Physiological and biological stud ies of some entomopathogenic nematode species of families ( steinernematidae and heterorabditidae )

Entomopathogenic nematodes of the genera Heterorhabditis and Steinernema are used as an insect biological control in agriculture. Numerous new species are being described but generally little information is provided on their ecological or physiological information. Therefore, this paper presents examination of virulence, penetration rate, reproduction, and some energy reserves (total lipids, total proteins and total carbohydrates) for six species of entomopathogenic nematodes, 3 species of Heterorhabditids ((HP2), (HP4) and H. indica) and 3 species of Steinernema ((S3), S. riobrave and S. rarum) are extracted from soil samples at different countries and places to refer the superlative one to unfavorable environmental conditions. The tested species differed in their penetration rate to Galeria mellonella larvae. Heterorhabditis sp. (HP2) recorded the highest penetration rate (56 %) where it recorded highest total lipids (35.8 %), highest total proteins (60.3%) and the highest virulence to G. mellonella (25%). The highest total carbohydrates were recorded to the steinernema rarum (26%) while the highest reproduction was recorded to Heterorhabditis indicus (149914 IJs/larva).


INTRODUCTION
The infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are currently used as biopesticides for controlling various insect pests (Hom, 1994).The infective juveniles are nonfeeding and free-living in soil and so are unable to compensate for energy consumption with energy intake, and depend solely on accumulated reserves for energy supply (Gaugler, 1988).Successful establishment of nematodes in diverse environments depends upon physiological, behavioral and biochemical adaptations (Nicholas, 1984).
Sixty species of entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae have been described (Nguyen, 2005;Qiu et al., 2005;Phan et al., 2005;Nguyen et al., 2006;Uribe-Lorio et al., 2007;Zhang et al., 2008 andNguyen et al., 2010).Descriptions of new entomopathogenic nematode species usually contain very little information on their biology and ecology and are not complemented by studies supplying this information (Koppenhöfer and kaya, 1999).There is little information concerning the energy metabolism and its relation to survival and infectivity of EPNs (Selevan et al., 1993).Entomopathogenic nematode with their associated Xenorhabdus spp.bacteria, lethal pathogens of soilinhabiting insects.Infective juveniles occur naturally in the soil where they infect and kill their insect host within 2 or 3 days and produce 2 or 3 generations in the host.Resulting infective juveniles emerge from host cadaver 1or 2 weeks later and search for new hosts (Akhurst, 1995).Entomopathogenic nematodes have been isolated from every inhabited continent and many islands (Poiner, 1990).These nematodes are faced with wide array of environmental conditions during the non-feeding infective stage.In this study, we report the differences in lipid, protein, carbohydrate contents, virulence, penetration rate and reproduction of entomopathogenic nematodes of 6 widely distributed, geographically diverse species.In all the following experiments, G. mellonella larvae (last instar) were exposed to freshly emerged nematode infecting stages, at a dose level of 20 IJ/larva in 300 µl of distilled water in 1.5 ml Eppendorf tubes, lined with double layer filter paper (Whatman No. 1) and kept at 25°C, in the dark.

A. Penetration Rate:
After 4-5days of the infection, according to the species of nematode, at least 10 dead G. mellonella larvae were washed twice with distilled water to remove any nematode juveniles that attached to them, dried and dissected under a stereomicroscope.The number of nematodes inside each larva was counted and the penetration rate was calculated as an average B. Reproduction rate: At least five dead insect larvae, after 2 days of infection, were washed twice with distilled water to remove any nematode juveniles that attached to them, and placed into modified White traps.For each of the tested nematode species, 5 replicates.After 15 days of infection all IJ that emerged from the host over this period in the water were harvested and the total nematode suspension was put in a 50 ml tissue culture flask.To assess the total production during the harvest period, the contents of the flask were mixed thoroughly with air bubbles from an aquarium pump and from this suspension 5 samples of 10 µl were counted under a stereomicroscope using a counting slide.

