Fatty Acid Composition of Eight Isolates of Entomopathogenic Nematodes from Five Egyptian Governorates

12 Arab Congress of Plant Protection ,ACPP ,4 -10 November, 2017 Hurghada Egypt Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Microbiology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the research across the whole spectrum of the subject. These including bacteriology, virology, mycology and parasitology. In addition, the journal promotes research on the impact of living organisms on their environment with emphasis on subjects such a resource, depletion, pollution, biodiversity, ecosystem.....etc www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.

The fatty acid composition of infective juveniles (IJs) related to five isolates of Heterorhabditis indica (EGAZ1, EGAZ2, EGAZ3, EGAZ4, and EGAZ5) and three isolates of Steinernema carpocapsae (EGAZ9, EGAZ10 and SA) collected from five Egyptian Governorates was assessed.Also, fatty acid composition of IJs from two commercially relevance strains of H. bacteriophora (HP88) and S. carpocapsae (All), was examined for comparison.Newly emerged IJs of all isolates had fatty acid number and pattern similar to that of the corresponding commercial species.Of the ten fatty acids identified, oleic, was the main fatty acid in all species and isolates.Unsaturated fatty acids were dominant and total amount of saturated fatty acids of H. indica isolates was significantly higher than that of S. carpocapsae isolates.Palmitic was the second most abundant fatty acid in IJs of H. indica isolates, while linoleic was the second most abundant fatty acid in IJs of S. carpocapsae isolates.Of the tested Egyptian isolates, IJs of EGAZ3 and EGAZ5 of H. indica had the highest amounts of fatty acids and their contents of saturated fatty acids/gram body weight are comparable to that of the commercial strain.It is suggested that isolates EGAZ3 and EGAZ5 of H. indica are the candidates for developing practical Egyptian bio-control product based on nematode formulation.

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The amount of energy provided by lipids depends on the amount of fatty acids and the degree of un-saturation of fatty acids since more energy is yielded by oxidation of saturated than of unsaturated fatty acids (Fitters et al., 1999).There are many species and isolates of EPN that vary enormously in their fatty acid contents, and consequently vary in their ability to maintain biological activity during formulation, storage, and application.Accordingly, to choose the appropriate nematode for formulation in applicable products the most logical approach is to understand the physiological chemistry of the candidate nematode isolates, particularly, their appropriate composition of fatty acids (Georgis and Kaya, 1998).
In the present study, the fatty acid composition of eight geographically diverse Egyptian isolates of Steinernema and Heterorhabditis was determined.Also, fatty acid contents of IJs from two commercially relevance strains of Steinernema and Heterorhabditis were examined for comparison.This will provide detailed information on the degree of variation in the fatty acid composition of these nematodes.The availability of nematode isolates with a higher fatty acid contents, especially saturated fatty acids would mean remarkable progress on the road to longer shelf life of nematode bio-pesticide formulations (Perry et al., 2012), which is an important requirement for Egyptian agriculture and environment.

Nematode Populations:
Eight EPN isolates collected randomly from several locations of five Egyptian governorates, and maintained at the Pest Physiology Department, Plant Protection Research Institute, Egypt, and two commercial nematode species were analyzed for their fatty acid composition (Table 1 All populations were cultured in the wax moth, Galleria mellonella larvae at 25°C following isolation as described by Kaya and Stock (1997).Freshly harvested IJs were rinsed in de-ionized water, three times and the number of IJs in each sample was estimated.Harvested IJs were concentrated, vacuum filtered, weighed and then stored in 2 ml microcentrifuge tubes at -20 o C. Three replicates of 100,000 nematodes from each nematode isolate were prepared for fatty acid analysis.

Extraction and preparation of Fatty acids:
The fatty acids were extracted and prepared for analyzing by gas chromatography coupled with mass spectrometer (GC/MS) using the methods of Sasser ( 2001) as described in details by Kyung et al., (2012).Briefly, fatty acids were extracted from IJs and saponified by adding 2 ml of extraction reagent (45 g sodium hydroxide, 150 ml HPLC grade methanol, and 150 ml distilled water) to 100 mg of IJs from each isolate in capped test tubes.The tubes were heated in a boiling water bath for 30 min with vortex every 10 min for 5-10 s.The cooled tubes were uncapped and 2 ml of methylation reagent (325 ml of 6.0 N hydrochloric acid and 275 ml methyl alcohol), was added to each tube.After vortexing, the tubes were heated for 10 min at 80 o C. Methylated fatty acids were extracted from the solution by adding 1.25 ml of hexane-methyl tert-butyl ether mixture (1:1) to each of the cooled tube and tumbling for approximately 10 min.The tubes were then uncapped and the aqueous (lower) phase was pipetted out and discarded.Each of the remaining organic phases in the tubes was washed by adding 3 ml of ca 1% sodium hydroxide in distilled water and tumbling for 5 min.The organic phase containing methylated fatty acids was then pipetted into a GC vial for GC-MS analysis.

