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Entomophilic nematodes
1
S. AJAY KARTHICK
2ND MSc entomology
Reg no: 081702

2. ENTOMOPHILIC NEMATOLOGY
Etymology
“ENTO” – insects
“PHILOS” – like or love
• Branch of parasitology that deals with nematodes associated with insects (either parasitic or non
parasitic).
• It may be intermediate or definite hosts
(H.E. Welch,1965)
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3. Types of association between nematodes
and insects
• Act as parasite – Romanomermis, Agamermis, Mermis,
Phasmarhabditis and Hermaphrodita
• Act as pathogen – Steinernema, Heterarhabditis, Neosterinernema
• Act as phoresy– Aphelenchus and Rhabditis
• Act as vector–
a) Red ring disease of coconut - Rhadinaphelenchus cocophilus
b) Pine wood nematode- Bursaphelenchus xylophilus
• Act as alternate hosts – Fergusobia spp.
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4. Entomophilic
• Nematodes which enter into
insect host and kill by direct
feeding.
• Single nematode
• Larger in size
• Eg: Mermithidae
Entomopathogenic
• Nematodes which enter into insect
host and kill by releasing pathogen
like bacteria.
• Many nematodes
• Smaller in size
• Eg: Heterorhabditis, Steinernema
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5. Role in biological control
• Entomopathogenic Nematodes as Biological control.
• The reasons - high reproductive potential, kill the host quickly, easy
invitro culture, broad host range, safe to vertebrates and other non
target organisms.
• Mainly three genera of EPN is used for bio control. They are:
Steinernema
Heterorhabditis
Neoplectana
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6. Entomopathogenic nematodes
• A group of nematodes (thread worms), causing death to insects.
• The term entomopathogenic has a Greek origin ‘entomon’, refers to
insect, and ‘pathogenic’, which denotes causing disease.
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7. Systematic position of EPN
Kingdom: Animalia
Phylum: Nematoda (roundworms)
Class: Chromadorea
Order: Rhabditidia
Family: Steinernematidae, Heterorhabditidae
Genus: Steinernema, Heterorhabditis, Neoplectana
• 64 species in Steinernema
• 8 species in Heterorhabditis
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8. DIFFERENCES BETWEEN STEINERNEMATIDS AND HETERORHABDITIDS
CHARACTERS STEINERNEMATID HETERORHABDITID
Secondary stage cuticle Easily lost Good retention
Secondary stage cuticle visibility Loosely fitted, Easily seen Tightly fitted, Difficult to see
Develops to Male or female Hermaphrodite
Bacterial symbionts Modified ventricular intestine Through the intestine
Luminescence No Yes
Colour of dead larvae Ochre, black Red, pink
Reproduction Amphimictic
Hermaphroditic- 1st &
Amphimictic - 2nd generation
Bursa in male No Yes
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10. Life cycle of EPN:
• Completes life cycle in insects – except Infective juveniles- Free-
living stage found in soil.
• IJ actively penetrate through midgut wall & gain entry into
haemolymph.
• IJ releases symbiotic bacteria from its intestine to the insect
haemolymph.
• Bacteria start multiplying in the nutrient-rich haemolymph and cause
septicemic death.
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11. • IJ recover from their arrested state (dauer stage) and start feeding on
multiplying bacteria
• Toxins are produced by developing nematodes and multiplying bacteria
in the body cavity
• It kills the insect host within 48 hours by causing Septicemia
• It produce a plethora of metabolites, toxins and antibiotics that ensures
development and reproduction of nematodes in insect cadaver.
• Life cycle of EPN - 12- 15 days at room temperature.
• The optimum temperature requirement is 25 - 300C
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13. Behaviour of EPN
• Behavior of EPN studied in three phases
1) Host search
2) Host selection and attachment
3) Host recognition
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14. 1)Host search
• Near the soil surface (E.g. S. carpocapsae)
• Deeper in the soil profile (E.g. H. bacteriophora).
• Two search strategies: Ambush or Cruise.
• Ambushers, like S. carpocapsae - Energy-conserving approach-
waiting for mobile surface-adapted hosts .
• Eg: Mole crickets
• Cruisers, like Steinernema giaseri, are highly mobile and respond
strongly to host chemoattractants.
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15. 2) Host selection and attachment
• Nictation – EPN select host by standing on its tail so that most of its
body is in the air
• Nictating species - ambushers
• whereas non nictating species - cruisers
(Campbell, 1993)
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16. 3) Host recognition:
• Surface carbohydrates – specificity of host recognition
• Amphid (olfactory sensory organs at base of lip) - role in recognition.