C. Virulence (one -on -one):
G. mellonella larvae were subjected to nematode infection at a dose level of 1 IJ/larva in 300 µl of distilled water and kept at 25°C, in the dark.For each of the tested nematode species, 5 replicates, each of 6 larvae, were made, in addition to a control containing only distilled water.Mortality records were taken after 48 hours and corrected according to Abbott's formula (Abbott, 1925).

2-Energy Reserves in Nematode Juveniles A-Total Lipids:
Nematode juveniles were washed and incubated in 15 ml of 80% ethanol at 75°C for 5 minutes to inactivate degradative enzymes such as phospholipases.The suspension was then cooled and stored in a tightcapped tube after flushing with N 2 , and stored at -70°C (Abu Hatab, and Gaugler 1997).Lipids were extracted and purified from frozen nematode samples (0.05-0.1g) according to Folch et al., (1957).Pure vanillin (1.2 g) was dissolved in 20 ml ethyl alcohol and completed to 200 ml with distilled water; 800 ml of concentrated phosphoric acid were added.The solution was stored in dark glass bottle at room temperature.The procedure described by Knight et al., (1972), is used for determination of Lipids and measured spectrophotometrically at 525 nm against a blank.

B-Total Protein
Protein was extracted from nematode tissues and prepared as described by Lewis et al., (1995).Nematode samples (0.05 g) were homogenized in 3 ml 30% potassium hydroxide (KOH) for 5 minutes.The homogenate was then washed by 4 ml 30% KOH, and boiled for 1 hour with occasional agitation; the sample was then cooled to room temperature.
Protein content of nematode samples was estimated spectrophotometrically by the method of Bradford (1976).

C-Total Carbohydrate:
Carbohydrates were extracted from nematode tissue and prepared according to Lewis et al., (1995).Nematode samples (0.05 g) were homogenized with 7 ml 30% KOH and boiled for 1 hour with occasional agitation.Carbohydrates content was determined by phenol-sulfuric acid reaction according to Dubois et al., (1956).

Statistical Analysis
Percentage values in the present study were normalized using arcsine transformation.The significance of the main effects was determined by analysis of variance (ANOVA).The significance of various treatments was evaluated by Duncan's multiple range test (P<0.05)(Colman, 2001) according to the statistical methods of Snedecor (1956).All analyses were made using a software package "Costat", a product of Cohort Software Inc., Berkeley, California .

1-Nematode Biological Activities A-Penetration Rate:
It is evident from the fig ( 1A) that the highest penetration rate of nematode (56%) was found in dead G. mellonella larvae infected with HP2.The penetration rate of S3, S. riobrave, S. rarum and H. indica reported (49.7%), (38.7%), (36%) and (32.7%), respectively.The least penetration rate (9.7%) was for HP4.No significant differences were found between the penetration rates of S. rarum and H. indica.Generally statistical analysis showed that there were high significant differences between the efficacy of tested EPNs species (F=13.62,df=5, P=0000).

B-Reproduction rate:
In fig ( 1B) it is clear that the largest cumulative production of juveniles for the H. indica where each cadaver produced (149914 IJs/L) through the 2 weeks after the day of infection, while S3, S. rarum, S. riobrave and HP2 were degraded in their reproduction as it recorded (41347 IJs/L), (39060 IJs/L), (28992 IJs/L) and (14066 IJs/L), respectively.(5943 IJs/L) was the least production recorded for the HP4.Statistical analysis showed that there were very high significant differences between the efficacy of entomopathogenic nematode species (F=547.2,df=5, P=0000).No significant differences were found between the reproduction rate of the tested species, S3 and S. rarum.

