Nematode Eggshell Research Articles

Characterization of the Serine Protease TlSP1 from Trichoderma longibrachiatum T6 and Its Function in the Control of Heterodera avenae in Wheat.

Serine protease is an extracellular protease secreted by biocontrol fungi that can effectively control nematode diseases by degrading nematode eggshells and enhancing plant resistance. Trichoderma longibrachiatum T6, an important biocontrol fungus, has been demonstrated to effectively parasitize and degrade Heterodera avenae cysts, eggs, and second-stage juveniles (J2s). However, the genes that encoding serine protease and their functions in T. longibrachiatum T6 have not been thoroughly investigated. In this study, we successfully cloned and sequenced the serine protease gene TlSP1 in T. longibrachiatum T6. Our results revealed that the expression level of the TlSP1 gene was induced and significantly increased in T. longibrachiatum T6 after inoculation with H. avenae cysts. The full-length sequence of the coding region (CDS) of TlSP1 gene was 1230 bp and encoded a protein consisting of 409 amino acids. Upon the transformation of the TlSP1 gene into Pichia pastoris X33, the purified recombinant TlSP1 protein exhibited optimal activity at a temperature of 50 °C and pH 8.0. Following 4-10-day of treatment with the purified recombinant TlSP1 protein, the eggshells and content were dissolved and exuded. The number of nematodes invading wheat roots was reduced by 38.43% in the group treated with both TlSP1 and eggs on one side (P1+N) compared to the control group, while the number of nematodes invading wheat roots was reduced by 30.4% in the TlSP1 and eggs two-sided treatment group (P1/N). Furthermore, both the P1+N and P1/N treatments significantly upregulated genes associated with defense enzymes (TaPAL, TaCAT, TaSOD, and TaPOD), genes involved in the lignin synthesis pathway (TaC4H, Ta4CL2, TaCAD1, and TaCAD12), and salicylic acid (SA)-responsive genes (TaNPR1, TaPR1, and TaPR2) and led to the high expression of jasmonic acid (JA)-responsive genes (TaPR4, TaOPR3, and TaAOS2). This study has highlighted the significant role of the TlSP1 gene in facilitating H. avenae eggshells' dissolution, preventing nematode invasion in the host plant, and boosting plant resistance in wheat.

In vitro assessment of the nematicidal potential of Streptomyces violascens strain AS2 against root-knot nematodes (Meloidogyne sp.)

Root-knot nematodes (RKNs, Meloidogyne spp.) present a major challenge to sustainable production. The detrimental environmental impacts and increasing resistance associated with synthetic nematicides have increased the need for biocontrol alternatives. Here, we report for the first time the nematicidal properties of Streptomyces violascens and its viability as a safe and effective solution against nematode infestation. In vitro assays showed that aqueous and ethyl acetate extracts of S. violascens significantly impeded Meloidogyne sp's reproductive and locomotive functions. The aqueous extract displayed remarkable efficacy, completely inhibiting egg hatching at a concentration of 600 ?g/mL, with an LC50 of 168.38 ?g/mL, and causing 99% immobility of second-stage juveniles (J2s) at 800 ?g/mL within 72 h. Conversely, the organic extract exhibited a 29.3% hatching rate (LC50 of 849.26 ?g/mL) and 79% immobility of J2s under comparable conditions. These findings point to the robust nematicidal potential of S. violascens extracts. Enzymatic profiles supported these results, with the selected isolate producing chitinase and protease enzymes capable of degrading nematode eggshells. Our results show that the S. violascens strain can be integrated into a sustainable nematode management practice as an important agent of RKN biocontrol.

Suppression of Meloidogyne incognita by co-application of chitin and Streptomyces nigrescens (AMV1033)

