Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

5.1 Introduction

The nematode cuticle is an extracellular coating that is secreted by the hypodermis and has a variety of important roles in nematode biology. The cuticle maintains the body shape, provides a strong layer against which muscles can act during locomotion and protects the nematode from the external environment. The cuticle is overlaid with a fuzzy coating material – the surface coat (SC). Substances on the surface of the cuticle are in direct contact with the outside world and therefore, also have important roles in terms of the interactions of nematodes during the various life-stages with their environment, including their host and other microorganism. Nematodes eggs generally are the most resistant nematode life-stage and many have a remarkable capacity for survival in stressful environments; therefore, the eggshell is a most important barrier at this stage.

5.2 Eggshell

Most nematode eggs are morphologically very similar and are similar in size (average of 53–133 μm in length and 17–79 μm in width) irrespective of the size of the adult. They are ellipsoidal in shape with a transparent shell (Bird and Bird 1991). The eggshell in nematodes is formed after fertilization and usually contains four layers. The outermost layer (uterine) consists of material that is secreted by the uterine epithelial cells and can be absent in some nematodes. In plant-parasitic nematodes the composition of the uterine layer resembles the gelatinous matrix (gm) secreted from various organs (Mackintosh 1960; Maggenti 1962; Bird and Rogers 1965). The next layer, vitelline, originates from the vitelline membrane (oolemma) which is formed after fertilization of the oocyte. In many eggs this retains a unit membrane-like structure and forms the outer layer of the eggshell. Carbohydrate residues have been detected on the surfaces of the eggs (Rao et al. 1988; Taylor et al. 1986; Spiegel and McClure 1991).

The underlying chitinous layer is made-up of chitin microfibrils embedded in a protein coat (Wharton 1983). Chitin is formed from the polymerization of N-acetyl glucosamine which is itself synthesized from glycogen (Preston and Jenkins 1985). The chitinous layer is often the thickest layer and provides structural rigidity to the eggshell. Protein is frequently present in this layer; it has been estimated that the eggshell of Globodera rostochiensis contains 59% protein and 9% chitin while that of Meloidogyne javanica contains 50% protein and 30% chitin (Clarke et al. 1967; Bird and McClure 1976). Perry and Trett (1986) suggest the sub-division of the chitinous layers of cyst nematodes into distinctive outer and inner components due to differences in their chemical composition on the basis of ultrastructural studies that showed that the external outer layer is thin and amorphous followed by a predominantly tetra-pentalaminate inner layer. Chitin is synthesised by chitin synthase and the gene encoding this enzyme has been shown to be expressed in the egg-producing adult stages and fertilized eggs of various nematodes including Caenorhabditis elegans, M. artiellia, Ascaris suum, Brugia malayi and Dirofilaria immitis (Veronico et al. 2001; Harris and Fuhrman 2002; Dubinsky et al. 1986a, b). These observations are consistent with a role for this enzyme in producing chitin for the eggshell. This has been confirmed with RNAi-mediated ablation of the C. elegans chitin synthase gene function which resulted in sterile hermaphodites that lay defective eggs (Hanazawa et al. 2001). RNAi of a chitin synthase gene expressed in the eggs of root-knot nematode M. artiella was achieved by soaking intact eggs within their gelatinous matrix in a solution containing dsRNA and led to a reduction in stainable chitin in eggshells and a delay in hatching of juveniles from treated eggs (Fanelli et al. 2005). So far, the eggshell is the only structure in nematodes in which the presence of chitin has been conclusively demonstrated (Bird and Bird 1991). A chitin synthase gene has been shown to be expressed in the cells that form the pharynx of C. elegans at a time that precedes a moult. It has also been suggested that this gene might be involved in the synthesis of the feeding apparatus which is replaced during each moult (Veronico et al. 2001). A secreted chitinase has been identified in the perivitelline fluid surrounding the infective larva of A. suum prior to hatching indicating that this enzyme might be responsible for the digestion of the eggshell during hatching of this nematode (Geng et al. 2002). The potential of using bacterial and fungal chitinases and chitin synthase inhibitors to control root-knot nematodes have been demonstrated (Spiegel and Chet 1985; Jung et al. 2002; van Nguyen et al. 2007).

The most internal layer is the lipid layer which is responsible for the extreme impermeability of the nematode eggshell. It is formed in the middle region of the uterus in M. javanica (Bird and McClure 1976) where proline-containing proteins are incorporated into both lipid and chitinous layers and the synthesis of the eggshell is then completed (Bird and Bird 1991). Nematode eggs are permeable to chemicals prior to the formation of the lipid layer and when this layer is broken down before hatching. Egg permeability changes are central to the hatching of cyst nematodes (Perry and Clarke 1981; Bird and McClure 1976) and work by Twomey et al. (2000) corroborated these studies and showed that the biological nematicide DiTera (fermentation product of killed Myrothecium verrucaria) induced a significant inhibition of hatch of G. rostochiensis by possibly preventing eggshell permeability change by competitively blocking the Ca2+ binding sites on the eggshell. DiTera showed a lack of inhibition of hatch on M. incognita and therefore do not affect second-stage juveniles (J2) directly during the hatch process or inhibit the action of enzymes. It has been suggested that the breakdown of the lipid layer is enzymatic and secretions have been shown to emanate from various structures of J2s of M. incognita and M. javanica, including the amphids, secretory-excretory pore and from around the mouth while still inside the egg. An increase in size of the nucleolus of the dorsal pharyngeal glands of G. rostochiensis J2 inside the eggs that were stimulated with root diffusate was demonstrated indicating that the oesophageal glands are activated and leucine amonipeptidase activity was identified in the supernatant of eggs of Heterodera glycines (Atkinson et al. 1987; Premachandran et al. 1988; Bird and Bird 1991). Perry and Trett (1986) observed that the eggs within H. glycines cysts internally contaminated with fungus, had no inner lipid layer and it was suggested that fungal lipases might have contributed to disrupt both inner and outer lipid layers.

As both the lipid layer and the juvenile epicuticle are derived directly from the secondary vitelline membrane of the embryo, both probably share the same protein moieties. Cross-reactivity of polyclonal and monoclonal antibodies (Fig. 5.1) produced against M. incognita J2 with eggshells support this suggestion (R. Curtis unpubl; Sharon et al. 2002, 2009a).

