26

CHAPTER III - MATERIALS AND METHODS

The study on Microsporidia as an entomopathogen, is always being

overlooked in India. Forests areas in India are almost unexplored for their

occurrence. In the present study, an attempt was taken towards accounting their

presence in Lepidopteran species collected from different forestry and

agroforestry areas from south Indian states. Also, the study was extended to

identify and characterize a selected Microsporidium and knowledge was gathered

by studying the role and relations of the Microsporidium with its original host as

well as some other major defoliators.

3.1. Occurrence of Microsporidia

3.1.1. Survey and collection of Lepidoptera

Surveys were conducted in selected forest and agro forestry areas of the

four south Indian states for collection of Lepidopterans. Different stages (larva,

pupa or adult) of Lepidopteran insects (moths and butterflies) were collected and

27

brought to the laboratory for screening and isolation of Microsporidia. Since,

moths are major defoliator pests, the present study was mainly concentrated on

folivorous moths, than butterflies. Following is the detailed description of 3 year

survey conducted from 2007-2009.

3.1.1.1. Study areas

Nurseries and plantations in forestry sectors and agricultural land in

agro-forestry sectors in both urban and rural areas were selected for the study.

The study was conducted in four states of south India - Karnataka, Kerala, Andhra

Pradesh and Tamil Nadu Fig 1. Atleast two study areas were selected from each

of the four states as a representative for the diversity of the insects and the

entomopathogenic Microsporidia. The places where the surveys were conducted

are furnished in Table 2.

3.1.1.2. Methods of Collection

Day collection was mainly by hand picking i.e. by using brush in case

of larvae and pupae (for both moths and butterflies), and, in case of adult

butterflies and diurnal moths, it was mainly by using sweep net (Plate 2). After

collection, the insects were transferred immediately to plastic jars containing

small piece of naphthalene. The naphthalene vapour was used as a fumigant

which makes the insects inactive, so that they can be easily carried to the lab

without further damage.

Different types of traps such as solar light trap, Robinson light trap,

sticky trap etc. were used to collect the nocturnal species of Lepidopterans. Light

traps were installed in Institute of Wood Science and Technology (IWST) campus

to collect the moth species throughout the year. One part of the campus is having

a natural forest that is rich with various species of moths. Though it was quite

difficult to get more moths in the monsoon season by the light traps, the traps

were generally useful for a reliable collection. Apart from IWST, a solar light trap

was installed in Doginal teak plantation which is near to Yellapur in northern

Karnataka. Two ordinary light traps were placed in Attapadi (Kerala) and in a

nursery in Doddbellapur for collection of moths. In addition to these methods,

PLATE – 2

Methods of Collection of Lepidoptera

A

B

C D

E F

A: Solar light trap; B: Robinson light trap; C: Moth

collection; D: Hand picking method; E: Butterfly collection

box; F: Sweep net

28

Lepidoptera specimens were collected by visiting different places where the host

plants were available.

3.1.1.3. General survey and collection

Among Lepidoptera, several moth species are potential pests of

agriculture and forestry. Many of them are defoliators; some are root, shoot, fruit,

seed and stem borers. Usually they are nocturnal (other than day flying moths) in

nature. Butterflies are generally diurnal in nature and available plenty in both

urban and rural areas. Most of them are defoliators and very few are considered

as pestiferous species.

General survey was carried out for collection of both moths and

butterflies. The frequency of the survey was once in three months in different

areas in and around Bangalore (including Ramnagara and Mysore) and once in six

months in the rest of the study areas. Apart from these, continuous collection was

made through light traps wherever it was installed.

3.1.1.4. Survey for selected pests

Elaborate surveys were carried out for the two teak pests, viz., Hyblaea

puera and Paliga machoeralis in selected study areas. The insects were seasonal

pests of teak. At the time of seasonal outbreak these pests were collected by hand

picking and by sweep net. H. puera insects were collected in monsoon season

from May to August from three places of Karnataka i.e. Gottipura and Kanakpura

that comes under Bangalore rural and Yellapur in the northern part of Karnataka;

Rangampet in Andhra Pradesh; and from Kannavam, Nilambur, and Mannarkkad

in Kerala for three years from 2007 to 2009. P. machoeralis larvae were collected

in pre and post monsoon periods and winter period, from April to May and

October to January from Gottipura (Bangalore rural) and Mysore in Karnataka;

Rangampet in Andhra Pradesh and Kannavam and Nilambur in Kerala from 2007-

2009.

Sr.

No.

State name Forest

division/range

Place name

1 Karnataka Bangalore urban Bangalore urban (IWST Campus)

2 Karnataka Bangalore rural Bangalore rural (Gottipura,

Devanahalli, Dodhbellapur,

Kanakpura)

3 Karnataka Ramanagaram Ramnagaram

4 Karnataka Mysore Mysore

5 Karnataka Yellapur Yellapura

6 Karnataka Sagar Sagar

7 Karnataka Mangalore Mangalore

8 Kerala Mannarkkad Mannarkkad

9 Kerala Mannarkkad Attapadi

10 Kerala Nilambur north Nilambur

11 Kerala Peechi Peechi

12 Kerala Kannur Kannavam

13 Andhra Pradesh Tirupathi Tirupathi

14 Andhra Pradesh Tirupathi Rangampet

15 Tamil Nadu Vellore Vellore

16 Tamil Nadu Salem Salem

17 Tamil Nadu Coimbatore Coimbatore

Table 2: State and Division wise selected study areas

Fig 1. Map of south India showing study

areas

30

3.1.1.5. Preservation of Lepidopteran specimens

Collected butterflies and moths were identified. To preserve,

specimens were pinned and oven dried at 35±1 OC for 24 hrs. The dried

specimens were kept with information tag, in insect boxes, with naphthalene balls/

Ethyl acetate for further work (Plate 2).

3.1.2. Screening for Microsporidia

3.1.2.1. Homogenizing

The collected Lepidopterans were individually homogenized in sterile

distilled water to examine the body fluid and tissues microscopically for the

presence of Microsporidia spores.

3.1.2.2. Examining under microscope

Under microscope (Nikon Eclipse E200), spores can be observed and

measured accurately without shrinkage and drastic distortion, often caused by

fixation and staining. Information about shape, color and refractivity of spores

was obtained from the study. One drop of homogenized tissue was placed on a

microscope slide, applied a cover slip, gently pressed and observed under bright

field optics at 40X magnification. Also, a drop of immersion oil was applied on

the coverslip and observed the slides under 100X magnification. Phase contrast

optics was also used for specific observations.

