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Entomopathogenic Nematode Virulence

It is well known fact that the infective juveniles of both Steinernema spp. and Heterorhabditis spp. enter their insect host through natural openings such as mouth, anus and spiracles and eventually reach in the insect body cavity.  As insects do not have a closed circulatory system like animals, their body cavity acts as an open circulatory system, which is filled with the blood that is technically called as hemolymph.  The infective juveniles of Steinernema spp. carry in their gut species specific symbiotic bacteria of the genus, Xenorhabdus whereas the infective juveniles of Heterorhabditis spp. carry in their gut species specific symbiotic bacteria of the genus, Photorhabdus. Once infective juveniles of both Steinernema spp. and Heterorhabditis spp are in the insect body cavity, they release several cells of symbiotic bacteria, Xenorhabdus spp. and Photorhabdus spp., respectively from their gut via anus in the insect blood. Insect blood is conducive for the multiplication of symbiotic bacteria. In the blood, multiplying nematode-bacterium complex causes septicemia and kill their insect host usually within 48 h after infection.  Based on color of insect cadaver, we can easily determine which entomopathogenic nematode-symbiotic bacterium complex was responsible for killing the insect pests. For example, as shown in a picture below, infection by entomopathogenic nematode, Steinernema spp. and symbiotic bacteria, Xenorhabdus spp. complex gives beige color to wax worm cadavers whereas infection by entomopathogenic nematode, Heterorhabditis spp. and symbiotic bacteria Photorhabdus spp. complex gives red color to wax worm cadavers.

To enlarge, click the photo 

Wax worms infected with entomopathogenic nematodes, Steinernema sp and Heterorhabditis sp: Photo by Ganpati Jagdale

Entomopathogenic nematodes- Mode of Infection

In the soil environment, infective juveniles of entomopathogenic nematodes (Figure 1.) are always searching for the insect hosts to infect, kill, feed and reproduce.  Once the infective juveniles of both Steinernematid (Steinernema spp.) and Heterorhabditid (Heterorhabditis spp.) nematodes locate any larval, pupal or adult stages of their insect host, they will rush to find any easy entry routes/points to enter into the insect host body.  As shown in Figure 2, the infective juveniles of both Steinernema spp. and Heterorhabditis spp. generally use natural openings such as mouth, anus and spiracles/breathing pores (usually one pair of spiracles per body segment located laterally along the thorax and abdomen of  insects) of their hosts as main points of entry.  However, the infective juveniles of only Heterorhabditis spp. can also enter into host’s body by puncturing the inter-segmental membranes of the cuticle (see Figure 2).  The infective juveniles that enter via mouth and anus will end up in digestive track (gut) whereas those enter through spiracles will reach in tracheal tubes.  However, to kill their host successfully for food and development, the infective juveniles of both Steinernematid and Heterorhabditid nematodes eventually need to penetrate by puncturing digestive track (gut) or tracheal tubules (currently, the process of puncturing is unclear) into insect’s body cavity (an open circulatory system) and release symbiotic bacteria, Xenorhabdus spp. and Photorhabdus spp., respectively from their gut in insect blood generally called hemolymph.  In the blood, multiplying nematode-bacterium complex causes septicemia and kill their insect host usually within 48 h after infection.

To enlarge, click the pictures.

Fig. 1. Infective juveniles of entomopathogenic nematodes- Photo by Ganpati Jagdale

Fig. 2. Points of infection by entomopathogenic nematodes into body of their insect hosts: Photo by Ganpati Jagdale

Entomopathogenic Nematodes- Nematode Information

Insect-parasitic nematodes that belong to both Steinernematidae and Heterorhabditidae families are also called as entomopathogenic nematodes because they cause disease to their insect hosts with the help of mutualistically associated symbiotic bacterial pathogens. The entomopathogenic nematodes that belong to families Steinernematidae (Steinernema spp.) Heterorhabditidae (Heterorhabditis spp.) are symbiotically associated with species specific bacteria, Xenorhabdus spp. and Photorhabdus spp., respectively.  The infective juveniles of entomopathogenic nematodes from both these families carry hundreds of specific bacterium cells in their guts and use them to cause disease and kill their insect host within 48 hours after infection.

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Infective juveniles of entomopathogenic nematodes- Photo by Ganpati Jagdale

Entomopathogenic nematodes and their symbiotic bacteria- Nematode Information

Molecular studies demonstrated that the closely related Photorhabdus, symbiotic bacteria of Heterorhabditis nematodes and Xenorhabdus, symbiotic bacteria of Steinernematid nematodes have developed totally different molecular strategies for the same objective of virulence to insects and symbiosis with the nematode.

