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Approximately 67% of the world's described fauna and flora which is nearly one million described species (May 2000) comprise the Order Insecta. Insects are central to the activities of many ecosystem processes. However, their role as herbivores that conflicts arise with agricultural production due to direct consumption of cultivated crops and indirect damage by plant virus transmission or spoilage of potential yield. The control of the insect pests has been primarily through the use of chemical pesticides. The indiscriminate use of conventional chemical insecticides has resulted in a number of serious problems, e.g. resistance to the available chemical insecticides, elimination of natural enemies, persistence in the environment, toxicity to humans and wildlife and higher cost of crop production (Khan and Selman, 1989). A large number of insects and mites are capable of tolerating virtually all pesticides available for their control as a result of cross and multiple resistances (Metcalf, 1980). Recognition of the harmful effects of pesticides has prompted the development of alternative, less harmful management strategies, such as the use of microbial control agents.
Entomopathogenic nematodes can be very effective biological control agents against a number of insect pests and possess several advantages over chemical pesticides (Kaya & Gaugler, 1993). For example, they can actively find their hosts, can recycle in the soil environment (Kaya and Gaugler, 1993), and are environmentally safe (Akhurst, 1990; Ehlers & Hokkanen, 1996). Moreover, they have a global distribution (reviewed by Hominick et al., 1996). Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae have great promise as biological alternatives to chemicals for the control of soil-inhabiting insect pests (Kaya and Gaugler,1993; ; Ehlers & Peters, 1995; Grewal and Georgis, 1998). Their impressive list of attributes including high virulence, ease of mass production, broad host range, and safety (exemption of registration by the U.S. Environmental Protection Agency) has generated a great deal of scientific and commercial interest in these insect-killing nematodes.
Steinernema feltiae is an effective bio-control agent against a wide range of lepidopteran (Kard et al. 1988; Williams & Walters, 2000), coleopteran (Kaya, 1985) and dipterans insects (Lindegren, et al. 1990; Peters & Ehlers, 1994) and pests (Premachandra et al. 2003). They are also effective to control some plant-parasitic nematodes (Perez and Lewis, 2004). Despite tremendous advances in research and development, implementation of these entomopathogenic nematodes under field conditions still remains hampered by the lack of predictability in efficacy of control (Georgis and Gaugler, 1991; Gaugler et al., 1997; Grewal and Georgis, 1998). Sensitivity to environmental stresses (heat, cold, UV, and desiccation) is one of the key factors attributed to the inconsistencies in the field performance of entomopathogenic nematodes (Kaya, 1990; Georgis and Gaugler, 1991; Gaugler et al., 1997). Genetic improvement has been proposed as a means of improving field efficacy of entomopathogenic nematodes (Gaugler, 1987; Kaya and Gaugler, 1993; Fodor et al., 1994; Burnell and Dowds, 1996). Entomopathogenic nematodes offer several advantages as subjects for genetic improvement including short generation time, small genome size, ease of culture, handling, and suitability for inundative applications (Gaugler et al., 1989b; Fodor et al., 1994; Ehlers et al.2001 and 2003) and has a world-wide distribution ( Hominick, 2002).
This study fulfils the following objectives:
Crossing of the most cold and desiccation tolerant strains of Steinernema feltiae.
Successive genetic selection of their cold and desiccation tolerance.
Investigation of the influence of cold and desiccation stress on fitness of the hybrid strains.
2. Literature review
In this review, information on the taxonomy, biology, life cycle of Steinernema feltiae has been summarized. Also the literatures on cross breeding, genetic selection of beneficial trait like heat, desiccation tolerance of entomopathogenic nematode, finally the influence of cold and desiccation stress on the fitness of hybrid strains have been discussed.