3.1.3. Isolation and purification of Microsporidian spores

If spores were present in the homogenized tissues, they were isolated

and purified by following standard procedures. First, the homogenized tissue was

filtered through multiple layers of cheese cloth. After removing the larger

particles, the isolated spores were transferred to centrifuge tube for further

purification.

The filtrate was centrifuged for 10 min. at 3500 rpm. Spores are denser

than most of the particles from the host and centrifugation concentrate them near

31

the bottom of the residue (Undeen, 1997). Due to the dense nature of the spores, a

series of washing and centrifugation was needed to remove dissolved substances,

but small particulate contaminants still remained. The supernatant was decanted

and the debris from the top of the residue was carefully re-suspended in a small

volume of water and discarded. The process was repeated for 3-4 times and the

sediment was re-suspended in distilled water and allowed to spin in 5000 rpm for

15 min.

To get completely purified spores, percoll gradient centrifugation was

performed. Percoll consists of colloidal silica particles. It is well suited for

density gradient experiments because it possesses a low viscosity compared to

others and no toxicity towards cells and their constituents. Spores were mixed

with percoll and centrifuged at 50,000 rpm for 30 minutes (Sato and Watanabe,

1980). Mature spores formed a band which was separated by syringe or

micropipette.

3.1.4. Preservation of Microsporidia

Purified Microsporidian spores were preserved in sterile distilled water

at 4°C. Water was replaced twice in a month to keep the spores viable. Also, the

spores were passed through their original host once in three months to sustain the

viability of the spores. Occasionally, spores were preserved in cadaver of their

host insects, which were partially dried. The process helps to prevent the insects

from other secondary infections and also keep the viability of the spores intact.

3.1.5. Identification and Characterization

Several techniques are being used in order to properly identify

Microsporidia. The following methods were adopted to identify the selected

isolated Microsporidium: 1) Light microscopy, 2) Electron Microscopy, 3)

Germination, 4) Life cycle, 5) Thermal and chemical tolerance and 6)

Phylogenetic analysis.

32

3.2.1. Morphological Characterization

Light microscopy

Microsporidia are best examined by phase-contrast microscopy. Nikon

Eclipse, E 200 was used for the study. The spores were measured by ocular

micrometer attached with the eye piece of the microscope. Also spores were

photographed and measured by using Image Pro Express, version 6.0. Data were

pooled to calculate the mean size of the spores.

3.2.2. Ultrastructural Study

Transmission Electron Microscopy

The TEM study provides the most important taxonomic information for

the micosporidiologist. Only ultra-thin section of the spore can provide the

information on the structure, number of polar filaments, polaroplasts and other

organelle that can serve the purpose for identification especially at the family and

generic level. TEM study was carried out for the spores isolated from Hyblaea

puera, by using the facilities at NIMHANS, Bangalore.

I. Protocol for Processing of spores for Electron Microscopy

Fixation

Fix the sample in 2.5% gluteraldehyde/ Kornavsky‟s (4% para-

formsldehyde + 3% gluteraldehyde) fixative in phosphate / sodium

cocodylate buffer pH 7.2 for 24 hours.

Wash the tissue with buffer till the same of the fixative is completely gone.

1% Osium tetraoxide for one and half hours.

Wash with phosphate/ cocodylate buffer.

Treat with 70% alcohol for 1 hr. or 1 overnight.

Treat with 80 % alcohol for 1 hour.

Put in 90% alcohol for 1 hour.

Treat with 2% Uranyl acetate in 95% ethanol (Enblock stain) for 1 hour.

Absolute alcohol for 1 hour.

33

Then, clean it with propylene oxide for 10-15 minutes – 2 changes (should

be over by 30 minutes)

Infiltration with 1:1 ratio of araldite and propylene oxide for overnight.

Infiltration with fresh araldite for 3 changes with a gap of 3-4 hours.

Embed and keep at 60° C for 48 hours.

Ultramicrotomy

To obtain a high resolution, the thickness of the specimen should be

about 500A˚ (50 nm). Such thin sections are obtained using ultramicrotome,

which are specially designed for the purpose.

Trimming

With the help of a razor blade the blocks were trimmed that made the

small cutting face free from extra resin (embedding medium). The trimmed blocks

were fitted to a specimen holder of the ultramicrotome.

Ultrathin sectioning

Ultrathin sections were cut by using a fresh glass knife. The ultrathin

sections show interference colours which makes it possible to determine the

thickness of the sections. The sections were golden in colour and having thickness

of 40-50 nm. The sections were collected in copper grid for further study.

Staining

Atomic density is introduced by staining the tissues with heavy metals

to obtain contrast for the cell materials. For the Microsporidia spore, double

staining procedure was used in which sections were stained with uranyl acetate

followed by lead citrate.

II. Photography

Digital photograph was taken by using TECHNAI G2 BioTwin

Electron microscope from FEI Neitherland and respective images were captured

34

by using CCD camera (mega view 3). The magnifications were different for

different photographs and were represented by scale bars.

3.2.3. Germination

Germination of the selected Microsporidium was studied by three

different methods, viz., priming, treating with buffered monovalent ions and

partial desiccation and rehydration method.

i) Priming:

The spores were treated with KOH (alkaline solution) at different concentrations

viz., 0.1, 0.3, 0.5, 0.7, 0.9 and 1% for 10, 20 and 30 min durations. Then the

spores solutions were neutralized (pH 7) rapidly with phosphate buffer saline (pH

7).

ii) Treating with monovalent ion:

KCl solution buffered to alkaline pH (9.0-11.0) was used to treat the spores for

germination.

iii) Desiccation and rehydration:

The spores were partially dehydrated and then rehydrated by using distilled water.

In all the cases, treated spores were observed under phase contrast microscope.

3.2.4. Life Cycle Study

Usually, all Microsporidia have similar pattern of life cycle, but with

specific variations, viz. the mode of divisions and/or the number of daughter cells.

Those variations have taxonomic value for classifying these organisms (Vávra

1976a). To perform the life cycle study the following methods were followed.

Dissection

Dissection of infected host is the primary step towards life cycle study

of a Microsporidium. Standard dissection method was followed to dissect the

infected tissues. 2-3 infected insects were dissected in every 24 hrs following the

initial infection, up to the completion of the life-cycle (Plate- 4).

35

Sample preparation

Smears of infected mid gut and fat body tissues were prepared at

specific intervals after infection as per standard procedure described by Undeen,

1997.

Sample of tissues from infected Hyblaea puera was dissected.

Small parts of tissues were smeared on slides.

The tissue smears were air dried.

