Molting Fluid Research Articles

Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis.

The location of midgut bacteria relative to meconial peritrophic membranes (MPMs) and changes in bacterial numbers during midgut metamorphosis were studied in Anopheles punctipennis (Say), Culex pipiens (L.), and Aedes aegypti (L.) pupae and newly emerged adults. After adult emergence in Aedes, Anopheles, and most Culex, there were few to no bacteria in the midgut. In most newly emerged adult mosquitoes, few bacteria were found in either the lumen or within the MPMs/meconia. In a few Culex specimens, high numbers of bacteria were found in the MPMs/meconia and low numbers in the lumen. In all three species bacterial counts were high in fourth instars, decreased after final larval defecation, increased in young pupae, and increased further in old pupae. A very effective gut sterilization mechanism is operating during mosquito metamorphosis and adult emergence. This mechanism appears to involve the sequestration of remaining larval gut bacteria within the confines of the meconium and one or two MPMs and the possible bactericidal effect of the exuvial (molting) fluid, which is ingested during the process of adult emergence.

L-Glutamate retrieved with the moulting fluid is processed by a glutamine synthetase in the pupal midgut of Calpodes ethlius

From apolysis until pupal ecdysis, the pharate pupa of the Brazilian Skipper ( Calpodes ethlius) lies wrapped in a prepupal shell composed of the larval cuticle and an ecdysial space (ES) filled with enzyme-rich moulting fluid (MF). In the 4 h before ecdysis the pharate pupa drinks the moulting fluid through its mouth and anus, and transfers the cuticular degradation products to its midgut (MG). At the same time, extra fluid passes across the body wall of the pharate pupa and flushes out the ES. The MF is recovered at an overall rate of 70 μl/h and reabsorbed across the pharate pupal midgut at about 26 μl/h. l-Glutamate was found to be the dominant amino acid in the moulting fluid. Total MF glutamate peaked at 850 nmol about 8 h before pupal ecdysis (P−8), but by ecdysis it had dropped to nearly zero as the MF became diluted with new fluid and was consumed. The drop in glutamate in the ES coincided with a rise in the glutamine content of the fluid in the midgut lumen. The highest rate of glutamine synthesis occurred in midguts isolated from pharate pupae actively drinking MF (P≤−4). The enzyme glutamine synthetase (GS) was found to be active in glutamate metabolism in the pharate pupal midgut. Glutamine synthesis in the midgut was l-glutamate-dependent and inhibited by two selective competitive inhibitors of GS activity, l-methionine sulfoximine (MSO) and glufosinate ammonium (GLA). Injection of GS inhibitors into the prepupal ES greatly reduced the glutamine content of the midgut epithelium by P+24. Although a corresponding increase in midgut glutamate levels was not seen, midgut serine levels in treated animals rose, suggesting that GS inhibitors shunted the MF-derived glutamate along an alternative metabolic pathway. GLA was much more toxic to pupae than MSO. Midgut GS appears to play a central role in the recycling of l-glutamate across the pupal MG epithelium at pupation.

Proteinase inhibitors from the molting fluid of the pharate adult tobacco hornworm, Manduca sexta

Proteinase inhibitors from the molting fluid of the pharate adult tobacco hornworm, Manduca sexta

Proteinase inhibitors from the molting fluid of the pharate adult tobacco hornworm, Manduca sexta.

The molting fluid of pharate adult Manduca sexta was found to contain at least two types of proteinase inhibitor activities. One inhibited the native cuticle degrading trypsin-like proteinase, MFP1, while the other was found to be highly specific for subtilisin-like enzymes. The developmental profiles of both these inhibitor activities were investigated. MFP-1 inhibitor activity was found to be present in the molting fluid of all stages of pre-ecdysial development, except stage 7, which possessed the highest levels of MFP-1 activity. The inhibitor was estimated to have a relative molecular mass of 14.5 k and was found to be heat stable. A role in regulation of cuticle degradation is suggested. Subtilisin inhibitor activity was found in molting fluid from all eight stages of pre-ecdysial development, although there was some variation observed between the stages when inhibitor activities were visualized using PAGE zymograms. A subtilisin inhibitor was purified using Sep-Pak cartridges and Reverse Phase HPLC. The inhibitor was found to be of low relative molecular mass (11 k), heat stable, and highly specific for fungal enzymes such as PR1 from the entomopathogen Metarhizium anisopliae. Therefore, a role in insect defense is suggested.

