J Arthropod-Borne Dis, September 2016, 10(3): 405–414 Afshin Salehi et al.: Detection of Bendiocarb … 405 http://jad.tums.ac.ir Published Online: January 06, 2016 Original Article Detection of Bendiocarb and Carbaryl Resistance Mechanisms among German Cockroach Blattella germanica (Blattaria: Blattellidae) Collected from Tabriz Hospitals, East Azerbaijan Province, Iran in 2013 Afshin Salehi 1, Hassan Vatandoost 2, Teimour Hazratian 3, Alireza Sanei-Dehkordi 4,5, Hossein Hooshyar 6, Mohsen Arbabi 6, Yaser Salim-Abadi 7, Reza Sharafati-Chaleshtori 8, Mohammad Amin Gorouhi 9, *Azim Paksa 6 1College of Paramedics, Kashan University of Medical Sciences, Kashan, Iran 2Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Departmemt of Parasitology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 4Department of Medical Entomology and Vector Control, Faculty of Health, Hormozgan University of Medical Sciences, Bandar Abbas, Iran 5Social Determinants in Health Promotion Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran 6Department of Parasitology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran 7Rafsanjan University of Medical Sciences, Rafsanjan, Iran 8Research Center for biochemistry and nutrition in metabolic diseases, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran 9Faculty of Health, Kerman University of Medical Sciences, Kerman, Iran (Received 7 Jan 2014; accepted 24 June 2015) Abstract Background: Insecticide resistance is one of the serious problems for German cockroach control program. This study was conducted to determine the bendiocarb and Carbaryl resistance mechanisms in German cockroaches using the piperonyl butoxide (PBO). Methods: Bioassay tests were conducted with 4 to 6 different concentrations of both insecticides with four replicates of 10 susceptible strain cockroaches per concentration to determine of discriminative concentration. After determining discriminative concentration, the result was compared to wild strain. The levels of susceptibility and resistance ratio (RR) and synergism ratio (SR) were calculated for each five wild strains. Moreover resistance mechanisms in wild strains were determined using PBO synergist in vivo. Results: Hospital strains showed different levels of resistance to bendiocarb and carbaryl compared to susceptible strain. The bendiocarb and carbaryl resistance ratios ranged from 2.11 to 7.97 and 1.67 to 2 at LD50 levels, respec- tively. The synergist PBO significantly enhanced the toxicity of bendiocarb and carbaryl to all strains with different degrees of synergist ratio, 1.31, 1.39, 3.61, 1.78, 1.62 and 2.1 fold for bendiocarb, 1.19, 1.18, 1.12. 1.29, 1.45 and 1.11- fold for carbaryl, suggesting monooxygenase involvement in bendiocarb and carbaryl resistance. Conclusion: The synergetic effect of PBO had the highest effect on bendiocarb and resistance level was significantly reduced, which indicates the important role of monoxidase enzyme in creating resistance to Bendiocarb. Piperonyl butoxide did not have a significant synergistic effect on carbaryl and did not significantly break the resistance. Keywords: German cockroach, Resistance mechanism, Carbaryl, Bendiocarb Introduction German cockroach is one of the most im- portant pests in terms of hygiene with a global spread. This insect is allergenic and lives in residential places, restaurants, hotels, hospi- *Corresponding author: Dr Azim Paksa, E- mail: a.paksa@yahoo.com J Arthropod-Borne Dis, September 2016, 10(3): 405–414 Afshin Salehi et al.: Detection of Bendiocarb … 406 http://jad.tums.ac.ir Published Online: January 06, 2016 tals, food warehouses, and can transmit many pathogens such as bacteria, viruses, fungi, protozoa and parasite eggs to human through mechanical methods or their digestive tract. Hence, it is necessary