Heat and desiccation tolerant

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Introduction

In the past two decades there has been immense growth of interest in the use of nematodes as biological pest control agents, with a lot of efforts being devoted to research that now focus on the potential of nematodes to control and manage destructive crop pest such as insect pests, molluscs, soil-borne plant pathogens and plant nematodes in general. Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditids are currently being used successfully as biological control agents against a wide group of pest insects, such as sciarid flies, weevils, scarab grub, thrips, mole crickets (Nyasani et al., 2008) and western corn root worm (Nadasy et al., 2008). These nematodes have also been reported to infect a number of lepidopterous species where they are able to recycle in the infected host and hence persist in the environment for longer periods of time (Tomalak, 2003). The entomopathogenic nematodes (Steinernema and Heterorhabditis) and slug-parasitic nematodes (Phasmarhabditis) have proven particularly successful and are now commercially mass produced worldwide to treat pest problems in agriculture, horticulture and veterinary and human husbandry (Grewal et al., 2004).

According to Grewal et al. (2005b), entomopathogenic nematodes and their symbiotic bacteria together are very crucial in the control of insect pests biologically, and they produce infective Dauer juvenile that are mostly in the soil (Sursurluk and & Ehlers, 2008) where they find a suitable insect host and finally invade through the natural body openings or the cuticle (Griffin et al., 2005) leading to infection, establishment (after overcoming the insects immunity) and reproduction. This is the only stage that can infect insect hosts that is well adapted for long term survival in the soil due to fat reserves, non-feeding and ambushing behaviour.

Heterorhabditid nematodes are obligate insect-parasitic that are highly specialised in their relationship with the mutualistic symbiont bacteria, Photorhabdus which they release (Boemare et al. 1993) after penetration and subsequent entry into the insect haemocoel. The released bacteria reproduce and increase rapidly, killing the host via septicaemia usually within 1 to 3 days (Dowds and & Peters 2002). These bacteria also provide essential nutritive source to the nematode infective juveniles (Han and & Ehlers, 2000) for growth and reproduction. The infective juveniles eventually mature to become self- fertile hermaphrodite females whose offspring contain both amphimictic males and females (Dix et al., 1992) and automictic stages (Strauch et al., 1994).The food source ultimately depletes over time, leading to the formation of infective dauer juveniles which exit the cadaver to search for new insect hosts (Poinar, 1990). The Dauer juveniles are more resistant to shear stress making their application using conventional spraying technology feasible (Wright et al., 2005).

However, the use of entomopathogenic nematodes as biological control agents is generally hampered by many biotic and abiotic related limitations. Desiccation tolerance is one important factor affecting commercial use of the nematodes at every stage, from their mass production to application in the field (Strauch et al., 2004). Variations in degrees of stress tolerance have been reported in natural wild EPN populations which may be insufficient to permit their commercialisation (Glazer, 2002). In addition, Grewal et.al (2005) noted the possibility of improving the potential of entomopathogenic nematodes through the enhancement of dauer juvenile longevity, bacterial retention, tolerance to heat, desiccation and resistance to encapsulation in the haemocoel of some key insect pests and trait stability.

Temperature is an important factor influencing host invasion and host mortality, development of the IJs to adults in the host and reproduction as earlier reported by Grewal et al. (1994), while studying eight different species of EPNs. They noted that Steinernema feltiae infected and established in Galleria mellonella (L.) larvae between 8 and 30 °C and reproduced between 10 and 25 °C, while Steinernema riobrave infected and established between 10 and 39 °C and reproduced between 20 and 35 °C. However, the lowest temperature for successful infection did not differ much among the nematode species with exception of S. feltiae which infected insects at 8 °C compared to 10 °C for all the other species.

