Cuticular adaptations in two parasitic copepods in relation to their modes of life

The structure, histochemistry, and possible functional properties of the cuticle in two parasitic copepods Pennella elegans Gnanamuthu and Caligus savala Gnanamuthu have been studied: the former is partially embedded in the host while the latter is an ectoparasite capable of free swimming.In Pennella elegans the cuticle of the embedded anterior region of the body is soft, colourless, and lacks an outer epicuticle while that of the posterior exposed part is pigmented and hard. Conspicuous in the cuticle of the ventral region of the head are pore canals which, though not chitinized, are functional even in the intermoult stage: these canals may be involved in the transport of nutrient materials from the host. The horns, which serve to fix the parasite firmly in the host tissues, are covered by cuticle in which the epicuticle and outer layers of the procuticle are hardened by formation of disulphide linkages. The cuticle of the neck region is not hardened and the procuticle in this region shows transverse regions of dense and light zones probably related to the coiling of the neck during penetration. The epicuticle is two layered in the cuticle of the exposed posterior region, the inner epicuticle and outer region of the procuticle being partially hardened by phenolic tanning so confer rigidity and resistance. The cuticle of the plumes is soft and devoid of an outer lipid epicuticle and so possibly adapted for a respiratory function.In Caligus savala, the epicuticle is two layered, and the procuticle has pigmented, calcified, and uncalcified layers. The cuticle is hardened by phenolic tanning as well as by calcification thus recalling the cuticular organization of decapod crustaceans.     

Die Feinstruktur des Integumentes und der Muskelansatzstellen vonEchiniscoides sigismundi (Heterotardigrada)

The structure of the integument and the muscle attachments of the marine heterotardigradeE. sigismundi (M. Schultze) was studied by electron microscopy. The cuticle consists of several layers: an outer tripartite (or multilayered) epicuticle, perhaps with an outermost coat; a homogeneous inner epicuticle; a trilaminated layer; an intracuticle; and a fibrous procuticle. These features resemble the cuticle described in Eutardigrada; in contrast, areas on the legs and near the claws, with an outer multilayered epicuticle and a striated layer (inner epicuticle), are — as far as investigated — more similar to the cuticle in Heterotardigrada. The epidermis consists of a single cell layer without glands. The muscle attachments are in line with the general pattern described in the eutardigradeMacrobiotus hufelandi and in Arthropoda.     

Molecular Organization of the Nanoscale Surface Structures of the Dragonfly Hemianax papuensis Wing Epicuticle

The molecular organization of the epicuticle (the outermost layer) of insect wings is vital in the formation of the nanoscale surface patterns that are responsible for bestowing remarkable functional properties. Using a combination of spectroscopic and chromatographic techniques, including Synchrotron-sourced Fourier-transform infrared microspectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS) depth profiling and gas chromatography-mass spectrometry (GCMS), we have identified the chemical components that constitute the nanoscale structures on the surface of the wings of the dragonfly, Hemianax papuensis. The major components were identified to be fatty acids, predominantly hexadecanoic acid and octadecanoic acid, and n-alkanes with even numbered carbon chains ranging from C₁₄ to C₃₀. The data obtained from XPS depth profiling, in conjunction with that obtained from GCMS analyses, enabled the location of particular classes of compounds to different regions within the epicuticle. Hexadecanoic acid was found to be a major component of the outer region of the epicuticle, which forms the surface nanostructures, and was also detected in deeper layers along with octadecanoic acid. Aliphatic compounds were detected throughout the epicuticle, and these appeared to form a third discrete layer that was separate from both the inner and outer epicuticles, which has never previously been reported.     

Photoaffinity cross-linking of acyl carrier protein to Escherichiacoli membranes

Acyl Carrier Protein (ACP) is a small acidic protein which interacts with the various enzymes implicated in the biosynthesis of fatty acids in E. coli. It also interacts with the inner membrane proteins implicated in the biosynthesis of phospholipids. Samples of radioactive ACP were prepared with high specific activities and bearing photoactivable aryl azide derivatives. Two photoactivable reagents were used: para azido phenacyl bromide (pAPA) which reacts with the SH of the ACP prosthetic group and the N-hydroxysuccinimide ester of 4-azido salicilic acid (NHS-ASA) which reacts with the amino groups of the protein. Various methods were used to demonstrate that ACP could be cross-linked specifically to an inner membrane protein of E. coli, most probably to the glycerol-3-phosphate acyl transferase (GPAT). This covalent link should provide a powerful tool for further analysis of the structure of GPAT and its role in phospholipid biosynthesis. These photoactivable aryl azide derivatives of ACP could also be very useful for studying the interaction of ACP with the soluble enzymes implicated in fatty acid biosynthesis.     

