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SUMMARYThe ability of certain species of parasitic nematodes to survive desiccation for considerable periods is a fascinating example of adaptation to the demands of fluctuating environments that occasionally can become extreme and life threatening. Behavioural and morphological adaptations associated with desiccation survival serve primarily to reduce the rate of drying, either to prolong the time taken for the nematode's water content to reach lethal low levels or, in true anhydrobiotes, to enable the structural and biochemical changes required for long-term survival to take place. Examples of these adaptations are reviewed, together with information on the factors involved in rehydration that ensure successful exit from the dormant state. Information on desiccation survival is central to effective management and control options for parasitic nematodes. It is also required to assess the feasibility of enhancing the longevity of commercial formulations of entomopathogenic nematodes, both before and after application; current research and future prospects for enhancing survival of these bio-insecticides are discussed.

Abstract Acridine orange staining was used to compare transcription patterns in hatched and unhatched second stage juveniles (J2s) of Globodera rostochiensis. Unhatched J2s showed little staining when compared with hatched J2s. Hatched J2s showed specific and reproducible staining of the nucleoli of the dorsal oesophageal gland cell and genital primordia. Some staining was also observed in the nucleoli of the subventral gland cells. Exposure of the unhatched J2s to sterile distilled water (SDW) did not result in any noticeable increase in staining with acridine orange. Prolonged periods in tomato root diffusate (TRD) were required to induce changes in dorsal oesophageal gland cell activity of unhatched J2s. Hatched J2s always showed staining of the dorsal oesophageal gland cell and genital primordia, regardless of their length of exposure to TRD. These results demonstrate that changes in transcription patterns of rRNA are induced in a range of structures during the hatching process. Activation de la transcription au cours de l'eclosion chez le nematode a kyste de la pomme de terre Globodera rostochiensis - La coloration a l'acridine orange a ete utilisee en vue de comparer les profils de transcription chez les juveniles de deuxieme stade (J2) de Globodera rostochiensis eclos et non eclos. La coloration des J2 non eclos est faible en comparaison de celle des J2 eclos. Ces derniers montrent une coloration specifique et reproductible des nucleoles de la cellule glandulaire oesophagienne dorsale ainsi que des primordiums genitaux. Une coloration des nucleoles des cellules glandulaires oesophagiennes subventrales est egalement observee. L'exposition des J2 non eclos a l'eau distillee sterile ne provoque aucune augmentation notable de la coloration a l'acridine orange. Des periodes prolongees de contact avec les exsudats radiculaires de tomate sont necessaires pour induire un changement dans l'activite de la cellule glandulaire oesophagienne dorsale des J2 non eclos. Les J2 eclos montrent toujours une coloration de la cellule glandulaire oesophagienne dorsale et des primordiums genitaux quelle que soit la longueur de l'exposition a ces exsudats. Ces resultats demontrent que les changements dans les profils de transcription du rRNA sont induits pendant l'eclosion par une serie de structures.

The total lipid content of the dry weight of whole cysts and 2nd-stage juveniles (J2) of Globodera rostochiensis was 17·1% in dry cysts, 20·9% in cysts soaked in distilled water (DW), 20·3% in cysts that had been in potato root diffusate (PRD) for 7 days, 7·3% in cysts that had been in PRD for 28 days and 29·2% for hatched J2. The fatty acid composition of the total lipid did not differ between dry cysts, cysts in DW and cysts in PRD for 7 days. However, major differences in the fatty acid composition of all lipid classes were found between rehydrated cysts in PRD and freshly hatched J2. After hatching, the degree of saturation and the percentage of monounsaturated fatty acids decreased and the percentage of polyunsaturated fatty acids increased considerably, especially in the free fatty acid fraction, where C20[ratio ]1 showed an 8-fold decrease and C20[ratio ]4 a 33-fold increase. There was a difference in the fatty acid composition of cysts in PRD for 7 days and cysts in PRD for 28 days (after most of the J2 had hatched); with increased time in PRD the percentage of polyunsaturated fatty acids increased and the percentage of monounsaturated fatty acids decreased in all lipid classes. Differences in the fatty acid profiles between cysts in PRD for 28 days and hatched J2 were found mainly in the free fatty acid and the non-acidic phospholipid fractions. The free fatty acid fraction of the cysts was dominated by monounsaturated fatty acids (52%), whereas the same fraction of hatched J2 contained large amounts of polyunsaturated fatty acids (60%). These polyunsaturated fatty acids, especially C20[ratio ]4, might play an important part in nematode–plant interactions during infection.