The origin, transport and cleavage of the molt-associated cuticular protein CECP22 from Calpodes ethlius (Lepidoptera, Hesperiidae)

CECP22 ( Calpodes ethlius Cuticular Protein 22 kDa) is a molt associated protein found in the cuticle of C. ethlius larvae and pupae. The mRNA for the CECP22 cuticular protein is expressed in the epidermis and fat body during the intermolt. The protein itself accumulates in intermolt hemolymph, but at molting, when the cuticle is being digested, it is also found in the cuticle of surface integument, tracheae, foregut and hindgut and in the molting fluid. CECP22 exists in two forms. The large form (19.17 kDa, p I 6.2) becomes smaller (16.1 kDa, p I 7.4) by cleavage at the proteolytic cleavage site (position 170) with amidation of the C-terminal. The small, more basic peptide, appears only at molting, first in the cuticle and then in the molting fluid. It is presumed to be the active form of an amidase involved in the earliest stages of cuticle degradation. The inactive form accumulates in the hemolymph during the long intermolt and probably represents an abundant source of precursor enzyme that can be provided to all cuticle containing organs for a precise initiation of cuticle degradation.

Mode of action of lufenuron on larval cat fleas (Siphonaptera: Pulicidae).

Adult cat fleas, Ctenocephalides felis (Bouché), were fed suboptimal in vitro concentrations of lufenuron in blood to allow hatching of flea larvae for cytological study. At concentrations of 0.125, 0.25, and 0.5 ppm, larval hatch was 64, 15, and 4%, respectively. Larvae hatching from eggs laid by adults fed lufenuron at concentrations of 0.025, 0.08, or 0.125 ppm did not differ significantly from the control. However, many larvae from the 0.08-ppm group and higher concentrations died during the 1st instar. Examination of these larvae revealed that they were dying from desiccation caused by bleeding from microscopic lesions in the cuticle or the inability to complete the molt to the next instar. Electron micrographs showed that lufenuron often disrupted formation of the endocuticle resulting in the deposition of an amorphous mass of randomly oriented chitin microfibrils. Other larvae formed normal endocuticle but were unable to digest the old endocuticle or produce new procuticle after apolysis. Failure of larvae to digest old cuticle or form new cuticle was caused by degeneration of the epidermal cells needed for the synthesis of molting fluid and chitin.

The role of the frontal ganglion in the feeding and eclosion behavior of the moth manduca sexta

We have examined the musculature and motor patterns of the foregut and the role of the frontal ganglion in the adult moth Manduca sexta. During adult development, the structure of the foregut changes from a simple straight tube to a pump consisting of a flexible-roofed chamber or cibarium, with dilator muscles that raise the roof to draw in fluids and a compressor to push it down and force the fluid down the thin-walled esophagus. The frontal ganglion drives the activity of this cibarial pump during feeding, which is triggered by the application of sucrose solution or water to the proboscis. The feeding motor pattern consists of coupled bursts of the pump dilators and shorter-duration, high-frequency bursts of spikes from the pump compressor. The pump is also activated at the adult molt. At this time, it is used both before the moth emerges from the pupal case for swallowing molting fluid and again after emergence for swallowing air. These behaviors are important for eclosion and are necessary for the expansion of the wings after eclosion. Their motor patterns are similar to the feeding program. Up to 24 h before adult ecdysis, this motor pattern can be triggered by the peptide eclosion hormone. The other eclosion-related peptide, Manduca sexta eclosion-triggering hormone, does not appear to trigger activity of the cibarial pump.