Expression of constitutive and tissue-specific acyl carrier protein isoforms in Arabidopsis

We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and 1 molar urea polyacrylamide gel electrophoresis revealed a complex pattern of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A Hloušek-Radojčić, JB Ohlrogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons II and III of the genomic ACP-1 clone and the mature coding sequences of the ACP-2 cDNA clone were subcloned into E. coli expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms.     

Penetration of the cuticular layers of elaterid larvae (Coleoptera) by the fungus Metarrhizium anisopliae, and notes on a bacterial invasion

The penetrant hyphae of Metarrhizium anisopliae in the exuvial cuticle of a molting wireworm can form secondary appressoria on the developing new cuticle. From these a new penetrant fungal apparatus can develop through the new cuticle toward the body cavity. The penetrant fungal apparatus in the ecdysial space of the host does not appear to be affected by the histolytic enzymes in the wireworm molting fluid. A mucoidlike substance that envelopes the fungus in the ecdysial space may be, in part, the protective mechanism involved. Bacteria from the soil often invade the ecdysial space of molting wireworms that have difficulty in shedding their exuvia. Pseudomonas aeruginosa can histolyze the proteinaceous exocuticle of the exuvium, the ecdysial membrane, and the dense inner epicuticle of the new cuticle, but not the epicuticle of the exuvium, when it invades the ecdysial space of a molting wireworm.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Structure of the cuticle of the alpine weta,Hemideina maori (Saussure) (Orthoptera: Stenopelmatidae)

Sclerotized cuticle segments from the thorax, dorsal abdomen, and ventral abdomen of the alpine, weta Hemideina maori (Saussure) (Orthoptera: Stenopelmatidae) were examined by light microscopy and by scanning and transmission electron microscopy. An epicuticle, exocuticle (outer and inner), mesocuticle, endocuticle, and deposition layer are present in transverse sections. The epicuticle is further composed of a cuticulin layer and inner epicuticle, the latter being finely laminated and containing narrow wax canals that terminate below the cuticle surface. Openings to dermal gland ducts are visible on the surface as are large setae and smaller sensory pegs. Frozen fractured cuticle reveals the presence of horizontal ducts or channels that run laterally within the cuticle. The structure of weta cuticle is compared with that of the common house cricket and arthropods in general.     

Ultrastructure of the penetration of the crayfish integument by the fungal parasite, Aphanomyces astaci, Oomycetes

In the crayfish, Astacus astacus, susceptible to the crayfish plague fungus, penetration of the cuticle by the parasite occurred in the soft cuticle. The zoospore lysed the surface lipid layer, tore it away, and formed an infection peg (germ tube) that penetrated through the epicuticle. A septum was formed in the infection peg, and a hypha was formed below the inner epicuticular surface. In the endocuticle, hyphae grew preferrentially parallel to the surface, occassionally perpendicular to it. Growth direction in relation to cuticle architecture is discussed. Subsequently, some hyphae started to penetrate out through the epicuticle. This process was preceded by the swelling of the hyphal tip touching the inner side of the epicuticle. The hypha penetrating out through the epicuticle was much thicker than the infection peg. Histolytic activity, combined with mechanical penetration, seems to be evident in all stages and levels except in the outward penetration of the epicuticular lipid surface layer, where only mechanical rupture could be seen. Differences in the protoplasmic ultrastructure were found between the spore and the penetrant hyphae. Penetration of the cuticle of a resistant crayfish was essentially identical to that in susceptible ones. However, inward penetration of intact epicuticle was too scarce to allow for ultrastructural studies.     

Cuticle secretion during larval growth in Drosophila melanogaster

The moulting cycle and growth of the larval integument of Drosophila melanogaster has been studied by light and electron microscopy. Growth during the first, second and third larval instars is accompanied by 3.0-, 3.4- and 3.7-fold increases in surface area, respectively. Growth in surface area occurs continuously during the larval stages, with no detectable relationship to the moulting cycle. Measurements of the thickness of the cuticular layers show that the endocuticle grows in thickness by apposition and in surface area by stretching. The pre-apolytic epicuticle remains at fairly constant thickness during the increase in surface area, indicating that it grows by intussusception of new components. Post-apolytic epicuticle becomes thinner and increases in surface area by stretching. The epicuticle and pre-ecdysial endocuticle are traversed by filaments, but these do not penetrate the endocuticle secreted after ecdysis. We suggest that the filaments transport breakdown products from the old cuticle inward to the epidermis for reutilization. The growth and deposition of cuticle in two larval growth mutants, lethal (2) giant larvae and Chubby Tubby, involves mechanisms similar to those found in wild-type larvae, but in Chubby Tubby the endocuticle contains inclusions which are ultrastructurally similar to dense epicuticle.     