Glazer et al. (1991) earlier found that heritability for heat tolerance for H. bacteriophora inbred lines was high (h2= 0.98). In addition they demonstrated that manipulation of temperature tolerance and temperature activity ranges through genetic selection at constant temperatures in the laboratory was possible. In recent research on different natural populations and strains of Heterorhabditis bacteriophora for their response to high temperature, Mukuka et al. (in press) demonstrated that, there is no correlation between tolerance assessed with or without adaptation to heat, implying that different genes are involved. They further noted that high variability in tolerance among strains and the relatively high heritability (h² = 0.68) as previously reported by Ehlers et al. (2005) for the adapted heat tolerance recorded for H. bacteriophora provide fundamental basis for future selective breeding with the objective to enhance heat tolerance of H. bacteriophora.

Similar work has been carried out for the desiccation tolerance showing that, selective breeding is a feasible approach to increase the tolerance of H. bacteriophora to desiccation only if infective juveniles are adapted to desiccation prior to exposure to desiccating conditions according to Strauch et al. (2004). They also found that the variability of desiccation tolerance within a population is positively correlated with the intensity of the stress during the initial adaptation phase increasing with the intensity of stress. Strauch et al. (2004) further exploited this increased variability of desiccation tolerance within a population for the improvement of desiccation tolerance through breeding and noted that all inbred lines were more tolerant than the hybrid strain PS7 from which inbred lines were produced. Glazer et al. (1991) similarly showed that the desiccation tolerance of most of the inbred lines produced from HP88 was higher than in the original population as highlighted by Grewal et al. (2006).

Objectives

Entomopathogenic nematodes are released with the aim of obtaining immediate pest suppression. Heterorhabditis bacteriophora has largely been used in pest control e.g. control of grubs and weevils (Grewal et al., 2005).

Recent field trials have proven its high potential to control larvae of the invasive pest Western Corn Rootworm (e.g., Toepfer et al., 2008). As such, they are expected to persist in the treated field at high numbers for at least 2-3 weeks (Georgis, 1994) to give effective pest control. Since long-term persistence is required for successful application, then the ability of the applied nematodes to tolerate the specific conditions should be determined (Grewal et al., 2006). The main objective of this study is therefore;

To develop specific primers for the identification of the heat and desiccation tolerant strains.This will include

  • Blast analysis of the heat and desiccation genes and comparison between different nematodes.
  • Selection of potential genes for amplification
  • Comparison of the sequences.
  • Design primers for distinguishing heat and/ or desiccation tolerance.

The null hypothesis

The null hypothesis is therefore that, "There is no genetic difference between the different strains of H. bacteriophora hence not possible to differentiate the heat/ desiccation tolerant strains from the rest" To test this hypothesis several set of primers will be developed and used for identification of different Heterorhabditis bacteriophora strains with low or high tolerance to heat and desiccation.

Heat tolerance

The effect of temperature on infectivity, survival and persistence of steinernematids and heterorhabditids has been clearly demonstrated (Grewal et al., 1993), with Somasekhar et al. (2000) further reporting survival between 37% and 82% among fourteen strains of S. carpocarpsae exposed to 400C for 2 hours. However as Koppenhofer (2000) noted, long time exposure to temperatures less than 00C and above 400C is lethal to most EPN species though the effect depends on overall time.

Most cells respond when exposed to high temperatures than their optimal physiological temperature (Hashimi et al.,1998), by production of heat shock proteins which function as chaperones with the ability to protect newly made proteins from misfolding or helping them to refold (Nollen and & Morimoto, 2002). The major heat-shock proteins that have chaperone activity belong to five conserved classes: HSP100, HSP90, HSP70, HSP60 and the small heat-shock proteins (sHSPs) as noted by Kyeong et al., 1998. However in view of Song et al. (2005), these proteins have been classified into several families according to their apparent molecular mass, such as HSP90 (85-90 kDa), HSP70 (68-73 kDa), HSP60, HSP47, and low molecular mass Hsps (16-24 kDa).