A technique for identifying the early-premolt stage in the male snow crab Chionoecetes opilio (Brachyura: Majidae) in Baie des Chaleurs, southern Gulf of St. Lawrence

New criteria for determining the early-premolt stage (D0) of the snow crab Chionoecetes opilio are described, based on histological observations. Identifying the early-premolt stage in snow crabs on the basis of observations of setagenesis poses difficulties for inexperienced observers. At the start of the premolt phase, numerous actively secreting cells beneath the epithelium and Leydig cells in the connective tissue can be observed using histological techniques. A semitransparent substance observed between the membranous layer and epithelium by histological means was further subjected to transmission electron microscope observations, which suggested that this was the molting fluid whose presence represents the beginning of the process of deposition of the epicuticle. At the time when the molting fluid is secreted in snow crabs, no morphological characteristics of premolt stage D1 are discernible, therefore these features can be used to identify D0. Using this technique, the molt stages of adolescent male crabs in the southern Gulf of St. Lawrence were examined, revealing the existence of two different types of molters that enter into the molting process in different times of the year. The majority of adolescent males go through the premolt stages in August-December and start to molt in March-April of the following year; another group of adolescent males with more epibionts on the carapace in the spring (adolescents that did not molt in the previous spring) are in the premolt stages in May-July and probably start to molt in December-January. The present study reveals that the molting patterns of snow crabs are far more flexible than the previous literature had suggested. The implications for long-term stock management are quite important, and further study on the structure and dynamics of molters is needed.

Hemagglutinating properties of apolipophorin III from the hemolymph of Galleria mellonella larvae

In search for factors that cause encapsulation of foreign bodies in insect hemolymph we discovered that larval hemolymph of Galleria mellonella caused aggregation of mammalian erythrocytes. The hemagglutinating agent was identified as an 18-kDa protein that did not react with lectins. The sequence of 81 amino acids in three protein fragments and the properties of the protein revealed that it was Galleria homologue of apolipophorin III (apoLp-III*). ApoLp-III was found in high amounts in the hemolymph of Galleria larvae, pupae, and adults, as well as in the molting fluid. The hemagglutinating action of the whole hemolymph or the purified apoLp-III was independent of the presence of sugars in the medium. This indicated that it was not mediated by carbohydrates on the erythrocyte surface. The hemagglutination was inhibited at low pH (3.0), in the absence of calcium ions, and in the presence of certain bacterial lipopolysaccharides or their essential component, the 2-keto-3-deoxyoctonate-3-deoxyoctulosonic acid (KDO). It is suggested that interaction of apoLp-III with lipopolysaccharides in bacterial cell walls may play a role in insect immune reactions. Arch. Insect Biochem. Physiol. 38:119–125, 1998. © 1998 Wiley-Liss, Inc.

Hemagglutinating properties of apolipophorin III from the hemolymph of Galleria mellonella larvae.

In search for factors that cause encapsulation of foreign bodies in insect hemolymph we discovered that larval hemolymph of Galleria mellonella caused aggregation of mammalian erythrocytes. The hemagglutinating agent was identified as an 18-kDa protein that did not react with lectins. The sequence of 81 amino acids in three protein fragments and the properties of the protein revealed that it was Galleria homologue of apolipophorin III (apoLp-III). ApoLp-III was found in high amounts in the hemolymph of Galleria larvae, pupae, and adults, as well as in the molting fluid. The hemagglutinating action of the whole hemolymph or the purified apoLp-III was independent of the presence of sugars in the medium. This indicated that it was not mediated by carbohydrates on the erythrocyte surface. The hemagglutination was inhibited at low pH (3.0), in the absence of calcium ions, and in the presence of certain bacterial lipopolysaccharides or their essential component, the 2-keto-3-deoxyoctonate-3-deoxyoctulosonic acid (KDO). It is suggested that interaction of apoLp-III with lipopolysaccharides in bacterial cell walls may play a role in insect immune reactions.

Cloning, expression, and hormonal regulation of an insect β- N-acetylglucosaminidase gene