ABSTRACT Root-knot nematodes, Meloidogyne spp., are among the most polyphagous and ubiquitous plant-parasitic nematodes. Among other management strategies, organic soil amendments and biological control agents are proposed. In this study, potential synergistic benefits of co-application of chitin and Streptomyces nigrescens (AMV1033) against M. incognita were determined. Hypothetically, chitinolytic activity of these microbes deteriorates nematode eggshells reducing nematode numbers. Additional chitin could enhance actinobacteria activity for nematode suppression. In in vitro and greenhouse bioassays, suppressive capacities of M. incognita by chitin amendment at 50 mg/g of soil (chitin-low) and at 500 mg/g of soil (chitin-high; only tested in the greenhouse), Streptomyces nigrescens (AMV1033) and the co-application of AMV1033 with the two chitin rates were tested. After four weeks of in vitro egg incubation, the number of extractable second-stage juveniles (J2s) was significantly reduced after co-application of chitin-low plus AMV1033, and less so after sole application of AMV1033. In a similar 2-week in vitro assay with M. incognita egg masses, the numbers of J2s were similarly reduced after the same treatments. In a watermelon greenhouse bioassay with M. incognita, root infection was decreased after co-application of AMV1033 plus chitin-high or chitin-low compared to the non-treated control. After these treatments, plants grew similarly as in the control, whereas sole chitin-high addition led to severe plant damage. Some amelioration of phytotoxicity of chitin-high by the co-application of AMV1033 was found. The results of the study demonstrated the potential to enhance suppression of M. incognita by S. nigrescens by co-application of chitin.

An eggshell-localised annexin plays a key role in the coordination of the life cycle of a plant-parasitic nematode with its host.

Host-specific plant pathogens must coordinate their life cycles with the availability of a host plant. Although this is frequently achieved through a response to specific chemical cues derived from the host plant, little is known about the molecular basis of the response to such cues and how these are used to trigger activation of the life cycle. In host-specific plant-parasitic cyst nematodes, unhatched juvenile nematodes lie dormant in the eggshell until chemical cues from a suitable host plant are detected and the hatching process is initiated. The molecular mechanisms by which hatch is linked to the presence of these chemical cues is unknown. We have identified a novel annexin-like protein that is localised to the eggshell of the potato cyst nematode Globodera rostochiensis. This annexin is unique in having a short peptide insertion that structural modelling predicts is present in one of the calcium-binding sites of this protein. Host-induced gene silencing of the annexin impacts the ability of the nematode to regulate and control permeability of the eggshell. We show that in the presence of the chemicals that induce hatching annexin lipid binding capabilities change, providing the first molecular link between a nematode eggshell protein and host-derived cues. This work demonstrates how a protein from a large family has been recruited to play a critical role in the perception of the presence of a host and provides a new potential route for control of cyst nematodes that impact global food production.

Nematode eggshells: A new anatomical and terminological framework, with a critical review of relevant literature and suggested guidelines for the interpretation and reporting of eggshell imagery.

A literature review for a recent ultrastructural study of a trichinelloid eggshell revealed consistently occurring errors in the literature on nematode eggshell anatomy. Examples included nematodes of medical, veterinary, and agricultural importance in several orders. Previous researchers had warned of some of these errors decades ago, but a comprehensive solution was not offered until 2012 when a clarifying new anatomical and developmental interpretation of nematode eggshells was proposed by members of the Caenorhabditis elegans Research Community. However, their findings were explained using arcane acronyms and technical jargon intended for an audience of experimental molecular geneticists, and so their papers have rarely been cited outside the C. elegans community. Herein we (1) provide a critical review of nematode eggshell literature in which we correct errors and relabel imagery in important historical reports; (2) describe common reporting errors and their causes using language familiar to researchers having a basic understanding of microscopy and nematode eggs; (3) recommend a new hexalaminar anatomical and terminological framework for nematode eggshells based on the 2012 C. elegans framework; and (4) recommend new unambiguous terms appropriate for the embryonated/larvated eggs regularly encountered by practicing nematodologists to replace ambiguous or ontogenetically restricted terms in the 2012 C. elegans framework. We also (5) propose a resolution to conflicting claims made by the C. elegans team versus classical literature regarding Layer #3, (6) extend the C. elegans hexalaminar framework to include the polar plugs of trichinelloids, and (7) report new findings regarding trichinelloid eggshell structure.

