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Michael Locke - One of the best experts on this subject based on the ideXlab platform.
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The characterization of ferritin in an insect
Insect Biochemistry, 2003Co-Authors: Helen Nichol, Michael LockeAbstract:Abstract In mammals, the iron storage protein ferritin is predominantly synthesized on free polysomes and accumulates in the cytosol but some is secreted and circulates in the blood as serum ferritin. In insect tissues, on the other hand, iron-containing holoferritin accumulates in the vacuolar system and can be secreted through the Golgi complex. The midgut can secrete it to the gut lumen and other tissues to the hemolymph. Ferritin was isolated from the midgut and hemolymph of fifth instar larvae of Calpodes ethlius , Lepidoptera, Hesperiidae. This holoferritin is stable to heat (75°C) or in the presence of SDS, proteinase K, or urea, has an Mr above 600,000, contains iron and resembles mammalian ferritins in appearance by electron microscopy. Calpodes ferritin is a glycoprotein having N-linked high-mannose oligosaccharides. It is not antigenically related to horse ferritin but is related to that from Manduca sexta , Lepidoptera, Sphingidae. In its native form, Calpodes ferritin has only 3 isoforms with a pI 6.5–7 suggesting a more uniform subunit composition than that in vertebrates. It has two principle subunits, with relative Mrs of 24,000 (L) and 31,000 (G) and two minor subunits with Mrs of 26,000 and 28,000 all of which cross-react with antibody to Manduca ferritin. The 24 kDa subunit is the only one that is not glycosylated. Iron injections induce an increase in the proportion of the 24 kDa subunit. We conclude that Calpodes has ferritin and that it is glycosylated like mammalian serum ferritin.
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The origin, transport and cleavage of the molt-associated cuticular protein CECP22 from Calpodes ethlius (Lepidoptera, Hesperiidae).
Journal of Insect Physiology, 1999Co-Authors: Oana Marcu, Michael LockeAbstract: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, pI 6.2) becomes smaller (16.1 kDa, pI 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.
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Secreted ferritin subunits are of two kinds in insects molecular cloning of cDNAs encoding two major subunits of secreted ferritin from Calpodes ethlius.
Insect Biochemistry and Molecular Biology, 1999Co-Authors: Helen Nichol, Michael LockeAbstract:Abstract In insects, holoferritin is easily visible in the vacuolar system of tissues that filter the hemolymph and, at least in Lepidoptera, is abundant in the hemolymph. Sequences reported for insect secreted ferritins from Lepidoptera and Diptera have high sequence diversity. We examined the nature of this diversity for the first time by analyzing sequences of cDNAs encoding two ferritin subunits from one species, Calpodes ethlius (Lepidoptera, Hesperiidae). We found that insect secreted ferritin subunits are of two types with little resemblance to each other. Ferritin was isolated from iron loaded hemolymph of C. ethlius fifth instar larvae by differential centrifugation. The N-terminal amino acid sequences for the nonglycosylated subunit with Mr 24,000 (S) and the largest glycosylated subunit with Mr 31,000 (G) were determined. The N-termini of the two subunits were different and were used to construct degenerate PCR primers. The same cDNA products were amplified from cDNA libraries from the midgut which secretes holoferritin and from the fat body which secretes iron-poor apoferritin. The G subunit most closely resembles the glycosylated ferritin subunit from Manduca sexta and the S subunit resembles the Drosophila small subunit. The S and G subunits from Calpodes were dissimilar and distinct from the cytosolic ferritins of vertebrates and invertebrates. Additional sequences were obtained by 5′ and 3′ RACE from separate fat body and midgut RACE libraries. cDNAs encoding both subunits had a consensus iron responsive element (IRE) in a conserved cap-distal location of their 5′ UTR. An integrin-binding RGD motif found in the G subunit and conserved in Manduca may facilitate iron uptake through a calreticulin (mobilferrin)/integrin pathway. Calpodes and other insect ferritins have conserved cysteine residues to which fatty acids can be linked. Dynamic acylation of ferritin may slow but not prevent its passage out of the ER.