• 12 sensory neurons
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17. Dispersal of juveniles
• IJ of Steinernematids and Heterorhabditids- both actively and
passively.
• They may be dispersed by rain, wind, soil, humans, or insects.
• Active dispersal - measured in cm
• passive dispersal- measured in km.
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18. Survival of juveniles
• The IJ do not feed but can live for weeks using stored reserves
• For months - anhydrobiotic state.
• The juveniles survive - factors such as temperature, humidity, natural
enemies, and soil type – absence of host.
• Survival is measured in weeks to months
• It is better in a sandy soil or sandy-loam soil than in clay soils with
low or high moisture and temperature.
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19. Inter specific competition
• Heterorhabditids and Steinernematids generally cannot coexist in the
same host.
• Whereas two Steinernematid species - parasitize the same host.
• The mutualistic bacteria - incompatibility of the Heterorhabditid and
Steinernematid species.
• one genus cannot feed on the mutualistic bacteria from another genus.
• Within a genus - can feed on the mutualistic bacterium of another
species.
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20. • Entomopathogenic fungus Beauveria bassiana and EPN - not
compatible within the same host
• The presence of both pathogens in the soil - higher mortality than
either pathogen alone.
• Eg: Scarabaeid larva with the milky disease bacterium, Bacillus
popilliae - increases its susceptibility to EPN
(Thurston & Kaya, 1993)
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21. Mass culturing of EPN:
Entomopathogenic nematodes are produced by different methods
 In-vivo culture method
 In-vitro culture method
solid Liquid
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22. In-vivo culture method
• Two dimensional system - trays and shelves
• Inoculated on a dish or tray lined with absorbent paper - soil or plaster
of Paris.
• After 2-5 days, infected insects are transferred to the White traps
• Cadavers rest surrounded by water in large area.
• As IJs emerge they migrate to the surrounding water trap where they
are harvested.
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24. In-vivo production of different entomopathogenic nematodes in wax moth larvae
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25. Factors affecting yield in In-vivo culture
• A dosage that is too low results in low host mortality
• A dosage that is too high may result in failed infections - secondary
invaders.
• Greater wax moth Galleria mellonella ( 25 to 200 IJs per insect)
• Yellow mealworm, Tenebrio molitor (100 to 600 IJs per insect).
• Environmental factors including
• optimum temperature
• adequate aeration,
• moisture
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26. In-vitro culture
Solid culture:
• Pure culture of their symbiont in a nutritive medium.
• Two dimensional arenas e.g, petri dishes, using various media
• .A liquid medium is mixed with foam, autoclaved, and then inoculated
with bacteria followed by the nematodes
• Media - animal product based (e.g., pork kidney or chicken offal)
• various ingredients including peptone, yeast extract, eggs, soy flour,
and lard
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28. In-vitro production of entomopathogenic nematodes
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29. Factors affecting yield in In-vitro solid culture
• Nematode inoculum rate (IJs per unit of media)
• Increasing inoculum size - Increase nematode growth and decrease
culture time.
• Increasing the lipid quantity and quality – increase yield
• Other media ingredients affects include proteins and salts.
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30. In-vitro culture medium
Liquid culture:
• Symbiotic bacteria are first introduced followed by the nematodes.
• Various ingredients - soy flour, yeast extract, canola oil, corn oil, thistle
oil, egg yolk, casein peptone, milk powder, liver extract and
cholesterol.
• Harvested from media via centrifugation
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31. Factors affecting yield in In-vitro liquid
culture:
• Steinernematids (except one species) occur only as males and females
– capable of mating
• Maximization of mating - achieved through bioreactor design and
regulation of aeration.
• Including media, nematode inoculum, and nematode species
• Nutrition include the content of glucose
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32. Comparison of entomopathogenic nematodes in production approach
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33. Application in field
• Equipment including pressurized sprayers, mist blowers, and electrostatic
sprayers.
• Large diameter nozzles (orifices) and high volumes (up to 400 gallons per
acre) are recommended.
• Filters, screens and swirl plates should be removed.
• It ensure adequate agitation during application - EPN settle quickly in
suspension.
• High pressures (> 300 psi) should also be avoided
• EPN - kept cool by adding ice packs to the spray suspension.
(Shapiro-Ilan et al. 2010)
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34. Methods of application
1. Spraying:
Spray with chemical pesticides on soil or foliage
2. Trickle irrigation:
Deliver the nematodes to the area on one side of the plant.