Fig. 5.1
figure 1_5

Monoclonal antibody raised to second-stage juveniles of Meloidogyne incognita shows reactivity with the egg-shell

Full size image

Cysteine proteinases are involved in a variety of biological functions and have been implicated in tissue remodeling in free-living and animal-parasitic nematodes. Cathepsin L has been shown to be present in the eggshell surrounding the embryos of C. elegans, Onchocerca volvulus and B. pahangi and is possibly involved in eggshell remodeling by processing of nutrients responsible for the synthesis and/or degradation of the eggshell in these nematodes (Hashmi et al. 2002; Guiliano et al. 2004). Transmission electron microscopy showed that the eggshells and cuticles layers of C. elegans and of the parasitic stages of Haemonchus contortus have an ABC transporter, P-glycoprotein. This might function as a membrane efflux ‘pump’ and may play a major role in the transport of antihelmintic drugs in parasitic nematodes of ruminants (Riou et al. 2005).

5.3 Cuticle Structure and Function

A detailed review describing the structure of the cuticle and contrasting the cuticular features of a wide range of nematode species has been published previously (Lee 2002) and only the salient points are summarized here. The cuticle of most nematodes can be subdivided into three main zones covered with an epicuticle. In addition, the cuticle is overlaid with a surface coat composed of mucins and other proteins (Fig. 5.2). The innermost cuticle zone, the basal zone, often has a striated appearance when sections through the cuticle are viewed under an electron microscope and is thought to be composed largely of collagens (see below), arranged in oriented layers of fibrils. The median zone varies in appearance in electron micrographs but often appears less electron-dense than the basal and cortical zones. It may appear to contain vacuoles and struts and has often been described as fluid-filled or gel-like. The appearance of this layer sometimes varies between nematode life-stages. For example, the median zone of unhatched second-stage juveniles (J2s) of G. rostochiensis appears to contain an electron dense material which is lost on hatching (Jones et al. 1993). It has been suggested that this zone is composed of a combination of collagens and soluble proteins, possibly destined for the nematode surface (Blaxter and Robertson 1998). The cortical zone varies enormously in thickness and may have regions that have different electron densities when viewed under an electron microscope. The cortical zone is composed of collagens and extremely insoluble cuticulins. The cuticle of many nematodes is annulated and may also carry a wide range of projections including hooks, bristles and papillae. Many of these projections are associated with mechanoreceptive neurons (Jones 2002). Lateral projections (alae) may also be present that extend along the length of the nematode body.

Fig. 5.2
figure 2_5

Transmission electron micrograph of a longitudal section of Meloidogyne javanica second-stage juvenile (X 40,000), showing surface coat (sc), epicuticle (e), cortical zone (c), median zone (m), basal zone (b) and hypodermis (h)

Full size image

The best characterised, and most abundant, of the cuticle proteins are the collagens. Collagens from a wide range of animal species have a conserved and characteristic triple helical tertiary structure. This region is conserved to such an extent that the first nematode collagen gene was identified using a probe derived from a vertebrate sequence (Kramer et al. 1982). The triple helical region of the protein is formed from large Gly-X-Y repeat regions in which every third amino acid is a glycine residue and where Y is frequently proline, which forces the helical turn. Collagen genes are present in extremely large gene families in a wide range of nematode species; over 170 genes are present in C. elegans (Page and Johnstone 2007) and 122 are predicted from the genome of M. incognita (Abad et al. 2008). This large suite of collagen proteins allows variation in the collagens present in different life-stages and thus variation in the structural and physical properties of the cuticle of different life-stages (Koltai et al. 1997; Liu et al. 2001). A number of conserved cysteine residues are present in the non Gly-X-Y, N- and C-terminal regions and within short stretches of amino acids that interrupt the Gly-X-Y regions. The number and spacing of these residues, along with other conserved residues, has been used to subdivide the C. elegans collagens into subfamilies (Johnstone 1994).The other major protein component of the cuticle is formed by cuticulins. Cuticulins contain cysteine rich regions and are extensively cross linked, particularly in the outer cortical layers and in the dauer cuticle, through tyrosine (Page and Johnstone 2007). It has been suggested on the basis of expression patterns of various cuticulin genes in C. elegans that cuticulins are also important in formation of lateral alae and other cuticular annulations and ridges (Sapio et al. 2005). Studies on the expression patterns during the development of M. artiellia indicate that there is a burst of expression of the cut-1 gene during moulting. Then, the expression rate is reduced in the infective juveniles, which migrate in the soil. In the sedentary females, in contrast, no expression is detected, while in the males which move freely through the soil, the gene is expressed and the transcript fully processed. These data strongly suggest that the gene is developmentally regulated. It is proposed that the production of cuticlin plays an important role in determining the mechanical properties of the cuticle (de Giorgi et al. 1997).

The cuticle itself is synthesized during a series of moults in the hypodermis, a syncytial cell layer immediately below the cuticle. The moulting process can be subdivided into several distinct phases and has been analysed in detail in C. elegans. Once the new cuticle has been resynthesised the nematode shows a decrease in activity and feeding (lethargus) followed by separation of the old cuticle from the new structure (apolysis). The nematode then moves rapidly in order to loosen the old cuticle which is shed during ecdysis. The new cuticle is synthesised at the end of each juvenile stage and the timing of the expression patterns of collagen genes during this process has been examined in detail (Johnstone and Barry 1996). Cuticle collagen genes are expressed in distinct waves and in an order that is repeated at each moult, with some genes always expressed early in the moulting cycle and others at a later stage. It is possible that these waves of gene expression exist to ensure that the collagen components required for each cuticle zone or substructure are expressed simultaneously, with different zones synthesised at different times in the moulting cycle.

5.4 The Surface Coat and Excreted/Secreted Antigens

The surface coat (SC) of nematodes contains various proteins, carbohydrates and lipids, as individual components or as glycoproteins/mucins, glycolipids, or lipoproteins. Studies have used antisera, lectins, biotin and neoglycoproteins to characterize and localize surface components and to learn about its nature. Binding of lectins indicated the presence of specific carbohydrates on the surface and amphids of different life-stages of plant-parasitic nematodes. Carbohydrate-recognition domains (CRDs) were demonstrated on the surface of plant-parasitic nematodes, suggesting the presence of lectins on the surface of the nematodes (Spiegel and McClure 1995; Sharon and Spiegel 1996). Carbohydrates or CRDs on nematodes surface and/or secretions might be involved in nematode-plant and nematode-microorganisms interactions (Spiegel and McClure 1995; Koltai et al. 2002). Surface coat proteins and glycoproteins from pre-parasitic J2s and adult females of Meloidogyne were labeled, extracted and partially characterized (Robinson et al. 1989; Lin and McClure 1996; Spiegel et al. 1997). Specific antibodies were raised against surface antigens and excretory-secretory (E-S) products of plant-parasitic nematodes and have been used to localize and characterize antigens on SC regions on the nematodes (Fig. 5.3), on amphids and secretory-excretory products. Antibodies served as a tool for studying interactions with plant hosts (e.g. Curtis 1996; Gravato-Nobre and Evans 1998; Lopez de Mendoza et al. 1999) and microorganisms (e.g. Spiegel et al. 1996; Davies and Danks 1992; Sharon et al. 2009a).