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A cuticular protein from the moulting stages of an insect
Insect Biochemistry and Molecular Biology, 1998Co-Authors: Oana Marcu, Michael LockeAbstract:A 22 kDa peptide was purified from prepupal cuticles of 5th instar Calpodes ethlius caterpillars. It was absent earlier in the stadium and from the egg and adult, i.e. it is related to cuticle turnover rather than cuticle structure. It was present at larval and metamorphic moults, showing that it is related to moulting not just metamorphosis. The cDNA corresponding to the 22 kDa peptide was isolated by antibody screening of an epidermal cDNA expression library. Hybridization to Calpodes genomic DNA showed that the gene was present as a single copy. The deduced amino acid sequence is not like any of the sequences of cuticular structural proteins that have been published, but has a 47 amino acid sequence similar to bacteriophage T7 N-acetylmuramoyl-L-alanine amidase (34% identical, 51% similar). The amino acid sequence, the timing of expression in development, and the similarity between the substrate of the bacteriophage amidase and components of insect cuticle, all suggest that the 22 kDa protein may have a role in cleaving chitin-peptide bonds as a prerequisite for digestion of the cuticle by chitinases and proteases.
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Integument and hemocyte peptides
Journal of Insect Physiology, 1994Co-Authors: Miklós Sass, Agnes Kiss, Michael LockeAbstract:Abstract There are four routing classes of integument peptide in the caterpillar of Calpodes ethlius. The epidermis secretes peptides apically into the cuticle (C), basally into the hemolymph (H) and in both directions (BD). Peptides in a 4th class (T), are presumed to be transported across the epidermis, because the epidermis does not synthesize them although they occur in both cuticle and hemolymph. In a search for the origin of the presumed transepidermal peptides we found that hemocytes contain some peptides from all four routing classes. Peptides prepared from washed hemocytes reacted in immunoblots to antibodies against integument peptides prepared from hemolymph and cuticle. These peptides are probably synthesized by hemocytes because they matched those from medium containing [35S]methionine in which hemocytes had been incubated. Calpodes hemolymph contains four hemocyte types. Immunogold labelling localized integument peptides in the secretory pathway of granulocytes and spherulocytes and in the cytosol of oenocytoids but not in plasmatocytes. Each peptide was localized in a particular kind or kinds of hemocyte. Granulocyte secretory vesicles reacted with antibodies to C180, C55 and BD82 kDa peptides. Spherulocytes secretory vesicles reacted with antibodies to C180, C55, BD89, BD82 and a 78 kDa peptide presumed to be the precursor of T66. Oenocyotoids reacted with antibodies to H45, 38, 32, 23 and BD89 kDa peptides. Spherulocytes were the only tissue to react with antibodies to the T66 kDa peptide that is found abundantly in cuticle and hemolymph. Spherulocytes are therefore presumed to secrete the 66 kDa peptide into the hemolymph from where it is transported to the cuticle. The C180 and C55 kDa peptides do not occur in hemolymph. Their presence in granulocytes and spherulocytes may be associated with hemocyte functions such as basal lamina formation, since immunogold localized them in that part of the basal lamina next to the hemolymph, as would be expected if hemocytes deposited components onto the exposed hemolymph surface. The presence of hemolymph peptides in oenocytoids is more difficult to interpret, since the antigenic reactions are localized in the cytosol rather than in the secretory pathway expected for exported proteins. We conclude that integument peptides are not secreted only by the epidermis, nor is the cuticle their only destination.
Helen Nichol - One of the best experts on this subject based on the ideXlab platform.
