Approximately 67 % of the universe ‘s described zoology and vegetation which is about one million described species ( May 2000 ) consist the Order Insecta. Insects are cardinal to the activities of many ecosystem procedures. However, their function as herbivores that conflicts arise with agricultural production due to direct ingestion of cultivated harvests and indirect harm by works virus transmittal or spoilage of possible output. The control of the insect pests has been chiefly through the usage of chemical pesticides. The indiscriminate usage of conventional chemical insect powders has resulted in a figure of serious jobs, e.g. opposition to the available chemical insect powders, riddance of natural enemies, continuity in the environment, toxicity to worlds and wildlife and higher cost of harvest production ( Khan and Selman, 1989 ) . A big figure of insects and touchs are capable of digesting virtually all pesticides available for their control as a consequence of cross and multiple oppositions ( Metcalf, 1980 ) . Recognition of the harmful effects of pesticides has prompted the development of alternate, less harmful direction schemes, such as the usage of microbic control agents.
Entomopathogenic roundworms can be really effectual biological control agents against a figure of insect plagues and possess several advantages over chemical pesticides ( Kaya & A ; Gaugler, 1993 ) . For illustration, they can actively happen their hosts, can recycle in the dirt environment ( Kaya and Gaugler, 1993 ) , and are environmentally safe ( Akhurst, 1990 ; Ehlers & A ; Hokkanen, 1996 ) . Furthermore, they have a planetary distribution ( reviewed by Hominick et al. , 1996 ) . Entomopathogenic roundworms in the households Steinernematidae and Heterorhabditidae have great promise as biological options to chemicals for the control of soil-inhabiting insect plagues ( Kaya and Gaugler,1993 ; ; Ehlers & A ; Peters, 1995 ; Grewal and Georgis, 1998 ) . Their impressive list of properties including high virulency, easiness of mass production, wide host scope, and safety ( freedom of enrollment by the U.S. Environmental Protection Agency ) has generated a great trade of scientific and commercial involvement in these insect-killing roundworms.
Steinernema feltiae is an effectual bio-control agent against a broad scope of lepidopterous insect ( Kard et al. 1988 ; Williams & A ; Walters, 2000 ) , coleopteran ( Kaya, 1985 ) and dipterans insects ( Lindegren, et Al. 1990 ; Peters & A ; Ehlers, 1994 ) and plagues ( Premachandra et al. 2003 ) . They are besides effectual to command some plant-parasitic roundworms ( Perez and Lewis, 2004 ) . Despite enormous progresss in research and development, execution of these entomopathogenic roundworms under field conditions still remains hampered by the deficiency of predictability in efficaciousness of control ( Georgis and Gaugler, 1991 ; Gaugler et al. , 1997 ; Grewal and Georgis, 1998 ) . Sensitivity to environmental emphasiss ( heat, cold, UV, and dehydration ) is one of the cardinal factors attributed to the incompatibilities in the field public presentation of entomopathogenic roundworms ( Kaya, 1990 ; Georgis and Gaugler, 1991 ; Gaugler et al. , 1997 ) . Familial betterment has been proposed as a agency of bettering field efficaciousness of entomopathogenic roundworms ( Gaugler, 1987 ; Kaya and Gaugler, 1993 ; Fodor et al. , 1994 ; Burnell and Dowds, 1996 ) . Entomopathogenic roundworms offer several advantages as topics for familial betterment including short coevals clip, little genome size, easiness of civilization, handling, and suitableness for inundative applications ( Gaugler et al. , 1989b ; Fodor et al. , 1994 ; Ehlers et al.2001 and 2003 ) and has a global distribution ( Hominick, 2002 ) .
Aims
This survey fulfils the undermentioned aims:
Crossing of the most cold and dehydration tolerant strains of Steinernema feltiae.
Consecutive familial choice of their cold and dehydration tolerance.
Probe of the influence of cold and dehydration emphasis on fittingness of the intercrossed strains.
2. Literature reappraisal
In this reappraisal, information on the taxonomy, biological science, life rhythm of Steinernema feltiae has been summarized. Besides the literatures on cross genteelness, familial choice of good trait like heat, dehydration tolerance of entomopathogenic roundworm, eventually the influence of cold and dehydration emphasis on the fittingness of intercrossed strains have been discussed.