Fig. 5.3
figure 3_5

Scaning electron micrograph of immunogold-labeled surface of Meloidogyne javanica second-stage juvenile; labeling was visualized by silver enhancement reaction, bar  =  1 μm

Full size image

One of the most interesting features of the nematode SC is its labile and dynamic nature and there is a continuous turn-over of surface-associated antigens that involves shedding and replacing of the antigens (Blaxter and Robertson 1998). Studies have shown that surface coat of plant-parasitic nematodes is shed (Fig. 5.4) both in vitro (Bird et al. 1988; Lin and McClure 1996; Spiegel et al. 1997; Robertson et al. 2000) and in the host (Curtis 1996; Gravato-Nobre et al. 1999; Sharon et al. 2002). Unlike other cuticle proteins surface coat molecules are readily secreted/released into the environment and this was demonstrated for the pre-parasitic juveniles of Meloidogyne spp., as the surface coat proteins of M. incognita were released when the J2 were incubated in water (Lin and McClure 1996). Also, when J2 of M. javanica were treated with detergents there was a reduction in the binding of red blood cells to the nematode surface but the binding properties were completely renewed after 24 h at 25°C, but not at 4°C, indicating that the sloughing-off and replacement of the nematode’s surface coat is an active event. This phenomenon was visualized also with detergent-extracted SC proteins, using gel-electrophoresis (Spiegel et al. 1997).

Fig. 5.4
figure 4_5

Antiserum (anti-rGR-TpX) binds to the surface of Globodera rostochiensis and to material shed in great quantities from the parasite surface

Full size image

Interestingly, binding of antibodies to M. javanica and G. pallida J2s affected their normal movement pattern on agar plates, regardless the binding pattern (i.e. SC regions, head, amphids, E-S products); this was reversed within several hours, probably due to renewal of the SC. However, continuous binding of antibodies did not enable the recovery and nematodes stopped moving after 2–3 days, subsequently inhibiting plants inoculation by the nematodes (Fioretti et al. 2002; Sharon et al. 2002). Some antibodies caused even more drastic lethal effects after less than one day of contact with antibodies (Sharon et al. 2009a).

There is evidence to suggest that some components of the surface coat are synthesised in the hypodermis (e.g. Jones et al. 2004). Further evidence to support this idea comes from the fact that antibodies reactive with the SC of M. incognita and M. javanica also show reactivity with the hypodermis (Sharon et al. 2002). The origin of surface-associated antigens on nematodes may differ for various antigens and is still not clear in most cases (Spiegel and McClure 1995; Blaxter and Robertson 1998). These non-structural proteins can originate from gland cells such as excretory cells, pharyngeal glands, amphids and phasmids as well as from the hypodermis (Blaxter and Robertson 1998; Page and Johnstone 2007).

In Meloidogyne spp., as well as in other species like Rotylenchulus, Tylenchulus and Heterodera, eggs are enveloped with a gelatinous matrix (gm) that contains glycoproteins (Sharon and Spiegel 1993; Agudelo et al. 2004). The eggs and the emerging J2s are exposed to these components and some of it probably attach to the surface of the J2s and affect their interactions with the environment. Labelling of M. javanica egg mass-originated eggs and J2s with the monoclonal antibody (MAb), MISC, presented different patterns and was more intense than on gm-free ones (from hypochlorite-treated eggs); labellings were inhibited by fucose (Sharon et al. 2009a). This MAb had also labelled M. incognita J2s SC, the gm and the rectal glands, where the gm originates, which suggests that there are mutual epitopes in the gm and SC (Hu et al. 2000). Actually, when gm-originated J2s are used, some of the surface components can be of gm origin. The gm plays a key role in attachment and parasitism of microorganisms, such as Trichoderma (see Sect. 5.5.3 and Chap. 8) on Meloidogyne J2s and eggs. The role of gm-originated components on the surface of nematodes and their fate during SC turn-over should still be further investigated.

5.4.1 Role of the Surface Coat in Host-Nematode Interactions

Nematodes can rapidly change their surface composition in response to environmental signals, which may enable animal-parasitic nematodes to escape host immune responses and free-living nematodes to escape pathogenic infections (Grenache et al. 1996; Olsen et al. 2007; Proudfoot et al. 1993). A growing body of evidence indicates that some molecules present at the nematode surface of parasitic nematodes serve as an active defense against host responses and are therefore important for nematode survival (Blaxter et al. 1992; Jones et al. 2004; Olsen et al. 2007).

5.4.2 Changes in the Surface Coat in Response to Host Derived Signal

The idea that nematodes switch surface composition in response to environmental signals has been based on rapid changes in surface lipophilicity (Modha et al. 1995; Proudfoot et al. 1993) or surface antigenicity (Philipp and Rumjaneck 1984; Politz and Philipp 1992) that occur during parasitic nematode infections. Therefore, the surface composition can change within a single stage of the life-cycle, during the entry of parasitic nematodes into a new host or host tissue; these surface changes are different from the moulting process as they occur more rapidly (Modha et al. 1995; Proudfoot et al. 1993). Some surface proteic epitopes present in the pre-parasitic Meloidogyne J2s were shown to be abundantly shed during root invasion (De Mendoza et al. 2002; Sharon et al. 2002; Curtis 2007b). In vitro, plant signals present in root exudates trigger a rapid modification of the surface cuticle of M. incognita and G. rostochiensis (de Mendoza et al. 2000; Akhkha et al. 2002). Increase in the surface lipophilicity was also induced by phytohormones, in particular indole-acetic acid (IAA) and kinetin in M. incognita but not G. rostochiensis (Akhkha et al. 2002, 2004). It has been suggested that the ability of M. incognita to respond to a general plant compound as opposed to a specific root diffusate is related to the broad host-range of this species. The increase in the lipophilicty of the SC of M. incognita J2, induced by plant signals might allow this nematode to adapt to and survive plant defence processes. By contrast, more specific host cues from root exudates of Solanaceous plants increase the lipophilicity of the surface cuticle of infective J2 of Globodera species (Akhkha et al. 2002; Curtis 2007a).