Fixed in absolute methanol for 4-5 minutes, drained the excess methanol

and allowed the smear to dry.

Stained the smears with fresh buffered Giemsa‟s staining solution with pH

7.4 (Hi-media) for 20 min.

The slides were washed in tap water, allowed to dry.

The smears were mounted in glycerine and observed under oil immersion

at 100X for the developmental stages of the parasite.

Cell Line Culture

Also, the life cycle study had been carried out in Antheraea

eucalypti ovarian cell line obtained commercially. First, the cell density was

counted in the culture by haemocytometer, and total number of spores was

estimated in the flask. Spores were primed before introducing into the culture.

100% Percoll purified spore suspension was added with 0.2 M KOH (usually, 1

part of KOH mixed with 1 part of spore suspension) and placed in a dry bath at

27°C for 40 min. After 40 min., the suspension was directly inoculated into the

culture flask containing the ovarian cells and medium IPL-41 and agitated gently.

Ovarian cell and inoculated spore ratio was maintained as 1:10. The culture flask

was centrifuged at 2000 rpm for 10 min to allow better germination of the spores.

After centrifugation flask was placed in an incubator at 27°C. The sample was

tested after 1h, 24h, 48h, 72h, 96h and 7days for the life stages of the

Microsporidia.

36

3.2.5. Response of the Microsporidium to temperatures and chemicals/

disinfectants

For the temperature and chemical response studies, spore suspensions

were prepared in sterile distilled water and the concentration of spores was

determined using a haemocytometer as per the method described below.

Purified spores were suspended in sterile distilled water.

Special coverslip was placed on a clean and dry haemocytometer.

Spore suspension was mixed thoroughly by using vortex mixer.

Small sample of spore suspension was taken (about 10 µl) using a pipette

and carefully deposited in the notch of the haemocytometer and allowed

the sample to be drawn by capillary action into the chamber. The chamber

was filled optimally.

Spores were allowed to settle for a minute.

Placed the haemocytometer under a microscope to count the spores.

The concentration of spores in the solution was determined using the

formula:

Spores/ml = No. of spore counted × 104

3.2.5.1. Response to Temperature

The spores of the Microsporidium preserved in sterilized distilled water

were treated at different temperatures for different periods of time to assess the

temperature tolerance. To study the tolerance, 108 ml

-1 concentration of spore

suspension was prepared. The spore solution was treated with nine different

temperatures viz., - 20°C, -5°C, 4°C, 25°C, 30°C, 40°C, 50°C, 60°C, 70°C and

one as a control which was maintained at room temperature (around 22±1°C). 1

ml spore suspension was taken for each treatment. Water bath was used to treat

the spores from 50°C onwards. BOD was used for 25°C, 30°C and 40°C, freezer

for 4°C and deep freezer for -5°C and -20°C. For each temperature, spore

suspension was kept for 5, 15 and 30 min. Viability of the spores after treatment

were determined by inoculating the treated spores to Hyblaea puera larvae. Each

of 10 insects with 3 replicates was inoculated with 50 µl of spore suspension from

37

each treatment group. Inoculated insects were reared individually till pupation and

sacrificed to determine the infectivity.

3.2.5.2. Response to Chemicals/ Disinfectants

Five different types of chemicals were used to conduct the study. The

purpose of the study was to examine the response of the Microsporidium to

various chemicals used as laboratory disinfectants. Formaldehyde (CH2O),

Sodium Hypochlorite (NaOCl), Hydrogen peroxide (H2O2), Phenol (C6H5OH) and

Benzalkonium chloride were used for the study. The spore suspension @ 1.5ml

having a concentration of 108 spores ml

-1 were treated with the chemicals at 0.1%,

0.5% and 1% concentrations for 2, 5 and 10 min. Spore suspensions were

centrifuged at 3500 rpm for 5 min and the supernatants were discarded before

treating with the chemicals. The pellet was suspended with 1.5ml of respective

chemicals. After exposure to the chemicals for the specified periods, spore

suspensions were centrifuged at 3500 rpm for 1min (inclusive of treatment time)

and the chemicals were discarded. The spore pellets were washed twice in sterile

distilled water to properly remove traces of the chemicals from the spore. Each of

10 Hyblaea puera larvae in each replicate (3 replicates for each treatment) was

inoculated with 50 µl of spore suspension from each treatment group of spore.

One “control” batch with 3 replicates was maintained separately for the study.

Inoculated insects were reared individually till pupation to sacrifice and examine

for the infection. Data were analyzed by performing multivariate analysis by

comparing means for different treatment groups (α=0.05).

Formaldehyde (CH2O)

Formaldehyde is an organic compound belongs to aldehyde group. It is

having widespread use as a disinfectant and preservation of biological specimens.

Laboratory grade 40% formaldehyde was taken and adjusted to 0.1%, 0.5% and

1% solution for the above study.

38

Sodium hypochlorite (NaOCl)

Sodium hypochlorite is chlorine compound, commonly known as

bleach and is frequently used as a disinfectant or a bleaching agent. 0.1%, 0.5%

and 1% solution was prepared from the laboratory grade of 4% NaOCl.

Hydrogen peroxide (H2O2)

H2O2 is an oxidizing agent and commonly used as a bleaching agent. It

is the simplest peroxide. Laboratory grade 30% H2O2 was used for the experiment

and was adjusted to predefined concentrations for further study.

Phenol (C6H5OH)

Phenol is an organic compound, consisting of phenyl group. It forms

crystal at room temperature and has high toxicity. Therefore, 0.1%, 0.5% and 1%

aqueous solution of phenol were prepared to conduct the experiment.

Benzalkonium chloride

Standard available concentration for laboratory grade Benzalkonium

chloride is 50%, which was purely aqueous and was adjusted to three required

concentrations for the study.

3.2.6. Molecular Study

3.2.6.1. 16S SSUrDNA Sequencing

Molecular study was done with the help of Dr. C. R. Vossbrinck, USA,

by sequencing the 16S Small Subunit Ribosomal DNA. Spore isolation and DNA

sequencing was by following standard procedures. Infected moths were

homogenized in water, filtered through 50 µM nylon cloths and centrifuged

through a 50% percoll cushion in a 1.5ml micro-centrifuge tube. The spores were

then washed twice in STE buffer. DNA was liberated by bead beating in STE

buffer. The solution was heated to 95°C for 5 minutes and 4µl of this solution

was added to a standard PCR reaction using cycling parameters and amplification

primers (18f 5‟CACCAGGTTGATTCTGCC3‟, 1492r 5‟

39

GGTTACCTTGTTACGACTT 3‟) as described previously by Vossbrinck et al.,

1998.

