ISBN 978-1-84626-xxx-x

Proceedings of 2010 International Conference on Agricultural and Animal Science

(CAAS 2010)
Singapore, 26-28 February, 2010, pp.

Pathogenecity of Bacillus thuringiensis var. kurstaki Against

Tobacco Caterpillar, Spodoptera litura (Fabricus)
Arti Prasad1 and Yogita Wadhwani2
1
Insect Microbial and Herbal Control Laboratory Department of Zoology, University College of Science
Mohanlal Sukhadia University, Udaipur (Raj.) 313001 INDIA
e-mail : artimlsu@yahoo.co.in ; Tel.: 91-9414809794

Abstract : Spodoptera litura (Fabricus) is one of the most notorious Lepidopteran pests of South Rajasthan affecting
cole crops, which are grown in majority. Indiscriminate use of pesticides has further aggravated pest problem. The
alternate at this juncture is to blend the Ancient Wisdom and Basic of Science and Modern Technology for sustainable
agriculture. Bio-pesticidal approach of pest control is eco safe and specific. Bacillus thuringiensis var. kurstaki. treated
larvae of tobacco caterpillar besides causing morphological abnormalities also resulted in histopathological alterations.
Histopathology of Bt. treated larvae exhibited alteration in the midgut, fat bodies, connective tissues and body wall.
Midgut epithelial cells were observed detached from basement membrane along with distorted shape of nuclei and cells
of midgut epithelium. Parietal and visceral fat layers became indistinct and fat cells and connective tissues lost there
identify. Different body layers of body wall were completely disintegrated forming space between exocuticle and
endocuticle. Fragile nature of larval integument was confirmed by loss of chitin content in the endocuticle. Hence, Bt.
not only affected visceral organs but cuticle of larvae as well thus causing two way effect..
Key words: Bacillus thuringiensis, Spodoptera litura, pathogenecity, histopathological alterations, disintegration.

1. Introduction
Inspite of increasing consumption of chemical fertilizers and pesticides, the agricultural production
in India is almost static since 1989 due to poor soil nutrition status and pests and diseases causing over
Rs.90,000 crore worth crop losses per annum.1 Soil quality is being deteriorated through continuous use of
chemical fertilizers. Indiscriminate use of pesticides has further aggravated pest problem. It has resulted in
resistance in pests resurgence of minor pests and high level of pesticide residues in commodities, which enter
food chain and can cause poisoning cases and death through organ dysfunctions, immunosupression,
neurotoxicity, impairment of reproductive functions, carcinogenecity, tumorogenecity, paralysis etc and
harm to non target beneficial fauna and flora. It is now realized that the usage of chemical pesticides is only
doing more harm than benefit to this biosphere2.
.
A balanced use of both bio-organic and chemical fertilizers in production cycle under Integrated
Nutrition Management (INM) and balanced use of both Bio-control agents and pesticides under Integrated
Pest Management (IPM) followed in a systematic manner leads to Integrated Crop Management System
(ICM)3. Bio-pesticidal approach of pest control involves the scientific use of living organisms, which can
intervene the life cycle of insect pests in such a way that the crop damage is minimized. Naturally occurring
entomopathogens are important regulatory factors in insect populations. The application of microorganisms
for control of insect pests was proposed by notable early pioneers in invertebrate pathology such as Agostine
Bassi, Louis Pasteur, and Elie Metchni Koff. 4,5 In addition to efficacy, the advantages of use of microbial
control agents are numerous which include eco-safety, specificity, reduced number of applications, no
resistance in pests, increase yields and quality improvement of crops and higher acceptability.
Today, a variety of entomopathogens are used for the control of invertebrate pests in glass-house and
row crops, orchards, ornamentals, range turf and lawn, stored products, and forestry and for the abatement of
pest and vector insects of veterinary and medical importance.6,7, Strategies for the use of entomopathogenic
organisms for insect control are basically the same as that for other biological control agents. Like other
natural enemies, insect pathogens can exert considerable control on target population.
The most widely used inundatively applied microbial control agent is Bacillus thuringiensis. As of
1998 about 200 Bacillus thuringiensis based products were registered in the United States alone.8 Today
large number of isolates of the bacterium are commercially produced for their activity against Lepidoptera,
coleoptera and Diptera, Effectiveness of Bacillus thuringiensis has been recorded against various pests for
eg. Plutella macullipennis (curtis)9 , Trichoplusia ni (Hubner)10, Prothetria dispar11,12, Although the entry of
Bacillus thuringiensis insecticide has been slow in market but now it is emerging as strong component of
IPM in present day scenario for the control of broad spectrum of pests.
Keeping the above facts in view, one of the most notorious Lepidopteran pest of South Rajasthan,
(India) affecting cole crops grown in majority, was chosen. Spodoptera litura (Fabricus) commonly known
as tobacco caterpillar was amongst minor pest nearly forty-fifty years ago but due to its polyphagous nature
it has now attained the status of major pest and inability to control the pest with chemical insecticide puts use
of biopesticides in fore front.
The study was carried out to find out the exact mode of action of bio-pesticide Bacillus thuringiensis
(kurstaki) by histopathological techniques so as to unveil the stories of effects of Bacteria on the insect
tissues.