These findings were presented by An, R. and Grewal, P.S. at the 50th annual meeting of the Society of Nematologists held in Corvallis, Oregon from July 17-20, 2011.

When the infective juveniles of entomopathogenic nematodes are applied to the soil surface in the fields or thatch layer on golf courses, they start searching for their insect hosts. Once insect larva has been located, the nematode infective juveniles penetrate into the larval body cavity via natural openings such as mouth, anus and spiracles. Infective juveniles of Heterorhabditis nematodes can also enter through the intersegmental membranes of the grub cuticle. Once in the body cavity, infective juveniles release symbiotic bacteria (Xenorhabdus spp. for Steinernematidae and Photorhabdus spp. for Heterorhabditidae) from their gut in insect blood. In the blood, multiplying nematode-bacterium complex causes septicemia and kill their insect host usually within 48 h after infection. Nematodes feed on multiplying bacteria, mature into adults, reproduce and then emerge as infective juveniles from the host cadaver to seek new larvae in the soil.

• Leafminers (Liriomyza spp.) are considered as economically important polyphagous pests of many indoor vegetable crops and flowering plants.
• Vegetable host crops included beans, beet, carrots, celery, cucumbers, eggplants, lettuce, melons, onions, peas, peppers, potatoes, squash and tomatoes.
• Flowering host plants included ageratum, aster, calendula, chrysanthemum, dahlia, gerbera, gypsophila, marigold, petunia, snapdragon, and zinnia.
• Leafminer maggots generally feed on leaf parenchyma tissues by tunneling/mining between the upper and lower epidermal leaf surfaces.
• Adults generally feed on sap exuding from the punctures caused by maggots during mining.
• Infested leaves appear stippled due to the punctures made by leafminers while feeding, mining and oviposition especially at the leaf tip and along the leaf margins.
• Widespread mining and stippling on the leaves generally decreases the level of photosynthesis in the plant leading towards the premature leaf drop reducing the amount of shade, which in turn causes sun scalding of fruits.
• Injuries caused by maggots on the foliage also allow entry of bacterial and fungal disease causing pathogens.
• Life cycle of leafminers contains four stages including egg, maggot, pupa and adult.
• Life cycle can be completed within 15-21 days depending upon the host and temperature.
• Adult females lay eggs in leaf tissues, eggs hatch within 2-3 days into maggots, hatched maggots starts feeding immediately and become mature within 3-4 days. Mature larvae eventually cut through the leaf epidermis and move to the soil for pupation and adults emerge within 3 weeks of pupation in the summer.
• Although, chemical insecticides are generally used to protect foliage from injury caused by leafminers, but development of insecticide resistance among leafminer populations is a major problem.
• Insecticides also are highly disruptive to naturally occurring biological control agents, particularly parasitoids.
• Therefore, biological control agents including Bacillus thuringiensis var. thuringiensis (Bt), parasitic wasps (Diglyphus begina, D. intermedius, D. pulchripes and Chrysocharis parksi) and entomopathogenic nematodes (Heterorhabditis spp, Steinernema carpocapase and S. feltiae) have been considered as alternatives to chemical pesticides.
• For successful control of leafminers, entomopathogenic nematodes can be easily applied in water suspension as spray application on plant foliage.
• Entomopathogenice nematodes including S. carpocapase and S. feltiae when applied at the rate of 5.3 X 108 nematodes/ha can cause over 64% mortality of leafminers but need at least 92% relative humidity.

How Entomopathogenic Nematodes kill leafminers

• When the infective juveniles are applied as spray to plant foliage, they enter the leaf mines through the leaf miner feeding punctures or exit holes made by the adults.
• Once inside the mine the nematodes swim to find a leafminer maggot, nematodes then penetrate into the maggot body cavity via natural openings such as mouth, anus and spiracles.
• Infective juveniles of Heterorhabditis also enter through the intersegmental members of the larval cuticle.
• Once in the body cavity, infective juveniles release symbiotic bacteria (Xenorhabdus spp. for Steinernematidae and Photorhabdus spp. for Heterorhabditidae) from their gut in the maggot blood.
• In the blood, multiplying nematode-bacterium complex causes septicemia and kills maggots usually within 48 h after infection.

For more information on the interaction between entomopathogenic nematodes and leafminers, please read following research and extension publications.

