Entomopathogenic nematodes (EPNs) are beneficial soil-dwelling roundworms that parasitize and kill insect pests. They have two life stages - an infective juvenile stage that seeks out host insects, and an adult stage that reproduces inside the insect cadaver. EPNs carry symbiotic bacteria that produce toxins to quickly kill the insect host. EPNs have been mass produced and formulated for use in biological control of agricultural insect pests. Their host range, safety for plants and animals, and ability to control resistant insect pests make EPNs a promising alternative to chemical insecticides.

1. Presented by
VIJAY JOSHI

2. 1
 Introduction
 History
 Classification
 Pathogenicity
 Biology/ life cycle
 Host range
 Mass production
 Advantages and limitations
 Conclusion
CONTENTS

3.  Entomopathogenic nematodes are soil-inhabiting, lethal insect
parasitoids that belong to the phylum Nematoda, commonly called
roundworms. The term entomopathogenic comes from the Greek word
entomon, meaning insect, and pathogenic, which means causing disease.
Although many other parasitic nematodes cause diseases in plants,
livestock, and humans, entomopathogenic nematodes, as their name
implies, only infect insects. Entomopathogenic nematodes (EPNs) live
inside the body of their host, and so they are designated endoparasitic.
They infect many different types of soil insects, including the larval
forms of butterflies, moths, beetles, and flies, as well as adult crickets
and grasshoppers. EPNs have been found in all inhabited continents and
a range of ecologically diverse habitats, from cultivated fields to deserts.
The most commonly studied genera are those that are useful in the
biological control of insect pests, the Steinernematidae and
Heterorhabditidae (Gaugler 2006).
2
• Nematodes are simple roundworms.
• These are colorless, unsegmented and lacking
appendages.
• Nematodes may be free-living, predaceous, or
parasitic.
INTRODUCTION
Gaugler(
2006).
• Nematodes pathogenic to insects are referred to
as Entomopathogenic nematodes.
• Possess an optimal balance of biological control
attributes.
 Entomopathogenic nematodes are extraordinarily
lethal to many important insect pests, yet are safe
for plants and animals

4. Fungi, 49
Baculo-
viruses, 16
Nematodes,
9 Bacteria, 36Micro-
sporidia, 2
Micro-organisms use in biocontrolMicro-organisms use in biocontrol

5. HISTORY
 The first entomopathogenic nematode was described by Steiner in 1923 as
.......Aplectana kraussei [now Steinernema kraussei]
 In 1928 Dutky found Neoaplectana carpocapsae
 The systematic position of the second entomopathogenic nematode, Neoaplectana
.........glaseri Steiner (1929)
 The infective juvenile (IJ) stage, were found to transmit a specific Gram-negative
.........bacterium in the anterior intestine to the hemocoel of insect hosts (Poinar et al.
.........1977).
 Heterorhabditis bacteriophora, was first described in 1976 by Poinar.

6. 6
CLASSIFICATION
Kingdom : Animalia
Phylum : Nematoda
Class : Secernentea
Order : Rhabditida
Family : Steinernematidae
Genus : Steinernema
Family : Heterorhabditidae
Genus : Heterorhabditis

7. 7
• There are 64 species in Steinernema and 8 species in
Heterorhabditis and 1 species of Neosteinernema)
• The two genera Steinernema and Heterorhabditis contain the most
important species of entomopathogenic nematode.

8. EPN -IMPORTANCE
Their demand is increasing due to concern
about development of –
 Pest resistance, resurgence
 Pesticide residue in crops
 Environmental hazards

9. 9
Pathogenicity

10. PATHOGENICITY
 EPNs enter through the insect's natural body openings, the mouth, anus or
respiratory inlets (spiracles) Poinar, 1990.
 Heterorhabditis species penetrate through the intersegmental membranes by
scratching away at these with a special tooth.
 In the insect's blood, infective juvenile releases a highly specialised symbiotic
bacterium.
 These symbiotic bacteria multiply rapidly produce toxins that cause
septicemia.
 Kills the insect within 24-48 hours

11.  Nematodes complete 2-3 generations inside the host
 Over 100,000 nematodes exit the insect
 The cycle of entry of infective juveniles in to the insect host to emerge of
new batch of infective juveniles it takes 10-14 days.