GC-MS Analysis:
Fatty acids extracted from the nematode isolates were measured by using Shimadzu GC/MS-QP2010 Plus (Japan) Gas Chromatography/mass spectrometer system equipped with a capillary column DB-5MS (30 m length, 0.25 mm thickness, 0.25m diameter).Injections (at 250°C) were made in split mode (50:1) with Helium as carrier gas at flow rate of 1ml/min.The temperature program was isothermal at 50°C for 2 min, 7 °C/ min to 200°C and 5°C/min to 220°C and held for 20 min.An external standard of fatty acid mixtures (PUFA-3, Supelco, Bellefonte, USA) was used for confirming the identity of the different peaks in nematode samples, and for determining the quantity of each fatty acid.Fatty acids were expressed as the percentage of total fatty acid contents of each isolate, nanograms (ng) fatty acids per nematode, and also as ng fatty acids per 100 ng of IJs fresh weight.

Statistical Analysis:
Percentage data were normalized using arcsine (square root) transformation and subjected to analysis of variance (ANOVA) using CoStat software, release 5.5.Duncan's multiple range test was used to compare means at p<0.05.Results were recorded as mean ± standard deviation (SD).
(1) show the relative percentage and pattern of fatty acids found in all studies EPN isolates.Although there were high variation in the percentage of each fatty acid among the studied EPN species and isolate, IJs of all isolates had the same ten fatty acids.Also, IJs of all isolates had fatty acid number and pattern similar to that of the corresponding commercial species.

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Quantitative analysis of total fatty acids content (Table 4) revealed that infective juveniles of the two commercial nematode species, H. bacteriophora (HP88) and S. carpocapsae (All) had approximately equal amounts of fatty acids (40.4 ± 3.5 and 36.0 ± 4.2 ng/IJ, respectively).However, such amount of total fatty acids is significantly much higher (P < 0.05) than the amount of total fatty acids detected in any of the studies Egyptian isolates that ranged from 16.6±2.2ng/IJfor isolate EGAZ3 of H. indica to 7.1±1 ng/IJ for isolate EGAZ9 of S. carpocapsae (Table 4).Also, the amount of saturated fatty acid per IJ was significantly higher (P < 0.05) in the two commercial nematode species than the amount of saturated fatty acids detected in each of the other eight Egyptian isolates.The amount of fatty acid contents in relation to nematode weight mirrored in general their amount per IJ despite the differences in IJ weight between species and isolates (Table 4).Means within the same column followed by the same letter are not significantly different (Duncan's multiple range test, P > 0.05).
The one obvious difference was in the amount of saturated fatty acids, as the two Egyptian isolates of H. indica, EGAZ3 and EGAZ5 showed an amount of saturated fatty acids comparable to that of the commercial species HP88 (3.1±0.26 and 3.5±0.33compared with 3.6±0.21ng/100ng nematode weight, respectively).The differences in the amount of saturated fatty acids between EGAZ3, EGAZ5 and HP88 were insignificant (P> 0.05).Fig. (2) shows that GC-MS peaks of the main fatty acids of HP88, EGAZ3 and EGAZ5 were comparable.

