Research Journal of Recent Sciences _______________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 313-316 (2012)
Res.J.Recent.Sci.

Short Communication

Levels of Glutathione S - Transferase In
Different Larval Tissues of Papilio Demoleus
Warade N. V.
Department of Zoology, Shankarlal Khandelwal College, Akola, M.S., INDIA

Available online at: www.isca.in
(Received 7th December 2011, revised 16th January 2012, accepted 28th January 2012)

Abstract
Papilio species are of economic importance, as their larvae are important defoliator of citrus known from almost all the citrus
growing areas of the world. As a result, the overall vigour and vitality of the citrus plant is reduced considerably. The aim of the
present article is to study the glutathione S-transferase in different larval tissues of Papilio demoleus. CDNB - conjugation
activity was compared in various fifth instar larval tissues of laboratory reared and field collected Papilio demoleus using CDNB
(1-chloro-2, 4-dinitrobenzene) as substrate. Different tissues estimated for GST include the midgut, fat bodies, cuticle,
haemolymph and the whole body of two days old fifth instar larvae. The GST activity was found highest (648.93 ± 0.08 µM mg
protein-1min-1) in fat bodies of field-collected larvae while the corresponding value in the laboratory-reared larvae was 80.19 ±
0.05 µM mg protein-1min-1, where it showed 8.09 fold increases in GST activity. The fat bodies showed highest GST activity
suggesting that the fat body is the major site of metabolism of insecticide, especially the organophosphate class, whiles the gut,
cuticle and haemolymph also plays an important role in the metabolism of insecticide.
Keywords: Glutathione S - transferase, Papilio demoleus, insecticide resistance, life cycle, GST

Introduction
Glutathione S - transferase (GST; E.C. 2.5.1.18) is a family
of multifunctional enzymes, which catalyze the conjugation
reaction of the xenobiotics (insecticides) with an endogenous
factor viz. reduced glutathione (GSH). The conjugates were
further metabolized to mercapturic acids and excreted1. GSTs
act by catalyzing the conjugation of large variety of
compounds bearing an electrophonic site, with reduced
glutathione. The conjugates were then eliminated from the
cell via the glutathione S - conjugate export pump2,3. It
detoxifies many insecticides including organophosphate
pesticides and acaricides4. It has been already studied by
various workers that indiscriminate use of insecticides,
multiple generations of insect per annum and year round
availability of host crops have contributed to the
development of resistance in different insect pest to almost
all kinds of insecticides5. The faster degradation of
insecticides by metabolic enzyme GST is one of the
mechanisms commonly associated with insecticide resistance
in different insects6.
Extensive indiscriminate use of organophosphate insecticides
(one of the most important insecticide in wide-scale use) has
led to the development of resistance in P. Demoleus to this
insecticide. In view of the importance of GST in the
insecticide resistance of many insects and their direct
involvement in their management, present studies were

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undertaken to characterize the GST mediated biochemical
mechanism of organophosphate resistance in P. demoleus.
For that the levels of GST enzyme were studied in different
tissues of laboratory reared and field collected P. demoleus.

Material and Methods
P. demoleus were reared in cages for many generations in
laboratory during three consecutive years. They were also
collected from the citrus orchards of Babulgaon block, Dr.
PDKV, Akola. The orchards were sprayed from time to time
with 0.3% dimethoate insecticide for the control of this pest.
The stages that were found on the plants on the tenth day
after spraying insecticide were collected and were used for
enzymological studies.
Estimation of protein using BSA as a standard 7: One mg /
ml solution of BSA was taken in test - tubes in different
amount ranging from 1 µl to 10 µl, to make the concentration
of BSA from 1 µg to 10 µg. Each concentration was prepared
in triplicate. Volume of the protein solution was adjusted to
20 µl by using 0.15 N NaCl. Ready to use Bradford reagent
(200 µl) was added to each test-tube and then each test-tube
was diluted five times. Test tubes were incubated for 15 min
at room temperature. The absorbance was measured at 595
nm. Calibration curve was plotted between mean value of
concentration on X-axis and mean value of absorbance on Yaxis.

