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Chapter 2Chapter 2Chapter 2Chapter 2
Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods
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2.1. The model pest of the study
Present investigations were carried out on biological control of an economic pest,
Spodoptera litura (Fabricius) (Order: Lepidoptera; Family: Noctuidae) commonly
known as the tobacco caterpillar, common cutworm, fall army worm, cluster caterpillar
or rice cutworm. This insect is a polyphagous pest of major status on cotton, rice,
tomato and tobacco. The larvae of S. litura feed on the leaves and foliage of many
plants. The insect has high reproductive capacity with an ability to migrate over large
distance in the adult stage. These characteristic have resulted in it becoming a pest of
many agricultural crops throughout its geographical range, which extends throughout
Asia and Oceania, from the borders of North Africa to Japan and New Zealand (Armes
et al., 1997). In India, it has become particularly noxious pest in most tobacco-growing
tracts of the country and causes extensive damage to tobacco plants (Nair 1986).
S. litura was one of the first insect pests of agricultural importance in India to develop
resistance to insecticides. By 1965 resistance to benzene hexachloride (BHC) was
reported in field populations from Rajasthan (Srivastava and Joshi 1965) and by the
early 1970s, S. litura was reported to develop resistance to endosulphan and carbaryl in
Haryana (Verma et al., 1971) and West Bengal (Mukherjee and Srivastava 1970) and
further in the early 1980s this insect pest was found to show resistance to lindane,
endosulphan, carbaryl and malathion in Andhra Pradesh (Ramakrishnan et al., 1984).
Hence, the present study was aimed at controlling S. litura by the use of egg parasitoids
T. chilonis Ishii and its interaction with F1 sterility (radio-genetic technique).
2.2. Maintenance of insect culture
The culture of S. litura was initiated in the insectary with a few pairs of freshly emerged
adults procured from the agricultural fields of Indian Agricultural Research Institute
(IARI), New Delhi. Mass rearing of this insect was established in an insectary (11x15
ft.). As this insect is vulnerable to the attack of microorganisms, the insectary was
sterilized with 10% formaldehyde fumigation prior to establishing the culture. The
room was illuminated with fluorescent light. Ambient environmental conditions like, 27
± 1.0 0C temperature, 75.0 ± 5.0 % relative humidity and 12 h light: 12 h dark, photo
period regimen were maintained.
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2.2.1. Adult pairing
The male and female adults were paired in groups in specially designed cages made of
perspex and nylon. 4-6 pairs were kept in cages of size: 20 x 20 x 20 cm. The cages
were specially designed with all the four walls made of perspex sheet, out of which the
front side of the cage was fitted with 30 cm long nylon net funnel for convenient
manipulation of moths (Figure 2.1). The two facing side walls of the cage were fitted
with nylon net windows to provide sufficient aeration. Cotton swabs soaked in 10-15 %
honey solution (w/v) were placed in small plastic containers on which adults were fed.
These swabs were replenished every day. A fresh castor (Ricinus communis) leaf with
its petiole dipped in water contained in a glass vial was introduced in the cage to
provide an ovipositional trap simulating the natural environment to the female moths
for oviposition. The eggs were generally laid in clusters of about 100-400 eggs on the
ventral surface of the leaves and sometimes on the nylon/perspex walls of the cage. The
eggs were collected with the help of a fine hairbrush (No.0) in small plastic containers
(7 x 7 x 5 cm). The eggs were treated with 0.2% sodium hypochlorite (NaOCl) or 2.0 %
formalin solution followed by a rinse with distilled water for proper surface
sterilization.
2.2.2. Rearing of S. litura on natural diet
The culture was maintained on castor leaves (Ricinus communis), the natural food for
this moth. The surface sterilized eggs were kept in a BOD incubator for incubation in
plastic containers (7 x 7 x 5 cm) at high relative humidity (80.5 ± 5.0 %). The eggs
containing containers were provided with fresh and tender castor leaves to provide
immediate food accessibility to newly hatched 1st instar (L1) hatchlings after the
embryogenesis was over. The newly hatched larvae were transferred to the glass jars
having a layer of filter paper at the base. Initially, about 50-100 L1 were placed in
each jar but as the larvae grew, gradually thinning was done to avoid overcrowding
and infection. These larvae were fed with tender and fresh leaves of castor. Castor
leaves were replenished at every 24 h interval till the larvae reached the 3rd instar.
Fourth instar onward, these larvae acquired a voracious feeding habit until pupa
formation occurred.
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Figure 2.1. Adult pairing cage (made up of Perspex and nylon) of size: 20 x 20 x 20 cm
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Thereby the food consumption increased and consequently sometimes, these leaves
were provided twice a day. Larvae were transferred daily to fresh jars. The rearing
jars were cleaned every day to remove all the excreta, dead and infected larvae
exuviae. The jars were washed and rinsed in 5% formaldehyde solution to avoid
infection. The 6th (last) instar larvae fed voraciously for 2-3 days and were found to
consume more than 70% of the total food they would devour in their entire larval life.