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The characterization of ferritin in an insect
Insect Biochemistry, 2003Co-Authors: Helen Nichol, Michael LockeAbstract:Abstract In mammals, the iron storage protein ferritin is predominantly synthesized on free polysomes and accumulates in the cytosol but some is secreted and circulates in the blood as serum ferritin. In insect tissues, on the other hand, iron-containing holoferritin accumulates in the vacuolar system and can be secreted through the Golgi complex. The midgut can secrete it to the gut lumen and other tissues to the hemolymph. Ferritin was isolated from the midgut and hemolymph of fifth instar larvae of Calpodes ethlius , Lepidoptera, Hesperiidae. This holoferritin is stable to heat (75°C) or in the presence of SDS, proteinase K, or urea, has an Mr above 600,000, contains iron and resembles mammalian ferritins in appearance by electron microscopy. Calpodes ferritin is a glycoprotein having N-linked high-mannose oligosaccharides. It is not antigenically related to horse ferritin but is related to that from Manduca sexta , Lepidoptera, Sphingidae. In its native form, Calpodes ferritin has only 3 isoforms with a pI 6.5–7 suggesting a more uniform subunit composition than that in vertebrates. It has two principle subunits, with relative Mrs of 24,000 (L) and 31,000 (G) and two minor subunits with Mrs of 26,000 and 28,000 all of which cross-react with antibody to Manduca ferritin. The 24 kDa subunit is the only one that is not glycosylated. Iron injections induce an increase in the proportion of the 24 kDa subunit. We conclude that Calpodes has ferritin and that it is glycosylated like mammalian serum ferritin.
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Secreted ferritin subunits are of two kinds in insects molecular cloning of cDNAs encoding two major subunits of secreted ferritin from Calpodes ethlius.
Insect Biochemistry and Molecular Biology, 1999Co-Authors: Helen Nichol, Michael LockeAbstract:Abstract In insects, holoferritin is easily visible in the vacuolar system of tissues that filter the hemolymph and, at least in Lepidoptera, is abundant in the hemolymph. Sequences reported for insect secreted ferritins from Lepidoptera and Diptera have high sequence diversity. We examined the nature of this diversity for the first time by analyzing sequences of cDNAs encoding two ferritin subunits from one species, Calpodes ethlius (Lepidoptera, Hesperiidae). We found that insect secreted ferritin subunits are of two types with little resemblance to each other. Ferritin was isolated from iron loaded hemolymph of C. ethlius fifth instar larvae by differential centrifugation. The N-terminal amino acid sequences for the nonglycosylated subunit with Mr 24,000 (S) and the largest glycosylated subunit with Mr 31,000 (G) were determined. The N-termini of the two subunits were different and were used to construct degenerate PCR primers. The same cDNA products were amplified from cDNA libraries from the midgut which secretes holoferritin and from the fat body which secretes iron-poor apoferritin. The G subunit most closely resembles the glycosylated ferritin subunit from Manduca sexta and the S subunit resembles the Drosophila small subunit. The S and G subunits from Calpodes were dissimilar and distinct from the cytosolic ferritins of vertebrates and invertebrates. Additional sequences were obtained by 5′ and 3′ RACE from separate fat body and midgut RACE libraries. cDNAs encoding both subunits had a consensus iron responsive element (IRE) in a conserved cap-distal location of their 5′ UTR. An integrin-binding RGD motif found in the G subunit and conserved in Manduca may facilitate iron uptake through a calreticulin (mobilferrin)/integrin pathway. Calpodes and other insect ferritins have conserved cysteine residues to which fatty acids can be linked. Dynamic acylation of ferritin may slow but not prevent its passage out of the ER.