12. Steinernema Infected –nematode are
exiting from wax moth pupa
Root weevil infected with
EPN

13. 13
Biology

14. 14
Biology of EPN
Simple life cycle includes the egg, four juveniles stage
and adult
Infective juvenile ( third stage specialized ) called dauer
larva is resistant to environmental conditions
EPN locate their insect hosts in soil by detecting
chemical cues such as excretory products and
carbondioxide gradient
One generation from egg to egg typically takes from 4 to 7
days.(Anonymous,2013)

15. Penetration
1st gen - Hermaphrodites
2nd gen- amphimictic male and female
IJ3 emergence
3rd stage Infective
Juvenile (IJ3)
Bacteria
Death in 24 - 48 h
15

16. General Life-cycle
Steinernema - Xenorhabdus
IJ3 Male-Female Male-Female IJ3
1st generation 2nd generation 8-10 days
Heterorhabditis - Photorhabdus
IJ3 Hermaphrodite Male-Female IJ3
1st generation 2nd generation 12 days
16

17.  The juvenile stage release cells of their symbiotic bacteria from
their intestines into the hemocoel of insect pest.
 The bacteria multiply in the insect hemolymph and the infected host
usually dies within 24 to 48 hours.
 Once released, nematodes actively seek out thei insect hosts. When
a host has been located, the nematodes penetrate into the insect
through body openings and release symbiotic bacteria that multiply
and rapidly kill the insect.
 Subsequently ,nematodes feed upon the host, and mature into
adults, which mate and produce the next generation.
 The life cycle is completed within a few weeks, and hundreds of
thousands of nematodes emerge in search of new hosts.
Mode of Action

18. Both Xenorhabdus and Photorhabdus are with peritrichous
flagella.
 It have primary and secondary phases
 Primary phase – optimizes nematode development
 Secondary phase –supplies nutrition and antibiotics to the
developing nematodes.
Nematode protect bacteria in their gut and acts as vector.
Bacteria convert the insect into suitable food for the nematodes
survival , reproduction and produces toxins for killing insect.
18

19. Nematode-Bacterium Complex
Nematode depends on bacterium for:
 Killing its host
 Creating a suitable environment for nematode development, and
prevent secondary infection by microbes
 Serving as food source
 Breaking down host tissues to serve as nutrient source
Bacterium depends on nematode for:
 Protection from the external environment
 Penetration into host’s haemocoel
 Inhibition of host’s antibacterial proteins
19

20. Nematode–arthropod associations can be divided into four categories:
1) Phoretic (Nematodes are transported by an insect),
2) Necromenic (Obtain nutrition from insect cadavers),
3) Facultative parasitism and
4) Obligate parasitism (Sudhaus, 2008)
Nematodes also interact with bacteria in at least three ways:
1) Trophism (Nematodes eat bacteria),
2) Parasitism (pathogens cause nematode diseases) and
3) Mutualism (Nematodes and bacteria cooperate)
(Dillman et al., 2012)
Nematodes – Insects – Bacteria Association

21. 21
 Anterior part of the intestine of the infective juvenile is
modified as a bacterial chamber.
 In this chamber the infective juvenile carries cells of a
symbiotic bacterium .
Nematode have
association
with bacteria

22. XENORHABDUS AND PHOTORHABDUS
 Produce enzymes and highly potent insect toxins
 Produce antibacterial and antifungal antibiotics
 Photorhabdus produces enzymes that cause it to glow in the darkness
dark

23. SURVIVAL OF INFECTIVE JUVENILES OF ENTOMOPATHOGENIC
NEMATODES UNDER STORAGE AND THEIR INFECTIVITYAGAINST
GALLERIA MELLONELLA AND SPODOPTERA LITURA
Species Stored in distilled
water
Infectivity on
Spodoptera
litura.
Infectivity on
Galleria
mellonella
Steinernema
carpocapsae
survived up to 20
days without
mortality
65%
75%
Heterorhabditis
indica
40 days without
mortality 80 % 85%
Fresh
Steinernema
carpocapsae
24 hrs 23% 25%
Fresh
Heterorhabditis
indica
24 hrs
35% 38%
Bangalore, Karnataka

24. ENTOMOPATHOGENIC NEMATODES IN RICE PEST
MANAGEMENT
EPN species
Time taken (h) for larval
mortality
C. cephalonica G. mellonella
Multiplication rate
(No. of times) on host
C. cephalonica G. mellonella
Optimum temp
(oC )
Rhabditis
(Oscheius sp.) 22 24 80 95 20-22
S. thermophilum 24 24 75 85 27-30
S. asiaticum 48 29 353 840 22-25
H. indica 51 36 371 1505 20-25
Padmakumari et al, 2008)

25. EFFECT OF EPN’S ON LATE INSTAR LARVAE OF
YELLOW STEM BORER
EPN Time taken for mortality
(h)
Recovery (IJs/ larvae)
Rhabditis (Oscheius sp.)
48 39331
S. thermophilum
135 41507
H. indica
46 56478
Padmakumari et al,( 2008)Andhra Pradesh

26. DISPERSAL OF JUVENILES
 They disperse actively and passively
 Passively they dispersed by rain, wind , soil, humans or insects.
 Active dispersal measured in centimeter and passive dispersal by
insects measured in Kilometeres

27. 27
 Infective juveniles do not feed but can live for weeks
on stored reserves
 Live for months by entering into anhydrobiotic states.
(Hussaini, et al. 2005
 Survival is better in a sandy soil at low moisture , 15-250C.
.

