A freeze-fracture study of the surface of the infective-stage larva of the nematode Trichinella

The surface layers of the cuticle of the infective, first-stage larva of the nematodes Trichinella spiralis and T. spiralis var. pseudospiralis have been studied by means of the freeze-fracturing technique. No obvious differences between the two nematodes were found. A double-layered structure covers the cuticle. Its outermost layer consists of particles embedded in an amorphous matrix; its inner layer is composed of a sheet of fine filaments which may be composed of globular subunits. This unique double layered structure is not like a normal cell membrane in structure. The surface of the cuticle beneath it is relatively smooth except for impressions from the inner surface of the double-layered structure. The cuticle surface did not fracture in the manner of a cell membrane.     

Direct evidence that the cuticle surface of Trichinella spiralis muscle larvae shares antigenicity with stichocyte alpha-granules and the esophagus-occupying substance.

Antibodies against the cuticle surface of Trichinella spiralis muscle larvae were purified by means of immunoaffinity chromatography and incubated with ultrathin sections of muscle larvae. Major constituents of the parasite reactive with the purified antibodies included the cuticle surface, stichocyte alpha-granules, and the esophagus occupying substance of the muscle larvae. Thus the present data suggest that the cuticle surface is an antigenically different entity from the cuticle inner layers and its origin is likely stichocyte alpha-granules.     

Trichinella spiralis: light microscope monoclonal antibody localization and immunochemical characterization of phosphorylcholine and other antigens in the muscle larva

A panel of monoclonal antibodies was used to examine the structure of the muscle larva of Trichinella spiralis under the light microscope. Immunofluorescence and, in some cases, immunoperoxidase staining were used. All four antibodies reacted with the cuticle of the organism, although differences in the staining pattern were observed for some of these. Interestingly, all the antibodies also reacted with the stichosome. One of the antibodies (Ts2Ab) is specific for the hapten, phosphorylcholine. In a binding assay, this antibody also reacted with extracts of Trichuris suis, Ascaris suum, and Fasciolopsis buski, but not with extracts derived from Cysticercus cellulosae, Candida albicans, Salmonella typhi, or Escherichia coli. This crossreactivity was confirmed microscopically in which the cuticle, oviduct and eggs of T. suis, the cuticle, muscle cells, and eggs of A. suum, and the cuticle and vitelline glands of F. buski were seen to be clearly stained by the antibody. In addition, Ts2Ab also reacted with the cuticle and stichosome of the adult T. spiralis worm. In Western blot analysis, Ts2Ab recognized a 43-kDa antigen from T. spiralis muscle larvae extracts, while a previously studied antibody (7C2C5Ab) identified four major antigens (48.5, 47, 43, and 39 kDa) in this preparation. Similar results were obtained when the 24-hr excretory-secretory (ES) antigens of T. spiralis were immunoblotted with the antibodies, although the reactivity shown by Ts2Ab was relatively weak. With the 72-hr ES material, on the other hand, major antigens of lower mol wt (44, 28, and 25 kDa) were revealed by 7C2C5Ab, and no reactivity was seen with Ts2Ab. However, this antigen preparation reacted well with both antibodies in an enzyme-linked immunoassay. Taken together, the findings suggest that the 72-hr ES antigens probably result from extensive degradation of material originally secreted or excreted by the worm. Similar binding studies on the 24-hr ES preparation indicated that this source may be relatively rich in 7C2C5Ab-reactive epitopes and relatively poor in the antigen identified by Ts2Ab. Other studies performed demonstrated that the antigens recognized by these two antibodies were distinct and physically unassociated.     

A spectrum of antibody response with time after Trichinella spiralis infection in rats

A chronology of class-specific antibody response against the Trichinella spiralis infection in Fischer rats was investigated. G-class antibodies against the cuticle inner layer(s), hypodermis, hemolymph, glycogen aggregates, discrete areas in genital primordial cells, intestinal gland cell granules, and cytoplasmic granules in the cords were detectable 2 wk after infection (the rapid-responding group), whereas G-class antibodies against the cuticle surface, stichocyte granules, and the esophagus-occupying substance were detected 6 wk after infection (the slow-responding group). M-class antibodies recognized a narrower spectrum of antigens than did G-class antibodies; M-class antibodies against hemolymph, cord granules, and intestinal gland cell granules were not detectable. M-class antibodies tended to decrease in titer with time after infection. This tendency was more striking with antibodies against the rapid-responding group than with those against the slow-responding group. This information sheds light upon antibody response against many antigenic components of T. spiralis muscle larvae.     