Up to 16 fatty acids were detected in total lipids from roots and leaves of two potato cultivars, Desiree and Maris Piper, and one tomato cultivar, Pixie. The fatty acid profiles from roots were similar for all three cultivars and were dominated by C18 : 2 (30-40%) and C 16 : 0 (about 22%). The fatty acid profiles from leaves were similar for all three cultivars and were dominated by C18 : 3 (about 52%). Both root and leaf lipids contained mainly C18 fatty acids. The lipid content found in dry cysts of Globodera rostochiensis rcared on different cultivars was 11.0%, 12.8% and 9.6% for potato cvs Desiree and Foremost and tomato cv. Pixie, respectively. The total lipid comprised 74-82% neutral lipid, 14-19% non-acidic phospholipid and 4-7% free fatty acid in the three samples. Hatched juveniles (J2) from cysts cultured on cvs Desiree and Foremost contained 26.1% and 24.3% lipid, respectively. The total lipid comprised 77% neutral lipid, 17-18% non-acidic phospholipid and 6-7% free fatty acid in the two samples. The fatty acid profiles of the main lipid classes of cysts of G. rostochiensis cultured on the three different cultivars were similar. The fatty acid composition of the main lipid classes of freshly hatched J2 from cysts of G. rostochiensis cultured on the two potato cultivars differed only in their free fatty acid fractions. A comparison of the fatty acid profiles of the total lipid of potato roots, cv. Desiree, and the total lipid of cysts of G. rostochiensis showed major differences. Root fatty acids and nematode fatty acids were 38% and 22% saturated, 10% and 44% monounsaturated and 52% and 35% polyunsaturated, respectively. The fatty acid profile of root lipids was dominated by C16: 0, C18: 2 and C18 : 3 but the nematode lipids consisted of mainly C18: 1, C20: 1 and C20: 4. The differences in the fatty acids of host and nematodes indicate that G. rostochiensis is capable of fatty acid chain elongation and/or desaturation.

Changes in chitinase activity were observed following infection of three potato clones (12380, P55/7 and Maris Piper) with four pathotypes of the potato cyst nematodes, Globodera rostochiensis and G. pallida. Following invasion, the intercellular fluid of the leaves of all the plants tested showed marked increases in exochitinase activity. In contrast, after removal of intercellular fluid the leaf homogenates showed only a limited increase in endochitinase activity. In roots, endochitinases were significantly increased in 12380 following invasion by G. pallida Pal and G. rostochiensis Ro1 and Ro5 but not by G. pallida Pa2/3. The largest increase in exochitinase activity in the roots of 12380 was observed after invasion by G. pallida Pa1. The possible role of these chitinases in potato-nematode interactions is discussed.

During hatching of cyst nematodes, fluid is released from the egg into the hatching medium. The presence of carbohydrates in the hatching medium of two populations of Heterodera avenae from India and the UK was determined using Western blots probed with different biotinylated lectins. Eighteen specific bands with molecular weights in the range 12-78 kDa were detected in the hatching media collected over the six weeks hatching period. There were no differences between the two populations. The majority of the lectin bands were inhibited by the appropriate competitive sugar, indicating sugar-specific binding. Four different sugars were detected. Of the bands detected with Con A, only two were not completely inhibited which may indicate non sugar-specificity. The possible origin of these carbohydrates during the hatching process is discussed. There were differences in the protein banding pattern of hatched second stage juveniles; an extra band was present at 20 kDa in the population from India, whereas the population from the UK had a band at 34 kDa.

Electrophysiological techniques and behavioural assays were used to determine the response of males of Globodera rostochiensis to 4 fractions of the homospecific sex pheromone from virgin females, separated using reverse-phase high-pressure liquid chromatography. Sex pheromone activity was confined to 2 of the fractions. The sex pheromone of G. rostochiensis appears to be composed of several weakly basic, polar compounds.

The desiccation survival and ability to control water loss during drying of individual infective juveniles (IJs) of populations ofSteinernema feltiaeandHeterorhabditis megidisfrom Greece and the UK were examined at different relative humidities. Survival at all relative humidities was for minutes only and there was no evidence of enhanced survival of the populations of either species from Greece compared with those from the UK. The survival of individuals of both populations ofH. megidiswas superior to that ofS. feltiaeand the rate of drying ofH. megidiswas significantly slower. The sheath surrounding the IJs ofH. megidisappears to be involved in slowing the rate of water loss of the enclosed nematodes.