3. Capsule:
Capsule prepared from wheat bran (5% w/w) with calcium alginate
containing 1000-2000 nematode/capsule is applied.
About 70-80 capsules per plant
4. Liquid Baits:
Desiccated nematodes (S. feltiae) are mixed with 56% sucrose
solution
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35. 5. Pellet Baits:
Wheat bran-wheat flour (50% ), locust bean gum (18mg/ml of
water) and corn oil.
6. Nylon pack cloth bands:
Cloth bands wrapped around tree trunks - Gypsy moth
larvae, Lymontria dispar
7. Cardboard band:
Trunk of apple trees as an artificial bark substrate - infected 23-
73% of Codling moth pre-pupae that moved under the barks
8. Punch and Syringe method:
Inoculation hole by hammer - 1 ml of nematode containing
medium (aerated 12% gelatin with 4000 nematodes/ml) - syringe
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36. Compatibility
• Compatible with many (but not all) insecticides, fungicides and
herbicides.
• Fresh manure or high rates of chemical fertilizers (e.g., urea) - EPNs
persistence and efficacy.
• EPN formulations - above ground applications
• Eg: Mixing EPN with particular surfactants and water dispersable
polymers
(Shapiro-Ilan et al. 2010)
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37. Storage
• Biochemical, behavioural and morphological differences -
Steinernematid and Heterorhabditid species were documented.
• Lipid composition - to regulate membrane function and to adjust for
environmental extremes
• . The relatively high lipid content - survive prolonged periods of
environmental stress.
• The selection of formulation type, ingredient, packaging size and
storage conditions depends upon oxygen, moisture and temperature
requirements for each species.
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38. Shelf life
• The first attempts at formulating EPN were initiated in 1979
• shelf-life as about 1 month.
• Sponge, vermiculite and peat require continuous refrigeration for
viability.
• Immobilization of the nematodes in a matrix increased shelf life
• which reduces energy utilization
• Extending their shelf-life.
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39. Formulation of EPN
1) Aqueous suspension:
• Storage temperatures between 4 and 15°C have produced survival times of 6–12
months for Steinernema spp. and 3–6 months for Heterorhabditis spp
2) Synthetic sponge:
• Survival time of 1–3 months at 5–10°C and the sponges are dipped in a bowl
with water.
3) Gels:
• Alginate capsules - EPN formulation.
• Steinernema feltiae - 99.8% survival after 6 months at 23°C and 100% relative
humidity.
• Heterorhabditis indica - 10°C, up to 1000 IJs per capsule and 90 days of storage
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40. 4) Clay and powder:
S. feltiae, Steinernema bibionis, Steinernema glaseri and
Heterorhabditis heliothidis in a hygroscopic attapulgite clay formulation
with survival time of 8 weeks at 23°C.
5) Infected cadavers:
• The insect cadaver serves as a reservoir to store the EPNs
• Coating facilitate storage and transportation - kaolin-starch mixture and
unflavoured gelatin
• Coating – protection and conservation of the insect cadavers.
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Product name Nematode species Target pests Producer
Ecomask
Steinernema
carpocapsae
Caterpillars BioLogic
Savior Weevil larvae
Steinernema
carpocapsae
Caterpillars Thermo-Trilogy
Guardian
Steinernema
carpocapsae
Caterpillars HydroGardens
J-3 Max
Steinernema
carpocapsae,
Heterorhabditis
bacteriophora
Caterpillars The Green Spot
Heteromask
Heterorhabditis
bacteriophora
Weevils, grubs BioLogic
Lawn Patrol
Heterorhabditis
bacteriophora
Weevils, grubs HydroGardens
Scanmask Steinernema feltiae Fungus Gnats BioLogic
Entonem Steinernema feltiae Fungus gnats Koppert
Nemasys Steinernema feltiae Fungus gnats E.C. Geiger
Commercially Available Entomopathogenic Nematodes 41

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44. Successful biological control using EPN
• Japanese beetle, Popillia japonica - Neoaplectana glaseri - Australia
(Glaser, McCoy, & Girth, 1940)
• Coffee mealy bugs (Homoptera, Pseudococcidae) - H. bacteriophora
– Cuba (Rodriguez et al.,1994)
• Colarado potato beetle, Leptinotarsa decimlineata (Coleoptera,
Chrysomelidae) - Heterorhabditis strains – USA (Berry et al., 1997)
• Sweet potato weevil - Cylas formicarius - H.bacteriophora HP88 in
USA (Jansson & Lacron,1997)
• Tea termite, Postelectrotermes militiaris - S.carpocapsae , S.feltiae
and Heterorhabditis spp. - Sri Lanka
(Amarasinghe & Hominick,1993)
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45. • Nymphs and adults of head lice, Pediculus humanus capitis -
Steinernema rara and S.glaseri and Heterorhabditis bacteriophora