Behavioral,Ultrastructural and Chemical Studies on the Honeydew and Waxy Secretions by Nymphs and Adults of the Asian Citrus Psyllid Diaphorina citri (Hemiptera: Psyllidae)

The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae) is the primary vector of the bacterium causing citrus huanglongbing (citrus greening), the most serious disease of citrus worldwide. Psyllids and other hemipterans produce large amounts of honeydew, which has been used previously as an indicator of phloem sap composition and insect feeding or metabolism. Behavioral, ultrastructural and chemical studies on ACP, its honeydew and waxy secretions showed important differences between nymphs, males and females, and suggested some mechanisms by which the psyllids, especially nymphs and adult females, can minimize their contamination with honeydew excretions. The anal opening in ACP, near the posterior end of the abdomen, is on the ventral side in nymphs and on the dorsal side in adult males and females. Video recordings showed that adult males produce clear sticky droplets of honeydew gently deposited behind their body on the leaf surface, whereas adult females produce whitish honeydew pellets powerfully propelled away from the female body, probably to get their excretions away from eggs and newly hatched nymphs. ACP nymphs produce long ribbons or tubes of honeydew that frequently stay attached to the exuviae after molting, or drop when feeding on the lower side of citrus leaves. Furthermore, honeydew excretions of both nymphs and adult females are covered with a thin layer of whitish waxy material ultrastructurally composed of a convoluted network of long fine filaments or ribbons. This material is extruded from intricate arrays of wax pores in the circumanal ring (around the anus) that is found in nymphs and females but not in males of ACP or other psyllid species. Infrared microscopy and mass spectroscopy revealed that, in addition to various sugars, honeydew excretions of ACP nymphs and females are covered with a thin layer of wax similar in profile to ester waxes.     

Structure of the cuticle of the common house cricket with reference to the location of lipids

Cuticle segments from the thorax, abdomen, and jumping legs of the house cricket. Acheta domesticus, were examined using histological techniques for light microscopy, scanning and transmission electron microscopy, and direct examination of frozen-fractured cuticle. The surface of untreated cuticle is covered by a lipid film which obscures fine surface detail. Standard EM preparative procedures, as well as washing the cuticle with ethanol before examination, remove this film exposing previously covered openings to dermal gland ducts and wax canals. An epicuticle, exocuticle, mesocuticle, endocuticle, and a deposition layer were present in all transverse sections of cuticle. Light microscopy showed that the exocuticle and mesocuticle are heavily impregnated with lipids, whereas there is little lipid associated with the endocuticle. Frozen-fractured cuticle clearly shows the ‘plywood’ structure of the meso- and endocuticle, while the exocuticle fractures as if it were a solid sheet. The epicuticle is composed of a dense homogeneous layer, cuticulin, outer epicuticle, and the outer membrane. Superficial wax was detected only in cuticle samples prepared using vinylcyclohexane dioxide as a polar dehydrant. The results were used to construct a comprehensive model of the cuticle of A. domesticus.     

A study on the cuticle of Stegoalpheon kempi Chopra (Isopoda: Crustacea) in relation to its parasitic mode of life

Histological, histochemical, and possible functional properties of the cuticle in the bopyrid isopod parasite Stegoalpheon kempi Chopra are described. In the male, the epi- and the procuticle show similar histochemical characters. The protein constituent of the cuticle suggests a similarity to collagen and its significance is discussed in relation to the parasitic mode of life.In the female, the epicuticle is fuchsin-positive in Mallory's Triple and Masson's Trichrome stains, positive to tests for aromatic amino acids, but negative to tests for phenol, phenol oxidase, and quinone. In addition, the epicuticle shows auto-fluorescence and investigations on the fluorescent compounds present in the epicuticle protein show the presence of di- and tri-tyrosine. The function of these two unusual amino acids as possible cross links is discussed.The cuticle covering the pleopods lacks epicuticle, but it has a simple protein positive to the biuret test, and also a protein containing sulphydryl groups, perhaps as an adaptation for providing respiratory exchanges of gases through cuticle.     