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Vacuolar apoferritin synthesis by the fat body of an insect (Calpodes ethlius)
Journal of Insect Physiology, 1991Co-Authors: Michael Locke, C. Ketola-pirie, Harry Leung, Helen NicholAbstract:Abstract Electron microscopy of insect fat body shows holoferritin to be rare or absent. This does not mean that the fat body does not have ferritin, since apoferritin lacks contrast and would not be observed. The fat body of Calpodes ethlius has therefore been studied to see if it could be a source of the ferritin found in haemolymph. After treatments that would be expected to load apoferritin with iron to convert it to holoferritin, abundant holoferritin-like particles appear in the rough endoplasmic reticulum (RER) and in microsomes prepared from fat body RER. The particles only occur in the secretory pathway and are absent from the cytosol. Insect ferritin antigens are localized in RER cisternae. In a cell-free system with microsomes and fat body mRNA, newly synthesized peptides having the same molecular weight as glycosylated ferritin subunits appear in the microsomal cisternae. The fat body is therefore probably a source of haemolymph ferritin.
Oana Marcu - One of the best experts on this subject based on the ideXlab platform.
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The origin, transport and cleavage of the molt-associated cuticular protein CECP22 from Calpodes ethlius (Lepidoptera, Hesperiidae).
Journal of Insect Physiology, 1999Co-Authors: Oana Marcu, Michael LockeAbstract: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, pI 6.2) becomes smaller (16.1 kDa, pI 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.
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A cuticular protein from the moulting stages of an insect
Insect Biochemistry and Molecular Biology, 1998Co-Authors: Oana Marcu, Michael LockeAbstract:A 22 kDa peptide was purified from prepupal cuticles of 5th instar Calpodes ethlius caterpillars. It was absent earlier in the stadium and from the egg and adult, i.e. it is related to cuticle turnover rather than cuticle structure. It was present at larval and metamorphic moults, showing that it is related to moulting not just metamorphosis. The cDNA corresponding to the 22 kDa peptide was isolated by antibody screening of an epidermal cDNA expression library. Hybridization to Calpodes genomic DNA showed that the gene was present as a single copy. The deduced amino acid sequence is not like any of the sequences of cuticular structural proteins that have been published, but has a 47 amino acid sequence similar to bacteriophage T7 N-acetylmuramoyl-L-alanine amidase (34% identical, 51% similar). The amino acid sequence, the timing of expression in development, and the similarity between the substrate of the bacteriophage amidase and components of insect cuticle, all suggest that the 22 kDa protein may have a role in cleaving chitin-peptide bonds as a prerequisite for digestion of the cuticle by chitinases and proteases.
Stanley Caveney - One of the best experts on this subject based on the ideXlab platform.
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Unusual Life History Characteristics of Elachertus scutellatus Howard (Hymenoptera: Eulophidae), a Koinobionic Ectoparasitoid
Environmental Entomology, 2004Co-Authors: Kimberley E. Macdonald, Stanley CaveneyAbstract:Elachertus scutellatus Howard is a synovigenic, koinobionic ectoparasitoid that preys on the Brazilian skipper caterpillar, Calpodes ethlius (Stoll) (Lepidoptera: Hesperiidae). This wasp exhibits a rare combination of life history strategies, some associated with being a koinobiont and others with being ectoparasitic. The female wasp lays large, inflexible, anhydropic eggs that are passed externally down the ovipositor. This process is uncommon and is considered the morphological intermediate between the use of the ovipositor as an egg-laying device and a stinger. The average clutch size and the oviposition frequency of E. scutellatus females are comparable with other wasps in the Eulophidae. Female wasps were observed to be nonconcurrent host-feeders. The sex ratios of E. scutellatus broods were consistently female biased. Adult male wasps lived longer than female wasps, which may have been a result of host size or the rearing technique used. E. scutellatus has potential as a biocontrol agent because of its female-biased sex ratios, destructive host-feeding, and synovigeny.
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EXTERNAL MORPHOLOGY AND DEVELOPMENT OF IMMATURE STAGES OF ELACHERTUS SCUTELLATUS (HYMENOPTERA: EULOPHIDAE) IN FLORIDA: THE FIRST NORTH AMERICAN RECORD
Florida Entomologist, 2004Co-Authors: Kimberley E. Macdonald, Stanley CaveneyAbstract:The first North American record for Elachertus scutellatus, a parasitoid of Calpodes ethlius Stoll, occurred August 1999, in Florida. A simple rearing protocol was established to allow the morphology and development of this wasp to be examined. The egg and larval morphology and development of E. scutellatus resemble other Elachertus species. The freshly-ecdysed pupa, on the other hand, is rare among parasitoids in that it secretes a fluid from its anus which, when dry, fastens the pupa to its substrate. The death of the colony after eight months has many possible explanations including a laboratory-induced castration, inappropriate food source(s), and pathogenic infection.