The phagoperiod of last instar larvae persisted for about first two days. This stage was
terminated by cessation of feeding; the larvae showed random movements and entered
in “wandering stage”. Subsequently, with a continuous purging out of material from
the alimentary canal, the larvae entered the prepupal stage, then transferred to jars
having a bed of filter papers and were covered with a castor leaf for pupation to occur.
The pupae were collected next day and kept in separate jar. After proper sclerotization
of the pupae, when the pupal skin was hard enough to be handled manually, i.e. two
day old pupal stage, the pupae were sexed accordingly to the location of gonopore
which is located on the eight abdominal sternum in female pupae and on the ninth
abdominal sternum in male pupae. After seven to eight days of pupae formation, the
adults eclosed out of the pupal cases and their reproductive pairing was resumed for
the continuation of culture.
2.2.3. Handling techniques to control microbial contamination in culture of S. litura
Following precautions were taken to check the microbial infection during the rearing of
insects, (i) Formaldehyde fumigation was done to disinfect the insectary before
introduction of insects. (ii) Glassware and plastic containers were washed with
detergents, followed by a rinse in 5.0% formalin and then water. The jars were then
oven dried at about 70-80 0C and cooled down to room temperature before use. (iii)
Castor leaves were washed with water or 0.001 % potassium permanganate (KMnO4)
solution followed by a second wash with water. (iv) Eggs were surface sterilized by
sodium hypochlorite (0.1-0.2 %) or 1.0-2.0 % formalin solution. (v) Pupae were surface
sterilized by 1.0% sodium hypochlorite or 3.0-4.0 % formalin solution. (vi) Remains of
uneaten castor leaves and excreta from the insect rearing jars were removed regularly.
(vii) Fresh food was replenished to the larvae daily or at times twice a day. (viii) No
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fresh leaves were introduced to the rearing jars at the wandering stage onwards as the
larvae in the late wandering and the early pre-pupae stages were observed to exude and
release moisture, otherwise that would create infection.
2.2.4. Life cycle of S. litura
S. litura is a polyphagous pest belonging to family noctuidae commonly known as the
tobacco caterpillar, is a foliage feeder. The male and female moths were observed to
attain sexual maturity about one day after emergence. Mating occurred during
scotophase. Eggs were laid on the lower surface of the castor leaves and on the walls of
the cage, in clusters of hundred to four hundred each. On an average a mated female
laid eighteen hundred to twenty two hundred eggs in its life span. The eggs were
arranged in rows and the egg mass was generally covered with buff colored hair shed
from the anal tuft of the female moth. Each egg was round and ridged, pearly white
when freshly laid but turned darker afterwards. The egg incubation period was about
three to five days.
S. litura showed growth and development through six larval stages (Figure 2.2). The
newly hatched caterpillars were tiny (averaging about 1.0 mm in length), blackish-green
in color. The body of the larva was distinctly divided into head, thorax and abdomen.
The abdomen consisted of ten segments. The functional legs were present on the
ventrolateral side of the thoracic segments. Five pairs of abdominals prolegs were also
located on the abdominal segments 3-6 and 10. The larvae grew in size with each
moult. The mature last instar caterpillar was stout and smooth; dull greyish and blackish
green in color with pale white dorsal and lateral stripes. The head capsule was black
with a typical inverted ‘V’ mark over it. The last instar caterpillar stopped feeding and
secreted fluid from the body, resulting in contracted size. The larvae became lethargic
and acquired somewhat ‘comma’ shape body forms. This stage lasted for 1-2 days and
it was termed as the pre-pupa, which moulted to form the pupa.
Freshly formed pupae were pale green in colour. With gradual melanization, they
turned dark reddish brown in colour with shiny surface. The skin covering the wing
pads was found to be darker in colour than rest of the body.
Figure
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Figure 2.2. Life cycle of Spodoptera litura
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Within 4 h of pupae formation, abdomen of pupa exhibited dark spiracles at the lateral
sides and medially placed inwardly curved spines on the last segment. The pupae
measured about 16-18 mm in length and 5-6 mm in breadth. Female pupae were
slightly larger than the male pupae. In addition, the fresh weight of female pupae was
greater than that of male (Seth et al., 1997). The adults were stoutly built. The females
were slightly larger than the males. The males were distinguished by the presence of a
steel gray shiny patch at the outer corner of the forewings, where as in females the patch
at the corresponding place was pale-straw to golden in colour.