In addition, Lindquisti et al. (1988) illustrated that eukaryotic cells respond to various stresses by trigerring the production of new sets of proteins including a group of heat shock proteins, among which small heat shock proteins form a diverse family of proteins produced in all organisms .e.g. through accumulation of heat shock proteins, Plants have been reported to develop many adaptive strategies to boost heat stress tolerance ability at molecular level (Rachmilevitch et al., 2006a) .It has been further shown that some heat stress responsive genes are heat shock-factor dependent genes in Arabidopsis thaliana (Busch et al., 2005), largely involved in physiological stress adaptation. In addition, isolation of genes that are responsive to high temperatures in plants like A. thaliana (Lim et al., 2006) revealed that several genes are required for metabolism, cell cycle and protein fate among others. It is documented that Heat shock factors (HSF) are the transcriptional regulator of the heat shock proteins, whereby, under normal conditions they operated as non-DNA binding monomer in the cytoplasm, but translocate to the nucleus when activated by heat shock or other stress where it trimerises and bind to heat shock elements (Devany, 2006),

The C. elegans genome contains an HSF- like gene (Y53C10A.12) on chromosome 1 encoding a predicted protein of 671 amino acids (Devany, 2006), 16 genes encoding 14 distinct small-heat shock proteins among them HSP-16.1 /HSP-16.48 and HSP-16.2/ HSP-16.41 which are major HSP-16 proteins, and these four Heat shock proteins (HSP) are the same in gene structure and amino acid sequence (Candido, 2000). In their experiments, Mingi et al. (2004) compared the responses of HSP-16.1, HSP-16.48, HSP-16.2, and HSP-16.41 to heat shock, alcohol, and hypoxia. None of the four genes were expressed under the normal condition, but were highly induced by heat shock and ethanol by using GFP based reporter transgenes for assaying gene expression.

In addition, during the comparisons of the promoter sequences a new conserved sequence pattern CAC (A/T) CT was revealed. This was shown to be important for the orientation-dependent hypoxia response but not for other response such as heat or ethanol (Hong et al., 2004). For further analysis of hypoxia response of the hsp-16genes in the nematodes, Hong et al. (2004) identified the C. briggsae hsp-16 genes available in the genome data base. It was reported that C. briggsae genome contains 10 hsp-16 genes (cb-hsp-16) unlike 6 in C. elegans, the cb-hsp-16 gene pairs had duplicated pattern in their genomic organisation similar to hsp-16 genes in C. elegans. (Hong et al., 2004). However the patterns were different with the C. elegans, resulting in almost identical pairs of genes encoding hsp-16.1 and hsp-16.48, those of cb-hsp-16 were duplicated in tandem orientation.

The variations in the number of genes in these closely related nematodes species indicates that the duplication of the heat shock proteins genes is a recent evolution event that occurred independently in each species. It's further documented that the heat shock elements (HSEs) for C. elegans hsp-16.1 genes are also conserved in the C. briggsae hsp-16 genes. Another conserved block was called ethanol and stress response element (ESRE) whose sequences were partially identified (GuhaThakurta et al., 2002) as a novel heat shock element. These conserved blocks had the following sequence; CAC (A/T) and GTG (A/G) GTG respectively. C. briggsae choice was due to its good characteristics as model for studying genetic functional conservation in nematodes. Partial sequencing of a cDNA library of infective juveniles of H. bacteriophora has revealed the presence of at least three temperature tolerance genes. Two of these genes, hsp 4 and hsp 6, belong to the hsp 70 family and one tre-1 (glycocyl hydrolase) is a precursor of trehalase (Sandhu et al., 2005).

Small Heat Shock Proteins (sHsps)

Small heat shock proteins are ubiquitous family of proteins with homologs present in eukaryotes, bacteria and archea, indicating that this family originated before the divergence of the three domains of life (Aevermann and & Waters, 2008). According to Nelly et al., (2002), many sHsps have been shown to be efficient at binding denatured proteins, and current models suggest that sHsps operate as molecular chaperones preventing irreversible protein aggregation (Horwitz et al., 1998). In addition to functioning as molecular chaperones to protect proteins from being denatured in high temperature stress (Van Montfort et al., 2001), sHSPs can also assist in protection in the conditions of other stresses, such as cold, drought, oxidation, hypertonic stress, UV, and heavy metals (Waters et al., 2008).

