Research Journal of Agriculture and Forestry Sciences __________________________________ ISSN 2320-6063 Vol. 3(1), 19-22, January (2015) Res. J. Agriculture and Forestry Sci. International Science Congress Association       
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Effect of Entomopathogenic Nematodes (Nematoda: Rhabitida) on Earthworms, Spiders and Ants Anes K.M. and Ganguly S. Division of Nematology, Indian Agricultural Research Institute, New Delhi -110012, INDIAAvailable online at: 
www.isca.in, www.isca.me
Received 18th  September2014, revised 29th October 2014, accepted 7th November 2014Abstract Steinernematids and Heterorhabditids are lethal insect pathogens with bio control potential for managing insect pests of agricultural importance.The non-target effects of entomopathogenic nematodes (EPNs) Steinernema thermophilum, S. glaseri and H. indica were evaluated on beneficial organisms like earthworm (Eiseinea foetida), Spider (Neoscona theisi) and ant (Messor himalayanus). These nematodes were found to be safe towards the earthworm species E. foetida as it did not cause any earthworm mortality up to 3 weeks of treatment when applied @ 500 Infective Juveniles (IJ)/gram compost. However, S. thermophilum, S. glaseri and H. indica induced mortality of 20, 10 and 30 %, respectively on Spider species Neoscona theisi when applied @ 500 IJs/spider, but no mortality was recorded @ 100 IJs/spider. The S. thermophilum and H. indica when applied on ant species M. himalayanus @ 100 IJs/ant could induce 100 % mortality, whereas S. glaseri caused 72 % ant mortality after 5 days. Our study revealed the safety of EPNs towards earthworms but possible adverse effects on spiders and ants.   Keywords:Steinernema, Heterorhabditis, Non target effect.Introduction Entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae are naturally occurring pathogens of insects1 2 which are environmental friendly alternative for insect pest management1 3. Most bio control agents require days or weeks to kill their hosts, yet nematodes, working with their symbiotic bacteria, kill insects within 24 to 48 hours. The non-feeding infective juvenile seeks out insect hosts, and penetrates into the insect body, usually through natural body openings (mouth, anus, spiracles) or areas of thin cuticle. These nematodes carry symbiotic bacteria (Xenorhabdus for steinernematids and Photorhabdus for herterorhabditids), which are released in the insect haemocoel where the bacteria multiply and cause septicemia, thus resulting in the mortality of the insect host. The nematodes feed upon the bacteria, liquefies insect and mature into adults. Thus, entomopathogenic nematodes are a nematode-bacterium complex. They are safe for the plant health, human health, soil and the environment and hence are exempted from registration in many developed countries. However, studies on the non target effects of entomopathogenic nematodes on various species of non target organisms showed a large range from complete harmlessness to pronounced harmful effect4-6. The results of some field trials showed a moderate influence of these nematodes on non target arthropods or even the absence of such an effect. Bathonreported that mortality can be observed among the non target organisms, but the influence of these agents should be temporary and local and so only a part of the population is under attack. Georgis et al demonstrated a negligible influence of entomopathogenic nematodes on non target organisms when used only in short-term pest control. Earthworms are one of the most important beneficial organism in the agricultural field as it improve soil conditions (aeration, drainage and organic matter content) and they are able to change soil structure, move large amounts of soil and affect micro floral and faunal diversity8 9. Besides, many associations (phoretic, paratenic intermediate or sole host) between nematodes and earthworms had been reported10-12, and some authors think that earthworms could be used as vectors to introduce and/or disperse beneficial organisms13-15. Most of the spiders are predators which feed on insect pests of agricultural importance. Most ant species present in agricultural field perform many ecological roles that are beneficial to humans, including the suppression of pest populations and aeration of the soil. Hence, the present study was carried out to evaluate the non target effect of indigenous entomopathogenic nematodes Steinernema thermophilum16, S. glaseri1718, and Heterorhabditis indica19 against epigeic earthworm (Eisenia fetida, spider (Neoscona theisi) and ant species (Messor himalayanus). Material and Methods Nematodes: The non target effect of different entomopathogenic nematodes (EPNs)namely Steinernema thermophilum, S. glaseri and Heterorhabditis indica were evaluated. The pure cultures of Infective juveniles (IJs) of these EPNs already available in our laboratorywere maintained on 4thinstar larvae of greater wax moth, Galleria mellonella (L.)20. The emerging IJs from the insect cadavers were harvested in 