3.2.6.2. Molecular phylogeny

Phylogenetic analysis was carried out by using sequenced data for 39

species including 8 Nosema Bombycis strains, 24 other Nosema sp., 5

Vairimorpha species and a single Amblyospora and Pleistophora sp. as an

outgroup (table 3) from the database of NCBI (http://www.ncbi.nlm.nih.gov).

MEGA 4 was used to construct the Maximum parsimony tree.

40

Table 3: List of Microsporidia and their accession numbers

Sr. No. Species Name Accession No.

1 Amblyospora connecticus AF025685

2 Nosema Apis U26534

3 N. bombi AY008373

4 N. bombycis NIS 408 AB093008

5 N. bombycis NIS 520 AB093009

6 N. bombycis NIS 611 AB093010

7 N. bombycis Y9101 AB093011

8 N. bombycis CGS AB093012

9 N. bombycis L39111

10 N. bombycis 1s AY017210

11 N. bombycis 2r AY017211

12 N. carpocapsae AF426104

13 N. ceranae U26533

14 N. furnacalis U26532

15 N. granulosis AJ011833

16 N. oulemae U27359

17 N. Portugal AF033316

18 Nosema. sp. 4m AY017213

19 Nosema sp. KU-9 AF141130

20 Nosema sp. NSD-2005 DQ323510

21 Nosema sp. C01 AY383655

22 Hp-M (Nosema sp.) GQ244502

23 Nosema sp. NIS-M11 D85501

24 Nosema sp. 3h AY017212

25 Nosema sp. AB009977

26 Nosema sp. isolate 1 AF240348

27 Nosema sp. isolate 2 AF240349

28 Nosema sp. isolate 3 AF240350

29 Nosema sp. 6 AF240353

30 Nosema sp. 7 AF240354

41

31 N. spodopterae AY211392

32 N. tyriae AJ012606

33 N. vespula U11047

34 Vairimorpha cheracis T1 AF327408

35 V. imperfecta 1 AJ131645

36 V. imperfecta 2 AJ131646

37 V. lymantriae AF033315

38 V. lymantriae Levishte AF141129

39 Pleistophora sp. (Sd-Nu-IW8201) D85500

42

3.3. Pathogenicity, Multiplication and Mode of Transmission

Hyblaea puera and Paliga machoeralis are two seasonal insect pests of

teak. They are not available in nature throughout the year. Therefore, for the

continuous study, it was necessary to maintain the insect cultures in the

laboratory. Whenever the insect cultures depleted in our laboratory, especially

Hyblaea puera, the experiments were conducted with the laboratory reared larvae

received from the Insect Rearing Centre of the Kerala Forest Research Institute at

Nilambur. Though no Microsporidian parasite was observed in P. machoeralis, as

it is also a serious pest of teak, many times co-existing with H. puera, the

experiments were conducted to observe the multiplication and transmission of the

Hp-Microsporidium in P. machoeralis. Pilot studies were carried out to finalize

the design of the detailed experiments.

Rearing of two major teak defoliators in the laboratory

Hyblaea puera Cramer

Hyblaea puera Cramer is the most prominent defoliator pest of teak

(Tectona grandis) belogs to the family Hyblaeidae. Other than teak, the insect

feeds on Vitex agnus, V. canescens, V. glabrata, V. penduncularis, Callicarpa

arborea, Heptapleurum venulosum etc. To rear this insect, batches of young

Hyblaea puera larvae were collected mainly from different teak plantations from

Bangalore rural district in Karnataka, Mundur in Palakkad district of Kerala and

also from KFRI (Kerala Forest Research Institute). The infection free larvae were

reared on teak leaves till 2nd

instar, and on artificial diet (Plate- 4) from 3rd

instar

onwards. For rearing on artificial diet, small quantity of diet cake was placed in

petriplates and individual larva was transferred to the plate. Fresh diet was

provided either on alternate days or after complete consumption of the given diet,

whichever was earlier. The rearing was conducted at room temperature. Adult

moths were kept in bottles and allowed to feed on sucrose solution or 10% honey.

Composition of artificial diet:

1. Agar: 20gm + 500 ml distilled water (80oC)

2. Teak leaf powder: 20 gm

43

3. Casein: 30 gm

4. Yeast extract powder: 10 gm

5. Kabuli gram powder: 100 gm

(2+3+4+5+500 ml dis. Water at 65oC)

6. Ascorbic acid: 3.5 gm

7. Sorbic acid: 1 gm

8. Methyl para-hydroxybenzoate: 1.5 gm

9. Evion: 400 mg, 1 tab

10. Multivitamin and mineral mixture: 2 tab

11. Streptomycin sulphate: 0.125 gm

12. Tetracyclin: 0.125 gm

13. Formaldehyde 10%: 2 ml

Life cycle of H. puera

Adults are small having 3-4 cm wing span and emerge more or less

simultaneously. Mating takes place within a couple of days. Eggs are laid on

tender new leaves. They are oval, flat and white in colour and measure about 1

mm in length. Larvae hatch in about 2 days. There are five larval instars. Each

instar is for approx. 2 days. Total larval period is 10-12 days. It may vary greatly

depending upon temperature and humidity (Beeson, 1934). The full grown larva

measures about 3.5 – 4.5 cm. The average pupal period lasts for six to eight days

under optimal conditions (Plate-3).

Paliga machoeralis Walker

Paliga machoeralis belongs to the family Pyralidae, is commonly

known as the teak skeletonizer, reach its peak population during pre-monsoon to

post-monsoon between May-September. Other than teak (Tectona grandis),

larvae feed on Callicarpa arborea, C. cana, C. macrophylla and Tectona

hamiltoniana. Paliga machoeralis larvae attack all types of teak leaves including

rough and mature ones. Paliga machoeralis larvae were collected from Gottipura

teak plantation in Karnataka, India, in the month of June, 2007 and were checked

for the Microsporidia infection. After confirmatory test, uninfected larvae were

PLATE-3

Life Cycle Stages of H. puera and P. machoeralis

H. puera

P. machoeralis

A: Egg; B: 4th instar larva; C: Late 5th instar larva; D:

Larva about to pupate; E: Pupa; F: Adult

A

A

B

B

C

C

D

E

F

E

F

44

reared in the laboratory to maintain continuous generations. Larvae were fed with

fresh teak leaves and the adults with 10% honey solution.

Life cycle of P. machoeralis

Eggs were first laid 1-4 days after pairing. The average number of eggs

laid by single female is around 250. Egg takes 2-3 days to hatch and larval

duration is about 8-27 days. The fifth instar larvae measures about 2.2-2.5 cm

(Plate-3). Emergence of moth varies from 14-41 days depending upon

temperature (Beeson, 1934).