2. Materials and Methods

Bacillus thuringiensis var kurstaki (Bt.k.) commercial preparation named HALT (Wockhart Com
Pvt. Ltd.) with 5×107 spores per mgm wettable powder was used for the present study. Doses were prepared
by serial dilution viz. 1.0, 0.1, 0.01 and 0.001 percent concentration of Bt.k. and fourth instar larvae were fed
with castor leaves, infested with experimental bio-pesticide (leaf discs were dipped in Bt.k. and dried in
shade). After 24 hours, observations were taken and fresh food was given to the larvae . The effects of
biopesticide, Bt.k. on larval tissues were studied by general histopathological techniques, which was carried
out by standard methods of microtomy. Whole body sections were stained in Haematoxylin and eosin, and
Mallory Triple Stain to visualise the exact effect of bio-pesticide on tissues and chitinous part of insect.

3. Result
3.1 Control:
Histopathological studies of untreated fourth instar larvae exhibited general body structures in
transverse section of whole body.
Body wall:
It is the outermost and cuticular covering of the body, which is divided into two main parts (a) outer cuticle
(b) inner epidermis. Cuticle is further divided to outer exocuticle and inner endocuticle. A very thin surface
layer called as epicuticle is present on exterior surface of exocuticle which appears as clear borderline. Both
exo and endocuticle are chitinous but due to other substances present in exocuticle it appears as a dense
structure (Fig. 2 Plate A). A continuous layer of epidermis is present in the form of simple epithelium. It
degenerates after the activity of cuticle formation and found as indistinct layer beneath the cuticle where cell
boundaries are indistinct (Fig. 2 Plate A). After body layer there is a basement membrane, which is indistinct
in section.
Body cavity:
The body cavity mainly consists of alimentary canal and visceral organs. A section through mid gut area
reveal outer visceral mass mainly consists of connective tissues, fat bodies, sections of trachea and circular
and longitudinal muscles (Fig.1PlateA). Fatty tissues are present in two layers: outer parietal and inner
visceral fatty layer. Fat bodies are identified as loosely aggregated mass of cells with characteristic
vacuolization and globules of oil and fat (Fig. 1&2, Plate-A). Midgut occupies the innermost part of body
cavity. It is made up of basal membrane, attached to it is highly folded layer of gut epithelial cells followed
by thin peritrophic membrane that ultimately surrounds the lumen of alimentary canal (Fig.3, Plate-A).
The epithelial cells of mid gut rest on basement membrane and are mostly columnar cells with irregular ends
having microvilli projecting into the gut lumen. Centrally placed large nucleus is observed with granular
cytoplasm (Fig.3-plate-A). In addition to columnar cells large spongy cells called as goblet cells or digestive
cells are seen in between the bases of columnar cells. Goblet cells have openings at their inner ends
projecting into the gut lumen and take active part in process of secretion (Fig.3, Plate-A). Smaller basal cells
called regenerative cells are found in small groups/ patches located at the bases of columnar epithelial cells
and their function is to propagate cells to replace digestive cells when exhausted or at the time of ecdysis.
3.2 Treated:
Bt.k. treated larvae of Spodoptera litura (Fabricus) showed various histopathological changes in the
different body tissues which were distinct and characteristic of pathogenic changes due to bacterial infection.
Since the infection in larva started after ingestion of Bt.k. infected diet and the bacterial crystal proteins were
activated after they were dissolved in alkaline pH of midgut and they released δ-endotoxin, mid gut tissues
were the first to be affected. .
Midgut:
Extensive changes in the gut epithelium followed by the gut paralysis can be fully supported by
histopathological alterations in the cells of the midgut.
Epithelial cells separated from each other and were observed detached from basement membrane (Fig.4-
PlateB) Cells had distorted shape and took very light stain (Fig.3,4 Plate-B). Highly disorganized midgut
epithelial cells with lesion formations in the basement membrane caused mixing of the gut content with
haemolymph which lead to easy transfer of infection. Overall shortening and shrinkage of epithelial cells
with distorted shape gave the appearance as if they had released their contents (Fig.3,4, Plate-B). In contrast
to coherent sheets and intact cell membrane observed in epithelial cells of control larvae Bt.k. treated larvae
exhibited lysis of midgut epithelial cells. Thus leading to sloughing off of damaged cells from the basement
membrane towards lumen was also pronounced. Nuclei of epithelial cells became distorted (Fig.4, Plate-B).