2.1. Entomopathogenic nematode: Steinernema feltiae Filipjev
Species: Steinernema feltiae
2.1.2. Life cycle and biology
A special developmental stage within the life cycle of all rhabditid nematodes is the dauer juvenile (DJ). This term dauer ( German for enduring ) was introduced by Fuchs (1915) and describes a morphologically distinct juvenile, formed as a response to depleting food resources and adverse environmental conditions. The third stage dauer juvenile occurs free in the soil that is well adapted to long-term survival in the soil (Susurluk & Ehlers, 2008) and seek suitable insect host ( Lewis, 2002; Torr et al. 2004). The DJ is the infective stage that carries 200-2000 cells of its symbionts in the anterior part of its intestine (Endo and Nickle 1994). . The symbiont of S. feltiae is X. bovienii (Akhurst & Boemare, 1988). Steinernema gains entry to the insect larva through natural openings (mouth, anus and spiracles). In the haemolymph, the nematodes encounter optimal conditions for reproduction. During recovery by the food signal (Golden & Riddle, 1982), the DJ release the symbiont cells into the insect's haemocoel. The bacteria produce toxins and other metabolites (Dunphy ans Webster 1988; Bowen et al. 1998), which contribute to overcome the insect's defence mechanisms and kill the insect within approximately 2 days after nematode invasion (Simoes and Rosa 1996). Some Steinernema also produce toxins that contribute to the pathogenicity of their symbionts (Ehlers et al. 1997). The bacteria proliferate and produce suitable conditions for nematode reproduction. Feeding on symbiont cells, they develop into adults and produce offspring. Nematode reproduction continues over two or three generations until the nutrient status of the cadavar deteriorates whereupon adult development is suppressed and DJ accumulate which retain the symbiotic bacteria in the intestine ( Popiel et al. 1989).
In Steinernema reproduction is amphimictic. Steinernematid DJ mature to become either a male or a female and sex determination appears to be of the XX/XO type, typical of nematode (Dix et al., 1994). Griffin et al., (2001) identified a hermaphrodite strain of Steinernema.
Growth and reproduction of adult stages is also influenced by the nutritional conditions.
Steinernematid adults responds to depleting food resources with the cessation of egg laying. Juveniles hatch inside the uterus and develop at cost of the maternal body content causing the death of the adult ( endotokia matricida). DJ yield in second and third generation adults of S. feltiae in monoxenic liquid cultures is less than S. carpocapsae probably due to the increasing bacterial population ( Hirao et al, 2010).
Fig. life cycle of Steinernema feltiae
2.2. Genetic Improvement
2.2.1. Genetic selection
Genetic selection can be a powerful tool to increase beneficial traits in biological control agents. The use of genetic selection for the improvement of beneficial traits to overcome limitations, was first suggested for EPN by Gaugler (1986) and first results were reported on improved host finding abilities of S. carpocapsae by Gaugler et al. (1989). Strauch et al. (20040, Ehlers et al. (2005) and Mukuka et al. (2010a, 2010b), demonstrated that desiccation and heat tolerance of Heterorhabditis bacteriophora can be improved by genetic selection.
Selection of proper candidate species, target traits, and provision of adequate genetic variability in base populations are the essential prerequisites for a sound genetic improvement program (Hoy, 1986; Gaugler et al., 1989b; Hastings, 1994). With sufficient variability for the desired traits in the natural isolates a systematic selection programme can be initiated by starting genetic selection (Fig. 1). Successful genetic selection depends on high heritability (h2) of the targeted trait in the population ( Hartl and clark 1989). The high h2 attributes to a high genetic variability. The h2 of heat and desiccation traits in Heterorhabditis bacteriophora have been determined already. Strauch et al. (2004) evaluated desiccation tolerance of a hybrid strain and found h2= 0.46 for desiccation tolerance of non-adapted populations and h2= 0.48 with inclusion of an adaptation phase. The h2 of heat tolerance was found to be 0.68 by Ehlers et al. (2005).
Collection and evaluation of wild-type populations from diverse locations are necessary for providing adequate genetic variability in target traits for selection. Biodiversity of natural isolates has been a rich source for genetic variants in domestication of crops and livestock. Hopper et al. (1993) worked on the natural isolate IS-5, demonstrates the importance of collecting and preserving the rich genetic diversity available in nature, with particular emphasis on sampling from as many ecologically and geographically diverse sites as possible.
It is critical that assessments on natural populations are made using newly field-collected populations. Grewal et al. (1994b) studied the thermal niche breadths for strains of Steinernema spp. Strains, cultured in the laboratory for several years, may lost some of the actual variability (Grewal et al. 1994b). Dramatic changes in biological traits of entomopathogenic nematodes during laboratory adaptation have been demonstrated in several studies (Grewal et al., 1996; Stuart and Gaugler, 1996; Wang and Grewal, 2002). So, all newly isolated populations should cultured once in the wax moth Galleria mellonella L. larvae at 25°C following isolation as described by Kaya and Stock (1997) and stored in liquid nitrogen as described by Curran et al. (1992) to prevent changes in field-adapted traits due to frequent culturing in the laboratory.
