Molting Fluid Research Articles

Biochemical and Physiological Studies of Certain Ticks (Ixodoidea). Phospholipid and Sterol Patterns in Biological Fluids of Nymphal and Adult Hyalomma (H.) Dromedarii Koch and H. (H.) Anatolicum Excavatum Koch (Ixodidae)1

In hemolymph, gut, and molting fluids of nymphal and adult Hyalomma (H.) dromedarii (Koch) and H. (H.) anatolicum excavatum (Koch) (family Ixodidae), the lipid classes were phospholipid (PL), cholesterol (CH), free fatty acids, triglycerides, and sterol esters, together with small amounts of mono- and diglycerides. PL, total sterols and percent CH contents were analyzed during specific physiological states of tick development. Hemolymph free sterols consisted exclusively of cholesterol. When fully fed nymphs and females dropped from the host, their gut PL and sterol concentrations were substantially higher than in host rabbit plasma and whole blood. Lipid concentrations on the day engorged nymphs dropped from the host, molting day and the female dropping day changed more consistently than for the other states. The results suggest that ticks have no requirement, and therefore lack the capacity, for sterol biosynthesis.

Cocoonase: III. PURIFICATION, PRELIMINARY CHARACTERIZATION, AND ACTIVATION OF THE ZYMOGEN OF AN INSECT PROTEASE

Abstract The inactive precursor (zymogen) of the insect protease cocoonase has been identified and purified from galeae of the silkmoth, Antheraea polyphemus. Prococoonase can be activated proteolytically, with either trypsin, subtilisin, active cocoonase, or a protease mixture isolated from the moulting fluid of A. polyphemus. The molecular weight of the zymogen is estimated as 33,000, whereas active cocoonase collected from moths has a molecular weight of approximately 24,500. Ion exchange chromatography and disc gel electrophoresis indicate that the zymogen is significantly more acidic than active cocoonase. The evolution of serine proteases is discussed.

General esterases of silkmoth moulting fluid: Preliminary characterization

The moulting fluid of silkmoths contains not only proteolytic enzymes with amino acid esterase activity, but also two naphthyl esterases (general esterases). These enzymes have been partially purified and separated by sucrose density-gradient centrifugation and DEAE-cellulose column chromatography. One is weakly anionic at pH 8 and has a molecular weight of 64,000; the other is cationic and has a molecular weight of 57,000. Both enzymes are most stable under the mildly alkaline pH conditions characteristic of moulting fluid but are irreversibly inactivated at acid pH. Both are inhibited by diisopropyl fluorophosphate, although at different rates. The enzymes hydrolyse low molecular weight esters of aromatic alcohols, but lack proteolytic activity; inhibition and specificity studies suggest that they are carboxyl esterases. They are found throughout development, in both moulting gel and moulting fluid; their rôle in the moulting process is as yet unclear.

Inactive proteinases in silkmoth moulting gel

The moulting gel of silkmoths lacks proteolytic activity but contains an inactive form of the proteinases which are later found in the moulting fluid. These inactive enzymes are activatable in vitro by dilution, activation proceeding most rapidly at low ionic strength. Activation proceeds as a first-order process and is not autocatalytic. Approximately the full amount of proteinases ultimately found in moulting fluid are already present in the gel. Moulting gel does not inhibit the active proteinases of moulting fluid; moreover, the proteolytic activity elicited by dilution of the moulting gel does not disappear upon reconcentration. These observations suggest that the proteinases in moulting gel are not inhibited by a stable, dissociable inhibitor; they may be present either as compartmentalized active enzymes or as proenzymes. Several possible mechanisms for the in vivo activation at the time of gel to fluid transformation are discussed.