Chitinolytic enzymes such as β- N-acetylglucosaminidases are major hydrolases involved in insect molting. By screening a Manduca sexta (tobacco hornworm) cDNA library with an antibody against β- N-acetylglucosaminidase from molting fluid of M. sexta pharate pupae, several putative cDNA clones for this enzyme were isolated. The longest of the cDNA clones has an insert of approximately 3 kb, and the complete nucleotide sequence was determined. Because this clone is missing the initiation codon and nucleotides corresponding to the leader peptide, the mRNA 5′-end sequence was determined by PCR (polymerase chain reaction) amplification and cycle sequencing. The sequence of the encoded protein from positions 23 to 35 is identical to the NH 2-terminal sequence of one of the β- N-acetylglucosaminidases isolated from pharate pupal molting fluid. The amino acid sequence is similar to those of silkworm, human, mouse, bacterial, and several other β- N-acetylglucosaminidases. Two highly conserved regions in the amino acid sequence were found in all members of this family. Southern blot analysis suggested that the number of genes in the Manduca genome closely related to the cDNA clone may be as few as one. The β- N-acetylglucosaminidase gene is expressed most abundantly in epidermal and gut tissues on days 6 and 7 of fifth instar larvae. Injection of 20-hydroxyecdysone induced expression of the β- N-acetylglucosaminidase gene, whereas topical application of the juvenile hormone analog, fenoxycarb, suppressed the inductive effect of molting hormone

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae

Baculovirus-mediated expression of a Manduca sexta chitinase gene: Properties of the recombinant protein

We constructed a recombinant nonoccluded baculovirus, Autographa californica nuclear polyhedrosis virus (AcMNPV), containing a 1.8 kb DNA fragment from a Manduca sexta (tobacco hornworm) chitinase cDNA under the control of the polyhedrin gene promoter. When Spodoptera frugiperda (fall armyworm) cells (SF9) were infected with this recombinant virus, a protein with an apparent molecular weight of 85 kDa was secreted into the culture medium. This protein hydrolyzed chitin and cross-reacted with a polyclonal antibody to M. sexta molting fluid chitinase. Tunicamycin treatment of infected SF9 cells and subsequent western blot analysis indicated that the secreted enzyme was a glycoprotein. GC-MS analysis revealed that carbohydrate accounted for approximately 25% of the mass of glycoprotein. The recombinant chitinase and the molting fluid enzyme were indistinguishable by N-terminal sequencing, polyacrylamide gel electrophoresis and carbohydrate analysis, indicating that the recombinant protein was similar, if not identical, to the molting fluid enzyme. Analysis of the expression level of recombinant chitinase in SF9, SF21 and Trichoplusia ni (Hi-5) cell lines showed that the yields were in the order Hi-5 > SF21 > SF9. Chitinase accumulated in hemolymph after injection of fourth instar M. sexta and S. frugiperda larvae with recombinant virus. The median time for mortality of S. frugiperda fourth instar larvae infected with the recombinant virus was approximately 20 h shorter than that for insects infected with a wild type virus. The results support the hypothesis that insect chitinase has potential to enhance the insecticidal activity of entomopathogens.

Activation Mechanisms of Pro‐Phenoloxidase on Melanosis Development in Florida Spiny Lobster (Panulirus argus) Cuticle

ABSTRACTPhenoloxidase (PO), pro‐PO [1] and pro‐PO [2], isolated from cuticular extracts of early premolt spiny lobster, resolved into doublets of molecular weights 156, 151, and 96.0, 93 kD, respectively. Trypsin‐activated forms of pro‐PO [1] and pro‐PO [2] were 123 and 83.6 kD, respectively. Apparently melanosis of shell and hyperdermal tissue in lobsters was related to stage of molt. The pro‐PO forms were activated in vitro by extracts of molting fluid which is secreted into the cuticle just before molt, and the activated PO co‐migrated in SDS‐PAGE with the endogenously activated PO. The molting fluid was hypothesized to be the source of the natural activator(s) of pro‐PO.

Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta

Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo- β- N-acetyl-glucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5–7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.

Moulting fluid enzymes of the tobacco hornworm, Manduca sexta: Inhibitory effect of 20-hydroxyecdysone on the activity of the cuticle degrading enzyme MFP-1

The moulting process of Manduca sexta is regulated by the ecdysteroid hormone 20-hydroxyecdysone. Injection of 20-hydroxyecdysone into pharate adult flight muscle delays eclosion, slows down the rate of thinning of the old cuticle and suppresses the cuticle degrading activity of moulting fluid. The effect of 20-hydroxyecdysone on the cuticle degrading activity of moulting fluid is dose-dependent. Also 20-hydroxyecdysone prevents the normal increase in activity of the enzyme moulting fluid protease 1 (MFP-1). The activity of another enzyme, β- N-acetylglucosaminidase, was reduced to a much smaller extent. The effects of 20-hydroxyecdysone on MFP-1 titre and ability of moulting fluid to solubilize labelled cuticle are consistent with the hypothesis that MFP-1 is the primary enzyme catalysing cuticle breakdown.

