For obligate parasitic species there are situations where persistence of a population requires survival of the infective stages outside the host. This may occur when the host is not available, requiring the nematode to survive in the absence of food, or environmental conditions exist that are not commensurate with life cycle progression, such as temperature extremes, osmotic stress and dehydration. Of the environmental extremes, desiccation survival has attracted most interest, partly because the ability to survive dry is implicit in the distribution of infective stages by wind and in dry plant material, such as seeds. Interest has also been generated by the spectacular abilities of some nematode to survive desiccation for periods considerably in excess of the normal life cycle. For example, D. dipsaci fourth-stage juveniles (J4) have been recorded as surviving for more than 20 years in the dry state, yet the whole life cycle of the nematode in favourable environmental conditions only takes about 4 weeks from egg to egg-laying adult; thus, the ability to survive in the dry state (anhydrobiosis) can prolong life by a factor exceeding 240 times!

Although some of the most astonishing survival attributes are found in species of nematodes, such as A. tritici, and D. dipsaci, that live within aerial parts of plants, the infective J2 of root-knot and, especially, cyst nematodes also survive for long periods as unhatched individuals inside the egg mass or cyst (Perry 1999). Morphological features such as the cyst, gelatinous matrix and eggshell all protect the J2 and ensure a slow rate of drying to ensure effective entry into anhydrobiosis. There is no one stage that can be termed the 'nematode survival stage'. Where species have the ability to suspend development and withstand environmental extremes, the stage(s) involved often differ. Similarly, there are variations in the behavioural and morphological adaptations that ensure desiccation survival, but all have the role of reducing the rate at which water is lost from the nematode as it experiences drying conditions (Perry and Moens 2011).

Galls induced by A. tritici in the tissues of host plants contain tightly packed aggregates of J2, each of which remains uncoiled when dry, yet the galls induced by A. amsinckia contain hundreds of desiccated adults and juveniles of all stages, many of which are coiled. Coiling and clumping are two behavioural responses that effectively reduce the surface area exposed to drying conditions (Perry 1999; Moens and Perry 2009). Reproduction of Aphelenchoides besseyi stops as rice grains ripen and adults coil in clumps beneath the hulls of grains, where the nematodes can remain

Fig. 1.4 Narcissus bulb with accumulation of Ditylenchus dipsaci fourth-stage juveniles (J4) as dry 'eelworm wool', and an inset showing a transmission electron microscope image of the eelworm wool with coiled, clumped, desiccated J4. (Photos courtesy of Roland N Perry, Rothamsted Research, UK; from Moens and Perry 2009)

Fig. 1.4 Narcissus bulb with accumulation of Ditylenchus dipsaci fourth-stage juveniles (J4) as dry 'eelworm wool', and an inset showing a transmission electron microscope image of the eelworm wool with coiled, clumped, desiccated J4. (Photos courtesy of Roland N Perry, Rothamsted Research, UK; from Moens and Perry 2009)

viable for 2-3 years in dry grains. As mentioned above, in D. dipsaci development stops at the J4 stage and hundreds of desiccated individuals coil and clump in masses termed 'eelworm wool', often associated with infected narcissus bulbs (Fig. 1.4) or inside bean pods. The death of the peripheral J4 apparently provides a protective coat that aids survival of the enclosed nematodes by slowing their rate of drying, in a manner that Ellenby (1969) termed the 'eggshell effect'. Survival of J4 of D. dipsaci and J2 of Anguina spp. is also associated with an intrinsic property of the cuticle, involving an outer lipid layer (Preston and Bird 1987; Wharton et al. 1988), to control the rate of water loss. The cuticle dries more rapidly than other layers and slows down the rate of water loss of internal, and perhaps more vital, structures. As dry individuals in galls, as 'eelworm wool', in plant tissue, or in cysts the nema-todes can survive for many years and withstand other adverse conditions, such as extremes of temperature; they are also more resistant to non-fumigant nematicides and can be dispersed effectively by wind.

Why is the rate of water loss important for survival even though the nematodes eventually lose all their body water? A slow rate of water loss appears to allow orderly packing and stabilization of structures to maintain functional integrity during desiccation. At water contents below about 20%, there is no free water in the cells. This 20%, usually referred to as 'bound water', is involved in the structural integrity of macromolecules and macromolecular structures, such as membranes. In desiccated, anhydrobiotic nematodes it is probable that the bound water has been lost and research has centred on molecules that might replace bound water and preserve structural integrity. The control of water loss enables biochemical changes to take place, including replacement of bound water, thus ensuring long-term survival of true anhydrobiotes (Barrett 1991; Perry 1999).