Molecular approaches for estimating the degree of genetic variation in a population, e.g. RAPD analysis, are now readily applicable to EPN (e.g. Hashmi et al., 1996; Shapiro et al., 1997b), and should facilitate selection studies.
Selection candidate nematode
Estimate heritability of desired trait
Maximize nematode genetic variability
Stress tolerance selection assay
Monitor selection progress
Characterize improved nematode
Green house trials
Evaluate stress tolerance performance
under field conditions
Fig.1. A systematic plan for the design of a genetic selection programme of entomopathogenic nematode (adapted from Gaugler et al. 1989, Burnell 2002).
2.2.2. Cold tolerance in Steinernema spp.
Temperature extremes are among the limiting factors affecting the survival of Heterorhadbitis spp. and Steinernema spp. Temperature has effect on infectivity, survival and persistence of steinernematids and heterorhabditis (Molyneux 1986; Griffin and Downes 1991; Kung et al. 1991; Wright 1992; Grewal et al. 1993, 2002). Extended exposure to temperatures below 00C and above 400C is lethal to most EPN species but the effect depends on exposure time (Koppenhofer 2000). Temperature influences the rate at which food reserves (lipids, proteins and carbohydrates) are utilized by nematode. When EPN are exposed to sudden rise (heat-shock) or fall (cold-shock) in temperature, they synthesize proteins called heat-shock proteins ( Jagdale et al. 2005).
Temperature had a direct effect on the time of death, penetration rate, emergence time of infective juveniles, and number of emerging infective juveniles of S. feltiae (Kaya et al. 2001). Somasekhar et al. (2002) reported survival between 37% and 82% among 14 strains of S. carpocapsae exposed to 400C for 2 h.
Screening for cold and desiccation tolerance among wild populations isolated from cooler region of the world is a better basis for selection process. Grewal et al. (1994b) studied the thermal niche breadths for infection, establishment, and reproduction for strains of Steinernema spp. Galleria mellonella (wax moth) larvae were infected by Steinernema riobravis at the widest temperature range (10-39°C), whereas S. feltiae at the narrowest (8-30°C). They found thermal niche breadth for establishment within hosts was the widest for S. glaseri, (10-37°C) and the narrowest for S. feltiae (8-30°C) and thermal niche breadth for reproduction was widest for S. glaseri (12-32°C) and the narrowest for S. carpocapsae (20-30°C). Steinernema scapterisci (20-32°C), S. riobravis (20-35°C), and Steinernema sp. (20-32°C) were more adapted to warm temperature reproduction, and S. feltiae to cooler temperatures (10-25°C)
Several H. bacteriophora strains have been isolated and their temperature preferences were described (Grewal et al. 1994; Glazer et al. 1996). Mukuka et al. (2010a) screened 36 natural populations and 18 hybrid or inbred strains of Heterorhabditis bacteriophora for their response to high temperature with or without prior adaptation to heat at 350C for 3 h. The assessment of the heat tolerance was done as described by Ehlers et al. (2005). Five cover-slide chambers containing 5 ml tap water were filled with 200 DJs each of one strain. The chambers were then distributed on a temperature gradient generated on an aluminium bar at temperatures between 320Cand 410C for 2 h. The temperature at the bottom of the chambers was recorded by a platinum Pt 100 thin layer sensors. They found mean tolerated temperature ranged from 33.30C to 40.10C for nonadapted and from 34.80C to 39.20C for adapted strain populations. They did not observe any correlation between tolerance assessed with and without adaptation to heat, implying that different genes are involved.
The thermal niche for entomopathogenic nematode are species-specific and do not generally relate to the mean temperature of the original locality of the isolate (Grewal et al. 1994; Hazir et al. 2001; Mukuka et al. 2010a).
2.2.2. Desiccation tolerance in Steinernema spp.
EPN use various strategies to maximize their survival in desiccated condition.
Behavioral adaptation like loose coiling and clumping has been observed among different Heterorhabditids and steinernematids. O' Leary et al. (2001) observed this phenomenon in H. bacteriophora, H. megidis, H. zealandica, H. indica and Solomon et al. (1999) in S. feltiae, S. carpocapsae and S. glaseri. This clumping and coiling assist in reducing the rate of water loss from the nematode during desiccation, thus allowing more time for suitable physiological changes in response to water loss. Desiccated nematode might live longer than non-desiccated nematode because of the reduced metabolism.