28. 28
HOST RANGE

29. HOST RANGE
In laboratory tests, S. carpocapsae alone infected more than 250 species of
insects from over 75 families in 11 orders.
 Some nematode species may search for hosts at or near the soil surface
“ambusher” (e.g., S. carpocapsae and S. scapterisci)
 Others are adapted to search deeper in the soil profile “cruiser” (e.g., H.
bacteriophora and S. glaseri).
(Campbell & Gaugler,1993)

30. MAJOR EPN SPECIES AGAINST VARIOUS INSECT
PESTS IN INDIA
CROP INSECT EPN
Rice, Sugarcane Scirpophaga incertulas S. carpocapsae
Maize Helicoverpa armigera S. riobrave
Cotton Spodoptera, Earias
insulana
S. carpocapsae
Tomato Agrotis ipsilon S. bicornutum
Brinjal Leucinodes orbonalis S.carpocapsae, H. indica
30

31. Pigeon pea H. armigera H. Indica
Cabbage Plutella
xylostella
H. bacteriophora
Apple Cydia pomonella S. carpocapsae
Ornamentals Root weevils,
Wood borers
H. bacteriophora,
H.megidis
Turf Army worm,
cut worm
S. carpocapsae
Sweet potato Cyras formicaius S.carpocapsae

33. Steinernema carpocapsae
S. feltiae
S. glaseri
S. kraussei
S. kushidai
S. riobrave
S. scapterisci
33
Heterorhabditis bacteriophora
H. indica
H. marelata
H. megidis
H. zealandica

34. CHARACTERISTICS OF KEY SPECIES
 Steinernema carpocapsae – the most studied, available and versatile of all
entomopathogenic nematodes.
 Steinernema feltiae - a parasite of immature flies including Mushroom flies,
fungus gnats and crane flies.
 Steinernema riobravis – highly pathogenic species with host range that runs
across multiple insect orders.
 Steinernema scapterisci – a classical biocontrol agent of adult mole crickets.
 Heterrorhabditis bacteriophora – highly parasitic on some lepidopterous and
coleopteran insect larvae.
 Heterrorhabditis megidis – a parasitic of black vine weevil and various other
soil insects.

35. Mass production
35

36. ISOLATION OF SYMBIOTIC BACTERIA
1.Symbiotic bacteria isolated from infected cadavers
2. Infected larva is surface sterilized by dipping into
95% ethanol
3. Larva cut open with sterile forceps and the
haemolymph is streaked on Macconkey agar
plates
4. Primary phase colonies appeared in red, bright pink colour
Secondary phase –light yellow to brown colour
36

37. ISOLATION OF EPN
• Infected host insect larvae collected from
field
• EPN Isolated using soil baiting technique
wax moth larvae
• Wax moth larvae exposed to soil in a
plastic container
• Incubate at 25o C, for 3 days
• Larvae examine, dead larvae keep in white
trap 10o c

38. STEPS INVOLVED (INVIVO MASS
PRODUCTION)
Bacterial suspension ( warm) at room temperature (20-24 °C).
Dilute 1-ml suspension in an appropriate quantity of sterile
distilled water add 200 nematodes/ml
Evenly distribute 1 ml of the nematode suspension on a 9 cm
Whatman paper
Add 10 conditioned Galleria larvae
38

39. Cover with lid (small petridish keep in large petri dish contain water)
Label the petri dishes and store them in a plastic bag
(to conserve moisture) at room temperature.
Place infected larvae into White traps (5-7 days
after infection)
Result :Steinernema-infected larvae will be
yellowish brown Heterorhabditis-infected larvae
turn brick-red and are also limp
39

40. Inivo production of different entomopathogenic nematodes species in
wax moth larvae using White traps; the different colors of cadavers.
Heather Headrick, USDA

41. REARING
In vivo production
Galleria mellonella is used as a host
WHY?
It is widely available, easily reared, very
susceptible and an excellent host for nematode
reproduction
Average production 30,000 to 50,000 IJs per insect.