3.3.1. Pathogenicity

For pathogenicity study infection free laboratory reared Hyblaea puera

larvae were used.

3.3.1.1. Bioassay of the Hp-Microsporidium in H. puera

Insects

A total of 960 laboratory reared infection free 3rd

instar H. puera larvae

of similar size were taken to conduct the experiments. Insects were reared at

24oC±1

oC and 60±5% RH under 12h L/12h D regime. The larvae were fed on

artificial diet, which was changed every alternate day. Precaution was taken to

maintain adequate hygiene to prevent from other infections. Each larva was kept

in separate petri plate to observe and record the growth and mortality.

Microsporidia

The Microsporidian spores isolated from Hyblaea puera, purified and

preserved by the method described earlier were used for bioassay.

Spore concentration was determined using a haemocytometer as mentioned

earlier. Serial dilutions were prepared as per the requirement with sterile distilled

water. The dilutions were of equally spaced concentrations of spore suspension.

First, 108 ml

-1 concentration of the spores was prepared as the stock solution and

45

then the solution was diluted 10 times in serial order to obtain concentrations up to

102 ml

-1 spore suspension.

Inoculation

Larvae were inoculated with the spore suspension by spreading 20 µl of

the serial spore concentrations (ranging from 108

to 102 of spores ml

-1) on the

surface of artificial diet. The actual infection dose (no. of spores inoculated) was

determined from the density of the spore in the sample and the volume inoculated.

Larvae were starved for 12 h before the inoculation. They were grouped into

eight batches for the treatments; each batch was with 30 individuals and 4

replicates. Seven batches were inoculated with different spore loads ranging from

2×106 to 2. One group was maintained as a “control”, treated with sterile distilled

water (Plate- 4).

3.3.1.2. Data recording and Statistical Analysis

Observations were made on mortality and growth till the end of the

larval period. Also, emergence of the adults was accounted. Larval weight and

mortality was recorded on daily basis. Percentage of growth was furnished for

every alternate day. The recording of growth data was continued till the sight of

first pupation in the experimented insects irrespective of the batches, but mortality

for the bioassay was recorded till all the surviving larvae pupated. Pupal mortality

was determined after eclosion of the adults from different treatment groups and

“control”. The experiment was terminated after emergence of all the live pupae.

Growth curve was prepared by calculating mean percentage of growth for each

treatment on every alternate day. Larval mortality and adult emergence from the

test and control groups are presented graphically. Probit analysis (Finney, 1971)

was done to determine the LC50 of the Microsporidium for H. puera larvae by

using SPSS 10.0.

3.3.2. Multiplication

Each individual Microsporidian spore that is present in the host‟s

habitat has a certain chance to enter a host, to multiply, and to produce offspring

PLATE – 4

Bioassay and Multiplication Study

A: Artificial diet for H. puera; B: Bioassay of Hp-

Microsporidium in H. puera; C: Leaf disc method

used for bioassay; D: Spore multiplication in P.

machoeralis; Dissected H. puera larva; E: Extraction

of spores from various infected tissues

A

D

B

C

F E

46

spores. Multiplication of the spores under different spore loads in relation to time

can be determined by sacrificing the hosts at regular intervals post inoculation

after a certain period of time, henceforth referred to as incubation period; i.e.

completion of one spore to spore cycle. Also, the expected reproductive rate of

spore can be estimated as the ratio of the average number of spores produced per

infected host (the spore yield in confirmed infections) and the concentration of

spore present in the medium at the time of host exposure (the dose) (Blaser and

Schmid-Hampel, 2005). Infections typically occur per os when the larva feeds on

medium contaminated with spores (Hanula and Andeadis, 1991) and the

penetration by the polar filament of the parasite into host gut cells starts within 10-

20 min after feeding of spores (Fisher and Sanborn, 1962a).

3.3.2.1. Spore multiplication in H. puera

An attempt was taken to observe the sequence of infection and to

determine the rate of multiplication of the Microsporidium in H. puera by

inoculating the spores to the larvae in the laboratory.

3.3.2.1.1. Sequence of infection in different organs of H. puera

A total of 150 larvae (5 replicates each with 30 individuals) were taken

to study the sequence of infection in different organs of H. puera. The larvae of

the first day of third instar were inoculated with 104 spores per larva to study the

sequence of infection in the various organs. Single larva from each replicate was

sacrificed every dpi to assess the infection. The tissues such as midgut, fat body,

tracheal membrane, malpighian tubules and reproductive organs (in case of pupae

and adults) were carefully dissected out and washed in a small quantity of sterile

distilled water and examined for the infection till 30 dpi.

3.3.2.1.2. Spore yield

Inoculation and recovery of spores were performed with healthy Hylaea

puera larvae by inoculating the spores of the Microsporidium. The replication of

spores with time was traced into different tissues of the inoculated insects and in

whole body. Spores were harvested from the larvae or pupae by sacrificing them

47

from each replicate of the different treatment groups. Spore yield was determined

per unit of weight of the tissues and the whole body.

The experiment was carried out at 24 ±1 °C with 55±5% relative humidity under

light regime 12h:12h (L:D). Larvae were inoculated with spore suspensions

through artificial diet. Spores were allowed to replicate inside the larvae. After a

certain time period spores were isolated from the body tissues by homogenizing

the tissues or the whole body in 0.5% K2CO3 solution with the help of mortar and

pestle. K2CO3 was used as a degrading solution for homogenizing the tissue

because it facilitates effective release of the spores from the tissues (Sasidharan et

al., 1994). The homogenate filtered through a single layer of thin cheese cloth

and the tissue debris collected in the cloth was washed again with a small quantity

of K2CO3 solution, vortexed and filtered to collect any trapped spore in the debris.

The filtrate was centrifuged at 3500 rpm for 5 min to sediment the spores. The

sediment was dispersed in 1 ml of sterile distilled water over a vortex and the

spore was counted using an improved Neubaur haemocytometer. Spore yield was

expressed per g of body weight or per g of examined wet-tissues (Plate- 4).

I. Spore yield from whole body

A total of 140 Hyblaea puera larvae were used for the study. They

were grouped into four batches among which three batches, each of which was

inoculated with the spore loads ranging from 2×103 to 2×10. One batch was

maintained as a “control”, treated with sterile distilled water.