to control this insect (Nasirian 2007, Behmanesh et al. 2010, Fakoo- rziba et al. 2010, Nejati et al. 2012). Development of resistance to insecticides in German cockroach due to excessive con- sumption and long-term insecticide spraying is one of the serious problems for controlling this pest (Fahiminia et al. 2010, Nasirian 2010). Many synergistic studies have been con- ducted in the world to study resistance mech- anism of German cockroach to phosphorous, carbamate and pyrethroid insecticides (Payne et al. 1984, Lee et al. 1996, Shinji Kasai et al. 1998, El-Merhibi et al. 2004, Chai and Lee 2010). Synergistic studies with piperonyl butox- ide (PBO) indicate the role of monoxidase system in creating resistance to these insecti- cides (Hemingway et al. 1993, Valles et al. 1994, Hodgson and Levi 1998, Tozzi 1998). Increasing resistance of German cockroach to organochlorine, organophosphate, carbamate, and pyrethroid insecticides has been reported (Cochran 1990, Ahad and Hollingworth 2004). The resistance mechanisms of German cock- roach to phosphorous, carbamate and pyre- throid insecticides have been determined using PBO and S, S, S-tributyl phosphorotri- thioate (DEF) synergists (Metcalf 1976, Rust 1987, Scott et al. 1990, Cochran 1990, Dong and Scott 1992, Lee et al. 1999). In Iran, German cockroach is resistant to Pyicom, diazinon, actylic, permethrin, cyper- methrin, deltamethrin, lambda-cyhalothrin and propoxur (Ladonni 1993, 1997). Resistance mechanisms to permethrin, DDT, chlorpyri- fos, malathion have been determined using PBO, DEF and DMC synergists (Ladonni and Sadegheyani 1998, Paksa et al. 2011, Limoee et al. 2011). Synergists are chemicals which lack pes- ticide effect but increase pesticide properties of their active ingredient. Piperonyl butoxide prevents activity of P-450 cytochrom. If re- sistance is due to increase of metabolism using MFO, one can overcome resistance- using PBO, which is MFO inhibitor. Syner- gists are widely used in insecticides contain- ing active ingredient of pyrethrin, pyre- throids and carbamates. Without PBO, en- zymes interfering in mechanism of the insect’s body particularly enzymes of P-450 cytochrom group can detoxify active ingre- dient of insecticide before affecting. Adding PBO to an insecticide reduces the dose of active ingredient required for production of the desired effect and increases the toxicity of insecticide (Hodgson and Levi 1998). Carbamate insecticides act in this way: when a nerve is stimulated, concentration of sodium and potassium ions changes inside and outside the nerve, which leads to neu- rotransmission. Neurotransmission from one nerve to another requires neurotransmitters such as acetylcholine, which acts in central nervous system (CNS) and in neuromuscular junction. Carbamate insecticides inhibit ace- tyl cholinesterase and in the absence of this enzyme, acetylcholine accumulates in synap- ses and neural transmission continues in synapses or neuromuscular junctions, which disrupts harmony and finally results in insect death (Talebi-Jahromi 2006). In Iran, no study has been conducted on resistance mechanisms of German cockroach to bendiocarb and carbaryl. Therefore, the present study was conducted to investigate and determine resistance mechanisms of Ger- man cockroach to these poisons in vivo in Tabriz City using PBO synergist. Conduct- ing this study and knowing resistance mech- anisms in this pest, we can manage defeating this pest, and the phenomenon of resistance in this pest compared with insecticide of carbamate group particularly bendiocarb and carbaryl. J Arthropod-Borne Dis, September 2016, 10(3): 405–414 Afshin Salehi et al.: Detection of Bendiocarb … 407 http://jad.tums.ac.ir Published Online: January 06, 2016 Materials and Methods In this experimental study, five