Research Journal of Agriculture and Forestry Sciences ____________________________________________ ISSN 2320-6063Vol. 3(1), 19-22, January (2015) Res. J. Agriculture and Forestry Sci. International Science Congress Association 
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sterile distilled water using a modified White’s trap, and stored at 15o C in BOD incubator. The IJs for the study were used within 2-4 weeks of storage. Beneficial organisms used: The impact ofentomopathogenic nematodes, S. thermophilum, S. glaseri and H. indica was evaluated on ant (Messor himalayanus), spider (Neoscona theisi; Family: Araenidae) and earthworm (Eisinea foetida). The earthworm species was procured from the Division of Entomology, IARI, New Delhi, whereas the spiders and ants were collected directly from IARI Research Farm. Effect of entomopathogenic nematodes on ant: The experiment was carried out in transparent plastic plates of 10 mm diameter lined with 2 layers of Whatman No. 1 filter paper. Each plate was provided with 50 µl of honey solution in small containers to feed the ants. The infective juveniles of entomopathogenic nematodes were released @ 1000 IJs/plate in 300 µl of sterile distilled water. Ten ants were released in each plate to yield a dosage of 100 IJs/ant. All the treatments were replicated five times and the experiment was held at 25+
1 C. The observations on mortality of ants were taken at every 24 h interval. Effect of entomopathogenic nematodes on spider: The experiment was carried out in 12-well plates lined with two layers of Whatman No. 1 filter paper. Two doses (100 IJs/well and 500 IJs/well) of the three species of entomopathogenic nematodes in 100 µl of sterile distilled water were applied in the each well of twelve well plates. After the release of IJs in the wells, the spiders were released as one spider/well of the 12-well plates. Ten spiders were included in each treatment and experiment was held at 25+
1 C. The observations on mortality of spiders were recorded in 24 h intervals. The cadavers were observed for the emergence of nematode progenies. Effect of entomopathogenic nematodes on earthworms: The experiment was carried out in 100 ml capacity plastic bottles containing 20 g of vermicompost. The EPN species were added @ 100 IJs/g compost and 500 IJs/g compost in each bottles and the earthworms were released @ 4 earthworms/bottle. All the treatments were replicated five times and held at 25+
1 C. The observations on survival of earthworms were recorded on 7th, 14th and 21st day of the treatment. Fourth instar larvae of G. mellenella @ 4 larvae/bottle were added on 10th day of treatment to confirm the presence and survival of IJs in the vermicompost. The dead larvae from each bottle were collected in 24 hour intervals. Results and Discussion Entomopathogenic nematodes, S. thermophilum, S. glaseri and H. indica were treated @ 100 IJs/ant on the adults of ant species Messor himalayanus. Among these three species, S. thermophilum observed to be more pathogenic followed by H. indica and least by S. glaseri, in all the time durations evaluated. The data on mortality are shown in table-1. A mortality level of 68 % after 24 hours and 100 % in 120 hours were induced by Steinernema thermophilum. Even though, H. indica induced only 40 % mortality in 24 h, it yielded 100 % in 120 h. However, S. glaseri was found to be least pathogenic against M. himalayanus as it induced only 24 % mortality after 24 h and a maximum of only 72 % in 120 h of treatment application. The levels of mortality induced by S. glaseri were found to be statistically significant as compared with the mortality induced by S. thermophilum in all the four time intervals evaluated. The data on mortality caused by S. glaseri after 120 h of treatment indicated significant difference from the treatments of both S. thermophilum and H. indica in the same time duration. Our study revealed, the possible threat of entomopathogenic nematodes to ant species inhabiting agricultural field, given the fact that the soil is the natural habitat of both EPNs and ants. Table-1 Mortality of ant species, Messor himalayanus by 3 entomopathogenic nematode species when applied at the rate of 100 Infective juveniles per ant Treatments % Mortality 