Common for all rhabditid nematodes are non-feeding and developmentally arrested third stage dauer juveniles (DJs). Survival of DJs during storage or in-transit to end-users is a vital factor limiting the use of EPN. Entomopathogenic nematologist community accept that prolongation of EPNs storage is best achieved by induction of a dormant state. Anhydrobiosis, "life without water" is a state of dormancy that is reversible and is caused by desiccation (Georgis et al. 1994; Georgis, 1990). Anhydrobiosis is an important means of achieving storage stability of EPN. This can be attained by evaporative or osmotic partial dehydration (Glazer, 2002; Perry, 1998).
Tolerance to desiccation in Heterorhabditis spp. is also due to the accumulation of glycerol, whereas trehalose synthesis was recorded in steinernema carpocapsae. During desiccation, presence of unsaturated fatty acids has been observed in Heterorhabditis bacteriophora (Selvan et al. 1993b). Chen et al. (2006) identified 10 desiccation-response proteins in Steinernema feltiae IS-6 infective juveniles by using Peptide mass mapping with MALDI-TOF mass spectrometry (MS) among among which several are known to be stress responsive including heat shock protein 60, coenzyme q biosynthesis protein, inositol monophosphatase and fumarate lyase that were found in both stresses.
In different formulations, the survival of DJs can vary significantly (Grewal, 2002; Strauch et al. 2002). At the beginning, on direct contact desiccation with the assistance of absorbents such as clay to achieve storage stability resulted in substantial success. The concentrated nematodes and an inert solid carrier, such as vermiculite or diatomaceous earth comprise the most commercial nematode product. In these formulations, nematodes are partially inactivated as a result of incomplete dehydration that induces a semi-dormant state considerably prolonging the nematodes' lifespan and enabling them to withstand the rigors of a fluctuating temperature regime that is typical when commercial products are shipped and applied (Perry, 1998). For example Steinernema carpocapsae survived up to seven months at 250C in a water-dispensible granular (WG) formulation compared to four months in tap water (Grewal 2000a). The WG formulation extended the shelf-life of S. carpocapsae with above 80% survival at 250C. The WG formulation facilitated transportation of EPN products at ambient temperatures as opposed to overnight shipments on ice (Grewal 2000a). This enhanced storage stability could have been attributed to the induction of partial anhydrobiosis that resulted in a reduced DJ metabolic activity at 250C.
Attempts to improve the desiccation tolerance were reported by Strauch et al. (2004). Strauch et al. (2004) assessed a heritability of the desiccation tolerance of h2 = 0.48 when populations had been adapted to desiccation prior to exposure to stress and of 0.46 without adaptation. The heritability of the trait 'desiccation tolerance' determined by using homozygous inbred lines.
They assessed the desiccation tolerance in liquid, hygroscopic poly (ethylene glycol) 600 solution. To produce different levels of desiccation stress they transferred DJ into different concentration of PEG 600. Desiccation stress was measured as water activity (aw value). The water activity is defined as the relative proportion of unbound water in a sample. The lower the water activity of the solution, the stronger is the removal of water from the DJ. The mean tolerated aw-value ranged between 0.89 and 0.81 when DJs had been adapted to desiccation prior to stress exposure.
Mukuka et al, (2010b) screened the desiccation tolerance of 43 strains of Heterorhabditis spp. and 18 hybrid/inbred strains of H. bacteriophora. They also measured the dehydrating condition as water activity (aw value) by treating DJ with different concentrations of the non-ionic polymer poly(ethylene glycol) 600. They found the mean tolerated aw value for 50% population ranged from 0.90 to 0.95 for non-adapted and 0.67 to 0.99 for adapted nematode populations and the lowest aw value tolerated by 10% of the population (MW10) ranged from of 0.845 to 0.932 for non-adapted nematode populations and 0.603 to 0.950 for adapted nematode populations.
Hybridization can be a powerful tool for genetic improvement of beneficial traits in the entomopathogenic nematode. The amphimictic nature of S. feltiae makes it possible to cross males and females for creation of hybrids. Most tolerant 10% individual of cold and desiccation tolerant strains of S. feltiae will selected for crossing to make a hybrid with more cold and desiccation tolerant.