2.3. Culturing of Corcyra cephalonica (Stainton)
In India, Corcyra cephalonica is being used as a laboratory host for multiplication of
Trichogrammatids. The production procedure for multiplication of Corcyra
cephalonica is detailed below:
1cc. (~20,000) of Corcyra eggs were collected from the Nuclear Research Laboratory
(NRL) of IARI, New Delhi, to establish the culture at the Radiation Biology and
Applied Entomology Laboratory of Department of Zoology, University of Delhi. The
following procedure was adopted for the mass production of Corcyra cephalonica
under laboratory conditions.
2.3.1. Facilities required for mass rearing of C. cephalonca
(i) Corcyra rearing boxes, (ii) Iron racks, (iii) Transparent polyvinyl 3 lits. Funnel, (iv)
Mosquito net, (v) Sieves (BSS 36), (vi) Cotton bundles, (vii) Measuring cylinders, (viii)
Glass tubes, (ix) Honey, (x) Formalin, (xi) Hot air Oven.
2.3.2. Production procedure
After sieving the cleaned eggs were collected in petridishes. Some of the eggs were
used for maintaining the culture and rest of eggs were used for rearing the parasitoids,
Trichogramma chilonis in the laboratory (Flow chart as given below).
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Sorghum with white bold grains meant for human consumption was procured
Sorghum was milled to make 3 to 4 pieces of each grain
Sorghum was heat sterilized in oven at 1000C for 30 min
Sorghum was sprayed with 0.1% formalin
Sorghum was air-dried
Sorghum was poured @2 Kg /Box
Boxes containing sorghum were infested with Corcyra eggs
Boxes are kept in racks and lid was closed
On 40th day onwards moths started emerging
Moths were collected daily and transferred to specially designed ovipositional cages (a thick transparent polyvinyl funnel whose wide bottom portion was covered with
mosquito net to prevent the escape of adult and the tip of inverted funnel was plugged with 20% honey cotton swab)
Eggs were collected and passed through 40, 30 and 15 mesh sieves and cycle continued
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2.3.3. Taxonomy and Life cycle of Corcyra cephalonica
The life cycle of C. cephalonica was completed in about 45 to 50 days (Figure-2.3).
The eggs were deserved to hatch after 4 -5 days of egg laying near the larval food. The
larvae were dull white in colour with brown heads and had long fine hairs covering the
body. They persisted for 15-20 days under favorable conditions. Rice moth’s larvae
produced large amounts of strong webbing and frass, before spinning a dense white
cocoon in which they pupated. The pupal stage lasted for 7-10 days. The adult survived
upto 7-8 days.
2.3.3.1. Features
The feature include, two pairs of well-developed membranous wings, with few cross
veins; Clothed with broad scales, generally suctorial mouth parts; metamorphosis
complete with egg, larval, pupal and adult stages; larvae frequently having eight pairs of
limbs, pest of grain (especially rice), oil seeds, cocoa beans, dried fruits, etc.
2.3.3.2. Systematic position
Phylum - Arthropoda
Sub-Phylum - Mandibulata
Class - Hexapoda
Group - Ectognatha
Division - Endopterygota
Order - Lepidoptera
Family - Pyralidae
Genus - Corcyra
Species - cephalonica (Rice meal moth)
(a) Eggs
(c) L2
(e) L4
(g) Pupae
Figure 2
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(b) L1
(d) L3
(f) L5 & PrePupae
(h) Adult- ♀ & ♂
2.3. Life cycle of Corcyra cephalonica
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2.4. Laboratory culture of egg parasitoid, Trichogramma chilonis Ishii
2.4.1. Mass production of Trichogramma chilonis
The strain of T. chilonis used in this test was obtained from the Nuclear Research
Laboratory, IARI, New Delhi where it was already mass reared for many generations
on eggs of the rice meal moth (C. cephalonica). This wasp’s culture was established at
Radiation Biology and Applied Entomology Laboratory of Department of Zoology,
University of Delhi, Delhi-7.
T. chilonis adults were reared for more than 30 generations on C. cephalonica eggs (as
factitious host) in a controlled environment, BOD was fixed at 27±10C, 65±5% relative
humidity (RH), and a 12-h photophase (0600 to 1800 h and 1800 to 0600h). Large
numbers of C. cephalonica eggs were obtained by placing 1 to 2 day old adults in
inverted 3.8-litres plastic funnel, closed with mosquito net at the bottoms, after a period
of 24 hrs. The collection of host eggs was followed by sieving with mesh size 15 (Mesh
opening-0.0496 inches), 30 (0.0234 inches) and 40 (0.0165 inches). Then these cleaned
eggs were exposed to ultra violet rays (15 W UV-tube) for 10 minutes in a closed
chamber maintaining a distance of 12.5 cm between the eggs and the tube to make these
eggs sterilized. The eggs could also be made inviable by exposing them to very low
temperature, 0-40C in the freeze chamber of a refrigerator for 3-4 hours (Singh, 1969).