Cuticular cycle and molting hormone levels during the metamorphosis of Tenebrio molitor (Insecta Coleoptera)

The cuticular cycle of Tenebrio molitor (apolysis, synthesis of outer and inner epicuticle, fibrous cuticle deposition) was studied during the last larval and pupal stages by electron microscopy. Concurrently, molting hormone (MH) titers in the hemolymph were determined by a radioimmunoassay method. It appears, both in larvae and in pupae, that the MH peak coincides with the initiation of pre-ecdysial cuticle deposition (i.e., outer epicuticle synthesis). Thus MH is involved in the induction of cuticular synthesis; however, its role in inducing larval-pupal apolysis is questionable. We note that this peculiar apolysis occurs long before MH release.     

Immunochemical characterization of human red cell acid phosphatase isozymes

An immunological study was performed on human red cell acid phosphatase (ACP1) isozymes encoded by different alleles, each of which is expressed as an electrophoretically fast (f) isozyme and a slow (s) isozyme. These isozymes reacted as two immunochemically different groups. Allele-specific reactions were not detected between either the f isozymes or the s isozymes. Quantitation of ACP1 isozymes in red cells by crossed immunoelectrophoresis revealed a phenotype-dependent variation in the concentration of isozyme protein. A simple gene dosage effect was indicated and the ordering of the ACP1 alleles (ACP1*A < ACP1*B < ACP1*C < ACP1*E) was identical to that found for enzyme activity levels. Also, an allele effect on the proportion between s and f isozymes (s/f) was observed; the ordering here was ACP1* B < ACP1*A < ACP1*, which is the same as that reported for the susceptibility to modulation with purines. These variations in isozyme protein levels appear to account for the phenotypic differences in the intensity of the isozyme bands, when activity-stained after electrophoresis, and in the red cell enzyme activity levels. Investigation of two carriers of a Null allele showed no evidence of an aberrant protein product, and half-normal concentrations of enzyme protein were observed in the red cells of these individuals.     

Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein

During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [³H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat.     

NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H: EFFECTS OF DIVALENT METAL IONS*

Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca²⁺ and Mg²⁺ and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by ¹⁵N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.     

Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel. Furthermore, KI mice exhibit dramatic differences in the enamel structure compared with wild type (WT) mice, including thinner enamel lacking enamel rods and ectopic surface calcifications. Here, we now demonstrate that amelogenin phosphorylation also affects the organization and composition of mature enamel mineral. We compared WT, KI, and heterozygous (HET) enamel and found that in the WT elongated crystals are co-oriented within each rod, however, their c-axes are not aligned with the rods’ axes. In contrast, in rod-less KI enamel, crystalline c-axes are less co-oriented, with misorientation progressively increasing toward the enamel surface, which contains spherulites, with a morphology consistent with abiotic formation. Furthermore, we found significant differences in enamel hardness and carbonate content between the genotypes. ACP was also observed in the interrod of WT and HET enamel, and throughout aprismatic KI enamel. In conclusion, amelogenin phosphorylation plays crucial roles in controlling structural, crystallographic, mechanical, and compositional characteristics of dental enamel. Thus, loss of amelogenin phosphorylation leads to a reduction in the biological control over the enamel mineralization process.     

<Superscript>1</Superscript>H NMR analysis of <Emphasis Type="Italic">Citrus macrophylla</Emphasis> subjected to Asian citrus psyllid (<Emphasis Type="Italic">Diaphorina citri</Emphasis> Kuwayama) feeding

The Asian citrus psyllid (ACP) is a phloem-feeding insect that can host and transmit the bacterium Candidatus Liberibacter asiaticus (CLas), which is the putative causative agent of the economically important citrus disease, Huanglongbing (HLB). ACP are widespread in Florida, and are spreading in California; they are the primary mode of CLas transmission in citrus groves. To understand the effects of ACP feeding, different numbers of ACP [0 ACP (control), 5 ACP (low), 15–20 ACP (medium), and 25–30 ACP (high)] were allowed to feed on Citrus macrophylla greenhouse plants. After 7 days of feeding, leaves were collected and analyzed using ¹H NMR. Metabolite concentrations from leaves of trees with ACP feeding had higher variability than control trees. Many metabolites were higher in concentration in the low ACP feeding group relative to control; however, leaves from trees with high ACP feeding had lower concentrations of many metabolites relative to control, including many amino acids such as phenylalanine, arginine, isoleucine, valine, threonine, and leucine. These results suggest ACP density-dependent changes in primary metabolism that can be measured by ¹H NMR. The implications in plant defense are discussed.