The composition of the nematode surface is also important for the survival of free-living nematodes (see chapter on C. elegans for more details) as specific surface-altered mutants of C. elegans are resistant to pathogen infections (Mendoza de Gives et al. 1999; Gravato-Nobre et al. 2005; Hodgkin et al. 2000; Ewbank 2002). C. elegans responds to environmental conditions by modifying its surface, a process similar to surface switching in parasitic nematodes and these environmental signals are detected by the nematode’s chemosensory organs (Grenache et al. 1996; Olsen et al. 2007). These studies suggest that surface switching in plant-parasites might also rely on chemosensation and it can be speculated that free-living and parasitic nematodes use their sensilla to detect environmental signals that lead to changes in the surface composition. Olsen et al. (2007) hypothesized that, like dauer formation, surface antigen switching in C. elegans is guided by chemical signals sensed by the amphid neurons. This behavioral adaptation may protect the nematodes from biological attack.

5.4.3 Protection of the Plant Nematode from Host Defence Responses

Endoparasitic nematodes spend a significant portion of their life-cycles within their hosts and are therefore exposed to host defence responses. All endoparasites will aim to minimise the effects of defence responses. In addition, many plant-parasitic nematodes, including cyst and root-knot nematodes, are biotrophic and it is particularly important for these nematodes to mask their presence from their host, or suppress any defence response that is mounted, as detection of the pathogen will lead to destruction of the feeding site and death of the pathogen. The nematode surface is in intimate contact with the host. Materials present on the surface of the nematode are therefore targets for detection by the plant and substances can be secreted to the surface that mask the presence of the nematode from the plant, suppress host defences or modulate the effects of any defence response that is mounted.

The ability of nematodes to continuously shed and renew the SC may help the nematode avoid recognition in the host-plant. It has been suggested that shedding of SC components may cause a defence response to be mounted against a region that the nematode has vacated, protecting the parasite from early host defences. However, this process of exudation continues once the nematode is established at the feeding site (e.g. Jones et al. 1993; de Mendoza et al. 2002; Curtis 2007b) as fibrillar exudates and secretion vesicles exuded as globules of different sizes (Fig. 5.5) have been observed on the cuticle surface of feeding nematodes, suggesting that other processes are also used by the nematode to mask its presence from the host. An abundant cuticular secretion was shown to envelop the adult females and the giant cells and it might have a role in protecting the nematodes from harmful root compounds (de Mendoza et al. 2002; Curtis 2007b).

Fig. 5.5
figure 5_5

Monoclonal antibody (IACR-CCNj.2a.15) shows reactivity with globules of different sizes exuded from the cuticle of adult female of Meloidogyne incognita

Full size image

Antibodies raised against nematode surface components cross react with host tissues; therefore it has been suggested that the SC may mimic host tissues in order to prevent a host reaction (Bird and Wilson 1994; Curtis 1996). Similarly, it has also been suggested that the presence of carbohydrate residues in glycoproteins of plant-parasitic nematodes SC, and therefore the binding of lectins to the nematode surface, is masking other components which may be recognised by the host. Numerous studies have shown that a range of lectins can bind to the surface of many plant-parasitic nematodes (reviewed by Lee 2002) but this idea has yet to be tested in vitro. The nematode surface may also play a role in suppressing or modulating the effects of host defences and striking parallels have been found between the proteins used by plant- and animal-parasites to suppress defence signalling. For plant-parasites these processes have been most thoroughly investigated in cyst nematodes. A range of enzymes that may neutralise active oxygen species produced by the host have been described on the nematode surface. A peroxiredoxin (thioredoxin peroxidase) secreted in great quantities from the surface of the potato cyst nematode G. rostochiensis was found to catalyse the breakdown of hydrogen peroxide but, unlike most proteins of this type, did not metabolise larger lipid hydroperoxides (Robertson et al. 2000). It is known that hydrogen peroxide is produced as part of the host response to cyst nematode infection (Waetzig et al. 1999) and it is therefore possible that the nematode peroxiredoxin has become modified to allow it to target host-derived hydrogen peroxide efficiently (Robertson et al. 2000). Another peroxidase, glutathione peroxidase, was also found on the surface of this nematode and this protein was shown to metabolise a wider range of hydroperoxides, including larger hydroperoxides (Jones et al. 2004). Like animal-parasites, plant-parasitic nematodes therefore have a range of peroxidases within their surface secretions that can metabolise a wide range of active oxygen species produced as part of the host defence response.

In contrast to root-knot nematodes which migrate between root cells, minimising disruption to host tissues, cyst nematodes migrate destructively through root cells to their chosen feeding site. This process and the damage caused to plant tissues are likely to provoke defence pathways that provide protection against herbivores. These defence pathways lead to production of antifeedants and are triggered by the plant hormone jasmonic acid (Kunkel and Brooks 2002). Jasmonic acid is produced via the octadecanoid pathway and peroxidation of linoleic and linolenic acid by lipoxygenase is an early step in this process. A surface localised retinol and fatty acid binding protein (GpFAR1) has been described from G. pallida which binds both linoleic and linolenic acids and it has been shown that FAR1 inhibits lipoxygenase mediated peroxidation of these fatty acids, presumably due to sequestration of the ligands (Prior et al. 2001). Cyst nematodes, therefore, contain proteins at their surface that can inhibit jasmonate signalling pathways and which can also metabolise active oxygen moieties produced as part of any defence response that is mounted. The genome sequencing projects for M. incognita and M. hapla (Abad et al. 2008; Opperman et al. 2008) show that similar proteins are also present in root-knot nematodes but functional studies examining localisation of these proteins and biochemical activities remain to be carried out.

5.4.4 Cross Reactivity of Surface Coats of Animal- and Plant-Parasitic Nematodes

Monoclonal antibodies (MAb) produced to excretory-secretory (E-S) products of plant-parasitic nematodes were shown to cross-react with E–S products and the SC of the animal-parasites Trichinella spiralis and Haemonchus contortus (de Mendoza et al. 1999). Glycosylated peptides have been reported to be present in abundance on the SC and in E–S products of several parasitic nematodes (Robertson et al. 1989; Schallig et al. 1994, 1995) and, in fact, most of the MAbs tested recognized carbohydrate epitopes. However, 2 out of 7 MAbs recognized proteic epitopes present in the SC and oral exudate of M. incognita, T. spiralis and H. contortus. One of these cross-reactive antigens was detected in the exudate present during ecdysis of H. contortus (de Mendoza et al. 1999). Whether this antigen plays any role in mediating ecdysis remains to be determined.

