This document provides information on mass production techniques for Trichogramma and nuclear polyhedrosis viruses (NPVs). It describes the steps for rearing the rice grain moth Corcyra cephalonica which is used as a host for producing Trichogramma. The techniques for mass rearing Trichogramma including parasitizing Corcyra eggs and storing the parasitized eggs are summarized. It also outlines the basic steps for mass producing NPVs which involve rearing host insects like Helicoverpa armigera on artificial diet, infecting the larvae with viruses, harvesting and purifying the viruses. Field application doses of different NPVs for crops are also mentioned.

1. DEPARTMENT OF ENTOMOLOGY
ALLAHABAD SCHOOL OF AGRICULTURE
SAM HIGGINBOTTOM INSTITUTE OF AGRICULTURE TECHNOLOGY &
SCIENCES
[Formerly-Allahabad Agricultural Institute]
(Deemed-to-be-University)
ALLAHABAD- 211007, U.P., INDIA BY,
RAKESH KR.
MEENA

2. MASS MULTIPLICATION OF

3. The egg of Sitotroga cerealella are
generally used as laboratory host for
production of Trichogramma in the
USA, France, Germany etc. Whereas in
India ,rice grain moth,Corcyra
cephalonica is used as the laboratory
host.

4. STEPS IN CORCYRA PRODUCTION:
The quantities of sorghum/pearl
millet/maize grain, free from
insecticides, are coarsely milled and
broken into 4-5 pieces in a milling
machine. The broken grains are heat
sterilized at1000C for 1 hour to
eliminate the residual population of
stored product insect.

5. After sterilization, the grains are cooled
and sprayed with 0.1% formalin to prevent
the growth of molds as well as to increase
the grain humidity lost during heat
sterilization.
2 kg of broken grain(provide
carbohydrate for developing larvae) are
then of transferred to plastic or Corcyra
cages along with 100 gm of roasted
groundnut powder(supply protein and
fat for developing larvae) and 5gm
yeast(regulate larval growth).

6. All the above gradients are properly
mixed and Corcyra eggs are sprinkled on
top of the mixture. The cages are tightly
closed with the lid.When plastic basins are
used, they are, covered with cloth and tied
with elastic band/rubber band and special
precautions are taken to protection from
Bracon sp infestation.

7. A total of 200 boxes/plastic or
metallic basins are charged with
rearing medium and Corcyra Eggs
in the first instance in march.
Similar sets of 500 boxes are
charged thrice at 45 days interval
in May, June and August.

8. MASS PRODUCTIO TECHNIQUES
FOR TRICHOGRAMMA
In india,about 26 Trichogramma
spp. Are recorded,of which
T.chilonis,T.japonicum and
T.achaeae are mortality factors for
many crops pest.These parasitoids
attack eggs of sugarcane
borers,scirpophaga insertulas;cut
worms,agrotis.

9.  spp;cotton bollworms,pectiniphora
gossypirlla and Erias spp.maize stem
borer,chilo pertellus etc.
 Angoumois grain moth,sitotroga
cerealella is used as fastitious host fr
mass production of trichogrammatids in
USA,USSR and many European
countries.
 In India,rice grain moth,Corcyra
cephalonica is used as the laboratory
host.

10. PRODUCTION OF TRICOGRAMMA
The production of Trichogramma involves
the following steps
1. The cards of 15 X 7.5 cm size prepared to
obtain 10/12 equal pieces are used. Name
of the laboratory, releasing instructions,
date of preparation of card and expected
date of parasitoid emergence are printed
on the backside of the cards. Dilute
Acacia gum is used for pasting the eggs.

12.  2. The egg are pasted on cards. The
extra loose egg laying on the cards can
be collected in Petri plates by tiling and
tapping the card with a finger. The
quantity of eggs laid by Corcyra is
assessed in measuring cylinder
volumetrically; about 16,000 to 18,000.

13. 3. The egg pasted on the cards are
sterilized by exposing the cards under
30 Watt UV tube at a distance of 35 cm
from the source for 10 minutes. UV
sterilized eggs can be stored in the
lower chamber of the refrigerator up to
5 days can be used for parasitization.

14. 4. The host eggs can also be made
nonviable by exposing the to very low
temp. of 0-20C in the freezer chamber
of a refrigerator for 3-4 hrs adopting the
freezing method the quality of the host
eggs is affected due to shrinkage.

15. 5. The eggs on cards are exposed to adult
Trichogramma in the ratio of 8 :1 for 24
hours at 28+20C in transparent plastic
container. In case the cards are exposed in
polythene bags the host egg to parasitized
egg ratio should be 30:1but in this method
the females are allowed to parasitize till
they die.
Two trichocards can be accommodated in
each polythene bag for parasitization.

16.  6. Parasitized eggs start turning black
on the 3day after parasitization and the
blacking is the life cycle of
Trichogramma is completed in 7-8 days
whereas in the case of Trichogramma it
is completed in 8-9days.

17. 7.The eggs when turned black can be
stored in the refrigerator or can be
transported for field releases. The
emergence of adults generally takes
places after 3-6 days after blacking of
the eggs.

18.  Parasitized egg cards showing blackening of
eggs can be stored in a refrigerator at 12-150C
for 10-15 days, the best stage for storage is
pupal stage.
 When eggs turn black. However, during the
storage the quality of emerging adults is
affected. Prolonged storage beyond 15 day
would impair emergence as well as longevity
and fecundity of the resulting progeny.