Ditylenchus dipsaci J4 and A. tritici J2 sequester trehalose, which has frequently been suggested as a desiccation protectant because of its role in preserving membrane stability, preventing protein denaturation and acting as a free-radical scavenging agent to reduce random chemical damage (Barrett 2011). However, there are contradictory reports about the importance of trehalose (Burnell and

Tunnacliffe 2011). For example, all stages of the mycophagous D. myceliophagus survive desiccation poorly, even at high humidities (Perry 1977). The survival of D. myceliophagus was unrelated to their trehalose content, and elevated levels of trehalose, generated by providing the nematodes with different food sources, did not enhance anhydrobiotic survival of this species (Womersley et al. 1998). Synthesizing trehalose during dehydration may indicate preliminary preparation for a period in the dry state, but it does not necessarily mean that survival during subsequent severe desiccation is assured. Following trehalose synthesis, it appears that other, at present unknown, adaptations are required at the cellular and subcellular levels for nematode survival, and rate of drying still has to be controlled (Higa and Womersley 1993).

Late embryogenesis abundant (LEA) proteins have been associated with survival in some nematodes, including C. elegans (Gal et al. 2004; Burnell and Tunnacliffe 2011), and LEA proteins may protect cellular components against the effects of desiccation (Goyal et al. 2005). Homologues of LEA genes have been identified in B. xylophilus (Kikuchi et al. 2007).

Aspects of the biochemical adaptations and the genes that are switched on during the induction of anhydrobiosis are likely to become evident when progress is made with comparative genomics and transcriptome analyses. In addition, the molecular information will also progress our understanding of the extent of the 'dauer' phenomenon in plant-parasitic nematodes. The dauer stage of the free-living nematode, Caenorhabditis elegans, represents a developmental arrest (Riddle and Albert 1997). Dauer larvae are enclosed by a dauer-specific cuticle and exhibit several characteristics associated with survival of adverse conditions, including reduced metabolism, elevated levels of several heat shock proteins and an enhanced resistance to desiccation (Kenyon 1997). Bird and Bird (1991) suggested that the survival forms of Anguina, may be regarded as dauers. In Ditylenchus dipsaci, the J4 that accumulate in response to adverse conditions are larger, have more lipid reserves and show a propensity to aggregate compared with J4 in a population feeding and developing under ideal conditions (Perry, unpublished), all properties that reflect the dauer state. In some species of the genus Bursaphelenchus a dauer form (J4) is present as a specialised survival and dispersal stage of the life cycle. Bursa-phelenchus xylophilus is a migratory endoparasitic nematode that has a complex life cycle involving beetles of the genus Monochamus as the vector (Mota et al. 2008). Bursaphelenchus xylophilus has a dauer stage, which uses the insect for transport to susceptible hosts where the nematodes enter the shoots of trees through the feeding wounds caused by the vector. In an analysis of more than 13,000 ESTs from B. xylophilus, Kikuchi et al. (2007) looked for homologues of 37 genes involved in dauer entry and maintenance in C. elegans. They identified 31 homologues of 18 C. elegans genes, including nine homologues for daf (dauer formation) genes.

Meloidogyne hapla carries 14 orthologues of C. elegans daf genes as well as three further matches that are weak (Abad et al. 2008; Abad and Opperman 2009) but it does not carry the daf-28 orthologue, which is key in the signal transduction pathway. Abad and Opperman (2009) conclude that basic development mechanisms are conserved, although signalling is not. Thus, there may be marked differences between free-living and parasitic nematodes in developmental response to adverse changes in the environment.

These studies provide initial evidence that the dauer phenomenon may be more widespread than currently recognised. Certainly, the indications in some species of plant-parasitic nematodes of an alternative developmental stage similar to a dauer larva are convincing (see Sect. 1.8 above). However, there are difficulties in relating information on dauer formation in C. elegans to parasitic nematodes. Comparison of expression profiles of dauer genes in C. elegans and in survival stages of parasitic nematodes (Elling et al. 2007) reveals marked differences in expression patterns between C. elegans and other nematodes; as yet, there is insufficient information to be able to link individual daf genes to specific survival traits.

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