wild strains of German cockroach were trapped or manu- ally collected from Behbood, Sina, Shafa, Taleghani and Noor Hospitals in Tabriz dur- ing summer 2013. Then, they were taken to laboratory and housed along with sensitive strains in special glass containers containing bread, starch, sugar and water under labora- tory conditions at 27±2 ˚C and humidity of 60±10% and lighting period of 12 h. Chemicals used in this study included formulation of technical insecticides such as bendiocarb 97% ai, (technical grade), car- baryl 98% ai, (technical grade), (Cyanamid Agro, India), piperonyl butoxide synergist (PBO) 94% ai, (Zeneca, Haslemere, UK), a monooxygenase inhibitor for synergism study. CO2 was used as anesthetic and acetone as solvent. Internal surface of the test contain- ers was measured in order to calculate the amount of insecticide needed (Schraft et al.1995). Standard dilutions of insecticide with ac- eton were poured at the bottom of the glass container. The solvent was evaporated through constant rotation of the container, so a uni- form layer of exterminator remained on the internal wall of the container. In this way, the desired concentration was used based on mg/m2. After specifying the concentrations for sensitive strains, the level and mecha- nisms of resistance for wild strains were determined using piperonyl butoxide in vivo. Synergistic tests were conducted in vivo using PBO synergist concurrently with ben- diocarb and carbaryl using Cochran contact method (Cochran 1990). These tests were performed in 4 replicates each with 10 male adult cockroaches. For each synergistic test, two controls were assigned to acetone and synergist. The data were analyzed using Probit software (Finny 1972). For this pur- pose, LD50 was calculated with confidence level of 95%. To calculate resistance ratio (RR), LD50 of resistant strain was divided by LD50 of sensitive strain and LD50 of insecti- cide was divided by LD50 (insecticide+ synergist) to calculate synergistic rate (SR). Results Resistance to Bendiocarb All five strains of German cockroach col- lected from hospitals showed different de- grees of resistance to bendiocarb with confi- dence level of 95% based on resistance ratio (RR) compared with the sensitive strain (Table 1). Lethal dose 50 of bendiocarb was calculated between 38.44 and 306.21 mg/m2 for sensitive strain and five collected strains with confidence level of 95% (Table 1). Resistance ratio of bendiocarb was obtained between 2.11 and 7.97 for five collected strains. The highest resistance ratio for ben- diocarb was found 7.97 in strain of Behbood Hospital (Table 1). Resistance to Carbaryl Different levels of resistance to carbaryl were observed in all strains of German cock- roach collected from hospitals (Table 2). LD50 of carbaryl was calculated between 280.87 and 560.81 for sensitive strain and five collected strains with confidence level of 95% (Table 2). RR of carbaryl was ob- tained between 1.67 and 2 for five collected strains. The highest resistance ratio (RR) for carbaryl was found 2 in strain of Behbood Hospital (Table 2). Synergistic effect of piperonyl butoxide on Bendiocarb As for the synergistic effect of PBO on Bendiocarb, LD50 for sensitive strain and five collected strains was obtained between 25.76 and 220.90 and the highest resistance ratio (RR) for bendiocarb was found to be 7.51 in strain of Behbood Hospital (Table 3). J Arthropod-Borne Dis, September 2016, 10(3): 405–414 Afshin Salehi et al.: Detection of Bendiocarb … 408 http://jad.tums.ac.ir Published Online: January 06, 2016 By studying the synergistic effect, synergism ratio of bendiocarb was calculated 1.31, 1.39, 3.61, 1.78, 1.62 and 2.12 fold in sensi- tive strain and strains from Behbood, Sina, Shafa, Taleghani and Noor Hospitals, re- spectively and the