20. PRECAUTION
1)Emergence date should be specified on
the cards to guide the user.
 2)The cards should be stapled on the inner
side of the leaves to avoid direct sunlight.
 3)The cards should be stapled in morning
hours and just before emergence to avoid
predation.
 4)Avoid application of insecticides in the
field where Trichogramma are released.if
need arises uses selective/safer
insecticides.

21. Ensure that insecticides are used 15
days after or before Trichogramma
release.
DOSES EGGS/ha. NO.OF
RELEASES
Cotton 1,50,000 06
Sugarcane 50,000 10
Maize 75,000 06
Tomato 50,000 06
Paddy 50,000 06

22. FIELD USE-FREQUENCY OF RELEASES
 Early shoot borer of sugarcane,chilo
infuscatalus-Trichogramma chilonis,4-6
releases@50,000/ha at 10 days interval
starting from 45th day after planting or
with appearance of the pest.
 Cotton bollworms,Helicoverpa
armigera;pectinophora gossypiella; Erias
spp.-T.Chilonis@1,50,000 eggs/ha from
45th day onwards,6weekly releases or
with the appearance of the pest.Moths
are to be monitored by pheromone
trapes.

23. Among viruses of the group
baculoviridae,nuclear
polyhedrosis virus is utilized for
the successful of various insect
pest.
 NPVs are obligating pathogens they
need their specific live hosts for
multiplication.so production if
viruses for use as insecticides
needs mass production of their
hosts as a first step.

24. Basic steps in the production of
NPVs of any insect are-
1.Mass production of/culturing of
host insects
2.Host inoculation with viruses
3.Harvesting of viruses
4.Purification
5.Storage

26. 1. Host insects viz. Helicoverpa armigera and
Spodoptera litura can be reared either on their
natural host plants(foliage, pod, fruits etc.) or on
artificial diet.
 2. Since natural host plants can not be found
throughout the year, maintenance of host insects
on artificial diet has the advantages 0f rearing
under sterile conditions, avoiding contamination,
saving space, time and labor. Thus the use of
artificial diet for mass production of host insects
is economical and easy.

27. 3. Mass culturing of host insects can be
started either from field collected adults
using light traps or from field collected
larvae. The male and female adults of
insects that are allowed in a oviposition jar
for mating should be provided 1% honey as
food in soaked cotton swabs.
 4. Egg laid are collected daily and inner
surface be sterilized by using 0.15% sodium
hypo chloride. Eggs so collected should be
incubated in Petri dishes.

28. 5. Rearing of newly hatched H.armigera
larvae is done by transferring
individually(since larvae are cannibalistic)
into rearing vials containing artificial diet.
6. In case of S.litura since the eggs are laid
in masses, the larval stages are not
cannibalistic; first two instars can be reared
in groups on castor leaves.

29. 7. Gram flour, yeast tablets, methyl
parahydroxy benzoate and ascorbic acid are
added to half the quantity of in a blender and
mixed for 2-3 minutes. Simultaneously, Agar-
Agar is boiled with the remaining quantity of
water and cooled down to 700C.
 Hot Agar- Agar liquid is added to the
blender and mixed with other ingredients.
Finally multivitaplax, vitamin E,Ascorbic acid
and formalin are added and blended for
about 2 minutes.

30. 8. Hot liquid diet is dispended into rearing
vials(approx. 20 ml vials) and allowed for
solidification at room temp. for about 20
minutes, Single larva should be introduced
in each vial on the diet surface and closed
with cotton plug.
9. While 20% of the larval population can be
allowed to pupate(can be collected after 20
days) for the continuous maintenance of the
host insect culture, the rest can be used for
virus production.

31. o Infection 8 to 9 days larvae are
introduced into vials inoculating the
surface of the diet poured in plastic
cues(as against the vials used for
healthy host insect culture) with a virus.
o Dose of 1.1 X104 polyhedral inclusion
bodies(PIB) per larva. Larvae are placed
individually on virus contaminated diet
cups and capped.

32. Observation on larval mortality
is made daily and the dead
larvae with viral symptoms
(cuticle of larvae becomes
fragile and ruptures easily when
touched;body colour changes
to blue or bluish purple)are
harvested and placed in conical
flask.

33. The disesed larvae are macerated in
a mixer using sterile distilled
water.The contents are allowed as
such in the conical flask for several
days at room temperature,during
which time the polyhedra settle
down as a white layer.

34. Alternatively macerated contents
can be sieved through a double
layer of muslin cloth and the filtrate
is standardized for the polyhedral
inclusion body/ml and packed in
white plastic cans for field use.

35. Ingredients Quantity
Kabuli gram flour 105 g
Sorbic acid 1g
Methyl parahydroxybenzoate 2 g
Ascorbic acid 3.25 g
Yeast tablets 10 g
Water 780 g
Agar-Agar 12.75 g
Formalin(10%) 2ml
Vitamin 2 capsules
Multivitaplex 2 capsules
Streptomycin sulphate 25 g

36. FIELD DOSE
 250 LE per hactare.
 One LE=6X109
 Cotton:-H.armigera-HaNPV-450LE/HA
with 5%jaggery and 0.1%Ranipal at ETL 7
ll instar larvae/20 plants.
 Chickpea:-H.armigera-HaNPV-250LE/ha
directed on the inflorescence,one or two
well timed applications to protect seed
crop.

37. Pigeon pea:-H.armigera-HaNPV-250
LE/ha,3-4 sprays required.
Crusiferous vegetables:-S.litura
SINPV-250-450 LE/ha;two-three
sprays at 7-10 days interval;virus in
200-400 lit.of water +1%crude sugar.

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