Chitosan induces differential transcript usage of chitosanase 3 encoding gene (csn3) in the biocontrol fungus Pochonia chlamydosporia 123

BackgroundPochonia chlamydosporia is an endophytic fungus used for nematode biocontrol that employs its cellular and molecular machinery to degrade the nematode egg-shell. Chitosanases, among other enzymes, are involved in this process. In this study, we improve the genome sequence assembly of P. chlamydosporia 123, by utilizing long Pacific Biosciences (PacBio) sequence reads. Combining this improved genome assembly with previous RNA-seq data revealed alternative isoforms of a chitosanase in the presence of chitosan. This study could open new insights into understanding fungal resistance to chitosan and root-knot nematode (RKN) egg infection processes.ResultsThe P. chlamydosporia 123 genome sequence assembly has been updated using long-read PacBio sequencing and now includes 12,810 predicted protein-coding genes. Compared with the previous assembly based on short reads, there are 701 newly annotated genes, and 69 previous genes are now split. Eight of the new genes were differentially expressed in fungus interactions with Meloidogyne javanica eggs or chitosan.A survey of the RNA-seq data revealed alternative splicing in the csn3 gene that encodes a chitosanase, with four putative splicing variants: csn3_v1, csn3_v2, csn3_v3 and csn3_v4. When P. chlamydosporia is treated with 0.1 mg·mL− 1 chitosan for 4 days, csn3 is expressed 10-fold compared with untreated controls. Furthermore, the relative abundances of each of the four transcripts are different in chitosan treatment compared with controls. In controls, the abundances of each transcript are nil, 32, 55, and 12% for isoforms csn3_v1, csn3_v2, csn3_v3 and csn3_v4 respectively. Conversely, in chitosan-treated P. chlamydosporia, the abundances are respectively 80, 15%, 2—3%, 2—3%. Since isoform csn3_v1 is expressed with chitosan only, the putatively encoded enzyme is probably induced and likely important for chitosan degradation.ConclusionsAlternative splicing events have been discovered and described in the chitosanase 3 encoding gene from P. chlamydosporia 123. Gene csn3 takes part in RKN parasitism process and chitosan enhances its expression. The isoform csn3_v1 would be related to the degradation of this polymer in bulk form, while other isoforms may be related to the degradation of chitosan in the nematode egg-shell.

Chitosan modulates Pochonia chlamydosporia gene expression during nematode egg parasitism.

SummaryClimate change makes plant‐parasitic nematodes (PPN) an increasing threat to commercial crops. PPN can be managed sustainably by the biocontrol fungus Pochonia chlamydosporia (Pc). Chitosan generated from chitin deacetylation enhances PPN parasitism by Pc. In this work, we investigate the molecular mechanisms of Pc for chitosan resistance and root‐knot nematode (RKN) parasitism, using transcriptomics. Chitosan and RKN modify the expression of Pc genes, mainly those involved in oxidation–reduction processes. Both agents significantly modify the expression of genes associated to 113 GO terms and 180 Pc genes. Genes encoding putative glycoproteins (Pc adhesives) to nematode eggshell, as well as genes involved in redox, carbohydrate and lipid metabolism trigger the response to chitosan. We identify genes expressed in both the parasitic and endophytic phases of the Pc lifecycle; these include proteases, chitosanases and transcription factors. Using the Pathogen—Host Interaction database (PHI‐base), our previous RNA‐seq data and RT‐PCR of Pc colonizing banana we have investigated genes expressed both in the parasitic and endophytic phases of Pc lifecycle.

Identification of novel insect β-N-acetylhexosaminidase OfHex1 inhibitors based on virtual screening, biological evaluation, and molecular dynamics simulation

Chitin can be widely found in the fungal cell wall, nematode eggshells, and the exoskeleton of arthropods; however, it is completely absent from higher plants and mammals. The process of chitin degradation is essential for both growth and maturation of insects. Thus, inhibiting chitin degradation is a promising strategy for the control and management of pests. The chitinolytic β-N-acetyl-D-hexosaminidase OfHex1 of Ostrinia furnacalis (one of the most destructive pests) has been suggested as a potential target for the design of eco-friendly pesticides. This study presents the sequential virtual screening of the ZINC library with 8 million compounds, targeting OfHex1. After confirmation via enzyme inhibition experiments, compound 5 exhibited potential inhibitory activity against OfHex1 with a K i of 28.9 ± 0.5 μM and significant selectivity (IC50 > 100 μM against HsHexB and hOGA). Molecular dynamics simulations combined with binding free energy and free energy decomposition calculations were conducted to investigate the molecular basis underlying the potency of these inhibitors toward OfHex1. The present work provides useful information for the future rational design of novel and potent OfHex1 inhibitors Communicated by Ramaswamy H. Sarma

Purification and Characterization of Chitinase from the Nematode – Fungus Paecilomyces sp. P1

Purification and Characterization of Chitinase from the Nematode – Fungus Paecilomyces sp. P1

Electropermeabilization of nematode eggs for parasite deactivation.