De Jong et al., (1998), Waters and Vierling, (1999) and Franck et al., (2004) noted that there is a high diversity of the sHSPs, with only a few of amino acid residues conserved in all small heat shock proteins. This is in contrast with the conservation seen among the other heat shock proteins including the HSP70s (Boorstein et al., 1994). However there is considerable structural conservation among the sHSPs despite this high level of amino acid sequence diversity (van Montfort et al., 2001).

Heat shock protein 70 (HSP 70)

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily that is very essential in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress (Song et al.,2005). Its direct function being the major heat-inducible protein in the development of thermo tolerance has been demonstrated in a variety of organisms (Li et al., 1991). It has been documented that most eukaryotes posses a high number of genes encoding a set of related HSP 70 proteins, some of which are always present under optimal growth conditions while others are expressed only after stress (Craig & Gross, 1991). For example, in C. elegans six different members of hsp 70 family have been reported (Snutch et al., 1988), some of them being heat shock inducible and the rest not.

Using the cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) that was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE), Song et al.(2005) noted that AIHSP70 gene shared high identity with other known HSP70 genes after doing BLAST analysis. Moreover, 3-dimensional structural prediction of AIHSP70 showed that its N-terminal ATPase activity domain and C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family (Song et al.,2005) as further revealed by sequence alignment, structure comparison and phylogenetic analysis. HSP70 can be stimulated by several chemicals and biological stresses such as heat shock, heavy metals, oxidative stress, and amino acid analogues, UV and irradiation, inhibitors of energy metabolism, , viral and bacterial infections, parasitism, alcohol, crowding and inflammation (Hartl et al., 1996). Furthermore analysis of the role played by promoter and terminator regions of HSP 70, using plasmid vector consisting of the GFP reporter gene flanked by these regulatory elements confirmed activity of GFP gene under the control of the hsp70 promoter (Wippersteg et al., 2002) after application of heat shock for 3h at 420C.In addition, this provided evidence for evolutionary conservation of heat shock regulation among the eukaryotes.

Leal et al. (2008) used heat shock protein 70 (HSP 70) from Bursaphelenchus xylophilus and B. mucronatus for comparison and designing of primers Bx701F and BOW R which amplify a 171 base pair fragment from B. xylophilus by polymerase chain reaction (PCR). They also used Bm701F and Bm701R primers as control for the amplification of the 168 base pair fragment from B. mucronatus. Consequently, it was noted that the B. xylophilus primers detected 23 target copies with high sensitivity or an equivalent of one nematode (Leal et al., 2008). In addition, a real- time PCR that was highly specific and sensitive was developed with a primer set and a specific Taqman(R) fluorescent probe for the amplification of B. xylophilus Hsp 70 sequences. It has been documented that this RT-PCR could detect at least 5 bp of B. xylophilus genomic DNA including DNA extracted from single nematodes (Leal et al., 2008). As for the Hsp70 primers and Taqman® probe design, Leah et al. (2007) did and reported the following;

"Specific primers and Taqman® probe were designed to the B. xylophilus and B. mucronatus Hsp70 sequence alignment, showing nucleotide differences between these two species (Leal et al., 2005), by using Beacon Designer 4 (Premier BioSoft International, Palo Alto, CA, USA). The addition of locked nucleic acids (LNAs) in all three sequences was performed using the LNA design tool available from Integrated DNA Technologies, IDT (http://www.idtdna.com/analyzer/Applications/lna/).

Primes and probes were obtained from IDT (Integrated DNA Technologies, Coralville, IA, USA). The Taqman® probe was dual-labelled: at the 5' end with fluorescent reporter dye (6-carboxy-fluorescein, FAM) and at the 3' end with a dark quencher dye (Iowa Black, FQ).

The sequences of the forward and reverse primers were;

5'-TAAGATGTC+-TTTT+AC+AGATGC+CAAG-3' and

5'-GCC+TGGACGAC+CTTGAAT-3', respectively.