24 Hrs. 48 Hrs. 72 Hrs. 96 Hrs. 120 Hrs. 
S. thermophilum 68.0 (55.6) 86.0 (68.1) 92.0 (73.6) 98.0 (81.9) 100.0 (90.0) 
S. glaseri 24. (29.3) 32.0 (34.5) 40.0 (39.3) 46.0 (42.7) 72.0 (58.1) 
H. indica 40.0 (39.3) 48.0 (43.9) 64.0 (53.2) 78.0 (62.1) 100.0 (90.0) 
Control 0.0 (0.4) 0.0 (0.4) 0.0 (0.4) 0.0 (0.4) 0.0 (0.4) 
SEM 6.1 4.5 5.4 3.9 2.8 
CD (P= 0.05) 17.0 12.5 15.1 10.8 7.8 
Adult stages of the spider, Neoscona theisi were treated with entomopathogenic nematodes at two different doses. Maximum of only 30, 20 and 10 % mortality was recorded by H. indica, S. thermophilum and S, glaseri respectively when applied @ 500 IJs/spider in 120 h after treatment. But there was no record of mortality of spiders up to 120 h in any of the treatments when these EPNs applied @ 50 IJs/spider. Hence, all the three species of EPNs evaluated were observed to be potentially pathogenic to spiders at higher doses, with least effect by S. glaseri. The data on the mortality of spiders in different treatments are given in table-2. The emergence of infective juveniles from the cadavers of spider confirmed the infection of entomopathogenic nematodes.Earlier Bathon reported the pathogenicity of entomopathogenic nematode, H. bacteriophora on spiders under laboratory conditions. But he could not observe any effect under natural conditions. This could be due to prohibition of natural habitat of these organisms from nematode infection. In the present study, we could observe only a maximum of 30 % spider mortality even at a higher dose of 500 IJs/spider, a dose which is unlikely to encounter in the natural habitat of spiders. So there is more possibility of safety of EPNs against spiders 
Research Journal of Agriculture and Forestry Sciences ____________________________________________ ISSN 2320-6063Vol. 3(1), 19-22, January (2015) Res. J. Agriculture and Forestry Sci. International Science Congress Association 
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under field conditions. But it indicated the need for precautions before the field application of EPNs, as we observed the mortality of spiders, even though at a lower level in very high dose. It would be better to go for field application at a time when spiders are least active.  Table-2 Mortality of Spider species, Neoscona theisi by two different doses of infective juveniles (IJs) of 3 entomopathogenic nematode speciesTreatment % Mortality 
24 Hours 48 Hours 72 Hours 96 Hours 120 Hours 
S. thermophilum@ 50 IJs 00 00 00 00 00 
S. thermophilum  @ 500 IJs 10 20 20 20 20 
S. glaseri  @ 50 IJs 00 00 00 00 00 
S. glaseri  @ 500 IJs 00 10 10 10 10 
H. indica  @ 50 IJs 00 00 00 00 00 
H. indica  @ 500 IJs 00 20 30 30 30 
The entomopathogenic nematodes, S. thermophilum, S. glaseriand H. indica were applied on the earthworms, Eisinia foetida in the vermicompost medium @ 100 IJs/g and 500 IJs/g compost and observed for the mortality/survival of earthworms up to 21 days. There was no record of mortality of earthworms in any of the treatments evaluated. The survival of infective juveniles of these nematodes was evaluated by putting 4th instar larvae of G. mellonella in each treatments after 10 days of starting the experiment. As 100 % of larval mortality was recorded in all the treatments within 48 h of exposure, this study confirmed the survival of infective juveniles