Spore yields from the inoculated insects (larvae/ pupae) were estimated from 15

dpi to 21 dpi. Daily, five larvae were sacrificed from each batch of inoculum to

determine the spore yield. The whole body of inoculated insects was macerated

and the spore count was taken by using haemocytometer. Data were analyzed by

performing two factor ANOVA with replications. (α=0.05, 0.01).

II. Spore yield from mid-gut tissue

270 Hyblaea puera larvae were used to assess the spore yield from

midgut tissue. They were grouped into five batches. The concentration of spores

in the dilutions prepared for the study ranged from 107

to 103

ml-1

. Each larva

48

from five batches was inoculated with the spore loads ranging from 2×105 to

2×10. One batch was “control”, treated with sterile distilled water.

Data were recorded from 13 dpi to 21 dpi. Per day five insects from each

treatment group were sacrificed for estimating the spore yield. Data were

analyzed by performing two factor ANOVA with replications (α= 0.05, 0.01).

III. Spore yield from fat body tissue

270 Hyblaea puera larvae were used to estimate the spore yield from

fat body tissue. They were grouped into five batches. Each batch was treated

with spore load ranging from 2×105 to 2×10 per larva. One batch was maintained

as “control”, treated with sterile distilled water.

Data were recorded from 13 dpi to 21 dpi. Per day five insects from each

inoculum were sacrificed. Data were analyzed by using two factor ANOVA with

replications (α=0.05, 0.01).

3.3.2.1.3. Spore concentration in excreta

A total of 96 larvae were used for the experiment. Each batch had 3

replicates with 4 larvae kept separately and inoculated with the Microsporidium

ranging from 2×106 to 2 spore loads, apart from a “control” batch that was treated

with sterile distilled water.

Excreta from inoculated larvae were examined daily for the presence of

the spores. Excreta samples were homogenized in 1 ml of sterile distilled water

and were examined under the microscope to determine: i) the time of the first

appearance of spores in the excreta (incubation period), and, ii) the number of

spores egested in the excreta. Presence of spores in excreta would indicate one

complete life cycle of Microsporidia in their host (Campbell et al., 2007).

The experiment was carried out at 20±1°C with RH of 50 ± 5% under

light regime 12h:12h (L:D). Data were taken from 8dpi to 20 dpi and were

analyzed by using two factor ANOVA with replications (α=0.05, 0.01).

3.3.2.1.4. Influence of temperatures on spore yield

49

The studies were carried out to determine the influence of temperature

on multiplication of the Microsporidium isolated from H. puera. Three different

temperatures viz., 20°C, 24°C and 28°C were chosen to rear the experimentally

infected H. puera larvae to conduct the study.

A total of 108 laboratory reared uninfected larvae were used for the

experiment. Larvae were inoculated with 3 different spore loads viz., 2×104, 2×10

3

and 2×102 and each of the three was maintained in three different temperatures

with a “control”group for each temperature. Each treatment had three larvae, each

of which served as a replicate. All insects were reared till 20 dpi and sacrificed to

estimate the multiplied spores in head and thorax combindly, in abdomen and in

the whole insect body. Spore yield was expressed per g of wet organ weight.

Data were analyzed by performing multivariate analysis by comparing

means for different treatment groups (α=0.05).

3.3.2.2. Spore multiplication in Paliga machoeralis

The Microsporidium isolated from H. puera was experimentally

inoculatd to P. machoeralis to assess the infectivity and spore production of the

Microsporidium in the insect. First day of 3rd

instar P. machoeralis larvae were

inoculated with spore loads ranging from 2×105 to 2×10

2. Four replicates were

maintained for each treatment group including “control” with the total of 20 larvae

(Plate- 4). Spores were recovered on 20 dpi and expressed per g body weight.

Data were represented graphically and one way ANOVA with replicates was

performed to compare the mean spore production for different doses (α=0.05,

0.01).

3.3.2.3. Comparative multiplication potential of Hp-Microsporidium

Spore yield from H. puera and P. machoeralis on 20 dpi was compared

by performing student‟s t-test (α=0.05).

3.3.3. Transmission

Mode of transmission of Microsporidia is one of the key factors of

parasitic virulence that is correlated with cost of host fitness, contributing towards

a parasite‟s overall fitness.

50

In this study, investigation was carried out through direct testing for the

mode of transmission of the Microsporidium in the teak defoliator Hyblaea puera

and the teak skeletonizer Paliga machoeralis.

3.3.3.1. Horizontal Mode of Transmission

i) Through silk:

Hyblaea puera

300 larvae were taken to investigate the release of the Microsporidium

spore in the silk of Hyblaea puera. First set of 150 larvae of H. puera were

inoculated with 20µl of spore suspension having a spore load of 2×103 and reared

individually on artificial diet with 5 replicates, each with 30 individuals. Second

set of 150 larvae were reared on artificial diet individually. Diet was replaced

everyday to keep the insects away from other infections.

a) Recovered silk was examined everyday for each of the inoculated larvae

from 2dpi till the end of the larval period (of survival) and were

microscopically examined for the presence of mature spores.

b) Second set of 150 larvae maintained with similar setup were daily exposed

to the silk of the corresponding inoculated larvae (individually), including

“control” batch till the end of the larval period. Test insects were

examined after death.

Paliga machoeralis

Though the silk produced by P. machoeralis larvae was comparatively

lesser than H. puera, the similar experiment was carried out to test the presence of

spores in the silk of this insect also. The insects were inoculated through teak leaf

disc instead of artificial diet and fed with the fresh leaves.

ii) Through excreta:

Hyblaea puera

a) To determine whether fecal pallets of infected larvae are possible sources

for Microsporidia infections, the following experiment was designed. A

51

total of 300 larvae were used for the experiment. First set of 150 3rd

instar

larvae were grouped into 5 batches each with 30 individuals, among which

4 batches were inoculated with a spore load of 2×103 per larva through

artificial diet. One batch was maintained without inoculation as “control”.

Larvae were reared in individual petriplates. Everyday fresh diet was

provided to the larvae to avoid contamination. The experiment was carried

out at room temperature (20±1°C) and RH (55±5%).

b) Excreta from inoculated larvae were examined daily from 2 dpi for the

presence of the spores.

c) The second set of 150 3rd

instar larvae were grouped into 5 batches and

similar set up was maintained for the experiment. These larvae were

experimentally introduced into the periplates containing the previous day‟s

excreta of the inoculated larvae from 1st batch (Plate- 5). The exercise

continued till pupation of either set of larvae. The test insects exposed to

the infected larval excreta were examined after death for the presence of

the spores.

Paliga machoeralis

Similar experiment was performed for Paliga machoeralis (Plate- 5).

