Indian Journal of Microbiology

, Volume 52, Issue 3, pp 316–324 | Cite as

Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora and Its Bacterial Symbiont Photorhabdus luminescens

  • Floyd L. InmanIII
  • Sunita Singh
  • Leonard D. Holmes
Review Article


Entomoparasitic nematodes (EPNs) are being commercialized as a biocontrol measure for crop insect pests, as they provide advantages over common chemical insecticides. Mass production of these nematodes in liquid media has become a major challenge for commercialization. Producers are not willing to share the trade secrets of mass production and by doing so, have made culturing EPNs extremely difficult to advance existing technologies. Theoretically, mass production in liquid media is an ideal culturing method as it increases cost efficiency and nematode quantity. This paper will review current culturing methodologies and suggest basic culturing parameters for mass production. This review is focused on Heterorhabditis bacteriophora; however, this information can be useful for other nematode species.


Beneficial nematodes Photorhabdus luminescens Heterorhabditis bacteriophora Mass production 


  1. 1.
    Bathon H (1996) Impact of entomopathogenic nematodes on non-target hosts. Biocontrol Sci Technol 6:421–434CrossRefGoogle Scholar
  2. 2.
    Bedding RA, Akhurst RJ, Kaya HK (1993) Future prospects for entomogenous and entomopathogenic nematodes. In: Bedding RA, Akhurst RJ, Kaya HK (eds) Nematodes and the biological control of insect pests. CSIRO, Melbourne, pp 157–170Google Scholar
  3. 3.
    Ehlers RU (2003) Entomopathogenic nematodes in the European biocontrol market. Commun Agric Appl Biol Sci 68(4a):3–16PubMedGoogle Scholar
  4. 4.
    Ehlers RU, Hokkanen HM (1996) Insect biocontrol with non-endemic entomopathogenic nematodes (Steinernema spp. and Heterorhabditis spp.): conclusions and recommendations of a combined OECD and COST workshop on scientific and regulatory policy issues. Biocontrol Sci Technol 6:295–302CrossRefGoogle Scholar
  5. 5.
    Gaugler R, Han R (2002) Production Technology. In: Gaugler R (ed) Entomopathogenic nematology. CAB International, Cambridge, pp 289–310CrossRefGoogle Scholar
  6. 6.
    Shapiro-Ilan DI, Gaugler R (2002) Production technology for entomopathogenic nematodes and their bacterial symbionts. J Ind Microbiol Biotechnol 28:137–146PubMedCrossRefGoogle Scholar
  7. 7.
    Williams RN, Fickle DS, Grewal PS, Dutcher J (2010) Field efficacy against the grape root borer Vitacea polistiformis (Lepidoptera: Sesiidae) and persistence of Heterorhabditis zealandica and H. bacteriophora (Nematoda: Heterorhabditidae) in vineyards. Biol Control 53:86–91CrossRefGoogle Scholar
  8. 8.
    Boemare NE, Laumond C, Mauleon H (1996) The entomopathogenic nematode–bacterium complex: biology, life cycle and vertebrate safety. Biocontrol Sci Technol 6:333–345CrossRefGoogle Scholar
  9. 9.
    Mahar AN, Jan ND, Mahar AQ, Mahar GM, Hullio MH, Lanjar AG (2008) Efficacy of entomopathogenic bacterium Photorhabdus luminescens and its metabolites against diamondback moth Plutella xylostella larvae on chinese cabbage and artificial diet. Pak J Nematol 26(1):69–82Google Scholar
  10. 10.
    Bowen D, Rocheleau TA, Blackburn M, Andreev O, Golubeva E, Bhartia R, Ffrench-Constant RH (1998) Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280:2129–2132PubMedCrossRefGoogle Scholar
  11. 11.
    Mohan S, Sabir N (2005) Biosafety concerns on the use of Photorhabdus luminescens as biopesticide: experimental evidence of mortality in egg parasitoid Trichogramma spp. Curr Sci 89(7):1268–1272Google Scholar
  12. 12.
    Wouts WM (1981) Mass production of the entomopathogenic nematode Heterorhabditid heliothidis (Nematoda: Heterorhabditidae) on artificial media. J Nematol 13(4):467–469PubMedGoogle Scholar
  13. 13.