A cross reactive cuticular proteic epitope was identified in T. spiralis and M. incognita and this antigen might play a role in the interaction of M. incognita with its host- plant and in the interaction of T. spirallis inside the nurse cell as they are secreted in planta and in vivo (Lopez-Arellano and Curtis 2002). In planta this proteic antigen was immunolocalized surrounding the cuticle of the adult females of M. incognita and in the plasma membrane of root cells of Arabidopsis thaliana, close to the feeding cell formed during infection with M. incognita. In vivo this antigen was localised on the nematode surface and as secreted droplets close to the collagen capsule surrounding the nematode nurse cell (Curtis 1996; De Mendoza et al. 2002). These common antigens might represent immunodominant epitopes which are secreted inside the hosts and may perform related functions in these parasitic nematodes.

5.4.5 The Role of the Surface Coat in Immune Evasion by Animal-Parasitic Nematodes

Animal-parasitic nematodes have evolved a mutiplicity of evasive strategies to survive in immunologically competent host. The parasite’s ability to exist for long periods of time in their host, has been attributed to a rapid turnover of their cuticle surface, shedding of surface antigens and membrane rigidity, which are likely to render the parasite less susceptible to immune attack (Simpson et al. 1984; Kusel and Gordon 1989). The mechanisms underlying surface antigen switching mechanisms are presently unknown but nematodes can alter their SC protein compositions at the moults between developmental stages or in response to host/environmental changes. As a rapid change in the surface lipophilicity of various animal-parasitic nematodes occurs during their transition from pre- to post-parasitic forms, these surface alterations may enable parasitic nematodes to evade host immune defenses during the course of infection (Jungery et al. 1983). Intracellular signalling and second messenger pathways involving cyclic nucleotides, calcium and intracellular alkalinisation participate in bringing about these surface changes (Modha et al. 1995, 1997).

Disguise of the parasite cuticle surface with the acquisition of host derived antigens (Smithers and Doenhoff 1982) and the action of parasite surface proteases that can cleave the Fc region of Immunoglobulins (Auriault et al. 1981) are some other mechanisms that may also help parasites to evade the host immune response by inhibiting important cellular functions.

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode SC, as this nematode is able to shed the entire SC in response to binding antibodies or eosinophils, thus permitting parasites to physically escape immune attack (Maizels and Loukas 2001a). The major constituent of the SC of this nematode is the O-linked TES-120 (Toxocara excretory/secretory) glycoproteins series, which has a typical mucin domain and may explain a generally nonadhesive property of this parasite. Membrane associated mucins are closely concerned with the adhesion status of cells through electrostatic charge and due to steric effects of long chains protruding from the surface. It has been shown that the inhibition of T-cell adhesion can interfere with the ability of eosinophils to bind to the surface cuticle and kill schistosome parasites in in vitro tests (Hayes et al. 1990). TES-120 is secreted in internal excretory glands and ducted to the surface via the oesophagus and excretory pore and it is also released from Toxocara surface. The overexpression of some membrane-associated mucins suggests a possible model for the role of SC in immune evasion by parasitic nematodes, through changing the nematode surface cuticle adherence to defence cells and/or by releasing soluble mucins that might interact with host cells and blocks defence responses (Gems and Maizels 1996). Toxocara canis also secretes large quantities of a C-type lectin thought to compete with host innate immune system receptors (Loukas and Maizels 2000).

Another important group of surface proteins which may act to promote immune evasion in B. malayi are the anti-oxidant products glutathione peroxidase and superoxide dismutase. Bm-GPX-1 is the major 29 kDa surface glycoprotein of adult Brugia (Cookson et al. 1992; Maizels et al. 1989), which is believed to act as a lipid hydroperoxidase, protecting parasite membranes from peroxidation caused by free-oxygen radicals (Tang et al. 1996). A minor surface-associated protein of similar molecular weight is a superoxide dismutase, allowing the parasite to detoxify superoxide radicals (Tang et al. 1994). Many other surface-associated molecules may contribute to immune escape in a less obvious manner. For example, the polyprotein antigen (variously named gp15/400 or Bm-NPA-1) has a very high affinity for fatty acids (Kennedy et al. 1995; Smith et al. 1998) which could sequester substrate required for host leukotriene synthesis. Non-protein filarial products are also likely to play a significant role: a novel lipid found in the cuticle of B. malayi acts as a sink absorbing oxidative attack, perhaps protecting essential membrane lipids and proteins from degradation (Smith et al. 1998). A further component prominently expressed by B. malayi is phosphorylcholine (PC). Not only has this been suggested as an immunosuppressive moiety in lymphatic filariasis (Lal et al. 1990), but it has been possible to demonstrate direct down-regulation of both B (Deehan et al. 1998) and T cell (Harnett et al. 1998) function by a PC-bearing protein secreted from the filarial parasite Acanthocheilonema viteae.

A proteinase inhibitors member of the cystatin (cysteine protease inhibitor) family located on the surface of both L3 and adult B. malayi, and secreted by these parasites in vitro blocks conventional cysteine proteases but also the aspariginyl endopeptidase involved in the Class II antigen processing pathway in human B cells (Maizels et al. 2001b)

5.5 Interactions of Nematode Surfaces with Microorganisms

Complex interactions are formed between microorganisms, nematodes, plants and the environment. Some of the pant-parasitic nematodes microbial antagonists are also root colonizers and this may affect their activity against the nematodes (Kerry 2000; Bordallo et al. 2002; Sharon et al. 2007). Microorganisms have a wide range of suppressive activities on different nematode species. In direct interactions, nematode surface is believed to be important in recognition events and determining the specificity of interactions or the defence mechanisms involving microbial antagonists (Spiegel and McClure 1995; Kerry and Hominick 2001; Morton et al. 2004). Interactions between nematodes and microorganisms (fungi and bacteria) have been described in several reviews (Kerry and Hominick 2001; Bird 2004; Chen and Dickson 2004a; Chen and Dickson 2004b; Morton et al. 2004; Davies 2005; Tian et al. 2007; Lopez-Llorca et al. 2008). We will refer to some examples of such interactions, regarding attachment and penetration aspects.

5.5.1 Interactions with Fungi

Fungal antagonists of nematodes can be grouped into predacious fungi (nematode - trapping fungi), endoparasites of vermiform nematodes, parasites of sedentary females and eggs, and fungi that produce antibiotic substances. Nevertheless, some fungi can belong to more than one category. Attachment of fungi to nematodes is either specifically to head and tail regions, or all over the body, or very sparse. Zoospores usually attach near natural body openings (Chen and Dickson 2004a).