In another study by Hashimi et al. (1998), transformation of plasmid vector containing C. elegans hsp70 encoding gene in H. bacteriophora HSP 90 was successful with the expression of C. elegans hsp70 gene in transgenic H. bacteriophora being verified by Northern blot hybridization, while their thermal resistance was correlated by the production of hsp70 mRNA transcripts after various heat treatments. Since that time nobody could ever repeat the transformation of H. Bacteriophora successfully..

Heat Shock Protein 90 (hsp90)

Heat shock protein 90 (Hsp-90) is a highly conserved (Devany, 2005) essential protein in eukaryotes (Gillan et al., 2009),. that is is essential in developmental switching due to its interaction with regulatory proteins such as signalling and receptor molecules with important roles in cell division, cell cycle and apoptosis (Reviewed in Young et al., 2001).This together with its ability to play a role in stress response provide a link between various developmental pathways and environmental change as demonstrated by Rutherford and Lindquist, (1998). Recent studies have also shown that it can be secreted by tumour cells (Eustace et al., 2004), and adult Brugia parasites cultured in vitro (Kumari et al., 1994).

The C. elegans genome contains a single hsp90 gene (daf-21, C47E8.5) located on chromosome V. It's hsp90 mRNA was originally shown to be 10-15-fold enriched in dauer larvae compared with other life cycle stages (Dalley & Golomb, 1992). Further studies showed that stimulation of the worms to exit dauer stage, led to a fast decrease in the level of hsp90 mRNA within 2 hours. Jones et al. (2001) further documented that hsp90 is an abundant transcript in dauers compared with other life cycle stages, while the expression of hsp70 was equal between stages.

In C. elegans, Hsp90 is not significantly up-regulated in response to heat-shock (Devaney et al., 2005), in contrast to other stress-inducible Hsps such as Hsp16 while others have reported that C. elegans Hsp90 is mostly expressed in germ cells and in embryos but upon heat shock, is detectable in other cells (Inoue et al., 2000). RNAi studies have also described a role for Hsp-90 in adult worms, as injection of double-stranded RNA resulted in cessation of egg production and an embryonic lethal phenotype (Inoue et al., 2006). In general, Hsp-90 is essential in most organisms, including nematodes, due to the nature of its client proteins. For example, Ce-daf-21 null mutants arrest at the J2/J3 stage, while worms carrying a single point mutation in Ce-daf-21, a weak gain of function mutation, are dauer-constitutive (Birnby et al., 2000).

Desiccation Tolerance

Nematodes, can survive unfavourable environmental conditions in a dormant or inactive state which substantially extends their life span enabling them to withstand the adverse effects of the fluctuating environment (Barrett, 1991; Wharton, 2003). Dormancy is classified into diapause and quiescence. Diapause is a state of arrested development that does not continue until specific requirements have been provided, even if suitable environmental conditions return as noted by Grewal et al. (2006). Quiescence is a spontaneous reversible response to unpredictable unfavourable environmental conditions and is readily reversible when favourable conditions return (Wright & Perry, 2006). Further persistence of the unfavourable environmental conditions trigger a state of cryptobiosis in some organisms, where metabolism is undetectable. These stressful conditions include absence of water, extreme temperatures, anaerobic conditions and osmotic stress which induce anhydrobiosis, thermobiosis and cryobiosis, anoxybiosis, and osmobiosis, quiescent states respectively (Barrett, 1991).

