INTRODUCTION
House flies (Musca domestica) are synanthropic and cosmopolitan in distribution. House flies lay eggs in moist organic matter so they are abundant in livestock, poultry ranches, dairy cattle sheds, horse corrals and pig ranches. House flies transmit various disease causing agents because they feed on decomposing matter, waste of humans and food (Deguenon et al., 2019; Malik et al., 2007; Suwannayod et al., 2019).
House flies secrete digestive juices, enzymes and saliva on food and then suck liquefy food with their proboscis. They can suck up food from any source of germs and a part of these germs stick to fly’s mouth and body parts and when these flies land on the human food they transfer the disease causing agents. More than 65 human and animal intestinal diseases are transmitted by house flies. House flies are also a vector of protozoan, bacterial, viral, helminthic infections and rickettsial infections (Attaullah et al., 2019; Malik et al., 2007).
House flies are mainly controlled by the use of chemical insecticides viz pyrethroids, carbamates, organophosphates, organochlorines neonicotinoids and others (Deguenon et al., 2019). These insecticides have different modes of actions, pyrethroids function by halting the closure of voltage gates of sodium channels present in membrane of axon (Scott et al., 2013); organophosphate obstruct the activity of cholinesterases and acetylcholinesterases, interrupt nerve impluses and result in killing or disabling the insect. House fly has successfully adapted to most insecticides (Ghosal, 2018).
From the estimated 10000 arthropod pests, 553 species are reported to have insecticide resistance. Pesticide resistance is the major issue in the control of agricultural and public health pest (Sharififard & Safdari, 2014). The house fly is one of 20 species that have shown the highest resistance to insecticides, and it is placed in the fifth row. This insect has become resistant to 44 different chemical insecticides, and its resistance is found to be due to a specific gene expression in the adult and larvae (Whalon et al., 2008).
Monitoring of the house fly population`s susceptibility to insecticides is required for the effective use of insecticides. Early detection of an insect pest’s resistance to chemical insecticides and selecting a more effective strategy to control them can efficiently reduce operational, financial, and social losses. (Sharififard & Safdari, 2014).
Increase frequency and application rate is a major cause of resistance to pesticides and consequently, effectiveness of insecticides is decreased. Farmers apply insecticides to control cotton, rice and wheat crops from pests. In Pakistan, farmers do not use insecticides according to their specificity to control various pests. These trends could be the reason of development of resistance in insect pests opened to insecticides ( Khan et al., 2013a,b; Abbas et al., 2015 ).
No data was available on resistance status of house flies against different insecticides in the area of Bhalwal. This study was done to assess resistance status of house flies against bifenthrin (pyrethroid) and dimethoate (organophosphate). Enzymes concentrations are also monitored in this study because enzymes are directly related to resistance of house flies.
The objectives of the present study were
- To collect houseflies from two different locations of Bhalwal and check their resistance status against bifenthrin and dimethoate.
- To collect laboratory strain from an area of low chemical use and then reared them in laboratory without exposure to insecticides and their resistance status was check against bifenthrin and dimethoate.
- To compare the level of detoxifying enzymes in field strain resistant house flies and laboratory strain resistant flies.
REVIEW OF LITERATURE
Kaufman et al. (2001) checked the resistance status of house flies against seven insecticides (dimethoate, tetrachlorvinphos, permethrin, cyfluthrin, methomyl, pyrethrins, fipronil) and these house flies were captured from different dairies of New York. Higher resistance was shown by house flies against tetrachlorvinphos, permethrin and cyfluthrin but against methomyl level of resistance was bit lesser. House flies showed less than 10 % survival rate against fipronil so flies were relatively susceptible to fipronil. House flies showed resistance against pyrethrins on lowest diagnostic concentrations. This study further showed that level of resistance found for insecticides at New York dairies in 1987 relatively increased for permethrin, decreased for dimethote.and remained same for tetrachlorvinphos.
Kaufman and Rutz (2001) studied the resistance level of house flies collected from dairies of New York against three commercial insecticides (Dimethoate, Permethrin and Cyfluthrin). This resistance was studied on painted and unpainted plywood panels. Dimethoate showed same results on the painted and unpainted ply wood panels. Permethrin EC (emulsified concentrate) killed more houses flies on gloss latex and unpainted panels as compared to flat latex panels. But permethrin WP (wetable powder) killed more house flies on flat latex and unpainted panels as compared to gloss latex panels. The cyfluthrin in both formulations had greater efficacy as compared to the dimethoate and permethrin. The cyfluthrin SC (suspension concentration) killed more house flies on flat latex whereas cyfluthrin WP kill more flies on flat latex and unpainted surface.