Molting in a parasitic nematode, Phocanema decipiens—VI The mode of action of insect juvenile hormone and farnesyl methyl ether

Both a synthetic preparation of insect juvenile hormone and farnesyl methyl ether inhibited ecdysis but left cuticle formation almost unaffected when present in the complete in vitro medium used to culture Phocanema through its final molt. When the worms were allowed to grow fo 2 1 2 days in 0.9% NaCl, where ecdysis does not normally occur, and then exposed to the compound, normal ecdysis occurred in many of the worms. In a similar experiment, the worms were examined for neurosecretory activity, which could be detected as little as 12 h after the addition of the compounds to the saline cultures. It is concluded that the compounds exert their effect by stimulating the neuroendocrine system of the nematode, which in turn brings about the production and release of the molting fluid.

Some properties of silkmoth moulting gel and moulting fluid

The moulting fluid of silkmoths contains numerous proteins, mostly in the range of 20,000 to 70,000 molecular weight. Only a fraction of these proteins have known enzymatic activity. Approximately ten bands are resolved by polyacrylamide disk gel electrophoresis. The moulting fluid is a distinct secretion differing from blood in almost every parameter studied: pH, visible absorption spectrum, protein content, enzymatic activities, and protein electrophoretograms; the u.v. spectrum and possibly the electrophoretic mobilities of a few proteins are similar in blood and fluid. Proteolytic activity appears in the moulting fluid at the beginning of the last week of the pharate adult stage (on day 12 of adult development in Antheraea polyphemus) and shows a time-activity profile that correlates well with the rate of digestion of pupal endocuticle. By contrast, general esterases of moulting fluid show a relatively constant level of activity. As adult development proceeds, the fluid becomes enriched in dialysable components. Endocuticle digestion was studied in vivo and in vitro. The amount of proteinase in moulting fluid ( ca. 9 μg trypsin equivalents in 150 μl, less than 1% of the total protein) is consistent with the quantity of protein in the pupal endocuticle ( ca. 85 mg) and the period of time (2–4 days) required for full digestion in vivo.

THE ORIGIN, DISTRIBUTION AND FATE OF THE MOLTING FLUID PROTEINS OF THE CECROPIA SILKWORM.

1. Molting in insects is always accompanied by the production of a molting fluid which fills the exuvial space between the new and the old cuticle and digests the inner layers of the old cuticle. In Hyalophora cecropia, molting fluid is secreted at the outset of adult development and persists until two days before eclosion, whereupon it is absorbed. 2. The present report examines the protein composition of the molting fluid of Cecropia, the origin of the molting fluid proteins, the relation of these proteins to blood proteins and the exchange of macromolecules between the molting fluid and the blood. It also examines the sites of absorption of molting fluid. 3. Disc electrophoresis on acrylamide gels reveals that the molting fluid of Cecropia contains about fifteen protein bands which can be resolved at pH 8.6. Some of these protein bands are detected in the molting fluid at all stages, whereas others appear only at specific times. About ten of the bands are peculiar to molting fluid and are not detected in the blood. About five bands are detectable in both blood and molting fluid, but none of these common bands appears to be a major component of the molting fluid, and only one is a major blood protein. In contrast, the epidermis contains most of the major protein bands found in molting fluid but lacks all but one of the major protein bands present in the blood. 4. Immunological analysis reveals that blood and molting fluid share five antigens. At least four of these common antigens also occur in the epidermis which appears to secrete these antigens into both the molting fluid and the blood. 5. Native and foreign proteins do not penetrate from the exuvial space into the blood or vice versa. Apparently the epidermis and cuticle act as a barrier to the exchange of most macromolecules between the blood and molting fluid. The exuvial space is clearly a separate fluid compartment. 6. In addition the exuvial space itself is compartmentalized and the fluids in the compartments do not admix several days before eclosion. 7. Absorption of molting fluid during the final two days of adult development occurs most readily through particular regions of the integument. In the abdomen the principal sites of absorption are pits which represent the points through which tonofibrils make attachment to the old cuticle. Two days before ecdysis, the attachments between the tonofibrils and the pupal cuticle rupture, exposing the points of attachment on the new cuticle. It is through these exposed surfaces that much of the molting fluid is absorbed. Molting fluid is also absorbed in the head and thorax through various flexible membranes at the bases of the appendages.