A freeze-fracture study of the body wall of adult, in utero larvae and infective-stage larvae of Trichinella (Nematoda)

The surface layers of the cuticle, the hypodermal membranes and the muscle membranes of the adult, the in utero larvae and the infective-stage larvae of the nematode Trichinella spiralis have been studied by means of the freeze-fracturing technique. The surface of the cuticle of both adults and larvae fractures in ways different from membranes of internal cells. The surface coat on top of the epicuticle is probably the layer that changes antigenically. Reticulate ridges, with associated particles, on the E face of the outer hypodermal membrane of the adult are probably sites of attachment of the hypodermis to the cuticle. Longitudinally arranged ridges, with associated particles, of the outer hypodermal membrane are probably points of attachment to the cuticle in the in utero and infective larvae. Rectilinear arrays of particles are present on the P face of the inner hypodermal membrane and the P face of the muscle membrane adjacent to the hypodermis of adults and larvae and probably play a role in adhesion of the muscle membrane to the hypodermis. Particle-free areas of membrane lie external to the Z bundles of the muscle cell and are similar to the sites of attachment of Z lines in insect muscles.     

Trichinella spp.: differential expression of two genes in the muscle larva of encapsulating and nonencapsulating species.

Kuratli, S., Lindh, J. G., Gottstein, B., Smith, D. F., and Connolly, B. 1999. Trichinella spp.: Differential expression of two genes in the muscle larva of encapsulating and nonencapsulating species. Experimental Parasitology 93, 153-159. The expression of the two genes tsmyd-1 and tsJ5 was studied in the muscle stage larva of three different species of Trichinella. T. spiralis and T. britovi are both encapsulating species, while T. pseudospiralis is a nonencapsulating species. Expression of tsJ5 is developmentally regulated in T. spiralis and has been shown in this study to be down-regulated in the T. pseudospiralis muscle larva compared with the other two species. Immunoblot analysis has also revealed that the relative abundance of the protein product of this gene, TSJ5, is lower in T. pseudospiralis muscle larvae. It has previously been shown that expression of tsmyd-1 is not developmentally regulated in T. spiralis (Connolly et al. 1996). In contrast, expression of this gene is slightly increased in the muscle larvae of T. pseudospiralis. Southern analysis of genomic DNA from the three Trichinella species shows that both genes are highly conserved.     

黄粉虫幼虫体壁硬化过程中酚氧化酶活性的变化

为研究酚氧化酶(PO)在昆虫蜕皮过程中的功能和作用, 采用微量测定法研究了黄粉虫Tenebrio molitor体壁硬化过程中血淋巴和表皮中的PO活性变化。结果表明：初蜕皮幼虫血淋巴中PO活性较高, 但随着体壁的不断黑化与硬化, 其活性呈现下降趋势, 在3～4 h内达到最低点, 而后PO活性逐渐上升, 7 h左右活性上升至最高, 并接近于正常幼虫的水平;在刚蜕完皮后的1 h内, 体壁中 PO活性基本无变化, 但随后即开始下降, 3 h左右降到最低点, 然后开始回升, 6~7 h左右恢复到正常水平, 并趋于稳定;以L-DOPA为底物, 通过双倒数曲线作图法求得黄粉虫血淋巴PO的Km＝1.176 mmol/L, 体壁PO的Km＝0.881 mmol/L, 表明体壁PO与底物L-DOPA的亲和力要高于血淋巴PO。研究表明两种来源的酚氧化酶均参与了黄粉虫幼虫的体壁硬化过程, 但在作用方式及与底物的亲和力方面存在差异。     

The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development

The cuticle of the free-living nematode Caenorhabditis elegans is a proteinaceous extracellular structure that is replaced at each of four postembryonic molts by the underlying hypodermis. The cuticles of the adult and three juvenile stages (L1, Dauer larva, L4) have been compared ultrastructurally and biochemically. Each cuticle has an annulated surface and comprises two main layers, an inner basal layer and an outer cortical layer. The adult cuticle has an additional clear layer which separates the basal and cortical layers and is traversed by regularly arranged columns of electron-dense material. The fine structure of the cortical layer is similar in cuticles from different stages while that of the basal layer is stage specific. Purified cuticles were obtained by sonication and treatment with sodium dodecyl sulfate (SDS) and their component proteins solubilized with a sulfhydryl reducing agent. The degree of cuticle solubility is stage specific and the insoluble structures for each cuticle were localized by electron microscopy. Analysis of ³⁵S-labeled soluble cuticle proteins by SDS-polyacrylamide gel electrophoresis yields unique banding patterns for each stage. Most proteins are of high molecular weight (100–200 K) and are restricted to particular stages. Sixteen of the nineteen major proteins characterized are specifically degraded by bacterial collagenase. The results indicate that the different molts are not reiterative, but require the integration of both unique and shared gene functions. The potential use of stage-specific cuticle differences to identify and characterize regulatory genes controlling cuticle-type switching during development is discussed.     