Electrophysiological techniques and behavioral bioassays were used to evaluate the responses of males of the potato cyst nematodesGlobodera rostochiensis and G. pallidato various chemicals. Exposure of all males of both species to acetylcholine elicited strong electrophysiological responses and positive movement towards the source. By contrast, neither glycine nor citric acid induced any marked electrophysiological responses and only males of G. pallida showed significant behavioral responses, moving toward glycine and away from citric acid. The electrophysiological and behavioral responses to GABA and α-aminobutyric acid were complementary, with G. rostochiensis showing significant response only to the latter and G. pallida responding significantly only to the former. The electrophysiological response of individuals of both species to L- and D-tryptophan varied from no response to a strong response with a significant mean increase in spike activity of G. rostochiensis to D-tryptophan and of G. pallida to L-tryptophan; behavioral bioassays showed L-tryptophan was a deterrent for G. pallida. Males of both species responded significantly in both electrophysiological and behavioral assays to L-glutamic acid but not to D-glutamic acid.

The esterase isozyme from Meloidogyne incognita with pI ∼4.95 was purified using a Rotofor electrophoresis system. The purified enzyme was then used to raise monoclonal antibodies. Hybridoma cell lines producing antibodies were cloned and screened against the purified esterase in an enzyme-linked immunosorbent assay (ELISA). A selected monoclonal antibody was then tested by western blot analysis of crude soluble protein extracts of all the major species of Meloidogyne separated by native and SDS-PAGE electrophoresis. Although the antibody was cross-reactive in western blots of denatured proteins in the presence of SDS and in ELISA, it was not cross-reactive in western blots of native protein. The results demonstrate that a monoclonal antibody to a diagnostic non-specific esterase enables M. incognita to be discriminated from M. javanica without the need to separate the esterases by electrophoresis first. This is an approach which could be adopted in a number of situations where organisms can be identified by a diagnostic protein which has already been identified by electrophoresis.

The activity of β-1,3-glucanases and β-D-glucosidases increased in the leaves of potato plants infected by species of the root parasite genusGlobodera. Enzyme assays revealed the appearance of both the extracellular and intracellularly targeted β-1,3-glucanases. The reactions of cultivars to nematode infection differed, indicating that different pathotypes of the same species or different species of potato cyst nematode elicited the appearance of different classes of β-1,3-glucanases. A polyclonal antibody against β-1,3-glucanases labelled enzyme bands only from intercellular fluid extracts. The analysis of infected roots showed no significant increase in β-1,3-glucanase activity. The importance of β-1,3-glucanases in the resistance response and other possible defence-related functions is discussed.

The response of individual adult males of the potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, to potato root diffusate (PRD) and to sex pheromones from adult females was investigated using electrophysiological techniques and behavioural bioassays. Males of both species responded to sex pheromones but PRD did not elicit any significant change in spike activity or movement. Males of PCN apparently require only sex pheromones to attract them towards the sessile females protruding from the root.

SUMMARY Six species of Pratylenchus indigenous to Great Britain, P. crenatus, P. fallax, P. neglectus, P. penetrans, P. pinguicaudatus and P. thornei were analysed by slab gel electrophoresis to compare protein patterns and isoenzyme phenotypes of esterase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, phosphoglucose isomerase and phosphoglucomutase. Multiple electromorphs were obtained from all enzymes examined. The results demonstrated that isoenzyme phenotypes are useful to supplement the morphological characterisation of these nematode species. Pair-wise comparisons of the six species were performed giving coefficients of similarity in the range 11–41%. A dendrogram of the six species, generated from the five enzyme banding patterns, gave two groups: group 1 contained P. pinguicaudatus, P. fallax and P. thornei and group 2 contained P. penetrans, P. neglectus and P. crenatus.

Three populations of Aphelenchoides besseyi, one population of A. bicaudatus, A. arachidis, A. fragariae, A. hamatus, two undescribed species of Aphelenchoides from rice and Ditylenchus angustus and D. myceliophagus were analyzed by native and SDS-PAGE electrophoresis. The highly reproducible banding patterns of all species differed in number, concentration and relative mobility. Certain enzyme bands were common between the species whereas other bands were specific. There were no detectable differences in the protein and esterase banding patterns of nematodes extracted from cultures of Botrytis cinerea or Rhizoctonia cerealis. The stability and taxonomic usefulness of the α, β esterase banding patterns are discussed.