Inoculation was done through leaf disc. Fresh teak leaves were used to feed the

larvae. The experiment was carried out at room temperature (22.5±1°C) and RH

(85±5%).

iii) Through infected meconium:

Hyblaea puera:

a) To detect the Microsporidia in meconia, the meconium remaining after

eclosion of inoculated adult was aseptically homogenized in a small

quantity of sterile distilled water (≈ 50µl), and was examined under the

microscope.

b) 30 individuals of 3rd

instar larvae were exposed to the meconium of 50

infected H. puera. The test larvae were reared till death and examined for

52

the presence of Microsporidia spores. The experiment was repeated for 3

times with a total of 90 test larvae.

iv) Through cadavers:

Hyblaea puera:

Cannibalism was observed in Hybaea puera culture maintained in the

laboratory. Therefore, to determine experimentally whether the cadaver is a

possible source for infection, a total 90 individuals of 4rd

instar uninfected larvae

in 3 batches were experimentally exposed to infected dead insects (Plate- 5). Each

batch was exposed to 20 dead insects for 2 days. Though fresh diet was provided,

cannibalism was observed in the culture. To accelerate cannibalism diet was not

provided to the culture on day 2. The larvae were placed individually in

petriplates. Exposed larvae (=test larvae) were examined after death or on adult

emergence, for the presence of Microsporidia.

3.3.3.2. Spread of infection

To determine the potency of individual infected larva to spread the

infection to the others in a colony under a given environmental condition, the

following experiments were designed. It was taken into consideration that density

of egested spores available for ingestion by the other larvae has a key role for

transmission, which again depends on the dose of inoculation of the infected

larva/larvae. Also, the probability of healthy individuals encountering pathogen

propagules depends on the density of uninfected larvae. Larvae were inoculated

with one or more doses and the experiments were performed with different sizes

of larval population. The spread of infection was calculated for both teak

defoliator Hyblaea puera and teak skeletonizer Paliga machoeralis (Plate- 5).

3.3.3.2.1. Hyblaea puera

To determine the spread of infection by a single H. puera larva,

infection free Hyblaea puera larvae were inoculated immediately after 2nd

moult

with different concentrations of the Microsporidium ranging from 2×10 to 2×105.

After 3 days, a single larva from each concentration was introduced into batches

PLATE – 5

Horizontal Transmission (H.T.) of

Hp-Microsporidium

A

F

E

D

B

A: H. puera exposed to infected excreta; B: P.

machoeralis exposed to infected excreta; C: H.T. in H.

puera; D: Cannibalism by H. puera; E-F: H.T. in P.

machoeralis

C

53

of 10 and 20, 3rd

instar infection free larvae. A total of 5 replications were

maintained for the study. The experiments were carried out at room temperature

(26±1°C) and RH (70±5%). Insects were reared on tender teak leaves and the

fresh leaves were provided only when it was required. The experiments were

terminated after eclosion of adults and the insects were sacrificed to examine for

the infection.

3.3.3.2.2. Paliga machoeralis

Infection free 3rd

instar Paliga machoeralis larvae were inoculated with

the Microsporidium (isolated from H. puera) with a spore load of 2×105, to

determine the spread of infection through a single individual. The infected larvae

were allowed to grow for 4 days and were introduced singly in a batch of 30 and

50 number of 3rd

instar larvae, each with 3 replicates. Insects were reared on fresh

mature teak leaves at room temperature (26±1°C) and RH (70±5%) till emergence

of adults and then sacrificed to calculate the spread of infection.

3.3.3.3. Vertical Transmission

In the present study vertical transmission of the isolated Hp-

Microsporidium was investigated in H. puera (Plate- 6).

Vertical Transmission in Hyblaea puera

A total of 720 laboratory reared infection free 3rd

instar Hyblaea puera

larvae of similar size were used for the study. Two sets of experiments were

carried out to study the vertical transmission of the Microsporidium. i) a set of

360 larvae in four batches including control, were taken to assess the degree of

vertical transmission when infected females were paired with infected males ii) a

second set of 360 individuals in four batches including control, were used for

assessing transmission when infected females were mated with healthy males.

Control was maintained in each case where healthy females were allowed to pair

with healthy males. Insects were reared on artificial diet at 26±1oC and 12h L:12h

D with 50-70% RH. Each larva was kept in a separate petriplate till pupation and

eclosion. Newly eclosed, third instar, infection free Hyblaea puera larvae were

PLATE – 6

Vertical Transmission (V.T.) of

Hp-Microsporidium

A

C D

B

A: Infected pupa in cage; B: Housing each pair

separately for vertical Transmission; C: Pairing of

H. puera; D: Eggs laid by infected insects

54

starved for 6 hrs before inoculation. Three spore loads, viz., 2×104, 2×10

3 and

2×102 were used for the experiment.

i) Infected female vs. Infected male

For this experiment, each of the three spore loads was inoculated

individually to 30 larvae with 3 replicates. One batch of “control” with 30

individuals in each replicate was maintained.

ii) Infected female vs. Healthy male

For this, the larvae were grouped into 4 batches (including a “control”

batch), each with 3 replicates of 30 individuals. 50% of the larvae from each of

the first three batches were inoculated individually with the three spore loads

mentioned earlier. Rest 50% was reared without inoculation to raise healthy

males.

Pairing of infected adults

After emergence, adult moths were housed for mating as per the

predetermined combinations.

Egg laying and hatching of larvae

i) The mated females from each treatment group (all the eight

categories) were housed in separate glass jars for oviposition. The egg masses

from each group were collected and kept separately. Eggs were treated briefly

with 0.5% of sodium hypochlorite to prevent neonate larvae getting infection

through the chorion contaminated with spores. The larvae were placed in mesh

boxes with tender teak leaves. Larvae hatched after three days. Each larva was

placed in separate petriplate to avoid contamination. Larvae were reared till 5th

instar and sacrificed to assess the extent transmission.

Examination of adults

55

After oviposition, adults were homogenized and examined for the

infection. Large numbers of spores were retrieved from each adult irrespective of

sex.

Success of parasite in vertically transmitted host

To gain an understanding of how the parasite and host populations

interact, the entire host population has to be considered, that is, estimates should

refer to all individuals that were inoculated. Two fitness measures as per Blaser

and Schmid-Hempel (2005) taking into account all exposed hosts were

considered: a) Survival upto eclosion, and b) Survival to mating; i.e. upto 4

days after eclosion. The period of 5 days were chosen because infected females

lay their first eggs 4-5 days after eclosion.

a) Survival upto eclosion

To meet the criteria for the first experiment, dead larvae and pupae

were counted. Individuals were not sexed during the experiment.

b) Survival to mating

To fulfill the criteria of expected survival to mating, the adults those were dead

within 4 days after eclosion were counted.