• Hara, A.H., Kaya, H.K., Gaugler, R., Lebeck, L.M. and Mello, C.L. 1993. Entomopathogenic nematodes for biological control of the leafminer, Liriomyza trifolii (Dipt.: Agromyzidae).  Entomophaga 38, 359-369.
• Head, J. and Walters, K.F.A. 2003.  Augmentation biological control utilising the entomopathogenic nematode, Steinernema feltiae, against the South American Leafminer, Liriomyza huidobrensis. Proceedings of the 1st International Symposium on Biological Control, (Hawaii, USA, 13-18 January 2002). USDA Forest Service, FHTET-03-05, 136-140.
• Olthof, T.H.A. and Broadbent, A.B. 1992.  Evaluation of steinernematid nematodes for control of a leafminer, Liriomyza trifolii, in greenhouse chrysanthemums. Journal of Nematology 24, 612.
• Tong-Xian Liu, Le Kang, K.M.Heinz, J.Trumble. 2008. Biological control of Liriomyza leafminers: progress and perspective. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources, 2009, 4, No. 004, 16 pp.
• Williams, E.C. and Walters, K.F.A. 1994.  Nematode control of leafminers: Efficacy, temperature and timing.  Brighton Crop Protection Conference – Pests and Disease. 1079-1084.
• Williams, E.C. and MacDonald, O.C., 1995.  Critical factors required by the nematode Steinernema feltiae for the control of the leafminers Liriomyza huidobrensis, Liriomyza bryoniae and Chromatomyia syngenesiae.  Annals of Applied Biology. 127, 329-341.
• Williams, E.C. and Walters, K.F.A. 2000.  Foliar application of the entomopathogenic nematode Steinernema feltiae against leafminers on vegetables. Biocontrol Science and Technology 10, 61-70.

• The shore fly, Scatella stagnalis (Fallén) (Diptera: Ephydridae) is an important insect pest of greenhouse plants.
• Larvae of these flies mainly feed on blue-green algae grown on the surface of plant growing media, walls, floors, benches, and pots.
• But larvae can also cause a serious damage to tender plant tissues thus reducing quality and productivity of plants.
• The adults are not considered as plant feeders but they are nuisance to people and disseminate pathogens such as Fusarium and Pythium from plant to plant as they disperse through the greenhouse.
• Currently, most growers rely on chemicals that kill host plants such as blue-green algae to reduce the incidence of shore flies. However, this method has not been proved effective in reducing shore fly incidence.
• Biological control agents including Bacillus thuringiensis var. thuringiensis (Bt) and entomopathogenic nematodes have been considered as alternatives to chemical pesticides.
• For successful control of shore flies, entomopathogenic nematodes can be easily applied in water suspension as spray application to the surface of plant growing medium.
• Entomopathogenice nematodes including Heterorhabditis megidis, Steinernema arenarium and Steinernema feltiae when applied at the rate of 50 nematodes/cm2 can cause 94- 100% mortality of shore flies.

How Entomopathogenic Nematodes kill Shore flies

• When the infective juveniles are applied to the surface of plant growing substrate, they start searching for their hosts, in this case shore fly larvae.
• Once a larva has been located, the nematode infective juveniles penetrate into the larval body cavity via natural openings such as mouth, anus and spiracles.
• Infective juveniles of Heterorhabditis spp also enter through the intersegmental members of the larval cuticle.
• Once in the body cavity, infective juveniles release symbiotic bacteria (Xenorhabdus spp. for Steinernematidae and Photorhabdus spp. for Heterorhabditidae) from their gut in the larval blood.
• In the blood, multiplying nematode-bacterium complex causes septicemia and kills shore fly larvae usually within 48 h after infection.
• Nematodes feed on multiplying bacteria, mature into adults, reproduce and then emerge as infective juveniles from the cadaver to seek new larvae in the potting medium/soil.

For more information on the interaction between entomopathogenic nematodes and leafminers, please read following research and extension publications.

• Foote, B.A. 1977.  Utilization of blue-breen algae by larvae of shore flies. Environmental Entomology 6, 812-814.
• Goldberg, N.P. and Stanghellini, M.E. 1990.  Ingestion-egestion and aerial transmission of Pythium aphanidermatum by shore flies (Ephydrinae: Scatella stagnalis). Phytopathology 80, 1244-1246.
• Lindquist, R., Buxton, J. and Piatkowski, J. 1994.  Biological control of sciarid flies and shore flies in glasshouses. Brighton Crop Protection Conference, Pests and Diseases, BCPC Publications 3, 1067-1072.
• Morton, A., Garcia del Pino, F., 2007.  Susceptibility of shore fly Scatella stagnalis to five entomopathogenic nematode strains in bioassays. Biocontrol 52: 533-545.
• Morton, A. and Garcia del Pino, F. 2003. Potential of entomopathogenic nematodes for the control of shore flies (Scatella stagnalis). Growing Biocontrol Markets Challenge Research and Development. 9th European Meeting IOBC/WPRS Working Group “Insect Pathogens and Entomopathogenic Nematodes”, Abstracts, 67.
• Vanninen, I., Koskula, H. 2000. Biological control of the shore fly (Scatella tenuicosta) with steinernematid nematodes and Bacillus thuringiensis var. thuringiensis in peat and rockwool. Biocontrol Sci. Technol.. 13: 47-63.
• Zack, R.S. and Foote, B.A. 1978.  Utilization of algal monoculture by larvae of Scatella stagnalis. Environmental Entomology 7, 509-511.

