the sheath cells begin to withdraw towards the proximal direction and to form microvilli on the apical membrane. The resulting outer receptor lymph space is bordered by envelope cell 4 and the trichogen and thecogen cells. The tormogen cell is absent in the sensilla of the imago.

Ultrastructure and early development of the pore plate sensilla ofGymnomerus laevipes (Shuckard) (Vespoidea, Eumenidae)

The oval pore plates (approx. 17 μm long) are separated from the antennal cuticle by a furrow, the inner wall of which is flexible. The thin perforated plates are strengthened by an encircling and a middle ledge, the latter of which branches into about 100 almost parallel rims. Each pore plate is innervated by about 20 sense cells. The dendrites fork into numerous branches occupying the outer receptor lymph cavity below the perforated plate. Each pore plate is associated with one thecogen cell, two trichogen cells, one tormogen cell and one “envelope cell 4”. A so-called “additional cell” surrounds the sensillum in the imaginal stage. The envelope cells in the later of the two pupal stages examined, have reached an arrangement which immediately precedes the secretion of the cuticulin layer. The surface of the duplicate trichogen cells is almost equal in area to the completed perforated plate. A dendritic sheath, entirely reduced in the imago, protrudes into the exuvial space, where it encloses a single dendrite.

The Disappearance of Moulting Fluid in the Tobacco Hornworm, Manduca Sexta

ABSTRACT In Manduca sexta, and other insects, moulting fluid (MF) accumulates after apolysis in the exuvial space which underlies the old cuticle, and then most of it disappears shortly before ecdysis (see reviews by Zacharuk, 1976; Jungreis, 1979). Moulting fluid digests and weakens the old endocuticle so that the animal may free itself at ecdysis, and the digested components of the old endocuticle are reabsorbed. In M. sexta, Jungreis (1979) found that MF is produced between 30 and 12 h before the larval-pupal ecdysis (LPE) and it begins to disappear about 9 h before the LPE (LPE –9h). By LPE –3 h, most, but not all, of the MF has disappeared. We have found that MF represents a significant fraction of the total body water, 14·7% body weight (bw) [0·579 ± ·042 (5) ml, mean ± S.E. (N)] at LPE –6h.

Potassium transport in the isolated pharate pupal integument of the tobacco hornworm,Manduca sexta

Molting fluid accumulates in the exuvial space beneath the old cuticle between 24 and 12 h before the larval-pupal ecdysis in pharate pupae ofManduca sexta. Just before the molt (9 to 3 h), however, molting fluid disappears from the exuvial space. The integument separates the molting fluid (K+=140 mM) from the hemolymph (K+=35 mM). We have studied potassium transport across isolated, short-circuited preparations of the integument. In 32 mM potassium saline, potassium is actively transported in the hemolymph to exuvial direction at 0.27 and 0.34 μEq cm−2h−1 in preparations isolated at 15 and 6 h before the molt, respectively. The open-circuit potential difference (exuvial side positive) is 5.6 and 14.4 mV at 15 and 6 h before the molt, respectively. The short-circuit current, which can be accounted for by net potassium transport at 15 h before the molt, can not be completely accounted for by net potassium transport at 6 h before the molt (short-circuit current is 7.5 and 13 μA cm−2 at 15 and 6 h before the molt, respectively). At 15 h before the molt the values forKm andJmax are 15.2 mM and 0.4 μEq cm−2 h−1 and at 6 h before the molt these values are 32 mM and 0.7 μEq cm−2 h−1. The short-circuit current is not inhibited by ouabain, thallous ions, and acetazolamide, but is inhibited by 100% N2. During the period between 15 and 6 h before the molt, the integument undergoes a number of developmental changes. These are reflected in the tanning of the cuticle, the changes in the electrical properties, such as the increase in resistance, and the changes in the kinetic properties of potassium transport. The integument appears to play an active role in regulating the composition of the molting fluid, but its role in fluid transport remains to be determined.