2.1. Entomopathogenic roundworm: Steinernema feltiae Filipjev
2.1.1. Taxonomy
Phylum: Aschelminthes
Class: Chromadorea
Order: Rhabditida
Family: Steinernematidae
Sub-family: Steinernematinae
Genus: Steinernema
Speciess: Steinernema feltiae
2.1.2. Life rhythm and biological science
A particular developmental phase within the life rhythm of all rhabditid roundworms is the dauer juvenile ( DJ ) . This term dauer ( German for digesting ) was introduced by Fuchs ( 1915 ) and describes a morphologically distinguishable juvenile, formed as a response to consuming nutrient resources and inauspicious environmental conditions. The 3rd phase dauer juvenile occurs free in the dirt that is good adapted to long-run endurance in the dirt ( Susurluk & A ; Ehlers, 2008 ) and seek suited insect host ( Lewis, 2002 ; Torr et Al. 2004 ) . The DJ is the morbific phase that carries 200-2000 cells of its symbionts in the anterior portion of its bowel ( Endo and Nickle 1994 ) . . The symbiont of S. feltiae is X. bovienii ( Akhurst & A ; Boemare, 1988 ) . Steinernema additions entry to the insect larva through natural gaps ( oral cavity, anus and spiracles ) . In the haemolymph, the roundworms encounter optimum conditions for reproduction. During recovery by the nutrient signal ( Golden & A ; Riddle, 1982 ) , the DJ let go of the symbiont cells into the insect ‘s haemocoel. The bacterium green goods toxins and other metabolites ( Dunphy ans Webster 1988 ; Bowen et Al. 1998 ) , which contribute to get the better of the insect ‘s defense mechanism mechanisms and kill the insect within about 2 yearss after nematode invasion ( Simoes and Rosa 1996 ) . Some Steinernema besides produce toxins that contribute to the pathogenicity of their symbionts ( Ehlers et al. 1997 ) . The bacterium proliferate and produce suited conditions for nematode reproduction. Feeding on symbiont cells, they develop into grownups and bring forth offspring. Nematode reproduction continues over two or three coevalss until the alimentary position of the cadavar deteriorates whereupon grownup development is suppressed and DJ accumulate which retain the symbiotic bacteriums in the bowel ( Popiel et al. 1989 ) .
In Steinernema reproduction is amphimictic. Steinernematid DJ mature to go either a male or a female and sex finding appears to be of the XX/XO type, typical of roundworm ( Dix et al. , 1994 ) . Griffin et al. , ( 2001 ) identified a hermaphroditic strain of Steinernema.
Growth and reproduction of grownup phases is besides influenced by the nutritionary conditions.
Steinernematid grownups responds to consuming nutrient resources with the surcease of egg laying. Juveniles hatch inside the womb and develop at cost of the maternal organic structure content doing the decease of the grownup ( endotokia matricida ) . DJ output in 2nd and 3rd coevals grownups of S. feltiae in monoxenic liquid civilizations is less than S. carpocapsae likely due to the increasing bacterial population ( Hirao et al, 2010 ) .
Fig. life rhythm of Steinernema feltiae
2.2. Familial Improvement
2.2.1. Familial choice
Familial choice can be a powerful tool to increase good traits in biological control agents. The usage of familial choice for the betterment of good traits to get the better of restrictions, was foremost suggested for EPN by Gaugler ( 1986 ) and first consequences were reported on improved host happening 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 dehydration and heat tolerance of Heterorhabditis bacteriophora can be improved by familial choice.
Choice of proper campaigner species, mark traits, and proviso of equal familial variableness in base populations are the indispensable requirements for a sound familial betterment plan ( Hoy, 1986 ; Gaugler et al. , 1989b ; Hastings, 1994 ) . With sufficient variableness for the coveted traits in the natural isolates a systematic choice programme can be initiated by get downing familial choice ( Fig. 1 ) . Successful familial choice depends on high heritability ( h2 ) of the targeted trait in the population ( Hartl and clark 1989 ) . The high h2 attributes to a high familial variableness. The h2 of heat and dehydration traits in Heterorhabditis bacteriophora have been determined already. Strauch et Al. ( 2004 ) evaluated dehydration tolerance of a intercrossed strain and found h2= 0.46 for dehydration tolerance of non-adapted populations and h2= 0.48 with inclusion of an version stage. The h2 of heat tolerance was found to be 0.68 by Ehlers et Al. ( 2005 ) .
Collection and rating of wild-type populations from diverse locations are necessary for supplying equal familial variableness in mark traits for choice. Biodiversity of natural isolates has been a rich beginning for familial discrepancies in domestication of harvests and farm animal. Hopper et Al. ( 1993 ) worked on the natural isolate IS-5, demonstrates the importance of roll uping and continuing the rich familial diverseness available in nature, with peculiar accent on trying from as many ecologically and geographically diverse sites as possible.