    de la Torre M (2003) Challenges for mass production of nematodes in submerged culture. Biotechnol Adv 21:407–416PubMedCrossRefGoogle Scholar
  14. 14.
    Tabassum KA, Shahina F (2004) In vitro mass rearing of different species of entomopathogenic nematodes in monoxenic solid culture. Pak J Nematol 22(2):167–175Google Scholar
  15. 15.
    Friedman MJ (1990) Commercial production and development. In: Gaugler R (ed) Entomopathogenic nematodes in biological control. CRC Press, Boca Raton, pp 152–172Google Scholar
  16. 16.
    Friedman MJ, Langston SE, Pollitt S (1991) Mass production in liquid culture of insect-killing nematodes. United States Patent, 5,023,183. Accessed on 3 Oct 2011Google Scholar
  17. 17.
    Gouge DH, Snyder JL (2006) Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria. J Invertebr Pathol 91:147–157PubMedCrossRefGoogle Scholar
  18. 18.
    Li XY, Cowles RS, Cowles EA, Gaugler R, Cox-Foster DL (2007) Relationship between the successful infection by entomopathogenic nematodes and the host immune response. Int J Parasitol 37:365–374PubMedCrossRefGoogle Scholar
  19. 19.
    Waterfield NR, Ciche T, Clarke D (2009) Photorhabdus and a host of hosts. Annu Rev Microbiol 63:557–574PubMedCrossRefGoogle Scholar
  20. 20.
    Nguyen KB, Smart GC Jr (1998) Morphology of the life stages of three Heterorhabditis spp. from the infective juvenile to the hermaphrodite. Proc Soil Crop Sci Soc Fla 57:101–107Google Scholar
  21. 21.
    Kahel-Raifer H, Glazer I (2000) Environmental factors affecting sexual differentiation in the entomopathogenic nematode Heterorhabditis bacteriophora. J Exp Zool 287:158–166PubMedCrossRefGoogle Scholar
  22. 22.
    Poinar GO, Thomas GM, Hess R (1977) Characteristics of the specific bacterium associated with Heterorhabditis bacteriophora. Nematol 23:97–102CrossRefGoogle Scholar
  23. 23.
    Forst S, Clarke D (2002) Bacteria-nematode symbiosis. In: Gaugler R (ed) Entomopathogenic nematology. CAB International, Wallingford, pp 57–77CrossRefGoogle Scholar
  24. 24.
    Munch A, Stingl L, Jung K, Heermann R (2008) Photorhabdus luminescens genes induced upon insect infection. BMC Genomics 9:229PubMedCrossRefGoogle Scholar
  25. 25.
    Akhurst RJ (1983) Antibiotic activity of Xenorhabdus spp. bacteria symbiotically associated with insect pathogen nematodes of the families Heterorhabditidae and Steinernematidae. J Gen Microbiol 128:3061–3066Google Scholar
  26. 26.
    Duchaud E, Rusniok C, Frangeul L et al (2003) The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens. Nat Biotechnol 21:1307–1313PubMedCrossRefGoogle Scholar
  27. 27.
    Somvanshi VS, Kaufmann-Daszczuk B, Kim K, Mallon S, Ciche TA (2010) Photorhabdus phase variants express a novel fimbrial locus, mad, essential for symbiosis. Mol Microbiol 77(4):1021–1038Google Scholar
  28. 28.
    Brugirard-Ricaud K, Duchaud E, Givaudan A, Girard PA, Kunst F, Boemare N, Brehelin M, Zumbihl R (2005) Site-specific antiphagocytic function of Photorhabdus luminescens type III secretion system during insect colonization. Cell Microbiol 7(3):363–371PubMedCrossRefGoogle Scholar
  29. 29.
    Daborn PJ, Waterfield N, Silva CP, Au CPY, Sharma S, Ffrench-Constant RH (2002) A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects. Proc Natl Acad Sci USA 99(16):10742–10747PubMedCrossRefGoogle Scholar
  30. 30.
    Ruby EG, Urbanowski M, Campbell J (2005) Complete gene sequence of Vibrio Fischeri: a symbiotic bacterium with pathogenic congeners. Proc Natl Acad Sci USA 102(8):3004–3009PubMedCrossRefGoogle Scholar
  31. 31.
    Brenner DJ, Farmer JL III (2005) Family I. Enterobacteriaceae. In: Garrity GM, Brenner DJ, Krieg NR, Staley TJ (eds) Bergey’s manual of systematic bacteriology, volume 2, the proteobacteria, Part B: the gammaproteobacteria, 2nd edn. Spinger, New York, pp 587–607Google Scholar
  32. 32.