C-Virulence (one -on -one):
Fig ( 1C) represented the tested isolates exhibited high virulence against larvae of G. mellonella was 25% mortality of HP2, while the second high mortality 20% was recorded for the S3.Both H. indica and S. rarum recorded 12.5% mortality of G. mellonella larvae.The other two isolates were less virulent recording (8.3% and 4.2%) mortality for S. riobrave and HP4, respectively, with non -significant differences among them (F=0.821df=5 P=0.5506).
Physiological and biological studies of some entomopathogenic nematode species 49 Total Carbohydrate: Results of total carbohydrate contents illustrated in fig ( 2C), revealed that a significant different between the two isolates S. rarum and S. riobrave for this energy reserve, it recorded (26%) and (21.8%), respectively.HP2 and S3 ranked in the third and fourth places with nonsignificant difference between them (16.4%) and (14.9%), respectively.The lowest total carbohydrate percentage were observed in case of HP4 and H. indica (12.1%) and (12%), respectively.Generally, statistical analysis showed that there were significant differences between the tested species of EPNs (F=47.302,df=5, P=0.0000).The quality of nematodes that survive the rigorous of manufacturing process is analyzed by determining their shelf-life, which is predicted from storage energy reserves (Grewal and Georgois, 1995).Nematode viability and infectivity may decline at different rates during storage, and prolonged survival of nematodes depends upon the conservation of energy reserves (Wijbenga and Rodgers, 1994).Ellakwah et al., (2008) used three simple methods to enhance Entomopathogenic nematodes efficacy.New progenies of S. riobrave and H. bacteriophora(ISk-2 strains) ,with higher penetration rate and virulence than in the original nematodes were obtained .The physiological and ecological properties are the basic information necessary for further studies of a new isolated nematode as a biological control agent.