Peritrophic membrane was completely damaged and seen as small fragments here and there near the lumen.
Completely damaged longitudinal and circular muscles could be observed. (Fig. 3 Plate-B).
Body Cavity:
Bt. treated fat cells and connective tissues were found to be adversely affected. Although parietal and
visceral fat layers could be seen distinctly but they were severely affected by Bt (Fig.1 Plate-B). There was
overall degeneration of fat cells and connective tissues and fat cells had lost identity due to general distortion
and disintegration of the cells (Fig.2 & 3, Plate-B). Nuclei of the fat cells were almost damaged and
deposited spores could be observed in the entire area of fatty tissues and at the edges of connective tissues
(Fig.1,3 and 4 Plate-B). In between the sections of fat tissues, sections of distorted trachea were also seen.
Body wall:
There was complete disintegrity between the different layers of body wall. Endocuticle became very thin and
delicate, there was no demarcation between exocuticle and loosening was evidenced by forming space in
between the two (Fig.1 Plate-B). Loss of chitin content in the endocuticle was evident by fragile nature of
treated larval integument.
4. Discussion
When whole body sections of fourth instar larvae treated with Bt.k. were cut and observed for the
pathogenecity of bacteria on different tissue of the insect, it was found that Bt.k. starts its infection from mid
gut tissue at its first stage of action from where the infection spreads into the haemocoel causing infection in
various tissues of the body cavity and finally to the cuticle as its last resort. The effect of the bacteria is
mainly due to its insecticidal activity, which is associated with the proteinaceous toxins located in the
parasporal inclusion bodies also known as parasporal crystals and produced at the time of sporulation. 13 It
accounts for total thirty percent protein of the bacterium and collectively the toxin present in the parasporal
body are known as δ-enndotoxin.14,15,16. As soon as the bacteria were ingested through infected diet, the toxic
protein called as protoxin which was inactive got activated in the alkaline mid gut which then started a
cascade of events leading to the disruption, disintegration and degeneration of various tissues causing death
of the insect within few hours (10-15 hours) of ingestion.
Degeneration and disorganization of gut epithelial cells and on different organs in insect pest due to
effect of various Neem derivativeshave been recorded17,18, and in the Nimbecidine treated Helicoverpa
armigera and Spodoptera litura were also observed by Ramarethinam et.al.2. The peritrophic membrane,
which faces the lumen of mid gut, reveals complete damage as degenerated peritrophic membrane could be
observed in some of our Bt. treated larvae (Fig.3 Plate 2). In the phytophagous Hymenoptera and
Lepidoptera after the development of characteristic symptoms of Bt. such as diarrhea, lethargy and atony
revealed when studied histopathologically that their was gradual destruction of peritrophic membrane along
with the mid gut epithelial cells.19 This provides an easy entry of the infected bodies into the mid gut
epithelial areas together with cellular degeneration of organs such as the salivary glands malpighian tubules,
muscles, nerve tissue and hypodermis. During sporulation Bt. produces parasporal crystalline protoxin
inclusion bodies.9 After insects eat these protoxin crystals, they are dissolved in the mid gut and are cleaved
by mid gut proteases to produce toxins. These toxins bind to target sites on mid gut cell membranes, which
leads to formation of pores in mid gut membranes, cell lysis and death of the insect.9
The next site of action considered as the target site was the columnar epithelial cells of mid gut,
various pathological changes could be observed in the mid gut epithelial cells. The epithelial cells revealed
an overall degeneration, which includes disruption and disintegration of cells. The cells became distorted and
lost their ability to take stain which indicated that content of the cell has been leaked out (Plate-B, Fig.3,4).
There are lesion formations the nuclei of epithelial cells became distorted losing their identity and it
appeared that the epithelial cells are being sloughed off from the basement membrane and moving toward
lumen (Plate-B Fig. 3, 4).
 Due to the loss of epithelial cells the entire mid gut area lost its identity. The toxin after getting
solubilized is cleaved by gut proteases which recognized the binding site at the brush border membrane
surface of columnar epithelial cells lining the gut lumen20,.
The proteolytically activated toxins then bind to a specific membrane receptor(s) in the gut, with
both membrane permeability andion transport ultimately being affected Consequences of toxin ingestion are:
distortion, enlargement of the mid gut epithelial cells, lifting of the cells from the basement membrane, loss
of the epithelia, and death of the insect.21
Activated toxin molecules bind to glycoprotein receptors on the mid gut epithelium microvillar
membrane and form pores or lesions leading to osmotic swelling, cell lysis and damage to the mid gut
haemocoel barrier, resulting in death.14,.
The activated toxin binds with high affinity to receptors on the apical membrane, and this is followed
by insertion of the toxin into the epithelial membrane. Toxin insertion is an irreversible step and is followed
by toxin oligomerisation and formation of an ion pore, which results in an osmotic imbalance. Insect
mortality occurs by several hours to days after ingestion of the toxin.22,,.
The next pathological observations revealed that their was a gradual loss of different tissues in a
step-wise manner i.e. after mid gut epithelial cells the basement membrane and the muscular layer were the
next sites for the effect of bacterial bio-pesticide. Since the bacteria have two important components, the
spores and the toxins. For their activation pH plays an important role. For the action of toxin alkaline
medium is essential whereas lower pH is favourable for spore germination. The secondary effect of the
toxicaemia is the reduction of pH to nearly neutral which allows the germination of spores and multiplication
of spores in the mid gut. As the peritrophic membrane and epithelial cells have been already destroyed due to
toxicaemia, they allow the infection to invade into the haemocoel through damaged basement membrane and
mid gut tissue (Fig. 2,3 Plate-B). The dissolution of the cells cementing the basement membrane due to toxic
crystal may be due to presence of hyalouronidase like substance in the protein crystal.23
The effect of Bt. on circular and longitudinal muscles is an indication of paralysis observed during
the poisoning symptoms. After invading the cells of epithelium, the bacteria germinate and penetrate the
peritrophic membrane and the infection spread to the lumen as evident by complete degeneration of tissues
of the mid gut. After ingestion by a susceptible insect, the endotoxin binds, to the receptor in the mid gut-
causing disturbance in ionic potential. If the rate of damage of mid gut exceeds rate of repair, lesions are
formed in the mid gut and the bacteria invade the hoemocoel. After ingestion by a susceptible insect,
crystalline N-endotoxin is solubilized and proteolytically cleaved from an inactive protoxin, to an active
toxin form within the insect mid gut. The activated toxin binds to receptors in the mid gut and is believed to
integrate into the lipid bilayer of the brush border membrane. Ion channels are formed, causing mid gut cells
to lose their membrane potential. If the rate of damage to mid gut exceeds the rate of repair, lesions form,
bacteria invade the haemocoel and death results from bacterial septicaemia.22
The tissues of the body cavity like fatty tissues and connective tissues also revealed significant effect
of bacteria. The fat body cells became swollen with damaged nuclear large vacuolization and finally losing
the integrity of the cells. The connective tissues also revealed the gradual loss with the deposition of spores.
(Plate-B Fig.2,3) As the haemolymph is the medium through which all chemical exchange between organs
are effected and regulated so the adversely effected haemolymph automatically disturbs the metabolism of
fatty tissues and other tissues of the body cavity.
Finally the infection spread on to the cuticular part of the body damaging various layers of the
cuticle. As the endocuticle was the most severely affected part of the cuticle and chitin being the most
important component of the cuticle the chitin synthesis and its deposition may be the target site of the
bacteria (Plate-B Fig. 1,2). Since the protein is the important component chitin and the haemolymph being
the important carrier of protein, after being effected by bacteria is not capable of transporting adequate
amount of protein for chitin deposition. The biochemical and immunological studies have shown conformity
between haemolymph and cuticle protein. High control of aromatic amino acid (Tyrosine and Phenyl aniline)
have led to the suggestion that haemolymph protein might be involved in cuticle sclerotization.23
Hence to conclude, it can be clearly explained that the effect of Bt. is two fold. At first step the toxic
crystal starts its effect on mid gut epithelium thereby making environment suitable for the spore germination
and its transmission. At thesecond step crystal along with germinated spore touch each and every part of the
insect’s body thus spreading the infection, which ultimately leads to the death of the infected larvae.

