1.        
ESTERASE ISOZYME VARIABILITY OF DIFFERENT STAGES OF BED BUG (CIMEX HEMIPTERUS FAB), SCREENING ADULTS AGAINST PESTICIDES (MALATHION, CYPERMETHRIN, BT TOXIN) AND ITS EFFECTS ON ESTERASES
Department of Zoology University of Dhaka Dhaka-1000
Presented by Md. Abdur Rashid, Sadia Islam, Zannatul Ferdous, Rowshan Ara Begum and Reza Md. Shahjahan

2. INTRODUCTION
Bed bugs are blood feeding ectoparasites of humans, chickens, bats and occasionally domesticated animals (Usinger, 1966).
It thrives under crowed and squalid living conditions and often associated with residential halls, army barracks, labor and prison camps and similar situations where they may readily contact a variety of hosts (Metcalf et al., 1973).
Play three distinct life stages (I.e; eggs, nymphs and adults).
Commonly used insecticides for bed bug control include DDT, Chlordane, Lindane, Diazinon, Malathion, Cypermethrin etc. (Metcalf et al., 1973).
Treated organisms may result sudden death due to inhibition of acetylcholine esterase.
Present study deals with the genetic status of bedbug in terms of esterase isozymes variability in different age group, toxicity levels of adults against chemo & biopesticides and its impact on esterases.

3. INTRODUCTION
Figure 1: Life cycle of .Cimex hemipterus

4. OBJECTIVES OF THE STUDY
To detect the esterase isozyme pattern in various stage of life cycle.
To determine the toxicity levels of adults bed bugs.
To observe the impact of the test insecticides on the esterase isozyme of C. hemipterus .

5. MATERIALS AND METHODS Sample collection & preparation
Steps of PAGE
Sample collection & preparation
Total protein estimation (Lowery method)
Electrophoresis (Shahjahan et al. 2008)
Staining and photography (Begum et al. 2008)
Bands identification (Richardson et al.1986)
Bands analysis (Image J)
Steps of Bioassay
Sample collection and rearing Dose Preparation (Islam 2007) Exposing in chalk powder) Effect recording PAGE
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6. MATERIALS AND METHODS Infected bed Rearing Feeding Bioassay
Electrophoresis
Protein estimation

7. RESULTS AND DISCUSSION
Bioassay
Table-1: Mortality rate of adult bedbug C. hemipterus at different concentration of Cypermethrin 10 EC, Bt soil-47 and Bt 1i. N
Cumulative mortality (%)
Cypermethrin (Avg)
Bt soil-47 (Avg)
Bt 1i (Avg) D
2 hrs
4 hrs
6 hrs 10
Control
0.0098 20
100
19.53
7.8
0.0196 40 47
39.06
15.6
0.0391 57
78.13 13
31.25 23 27
0.0782 50 67
156.25 30
62.5 17 33
0.1563 72
321.5
125
0.3125 60 77
625
250 21 43
0.625 63 78
1250 42
500
1.25 65 80
2500
1000
2.5 70 90
5000 87
2000 88 97 5
N= Number of insect exposed
D= Concentration of doses (ppm)
Avg= Averages of 3 replica

8. RESULTS AND DISCUSSION
Bioassay
Table-2: Comparative LC50 and LC90 values (in ppm) at 95% confidence limits of the test insecticides against the adults of C. hemipterus
Exposure hours
Observed LC50 (ppm)
Calculated LC50 (ppm)
95% confidence limits for LC50
Observed LC90 (ppm)
Calculated LC90 (ppm)
Lower
Upper
Cypermethrin 2
0.0782
0.1223
0.0452
0.2623
<10
27.81 4
<0.0391
0.0383
0.0172
0.068
2.5
1.46
Bt soil-47
2500
3012
2419
4246
>5000
76729
625
1188
541
1989
26726 6
765
398
1207
12535
Bt 1i
1000
579
116
1044
>2000
13263
500
375
229
702
6218
>125
173
117
262
<2000
1905
Cypermethrin was found more active than other two BT toxins.

9. RESULTS AND DISCUSSION
Bioassay
Figure 2: Probit graphs at different mortality hours in response with various dose concentration of Cypermethrin, Bt soil-47 and Bt 1i against adult C. hemipterus.

10. RESULTS AND DISCUSSION
Esterase variability
Figure 1: Esterase banding pattern of different developmental stages of C. hemipterus on 7.5% PAGE stained with both α and β naphthyl acetates (Plate-A: Lane 1 eggs, 2-6 1st, 2nd, 3rd, 4th and 5th nymph stages, 7-8 adult male and female, 9 blood feed and 10 control), Untreated adults (Plate-B), Malathion treated adults (Plate C-D), Cypermethrin treated adults (Plate E-F), Bt soil 47 treated adults (Plate G-H) and Bt 1i treated adults (Plate I-J).

11. RESULTS AND DISCUSSION
Esterase variability
Altogether 3 bands (Est-10.99, Est and Est ) were observed with different relative mobility.
No esterase activity was found in eggs.
All the nymphal stages showed only one esterase band (Est-2) showing a gradual increase in intensity with aging.
But in adults (both male and female) two esterase bands (Est-2 & Est-3) were found. All 3 bands were observed only in blood sucked adults.
Adult bedbugs were treated with malathion to see the changes of esterase activity due to insecticidal action where instantly dead showed lesser esterase activity. Est-1 band was mostly absent, found in only few individuals and Est-2 showed less intensity than untreated organism.
In cypermethrin, the bands become gradually faint when an increased dose concentration.
But in BT treatments, the bands did not become faint in general.

12. SUMMARY AND CONCLUSION
Chemical pesticides are often used to control the harmful infestation of insects but some of these chemicals are non-degradable and thus magnified biologically in the food chain.
Use of biopesticide may be a potential solution when used safely.
Enzymes study associated with pesticide resistance help us to understand the development strategy of resistance and to endeavor control measures but it needs further investigation.

13. REFERENCES
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Islam, Bioassay of five organophosphates and two pyrethroids against the larvae of Cx. Quinquefasciatus Say (Diptera:Culicidae). Unpublished M Sc. Thesis, Dept. Zool, Dhaka University, Dhaka, Bangladseh. 22 pp.
Lowry, O. H., N. J. Rosebrough, A.L. Farr and R. J. Randall. 1951. Protein measurement with the Folin-Phenol reagents. J. Biol. Chem. 193:
Metcalf, C.L. and Flint, W.P Destructive and Useful Insects- their habits and control (4th edition). Tata Mc Graw-Hill Publishing Company Ltd. New Delhi, pp.
Richardson, B.J.; Baverstock, P.R. and Adams, M Allozyme Electrophoresis; A hand book for Animal Systematics and Population studies. Academic press Inc.
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