EPNs are not fully anhydrobiotic hence are only able to withstand m oderate levels of desiccation ( O'Leary et al., 2004) . However they can be subjected to a gradual loss of water in their natural environment . While researching on the anhydrobio tic- effects on longevity and infectivity of dauer juveniles ( D Js) , using three species of entomopathogenic nematodes namely ; Steinernema carpocarpsae , Steinernema feltiae, and Steinernema riobrave at 5 0 C and 25 0 C , Grewal (2002) found out that , the longevity of S. carpocarpsae D Js was increased by three months and of S. riobrave by one month in Water -dispersible granules at 0.966±0.971 water activity and 25 0 C as compared with D Js stored in water as control. Additionally, comparison of s urvival and infectivity of the desiccated (anhydrobiotic) D Js with non-desiccated ones stored in water for different periods showed variations among the tested species . In summary , a shelf-life of five months for S. carpocarpsae at 25 0 C and nine months at 5 0 C in WG with over 90% IJ survival was attained. In contrast , more than 90% survival was recorded only for two months at 25 0 C and five months at 5 0 C in WG f or S. feltiae while S . riobrave had a survival rate of over 90% only for one month and dropped to 85% survival rate within one month at 25 0 C and 5 0 C respectively . He further conclusively noted that desiccation had no adverse effect on the performance of the three species in infection. The differences in IJ longevity and desiccation survival at different temperatures were linked to differences in foraging and temperature adaptation (Grewal , 2002).

According to Goyal et al. (2005), the molecular mechanisms controlling anhydrobiosis that is linked to desiccation, are not well understood. However there is considerable interest in the functions of the non-reducing disaccharide trehalose. Vogel et al. (2001) reported the presence of sugar trehalose in many microorganisms, nematodes, fungi, plants, insects and invertebrates. It has been further demonstrated that this disaccharide is present specifically in plant parasitic (Goodman et al., 1993), animal parasitic (Learmonth et al., 1987), entomopathogenic and free-living nematode species. This sugar is therefore thought to be essential in the nematode's physiology playing a role in sugar transport (Barrett, 1981), energy storage (Powell et al., 1986a, b), in the hatching mechanism of eggs (Perry, 1989) and protection against unfavourable environmental conditions (Wharton et al., 2002). Trehalose offers protection against stress through the preservation of lipid membranes and proteins stabilisation (Guo et al., 2000).

Laboratory studies have shown a conversion of up to 20% of the nematode Aphelenchus avenae dry weight to trehalose, after induction of anhydrobiosis through slow drying over time. This correlated with attainment of desiccation tolerance (Browne et al., 2004). Similarly in the budding yeast Saccharomyces cerevisiae accumulation of high levels of trehalose correlates with increased survival of desiccation as noted by Gadd et al. (1987) and Hottiger et al. (1987). It has been further argued that, the correlation of the acquisition of desiccation tolerance with disaccharide biosynthesis in naturally anhydrobiotic organisms is weak (Tunnacliffe and & Lapinski, 2003). In anhydrobiotic nematodes, production of trehalose in the intial stages of dehydration reaches maximum concentration before the achievement of full desiccation tolerance indicating that additional physiological adaptations are necessary (Higa and & Womersley, 1993; Womersley and & Higa, 1998; Perry, 1999). In addition, it has been documented that up-regulation of trehalose synthase genes can be used to enhance trehalose accumulation associated with anhydrobiosis (Goyal et al., 2005).

Trehalose synthase genes

D auer Juveniles of both Steinernema feltiae and Steinernema carpocapsae synthesise trehalose as a result of desiccation (O'Leary et al., 2001). It is purported that trehalose fills the space between the phospholipids replacing water in the cell membranes of desiccated cells , (Crowe et al. , 1984 ) , hence maintaining the integrity of the cells. O'Leary et al . ( 200 1 ) illustrated that preconditioning of two species of Heterorhabditis ( megidis and indica ) at 98% relative humidity triggered the synthesis of glycerol and not trehalose , whereas similar preconditioning induced trehalose synthase in Steinernema carpocarpsae and Aphelenchus avenae. They further concluded that this phenomenon indicate d that the probability of Heterorhabditis hav ing the essential metabolic responses to desiccation enabling it to enter into a fully anhydrobiotic state was low . Another study was carried out by Jagdale and Grewal (200 3 ) demonstrating the induction of trehalose accumulation in three Steinernema species in response to warm temperature acclimatisation , showing interspecific variations in the amount of trehalose accumulated at 35 0 C . S. feltiae had the highest percentage increment in trehalose accumulation, followed by S. c arporcapse and finally S. r iobrave . They further reported the same trend of trehalose increase in all the three species throughout the cold acclimation at 5 0 C .