The eggs of parasitic helminth worms are incredibly resilient - possessing the ability to survive changing environmental factors and exposure to chemical treatments - which has restricted the efficacy of wastewater sanitation. This research reports on the effectiveness of electroporation to permeabilize ova of Caenorhabditis elegans (C. elegans), a helminth surrogate, for parasite deactivation. This technique utilizes electric pulses to increase cell membrane permeability in its conventional application, but herein is used to open pores in nonparasitic nematode eggshells - the first report of such an application to the best knowledge of the authors. A parametric evaluation of electric field strength and total electroporation duration of eggs and worms in phosphate-buffered saline was performed using a 1 Hz pulse train of 0.01% duty cycle. The extent of pore formation was determined using a fluorescent label, propidium iodide, targeting C. elegans embryonic DNA. The results of this research demonstrate that electroporation increases eggshell permeability. This treatment, coupled with existing methods of electrochemical disinfection, could improve upon current attempts at the deactivation of helminth eggs. We discuss electroporation treatment conditions and likely modification of the lipid-rich permeability barrier within the eggshell strata.

Nematode Chitin and Application.

Plant-parasitic nematode infection is a global problem for agriculture and forestry. There is clearly a need for novel nematicides, because of the pitifully small repertoire of nematicides available and the effectiveness of losing or environmental prohibition of these nematicides. Chitin is the essential component of nematode eggshell and pharynx. The disturbance of chitin synthesis or hydrolysis led to nematode embryonic lethal, laying defective eggs or moulting failure. Thus, the key components in the chitin metabolic process are promising targets for anti-nematode agent's development. In this chapter, we focus on chitin and chitin synthase of nematodes, chitinases and their roles in nematode survival and application of chitin in nematode control.

Implications of molecular diversity of chitin and its derivatives.

Chitin is a long unbranched polysaccharide, made up of β-1,4-linked N-acetylglucosamine which forms crystalline fiber-like structure. It is present in the fungal cell walls, insect and crustacean cuticles, nematode eggshells, and protozoa cyst. We provide a critical appraisal on the chemical modifications of chitin and its derivatives in the context of their improved efficacy in medical applications without any side effect. Recent advancement in nanobiotechnology has helped to synthesize several chitin derivatives having significant biological applications. Here, we discuss the molecular diversity of chitin and its applications in enzyme immobilization, wound healing, packaging material, controlled drug release, biomedical imaging, gene therapy, agriculture, biosensor, and cosmetics. Also, we highlighted chitin and its derivatives as an antioxidant, antimicrobial agent, anticoagulant material, food additive, and hypocholesterolemic agent. We envisage that chitin and chitosan-based nanomaterials with their potential applications would augment nanobiotechnology and biomedical industries.

Macrocyclic lactones differ in interaction with recombinant P-glycoprotein 9 of the parasitic nematode Cylicocylus elongatus and ketoconazole in a yeast growth assay.

Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the presence of ivermectin, moxidectin, daunorubicin and ketoconazole but not selamectin. These results demonstrate direct effects of MLs on a recombinant nematode Pgp in an ML-specific manner.

Pharyngeal Polysaccharide Deacetylases Affect Development in the Nematode C. elegans and Deacetylate Chitin In Vitro

Chitin (β-1,4-linked-N-acetylglucosamine) provides structural integrity to the nematode eggshell and pharyngeal lining. Chitin is synthesized in nematodes, but not in plants and vertebrates, which are often hosts to parasitic roundworms; hence, the chitin metabolism pathway is considered a potential target for selective interventions. Polysaccharide deacetylases (PDAs), including those that convert chitin to chitosan, have been previously demonstrated in protists, fungi and insects. We show that genes encoding PDAs are distributed throughout the phylum Nematoda, with the two paralogs F48E3.8 and C54G7.3 found in C. elegans. We confirm that the genes are somatically expressed and show that RNAi knockdown of these genes retards C. elegans development. Additionally, we show that proteins from the nematode deacetylate chitin in vitro, we quantify the substrate available in vivo as targets of these enzymes, and we show that Eosin Y (which specifically stains chitosan in fungal cells walls) stains the C. elegans pharynx. Our results suggest that one function of PDAs in nematodes may be deacetylation of the chitinous pharyngeal lining.

