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

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.

INTRODUCTION Figure 1: Life cycle of .Cimex hemipterus

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 .

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 ccc

MATERIALS AND METHODS Infected bed Rearing Feeding Bioassay Electrophoresis Protein estimation

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

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.

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.

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).

RESULTS AND DISCUSSION Esterase variability Altogether 3 bands (Est-10.99, Est-20.45 and Est-30.18 ) 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.

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.

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