1. Steinernema abbasi- undescribed
2. S. aciari- undescribed
3. S. affine-Xenorhabdus bovienii
4. S. akhursti- undescribed
5. S. anatoliense- undescribed
6. S. apuliae- undescribed
7. S. arenarium- X. kozodoii
8. S. ashiuense- undescribed
9. S. asiaticum- undescribed
10. S. backanense- undescribed
11. S. beddingi- undescribed
12. S. bicornutum- X. budapestensis
13. S. carpocapsae- X. nematophila
14. S. caudatum- undescribed
15. S. ceratophorum- undescribed
16. S. cholashanense- undescribed
17. S. cubanum- X. poinarii
18. S. cumgarense- undescribed
19. S. diaprepesi- undescribed
20. S. eapokense- undescribed
21. S. feltiae- X. bovienii
22. S. glaseri- X. poinarii
23. S. guangdongense- undescribed
24. S. hebeiense- undescribed
25. S. hermaphroditum- undescribed
26. S. intermedium – X. bovienii
27. S. jollieti-undescribed
28. S. karii- undescribed
29. S. khoisanae- undescribed
30. S. kraussei- X. bovienii
31. S. kushidai- X. japonica
32. S. leizhouense- undescribed
33. S. litorale- undescribed
34. S. loci- undescribed
35. S. longicaudum- undescribed
36. S. monticolum- undescribed
37. S. neocurtillae- undescribed
38. S. oregonense- undescribed
39. S. pakistanense- undescribed
40. S. puertoricense- X. romanii
41. S. rarum- X. szentirmaii
42. S. riobrave- Xenorhabdus sp
43. S. ritteri- Xenorhabdus sp
44. S. robustispiculum- undescribed
45. S. sangi- undescribed
46. S. sasonense- undescribed
47. S. scapterisci- X. innexi
48. S. scarabaei- X. koppenhoeferi
49. S. serratum- X. ehlersii
50. S. siamkayai- X. stockiae
51. S. sichuanense- X. bovienii
52. S. silvaticum- undescribed
53. S. tami- Xenorhabdus sp
54. S. texanum- undescribed
55. S. thanhi- undescribed
56. S. thermophilum- X. indica
57. S. websteri- undescribed
58. S. weiseri- undescribed
59. S. yirgalemense- undescribed

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known species of symbiotic bacterial genus Xenorhabdus Thomas and Poinar 1979 associated with a nematode genus Steinernema.

Identification based on colony morphology and molecular techniques

1. Xenorhabdus beddingii (Akhurst 1986) Akhurst and Boemare 1993
2. X. bovienii (Akhurst 1983) Akhurst and Boemare 1993
3. X. budapestensis Lengyel, Lang, Fodor, Szállás, Schumann, Stackebrandt, 2005
4. X. cabanillasii Tailliez, Pagès, Ginibre & Boemare, 2006
5. X. doucetiae Tailliez, Pagès, Ginibre & Boemare, 2006
6. X. ehlersii Lengyel, Lang, Fodor, Szállás, Schumann, Stackebrandt, 2005
7. X. griffiniae Tailliez, Pagès, Ginibre & Boemare, 2006
8. X. hominickii Tailliez, Pagès, Ginibre & Boemare, 2006
9. X. indica Somvanshi, Lang, Ganguly, Swiderski, Saxena, & Stackebrandt 2006
10. X. innexi Lengyel, Lang, Fodor, Szállás, Schumann, Stackebrandt, 2005
11. X. japonica Nishimura et al. 1995
12. X. koppenhoeferi Tailliez, Pagès, Ginibre & Boemare, 2006
13. X. kozodoii Tailliez, Pagès, Ginibre & Boemare, 2006
14. X. mauleonii Tailliez, Pagès, Ginibre & Boemare, 2006
15. X. miraniensis Tailliez, Pagès, Ginibre & Boemare, 2006
16. X. nematophila (Poinar and Thomas 1965) Thomas and Poinar 1979
17. X. poinarii (Akhurst 1983) Akhurst and Boemare 1993
18. X. romanii Tailliez, Pagès, Ginibre & Boemare, 2006
19. X. stockiae Tailliez, Pagès, Ginibre & Boemare, 2006
20. X. szentirmaii Lengyel, Lang, Fodor, Szállás, Schumann, Stackebrandt, 2005

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