The egg cards used for pasting the eggs comprised of 6 cm height × 1.5 cm width
(Figure 2.5). These eggs were sprinkled on cards smeared with uniform layer of gum so
as to enable uniform spreading of the eggs on the cards. The “egg card” after drying
was kept in a container, a glass vial of size 10cm height × 3 cm diameter and exposed to
freshly emerged Trichogrammatids under tube light (40W). T. chilonis which were
about to emerge from Tricho - cards, (hereafter referred as ‘mother cards’) inside the
glass vial and freshly prepared egg cards (hereafter referred as ‘daughter cards’) were
kept just opposite to that of the mother card in the same vial. Fine streaks of 50%
honey solution was provided regularly as food source on the inner wall of the Vial till
the death of the Trichogramma adults. After 24h- 48h of exposition of daughter cards
were transferred to the new vials, the parasitized eggs started turning black on the 3rd
day of parasitization and the blackening was completed on the 4th day, normally 80-
90% successful parasitization was observed to occur.
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a) Culture Jar & Diet b) Damaged Sorghum grain
c) Pairing cage (PVC funnel &Net) d) Pairing cage with sieve
Figure 2.4. Culture of Corcyra cephalonica with natural diet, Sorghum grain and collection of host eggs from the adult pairing cage
a) Parasitised host eggs b) Culture vials with egg card
Figure 2.5. Culture of Trichogramma chilonis on factitious host eggs, Corcyra cephalonica
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2.4.2. Life history and biology of T. chilonis
The parasitoid, Trichogramma species are free living tiny wasps usually found in most
of the crops except tobacco, chick pea and pegion pea. The female Trichogramma is
able to locate the pest egg on the crop and parasitizes it by inserting her egg in it. As a
result of parasitism, the pest larva does not hatch. Instead, Trichogramma wasp
completes the development within the pest egg and emerges out after 7 days (Figure
2.6). Thus, the pest is killed in its egg stage itself. One female Trichogramma is capable
of parasitizing upto 120 eggs of the pest in her life span of 4 to 5 days.
2.5. Irradiation of insects
Irradiation facility was used in the INMAS, New Delhi. A sublethal dose range of 100-130
Gy was used to irradiate freshly emerged adult male moths Spodoptera litura by the source
Cobalt-60, (Gamma-5000, BARC) (Figure 2.7). The dose rate was 2.16-3.77KGy/hr.
2.6. Locomotor activity egg parasitoid, Trichogramma chilonis
2.6.1. Locomotor assay chambers
Tests were conducted in a vertically held clear poly-acetate cylindrical chamber (200
mm high, 90 mm dia.), having a tightly fitting, removable clear plastic Petri dish as its
bottom and another similar dish forming its lid at the top. The lid was illuminated by a
fluorescent tube light (40 W) at 6 inches above the chamber. The locomotor assay
chambers used for experiments were of three types, viz., Version I chamber, Version II
chamber and Version III chamber (Figure 2.8). In Version I chamber (Figure 2.10), the
cylinder was marked off into a 4-cm high ‘lower zone’ (from 0-cm to 3-cm marked as
A-zone in addition to sticky Vaseline ring from 3-cm to 4-cm) and a 16-cm high ‘upper
zone’ (i.e. from B-zone to Top lid). The section of its vertical wall in the lower zone
was designated as the ‘lower wall’ and that in the upper zone as the ‘upper wall’.
Version I chamber had a coating of odourless petroleum jelly (brand name ‘Vaseline’,
Hindustan Lever, India) as a ring around the inner surface of the upper 1-cm border of
the lower wall. The lower zone of the cylinder was thus comprised of its bottom and
lower wall including the Vaseline ring whereas its upper zone included the upper wall
and the lid. The Vaseline ring would trap and prevent the wasps from walking, but not
from flying, from the chamber’s bottom or lower wall to the upper wall or the lid.
Figure 2.6
Figure 2.
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6. Life cycle of Trichogramma chilonis
.7. Gamma Irradiator (Gamma-5000)
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Version-I
Figure 2.8. Modified versions of the assay chambers, as described by Forsse Dutton & Bigler, 1995; van Lenteren,
Version
Figure 2.9. During experiment the chamber was surrounded by an equal sized cylindrical sleeve of opaque black paper to prevent entry of light from its sides in case of V
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Version-II
Modified versions of the assay chambers, as described by Forsse Dutton & Bigler, 1995; van Lenteren, et al. 2003.
Version-II Version-III
During experiment the chamber was surrounded by an equal sized cylindrical sleeve of opaque black paper to prevent entry of light from its sides in case of Version II & Version III test cylinders.