Marcon et al. (2003) checked the efficacy of three different insecticides (permethrin, stirofos and methoxychlor) against house flies collected from Southeastern Nebraska. Results showed that houseflies had moderate resistance against permethrin but showed very high resistance against stirofos and methoxychlor. The study further showed that freely dispersed house fly populations had greater level of resistance as compared to the stable flies populations and co-habitation of resistant and susceptible house fly populations increased the chance of dispersal of house flies with high level of resistance.
Akiner and Cagar (2005) collected houseflies from cow farms in Antalya and Izmir and garbage dumps in Adana, Ankara, Istanbul, and Sanlıurfa in Turkey during the months of April and September assessed their resistance against six insecticides (cypermethrin, cyphenothrin, deltamethrin, permethrin, resmethrin, fenitrothion). House flies resistance level ranged from 23.27 (fall strain) to 633.09 (spring strain) against pyrethroid group of insecticides and resistance level ranged from 5.78 (fall strain) to 51.04 (spring strain) against fenitrothion insecticide. House flies had lower level of resistance against fenitrothion than pyrethroids but this level was still high for effective control of house flies.
CAO et al. (2006) collected the house flies from 17 urban trash heaps from the cities of Beijing, Tianjin and Zhangjikou to checked their resistance against deltamethrin. Results showed that houseflies of Beijing city were more resistant as compared to house flies of Tianjin and Zhangjiakou. Houseflies of Beijing city were more resistant because of higher frequency of kdr allele.
Deacutis et al. (2006) collected house flies from United States in 2005 and evaluated their susceptibility before and after one season use of Spinosad. Spinosad was a new and highly promising insecticide and it had very unique mechanism of action against house flies. Results showed that there was no evidence of Spinosad resistance evolving after one season use. This study also indicated that selection of field house flies for eight generations produced highly resistant strain but one season use did not show significant differences in survival rate of houseflies.
Zhang et al. (2007) collected house flies from heaps of Beijing against beta cypermethrin. These house flies were treated with beta cypermethrin for 25 consecutive generations to produce a resistant strain (CRR). Resistance level increased with each generation in CRR (cyclic resistance ratio) strain as compare to a laboratory susceptible strain. This study further showed that CRR strain had high level of carboxylesterase activity as compared to CSS strain (consmic strain). Carboxylesterase was the main cause of high level of resistance in CRR strain as level of glutathione S – transferase activity and level of cytochrome P450 was almost same in both CRR and CSS strains.
White et al. (2007) stuided the efficacy of three insecticides, namely Spinosad, imidacloprid and methomyl against houseflies. Spinosad showed most effective results, it was 2.7 times more effective than methomyl and eight times more effective than imidacloprid. This study also revealed that high temperature slowed down the activity of Spinosad, imidacloprid and methomyl in the period of 24 hours. It was observed that house flies exposed to imidacloprid were swiftly demolished but they remained alive which showed that house flies recovered from initial exposure to insecticides.
Kristensen and Jespersen (2008) checked the efficacy of thiamethoxam in comparison to azamethiphos, methomyl, spinosad and imidacloprid in house flies collected from Danish livestock farms. Results showed that thiamethoxan was respectively 19-fold, 11-fold and threefold more toxic to house flies as compared to azamethiphoa, methomyl and Spinosad. Imidacloprid was 19 times less toxicity as compared to thiamethoxam. This study further showed that a cross resistance was present between thiamethoxam and imidacloprid, because these both insecticides had common mechanism of action and house flies developed common mechanism of resistance against them.
Cakir et al. (2008) studied the effect of piperonyl butoxide and tetramethrin on the efficacy of three pyrethroid namely cypermethrin, deltamethrin and permethrin against different populations of house flies collected from different cities of Turkey (Ankara, Istanbul, Izmir, and Adana). Results showed that death efficacy of all insecticides increased with addition of PBO. Synergistic effect of PBO was effective for all populations of house flies and for all insecticides, but it was more prominent in deltamethrin.
Acevedo et al. (2009) collected three house flies populations from Argentinean poultry farms and checked their resistance against three insecticides which included one larvicide (cyromazine) and two adulticides (dichlorovinyl dimethyl phosphate (DDVP) and permethrin). Results showed that efficacy of all three insecticides was very low and no significant differences were found among the studied populations because insecticides were widely overused in Argentina. This overuse of insecticides was the main cause for development of resistant populations. This study further suggested that recommendations of insecticide monitoring programs of Argentina about poultry farms should be executed to get better results.
Kaufman et al. (2009) collected house flies from Florida dairies and assessed their resistance against Nicotinoid and Pyrethroid insecticides. Four insecticides were evaluated namely beta – cyfluthrin, permethrin, imidacloprid and nithiazine. Resistance ratios were calculated at LC90 level. Three field strains for beta-cyfluthrin had greater than tenfold of resistance, two field strains for permethrin and one field strain for imidacloprid had greater than 20-fold resistance and in one field strain had four fold resistance against nithiazine. This study also showed that to retain efficacy of these insecticides their used must be restricted.