The Dermal Glands of Rhodnius Prolixus

Dermal glands are epidermal derivatives which are reported to secrete either the cement layer, which is the outermost layer of the epicuticle or some component of the moulting fluid which digests the endocuticle. The secretions do not show well-defined staining reactions and therefore they have not been positively identified. This has contributed to another difficulty, namely, that of determining the time of secretory activity. This description of the fine structure of the developing glands in Rhodnius was undertaken to determine the time of activity, with a view to investigating their function.

Effect of Heterologous Antibody on Haemonchus contortus Development In vitro

Double diffusion tests of sera obtained from rabbits immunized with somatic extracts of the free-living nematodes, Caenorhabditis briggsae and C. elegans, resulted in cross-reactions with metabolic and somatic extracts of the sheep parasitic stomach worm, Haemonchus contortus. In vitro culture experiments demonstrated that H. contortus development was retarded by the same heterologous antisera. The possibility of complement playing a suggested. Indirect evidence strongly suggests that the molting process of histotropic stages of trichostrongylid nematodes is closely associated with the immune resistance response (Soulsby, 1963). The evidence indicates that important antigens are released (and probably elaborated) during exsheathment of third-stage larvae and during ecdysis to the fourth and fifth stages. It also appears that the parasitic nematode is particularly susceptible to the action of antibody during molting. If molting fluids of parasitic nematodes are involved in the stimulation of host resistance, similar fluids from free-living forms may have the same effect, since it is believed that the process of ecdysis will not differ greatly among nematodes. The substances responsible for controlling and initiating molting in insects, for example, have been shown to be the same and to act similarly when a wide variety of insect species have been compared. This study was initiated to examine the possibility of immunologic cross-reactions between the parasitic nematode, Haemonchus contortus, and the free-living nematodes, Caenorhabditis briggsae and C. elegans. These free-living nematodes are especially suited for such an investigation because of the ease with which they can be cultured axenically in large quantities (Buecher et al., 1966). MATERIALS AND METHODS Antigen preparations Free-living nematodes C. briggsae and C. elegans were cultured under identical conditions at 20 C in C. briggsae mediumReceived for publication 4 November 1968. * Part of a dissertation submitted to the Graduate College of the University of Illinois in partial fulfillment of the requirements for the Ph.D. degree by Birute P. Jakstys. role in the heterologous immune cross-reactions is 75 (Grand Island Biological Co.) (Buecher et al., 1966; Bryant et al., 1967). C. briggsae culture-75 (EM) was supplemented with 10% chick embryo extract (CEE) which was prepared in accordance with the method of Nicholas et al. (1959). C. elegans cultures, due to the worms' prolific reproduction, were harvested every 2 weeks, while C. briggsae were collected every 4 weeks. The worms were washed three times in 40 ml of sterile phosphate-buffered 0.9% saline (pH 6.8 to 7.0) and incubated overnight at 20 C in a fourth change of fresh saline. The following day, the incubation saline was discarded and for every 1 ml of settled worm volume, 4 ml of fresh saline were added. The worm-saline suspension was passed through the French press (French and Milner, 1955) at room temperature and extrusion pressures of 20,000 to 25,000 p.s.i. A drop of the homogenate was inspected under the light microscope to ensure that the worms were completely disintegrated. The homogenate was centrifuged at 5,000 g for 1 hr at 4 C. The residue was discarded and the supernatant was used as the antigen. Protein concentration of the extracts was determined according to the technique of Lowry et al. (1951). The antigens were lyophilized and stored at 4 C until needed.