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Version-III
Modified versions of the assay chambers, as described by Forsse et al. 1992;
III
During experiment the chamber was surrounded by an equal sized cylindrical sleeve of opaque black paper to prevent entry of light from its
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For locomotor assays, the Tricho cards were made of Spodoptera litura eggs and the
parasitoid emerging out from there Tricho cards (~50-60 parasitoids) were used for
ascertaining walking, hops and flight activity.
2.6.2. Flight tests with single wasps
Adults emerging from the parasitized host eggs during the periods (4.00AM-9.00AM)
were transferred singly to the bottom of the Version I chamber. Each wasp was
observed continuously for 15 min. Aerial movements of the wasps for distances shorter
than ≈2 cm looked like hops and were regarded as such whereas longer ones were
regarded as flights, as distinguished by King et al. (2000) for Nasonia vitripennis
(Hymenoptera: Pteromalidae). The number of flights shown by each individual was
recorded. The site of origin of each flight from the bottom, lid, lower or upper wall of
the chamber and its destination on any of these sites were also recorded. As the
parasitoids were minute to distinguish the sexes before the experiment; here at the end
of the observation period, sex of the tested wasp was identified under microscope.
As female adults of Trichogramma chilonis are responsible for locating host eggs and
ovipositing therein so the walking and hopping (low level flight) activity of female
wasps were reported here. The wasps released singly on the bottom of the, ‘Version-I’
test chamber were observed to move about in various directions for varying distances
by walking and hopping at different times. Of the fifteen wasps tested for each regimen,
some individuals might not hop at all but walked only, whereas others walked and
hopped one or more times during 15-minute observation periods.
2.6.3. Locomotor assay chamber for group wasps, Trichogramma chilonis
Version-II cylinder (Figure 2.8 and 2.11)
Group locomotor assay was performed in Version-II polyacetate cylinder, taking
readings on locomotor activity (number of walks, hops, flight) of a cohort of parasitoids,
Trichogramma chilonis. Version-II cylinder (20 cm ht x 9.0cm dia.) was divided into 5
zones (from bottom upwards- Zone A : 0-1.5cm, Zone B: 2.5-7.0cm, Zone C: 7.0-
11.5cm, Zone D: 11.5-16.0cm, Zone E: 16.0-20.0cm) with Vaseline coated sticky ring
(1.5-2.5cm) in between Zone A and Zone B. Vaseline (odorless petroleum jelly, brand
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name ‘Vaseline’, Hindustan Lever, India) was coated on floor, ‘sticky’ ring (1.5 – 2.5cm
from bottom) and top petridish (roof). Releasing site small Petridish (dia-3.5cm) was
unglued and it was placed on the center of the bottom petridish (dia-9.0 cm).
Version-III cylinder (Figure 2.8 and 2.12)
Group locomotor assay was also performed in Version-III polyacetate cylinder, taking
readings on locomotor activity (walks, hops, flight) of a cohort of male and female
Trichogramma chilonis. Version-III cylinder (20 cm ht x 9.0cm dia.) was divided into 5
zones (from bottom upwards- Zone A: 0-1.5cm, Zone B: 2.5-7.0cm, Zone C: 7.0-
11.5cm, Zone D: 11.5-16.0cm, Zone E: 16.0-20.0cm) with no Vaseline coated sticky
ring. Instead the vaseline (odorless petroleum jelly, brand name ‘Vaseline’, Hindustan
Lever, India) was coated on floor, all walls and top Petridish (roof). Releasing site
Petridish (dia-3.5cm) was unglued and it was placed on the center of the bottom
petridish (dia-9.0 cm).
2.6.3.1. Flight tests with wasps in groups
These tests were conducted in the cylindrical chamber versions II and versions III. For
each test, a 2.5 cm2 card bearing approximately 100 parasitised host eggs was placed in
a Petri dish (3 cm dia.). After allowing adults to emerge during the periods (4:00 A.M.
to 9:00 A.M.), the unhatched host eggs were removed, and the Petri dish with the wasps
of both sexes was placed in the chamber on its bottom. The chamber in its black paper
sleeve was placed under the light source (Figure 2.9) to start the test at 9:30 A.M. The
wasps could move about and fly to various sites in the chamber, getting stuck to the
sites with Vaseline coating or moving out again from bare sites. After 8 h, the black
paper sleeve of the cylinder was taken off. The numbers of wasps present in the
chamber on the release dish, bottom, lower wall, upper wall and the lid were recorded.
The total number of wasps introduced in the chamber was obtained for each test by
adding all the numbers on its different sites together with the dead ones, if any. Mean
numbers of wasps used in different tests are given in Tables footnotes. Group of about
50-60 parasitoids were tested in Version II and Version III assay. Percentages of wasps
present on different sites were then calculated. The tests were replicated 15 times in the
assay chamber version II and 15 times in the assay chamber version III.