DISCUSSION
Lipid considered as a major energy reserve.The amount of lipid varies with nematode species (Selevan et al., 1993).In the present study, the total lipids of the six species (S. rarum, S. riobrave, HP4, S3, H. indica and HP2) varied significantly where it recorded (27.3%, 29.2%, 30.4%, 34.3%, 34.5%, and 35.8%). Fitters et al., (1999) found that lipids represented 34-43% of the dry weight of three isolates of Heterorhabditis sp.(UK211 from England, HF85 from the Netherlands & EU17 from Estonia).Lewis et al., (1995), in another strain of the same species, where total lipids comprised was 36-40% for Heterorhabditis sp.We recorded the highest total lipid percentage (35.8%)for the HP2 and as well it obtained highest virulence and penetration rate to G. mellonella (25% &56%, respectively), but its reproduction (14065IJs/L) arranged in the fifth rank correlated to the six tested species.Pate1 et al., (1997) declared a decline in neutral lipids was closely connected with the observed decline in infectivity and survivorship of three steinernematids.Moreover, in the study of Lewis et al., (1995) declared that stored lipids are the major component of nematode energy reserves in members of Steinernematids sp.This indicates the role played by lipids as an energy reserve, in these nematode species.Also, HP2 was recorded highest percentage of total protein (60.3 %).(Qiu and Bedding 2000) mentioned that, Proteins were about half dry weight of fresh IJs then asked, why IJs store large amounts of proteins instead of extra lipids which provide more energy per unit mass is still unknown but proteins might provide extra muscle for locomotion and infection for as long as possible prior to be used as an energy source.This answer may explain that, S. rarum was obtained the lowest total lipids percentage (27.3%),although that it did not have the lowest penetration rate or virulence but it located in the fourth rank for both and its reproduction (39060 IJs/L) arranged in third, however it recorded highest total carbohydrate (26%) and located in the second rank of protein content according to the six species studied.
We observed that S. riboravis has total lipids percentage (29.19%).Abu Hatab and Gaugler (1997) assured that lipid content in the nematode S. riboravis was up to 43.4% per dry weight.Abu Hatab et al., (1998), who asserted that total lipids ranged from 50.7-64.4% in S. glaseri, depending upon the culturing method.On the other hand, S. riboravis reared in the laboratory for several years and changes in its natural traits are expected.El-Assal et al., (2008) established progenies of S. riboravis and H. bactertophora have total lipids, total proteins and total carbohydrates higher than the origin after passing in optical laboratory condition.In our result, S. riboravis obtained penetration rate (38.7%), this result related to that was reported by Koppenhöfer and kaya, (1999), where they mentioned that the average number of nematodes which had penetrated during the 24-h expose at 25°C was (40.5±1.7).The reproduction of S. riboravis (28992IJs/L) arranged in the fourth rank comparing to the reproduction of the other tested species.And its virulence (8.33%) to G. mellonella arranged in the fifth rank in correlation to the other species.(Qiu and Bedding 2000) affirmed that the infectivity of the different species of EPN respond differently to changes in energy reserve levels.As we show the S3 recorded penetration rate (49.7%), virulence (20.9%) and reproduction (41347 IJs/L) these arranged in the second rank according to the other studied species, even though it recorded total lipids (34.3%), total protein (50.6%) and carbohydrate (14.9%).Lewis et al., (1995) estimated the percentage of lipid (34.53%) for H. bacteriophora, 32.40% for S.carpocapsa, and 36.13% for S. glaseri, even so, the author assured that the percentage of lipid was similar for all species when freshly emerged from cadaver.By looking to our result the total lipid percentage ranged from 27.30 to 35.72%, whereas the protein percentage ranged from 42.67 to 60.28%.thatmatched with Selvan et al., (1993) who projected chemical composition of inflictive juvenile entomopathogenic nematodes varied between species.Simoes et al., (1993) indicated to the climatic conditions of the nematode original locality, where each species has an optimum temperature for activity and reproduction of the species.Entomopathogenic nematodes isolated from diverse geographic regions and climates provide an opportunity to compare the biochemical adaptations of morphologically similar animals with similar life histories to a wide variety of environmental challenges Selvan et al., (1993) 149914IJs/L).The rank of virulence(12.5%)for H. indica was the third and its penetration rate(32.66%)was the fifth according to the studied species.In the present study, the recorded penetration rate and virulence of tested nematode species were not associated with their energy reserves.This may be due to the pathogenicity of the bacterial symbiont, as the virulence was determined by the pathogenicity of the bacterium and by the interaction between the nematode and bacteria (Gerritsen and Smits, 1994).
Free living nematodes accumulate and utilize lipid as their major source of energy, whereas in the absence of oxygen, nematodes accumulate and utilize glycogen.Glycogen is critical for the IJ to survive under the anaerobic conditions where IJs are likely to encounter periodically in the soil (Qiu and Bedding 2000).Glycogen and trehalose is the principle carbohydrate energy stored in many nematode species (Behm, 1997&Qiu et al., 2000).Glycogen and protein play a role as alternative energy reserves which are only used on a significant scale when lipids are depleted to a low level.Proteins were about half dry weight of fresh IJ and 40% were consumed during 6 weeks incubation at 28°C indicating that proteins may be significant energy reserves (Qiu and Bedding 2000).Lewis et al., (1997) provided some evidence that in some cases, Steinernema species change their foraging strategy for infecting a host, from ambushers to cruisers (active seekers for host).It seems that nematode juveniles during the ambushing state depend mainly upon carbohydrates as energy reserves because they, actually, stay motionless, waiting for passing by hosts.When they change their searching strategy to be cruisers, where richer sources of energy are needed, they begin to utilize their lipid content.The types of nematodes differ in their energy reserves and their biological activity, such as reproduction and penetration rate, virulence and survival alive in the soil until they find the host.The nematode, which is characterized by high levels of these properties is good nematodes for biological control, if it had some deficiencies in one of these properties, it could be enhanced and an average increase as was did by (El-lakwah et al., 2008),who introduced progenies of nematodes have a better ability to penetrate and virulence than their parents.Therefore, we hope in the future to offer some of these types of nematodes on one method for improvement.