    Gerritsen LJM, Smits PH (1997) The influence of Photorhabdus luminescens strains and form variants on the reproduction and bacterial retention of Heterorhabditis megidis. Fundam Appl Nematol 20(4):317–322Google Scholar
  33. 33.
    Plichta KL, Joyce SA, Clarke D, Waterfield N, Stock SP (2009) Heterorhabditis gerrardi n. sp. (Nematoda: Heterorhabditidae): the hidden host of Photorhabdus asymbiotica (Enterobacteriaceae: γ-Proteobacteria). J Helminthol 83:309–320PubMedCrossRefGoogle Scholar
  34. 34.
    Han R, Wouts WM, Li L (1990) Development of Heterorhabditis spp. strains as characteristics of possible Xenorhabdus luminescens subspecies. Rev Nematol 13(4):411–415Google Scholar
  35. 35.
    Bedding RA (1981) Low cost in vitro mass production of Neoaplectana and Heterorhabditis species (Nematoda) for field control of insect pests. Nematologica 27:109–114CrossRefGoogle Scholar
  36. 36.
    Ehlers RU, Stoessel S, Wyass U (1990) The influence of phase variants of Xenorhabdus spp. and Escherichia coli (Enterobacteriaceae) on the propagation of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis. Rev Nematol 13(4):417–424Google Scholar
  37. 37.
    Strauch O, Ehlers RU (2000) Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures. Appl Microbiol Biotechnol 54:9–13PubMedCrossRefGoogle Scholar
  38. 38.
    Strauch O, Ehlers RU (1998) Food signal production of Photorhabdus luminescens inducing the recovery of entomopathogenic nematodes Heterorhabditis spp. in liquid culture. Appl Microbiol Biotechnol 50:369–374CrossRefGoogle Scholar
  39. 39.
    Boemare NE, Akhurst RJ (1988) Biochemical and physiological characterization of colony form variants in Xenorhabdus spp. (Enterobacteriaceae). J Gen Microbiol 134:751–761Google Scholar
  40. 40.
    Forst S, Nealson K (1996) Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol Rev 60:21–43PubMedGoogle Scholar
  41. 41.
    Lango L, Clarke DJ (2010) A metabolic switch is involved in lifestyle decisions in Photorhabdus luminescens. Mol Microbiol 77(6):1394–1405PubMedCrossRefGoogle Scholar
  42. 42.
    Rosner BM, Ensign JC, Schink B (1996) Anaerobic metabolism of primary and secondary forms of Photorhabdus luminescens. FEMS Microbiol Lett 140:227–232CrossRefGoogle Scholar
  43. 43.
    Salaün L, Snyder LAS, Saunders NJ (2003) Adaption by phase variation in pathogenic bacteria. Adv Appl Microbiol 52:263–301PubMedCrossRefGoogle Scholar
  44. 44.
    Clarke DJ (2008) Photorhabdus: a model for the analysis of pathogenicity and mutualism. Cell Microbiol 10(11):2159–2167PubMedCrossRefGoogle Scholar
  45. 45.
    Crawford JM, Kontnik R, Clardy J (2010) Regulating alternative lifestyles in entomopathogenic bacteria. Curr Biol 20(1):69–74PubMedCrossRefGoogle Scholar
  46. 46.
    Wouts WM (1991) Steinernema (Neoaplectana) and Heterorhabdititis species. In: Nickle WR (ed) Manual of agricultural nematology. Marcel Dekker, New York, pp 855–897Google Scholar
  47. 47.
    Inman FL III, Holmes LD (2012) The effects of trehalose on the bioluminescence and pigmentation of the phase I variant of Photorhabdus luminescens. J Life Sci 5:454–465Google Scholar
  48. 48.
    Wyatt GR, Kalf GF (1957) The chemistry of insect hemolymph. Pt II. Trehalose and other carbohydrates. J Gen Physiol 40(6):833–847PubMedCrossRefGoogle Scholar
  49. 49.
    Han R, Ehlers RU (2001) Effect of Photorhabdus luminescens phase variants on the in vivo and in vitro development and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae. FEMS Microbiol Ecol 35(3):239–247PubMedCrossRefGoogle Scholar
  50. 50.
    Poinar GO (1975) Description and biology of a new insect parasitic rhabditoid, Heterorhabditis bacteriophora n. gen., n. sp. (Rhabditida; Heterorhabditidae n. fam.). Nematologica 21:463–470CrossRefGoogle Scholar
  51. 51.
    Adams BJ, Fodor A, Koppenhofer HS, Stackebrandt E, Stock SP, Klein MG (2006) Biodiversity and systematics of nematode–bacterium entomopathogens. Biol Control 37:32–49CrossRefGoogle Scholar
  52. 52.