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2.6.4. Effect of hosts, Corcyra cephalonica (factitious host) and Spodoptera litura
(model pest of our study) on the locomotor behavior of female egg
parasitoid, Trichogramma chilonis
2.6.4.1. Locomotor behaviour of Trichogramma chilonis derived from Corcyra
cephalonica and Spodoptera litura eggs, assayed in Version- I cylinder
The mean number of walks (94.67) for the parasitoids, T. chilonis females derived
from normal host eggs, S. litura (N♂×♀N) was little more (F=6.9* at p≤ 0.05) than
the walk activity (82.7) of the parasitoids derived from normal host eggs C.
cephalonica (Table 2.1). Whereas, there was no significant variation (F=0.01ns at p≤
0.05) in between the mean number of hops (10.2) for the parasitoids, T. chilonis
females derived from normal host eggs as compared to the mean number of hops
(10.3) of parasitoids (♀) derived from C. cephalonica (Table 2.1).
Table 2.1. Locomotor behaviour of Trichogramma chilonis derived from Corcyra
cephalonica and Spodoptera litura eggs, assayed in Version-I cylinder (Single insect test)
S.No. Nature of parasitoid Walks Hops
1. Normal T. chilonis derived from C. cephalonica eggs#
82.7 a ± 3.09
10.3 a ± 1.17
2. Normal T. chilonis derived from S. litura eggs
94.67 b ± 3.33
10.2 a ± 0.51
F-value 6.9*
P ≤ 0.05 0.01ns
P ≤ 0.05
Single locomotor assay was performed in ‘Version-I’ polyacetate cylinder, taking readings on locomotor activity (number of walks, hops and flight) of individual female Trichogramma chilonis. Version-I cylinder (20 cm ht x 9.0cm dia.) was divided into 5 zones (from bottom upwards- Zone A: 0-3.0cm, Zone B: 4.0-8.0cm, Zone C: 8.0-12.0cm, Zone D: 12.0-16.0cm, Zone E: 16.0-20.0cm) with sticky ring (3.0-4.0cm) in between Zone A and Zone B. †Hops (short flight up to ~2 cm) too; # treated with UV rays. Means ± SE followed by the same letter in a column are not significantly different at P < 0.05 (ANOVA followed by LSD post-test); n=15
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2.6.4.2. Locomotor behaviour of Trichogramma chilonis derived from Corcyra
cephalonica and Spodoptera litura eggs, assayed in Version-II cylinder
(i) Percentage of parasitoids attempting flight (Total % of flyers)
There was a little (but insignificant, F=0.03ns at p≤ 0.05) difference of mean
percentage of parasitoids attempting flight activity among the parasitoids (56.98%)
derived from Corcyra cephalonica host than the percentage of parasitoids attempting
flight (57.69%), in case of normal host eggs, S. litura (N♂×♀N) (Table 2.2).
Table 2.2. Locomotor behaviour of Trichogramma chilonis derived from Corcyra
cephalonica and Spodoptera litura eggs, assayed in Version-II cylinder (Group insect test)
S.No. Nature of parasitoids (Activity of parasitoid)
Total percentage of parasitoids
found at B &C ( % low flyers )
Total percentage of parasitoids found at
D, E & Top (% high flyers)
Total % of flyers
1. Normal T. chilonis
derived from C. cephalonica eggs#
12.22a ± 3.1
44.75a ± 6.16
56.98 a ± 5.03
2. Normal T. chilonis
derived from S. litura eggs
14.2a ± 1.80
43.4a ± 2.45
57.69a ± 3.1
F-value 0.53ns
P ≤ 0.05 0.07ns
P ≤ 0.05 0.03ns
P ≤ 0.05
Group locomotor assay was performed in Version-II polyacetate cylinder, taking readings on locomotor activity (walks, hops, flight) of a cohort of male and individual female Trichogramma
chilonis. Version-II cylinder (20 cm ht x 9.0cm dia.) was divided into 5 zones (from bottom upwards- Zone A : 0-1.5cm, Zone B: 2.5-7.0cm, Zone C: 7.0-11.5cm, Zone D: 11.5-16.0cm, Zone E: 16.0-20.0cm) with Vaseline coated sticky ring (1.5-2.5cm) in between Zone A and Zone B. Vaseline was coated on floor, ‘sticky’ ring (1.5 – 2.5cm from bottom) and top lid (roof). Releasing site Petridis (dia-3.5cm) was unglued.1Parasitoid exhibiting flight from 2.5 cm up to 11.5 cm height and 2 Parasitoid exhibiting flight from 11.5 cm up to 20.0 cm height; # treated with UV rays. Means ± SE followed by the same letter in a column are not significantly different at P < 0.05 (ANOVA followed by LSD post-test); percentage data were arcsine transformed before ANOVA, but data in table are back transformations. n=15
(ii) Percentage of parasitoids as low flyer
There was no significant difference (F=0.53ns at p≤ 0.05) of mean percentage of
parasitoids exhibiting low flyer activity between the parasitoids (12.2%) those were
derived from C. cephalonica host eggs and the parasitoids (14.2%) derived from host
eggs S. litura (N♂×♀N) (Table 2.2).