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.     

Feeding behavior of Trichinella as a leading factor of adaptation to the body of the mammals]

Studies were carried out of the permeability of the cuticle of Trichinella spiralis (Owen, 1835) for fluorochromes and histidine, the filling of the mid-gut and the amino-acids contents in it. The quantity of glicogene in different organs was estimated. A conclusion has been grown that a larva consumes food through the integuments of its body; intestinal Trichinella use within the first 20 hours of their development the reserves of glicogene and later consume food monomeres and oxygen from the host's mucous membrane through their integuments. The mode of feeding of Trichinella was formed during their evolution as an adaptation to parasitism on the mucous membrane of the host.     

Ultrastructure, development, and homology of insect embryonic cuticles

Ultrastructure and deposition of the cuticles secreted by embryos representing eight insect orders were examined by transmission and scanning electron microscopy. Embryos of the apterygote silverfish Thermobia domestica deposit two embryonic cuticles. Deposition of the first (EC1) is initiated at the beginning of appendage development when the intercalary segment and the neural groove are clearly visible. This cuticle lacks surface microsculpture and consists of an outer epicuticle and an underlying fibrous layer, thought to represent procuticle. At the time of dorsal closure, deposition of a second embryonic cuticle (EC2) begins; this bears sensilla and functions in the first instar larva. In representative embryos of seven pterygote orders (Ephemeroptera, Odonata, Plecoptera, Neuroptera, Coleoptera, Lepidoptera, and Mecoptera), three cuticles were found to be secreted. The first cuticle in pterygotes is homologous to EC1 of T. domestica, but consists solely of outer epicuticle. EC2, the "prolarval cuticle," bears a characteristic surface microsculpture in embryos of some species and egg-teeth and other hatching devices, and consists of outer and inner epicuticles and a more or less reduced procuticle. EC2 is reduced in the embryos of derived endopterygotes, where a procuticle is lacking and the inner epicuticle is reduced. After hatching, when EC2 is shed, the first instar larva is covered by a third embryonic cuticle (EC3), whose deposition was initiated while the insect was still within the egg. Presence of only two embryonic cuticles in cyclorrhaphous flies is due to the total loss of prolarval cuticle. Investigated exopterygote and endopterygote insects excluding flies thus deposit three embryonic cuticles, and their juveniles (exopterygote "nymphs"; endopterygote "larvae") seem to hatch at equivalent stages of development. Differences between the modes of cuticulogenesis in silverfish and pterygote embryos suggest that the apterygote first larval instar was embryonized and became a fully embryonic prolarva in pterygotes.     

Trichinella spiralis: modifications of the cuticle of the newborn larva during passage through the lung.

A scintigraphic method was developed to study the distribution of radioactivity after iv injection of 131I-labeled Trichinella spiralis newborn larvae into normal rats. It was found that the radioactivity was immediately retained in the lungs and thereafter slowly released, with a mean transit time in excess of 9 hr, as calculated by image analysis. At various times after iv injection of newborn larvae into normal mice, the lungs were removed and parasites were recovered and counted. Fifty to seventy percent of the larvae injected were recovered after 30 sec, between 10 and 30% after 1 min, and less than 4% at 15 min. These results indicate that during the very rapid passage of newborn larvae through the lungs, labeled components of the cuticle are detached and retained. It is suggested that the modifications produced in the cuticle of the newborn larva during its passage through the lung may increase its resistance to the nonspecific defense mechanisms of the host.     

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.     