It is critical that appraisals on natural populations are made utilizing freshly field-collected populations. Grewal et Al. ( 1994b ) studied the thermic niche comprehensivenesss for strains of Steinernema spp. Strains, cultured in the research lab for several old ages, may lost some of the existent variableness ( Grewal et al. 1994b ) . Dramatic alterations in biological traits of entomopathogenic roundworms during laboratory version have been demonstrated in several surveies ( Grewal et al. , 1996 ; Stuart and Gaugler, 1996 ; Wang and Grewal, 2002 ) . So, all freshly isolated populations should cultured one time in the wax moth Galleria mellonella L. larvae at 25A°C following isolation as described by Kaya and Stock ( 1997 ) and stored in liquid N as described by Curran et Al. ( 1992 ) to forestall alterations in field-adapted traits due to frequent culturing in the research lab.
Molecular attacks for gauging the grade of familial fluctuation 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 ease choice surveies.
Choice campaigner roundworm
Estimate heritability of coveted trait
Maximize nematode familial variableness
Foundation strain
Cryopreserve foundation
strain
Stress tolerance choice check
Monitor choice advancement
Lab tests
Characterize improved roundworm
Green house tests
Evaluate emphasis tolerance public presentation
under field conditions
Fig.1. A systematic program for the design of a familial choice programme of entomopathogenic roundworm ( adapted from Gaugler et Al. 1989, Burnell 2002 ) .
2.2.2. Cold tolerance in Steinernema spp.
Temperature extremes are among the restricting factors impacting the endurance of Heterorhadbitis spp. and Steinernema spp. Temperature has consequence on infectivity, endurance and continuity 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 consequence depends on exposure clip ( Koppenhofer 2000 ) . Temperature influences the rate at which nutrient militias ( lipoids, proteins and saccharides ) are utilized by roundworm. When EPN are exposed to sudden rise ( heat-shock ) or autumn ( cold-shock ) in temperature, they synthesize proteins called heat-shock proteins ( Jagdale et Al. 2005 ) .
Temperature had a direct consequence on the clip of decease, incursion rate, outgrowth clip of morbific juveniles, and figure of emerging morbific juveniles of S. feltiae ( Kaya et al. 2001 ) . Somasekhar et Al. ( 2002 ) reported endurance 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 part of the universe is a better footing for choice procedure. Grewal et Al. ( 1994b ) studied the thermic niche comprehensivenesss for infection, constitution, and reproduction for strains of Steinernema spp. Galleria mellonella ( wax moth ) larvae were infected by Steinernema riobravis at the widest temperature scope ( 10-39A°C ) , whereas S. feltiae at the narrowest ( 8-30A°C ) . They found thermic niche comprehensiveness for constitution within hosts was the widest for S. glaseri, ( 10-37A°C ) and the narrowest for S. feltiae ( 8-30A°C ) and thermic niche comprehensiveness for reproduction was widest for S. glaseri ( 12-32A°C ) and the narrowest for S. carpocapsae ( 20-30A°C ) . Steinernema scapterisci ( 20-32A°C ) , S. riobravis ( 20-35A°C ) , and Steinernema sp. ( 20-32A°C ) were more altered to warm temperature reproduction, and S. feltiae to cooler temperatures ( 10-25A°C )
Several H. bacteriophora strains have been isolated and their temperature penchants were described ( Grewal et al. 1994 ; Glazer et Al. 1996 ) . Mukuka et Al. ( 2010a ) screened 36 natural populations and 18 loanblend or inbred strains of Heterorhabditis bacteriophora for their response to high temperature with or without anterior version to heat at 350C for 3 h. The appraisal of the heat tolerance was done as described by Ehlers et Al. ( 2005 ) . Five cover-slide Chamberss incorporating 5 ml tap H2O were filled with 200 DJs each of one strain. The Chamberss were so distributed on a temperature gradient generated on an aluminum saloon at temperatures between 320Cand 410C for 2 h. The temperature at the underside of the Chamberss was recorded by a Pt Pt 100 thin bed detectors. 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 non detect any correlativity between tolerance assessed with and without version to heat, connoting that different cistrons are involved.
The thermic niche for entomopathogenic roundworms are species-specific and do non by and large associate to the average temperature of the original vicinity of the isolate ( Grewal et al. 1994 ; Hazir et Al. 2001 ; Mukuka et Al. 2010a ) .