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(iii) Percentage of parasitoids as high flyer
The mean percentage of parasitoids as high flyers (44.75%), derived from normal host
eggs, C. cephalonica was similar (F=0.07ns at p≤ 0.05) with the percentage of
parasitoids showing high flyer activity (43.4), were hatched from host eggs, S. litura
(Table 2.2).
2.6.4.3. Locomotor behaviour of Trichogramma chilonis derived from Corcyra
cephalonica and Spodoptera litura eggs, assayed in Version-III cylinder
(i) Percentage of parasitoids attempted to flight (Total % of flyers)
An overall percentage of parasitoids (62.2%) attempting flight activity among the
parasitoids those were hatched from normal host eggs, S. litura (N♂×♀N) was not
significantly different (F=0.33ns at p≤ 0.05) as compared to these mean percentage of
parasitoids attempting flight (60.35%) of parasitoids T. chilonis derived from
C. cephalonica host eggs (Table 2.3).
Table 2.3. Locomotor behaviour of Trichogramma chilonis derived from Corcyra
cephalonica and Spodoptera litura eggs, assayed in Version-III cylinder
S.No. Nature of parasitoids (Activity of parasitoid)
Total percentage of parasitoids
found at B &C ( % low flyers )
Total percentage of parasitoids found at
D, E & Top (% high flyers)
Total % of flyers
1. Normal T. chilonis derived from C. cephalonica eggs#
14.35 a ± 2.6
45.99 a ± 4.7
60.35 a ± 2.6
2. Normal T. chilonis derived from S. litura eggs
23.7 b ± 1.4
38.5 b ± 2.04
62.2 a ± 3.3
F-value 17.05** P ≤ 0.01
7.06* P ≤ 0.05
0.33ns
P ≤ 0.05
Group locomotor assay was performed in Version-III polyacetate cylinder, taking readings on locomotor activity (walks, hops, flight) of a cohort of male and female Trichogramma chilonis. Version-III cylinder (20 cm ht x 9.0cm dia.) was divided into 5 zones (from bottom upwards- Zone A: 0-1.5cm, Zone B: 2.5-7.0cm, Zone C: 7.0-11.5cm, Zone D: 11.5-16.0cm, Zone E: 16.0-20.0cm) with no Vaseline coated sticky ring. Vaseline was coated on floor, walls and top lid (roof). Releasing site Petridish (dia-3.5cm) was unglued.1Parasitoid exhibiting flight from 2.5 cm up to 11.5 cm height and 2 Parasitoid exhibiting flights from 11.5 cm up to 20.0 cm height; # treated with UV rays. Means ± SE followed by the same letter in a column are not significantly different at P < 0.05 (ANOVA followed by LSD post-test); percentage data were arcsine transformed before ANOVA, but data in table are back transformations. n=15
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(ii) Percentage of parasitoids as low flyer
There was a significant difference (F=17.05** at p≤ 0.01)of mean percentage (23.7%)
of parasitoids as low flyer among the parasitoids, derived from normal host eggs, S.
litura (N♂×♀N) than these mean percentage of parasitoids as low flyer (14.35%) of
parasitoids (T. chilonis) derived from Corcyra cephalonica host eggs (Table 2.3).
(iii) Percentage of parasitoids as high flyer
The mean percentage (45.99%) of parasitoids as high flier among the parasitoids,
hatched from normal host eggs, C. cephalonica (N♂×♀N) was little more (F=7.06* at
p≤ 0.05) than the mean percentage of parasitoids as high flyer (38.5%) (T. chilonis)
derived from S. litura host eggs (Table 2.3).
This experiment was conducted in order to assess the differential activity of
Trichogramma chilonis derived from factitious host Corcyra cephalonica and the
target host (model pest, Spodoptera litura). This study was also intended to habituated
the parasitoids on eggs of Spodoptera litura as the parasitoids would interact with
Spodoptera litura (as a target pest) in the field.
Activity of parasitoids hatched from Corcyra host eggs and from Spodoptera host
eggs were almost similar in test assay chamber Version-II and III, although % of high
flyers was more in case of parasitoids hatched from Corcyra cephalonica eggs
although the difference the overall % of flyers were quite similar among the
parasitoids hatched from Corcyra host eggs and from Spodoptera host eggs.