5. References
[1] Singhal, Vivek., 2005. Uses and benefits of bio-pesticides and bio-fertilizer. Agriculture today, 16-17.
[2] Ramrethinam, S., Marimuthu,S. and Murugesan,N.V. 2000. Effect of a Neem oil formulation on the growth,
development and histomorphology of some Lepidopteran pest (Helicoverpa armigera H.; Spodoptera litura F.
& Gangara thyrsis) Pestology Vol. XXIV No. 10.
[3] Kakkar,A. 2000. Integrated crop management – A Holistic Approach Pestology Vol. XXIV No. 10.
[4] Steinhaus, E.A.,1956 Microbial control: The emergence of an idea. Hilgardia 26, 107-160.
[5] Steinhaus, E.A.1975 ‘Disease in a Minor Card”. Ohio State University Press, Columbus DH.
[6] Burges H.D., 1981. (Ed.) Microbial control of pests and plant diseases 1970-1980. Academic Press, London.
[7] Tanada, Y. and Kaya, H.K., 1993. “Insect Pathology” Academic press, New York.
[8] Narayanan, K. Jayaraj, S. and Subramaniam, T.R., 1970. Control of three species of Lepidopterous insects
with the pathogen Bacillus thuringiensis (Berliner). Madras Agric. J., 57 (11): 665-673.
[9] Dulmage, H.T., Walfenbarger, D.A. Lukefahr. M.J. and Corea, J.A., 1981. Field tests with HD-1 formulation
of the δ-endotoxin of Bacillus thuringiensis. J. Econ. Ent., 64(6):1421-1422.
[10] Federici, Brian. A. and Bauer, Leah. S., 1998. Cyt1 Aa Protein of Bacillus thuringiensis is toxic to the
cottonwood leaf beetle, Chrysomela scripta, and suppresses high levels of resistance to Cry 3 Aa. Applied and
Environmental Microbiology. 64 (11): 4368-4371.
[11] Hofte, H. and Whiteley, H.R., 1989. Microbiol. Rev. 53, 242-255.
[12] Aronson, A.I., 1993. The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential
survival. Mol. Microbiol. 7: 489-499.
[13] Agaisse, H., and Lereclus, D., 1995. J. Bacteriol.177: 6027-6032.
[14] Schumutterer, H and Rambold, H.1980. Zur wirkuny eniger reinfraktionen aus samen von Azadirachta indica
aufn Frebaktinitat and Meatamorphase. Vol. Epilachna varivestis (Col.: Coccinelidae). Z. augen. Ent. 89:179-
188.
[15] Schultz, W.D. 1980. Pathological alteration in the ovaries of Epilachna varivestis induce ny extract from neem
kernels. In “Natural Pesticides from Neem tree, Azadirachta indica A.Juss”. Proceedings of the Ist
International Neem Conference. Eds. Schmutterer, H.Aschler, K.R.S. and Rambold,H. pp:81-93.
[16] Mayas, I., 1970. These Fac. Sci. Paris, pp 92.
[17] Hofmann, C., Luthy, P., Hutter, R., and Pliska, V., 1988b. Eur. J. Biochem. 173, 85-91.
[18] Keeton-Timothy, P. and Bulla-Lee, A-Jr., 1997. Ligand specificity and affinity of Bt. R-1, the Bacillus
thuringiensis Cry 1A toxin receptor from Manduca sexta, expressed in mammalian and insect cell cultures.
Applied and Environmental Microbiology, 63(9): 3419-3425.
[19] Gill, S.S., Cowles, E.A. and Pietrantonio, P.V., 1992. The mode of action of Bacillus thuringiensis endotoxins.
Annu. Rev. Entomol. 37:615-636.
[20] Knowles, B.H., 1994. Mechanism of action of Bacillus thuringiensis insecticidal data- endotoxin. Adv. Insect
Physiol., 24: 275-308.
[21] Burges, H.D. and Hussey, N.W., 1971. Microbial control of Insects and mites. Academic Press,
[22] Wu-Sheng-Jiun, Koller,C- Noah.,Miller-Deborah-L., Bauer- Leah, S., Dean-donald, H.,2000. Enhanced
toxicity of Bacillus thuringiensis Cry 3A delta- endotoxin in coleopterans by mutagenesis in a receptor binding
loop. FEBS- letters, 473 (2): 227-232.
[23] Roberts, D.W. and Brock, 1981. Toxins of entomopathogenic fungi. In: Microbial Control of Pests and Plant
Diseases, 1979-1980, H.D. Burges, (ed.) London: Academic Press. pp 57.
 PLATE - A