5.5.2 Predacious Fungi

Nematophagous fungi can capture, kill and consume their prey, and have evolved special devices for capturing vermiform nematodes: adhesive hyphae, branches, nets or knobs, non-constricting or constricting rings, and stephanocyts. Adhesive hyphae or branches are usually produced by lower fungi and Deuteromycetes such as Arthrobotrys and Dactylaria (Chen and Dickson 2004a), (see also Chap. 6).

In nematode-trapping fungi, parasitism begins with the induced formation of traps or other parasitism structures. Despite the variation in trap morphology, majorities of nematode-trapping fungi are closely related and the infection mechanism appears to be rather similar. Following traps development, the infection process proceeds through a sequence of events: attachment of the trap cells to nematode surface, penetration of the cuticle, digestion and assimilation of the nutrients from the killed nematode (Fekete et al. 2008). Free-living nematodes such as Panagrellus redivivus and C. elegans serve as model host nematodes in research of these fungi. Involvement of a Gal-NAc-specific lectin of A. oligospora (Nordbring-Hertz and Mattiasson 1979) in nematode recognition has been suggested. Subsequently, a carbohydrate-binding protein from the capture organs of the fungi, not present on hyphae, was isolated and partially characterized (Borrebaeck et al. 1984). These early infection events lead to signaling cascades necessary for penetration and colonization of the nematode prey (Tunlid et al. 1992). After contact, an extracellular material, or adhesive, is formed which adhere the fungus to the nematode surface. The adhesive layer has a fibrillar structure containing residues of neutral sugars, uronic acid and proteins (Tunlid et al. 1991). The adhesive on the traps of A. oligospora changes from an amorphous to a fibrillar appearance after contact with a nematode (Jansson and Nordbring-Hertz 1988).

Initial contact with the host cuticle is probably followed by a cascade of interactions with specific receptors, reorganization of surface polymers to strengthen the adhesions, changes in morphology, and the secretion of specific enzymes. Trapped nematodes become immobilized after adhesion, when the fungus starts to penetrate the nematode cuticle (Dijksterhuis et al. 1994). A narrow penetration tube develops from the trap cells and the nematode cuticle is breached by a combination of physical force and enzymatic degradation (Kerry and Hominick 2001). The nematode-trapping fungus, A. oligospora, produces a subtilisin-like serine protease (designated PII) that immobilized P. redivivus and hydrolyzed proteins of purified cuticle (Tunlid et al. 1994; Åhman et al. 2002). Once inside the nematode, the penetration tube swells to form an infection bulb from which hyphae are growing inside the infected nematodes. At this stage the internal tissues of the nematode are rapidly degraded (Dijksterhuis et al. 1994). An extracellular 35 kDa alkaline serine protease (Ds1) was purified and characterized from the nematode-trapping fungus Dactylella shizishanna. The purified protease could degrade purified cuticle of P. redivivus and a broad range of protein substrates. It showed a high homology with Aozl and PII, two serine proteases purified from A.oligospora (Wang et al. 2006).

Monacrosporium haptotylum traps nematodes using a spherical structure called knob, which develops on the apex of a hyphal branch. The transcriptional response in the parasitic fungus M. haptotylum and its nematode host C.elegans were analyzed during infection using cDNA microarrays (Fekete et al. 2008). Among the infection-induced C. elegans genes were those encoding antimicrobial peptides, protease inhibitors and lectins. C. elegans mount protective responses against bacterial and fungal pathogens by activating several intracellular signalling pathways that lead to the production of compounds that limit the infection or destroy invading microorganisms (Gravato-Nobre and Hodgkin 2005). C-type lectin domains (CTLD) might act as pathogen recognition molecules, or may mask the virulence factors of the pathogen. CTLD-containing genes were induced by bacterial infections of C. elegans (Mallo et al. 2002; O’Rourke et al. 2006), but were down-regulated during infection with M. haptotylum (Fekete et al. 2008).

5.5.3 Endoparasites of Vermiform Nematodes, Sedentary Females and Eggs

The fungal species, most commonly isolated from sedentary stages, include Acremonium, Fusarium, Gliocladium, Nematophthora, Paecilomyces, Penicillium, Phoma and Pochonia (Verticillium) (Chen and Dickson 2004a). The infection of root-knot and cyst nematode eggs by parasitic fungi involves formation of appressoria when the hyphae encounter the eggshell. This depends on recognition of host surface, where the hydrophobicity of the surface is an important factor (Morton et al. 2004).

Pochonia chlamydosporia (Verticillium chlamydosporium), which colonizes cysts or egg-masses, can also attack J2s. The infection of nematode eggs by Pochonia rubescens and P. chlamydosporia starts with contact of the hyphae and formation of an appressorium. Extracellular material, or an adhesive, formed on the appressorium, could be labeled with the lectin Concanavalin A (Con A), indicating the presence of glucose/mannose residues (Lopez-Llorca et al. 2002). The fungus penetrates the nematode eggshell mechanically and by enzymatic hydrolysis. P. rubescens protease P32 was immunolocalized on appressoria that were infecting eggs of the beet cyst nematode H. schachtii (Lopez-Llorca and Robertson 1992).

Conidia of the endoparasitic fungus, Drechmeria coniospora, adhere to the chemosensory organs natural body openings of the nematodes (Jansson and Nordbring-Hertz 1983). Conidial adhesion to P. redivivus was suggested to involve a sialic acid-like carbohydrate since treatment of nematodes with the lectin Limulin, and treatment of the spores with sialic acid, decreased adhesion (Jansson and Nordbring-Hertz 1984). Adhesion of conidia to C. elegans was reduced after treatment of the nematodes with Pronase E. The process was reversible within 2 h, indicating that the proteinaceous material emanating from the sensory structures was rapidly replaced (Jansson 1994). Conidia of D. coniospora adhere to the chemosensory organs of Meloidogyne spp., but do not penetrate and infect the nematodes. Nevertheless, the fungus reduced root galling in tomato (Jansson et al. 1985), probably due to behavior interference. A chymotrypsin-like, was partially characterized from D. coniospora conidia (Jansson and Friman 1999).

The fungus Clonostachys rosea (syn. Gliocladium roseum) is a widely distributed facultative saprophyte in the soil. Observations reveal that the pathogenesis on P. redivivus started from the adherence of conidia to nematode cuticle for germination, followed by the penetration of germ tubes into the nematode body and subsequent death and degradation of the nematodes. The conidia excrete a mucous liquid that can stick to the cuticle of nematodes. Microscope observations suggested that the glutinous substance could prevent the nematodes escape from the adhesive areas formed by the secretions of the conidia (Zhang et al. 2008). Two extracellular serine proteases (Lmz1 and PrC) were isolated from C. rosea and identified as important factors in fungal pathogenicity. Chemicals isolated from the fungus showed strong nematicidal activities against several nematodes: C. elegans, P. redivivus, and Bursaphelenchus xylophilus (Zhang et al. 2008).