A Histological and Bionietrical Study of Wing Development in the Grasshopper Melanoplus lakinus1,2

Melanoplus lakinus Scudder (Orthoptera: Acrididae) is a brachypterous grasshopper of Arizona rangeland that occasionally shows macropterism. Wing development is similar histologically to that in holometabolans, although the epidermal cells are less attenuated in this species. The cells extend to the middle membrane and generally are not continuous with opposing cells. They become distorted after the procuticle is secreted. Nuclei elongate and migrate toward the middle membrane to varying degrees. The middle membrane may be as much as 3 µ thick. Granular molting fluid occurs in large quantity and is prevalent during the last half of each instar. Chromatolysis may occur in the last stage of growth in the hindwing. On the surface of the wing pads polygonal cuticular plaques change to “shingled” rows in the last 2 instars. Two characteristics of mitotic activity in wing pads are revealed: (1) mitoses occur in distinct bursts and at fluctuating low levels, and (2) the process begins while the epidermis is still attached to the endocuticle and continues after deposition of the procuticle. A mitotic burst could be followed from its initiation to its termination, over a 6-hr period, by successive sampling of the 4 wings of 1 specimen. Mitotic activity corresponds well with the increase in nuclei during each instar. There are no obvious structural differences between nymphs of brachypterous and macropterous adults. The regression coefficient of wing length on hind femur length increases significantly during most instars. The large increase in wing length of the macropterous form occurs in the last instar and deviates greatly from simple allometric growth. Thus far, mating of macropterous forms in cages has increased their frequency from less than 4% to 39% in the F1 generation.

Muscle attachment related to cuticle architecture in Apterygota.

ABSTRACT In Apterygota muscles are attached to the cuticle by a series of discrete structures. The junction of the muscle and epidermal cells is demarcated by regular interdigitations of the two tissues, with desmosomes lining these processes. Within the epidermal cells, microtubules link up the desmosomes of the interdigitated region with dense material associated with cone-like depressions in the apical plasma membranes of the epidermal cells. Each of these ‘conical hemidesmosomes’ is situated opposite a pore canal. From within each cone, an electron-dense ‘muscle attachment fibre ‘extends up the corresponding pore canal through the procuticle and is inserted on the epicuticle. There is no direct link between the microtubules and the muscle attachment fibres. The muscle attachment fibres are slightly elliptical in cross-section, and are twisted, this twist being in phase with the orientation of the chitinprotein microfibrils forming the lamellae of the procuticle. The attachment fibres are straight, and not helically arranged; patterns obtained in oblique sections of procuticle including these structures support the twisted ribbon model of pore canal shape. The cuticle, particularly in Thysanu?an and Dipluran intersegmental membrane, displays the parabolic patterning typical of softer insect procuticle and procuticle deposition zones. The epicuticular insertion of the muscle attachment fibre is characterized by a pit in the cuticulin layer, the fibre passing through the middle of this pit. The microtubule-associated conical hemidesmosomes appear to be cytoskeletal in function. The muscle attachment fibres are rigid structures which are not digested by the moulting fluid enzymes. New muscle attachment fibres may only become attached to the epicuticle during its formation. The structures described in regions of muscle attachment in Apterygota are similar to those recorded for other arthropods.

The structure and development of a surface pattern on the cuticle of the green vegetable bug Nezara viridula

Adult Nezara possess an area of cuticle on the metathoracic sternite with a surface pattern of minute mushroom-like projections. The surface pattern is formed by two types of cell, one forming projections and the other depressions. The surface pattern is largely determined before epicuticle deposition by deformation of the epidermal surface. This is brought about by interactions between the moulting fluid secretions and the cells. Maintenance of shape of the cells may be associated with the presence of oriented cytoplasmic microtubules. The mushroom area may function as an external reservoir causing retarded evaporation of defensive secretions.