    Nguyen KB, Hunt DJ (2007) Heterorhabditidae: species descriptions. In: Nguyen KB, Hunt DJ (eds) Entomopathogenic nematodes: systematics, phylogeny and bacterial symbiosis. Koninklijke NV, Leiden, pp 611–692Google Scholar
  53. 53.
    Zioni S, Glazer I, Segal D (1992) Life cycle and reproductive potential of the nematode Heterorhabditis bacteriophora strain HP88. J Nematol 24(3):352–358Google Scholar
  54. 54.
    Ehlers RU, Lunau S, Krasomil-Osterfeld K, Osterfeld KH (1998) Liquid culture of the entomopathogenic nematode–bacterium-complex Heterorhabditis megidis/Photorhabdus luminescens. Biocontrol 43:77–86CrossRefGoogle Scholar
  55. 55.
    Stanbury PF, Whitaker A, Hall SJ (2003) Principles of fermentation technology, 2nd edn. Oxford Press, Butterworth, HeinemannGoogle Scholar
  56. 56.
    Bleakley B, Nealson KH (1988) Characterization of primary and secondary forms of Xenorhabdus luminescens strain Hm. FEMS Microbiol Ecol 53:241–250Google Scholar
  57. 57.
    Dunphy GB (1995) Physicochemical properties and surface components of Photorhabdus luminescens influencing bacteria interaction with non-self response systems of nonimmune Galleria mellonella larvae. J Invertebr Pathol 65:25–34CrossRefGoogle Scholar
  58. 58.
    Pellerone FI, Archer SK, Behm CA, Grant WN, Lacey MJ, Somerville AC (2003) Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes. Int J Parasitol 33:1195–1206PubMedCrossRefGoogle Scholar
  59. 59.
    Chandra GK, Chater F, Bornemann S (2011) Unexpected and widespread connections between bacterial glycogen and trehalose metabolism. Microbiol 157:1565–1572CrossRefGoogle Scholar
  60. 60.
    Jain NK, Roy I (2008) Effect of trehalose on protein structure. Protein Sci 18:24–36Google Scholar
  61. 61.
    Iturriaga G, Suarez R, Nova-Franco B (2009) Trehalose metabolism: from osmoprotection to signaling. Int J Mol Sci 10:3793–3810PubMedCrossRefGoogle Scholar
  62. 62.
    Ohtake S, Wang YJ (2010) Trehalose: current use and future applications. J Pharm Sci 100(6):2020–2053CrossRefGoogle Scholar
  63. 63.
    Krasomil-Osterfeld KC (1995) Influence of osmolarity on phase shift in Photorhabdus luminescens. Appl Environ Microbiol 61(10):3748–3749Google Scholar
  64. 64.
    Moreau E, Inman FL III, Singh S, Walters H, Holmes LD (2011) Remote control of fed-batch fermentation systems. J Chem Chem Eng 5:897–902Google Scholar
  65. 65.
    Singh S, Moreau E, Inman III FL, Holmes LD (2011). Characterization of Photorhabdus luminescens growth for the rearing of the beneficial nematode Heterorhabditis bacteriophora. Indian J Microbiol. doi:10.1007/s12088-011-0238-7
  66. 66.
    Yoo SK, Brwon I, Gaugler R (2000) Liquid media development for Heterorhabditis bacteriophora: lipid source and concentration. Appl Microbiol Biotechnol 54:759–763PubMedCrossRefGoogle Scholar
  67. 67.
    Garcia-Ochoa F, Gomez E (2009) Bioreactor scale-up and oxygen transfer rate in microbial processes: an overview. Biotechnol Adv 27:153–176. doi:10.1016/j.biotechadv.2008.10.006 PubMedCrossRefGoogle Scholar
  68. 68.
    Baliadi Y, Yosiga T, Kondo E (2001) Development of endotokia matricida and emergence of originating infective juveniles of Steinernematid and Heterorhabditid nematodes. Jpn J Nematol 31(1/2):26–35Google Scholar
  69. 69.
    Surrey MR, Davies RJ (1996) Pilot-scale liquid culture and harvesting of an entomopathogenic nematode, Heterorhabditis bacteriophora. J Invertebr Pathol 67:92–99Google Scholar

Copyright information

© Association of Microbiologists of India 2012

Authors and Affiliations

  • Floyd L. InmanIII
    • 1
  • Sunita Singh
    • 2
  • Leonard D. Holmes
    • 1
  1. 1.Sartorius-Stedim Biotechnology Laboratory, Biotechnology Research and Training CenterUniversity of North Carolina at PembrokePembrokeUSA
  2. 2.Central Institute of Agricultural EngineeringBhopalIndia

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