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Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 313-316 (2012)
Res.J.Recent.Sci
Isolation of GST enzyme8: Two days old first to fifth instar
larvae reared in the laboratory and collected from field were
chilled in refrigerator, separately. The larvae were pinned
dorsally at head and anal region in wax plate and their
midguts were dissected out. Dissections were carried out
with the help of sterilized dissecting needles in ice-cold
sodium phosphate buffer (SPB) (0.1M pH 6.5). Fat bodies
and food particles were removed from the midguts with the
help of soft brush. Removed midguts were placed in glass
homogenizing tubes containing 1ml ice- cold SPB (0.1M pH
6.5) containing 0.1 M of EDTA, PTU, PMSF each and 10%
Glycerol. The different developmental stages of P. demoleus
were homogenized in ice-cold condition in the Teflon tissue
homogenizer at 1500 rpm, separately. The homogenate thus
obtained were centrifuged at 15,000 rpm for 15 min at 4°C in
high-speed refrigerated centrifuge. Solid debris and cellular
material was discarded. The resultant post - mitochondria
supernatant obtained was stored at –20°C and used as the
enzyme source.
Estimation of GST9: 50 µl of 50 mM 1 chloro-2, 4
dinitrobenzene (CDNB) and 150 µl of 50 mM reduced
glutathione (GSH) were added in 2.77 ml (2770 µl) of
phosphate buffer (100 mM pH 6.5, 0.1 mM PTU). 30 µl of
enzyme stock was added in the above mixture. Reaction was
carried out in triplicate set. The content were gently shaken
and incubated for 2 minutes at 24oC and then transferred to
cuvette placed in sample cuvette slot of the UV
Spectrophotometer. 3 ml of the reaction mixture without
enzyme was placed in the cuvette present in reference slot.
Absorbance was read for 5 min at 340 nm by employing time
scan menu of the spectrophotometer. The GST activity was
calculated as follows:
Abs (increase in 5 min) x 3 x 1000

CDNB - GSH conjugate = --------------------------------------(µM min-1mg proten-1)
9.6* x 5 x mg of protein

9.6 mM / cm – extinction coefficient for CDNB – GSH
conjugate.

Materials and Disscussion
CDNB - conjugation activity was compared in various fifth
instar larval tissues of laboratory reared and field collected P.
demoleus using CDNB as substrate. Different tissues
estimated for GST include the midgut, fat bodies, cuticle,
haemolymph and the whole body of two days old fifth instar
larvae. The GST titers were found to be 101.13, 80.19, 17.87,
10.77 and 87.36 µM mg protein-1min-1 in the midgut, fat
bodies, cuticle, haemolymph and whole body of the
laboratory reared fifth instar larvae, respectively. Different
tissues of fifth instar larvae collected from field showed
616.81, 648.93, 53.60, 42.15 and 579.16 µM mg protein 1
min-1 in the midgut, fat bodies, cuticle, haemolymph and
whole body, respectively. table – 1
The field collected P.demoleus showed high levels of GST. It
showed 6.09, 8.09, 2.99, 3.91 and 6.62 fold increase of GST
activity in midgut, fat bodies, cuticle, haemolymph and
whole body, respectively. Among these tissues, the highest
activity was found in the fat bodies (figure - 1). In the present
studies, GST activity was determined in the fat bodies, whole
body, midgut, haemolymph and cuticle of P. demoleus.
Among different tissues, the highest activity was found in the
fat bodies of P. demoleus. The field collected P. demoleus
showed high levels of GST than the laboratory reared P.
demoleus. It showed 8.09, 6.62, 6.09, 3.91 and 2.99 fold
increase of GST activity in fat bodies, whole body, midgut,
haemolymph and cuticle, respectively. In Lucilia cuprina
also elevated GST level were found in the larval fat body10.
The blood component probably included some enzyme
released from the fat body, gut and cuticle as they were cut
during dissection.

