Mass Production

The two different techniques for mass production of entomopathogenic nematodes are (i) in vivo, and (ii) in vitro. Production of entomopathogenic nematodes depend upon the area to be applied as well as the type of nematode species used. If a small plot is to be applied as for research purpose, the in vivo production technique would be appropriate, otherwise for fields in vitro methods are used. In Vivo Production

White trap (White 1927) is one of the most common methods to produce entomo-pathogenic nematodes. Insects are inoculated with entomopathogenic nematodes on a petridish lined with filter paper. After 2-5 days, the infected insects are transferred to the White trap. The White trap consist of an inverted watch glass placed in a petridish on which Whatman paper of appropriate size is placed and moistened with sterilized distilled water. Adequate amount of distilled water is also maintained on and around the watch glass. As the infective juveniles emerge from the cadaver they migrate to the surrounding water and get trapped. The nematodes are harvested from the White trap and collected in a beaker. The concentration of nematodes can be accomplished by

Entomopathogenic Nematodes
Fig. 13.1 Entomopathogenic nematode Steinernema masoodi multiplying over the body of Galleria mellonella larva (Reproduced from Ali et al. 2005b)

gravity settling (Dutky et al. 1964) and/or vacuum filtration (Lindergen et al. 1993). Entomopathogenic nematodes produced in vivo are highly virulent and infective. The last instar of the greater wax moth, G. mellonella, is generally used for in vivo production of entomopathogenic nematodes as this insect is highly susceptible, easily available and produces high yields (Fig. 13.1) (Woodring and Kaya 1988).

Other Lepidopterans and Coleopterans have also been used for in vivo production of nematodes (Shapiro-Ilan and Gaugler 2002). Nematode yield depends upon the insect host size. In general yield of nematode is proportional to the size of the insect host (Blinova and Ivanova 1987; Flanders et al. 1996), however, yield per milligram insect (within host species) and susceptibility to infection is inversely proportional to size or age of host (Dutky et al. 1964; Shapiro-Ilan et al. 1999). The major drawback of in vivo technique is cost of production, which tilts towards the higher side, as two different organisms, host insect and entomopathogenic nematode are to be cultured simultaneously. But such limitation has not restricted the production technology to sustain itself as a cottage industry (Gaugler et al. 2000; Gaugler and Han 2002). In vivo production of entomopathogenic nematodes is likely to continue as small ventures for niche markets or in those countries where labour cost is low. The production and application of entomopathogenic nematodes in infected host cadaver is also an alternative to encourage this technology (Shapiro-Ilan et al. 2001, 2003). In Vitro Production

Bedding (1984) developed a technique whereby huge number of infective juveniles may be economically produced using a chicken, duck or turkey offal medium on a porous polyurethane foam substrate. The rearing container used in this method is a glass flask or autoclaved plastic bags aerated with aquarium pumps and inoculated with approximately 2,000 infective juveniles per gram medium. This method can be used to produce on an average one billion infective juveniles per bag of flask of 500 ml capacity (100 g medium). Currently, some companies, viz., Andermatt (Switzerland), Bionema (Sweden), Oviplant (Poland) and Biologic (USA) are using this technology of nematode production (Ehlers and Shapiro-Ilan 2005). This technique involves the following steps.

Preparation of Rearing Flasks/Bags

Small foam pieces are impregnated with chicken, duck or turkey offal homogenate at the rate of 12.5 parts medium to one part foam by weight. A wide mouthed Erlenmeyer flask of 500 ml capacity is filled with this foam homogenate mixture to the 250-300 ml mark (about 100 g). The mouth of the flask is wiped, plugged with cotton, wrapped with cheese muslin cloth and autoclaved at 121°C for 20 min.

Inoculation with Bacteria

Appropriate Xenorhabdus or Photorhabdus bacterial cells are aseptically transferred to 5 ml of nutrient broth in a test tube and kept overnight on a shaker. The flasks containing autoclaved material are inoculated with the bacterial culture by pouring the contents of one culture tube. The flask is shaken well and stored for 2-3 days at 25°C to allow multiplication of the bacteria.

Inoculation with Nematodes

Each flask colonized with the bacteria is inoculated with surface sterilized 500-1,000 infective juveniles of an appropriate species in 5 ml sterilize distilled water and are incubated at 25°C. The flask after inoculation should not be shaken vigorously to enable better feeding and reproduction of the nematode.


The nematodes can be harvested from the flask in about 15 days. A 20 mesh sieve is taken and foam pieces are piled 5 cm deep on it. The sieve is then placed in a pan and brought near water tap with water level adjusted so that the foam pieces are just submerged. It is left for 2 h. During this period infective juveniles will migrate into the water. The nematodes may be sedimented and rinsed to remove particulate matter and inactive or dead juveniles. The infective juveniles thus obtained should be rinsed with specialized distilled water for several times to make the suspension clear. Various other synthetic media tested to mass culture of entomopathogenic nematodes have been enlisted (Table 13.3).

Table 13.3 Different media recommended for production of entomopathogenic nematodes

Synthetic medium

Nematode species

Incubation period


Nematode harvested


Beef extract, peptone, corn meal, water






on sponge


Dogfood agar medium

S. feltiae



10-Vg medium

Hara et al. (1981)

Kidney/fat homogenate

S. feltiae S. bibionis S. glaseri Heterorhabditis bacteriophora H. heliothidis

2-3 weeks

25 °C

3.8 x 10730 flasks 2.9x10773 flasks 8x10711 flasks 3.6x10710 flasks

3.2x10715 flasks

Bedding (1981)

3% Soyapeptone + 3% yeast extract + 10%

S. glaseri



lOVweek for 93 days

Tarakanov (1980)

chick embryo extract medium

Nutrient broth yeast extract vegetable oil.

H. heliothidis

4 weeks

25 °C

107250 ml flask

Wouts (1981)

flour coated on sponge

Wheat bran+ salad oil

S. feltiae

3 weeks

25 °C

107g medium

Abe (1987)

Wheat bran

S. feltiae

3 weeks

25 °C

107g medium

Abe (1987)

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