T.S. of fourth instar through mid gut Magnified view of a part of cuticle
region showing following layers from along with fat bodies of fourth instar
outer to inner. EPI–Epicuticle, larva. Epicuticle (EPI), Exocuticle
ENDO- Endocuticle, EPD- (Exo), Endocuticle (ENDO) and
Epidermis, FB-Fat bodies, CT Epidermis (EPD) are very well
-Connective tissues, LM- distinguished. The internal extension
Longitudinal muscles, CM- Circular of epicuticle in the form of Apodeme
muscles, BM- Basement membrane, (APO) is also significant. Fat body
MGE- mid gut epithelium, and L- cells(FB) are clearly marked with
Lumen. × 10 vacuoles(V) and nucleus(N). × 100
Magnified view of one complete crypt
(CR) and villi (V) with large distinct
goblet cells (GC), regenerative cells
(RC), columnar epithelial cells (CC)
peritrophic membrane (PM) muscular
layer (ML), basement membrane
(BM), lumen of villi (LV) and lumen
(L) of mid gut. × 40

PLATE - B

Magnified view through body wall and mid gut depicting

Magnified view of mid gut showing hypertrophied
thin and almost degenerated cuticle (DC) space formation
epithelial cells (HEC), longitudinal muscles (LM) circular
(SF), infected connective tissues (ICT), degenerated mid
muscles (CM) basement membrane (BM) with bacterial
gut epithelium (DMGE), basement membrane (BM) and
spore deposition (BS) in fat cells. ×40
lumen (L)×10.

Highly magnified view of damaged mid gut epithelium
showing sloughing off of mid gut epithelial cells (SE)
with disintegrating nuclei (DN), longitudinal and circular
muscles (LM and CM) along with basement membrane
(BM), are also at the verge of disintegration. ×100
T.S. of body section showing damaged connective tissues
(DCT) and fat cells (DFB). Mid gut epithelial cells are
completely degenerated (DMGE) forming lesions (L)
along with degenerated longitudinal and circular muscular
layer (LM and CM) and basement membrane (BM).
Disintegrating trachea (DT) is also visible. × 100