highest synergism ratio of carbaryl (SR) was calculated 3.61 in Sina strain (Table 3). Synergistic effect of piperonyl butoxide on carbaryl Lethal dose 50 of carbaryl+PBO for all col- lected strains was obtained between 235.77 and 480.64 and the highest (RR) for carbaryl was found to be 2.04 in Noor Hospital. As for synergistic effect, synergism ratio (SR) of carbaryl was calculated 1.19, 1.18, 1.12, 1.29, 1.45 and 1.11 fold in the sensitive strain and strains from Behbood, Sina, Shafa , Taleghani and Noor Hospitals, respectively and the highest synergism ratio (SR) of car- baryl (SR) was calculated 1.45 in Taleghani strain (Table 4). Table 1. Lethal dose values for bendiocarb on susceptible and five hospital-collected strains of German cockroach Strain N Y-intercept Slope(SE) X 2(df) LD50 , 95% C.L RR Susceptible 200 -9.17 5.78±0.778 5.89 (3) 38.44 1 Behbood-H 200 -8.79 3.54±0.501 2.94 (2) 306.21 7.97 Sina-H 200 -3.93 1.1±0.265 5.71 (3) 92.96 2.42 Shafa-H 200 -4.92 2.58±0.305 3.03 (3) 80.92 2.11 Talaghani-H 200 -4.86 2.54±0.301 4.9 (3) 81.32 2.12 Nour-H 200 -6.10 2.72±0.434 4.37 (2) 175.61 4.57 RR: Resistance Ratio H: Hospital N: Number Table 2. Lethal dose values for Carbaryl on susceptible and five hospital-collected strains of German cockroach Strain N Y-intercept Slope(SE) X 2(df) LD50 , 95% C.L RR Susceptible 200 -11.33 4.63±2.015 20.06 (3) 280.87 1 Behbood-H 200 -45.84 16.68±2.780 4.05 (2) 560.81 2 Sina-H 200 -69.88 25.86±11.740 18.28 (2) 504.06 1.8 Shafa-H 200 -41.90 15.68±2.592 2.10 (2) 469.1 1.67 Talaghani-H 200 -48.0365 17.71±2.145 1.680 (3) 514.96 1.83 Nour-H 200 -93.09 34.14±10.015 9.45 (2) 533.47 1.9 RR: Resistance Ratio H: Hospital N: Number J Arthropod-Borne Dis, September 2016, 10(3): 405–414 Afshin Salehi et al.: Detection of Bendiocarb … 409 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 3. Toxicity bendiocarb with and without piperonyl butoxide to five bendiocarb resistant strains of the German cockroach compared to the susceptible strain Insecticide alone Insecticide + PBO Strain N LD50 , 95% C.L RR N Y-intercept Slope(SE) X2(df) LD50 , 95% C.L SR RR Susceptible 200 38.44 1 200 -8.37 5.70±0.753 2.50 (2) 29.42 1.31 1 Behbood-H 200 306.21 7.97 200 -9.33 3.98±0.990 7.57 (2) 220.90 1.39 7.51 Sina-H 200 92.96 2.42 200 -4.13 2.92±0.444 2.61 (2) 25.76 3.61 0.88 Shafa-H 200 80.92 2.11 200 -8.69 5.24±0.708 1.36 (2) 45.46 1.78 1.55 Talaghani-H 200 81.32 2.12 200 -6.62 3.89±0.493 5.08 (2) 50.11 1.62 2.81 Nour-H 200 175.61 4.57 200 -9.69 5.05±1.917 13.68(2) 82.65 2.12 2.12 H: Hospital N: Number Resistance Ratio: LD50 of resistant strain/ LD50 of susceptible strain Synergist Ratio: LD50 of insecticide alone/ LD50 of insecticide + PBO Table 4. Toxicity Carbaryl with and without piperonyl butoxide to five Carbaryl resistant strains of the German cockroach compared to the susceptible strain (SUS) Insecticide alone Insecticide + PBO Strain N LD50 , 95% C.L RR N Y-inter- cept Slope(SE) X2(df) LD50 , 95% C.L SR RR Susceptible 200 280.87 1 200 -12.42 5.23±2.404 32.03 (3) 235.77 1.19 1 Behbood-H 200 560.81 2 200 -65.17 24.36±3.526 3.78 (2) 472.89 1.18 2.01 Sina-H 200 504.06 1.8 200 -46.90 17.69±4.519 6.34 (2) 448.22 1.12 1.9 Shafa-H 200 469.1 1.67 200 -70.68 27.61±7.532 7.41 (2) 362.98 1.29 1.53 Talaghani-H 200 514.96 1.83 200 -49.51 19.42±2.535 2.15 (2) 354.42 1.45 1.5 Nour-H 200 533.47 1.9 200 -67.09 25.02±7.164 9.34 (2) 480.64 1.11 2.04 H: Hospital N: Number Resistance Ratio: LD50 of resistant strain/ LD50 of susceptible strain Synergist Ratio: LD50 of insecticide alone/ LD50 of insecticide+PBO J Arthropod-Borne Dis, September 2016, 10(3): 405–414 Afshin Salehi et al.: Detection of Bendiocarb … 410 http://jad.tums.ac.ir Published Online: January 06, 2016 Discussion The results of PBO synergistic tests on ben- diocarb insecticide showed