The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting fluid in an insect

AbstractA structure for a generalized insect epidermal cell during the formation of the epicuticle is proposed, based on studies of several different epidermal cell types. The protein epicuticle is defined as the dense homogeneous layer below the cuticulin. The formation of the protein epicuticle involves secretory vesicles arising in Golgi complexes, and marks an interlude in the involvement in cuticle formation of plasma membrane plaques. The plaques are concerned in cuticulin formation before and in fibrous cuticle formation after the deposition of the protein epicuticle.The epidermis is characterized by the possession of a cytoskeleton of microtubules and a matrix of microfibers. In the elongated cells forming bristles and spines, the microfibers are often oriented in bundles with an axial banding which repeats every 120 Å. The microtubules are also arranged in columns with a trigonal packing and center to center spacing of about 800 Å. These cytoskeletal structures separate the other organelles into channels which may restrict the pathways open for the movement of secretory and pinocytotic vesicles. The protein epicuticle arises from the secretory vesicles which discharge at the apical surface. The contents disperse and reaggregate below the cuticulin. The Golgi complexes in the basal and central regions have many secretory vesicles and a small saccular component, differing from those nearer the apex which are smaller and have fenestrated saccules. The small coated vesicles (800 Å in diameter) associated with both sorts of complex, probably move to the apical and basal faces of the cell where they may give rise to the large coated vesicles (2000 Å in diameter) inserted in the plasma membrane. Pinocytosis occurs from both apical and basal faces but most lytic activity is in the apical region. Plant peroxidase injected into the haemocoel is taken up basally and transported to the apical MVBs. The large coated vesicles on the apical face may be concerned in the control of the extracellular subcuticular environment. They appear to fill up and detach, fusing to become the apical MVBs.

Sequestration of protein by the fat body of an insect.

THE fat body in an insect commonly contains a single cell type which is functionally diverse. At various times it stores and mobilizes fat, protein and glycogen which it may have synthesized or sequestered. In general the fat body becomes filled with reserves as metamorphosis approaches1. Larvae of Aedes fed on casein build up reserves of protein granules in the fat body2, and in Drosophila larvae, protein granules are induced to form in the middle of the third stadium by a hormone from the ring gland3. In Calpodes larvae the protein granules are probably translocated endocuticle. They arise suddenly within the Golgi vesicles 30–35 h before pupation, at a time when the thick larval endocuticle is being resorbed by the moulting fluid. At about 10 h before pupation other granules containing both protein and ribosomes arise from the isolation of endoplasmic reticulum in cytolysomes4. Although the formation and occurrence of protein granules in the fat body are well documented, there is little information on the origin of the protein.

The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae).

AbstractThe cuticulin layer is defined as the dense lamina (120–175 Å thick in Calpodes larvae, depending upon the stage) forming the outer part of the epicuticle in insects. It completely invests an insect except for the gut and the openings of some sense organs. It is the first layer to be secreted during the formation of new cuticle. The formation of the cuticulin membrane may be a useful model for studying the origin of membranes in general. It arises as a triple layer de novo and is not a modified plasma membrane. Growth is by accretion at the edges of patches of cuticulin which increase in area until they cover the new surface. The triple layer (i.e. three dense laminae) may develop striations about 30 Å apart transverse to the membrane, which perhaps form a sieve allowing small molecules to pass while protecting the cell from enzymes in the molting fluid. A similar porous structure persists in the tracheoles. After the resorption of molting fluid the triple layered structure again becomes obvious and the outermost layer separates from the other two to become what may be the surface lipid monolayer. The surface patterns in cuticle of various sorts probably arise by buckling of the cuticulin layer as it increases in surface area.

Perivascular leucocytosis and other types of cellular reactions in the oyster Crassostrea virginica experimentally infected with the nematode Angiostrongylus cantonensis

Histopathologic studies on the oyster, Crassostrea virginica, experimentally infected with first-stage larvae of the nematode Angiostronglus cantonensis revealed conspicuous leucocytic response in the host 10 to 14 days after infection. Furthermore, intraluminal nematode larvae attracted extraluminal leucocytes after the 10th day so that heavy perivascular capsules of leucocytes were consistently found. The leucocyte-attracting substance (LAS) is believed to be the nematode's molting fluid. Similar types of leucocytic response have been observed in experimentally infected rats and monkeys. Infection of oysters only occurred when first-stage larvae were ingested, followed by successful penetration of the alimentary wall. Evidence indicated that larvae can be transported to the oyster's Leydig tissues in blood vessels.