of these EPNs in the vermicompost medium without causing any harmful effect on the earthworms. The data on the study is presented in Table-3. Earlier several authors have observed the non-susceptibility of earthworms to different steinernematids14,21,22 and to the slug-parasitic nematode Phasmorhabditis hermafrodita Schneider (Nematoda: Rhabditidae)23. Some authors think that earthworms could be used as vectors to introduce and/or disperse beneficial organisms including EPNs13-15. Raquel et al24 studied the interaction of S. feltiae with earthworm species Eisenia fetida and reported about 20 – 90 % of the earthworms as transmitting nematodes through their gut without any pathogenic effect on earthworms. However they observed a reduction in the mobility of infective juveniles of S. feltiae after passage through the E. fetida gut. The present study concluded the safety of EPN species studied towards earthworms as we could not observe any harmful effect of earthworms exposed to very high dose of EPNs for a period of 3 weeks. Table-3 Mortality/survival of earthworm species, Eisenia foetida and fourth instar larvae of Galleria mellonella (Greater wax moth) by infective juveniles (IJs) of 3 entomopathogenic nematodes speciesTreatment % Mortality 
Earthworms Greater wax moth larvae 
7 Days 14 Days 21 Days 24 Hrs 48 Hrs 72 Hrs 
S. thermophilum  @ 250 IJs/g 00 00 00 5 100 100 
S. thermophilum  @ 100 IJs/g 00 00 00 00 100 100 
S. glaseri  @ 250 IJs/g 00 00 00 00 100 100 
S. glaseri  @ 100 IJs/g 00 00 00 00 100 100 
H. indica  @ 250 IJs/g 00 00 00 00 100 100 
H. indica  @ 100 IJs/g 00 00 00 00 100 100 
Control 00 00 00 00 00 00 
Conclusion Non target effect of entomopathogenic nematodes namely S. thermophilum, S. glaseri or H. indica were evaluated on ant, spider and earthworm. The study observed no pathogenic effect of these EPNs towards earthworm species, E. foetida even at very higher dose. So, we concluded the safety of earthworms by these nematodes. However these EPNs at higher doses were found to be lethal to spider species, N. theisi and ant species M. himalayanus at higher doses. Even though spiders could potentially escape the infection of EPNs, because of the habitat characteristics, it could be a potential threat to ants. So these EPN species should be applied at the time when beneficial ants or spiders are least active.  AcknowledgementWe sincerely acknowledge Indian Agricultural Research Institute for funding the research work in the form of Senior Research fellowship. References 1.Kaya H.K. and Gaugler R., Entomopathogenic nematode, Annu. Rev. Entomol., 38, 181-206 (1993) 2.Hominick W.M., Reid A.P., Bohan D.A. and Briscoe B.R., Entomopathogenic nematodes: Biodiversity, geographical distribution and convention on biological diversity, Biocontrol Sci. Techn., 6, 317-331 (1996)3.Kaya H.K., Soil ecology. In: Gaugler, R. and Kaya, H.K. (eds.). EntomopathogenicNematodes in Biological Control, pp: 93-115, CRC Press, Boca Raton, FL. (1990)