The structure of the cuticle and sheath of the infective juvenile of Trichostrongylus colubriformis

The infective third-stage juvenile of Trichostrongylus colubriformis is surrounded by its own cuticle as well as the incompletely moulted cuticle of the second-stage juvenile, which is referred to as the sheath. The sheath comprises an outer epicuticle, an amorphous cortical zone, a fibrous basal zone and an inner electron-dense layer. The basal zone of the sheath consists of three layers of fibres; the fibres are parallel within each layer, but the fibre direction of the middle layer is at an angle to that of the inner and outer layers. The cuticle comprises a complex outer epicuticle, an amorphous cortical zone and a striated basal zone. The lateral alae of the cuticle and the sheath are aligned and overlie the lateral hypodermal cords. The lateral alae of the sheath consist of two wing-like expansions of the cortical zone with associated specializations of the inner electron-dense layer which form a groove. The cuticular lateral alae consist of two tube-like expansions of the cortical zone. The lateral alar complex of the cuticle and the sheath may maximise locomotory efficiency and prevent rotation of the juvenile within the sheath.     

Cuticle differentiation in the embryo of the amphipod crustacean Parhyale hawaiensis

The arthropod cuticle is a multilayered extracellular matrix produced by the epidermis during embryogenesis and moulting. Molecularly and histologically, cuticle differentiation has been extensively investigated in the embryo of the insect Drosophila melanogaster. To learn about the evolution of cuticle differentiation, we have studied the histology of cuticle differentiation during embryogenesis of the amphipod crustacean Parhyale hawaiensis, which had a common ancestor with Drosophila about 510 million years ago. The establishment of the layers of the Parhyale juvenile cuticle is largely governed by mechanisms observed in Drosophila, e.g. as in Drosophila, the synthesis and arrangement of chitin in the inner procuticle are separate processes. A major difference between the cuticle of Parhyale and Drosophila concerns the restructuring of the Parhyale dorsal epicuticle after deposition. In contrast to the uniform cuticle of the Drosophila larva, the Parhyale cuticle is subdivided into two regions, the ventral and the dorsal cuticles. Remarkably, the boundary between the ventral and dorsal cuticles is sharp suggesting active extracellular regionalisation. The present analysis of Parhyale cuticle differentiation should allow the characterisation of the cuticle-producing and -organising factors of Parhyale (by comparison with the branchiopod crustacean Daphnia pulex) in order to contribute to the elucidation of fundamental questions relevant to extracellular matrix organisation and differentiation. This work was supported by the German Research Foundation (DFG, grant number MO 1714/1-1).     

Trichinella spiralis: secreted antigen of the infective L1 larva localizes to the cytoplasm and nucleoplasm of infected host cells

Antibodies were elicited against a purified antigen with an apparent molecular weight of 43K. This antibody preparation also detected a second antigen consisting of a group of closely related components of 45-50K. These antigens are stage specific for the infective first stage larva of Trichinella spiralis and are among the repertoire of secreted antigens originating from the stichosome. Antibody raised against the 43K antigen reacted with the stichosome and cuticle of the mature larva and the cytoplasm and nucleoplasm, but not nucleolus, of all nuclei of infected host cells (Nurse cells) in sections of infected tissues. Studies on sections of synchronously infected muscle tissue revealed that antigen was present only within the worm on Day 7 of the infection. On Day 9 after infection, the stichosome and cuticular surface of the larva and the cytoplasm and nucleoplasm of each nucleus of the Nurse cell reacted with antibody. Nurse cell cytoplasmic and nuclear reactivity increased in intensity until Day 18 after infection. These results suggest that stichocyte-specific antigens are synthesized during the early phase of infection in the muscle, and that as the Nurse-parasite complex develops, some of the antigen is secreted into the milieu of the Nurse cell. The presence of antigen in the cytoplasm and nucleoplasm of the infected host cell is discussed in relation to Nurse cell formation and maintenance.     

Digestion of free-living nematodes fed to fish

Free-living nematodes— Panagrellus sp., Turbatrix aceti, Caenorhabditis elegans and C. briggsae—were each fed to the fish Danio sp. and the process of their digestion, in the fish alimentary canal, was studied by light and electron microscope.
Almost no identifiable nematodes were found in the fish gut when the digestion period was 3 h or more, except for buccal capsules of the four studied species, males' spicules of Panagrellus sp. and Turbatrix aceti and egg-capsules of the Caenorhabditis species. These structures could serve as indicators that the nematodes had been preyed on and digested by the fish.
Differences in the mode of digestion were noticed between the various species of nematodes studied, after a period of 0·5–1 h, in the fish gut. In Panagrellus sp. and T. aceti disintegration of the soft inner tissues occurred mostly at the anterior or posterior ends of the nematode's body, while in Caenorhabditis the majority of digested nematodes were affected at both ends or evenly along the entire body. Digestion seemed to be initiated mostly at the nematodes' body apertures: mouth, anus or cloaca, and vulva which could be due to a more vulnerable cuticle around those areas. Disintegration proceeded from the soft inner parts to the more resistant cuticle that was finally disintegrated. Of the three layers of cuticle the most resistant were the external cortex and the basal layers.     