Effect of Hp-Microsporidium infection in fecundity of insects

Oviposition by individual females from different treatment groups and

“control” were documented. The study was repeated trice to get the mean value.

Data were analyzed by using single factor ANOVA with replications (α=0.05,

0.01) to observe the effect of parasite doses on fecundity of H. puera.

Assessment of vertical transmission

The extent of vertical transmission was assessed in the two categories

of mating as described earlier.

56

3.4. Cross-infectivity of the Hp-Microsporidium to other major defoliators

The objective of the study is to identify target insects, other than the

original host those are serious pests in forestry. According to Cate and Maddox

(1994), if the biology, ecology and taxonomy of a pathogen and its natural hosts

are known, physiological host specificity testing can supply the additional

information needed to make predictions regarding the ecological host range of the

pathogen when it is introduced into a new habitat.

The target host-pathogen interactions can be categorized as follows as

described by Solter and Maddox (1998a).

1) No infection

2) Atypical infections, including severely reduced or no production of infectous

spores

3) Moderate or high numbers of spores in one or more individual test insects

Bioassays to assess the cross-infectivity of the Hp-Microsporidium

were carried out with 6 different species of defoloiator moths by inoculating the

Microsporidium. Four other species of moths were also checked for susceptibility

to the Microsporidium.

3.4.1. Rearing of Defoliator pests

The larvae of different species that were taken for cross-infectivity

study were reared in the laboratory at least for one generation before inoculation.

After getting the progeny, individuals from parental generation were screened for

Microsporidia infection. Only infection free larvae were taken for obtaining the

progeny. Other than Paliga machoeralis and Spodoptera litura, rest of the species

were collected from the field and then reared in the laboratory for one generation.

To study the cross-infectivity in P. machoeralis laboratory cultured larvae were

used which was maintained in our lab from 2007.

Paliga machoeralis Walker

Described under the section 3.3.

57

Sylepta derogata Fabricius

Sylepta derogata is commonly known as Cotton leaf roller, belongs to

the family Pyralidae. Larvae seen in groups during initial stages in folded leaves

amidst fecal material. Late/ last instar larvae move out and pupate individually.

The experimental insects were collected from Gossypium sp. (cotton plant) from

Institute of Wood Science and Technology Campus, Bangalore and from

Agricultural field in Tamilnadu, India. The insects were screened for

Microsporidia before starting the rearing. Also, parental insects were screened

after getting next generation larvae. The larvae were reared on cotton plant

leaves.

Eupterote sp.

This insect belongs to the family Eupterotidae. The insects were

collected from Andhra Pradesh, India, from their host plant Gmelina arborea.

One of the other major host plants of the insect is Tectona grandis. Larvae were

checked for the presence of Microsporidia by examining the body fluid. Once it

was confirmed that larvae were free of infection, they were reared in the

laboratory to get the progeny to conduct the experiment. The larvae were reared

on teak leaves.

Atteva fabriciella Swederus

It is a serious pest of Ailanthus excelsa and belongs to the family

Yponomeutidae. They were collected from Vellore in Tamilnadu. Sample larvae

were screened for any infection by Microsporidia before rearing the insects.

Infection free larvae were reared on their host plant leaves.

Spilarctia obliqua Walker

It is a polyphagous pest belonging to the family Arctiidae, initially

collected from teak plantation at Mannarkkad, Kerala. The larvae were examined

for the Microsporidian infection by random sampling and infection free larvae

were cultured on teak leaves in the laboratory for further study. After egg laying

by the adults all the parental insects were screened for the infection. Once it was

58

confirmed that the batch was mirosporidia free, insects were reared for few

generations for taking up the cross-infectivity study.

Spodoptera litura Fabricius

The moth belongs to family Noctuide; a pest of various plants.

Laboratory reared larvae were obtained from National Bureau of Agricultural

Important Insects, Bangalore to conduct the bioassay. Larvae were maintained on

artificial diet.

Composition of artificial diet of Spodoptera litura

Ingredients:

Kabuligram: 100g

Methyl para-hydroxybenzoate: 2g

Sorbic acid: 1g

Yeast tablets: 10g/30tablets

Agar agar: 12.75 g

Ascorbic acid: 3.25g

Multivitamin complex: 4 cups

Streptomycin sulphate: 0.25g

10% Formaldehyde: 5ml

Distilled water: 800 ml

Standard protocol was followed to prepare the diet.

3.4.2. Bioassay with the Hp-Microsporidium

For cross-infectivity study, 720 third instar larvae of each species were

used. They were grouped into 8 batches to inoculate the Microsporidium isolated

from Hyblaea puera. The concentration of spores ranged from 108 to 10

2 with the

spore load of 2×106 to 2 per larva. In case of Eupterote sp., the inoculation ranged

from 5×106 to 5. Each batch was with 3 replicates of 30 individuals including

“control” for each species. Larvae of all the 6 species were maintained at 24 ± 1°C

with RH 60-70% and light regime 12h L:12h D. Larvae were inoculated by

spreading the spore suspension on the surface of their host plant leaves. 15 mm

59

diameter leaf disc was taken to spread spore suspension for each larva of 5

species, while 20mm disc was used for inoculating larvae of Eupterote sp. Each

of the inoculated larvae for all 6 species was maintained in separate petriplates till

the end of the experiment. Larvae were starved for 6 hrs prior to inoculation.

3.4.3. Statistical Analysis

Statistical analyses of the data were carried out and the LC50 values

determined for each species by using probit analysis as per the method of Finney

(1971), with the help of SPSS software. The calculated value is a predicted dose,

which can cause 50% mortality of the experimental insect population. There was a

single mortality observed in one of the replicates of “control” batch of Eupterote

sp. The LC50 value was corrected by using Abbott‟s correction formula (Abbott,

1925).

3.4.4. Other moths tested for cross-infectivity

The cross-infectivity study was extended to four other moth species

viz., Achaea janata Linnaeus (family: Noctuidae), Bombyx mori Linnaeus (family:

Bombycidae), Lamprosema nephealis Walker (family: Crambidae) and Parasa

lepida Cramer (family: Lymacodidae) to determine the cross-infectivity of the

Microsporidium species. Insects were reared in the laboratory in a small scale

before inoculation. Insects were inoculated with a spore load of 2×104

individually. LC50 values were not calculated owing to less number of larvae

involved in the experiment. The insects were reared on their host plant leaves

throughout the experiment.