DISCUSSION
When aiming for nematode strains with superior stress tolerant improved traits for formulation in applicable products, first it is preferable to look for physiologically preadapted wild types as such genotypes are likely to be more stable and more tolerate to environmental and climatic conditions in the geographic regions from where the nematode population was isolated (Somasekhar et al., 2002 andDolinski et al., 2008).It is well established that fatty acid contents and profile are important in several physiological processes, such as desiccation and osmotic tolerance of IJs (Patel andWright, 1997 andQui et al., 2000) which in turn affect the quality and shelf life of nematode formulations (selvan et al., 1993a&b andWright et al., 1999).Therefore, we analyzed the fatty acid contents of eight isolates of S. carpocapase and H. indica collected from different Egyptian geographical regions.We also included two relevant nematode species extracted from imported commercial formulations for comparison.
Our results demonstrated that IJs of all isolates tested in this study, had fatty acid number and pattern similar to that of the corresponding commercial species.Oleic, was the main fatty acids in all species and isolates, accounting for 50-60% of total fatty acid contents in newly emerged IJs.Unsaturated fatty acids dominated as their proportion was at least three-fold greater than the proportion of saturated fatty acids.These results are in agreement with the general consensus that oleic, as well as unsaturated fatty acids, predominate in EPNs (Fitters et al., 1999, Abu Hatab and Gaugler, 2001, and Andalo et al., 2011).
Results of this work have shown that total amount of saturated fatty acids of all the studied Heterorhabditis isolates was significantly higher than that of Steinernema isolates.This difference was due mainly to the higher amount of the saturated palmitic (C16:0) in IJs of Heterorhabditis isolates, as it was the second most abundant fatty acids while in IJs of Steinernema isolates the unsaturated linoleic fatty acid was the second most abundant fatty acids.Patel andWright (1997), andEl-Badawyet al., (2011) also found the same fatty acid patterns in IJs of Heterorhabditis and Steinernema species they studied.IJs of H. bacteriophora and H. indica had higher motility nature than the S. carpocapsae (Campbell and Gaugler, 1992), thus Heterorhabditis species, in general, need more energy reserves.IJs rely on fatty acids as their sole energy source and saturated fatty acids provide more energy than unsaturated fatty acids (Selvan et al., 1993a, andAndalo et al., 2011).This explains our finding that amount of saturated fatty acids of Heterorhabditis isolates was significantly higher than that of Steinernema isolates as an increase in saturated fatty acid content in IJs will increase their energy reserve.
The main finding of our study is that IJs of the two commercial strains contained more than three-fold greater total fatty acid/IJ than the local Egyptian isolates.Also, total amount of saturated fatty acids was significantly higher in commercial strains than Egyptian isolates.Selvan et al. (1993a) suggested that increase quantity of fatty acid composition and proportion of their saturation among infective juveniles in commercial products likely to improve their shelf life.This could be achieved by feeding nematode with certain oils such as olive oil in combination with a fat such as beef fat (rich in palmitic 16:0 and stearic 18:0 acids) (Abu Hatab and Gaugler, 2001).Rouse et al. (1992) increased both fatty acids content (5fold) and double the percentage of unsaturated FAs in the free-living soil nematode Panagrellus redivivus by adding fish oil to their diet.Accordingly, the high fatty acid contents of commercial IJs in this study could be attributed to the fact that certain techniques, mainly selection, and manipulation of dietary lipid content has been used to increase lipids and fatty acid contents of such IJs and in consequence improve longevity of commercial formulations (Abu Hatab andGaugler, 2001 andPerry et al., 2012).However, as natural populations are preferable for preparing effective formulation as mentioned above, screening among natural populations for higher fatty acid contents is the logical first step in formulation of EPN for practical use.
Our results shows that of the tested Egyptian isolates, IJs of EGAZ3 and EGAZ5 of H. indica had the highest amounts of fatty acids.Also, the amount of saturated fatty acids/gram IJs body weight of EGAZ3 and EGAZ5 was comparable to that of the imported commercial strain.Accordingly, it is suggested that isolates EGAZ3 and EGAZ5 of H. indica are the candidate for further improvements through selection and dietary manipulation for developing practical Egyptian bio-control product based on entomopathogenic nematode.

Table 1 :
Locality, crop, and source of entomopathogenic nematode populations used in the present study.
*Nematodes were isolated from soil using last instar larvae of Galleria mellonella as bait

Table 2 :
Fatty acid composition (% of total FA) of freshly harvested infective juveniles of Heterorhabditis bacteriophora HP88 (commercial) and Heterorhabditis indica isolates:

Table 3 :
Fatty acid composition (% of total FA) of freshly harvested infective juveniles of Steinernema carpocapase isolates.

Table 4 :
Fresh weight and total fatty acid contents of freshly harvested infective juveniles of the tested Heterorhabditis and Steinernema isolates.