Table – 1
Levels of GST in different tissues of fifth instar larvae
GSH-CDNB CONJUGATION (µM mg protein-1min-1)
Name of the tissue
Laboratory reared

Field collected

Field collected/Lab. reared*

Midgut

101.13 ± 0.02

616.81 ± 0.05

6.09

Fat bodies

80.19 ± 0.05

648.93 ± 0.08

8.09

Cuticle

17.87 ± 0.02

53.60 ± 0.06

2.99

Haemolymph

10.77 ± 0.06

42.15 ± 0.04

3.91

Whole body

87.36 ± 0.12

579.16 ± 0.33

6.62

Field collected / Lab. reared: fold increase in enzyme activity

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activity-----GST

(μ M mg protein -1 min-1)

Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 313-316 (2012)
Res.J.Recent.Sci

700
600
500
400
300
200
100
0

MIDGUT

FAT BODIES

CUTICLE

HAEMOLYMPH

WHOLE BODY

---Tissues---

Laborato…
Figure – 1
GST activity in different tissues of
Fifth instar larvae of P. demoleus

Similarly, it was found that GST activity was highest in fat
bodies of H. armigera indicating the major site of
metabolism of organophosphate insecticide11. It is a common
feature that the fat body is the major site of metabolism of
many insecticides, especially the organophosphate class,
while the gut, cuticle and haemolymph also plays an
important role in the metabolism of xenobiotics. Low
enzyme activity in whole body extract is probably due to the
effect of endogenous inhibitors such as polyphenols and
quinines released during homogenization12.

Conclusion
Among different tissues, the highest activity was found in the
fat bodies of P. demoleus collected from the field, suggesting
that fat body is the major site of metabolism of insecticide
while the gut, cuticle and haemolymph also plays an
important role in the metabolism of insecticide.

Acknowledgement
I am very much grateful to Dr. P.S. Watane, Reader and
Head, Department of Zoology, Shri Shivaji college of Arts,
Commerce and Science, Akola and Dr. Mangesh Moharil,
Dr. PDKV, Akola for his valuable guidance and constant
encouragement throughout the progress of this work.

3.

Jovanovic Galovic A., Blagojevic D.P., Grubor Lajsic
G., Worland R. and Spasic M.B., Role of antioxidant
defense during different stages of preadult life cycle in
European corn borer (Ostrinia nubilalis, Hubn.)
diapause and metamorphosis, Archives of Insect
Biochem. and Physio., 55 (2), 79–89 (2004)

4.

Wu J.Q., Acaricide resistance as mediated by
detoxification and target site insensitivity, Resistant
Pest Management Newsletter, 13 (1), 65 – 70 (2003)

5.

Mohan M. and Gujar G.T., Local variation in
susceptibility of the diamondback moth Plutella
xylostella (L.) to insecticides and role of detoxification
enzymes, Crop Prot., 1, 10 (2003)

6.

Yu S.J., Biochemical characterization of microsomal
and cytosolic glutathione –S transferases in larvae of
the fall armyworm, Spodoptera frugiperda (Smith),
Pesti. Biochem. and Physio., 72, 100 – 110 (2002)

7.

Bradford M.M., A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein dye binding, Anal. Biochem.,
72, 248 (1976)

8.

Kranthi K.R., Armes N.J., Rao N.G.V., Raj S. and
Sunderamurthy V.T., Seasonal dynamics of metabolic
mechanism mediating pyrethroid resistance in H.
armigera in Central India, Pesticide Sci., 50, 91- 98
(1997)

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Kao C.H., Hung C.F. and Sun C.N., Parathion and
methyl parathion resistance in diamondback moth (Lep:
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Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
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Res.J.Recent.Sci
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