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.

Understanding the desiccation survival attributes of infective juveniles of entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis, is central to evaluating the reality of enhancing the shelf-life and field persistence of commercial formulations. Early work on the structural and physiological aspects of desiccation survival focused on the role of the molted cuticle in controlling the rate of water loss and the importance of energy reserves, particularly neutral lipids. The accumulation of trehalose was also found to enhance desiccation survival. Isolation of natural populations that can survive harsh environments, such as deserts, indicated that some populations have enhanced abilities to survive desiccation. However, survival abilities of EPN are limited compared with those of some species of plant-parasitic nematodes inhabiting aerial parts of plants. Research on EPN stress tolerance has expanded on two main lines: i) to select strains of species, currently in use commercially, which have increased tolerance to environmental extremes; and ii) to utilize molecular information, including expressed sequence tags and genome sequence data, to determine the underlying genetic factors that control longevity and stress tolerance of EPN. However, given the inherent limitations of EPN survival ability, it is likely that improved formulation will be the major factor to enhance EPN longevity and, perhaps, increase the range of applications.

The life cycle of parasitic nematodes essentially consists of two phases, the preparasitic and parasitic. The pre-parasitic phase, which may equate to the infective stage, occurs either as a free-living stage or inside, or transported by, an interrnediate host. On locating and invading the definitive host, the parasitic phase commences. For obligate parasitic species there are situations where persistence of a population requires survival of the free-living stages. This may occur when the host is not available or environmental conditions exist that are not commensurate with continuing development. The requirements, first, to survive long enough to infect a host and, second, to ensure the survival of progeny when the host is no longer supportive, are the essential non-parasitic tasks of the life cycle. Survival of adverse environmental conditions may involve enduring temperature extremes (see Wharton, Chapter 8, and Devaney, Chapter 10, this volume), osmotic stress (see Wharton and Perry, Chapter 11, this volume) and dehydration, in addition to withstanding the absence of food. The ability of some species of nematode to survive desiccation for periods considerably in excess of the duration of the normal life cycle has been studied in detail, in part because in species with a direct life cyele this attribute is hnked to effective dispersion of nematodes. In the past, research has focused primarily on the remarkable structural, physiological and behavioural adaptations that facilitate desiccation survival (Perry, 1999). However, more recently the molecular aspects have received considerable attention, and these ar reviewed by Burnell and Tunnaeliffe, Chapter 6, and Adhikari emd Adams, Chapter 9, this volume. In this chapter, we examine the morphologicaI, physiological and behavioural adaptations, focusing principallyon desiccation survival and the link to nematode dispersion. This link and the need to understand the temporaI factors involved in survival are elearly vital for effective management and control ophons for parasitic nematodes. The pre-adult stages of nematodes are ca lied juveniles by plant nematolof ogists, and the term infective juvenile (IJ) is favoured by researchers working with entomopathogenic nematodes. However, the term larva(e) is the term of choice for animal nematologists and the Caenorhabditis efegans community. To ensure consistency throughout this chapter, larva(e) will be used.

Nematodes are a remarkable group of invertebrates with enormous economic and social importance. Free-living nematodes are found in a wide variety of habitats, from the Antarctic to the hot springs in New Zealand. Parasitic species of nematodes parasitize almost every species of plants and animals, including humans. The success of nematodes depends partly on sophisticated adaptations to withstand environmental extremes and, for parasitic forms, to overcome the switch between the external environment and the internal environment of the host, and the ensuing antagonistic response of the host. This review briefly outlines the astonishing abilities of some species of nematodes to survive environmental extremes, including desiccation, and to withstand host responses. Understanding the mechanisms for survival has relevance to many other biological fields and the importance of molecular approaches to unravel the mechanisms is discussed.

Nematodes are renowned for their ability to survive severe environmental fluctuations. Their mechanisms to withstand temperature extremes, desiccation, and osmotic and ionic stress are presented here together with information on the underlying biochemical basis contributing to survival. Highlighting parallels and contrasts between parasitic and free-living nematode groups, this book integrates strategies that enable nematodes to persist in the absence of food with tactics used by parasitic forms to survive the defence responses of a plant or animal host. This functional study is an essential resource for researchers in nematology, parasitology and zoology.