Title and Certificate - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/31606/7/07_chapter 2.pdf(IARI), New Delhi. Mass rearing of this insect was established in an insectary

Chapter 2Chapter 2Chapter 2Chapter 2

Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods

Materials 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)


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.


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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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).


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).


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).


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).

Documents

Title and Certificate - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/31606/7/07_chapter 2.pdf(IARI), New Delhi. Mass rearing of this insect was established in an insectary