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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 cuticular covering of the body, w divided into two main parts (a) outer cuticle (b) inner epidermis.
Cuticle is further divided to outer exocuticle and inner endocuticle with a very thin surface layer called as
epicuticle. Both exo and endocuticle are chitinous (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 (Fig. 2 Plate A).
Body cavity:
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). 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, made up of basal membrane and folded layer of gut
epithelial cells followed by thin peritrophic membrane facing the lumen of alimentary canal (Fig.3Plate-
A).The epithelial cells of mid gut rest on basement membrane with columnar cells having irregular ends and
microvilli projecting into the gut lumen(Fig.3-plate-A). In addition to columnar cells, large spongy goblet
cells are seen in between the bases of columnar cells taking active part in the 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.
3.2 Treated:
Characteristic histopathological changes in the different body tissues were observed after treatment.
Since the infection started after ingestion of Bt.k. infected diet , 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 were distorted and separated from each
other and were observed detached from basement membrane (Fig.4-PlateB&C) Highly disorganized midgut
epithelial cells with lesion formations , caused mixing of the gut content with haemolymph leading to easy
transfer of infection (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 leading to
sloughing off of damaged cells from the basement membrane towards lumen. Nuclei of epithelial cells
became distorted (Fig.4, Plate-B). Peritrophic membrane was completely damaged seen as small fragments
here and there near the lumen. Completely damaged muscular layers could be observed. (Fig. 3 Plate-B).
Body Cavity:
Bt. treated fat cells and connective tissues were found to be adversely affected (Fig.1 Plate-B). There was
overall degeneration of fat cells and connective tissues. Fat cells 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 spore
deposition 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 with 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 , it was found that Bt.k. starts its infection from mid gut tissues 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,. 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.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.21Activated 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 in, 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 septicaemia22. 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 area
with 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 of chitin and the haemolymph
being the important carrier of protein, after being effected by bacteria ,it is not capable of transporting
adequate amount of protein for chitin deposition. The biochemical and immunological studies have shown
conformity between haemolymph and cuticle protein23. 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 the second 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
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[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
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[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. . 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

Fig – 1 : T.S. of fourth instar through Fig -2 :View of a part of cuticle along Fig – 3:Magnified view of one
mid gut region x 10. with fat bodies of fourth instar x 100. complete crypt and villi x 40.

PLATE - B

Fig–2:Highly magnified view of Fig – 3: T.S. of body section showing


Fig – 1: Magnified view of treated
damaged mid gut epithelium cells damaged connective tissues × 100.
midgut and body cavity x 10.
×100.
EP- epicuticle, EXO-exocuticle, END-endocuticle, FB-fat bodies, CT-connective tissues, LM-Longitudinal
muscles, CM-circular muscles, BM-basement membrane, L-lumen, V-vacuoles, N-nucleus, R-regenerative
cells, GC-goblet cells, V-villi, CC-columner epithelium, BM-basement membrane, DC-degenerating cells,
DMGE-damaged midgut epithelium, BS-bacterial spores, DN-degenerating nuclei, HEC- hypertrophied cells