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4.Bathon H., Impact of entomopathogenic nematodes on non-target hosts, Biocontrol Sci. Techn., 6, 421-434 (1996)5.Farag N.A., Impact of two entomopathogenic nematodes on the ladybird, Coccinella undecimpunctata and it’s pray, Aphis fabae. Ann. of Agricult. Sci. (Cairo). Fac. Agric., Ain Shams Univ., Cairo, Egypt. 47, 431-443 2002) 6.Powell J.R and Webster J.M., Interguild antagonism between biological controls: impact of entomopathogenic nematode application on an aphid predator, Aphidoletes aphidimyza (Diptera: Cecidomyiidae), Biol. Control,30, 110-118 (2004) 7.Georgis R. Kaya H.K. and Gaugler R., Effect of steinernematid and heterorhahditid nematodes (Rhahditida: Steinernematidae and Heterorhahditidae) on non target arthropods, Environ. Entomol.,20, 815-822 (1991)8.Brown G.G., How do earthworms affect microflora and faunal community and diversity, Plant Soil,170, 209-231 (1995)9.Doube B.M. and Brown G.G., Life in a complex community: functional interactions between earthworm, organic matter, microorganisms and plant growth. In: Edwards, C.A. (Ed.), Earthworm Ecology, 179–211. St. Lucie Press, Boca Raton, FL. (1998) 10.Gunnardson T. Rundgren S., Nematode infestation and hatching failure of lumbricid cocoons in acidified and polluted soils, Pedobiologia,29, 165-173 (1986) 11.Poinar G.O., Associations between nematodes (Nematoda) and Oligochaetes (Annelida), Proceedings of Helminthological Society of Washshington,45, 202-210 1978) 12.Timper P. and Davies K. G., Biotic interactions. In: Gaugler, R., Bilgrami, A.L. (Eds.)., Nematode behavior, 277-307, CABI Publishing, Wallingford, OX, UK., (2004) 13.Eng M. Sr., Peisser E.L. and Strong D.R., Phoresy of the entomopathogenic nematode Heterorhabditis marelatus by a non-host organism, the isopod Porcellio scaber, J. Invertebr. Pathol., 88, 173-176 (2005) 14.Shapiro D.I., Berry E.C. and Lewis L.C., Interactions between nematodes and earthworms: enhance dispersal of Steinernema carpocapsae, J. Nematol.,25(2), 189-192 (1993)15.Shapiro D.I., Tylka G.L., Berry E.C. and Lewis L.C., Effects of earthworms on the dispersal of Steinernema spp, J. Nematol.,27, 21-28 (1995) 16.Ganguly S. and Singh L.K., Steinernema thermophilumsp. n (Rhabditidae: Steinernematidae) from India, Int. J. Nematol., 10(2), 183-191 (2000) 17.Steiner G., Neoaplectana glaseri n. g., n. sp. (Oxyuridae), a new nemic parasite of the Japanese beetle Popilli japonica (Newm.), J. Washington Acad. Sci.,19, 436-440 (1929) 18.Wouts W.M, Mracek Z., Gerdin S. and Bedding R.A., Neoaplectana Steiner, 1929 a junior synonym of Steinerrnema Travassos, 1927 (Nematoda: Rhabditida), Syst. Parasitol.,4, 147-154 (1982) 19.Poinar G.O., Karunakar G.K. and David H., Heterorhabditis indicaus n.sp. (Rhabditida, Nematoda) from India: separation of Heterorhabditis spp. by infective juveniles, Fund. Appl. Nematol., 15, 467-472 (1992)20.Kaya H.K. and Stock S.P., Techniques in insect nematology. In:Lacey, L.A. (ed.). Manual of techniques in insect pathology, pp: 281-324, San Diego: Academic Press, (1997)21.Capinera J.L., Blue S.L. and Wheeler G.S., Survival of earthworms exposed to Neoaplectana carpocapsae nematodes, J. Invertebr. Pathol.,39, 419–421 (1982)22.Nguyen K.B., Smart Jr. G.C., Pathogenicity of Steinernema scapterisci to selected invertebrates, J. Nematol.,23, 7-11 (1991)23.Grewal S.K. and Grewal P.S., Survival of earthworms exposed to the slug-parasitic nematode Phasmarhabditis hermafrodita, J. Invertebr. Pathol.,82, 72-74 (2003) 24.Raquel C.H., Dolores T. and Carmen G., Phoresy of the entomopathogenic nematode Steinernema feltiae by the earthworm Eisenia fetida, J. Invertebr. Pathol., 92, 50-54(2006)