Purification and characterization of a biliverdin-binding protein from the molting fluid of the eri silkworm, Samia cynthia ricini (Lepidoptera: Saturniidae)

1. 1. The biliverdin-binding protein (BBP) was found in the molting fluid of the saturniid silkmoth, Samia cynthia ricini at the time of the larval-pupal ecdysis. 2. 2. It was purified from the molting fluid using hydrophobic interaction chromatography, gel-filtration and high-performance liquid chromatography (HPLC). 3. 3. The highly purified BBP showed a M r of approximately 49,000 by gel permeation of HPLC analysis using a TSKgel G3000SW column. 4. 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of M r approximately 24,000, indicating that the native BBP is dimer. 5. 5. The blue coloration of BBP from the molting fluid was due to the presence of biliverdin type pigments, as judged from its absorption spectra. 6. 6. These results suggested that BBP from Samia molting fluid may be a new type of insecticyanin (INS).

An aminopeptidase from the moulting fluid of the tobacco hornworm, Manduca sexta

A neutral metalloprotease (moulting fluid protease 2; MFP-2) from the moulting fluid of Manduca sexta (Lepidoptera: Sphingidae) pharate adults has been purified and partially characterized. The enzyme has a native molecular mass of 240 kDa as determined by HPLC gel filtration. SDS-PAGE of MFP-2 gave a single band of molecular mass 39.4 kDa indicating that the native enzyme probably exists as a hexamer. MFP-2 degrades a broad range of synthetic amino acid-β-naphthylamide substrates with a preference for methione-, leucine- or alamine β-naphthylamides. Activity is inhibited by amastatin, 1,10-phenanthroline and, to a lesser extent, ethylenediaminetetraacetic acid (EDTA). The inhibitory effects of divalent metal ion chelation are most effectively overcome by the addition of cobalt ions. MFP-2 alone has low cuticle degrading activity but acts with another moulting fluid enzyme, MFP-1, to degrade Manduca pupal cuticle.

A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta

Moulting fluid of pharate adult tobacco hornworm moths, Manduca sexta, contains a novel cuticle-degrading proteinase, designated as MFP-1. The enzyme has been purified using heparin affinity chromatography and partially characterized. Before purification MFP-1 is associated with a large complex having an apparent native molecular mass > 669 kDa. After purification MFP-1 has a molecular mass of 41 kDa. The p I of the enzyme is 5.54. MFP-1 can be classified as generally trypsin-like on the basis of its substrate specificity and inhibition by soybean trypsin inhibitor. The enzyme's preferred substrate, Tos-Gly-Pro-Arg- p NA, its inhibition by hirudin, and its affinity for herapin, all indicate that MFP-1 has some characteristics in common with the vertebrate blood-clotting enzyme thrombin. MFP-1 is probably a serine protease, since it is inhibited by both DFP and PMSF (specific inhibitors of serine proteinases). However, the enzyme was also inhibited by a number of agents that effect cysteine proteinases. Purified MFP-1 degrades Manduca cuticle in vitro. We suggest that the enzyme may act as the first step in the degradation of the cuticle during the moulting process.

Molting fluid enzymes of the tobacco hornworm, Manduca sexta: Timing of proteolytic and chitinolytic activity in relation to pre‐ecdysial development

AbstractDevelopmental profiles for a number of molting fluid (MF) enzyme activities were established and related to the progress of pupal cuticle degradation during the four days that precede the eclosion of adult tobacco hornworms. Cuticle degrading activity, molting fluid protease 1 (MFP‐1), and molting fluid protease 2 (MFP‐2) all increased in activity at the time that loss of material from the old cuticle occurred. In contrast, chitinase and β‐acetylglucosaminidase activities did not parallel weight loss from the old cuticle. These results are consistent with the hypothesis that proteolytic activity is a prerequisite for the action of chitinase on cuticle chitin. © 1993 Wiley‐Liss, Inc.