Correlations between integument structure and ecdysteroid titers in fifth-stage nymphs of the tick, Ornithodoros moubata (Murray, 1877; sensu Walton, 1962)

The integument structure of fifth-stage nymphs of the tick Ornithodoros moubata was studied during the molting processes which are induced by the blood meal. In parallel, the ecdysteroid titers in hemolymph and in extracts from whole animals were determined by a radioimmunoassay (RIA). Between Days 2 and 3 after the meal (a.m.) a few procuticle lamellae were deposited and the mitotic period initiated. Ecdysteroid titers were low during the first 3 days a.m. They began to increase between Days 3 and 4 a.m. with the mitotic period ending. The titers rose sharply when apolysis and formation of the exuvial space took place (Day 4–5 a.m.). Highest ecdysteroid concentrations were measured during the deposition of the new epicuticle (Day 5–6 a.m.). Concomitant with the beginning of procuticle deposition and digestion of the old cuticle (Day 6 a.m.) the titers began to drop and reached low values again shortly before ecdysis (Day 9–10 a.m.). A preliminary chemical identification by thin-layer and high-performance liquid chromatography revealed that the majority of the RIA-positive ecdysteroids were composed of ecdysone and mainly ecdysterone. This was confirmed by gas chromatography of the silylated ecdysteroids. The results demonstrate that the titer changes in relation to the molting events in both ticks and insects appear to be very similar. It is thus likely that ecdysteroids are important in the control of tick molting.

Hair regeneration in a solfugid chemotactile sensillum during moulting (Arachnida: Solifugae).

The hair regeneration of a chemotactile sensillum was studied in the sunspiderGluvia during moulting. The sensilla in the old cuticle remain connected to the epidermis by dendrites which extend outwards during apolysis. The trichogen cells forming the new hairshaft in the exuvial space grow along the chemoreceptive dendrites, while the mechanoreceptive dendrites run separately. Morphogenetic aspects are discussed in comparison to results from other arthropods.

Fine structure of sensilla during moulting inNeomysis integer (Leach) (Crustacea, Mysidacea)

During the moulting cycle the sensory cells and the newly formed hair shaft remain connected to the old cuticular receptive apparatus of the sensillum by the elongated outer segments of the dendrites, which run through the exuvial space. A surface coat covering the outer dendritic segments protects them against the chemical influences of the exuvial fluid. The findings suggest that the receptors remain functional until ecdysis. This moulting type is considered to be a transitional form between the moulting types so far known.

Dependence of the parasitoid Gonia cinerascens on the hormones of its lepidopterous hosts

Development of first instar larvae of Gonia cinerascens, which rest in the muscles of host caterpillars, is triggered by the release of the host's ecdysteroids when the juvenile hormone is absent. Ecdysteroids act on the parasitoid directly and at the same time induce physiological and biochemical changes in the host, which are indispensable for the parasitoid's development. These changes do not occur when metamorphosis of the host is suppressed with the juvenile hormone. Normally the parasitoids initiate development at the larval-pupal transformation of the host, but under experimental conditions, they do so whenever a high ecdysteroid titre is coupled with the proper internal environment in the host, that is in decapitated caterpillars, isolated host abdomens, and when implanted into host pupae. Activated parasitoids moult into the second instar and migrate to the exuvial space of the host; this migratory behaviour is also triggered by ecdysteroids and may be induced experimentally in the first instar parasitoids. Unknown clues direct the migrating parasitoids under the wings and appendages of the host pharate pupal stage. The second instar parasitoids, which anchor to the integument of the host pupae, apparently develop independently of the host's hormones: they can produce third instar larvae, pupae, and adult flies when cultured in vitro.

COMPARISON OF THE GRADIENT OF SETAL DEVELOPMENT OF UROPODS AND OF SCAPHOGNATHITES INASTACUS LEPTODACTYLUS

1. Characterization of the different stages of the molt cycle of Astacus leptodactylus has been established using morphological criteria of the setae observed through the uropod. This method is paraticularly useful for the scientist who wants to get replicable, physiological data without disturbing the animals. 2. Stages determined from uropod studies agree with those obtained from comparative studies of scaphognathites. 3. According to the seasons, a more or less quiet intermolt phase is observed. The animals either directly enter the premolt phase or are blocked in stage C4 or early DO. Therefore, the morphological stage DO seems to be heterogenous, and apolysis is not necessary to the onset of premolt. 4. A single mode of setal formation seems evidenced. Moreover, setae with compound or connected matrices are seen in the scaphognathite. 5. Number and mobility of blood cells in the uropods are related to the investigated stage. During the premolt stages hemocytes, especially granulocytes, are observed within the exuvial space.