Three major enzymes have been shown to be involved in catalytic metabolism of trehalose in eukaryotes namely ; trehalose-6-phosphate synthase ( TPS; EC2.4.1.15) and trehalose-6-phosphate phosphatise (TPP; EC3.1.3.12) both responsible for trehalose synthesis , which entails ch ange of glucose from U ridine d iphosphate -glucose to glucose-6-phosphate giving trehalose-6-phosphate, there after the removal of the phosphate to give trehalose . T rehalase (TRE; EC3.2.1.28) catalyses the hydrolysis of the sugar ( P ellerone et al. , 2003 ) . A lot of study has been done in y east on synthesis and hydrolysis of trehalose e.g. by ( Nwaka and Holzer, 1998) while Kaasen et al . ( 1994) ha ve used b acteria yeast to characterised tps genes . Others have done similar studies using insects (Chen et al., 2002) and plants (Vogel et al ., 2001). Using the yeast model Saccharomyces cerevisiae , N waka and Holzer ( 1998) described trehalose breakdown in eukaryotes while TRE activity was detected in nematodes (reviewed in Beh m, 1997) and insects (Becker et al ., 1996) besides being reported in mammals which do not synthesise trehalose . Goyal et al.,(2005) identified two trehalose-6-phosphate synthase ( tps ) genes in the anhydrobiotic nematode A . avenae which encode d very similar proteins comprising of the catalytic domain like that of the GT-20 family of glycosyltransferases similar to tps-2 of C. elegans . In order to generate gene-specific real-time PCR standards for the production of cDNA , " primers were designed that amplified a small region (~125 bp) of the target ( Aav-tps-1 and Aavtps- 2 ) and control ( Aav-ama-1 ) genes. Specific forward primers were used for Aav-tps-1 (5'-GAG CAG CAT TTG CAT ACA AAA AC-3') and Aav-tps-2 (5'-GAG TTT ACG TAC GAA CAA ATT GG-3') together with a common reverse primer 5'-GTT GTG CTG ACC TTA TTC GTC T-3'). " By further determining the f ull length cDNA sequences and comparing with genomic sequences, they found that there are at least 17 and 15 exons for Aav-tps-1 and Aav-tps-2 respectively.

Late embryonic abundant (LEA)-related proteins

Through the analys i s of seven different ESTs encoding LEA proteins , Tyson etal . (2007) reported the ir upregulation in response to desiccation stress in S. C c arpocarpsae dauer juveniles . LEA proteins had previously been reported in the last stage of desiccation during seed maturation where they were highly expressed (Dure et al., 1989), besides their presence in plant vegetative tissues due to dehydration, osmotic stress or low temperatures (Close, 1997) , and in anhydrobiotes during desiccation (Bockel et al., 1998). Similarly, it was discovered that during the induction of anhydrobiosis in A. avenae a LEA Group 3 gene Aav-lea-1 was highly induced (Browne et al., 2002) in addition to EST transcript upregulation in S. feltiae in response to desiccation (Gal et al., 2003). Moreover , for the model C. elegans , its genome encodes three lea genes and that of Drosophila melanogaster one lea gene (Browne et al., 2004). Gal et al. (2004) have also demonstrated the upregulation of the lea-1 gene in response to desiccation stress in C. elegans dauer juveniles and the resultant reduction in dauer juvenile survival during induction of desiccation, osmotic and heat stress after partial silencing of the C. elegans lea-1 gene by RNA interferenc e (RNAi) . The availability of genes encoding LEA3 proteins in microorganisms, animals and plants indicates its importance in managing water stress and is phylogenetic relations (Tyson et al., 2007).

Entomopathogenic nematode infective dauer juveniles have also been reported to manifest physiological mechanisms such as alteration in the proportion of saturated and unsaturated fatty acids, variations in the activity of metabolic enzymes and synthesis of novel isozymes during survival under cold or warm conditions (Grewal et al., 2006).