Species of Trichoderma (see Chap. 8) are also facultative saprophytic fungi in soil and posses parasitism abilities against plant-pathogenic fungi and plant-­parasitic nematodes, such as Meloidogyne spp. (Sharon et al. 2007, 2009b) and Xipinema spp. (Fig. 5.6) (Spiegel, Sharon, Chet unpubl.). Mechanisms involved in the attachment and parasitism processes on M. javanica were investigated, mainly with T. asperellum-203 and T. atroviride. It was found that the gelatinous matrix (gm) enables fungal attachment and enhances parasitic abilities of most isolates, which could also utilize it as a nutrient source. Fungal conidia can attach to nematode egg masses and to eggs and J2s that had contact with the gm, whereas gm-free J2s and eggs are almost unattached by fungal conidia; those were penetrated only by few fungal hyphae and colonized. Observations showed typical fungal parasitic behavior, including tight attachment of spores and hyphae to the J2s, coiling of hypae around J2s and appressoria-like structures formation upon egg penetration. Conidia were agglutinated by a gm suspension (enhanced in presence of Ca2+) and their germination was improved. A model for fungal conidia attachment to nematodes suggests that carbohydrate-lectin-like interactions might be involved in this process (Sharon et al. 2007, 2009a).

Fig. 5.6
figure 6_5

Parasitism of Trichoderma asperellum-203 (constitutively expressing green fluorescent protein (GFP) construct) on Xipinema index. The image shows the posterior part of a female, colonized by the fungus, 4 days after inoculation with conidia, bar  =  50 μm

Full size image

Fungi of Catenaria spp. are known as obligate parasites of vermiform nematodes; however, C. anguillulae is a facultative parasite of females and eggs of root-knot nematodes (Wyss et al. 1990). Uniflagellate zoospores are attracted to natural openings of the nematode and show “amoeboid movement” upon contact with the nematode cuticle, before encystment. During encystment a cell wall is formed, covered by an adhesive, and the flagellum is withdrawn. The encysted zoospore forms an infection peg which penetrates the nematode cuticle and the nematode content is digested.

5.5.4 Fungi That Produce Antibiotic Substances and Toxins

Toxic effects of fungal culture filtrates on vermiform nematodes and eggs have been reported for several fungi, such as species of Paecilomyces, Verticillium, Fusarium, Aspergillus, Trichoderma, Myrothecium and Penicillium (Chen and Dickson 2004a; Morton et al. 2004). Toxins are important for parasitic microorganisms because they facilitate infection by debilitating the host. The combination of lytic enzymes and nematicidal compounds can improve the efficacy of the biocontrol agent by increasing the permeability of the nematode surfaces and eggshells. In biocontrol, our interest is to avoid damage to non-target organisms, such as beneficial nematodes. For example, nematicidal activity of T. atroviride culture filtrates was restricted to several plant parasitic species, and did not harm non-target and beneficial nematode species (Spiegel, Sharon, Chet unpubl.). The wide differences among the structures and compositions of the various nematode surfaces might determine the different permeability of the nematodes.

5.5.5 Interactions with Bacteria

Due to the chemical nature of the nematode’s cuticle, few bacteria and fungi could utilize and degrade the extracellular exoskeleton. The physical body design of plant-parasitic nematodes prevents bacteria from entering through body openings; therefore, to become an endoparasite of nematodes, the bacterium must overcome the nematode surface barriers (Chen and Dickson 2004b). Most nematophagous bacteria, except for obligate parasites, are saprophytes that can also penetrate the nematodes and use them as a nutrition resource. Members of the genera Pasteuria, Pseudomonas and Bacillus have shown great potential for biological control of nematodes (Tian et al. 2007).

5.5.6 Obligate Bacterial Parasites

Species of Pasteuria are bacterial obligate hyperparasites of nematodes. The attachment process of endospores and its host specificity have been extensively studied mainly with P. penetrans on root-knot nematodes (recently reviewed by Bird 2004; Davies 2005, 2009; Tian et al. 2007). The involvement of surface coat components in the attachment has been demonstrated and antibodies raised against Meloidogyne surfaces, could inhibit spore attachment (Davies and Danks 1992; Spiegel et al. 1996). Monoclonal antibodies, raised against H. cajani, could also reduce spore attachment, whereas one antibody increased the attachment (Sharma and Davies 1997). There is high heterogeneity in bacterial surfaces, both within and among different P. penetrans populations (Davies and Redden 1997; Davies et al. 2000). The initial binding of endospores to their nematode hosts has been studied (Stirling et al. 1986; Persidis et al. 1991; Davies and Danks 1993; Spiegel et al. 1996). The results suggested a model in which a carbohydrate ligand on the surface of the endospore binds to a lectin-like receptor on the cuticle of the nematode host; however, binding of lectins to nematode surface also reduced the spore attachment (Spiegel et al. 1996). The fibres on the Pasteuria endospore are thought to be responsible for the adhesion to the host cuticle through a Velcro-like mechanism (Davies 2009). These fibres were shown to be glycoproteins, containing a high level of N-acetyglucosamine (Persidis et al. 1991). Collagen on the nematode’s cuticle has been suggested to be involved in recognition process, because attachment was reduced by trypsin and endoglycosidase F, and because gelatin (denatured collagen) could inhibit spore attachment (Persidis et al. 1991; Mohan et al. 2001). However, treatments of J2s with collagenases did not inhibit endospore attachment (Davies and Danks 1993). Davies and Opperman (2006) identified several collagen-like proteins in P. penetrans that contain G-x-y repeats. Pretreatment of endospores with collagenase or the collagen-binding domain of fibronectin inhibited endospore binding to nematode cuticle (Mohan et al. 2001), suggesting that collagen-like proteins are also present on the P. penetrans exosporium surface and are involved in attachment.

5.5.7 Opportunistic Bacterial Parasites

Various isolates of Brevibacillus laterosporus have been reported as parasites of nematodes. The plant-parasites Heterodera glycines, Trichostrongylus colubriformis and Bursaphelenchus xylophilus, and the saprophytic nematode P. redivius could be killed by the bacterium (Tian et al. 2007). After attachment to the cuticle, the bacterium can multiply and form a single clone in the epidermis of the nematode cuticle. A circular hole can appear following the continuous degradation of the cuticle and tissue, until bacteria enter the nematode’s body and digest it all for nutrition (Huang et al. 2005). The degradation of all the nematode cuticle components around the holes suggests the involvement of hydrolytic enzymes; the major pathogenic activity could be attributed to an extracellular alkaline serine protease, designated BLG4 (Huang et al. 2005; Tian et al. 2006).