2.2.2. Dehydration tolerance in Steinernema spp.
EPN use assorted schemes to maximise their endurance in dried-out status.
Behavioral version like loose coiling and clip-clop 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 clip-clop and gyrating aid in cut downing the rate of H2O loss from the roundworm during dehydration, therefore leting more clip for suited physiological alterations in response to H2O loss. Desiccated nematode might live longer than non-desiccated roundworm because of the decreased metamorphosis.
Park for all rhabditid roundworms are non-feeding and developmentally arrested 3rd phase dauer juveniles ( DJs ) . Survival of DJs during storage or in-transit to end-users is a critical factor restricting the usage of EPN. Entomopathogenic nematologist community accept that protraction of EPNs storage is best achieved by initiation of a hibernating province. Anhydrobiosis, “ life without H2O ” is a province of quiescence that is reversible and is caused by dehydration ( Georgis et al. 1994 ; Georgis, 1990 ) . Anhydrobiosis is an of import agencies of accomplishing storage stableness of EPN. This can be attained by evaporative or osmotic partial desiccation ( Glazer, 2002 ; Perry, 1998 ) .
Tolerance to dehydration in Heterorhabditis spp. is besides due to the accretion of glycerin, whereas trehalose synthesis was recorded in steinernema carpocapsae. During dehydration, 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 morbific juveniles by utilizing Peptide mass mapping with MALDI-TOF mass spectroscopy ( MS ) among among which several are known to be stress antiphonal including heat daze protein 60, coenzyme q biogenesis protein, inositol monophosphatase and fumarate lyase that were found in both emphasiss.
In different preparations, the endurance of DJs can change significantly ( Grewal, 2002 ; Strauch et Al. 2002 ) . At the beginning, on direct contact dehydration with the aid of absorbents such as clay to accomplish storage stableness resulted in significant success. The concentrated roundworms and an inert solid bearer, such as vermiculite or diatomaceous Earth consist the most commercial roundworm merchandise. In these preparations, roundworms are partly inactivated as a consequence of uncomplete desiccation that induces a semi-dormant province well protracting the roundworms ‘ lifetime and enabling them to defy the asperities of a fluctuating temperature government that is typical when commercial merchandises are shipped and applied ( Perry, 1998 ) . For illustration Steinernema carpocapsae survived up to seven months at 250C in a water-dispensible granular ( WG ) preparation compared to four months in tap H2O ( Grewal 2000a ) . The WG preparation extended the shelf-life of S. carpocapsae with above 80 % endurance at 250C. The WG preparation facilitated transit of EPN merchandises at ambient temperatures as opposed to nightlong cargos on ice ( Grewal 2000a ) . This enhanced storage stableness could hold been attributed to the initiation of partial anhydrobiosis that resulted in a decreased DJ metabolic activity at 250C.
Attempts to better the dehydration tolerance were reported by Strauch et Al. ( 2004 ) . Strauch et Al. ( 2004 ) assessed a heritability of the dehydration tolerance of h2 = 0.48 when populations had been adapted to desiccation prior to exposure to emphasize and of 0.46 without version. The heritability of the trait ‘desiccation tolerance ‘ determined by utilizing homozygous inbred lines.
They assessed the dehydration tolerance in liquid, hygroscopic poly ( ethylene ethanediol ) 600 solution. To bring forth different degrees of dehydration emphasis they transferred DJ into different concentration of PEG 600. Desiccation emphasis was measured as H2O activity ( aw value ) . The H2O activity is defined as the comparative proportion of unbound H2O in a sample. The lower the H2O activity of the solution, the stronger is the remotion of H2O from the DJ. The average tolerated aw-value ranged between 0.89 and 0.81 when DJs had been adapted to desiccation prior to emphasize exposure.
Mukuka et Al, ( 2010b ) screened the dehydration tolerance of 43 strains of Heterorhabditis spp. and 18 hybrid/inbred strains of H. bacteriophora. They besides measured the desiccating status as H2O activity ( aw value ) by handling DJ with different concentrations of the non-ionic polymer poly ( ethylene ethanediol ) 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.
2.2.3. Cross-breeding
Hybridization can be a powerful tool for familial betterment of good traits in the entomopathogenic roundworm. The amphimictic nature of S. feltiae makes it possible to traverse males and females for creative activity of loanblends. Most tolerant 10 % person of cold and dehydration tolerant strains of S. feltiae will selected for traversing to do a loanblend with more cold and dehydration tolerant.