Chitin synthesis inhibitors: old molecules and new developments

Abstract Chitin is the most abundant natural aminopolysaccharide and serves as a structural component of extracellular matrices. It is found in fungal septa, spores, and cell walls, and in arthropod cuticles and peritrophic matrices, squid pens, mollusk shells, nematode egg shells, and some protozoan cyst walls. As prokaryotes, plants and vertebrates including humans do not produce chitin, its synthesis is considered as an attractive target site for fungicides, insecticides, and acaricides. Although no chitin synthesis inhibitor has been developed into a therapeutic drug to treat fungal infections in humans, a larger number of compounds have been successfully launched worldwide to combat arthropod pests in agriculture and forestry. This review summarizes the latest advances on the mode of action of chitin synthesis inhibitors with a special focus on those molecules that act on a postcatalytic step of chitin synthesis.

Kinetic parameters of N-acetylglucosaminidase in adult femaleNippostrongylus brasiliensisby a quantitative colorimetric micromethod

Chitin, the 1,4-β-linked homopolymer of N-acetylglucosamine, is a component of Nematode eggshell. Chitin is hydrolyzed to its constituent monomer by the binary chitinase enzyme system. Chitinase (EC 3.2.1.14) hydrolyses first the polymer to N,N'- diacelylchitobiose which is then split to N-acetylglucosamine by N-acetylglucosaminidase (EC 3.2.1.30). N-acetylglucosaminidase is also able to release N-acetylglucosamine monomers directly from chitin. This study reports the identification of N-acetylglucosaminidase in adult female Nippostrongylus brasiliensis. A quantitative colorimetric micromethod has been developed to detect N-acetylglucosaminidase activity from N. brasiliensis using p-nitrophenyl-N-acetylglucosaminide as substrate. The assay conditions were optimized as follows: 50 μl of protein extract were incubated at 37 °C for 2 h in the presence of 50 μl of 19 mM substrate solution and 50 μl of 0.2 M potassium phosphate buffer pH 6.0. The reaction was stopped by the addition of 50 μl 0.1 N NaOH and the absorbance was measured on a Multiskan MS spectrophotometer at 405 nm within 30 min. after the reaction was stopped. The optimal pH was 6.0. Specific activity of protein extract was found at 94.4 ± 5.6 pmol/min/mg protein (n = 4). Vmax app. and Km app. for p- nitrophenyl-N-acetylglucosaminide were respectively 280 ± 11 pmol/min/mg protein (n = 3) and 1.8 ± 0.2 mM (n = 3). This test can now be proposed as a tool for screening anthelmintic compounds in the field of new inhibitors of chitin metabolism.

Chitin synthesis and degradation as targets for pesticide action

Various pesticides are being used to destabilize, perturb, or inhibit crucial biochemical and physiological targets related to metabolism, growth, development, nervous communication, or behavior in pestiferous organisms. Chitin is an eukaryotic extracellular aminosugar biopolymer, massively produced by most fungal systems and by invertebrates, notably arthropods. Being an integral supportive component in fungal cell wall, insect cuticle, and nematode egg shell, chitin has been considered as a selective target for pesticide action. Throughout the elaborate processes of chitin formation and deposition, only the polymerization events associated with the cell membrane compartment are so far available for chemical interference. Currently, the actinomycetes-derived nucleoside peptide fungicides such as the polyoxins and the insecticidal benzoylaryl ureas have reached commercial pesticide status. The polyoxins and other structurally-related antibiotics like nikkomycins are strong competitive inhibitors of the polymerizing enzyme chitin synthase. The exact biochemical lesion inflicted by the benzoylaryl ureas is still elusive, but a post-polymerization event, such as translocation of chitin chains across the cell membrane, is suggested. Hydrolytic degradation of the chitin polymer is essential for hyphal growth, branching, and septum formation in fungal systems as well as for the normal molting of arthropods. Recently, insect chitinase activity was strongly and specifically suppressed by allosamidin, an actimomycetes-derived metabolite. In part, the defense mechanism in plants against invasion of pathogens is associated with induced chitinases. Chitin, chitosan, and their oligomers are able to act as elicitors which induce enhanced levels of chitinases in various plants. Lectins which bind to N-acetyl-D-glucosamine strongly interfere with fungal and insect chitin synthases. Plant lectins with similar properties may be involved in plant-pathogen interaction inter alia by suppressing fungal invasion.