42. Variety of formulation of EPN using carriers like Polyurethane
foams,
 Vermiculate,
 Clay
 Aliginate gels
 Flowable gels
 Water dispersible granules
Concentration:
1 billion IJs/ha or 400 million IJs/ac
Formulation

43. COMMERCIAL PRODUCTS AVAILABLE IN
INTERNATIONAL MARKET
Nematode species Product formulation Country
Steinernema carpocapsae ORTHO biosafe,
bio vector, X GNAT
USA
S feltiae Magnet, Nemasys UK
S.riobrave Vector MG
Bio vector
USA
Heterorhabditis
bacteriophora
Otinem USA
H.megidis Nemasys UK
S. carpocapsae Green commandos
Soil commandos
India

44. 44

45. 45

46. APPLICATION
 EPN- initial sign of pest damage
 Their survivorship and success are based on
environmental condition.
 Light irrigation after application
 Apply in the late evening (avoid sunlight and UV)
 Add adjuvants like glycerol, liquid praffin.
 Nematodes should be reapplied on seven day intervals if damage
continues.
 Relative humidity is high, ambient temperature is neither extremely
hot or cold, soil temperature is between 10 to 35 °C.

47. FACTORS AFFECTING BIOEFFICACY
Environmental factors
Temperature (20-300C)
Heat tolerent – S. riobrave, H. indica, S. glasseri
Cold tolerent – S. feltiae, H. megidis, H. marelatus
Ultraviolet radiation, Relative humidity.
Soil : Sandy soil with amendments is good.
(Raveendranath et al. 2007)

48. BIOTIC FACTORS
Natural enemies
 Population of EPN in soil are reduced by bacteria, fungi, mites
and predatory nematode.
 Mites appear to be especially voracious nematode feeders.
 Survival is better in sterilized soil
Hypoaspis aculeifer Beauveria bassiana

49. PREDATORY NEMATODE EATING
ANOTHER NEMATODE
Mononchus sp

50. Combined Application of two Entomopathogenic Nematodes,
Heterorhabditis indica and Steinernema asiaticum to Control The
Rice Leaf Folder, Cnaphalocrosis medinalis (Goen.)
Treatments
Duration
(hrs)
Dosage
IJs/larva
The percent
larval
mortality (%)
EPN emerged
from leaf
folder larvae
Heterorhabditis
indica +
Steinernema
asiaticum
24 75 90 6134 IJ/larvae
Heterorhabditis
indica 48 75 60 4843 IJ/larvae
Steinernema
asiaticum 48 75 30
4330 IJ/larvae
( Sankar M.et al,2009)

51. Dosage Duration (hrs) Glasshouse
(% mortality)
Microplot
(% mortality)
1.25 x 109 24 36 28.7
2.5 x 109 e 48 45.3 38.3
5 x 109 72 75.6 56.6
(Umamaheswari, et al. 2006)

52. INTEGRATION OF EPN IN IPM SCHEDULE
 Heterorhabditis sp. are more compatible than
Steinernema sp. with neem, endosulfan and
fenvalerate were proved safe to nematodes
 Tolerate malathion, mancozeb, carbofuran
etc.
 S. carpocapsae in combination with NPV
produced additive effect for control of S.
exigua in soyabean .
 H. bacteriophora and B. bassiana in
combination resulted higher mortality S.
exigua in soil than individual treatments

53. ADVANTAGES
 High reproductive potential
 Highly virulent ( kill the host within 48 hrs)
 Viable up to 3-6 months (refrigerated at 100C)
 Broad host range ( safe to vertebrates)
 Easy application using spray equipments
 Compatible with many chemical pesticides
 Environmentally safe and acceptable.
 EPN seek out and kill all stages of harmful soil-
dwelling insects.
53

54. LIMITATIONS
 Nematode based insecticides –inactivated if stored in hot
 Cannot left in spray tanks for long periods
 Incompatible with many of the chemicals
 Certain species cannot applied in high pressure equipment
 Different species require different screen sizes

56. Global warming, Anthropogenic causes, adverse environmental
conditions
To increase EPN market share, it is necessary to develop strains of
nematodes and bacteria having enhanced efficacy
Subject to genetic improvement- short generation, small genome
Awareness among researchers of potential for genetic
improvement of these organisms
Need for improvement
56

57.  EPNs
-Efficacy,
-Resistance to environmental extremes,
nematicides
-Development of anhydrobiotic strains
-Tolerance to salinity
 Symbiotic Bacteria
-Pathogenicity ,
-Host specificity,
-Symbiont specificity,
-Resistance to environmental extremes such as cold or
desiccation
-Control of phase variation
Targets For Genetic Improvement
57

58. .
 EPN show great promise as biological control agents of insect pests
as they possess several attributes of ideal biocontrol agents
 Suggests that several insect pests are susceptible to these nematode
and result of preliminary field studies encouraging results
 Due to hazardous insecticides, In IPM particularly organic farming,
nematodes can be used as an alternative.
Conclusions

59. 59