INTEGUMENTARY CHANGES DURING MOULTING OF ARTHROPODS WITH SPECIAL REFERENCE TO THE SUBCUTICLE AND ECDYSIAL MEMBRANE.

AbstractAt or shortly prior to the separation of the cuticle and the epidermis a cuticular layer appears to be added to the inner surface of the old cuticle. The term interzone cuticle is applied to this layer. The interzone cuticle appears to give rise to the ecdysial membrane.Ecdysial membranes are characterized by the fact that they (1) vary in position with respect to the moulting fluid and old cuticle, (2) are generally quite thin, transparent, delicate membranes, (3) contain chitin, (4) vary in their resistance to the action of the moulting fluid, and (5) generally have the same stainng properties as the subcuticle, due primarily to the presence of a mucous material present in the subcuticle which becomes attached to the inner surface of the ecdysial membrane and which is believed to function as a lubricant at ecdysis.The ecdysial membrane and subcuticle are structurally quite different when examined under the electron microscope.No particular evolutionary significance can be ascribed to the existence of an ecdysial membrane — such membranes being found all the way from the primitive Collembola to the advanced Hymenoptera. Any possible function(s) of the ecdysial membrane has yet to be discovered. Various functions are suggested for the subcuticle, all of which are consistent with its reactions to various histochemical reagents.Additional observations were made and discussed concerning (1) the reactions of various portions of the cuticle at various stages to the PAS test, (2) the staining reactions of cuticle undergoing enzymatic degradation by the moulting fluid, and (3) the distinct staining reactions of the intersegmental membranes and basement membranes.

Eine einfache Methode zur Gewinnung von Exuvialflüssigkeit bei der Honigbiene (Apis mellifica L.)

A simple method is described for obtaining the ecdysial fluid of the honeybee. For this purpose, full-grown worker and drone larvae are taken out of the cells and their cocoons as young prepupae shortly after termination of spinning. They are raised at a temperature of 34.5° to 35° C. The secretion of the moulting fluid begins 12 to 24 h later in all larvae which are ready to pupate. In larvae without a cocoon, the casting of the old cuticle does often not occur, or is incomplete. The secretion gathers abundantly in the body region under the larval skin and can be sucked off without difficulty with fine glass pipettes in a completely pure state for the biochemical analysis.

On the tissue isolated in some of the larval appendages of Sialis lutaria L. at the larval-pupal moult

Blood and epidermal cells are isolated in the old cuticle during the larval-pupal moult and are shed with that cuticle. The cells are left behind in the isolated fluid, which is a mixture of moulting fluid and haemolymph. No cells have been observed in the moulting fluid at any other moult. The number of cells cut off seems to be directly proportional to the size of the larva and hence to the size of the structure withdrawn. The blood cells in the isolated fluid are capable of clotting and forming tanned wound plugs at a time when the haemolymph of the pupa scarcely clots. The activity of the blood cells within the isolated fluid declines with time. During the retraction of the epidermis in the gills, a new cuticle, or moulting membrane, is continuously secreted. Where this is exposed to activated moulting fluid it is dissolved immediately before the larval-pupal ecdysis except for the innermost layer, secreted immediately before the production of the new pupal cuticle. The latter layer envelops the whole body of the insect and seems to be homologous to the ecdysial membrane of other insects. The thick new cuticle within the gills does not appear to have any distinct functional significance for the pupa beyond protecting the retracting gill tip.

The origin and nature of the ecdysial membrane in Schistocerca gregaria (Forskål)

The few innermost laminae of the fully formed larval endocuticle of Schistocerca gregaria show remarkable resistance to the action of the moulting fluid as well as to concentrated mineral acids. These laminae become subsequently isolated during moulting through the dissolution by the moulting fluid of the less resistant outer laminae of the endocuticle. They give rise to a discrete ecdysial membrane which persists throughout the premoult period and is ultimately cast with the inner lining of the larval exuvium. In Schistocerca an ecdysial membrane is formed at the larval adult moult as well as at all the preceding larval moults.