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l-Glutamate retrieved with the moulting fluid is processed by a glutamine synthetase in the pupal midgut of Calpodes ethlius
Journal of Insect Physiology, 2000Co-Authors: C Yarema, H Mclean, Stanley CaveneyAbstract: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 4h 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 70µl/h and reabsorbed across the pharate pupal midgut at about 26µl/h. L-Glutamate was found to be the dominant amino acid in the moulting fluid. Total MF glutamate peaked at 850nmol about 8h 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
Scott C. Henderson - One of the best experts on this subject based on the ideXlab platform.
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A shell of F-actin surrounds the branched nuclei of silk gland cells
Cell Motility and the Cytoskeleton, 1992Co-Authors: Scott C. Henderson, Michael LockeAbstract:The branched nuclei from silk gland cells of larvae of Calpodes ethlius label with antibodies to actin and myosin and with rhodaminyl-phalloin, which is specific for f-actin. Optical sectioning localizes this actin and myosin to the nuclear periphery. Residual nuclear-associated fractions prepared from these cells contain sheets of nuclear lamina-like structures that bind heavy meromyosin and gold-tagged antibodies to actin and myosin. The results suggest that both actin and myosin, or a myosin-like protein, are components of a layer at the nucleocytoplasmic boundary that we call the nuclear shell. The nuclear shell appears to be associated with the nuclear envelope and may correspond to a zone on the cytoplasmic face of the envelope seen in electron micrographs of unextracted cells. The residual nuclear-associated fraction has a unique isoform of actin (43 kD, pl 6.45) that might allow the nuclei to associate with an actin network structurally and developmentally distinct from that of the cytoplasm. © 1992 Wiley-Liss, Inc.
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Regional differences in the apical F-actin cytoskeleton of silk gland cells of Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae)
International Journal of Insect Morphology and Embryology, 1991Co-Authors: Scott C. Henderson, Michael LockeAbstract:Silk glands of the larvae of Calpodes ethlius, Stoll (Lepidoptera : Hesperiidae) were labelled with rhodaminyl-phalloin, which is specific for f-actin. Most f-actin lies below the apical cell surface in bundles oriented in rings around the lumen of the gland. By the fifth larval stadium, the density of bundles differs in the 5 regions of the gland, the greatest density occurring in the narrower anterior regions. The greater bundle density may be correlated with the increased hoop stress where the gland has the smallest diameter. F-actin from the bundles redistributes during larval moults to coat vacuoles in the cytoplasm, except in the 2 most anterior regions where it keeps its arrangement from stage to stage, even through the moult.
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The development of branched silk gland nuclei.
Tissue and Cell, 1991Co-Authors: Scott C. Henderson, Michael LockeAbstract:Nuclei in the giant polyploid silk gland cells of Calpodes ethlius grow by endomitosis and can develop hundreds of branches during larval life. The shape of the these nuclei is characteristic for each region of the gland. We have found shape to be correlated with arrangement of the nuclear matrix. Scanning electron microscopy showed nuclear matrices with shapes similar to those of feulgen stained nuclei. Profiles of isolated matrices seen by transmission electron microscopy had filaments aligned parallel to the long axis of nuclear branches. DNA stained by Hoechst had a similar parallel alignment within the branches. Nuclear shape may be maintained by a small number of components, since electrophoretic analysis showed only a few abundant polypeptides in the matrix fraction. Silk gland nuclei have some of the same nuclear matrix antigens found in smaller, more regularly shaped, eukaryote nuclei.