(Doucet et al.,1992)
• Ticks Boophilus annulatus - S.carpocapsae (Glazer & Smith.,1993)
• Cassava root mealy bug, Dysmicoccus sp. - NEPET11 (Heterorhabditis
sp.) and RSCO5 (Heterorhabditis amazonensis) - Brasil
(Bruna and Soares, 2016)
• Black vine weevil, Otiorhynchus sulcatus, and Strawberry root weevil,
O. ovatus - S. carpocapsae and H. bacteriophora - Western Europe and
North America (Georgis et al., 1991)
• Billbugs, Sphenophorus spp. - S. carpocapsae in North America and
Japan (Smith,1994)
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46. • Citrus root weevil, Pachnaeus litus - S. carpocapsae - USA
• Pupae of Western flower thrips - Frankiniella occidentalis control by
nematode Steinernema feltiae
(Lello et al., 1996)
• S. carpocapsae is effective against mole crickets and H. bacteriophora
can be as effective against white grubs
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47. Success stories of EPN in India
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48. Impact of EPN technology in India
1. 1200 tonnes of WP formulation
2. Area of 20,000 ha covered under EPN Green Tech - Management of
white grubs & other soil borne insect pests
3. Area under major crops with EPN Greentech for crop care and
control of white grubs (2017) - Arecanut (14,000ha), banana (500ha),
brinjal (200ha), cardamom (400ha), groundnut (800ha), sugarcane
(14,000ha); others (800ha)
4. Mitigated of cost of production and use of pesticides (fipronil,
chlorpyriphos, phorate) in cardamom, arecanut, groundnut, sugarcane
and vegetables
(ICAR-NBAIR,2017)
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49. EPN isolated from their original locality and source
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50. EPN isolated from original localities and their sources in India
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51. 1)Case study
• Three lepidopterans, Helicoverpa armigera, Spodoptera litura and Galleria
mellonella under laboratory and greenhouse conditions.
• In laboratory bioassay experiment, the pathogenicity on 2nd , 3rd , 4th and final
instars were evaluated with 100, 200 and 300 IJs of H. indica/larva exposed for
12, 18 and 24 h period.
• In the green house experiment, nematode spray [@ 25 ml per plant (at 1000
IJs/ml)] was given on potted cotton leaf harbouring larval stages of H. armigera
and S. litura.
• In the dose response bioassay on 2nd and 3rd instar larvae more susceptible to
300 IJs of H. indica/larva than 4th and 5th instars larvae exposed for 24 h.
• The percent larval mortality of H. armigera and S. litura on cotton plant
influenced by H. indica - final instar larva (62.87 and 56.75% respectively) at 60 h
- superior to the other instars.
(Divya et al.,2010)
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52. 2)Case study
• Actual field tests with the DD-136 N. carpocapsae against agricultural
and forest pests
• These recorded mortality in excess of 60% against the codling moth,
the tobacco budworm, Heliothis virescens (F.),
• The initial field trials with members of the genus Neoaplectana
utilized Neoaplectana glaseri - The Japanese beetle, P.japonica
• It indicated grub reduction of as high as 40 %
(Mertin & Coppel,1977)
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53. 53
ROLE IN IPM
(Hussaini, 2014)
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54. Advantages
• Broad host range
• Able to seek or ambush the host and can kill rapidly
• Mass produced by in-vivo and in-vitro (solid and liquid culture
method)
• Can be used with conventional application equipment
• Safety for all vertebrates, most non target invertebrates and the food
sources.
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55. Disadvantages
• High cost in production
• Lack of labour, knowledge and skills required in nematology
• Limited shelf life and refrigerated storage required
• Difficulties in formulation and quality control
Environmental limitations:
• For survival and infectivity adequate moisture and temperatures are needed
• Sensitive to UV radiation
• lethal effect of several pesticides
• lethal or restrictive soil properties
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56. Take home messages
• Entomopathogenic Nematodes can also be viable alternative in the
near future.
• Three genera Steinernema, Heterorhabditis and Neoplectana
• Neoplectana carpocapse available in market as strain DD 136
• DD 136 strain is being mass cultured in Galleria mellonella.
• It is produced by NBAIR, Bangalore in India
• Other nematodes are not commercially mass multiplied because of low
reproductive potential and low capacity to kill the host.
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