Improvement of heat and dehydration tolerance in H. bacteriophora through cross-breeding was done by Mukuka et Al, ( 2010c ) . To accomplish this end, most tolerant strains which have been identified within testing for heat ( Mukuka et al. 2010a ) and dehydration tolerance ( Mukuka et al. 2010b ) were hybridized and checked for their tolerance. Crosses were done harmonizing to Iraki et Al. ( 2000 ) .
2.2.3.1. Determination of and familial choice 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 15A°C. They found that cold choice enhanced nematode virulency at the ice chest temperatures. Virulence measured as entire insect-mortality at 8A°C improved by 5.3- and 6.6-fold after six and 12 transitions, severally. They observed nematode constitution improved at all temperatures after 12 transitions and the highest addition of 9-fold was observed at 8A°C.
Ehlers et Al. ( 2005 ) increased the mean tolerated temperature from 38.5 to 39.20C in H. bacteriophora by 4 choice stairss and decreased average temperature at which the dauer juveniles of H. bacteriophora were active, from 7.3 to 6.1 0C during the first 5 choice stairss through selective genteelness.
Mukuka et Al. ( 2010c ) improved the heat tolerance through cross genteelness of tolerant strains of H. bacteriophora and consecutive familial choice. The heat tolerance of intercrossed strains was assessed harmonizing to Ehlers et Al. ( 2005 ) and Mukuka et Al. ( 2010a ) . After 11 choice, they observed average heat tolerance addition 5.50C when roundworms had been adapted to heat emphasis. For non-adapted tolerance an addition of 3.00C from 40.10C to 43.10C was recorded
2.2.3.2. Determination of and familial choice of dehydration tolerance
Cross-breeding of most dehydration tolerant strains of H. bacteriophora ( Mukuka et al, 2010b ) reduced the aw-value from 0.67 to 0.65 after version and from 0.9 to 0.7 without anterior version after six choice stairss ( 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 heterogenous population Steinernema feltiae for dehydration tolerance and enhanced host-seeking ability. Survival rate of 80-90 % was reached after 10 choice rhythms for tolerance of rapid dehydration by exposing morbific juveniles ( IJs ) to ambient conditions ( 22-25A°C ; 50-65 % r.h. ) for 100 min and survival rate of 80-90 % was reached after 10 choice rhythms for tolerance of slow dehydration by open IJs to 97 % r.h. for 72 h. Host happening ability of the nematode addition & gt ; 75 % after 25 choice rhythms.
2.2.4. Fitness of cold and dehydration loanblend strain
Choice of any trait can be followed by alterations in the genome that can impact look of 2nd trait ; in most instances consequences in decrease of fittingness ( Falconer, 1981 ) . There is demand to mass bring forth the attendant intercrossed strain so that the gained traits of cold and dehydration can be stabilized ( Bai et al. 2005 ) . So, monitoring of good traits like infectivity, virulency, host incursion, generative potency are indispensable during efforts to genetically better other traits by traversing tolerant strains or selective genteelness.
When a desired phenotype is obtained, there is a inclination of selected strains to return bit by bit towards the unselected province ( Hastings, 1994 ) . This can be prevented by cryopreserving and reapplying choice force per unit area at regular interval on the selected loanblend strains.
Mukuka et Al. ( 2010e ) compared the fittingness of heat and dehydration tolerant intercrossed strains of Heterorhabditis bacteriophora to the commercial strain EN01 and found that merely heat tolerant strains were superior or similar in fittingness in virulency, host incursion and generative potency to strive EN01 but strains with increased dehydration tolerance were normally less fit. There was high opportunity that cistrons involved in the dehydration tolerance might be dissembling the look of the virulency, generative potency and host incursion traits. They besides found that the intercrossed strains with inclusion of an version stage showed more fittingness than those hybridized without including an version stage.
Desiccation emphasis can impact to the infectivity of the entomopathogenic roundworm. Mukuka et Al. ( 2010e ) found that dried-out Heterorhabditis bacteriophora are less deadly in Galleria mellonella than undesiccated 1s. But Shapiro & A ; Lewis ( 1999 ) ; serve-Rodriguez ( 2004 ) found that dried-out Steinernema carpocapsae to be more deadly in G. mellonella that undesiccated 1s. In add-on Solomon et Al. ( 1999 ) found no difference in infectivity between desiccated and nondesiccated S. feltiae strains that were exposed to meal worm, Tenebrio molitor.