Trends and Perspectives in Parasitology 1

This small volume is a collection of six articles published in the 1980 volume of Parasitology. It is the first of a projected series of such volumes to contain the articles published under the title “Trends and Perspectives in Parasitology” as a regular part of the six individual issues of each volume of the journal. Authors of these articles are asked to present “a personal view about their own interest in a subject” in a way that will focus attention on advances in parasitology, call attention to problems in need of research, suggest how research in other areas of biology might relate to studies in parasitology, and provide summaries of recent discoveries and new theories. In this volume are “Recent developments in coccidian biology …” by John F. Ryley of Imperial Chemical Industries, “Invertebrate immunity” by Ann M. Lackie of University of Glasgow, “Some implications of a sexual cycle in Theileria” by A. D. Irvin and C. D. H. Boarer of the International Laboratory for Research on Animal Diseases at Nairobi, Kenya, “The contribution of Ascaris lumbricoides to malnutrition in children” by Lani S. Stephenson of Cornell University, “Nematode eggshell” by David Wharton of University College of Wales, and “Prospects for the biological control of plant-parasitic nematodes” by H. T. Tribe of University of Cambridge.

The tylenchid (Nematoda) egg shell: structure, composition and permeability

SummaryThe fine structure of egg shells of four different genera belonging to the order Tylenchida has been examined. The species examined were Meloidogyne javanica, Rotylenchulus reniformis, Tylenchulus semipenetrans and Pratylenchus minyus. They are all similar in their basic structure, being composed of vitelline membrane, chitin and lipid layers, but there is considerable variability in the thickness of these layers.We have retained the conventional nomenclature because of its convenience, but it is clear that these layers have a variety of chemical components. However, they do appear to contain the compounds from which they take their name. Thus chitin occurs in the chitin layer, and lipid in the lipid layer. The latter is removed by the technique used in isolating the shell from the egg. Chemical analysis of the hydrolysis products of these shells has revealed a high (35 %) proline content which appears to be a characteristic of those nematode egg shells which have been examined so far. These analyses and treatment with enzymes indicate that the chitin layer is a chitin–protein complex.Experiments on the permeability of eggs of M. javanica at different temperatures indicate that changes in permeability are not due to the melting of a single lipid with a distinct melting point as had been thought in the past. We have found that Arrhenius activation energies calculated from the two slopes of an Arrhenius plot were 17·8 kcal/mol and 43·0 kcal/mol respectively, the transition from one to the other taking place at 62°C. We think that these changes are due to changes in the properties of lipoprotein membranes in the lipid layer. These membranes appear to be of paramount importance in controlling the permeability of the nematode egg shell.

The tylenchid (Nematoda) egg shell: structure, composition and permeability

The fine structure of egg shells of four different genera belonging to the order Tylenchida has been examined. The species examined were Meloido-gyne javanica, Rotylenchulus reniformis, Tylenchulus semipenetrans and Pratylenchus minyus. They are all similar in their basic structure, being composed of vitelline membrane, chitin and lipid layers, but there is considerable variability in the thickness of these layers.We have retained the conventional nomenclature because of its convenience, but it is clear that these layers have a variety of chemical components. However, they do appear to contain the compounds from which they take their name. Thus chitin occurs in the chitin layer, and lipid in the lipid layer. The latter is removed by the technique used in isolating the shell from the egg. Chemical analysis of the hydrolysis products of these shells has revealed a high (35 %) proline content which appears to be a characteristic of those nematode egg shells which have been examined so far. These analyses and treatment with enzymes indicate that the chitin layer is a chitin-protein complex.Experiments on the permeability of eggs of M. javanica at different temperatures indicate that changes in permeability are not due to the melting of a single lipid with a distinct melting point as had been thought in the past. We have found that Arrhenius activation energies calculated from the two slopes of an Arrhenius plot were 17·8 kcal/mol and 43·0 kcal/mol respectively, the transition from one to the other taking place at 62°C. We think that these changes are due to changes in the properties of lipoprotein membranes in the lipid layer. These membranes appear to be of paramount importance in controlling the permeability of the nematode egg shell.