In a recent study undertaken by Somvanshi et al. (2008), four different genes (aldehyde dehydrogenase, nucleosome assembly protein 1, glutathione peroxidise and heat shock protein 40) were characterized during desiccation stress in five entomopathogenic species with differing stress tolerance. Heterorhabditis bacteriophora showed the highest expression of the all genes studied despite the fact that it was the most susceptible to desiccation. Furthermore, the study showed negative correlation between the survival ability of nematodes and the degree of expression of these genes whereby there was no induction of gene expression in stress tolerant nematodes while gene expression in stress-susceptible nematodes was effectively triggered by stress, with different levels of gene expression being related to the different stress-tolerance capabilities of the nematodes. These gene-expression ratios can potentially be used as markers of desiccation tolerance in entomopathogenic nematodes (Somvanshi et al., 2008).

As earlier highlighted, desiccation tolerance is a very important limiting factor in commercial production and use of the nematodes for biological control in general (Strauch et al., 2004). Moreover, desiccation of nematodes is not an isolated phenomenon but is usually accompanied by pre-exposure to osmotic stress, since solute concentrations increase upon drying of the soil (Grewal et al., 2006). Compared to the developmental EPN stages inside a host insect, the dauer juveniles are able to withstand stressful environmental conditions, like high temperatures and desiccation better (Glazer, 2002).

Population studies using molecular tools

The use of molecular techniques to identify intraspecific variations that may discriminate different biological characters might provide foundation populations for selecting positive characteristics (Liu et al., 2000). However, the lack of suitable molecular markers to aid in these selection/breeding studies makes the procedure difficult, long and tedious (Segal and & Glazer, 2000; Strauch et al., 2004).

Generally, molecular techniques are being used with increasing frequency to infer interspecific entomopathogenic nematode phylogenetic relationships (e.g., Szalanski et al., 2000; Guyen et al., 2001). However, it is rare that phylogenetic patterns or hierarchical structures have been inferred for same-species isolates as noted by Dillon et al., 2008) among others.

Exon-primed introns crossing polymerase chain reaction (EPIC-PCR)

Introns are present in all of the eukaryotic genomes that have been sequenced, including those of parasitic, unicellular eukaryotes (Embley and & Martin, 2006; Nixon et al., 2002; Simpson et al., 2002). These are the non- coding regions of the gene that are spliced post transcriptionally from the mRNA before translation into a protein. These non-coding sections are transcribed to precursor mRNA, and there after removed by a process called splicing during the processing to mature RNA. After intron splicing (i.e. removal), the mRNA consists only of exon derived sequences, which are translated into a protein (http://en.wikipedia.org/wiki/Intron).

The targeting of these introns in highly conserved nuclear genes, such as ß-tubulin, very essential for identification of high levels of variation within intraspecific populations (Lessa, 1992; France et al., 1999). Introns are characterised by high levels of sequence and length polymorphisms (Palumbi& Baker, 1994; Graur & Li, 2000), hence they are suitable molecular markers for studies of population structure within and among species, and also for reconstructing relationships among closely relate species (Regeai et al., 2008).

Stoltzfus et al. (1997) noted that the positions of introns in homologous genes do not always match; the percentage of common intron positions between these genes has declined with increased evolutionary distance. They finally concluded that most intron positions are a recent phenomenon, by intron sliding, intron gain, or both, since the introns observed in the existing genes occurred at many varying positions to have originated from the same ancestral gene, therefore showing high diversity.

In their study, Regeai et al. (2009) recently developed novel primer sets for the amplification of introns from 24 structural and housekeeping genes from H. bacteriophora Their results using exon-primed introns crossing (EPIC) polymerase chain reaction (PCR) primers showed variability in length and splice site of nucleotide interspecifically in the sequenced introns and for one gene an intron gain was observed. They further concluded that these intron primers would be useful molecular marker tools for studying population genetics, genetic diversity within the genus Heterorhabditis and other genera of rhabditid nematodes.

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