that resistance level was reduced in all tested strains. Syner- gism rate (SR) for bendiocarb on suscepti- ble, Behbood, Sina, Shafa, Taleghani and Noor Hospitals of strain was 1.31, 1.39, 3.61, 1.78, 1.62, 2.12 fold, respectively. Based on the obtained results in strain of Sina Hospital and Noor Hospital, the syner- getic effect of PBO on bendiocarb insecti- cide showed the highest effect and resistance level was significantly reduced, which indi- cates the important role of monoxidase en- zyme (MFO) in creating resistance to bendi- ocarb exterminator. In strain of Behbood, Taleghani and Shafa Hospitals, the synergis- tic effect of PBO was found to be low to medium. These results indicated that monoxidase was not among the main factors of resistance but it was a cofactor of resistance compared with Bendiocarb. PBO did not have a signifi- cant synergistic effect on carbaryl insecticide and did not significantly break the resistance. This result shows that monoxidase en- zyme does not have a main role in creating resistance to carbaryl in this strain. A major part of resistance was created probably due to mechanisms such as reduced Cuticle pen- etration, esterase and other factors created due to fighting with German cockroach. Syn- ergism rate (SR) for carbaryl no susceptible, Behbood, Sina, Shafa, Taleghani and Noor Hospitals of strain were 1.19, 1.18, 1.12, 1.29, 1.45, 1.11 fold respectively. In strains of Shafa and Taleghani hospitals, the syn- ergistic effect of PBO on carbaryl, these two are different. These results are consistent with our previous studies on resistance mech- anisms among German cockroach strains in vivo in Tehran (Limoee et al. 2006). Besides, the results of the present study are in line with those of another study on resistance mechanisms in some populations of the German cockroaches from kashan (Paksa et al. 2014). According to Reierson, 10-fold resistance measured by topical application is the criti- cal point above which operational control failures are likely to occur while; resistance ratio at 5x and below may still achieve a good control of the German cockroach pop- ulation. Thus, the low resistance ratios of the three strains to chlorpyrifos compared with the susceptible strain probably reflect a rela- tively low levels of resistance to this com- pound because these strains have not been selected by chlorpyrifos for several genera- tions. Hence, bendiocarb and specially chlorpyr- ifos may still provide adequate control of these strains (Reierson et al. 1998).The find- ings show involvement of monooxygenase in cyfluthrin resistance. In 1987, first Rust recognized resistance to diazinon and bendiocarb insecticides in different strains of German cockroach (Rust 1987). A study was conducted by Hemingway in 1993 to investigate resistance of German cock- roach to chlorpyrifos and propoxur compared with sensitive strain. Thirteen out of 14 strains showed resistance to chlorpyrifos and 12 strains showed resistance to propoxur. Syn- ergistic studies with PBO also showed role of monoxidase system in creating resistance to these insecticides (Hemingway et al. 1993). In this study, an increase in activity of ester- ase was observed in strains resistant to a broad spectrum of organophosphates. Lee et al. collected 12 strains of German cockroach from Malaysia in 1996 to provide direct evidence for mechanisms involved in resistance of German cockroach to propoxur, chlorpyrifos, cypermethrin and permethrin and performed synergistic tests after performing sensitivity tests and specifying their resistance ratio. In these studies, PBO and DEF syner- gists reduced resistance to propoxur, which J Arthropod-Borne Dis, September 2016, 10(3): 405–414 Afshin Salehi et al.: Detection of Bendiocarb … 411 http://jad.tums.ac.ir Published Online: January 06, 2016 indicated involvement of monooxygenase resistance (Lee et al. 1996). However, these synergists did not affect resistance to cy- permethrin and permethrin. Scott et al. conducted a study in 1990 to determine sensitivity of 4 strains of German cockroach and the effect of synergists on increasing toxicity of pyrethrin, propoxur, mal- athion, deltamethrin, chlorpyrifos and ben- diocarb insecticides using topical application method and showed that bendiocarb insec- ticides along with PBO and DEF synergists were applied in Kenly strain (resistant to ben- diocarb and permethrin) and Rugers strain (resistant to permethrin) and as a result of this test, resistance was reduced indicating the presence of hydrolytic and oxidase me- tabolism in creating resistance in the above strains (Scott et al. 1990). Lee et al. collected 23 strains of German cockroach in 1999 and calculated their sen- sitivity to propoxur, baygon, bendiocarb and chlorpyrifos. Resistance in pesticides of car- bamate group was incompletely removed by PBO and DEF synergists and probably, re- sistance was controlled by monooxygenase and esterase. Resistance to phosphorous in- secticides is neutralized by DEF (Lee et al. 1999). Generally, PBO reduces resistance level of but it does not remove resistance com- pletely in the present study like the studies conducted in the world, which indicated en- zymes of cytochrom P540 and monooxygen- ase as the factor helping create resistance to bendiocarb and carbaryl in the above strains. Monooxygenase P540 interferes in resistance of B. germanica to carbamate insecticides and confirms the present study which is conducted for the first time in Iran (Rust 1987, Scott et al.1990, Hemingway et al. 1993, Lee et al. 1996, Lee et al. 1999). To succeed in controlling German cock- roach in management of resistance to exter- minators, it seems vital to recognize resistance in the early stages and the mechanism of pest resistance using bioassay tests. Such tests measure the sensitivity of insects. Therefore, knowing of resistance in order to control pests desirably is based on measurement of pest resistance before its occurring or de- velopment in conventional insecticides or new compounds. Considering the obtained results of this study and receiving information about the re- sistance mechanisms, it is recommended not to use bendiocarb and carbaryl exterminators or to use them periodically and in places which have not been sprayed with them, or use new insecticides such as spinosad or poisonous baits with different mechanism of action from those of carbamate group such as poisonous baits of imidacloprid and fipronil (Nasirian 2007, Nasirian et al. 2011). Furthermore, it will not be possible to man- age resistance to insecticides unless re- sistance mechanisms neutralizing the effect of insecticide inside body of German cock- roach are studied comprehensively. Studies that are more comprehensive are recommend- ed in this field to investigate other mecha- nisms such as reduced penetration of cuticle and insensitivity to acetylcholinesterase. Conclusion All five collected strains showed different levels of resistance compared with sensitive strain. As a result, RR of all strains was sig- nificant compared with sensitive strain. Pip- eronyl butoxide significantly increased tox- icity of bendiocarb and carbaryl almost in all strains at different degrees, but resistance was not completely removed by PBO. These results, reduction of resistance, show involve- ment of monoxidase enzyme in creating re- sistance to these insecticides. Considering that resistance was not removed by PBO synergist, other mechanisms such as reduced Cuticle penetration and insensitivity to ac- etylcholinesterase may be involved. 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