Bong et al. (2010) collected the houses flies from Malaysia in April 2007 to April 2008 and checked the fluctuations of insecticides resistance with time against DDT, malathion, propoxur and permethrin. The results showed that resistance level in house flies for propoxur, malathion and DDT changed broadly through the 13 months. Resistance level for permethrin also changed but this change was in limited range. Among these four insecticides, permethrin was most effective insecticide. Resistance ratio (RR) of house flies for propoxur, malathion, DDT and permethrin ranged from 10.28 to 99.00, 7.83 to 47.01, 6.05 to 31.10 and 0.50 to 1.96 respectively. Humidity also played most important role in changing resistance, because with high humidity and warm temperature breeding rate was increased, more generations of house flies were produced, that’s why resistance increased from generation to generation.
Pezzi et al. (2011) collected house flies strain OcRo from intensive chicken farm in Northern Italy and checked their resistance against four insecticides in comparison to a susceptible strain ( s-DBF). Results showed that resistance level for Spinosad and imidacloprid was low and very effective for the control of OcRo strain. Enzyme analysis of OcRo strain showed that hydrolases were involved in phosphorganic detoxification and cytochromes p 450 monoxygenases were involved in resistance against pyrethroid. Enzyme analyses also showed that OcRo was multi resistant to pyrethroids and organophosphates, so OcRo was very important for an environmentally safe pest management.
Akıner and Caglar (2012) collected houses flies during 2004- 2006 from cow farm and trash heaps from different cities of Turkey (Antalya, Izmir Adana, Ankara, Istanbul, Şanliurfa) and checked their resistance against five insecticides namely cyphenothrin, cypermethrin, permethrin, deltamethrin and fenitrothion. Result showed that Antalya 2005 strain had highest resistance against pyrethroids and Sanliurfa 2004 strain had lowest resistance. Sanliurfa 2004 strain had highest resistance against fenltrothion insecticide and Adana 2004 strain had lowest fenitrothion resistance. Flies collected from cow farms were more resistant in comparison to the trash heaps.
Scott et al. (2013) checked the resistance status in house flies against six different insecticides (permethrin, methomyl, cylluthrin, pyrethrin, imidacloprid and piperonyl butoxide), house flies were collected from dairies in nine different states of USA. Results showed that house flies had highest and consistent resistance against permethrin across all the states. Resistance against methomyl was consistent but resistance against cylluthrin and pyrethrins mixed with piperonyl butoxide varied considerably. House flies showed modest resistance against imidacloprid and showed no signs of increasing resistance.
Khan et al. (2013) collected house flies from dairies and checked their resistance against pyrethroids separately and in mixtures. When pyrethroid insecticides were used in mixture their toxicity increased significantly, particularly in cases when synergistic interaction occured between insecticides. Toxicity of bifenthrin, deltamethrin, cypermethrin and emamectin increased notably when used in combination with PBO and DEF enzymes inhibitors. These findings were very useful for the management of resistance in house flies.
Peck et al. (2013) studied the efficacy of pyrethroid treated netting exposed to sunlight for control of housefly. The results showed that β- cyfluthrin treated nettings were stilled effective against house flies and gave 100% mortality after 12 weeks of direct sunlight exposure, whereas the efficacy of λ- ghalothrin treated netting decreased and 50% house flies survived after sunlight exposure for 10 weeks. The efficacy of Bifenthrin treated netting reduced greatly 50% house flies survived after 3 weeks sunlight exposure.
Sharififard and Safdari (2014) collected the house flies from three livestock farms near the city of Ahvaz to checked their resistance against pyrethroid insecticides. Results showed that pyrethroid resistance was present in all three populations. House flies showed highest resistance against cypermethrin. This study further showed that use of insecticides those mode of action was same may developed cross resistance in target pest. But by the used of insecticides those mode of action was different, we can destructed resistant populations of house flies.
Khan et al. (2014) studied the change in toxicity of insecticides with temperature and implicated it for effective management of diarrhea. Study results indicated that with increases in temperature toxicities of chloropyrifos, profenofos, emamectin and fipronil also increased showing positive temperature co- efficient. But the toxicities of cypermethrin, deltamethrin and Spinosad decreased with increased in temperature showing negative temperature co-efficient. So, for the effective control of houses flies for reducing diarrheal cases insecticides were chosen according to the existing environment temperature. To control diarrhea in hot season insecticides with positive temperature co- efficient should be used and vice versa.
Abbas et al. (2015) collected the house flies from poultry facilities of Khanewal in three different seasons July, November and March and studied the fluctuations in house flies resistance in different seasons against conventional organophosphate, pyrethroids and against novel chemistry spinosyn, oxadiazine and neonicotinoid insecticides. Results showed that highest level of resistance was present against organophosphates as compared to other tested insecticides. Resistance to organophosphate and pyrethroid decreased considerably in March as compare to July and November. But resistance against oxadiazine and avermectins decreased considerably in November. Resistance in Spinosad and imidacloprid remained stable throughout the seasons. This study showed that for effective control of flies seasonal changes must be considered.