A neutral protease (npr) (designated Bae16) of 40 kDa, toxic to nematodes, was purified to homogeneity from the strain Bacillus nematocida. The activity was tested against P. redivivus and B. xylophilus. This purified protease could destroy the nematode cuticle and its hydrolytic substrates included gelatin and collagen. The gene encoding Bae16 was cloned and expressed in Escherichia coli, confirming its nematicidal activity (Niu et al. 2006).

5.5.8 Other Bacterial Interactions

Bacterial antagonists that have been studied, such as Telluria chitinolytica (Pseudomonas chitinolytica) (Spiegel et al. 1991; Bowman et al. 1993) were able to attach to M. javanica juveniles, especially to head and tail regions, but no direct parasitism or degradation was observed. The bacteria and its culture filtrates could immobilize the nematodes and produce nematicidal compounds and hydrolytic enzymes such as chitinases, proteases and collagenases, which might contribute to the biocontrol process. These bacteria can colonize root surface and this might affect the nematode-plant interactions.

Bacillus cereus had also presented biocontrol activity against the root-knot nematode (Oka et al. 1993). An enzyme with collagenolytic/proteolytic activities that could degrade M. javanica cuticular collagens was purified from this bacterium (Sela et al. 1998).

Nematodes can serve as a vector of microorganisms that attach to their surface and enter the plants. Nematode species of Anguina are parasites of cereal grasses. They are vectors of Rathaybacter bacteria (formerly referred as Clavibacter and Corynebacterium). These bacteria produce toxins that can harm the animals feeding on the cereals. A positive correlation was found between a bacteriophage presence in R. toxicus and the toxin production (reviewed by Bird 2004). McClure and Spiegel (1991) showed that the bacteria are attached to the surface coat on the nematode’s cuticle; however, the mechanism of the attachment remains unknown. Normal adhesion leads to fusion of the bacterium capsular material with the surface coat of the nematode. Slight swelling of nematode’s epicuticle and cuticular damage, with loss of nematode’s mobility may occur (Bird 2004).

Anguina funesta is also a vector of the fungus Dilophosphora alopecuri, a pathogen of cereals and grasses (reviewed by Bird 2004). The fungus has an inhibitory effect on nematode development in the plant. It shows also antagonism to R. toxicus and was suggested as a potential biocontrol agent. During adhesion, the fungal conidium secrets adhesive material that covers the surface of the nematode and fills the surface annulations, but no morphological changes were observed in the cuticle.

5.5.9 Role of Lytic Enzymes in Nematode Penetration

Enzymes degrade the host’s barriers to infection and, therefore, play an important role in infection of nematodes by fungi and bacteria. The structure of nematode’s cuticle and eggshells indicates that proteases and chitinases are necessary for their degradation. Such enzymes that have been identified and are associated with anti-nematode activity were described (Morton et al. 2004; Casas-Flores and Herrera-Estrella 2007; Lopez-Llorca et al. 2008; Tian et al. 2007; Gortari and Hours 2008). A cooperative effect of proteolytic and chtinolytic enzymes in eggshells degradation was demonstrated (Tikhonov et al. 2002). In nematophagous fungi, Pr1-like alkaline serine proteases act on the collagen-like proteins of the nematode cuticle and the protein-containing elements of the eggshell; such enzymes have been identified in several fungi. The major proteases identified from nematophagous fungi belong to the proteinase K family of subtilases (from Peptidase S8 Subtilase family) (Morton et al. 2004). This family includes also the proteinase Prb1, produced by T. atroviride and is also active against nematodes (Sharon et al. 2009b). Bacterial serine protease genes from nematophagous bacteria (Brevibacillus and Bacillus strains) have shown high sequence identity, indicating that these genes are highly conserved in these bacteria (Tian et al. 2007). Collagenases might also be important in cuticle degradation, since the majority of the proteins in the nematode cuticle are collagens. Collagenase was identified in Arthrobotrys spp (Tosi et al. 2002). Galper et al. (1991) evaluated a collagenolytic fungus, Cunninghamella elegans, for biocontrol activity against plant-parasitic nematodes. Lipases have been implicated in the infection of nematode eggs: Heterodera schachtii eggs, infected by fungi, appeared to have their inner lipid layers degraded (Perry and Trett 1986).

5.6 Concluding Remarks and Future Directions

Plant nematodes must survive the hostile environment that runs from the bulk soil through to the rhizosphere which contains numerous microorganisms and then onto the host plant. Plant nematode surfaces are targets for passive and active environmental attack by the plant immune system, the attachment of bacterial spores and fungal traps. The nematode egg surface is a target for fungal attack and the understanding of this fungus-nematode interaction might lead to the development of biological control methods for soil nematodes. The cuticle and its surface coat have a central importance not only in the biosynthesis and maintenance, but also play a part in defence against their host innate immunity and pathogens. The cuticle, and in particular its surface, can be looked upon as a major part of the nematodes immune system as can be seen from the specifity observed between Pasteuria penetrans and root-knot nematodes. It is likely that this variation, as observed by endospore attachment to the nematode cuticle may also play an important role in nematode-plant interactions.

Nematodes respond to environmental conditions by modifying their surfaces, a process formally similar to surface antigen switching in animal-parasitic nematodes. Now that the full genome of various plant-parasitic nematodes and their host plants are available it should be possible to perform detailed study of the genes involved in these interactions and the molecular nature of the surface modification response. It is now possible to study the tritrophic interactions between both the plant and nematode, and the nematode and the microbe, so including all sides of the pathosystem (see Chap. 12). It is also important to identify the environmental factors in the soil, rhizosphere and plants inducing changes in the nematode surface and behaviour.

Another important aspect of research in plant nematology is the identification of nematode effector proteins present in the surface cuticle and also the plant compounds being suppressed by the nematode. There is a great interest in the development of high throughput method for identifying putative nematode effector proteins, which are essential parasitism genes.

This knowledge should lead to a better understanding of interactions between plants and would undoutdly contribute to the development of novel environmentally friendly methods to control plant nematodes which could be incorporated into an integrated pest management programme. Plant nematode surfaces are easily accessible to control measures and constitute an important target for any anti-nematode gene therapy.