Therefore, it was reasonable to use this biocontrol agent, Trichogramma chilonis
(Ishii) against Spodoptera litura (Fabricius) as target host in the present study.
The locomotor assays of the parasitoid in all three versions (Figure-2.10, 2.11 and 2.12)
were conducted in the following regimens.
(i) Locomotor behaviour of Trichogramma derived from normal host eggs (N ♂ x N ♀)
(ii) Locomotor behaviour of Trichogramma derived from F1 host eggs (P1♂ x N ♀)
[100Gy/130Gy]
(iii) Locomotor behaviour of Trichogramma derived from F2 host eggs (F1♂x N♀;
N♂x F1♀; F1♂ x F1 ♀) [100Gy/ 130Gy]
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Figure 2.10. Version-I cylinder
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Figure 2.11. Version-II cylinder
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Figure 2.12. Version-III cylinder
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2.7. Bioefficacy of egg parasitoid, Trichogramma chilonis
2.7.1. Exposure of host eggs to T. chilonis
In the glass vials (Homeopathic vials 1drum size) containing S. litura egg cards (100 eggs,
for first day, 50 eggs for second day and 30 eggs for third day respectively) were allowed a
pair of T. chilonis with the help of brush, while they were mating, from the nuclear culture.
Then 50% diluted honey strips at the inner wall of the glass vial were provided and then the
vials were closed by cotton plugs. After 24 h of exposition for parasitization the old cards
were transferred to new vials, and another fresh cards were replaced for further
parasitization by T. chilonis on 2nd day, like-wise on 3rd day, it was repeated.
2.8. Parasitization behavior of Trichogramma chilonis was ascertained on
host eggs of Spodoptera litura having irradiation background in various
regimens
(i) Parasitization behavior of normal T. chilonis on normal host eggs (N ♂ x N ♀) &
on F1 host eggs (P1 ♂ x N ♀) [100Gy/ 130Gy] [N-Tc vs F1-host].
(ii) Parasitization behavior of normal T. chilonis on normal host eggs (N ♂ x N ♀) &
on F2 host eggs (F1♂x N♀; N ♂x F1♀; F1♂x F1♀) [100Gy/ 130Gy] [N-Tc vs F2-
host].
(iii) Parasitization efficacy of F1 T. chilonis, derived from F1 sterile host eggs
(oviposited by N-female x T- male S. litura) were ascertained on N-host eggs [F1-
Tc vs N-host].
(iv) Parasitization efficacy of F1 T. chilonis, derived from F1 sterile host eggs
(oviposited by N-female X irradiated male with 100Gy / 130Gy of S. litura) on F1
sterile host eggs [F1-Tc vs F1-host].
2.8.1. The parasitization efficacy of Trichogramma chilonis was studied on host
eggs having irradiation history in the following manner
2.8.1.1. Parasitization capacity of parasitoids
The parasitizing behavior of parasitoid, T. chilonis was observed for three days of
parasitization in the respective vial having host egg cards. The data was recorded only
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for three consecutive days. The identifying characteristic for parasitization was the
change of colouration from light-greenish to dark for S.litura eggs. Then the darkened
host eggs were recorded.
2.8.1.2. Emergence of parasitoids from host eggs
The emergence of parasitoids from the parasitized eggs were noted down daily, and the
per cent emergence of parasitoids from host eggs was recorded after 7 days of
parasitization.
2.8.1.3. Developmental period of T.chilonis
The developmental period of T. chilonis within parasitized eggs of lepidopteran host,
Spodoptera litura was recorded in days.
2.8.1.4. Growth Index (GI) of T. chilonis
The Growth index (GI) of parasitoids was calculated by the ratio of percentage
emergence of parasitoids from parasitized host eggs and developmental period (days) of
the emerged parasitoids from the parasitized host eggs.
2.8.1.5. Longevity of parasitoids
The adult pairs which were allowed for oviposition leading to parasitization in the
different host eggs were daily observed and provided with 50% honey solution also.
The male and female longevity was noticed in days, as computed by daily observations.
2.8.1.6. Female emergence
Out of the emerging parasitoids, the relative number of male and female parasitoid was
recorded, and the percentage of female emergence was computed to assess the sex ratio.
2.9. Photography
Photography was conducted by using Sony digital camera and Motic microscope
version 2.0.
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2.10. Statistical Analysis
The data obtained in the above experiments were usually replicated fifteen times; and
any variation in replicate number has been specified at an appropriate place in the text.
The data were subjected to appropriate analysis of variance (ANOVA). Percentage data
was transformed using arcsine √x value before ANOVA, but data shown in tables are
back transformations. LSD post test was then performed to determine significant
differences among the different treatments (Snedecor and Cochran 1989).