Improvement of heat and desiccation tolerance in H. bacteriophora through cross-breeding was done by Mukuka et al, (2010c). To achieve this goal, most tolerant strains which have been identified within screening for heat (Mukuka et al. 2010a) and desiccation tolerance (Mukuka et al. 2010b) were hybridized and checked for their tolerance. Crosses were done according to Iraki et al. (2000).
126.96.36.199. Determination of and genetic selection of cold tolerance
Improvement of cold tolerance of the SN strain of Steinernema feltiae together with its bacterial symbiont, Xenorhabdus bovenii Grewal et al. (1996) repeatedly passage the strain through the wax moth Galleria mellonella larvae at 15°C. They found that cold selection enhanced nematode virulence at the cooler temperatures. Virulence measured as total insect-mortality at 8°C improved by 5.3- and 6.6-fold after six and 12 passages, respectively. They observed nematode establishment improved at all temperatures after 12 passages and the highest increase of 9-fold was observed at 8°C.
Ehlers et al. (2005) increased the mean tolerated temperature from 38.5 to 39.20C in H. bacteriophora by 4 selection steps and decreased mean temperature at which the dauer juveniles of H. bacteriophora were active, from 7.3 to 6.1 0C during the first 5 selection steps through selective breeding.
Mukuka et al. (2010c) improved the heat tolerance through cross breeding of tolerant strains of H. bacteriophora and successive genetic selection. The heat tolerance of hybrid strains was assessed according to Ehlers et al. (2005) and Mukuka et al. (2010a). After eleven selection, they observed mean heat tolerance increase 5.50C when nematodes had been adapted to heat stress. For non-adapted tolerance an increase of 3.00C from 40.10C to 43.10C was recorded
188.8.131.52. Determination of and genetic selection of desiccation tolerance
Cross-breeding of most desiccation tolerant strains of H. bacteriophora (Mukuka et al, 2010b) reduced the aw-value from 0.67 to 0.65 after adaptation and from 0.9 to 0.7 without prior adaptation after six selection steps ( Mukuka et al. 2010c). Desiccation tolerance was assessed as described by Strauch et al. (2004) and Mukuka et al. (2010b).
Salame et al. (2010) bred a heterogeneous population Steinernema feltiae for desiccation tolerance and enhanced host-seeking ability. Survival rate of 80-90% was reached after ten selection cycles for tolerance of rapid desiccation by exposing infective juveniles (IJs) to ambient conditions (22-25°C; 50-65% r.h.) for 100 min and survival rate of 80-90% was reached after ten selection cycles for tolerance of slow desiccation by exposed IJs to 97% r.h. for 72 h. Host finding ability of the nematode increase >75% after 25 selection cycles.
2.2.4. Fitness of cold and desiccation hybrid strain
Selection of any trait can be followed by changes in the genome that can affect expression of second trait; in most cases results in reduction of fitness (Falconer, 1981). There is need to mass produce the resultant hybrid strain so that the gained traits of cold and desiccation can be stabilized (Bai et al. 2005). So, monitoring of beneficial traits like infectivity, virulence, host penetration, reproductive potential are essential during attempts to genetically improve other traits by crossing tolerant strains or selective breeding.
When a desired phenotype is obtained, there is a tendency of selected strains to revert gradually towards the unselected state (Hastings, 1994). This can be prevented by cryopreserving and reapplying selection pressure at regular interval on the selected hybrid strains.
Mukuka et al. (2010e) compared the fitness of heat and desiccation tolerant hybrid strains of Heterorhabditis bacteriophora to the commercial strain EN01 and found that only heat tolerant strains were superior or similar in fitness in virulence, host penetration and reproductive potential to strain EN01 but strains with increased desiccation tolerance were usually less fit. There was high chance that genes involved in the desiccation tolerance might be masking the expression of the virulence, reproductive potential and host penetration traits. They also found that the hybrid strains with inclusion of an adaptation phase showed more fitness than those hybridized without including an adaptation phase.
Desiccation stress can affect to the infectivity of the entomopathogenic nematode. Mukuka et al. (2010e) found that desiccated Heterorhabditis bacteriophora are less virulent in Galleria mellonella than undesiccated ones. But Shapiro & Lewis (1999); serve-Rodriguez (2004) found that desiccated Steinernema carpocapsae to be more virulent in G. mellonella that undesiccated ones. In addition Solomon et al. (1999) found no difference in infectivity between desiccated and nondesiccated S. feltiae strains that were exposed to meal worm, Tenebrio molitor.