大蜂螨对一些气味物质的趋性

狄斯瓦螨Varroa destructor Anderson & Trueman是意大利蜜蜂Apis mellifera Spinola的主要外寄生螨。雌成螨在幼虫巢房封盖前不久侵入幼虫巢房,并开始繁殖为害。从雌成螨在一个很短的时间内进入蜜蜂幼虫巢房,以及雄蜂幼虫巢房蜂螨的寄生率明显高于工蜂幼虫巢房的现象,表明蜜蜂幼虫体表一些信息素(semiochemicals)可能起着重要的引诱作用。作者对与大蜂螨相关的19种气味物质进行筛选,并对封盖前工蜂幼虫和雄蜂幼虫表皮挥发物进行气谱及气-质联谱测定。结果表明:雄蜂6龄幼虫对大蜂螨的引诱作用显著高于丁香水等10种气味物质。工蜂和雄蜂末龄幼虫体表挥发物的共有组份是9-二十三烯(C23H46),但它在雄蜂幼虫中所占的比例要明显高于工蜂幼虫。工蜂幼虫的特有主要组分是十八烷(C18H38)和9-甲基十九烷(C19H40);而雄蜂幼虫的特有主要组分是二十五烷(C25H52)和二十三烷(C23H48)。     

Structure of the stomach cuticle in adult and larvae of the spider crab Maja brachydactyla (Brachyura,Decapoda)

The stomach of decapods is a complex organ with specialized structures that are delimited by a cuticle. The morphology and ontogeny of the stomach are largely described, but few studies have focused on the morphology of its cuticle. This study examined the morphology of the stomach cuticle of cardiac sacs, gastric mill ossicles, cardio-pyloric valve and pyloric filters, and during various stages (zoea I and II, megalopa, first juvenile, and adult) of the common spider crab Maja brachydactyla using dissection, histology and transmission electron microscopy. The results show that cuticle morphology varies among structures (e.g., cardiac sacs, urocardiac ossicle, cardio-pyloric valve, pyloric filters), within a single structure (e.g., different sides of the urocardiac ossicle) and among different life stages. The cuticle during the larval stages is very thin and the different layers (epicuticle, exocuticle, and endocuticle) are infrequently distinguishable by histology. Major changes during larval development regarding cuticle morphology are observed after the molt to megalopa, including the increment in thickness in the gastric mill ossicles and cardio-pyloric valve, and the disappearance of the long thickened setae of the cardio-pyloric valve. The cuticle of all the stomach structures in the adults is thicker than in larval and juvenile stages. The cuticle varies in thickness, differential staining affinity and morphology of the cuticle layers. The structure–function relationship of the cuticle morphology is discussed.     

Trichinella spiralis: activation of complement by infective larvae, adults, and newborn larvae.

The ability of Trichinella spiralis to activate complement (C) has been addressed by several investigators. However, these investigators employed methods in which either detection of C fragments on the parasite surface or the adherence of leukocytes to the parasite was considered an indication of C activation. The present studies were undertaken to examine: (a) whether activation of C occurs via the classical and/or alternative pathway, (b) at which stage(s) of the parasite C activating capacity is acquired, and (c) what molecular entities of the epicuticle and/or cuticle are responsible for initiating C activation. Our studies indicate that T. spiralis activates C primarily via the alternative pathway (and weakly via the classical pathway) since incubation of parasites obtained from infected mice with either normal human serum (NHS) or Mg.EGTA-NHS, followed by incubation (1 hr, 37 degrees C) with antibody-sensitized sheep erythrocytes or rabbit erythrocytes, respectively, showed a time-and parasite number-dependent depletion of C. Although the three stages of T. spiralis, i.e., infective larvae, adults and newborn larvae, are capable of activating C, the newborn appears to be the most potent activator, especially when parasite number and size are taken into consideration. Further evidence of C activation is obtained from SDS-PAGE and Western blot analysis in which homogenates of parasites preincubated with NHS showed the presence of C3, C9, and C1q, whereas controls without serum were negative. Since isolated C1q was also capable of directly binding to the surface of adults and infective larvae, it is postulated that their cuticle and/or epicuticle may possess surface structures which serve as binding sites for C1q.(ABSTRACT TRUNCATED AT 250 WORDS)