Ong et al. (2016) studied the patterns of degradation of insecticides. These degradation patterns were very important for the control programs of house flies on poultry farms. Insecticides were spiked on the poultry manure and then insecticide residues analyzed quantitatively. Results showed that cyromazine degraded rapidly its half-life was 3.01 days. Half-life of chloropyrifos was 4.36 days and half- life of cyermethrin was 3.74 days. This study showed that we should pay attention what effects were caused by insecticide metabolites on houses and we should pay attention on effects of metabolites insecticide on house flies and environment of poultry manure.
Kustiali et al. (2016) collected the house flies from 26 capital of provinces of Indonesia in 2013 and 2014 and assessed their resistance against permethrin and imidacloprid. Results showed that majority of strains of flies showed very high levels of resistance against permethrin as compare to susceptible strains. But few strains showed no to high level of resistance against permethrin. Resistance for imidacloprid was no to very low in all strains. Results also showed that when PBO was used with permethrin LD value decreased considerably.
Khan et al. (2017) evaluated the resistance status of four pyrethroid insecticides namely beta-cyfluthrin, deltamethrin, permethrin, transfluthrin in house flies collected from urban areas of Punjab . Results showed that house flies collected from city areas of Punjab had resistance against pyrethroid insecticides, lethal dose (LD50) level were in the range of 5.25- to 11.02-fold for betacyfluthrin, 7.22- to 19.31-fold for deltamethrin, 5.36- to 16.04-fold for permethrin and 9.05- to 35.50-fold for transfluthrin. This study also revealed that to maintain the efficacy of insecticides for longer period of time regular resistance monitoring was necessary.
Asid et al. (2017) studied the synergistic effect of 3 pyrethroid and 3 organophosphorus insecticides against field strain of the females and 2nd larval instars of the houseflies. Results showed that mixture of chemicals had better insecticidal effect against the housefly as compare to sole use of insecticides. So, we can used mixture of insecticides for the control of houseflies.
Levchenko et al. (2017) checked the resistance status of houseflies against six insecticides (deltamethrin, cypermethrin, thiamethoxam, permethrin, fipronil and chlorfenapyr) collected from livestock farms of the Tyumen region of Russia. Results showed that house flies resistance increased in following order: fipronil, chlorfenapyr, permethrin, deltamethrin, cypermethrin and thiamethoxam.
Cetin et al. (2019) collected four Turkish house flies populations from Ankara, Antalya, Gaziantep and Sanliurfa and evaluated their resistance against synthetic pyrethroid combined with different concentration of piperonyl butoxide (PBO). Results revealed that addition of PBO to the synthetic pyrethroid insecticides increased their biological efficiency and lower the KDT (Knockdown time) values. Optimum concentration of PBO in pesticide formulations should be determined for best results, with the help of efficacy tests according to region and pest species.
Wang et al. (2019) collected house flies from 12 districts of Zhejiang Province in 2011, 2014 and 2019 and studied their resistance status against permethrin, deltamethrin, beta-cypermethrin, propoxur and dichlorovos. Results showed that resistance was present in propoxur, beta-cypermethrin, deltamethrin and permethrin. But level resistance in house flies against dichlorvos was found to be decreasing. This study further showed that constant use of certain insecticides would produce resistance in target pest, so use of insecticides with no cross resistance was an effective approach in control of house flies.
MATERIALS AND METHODS
Insects
Adult house flies were collected by using sweep-netting from two different locations of Bhalwal, Punjab, Pakistan.The first locality was Chak No. 07 SB Bhalwal (32°14’52.3″N 72°55’25.5″E) while second locality was Chak No. 02 NB Bhalwal (32°19’20.1″N 72°54’56.5″E). After collection house flies were brought to laboratory for colonization and were kept separately. The adult flies were enclosed in mesh cages with volume of 40 × 30 × 30 cm3. Adult flies were fed on mixture of powdered milk and icing sugar in 1: 1 ratio and water. The larvae were reared on a medium caster sugar, powdered milk, yeast and water 0.3:0.3:1:4 by weight respectively as described by Bell et al. (2010). The insects were kept in maintained conditions such as at 60 ± 5 % RH, 25 ± 2 ℃ and 12:12 light : dark photoperiod. F1 generation of field collected flies were used for bioassays. The lab strain or laboratory susceptible strain (lab) was collected from such area in which use of insecticides is low and kept in laboratory without exposure of any kind of insecticide. Lab strain was not completely susceptible but values of LC50 of this particular strain were much less and that’s why used as a baseline for resistance determination in future (Ahmed and Arif 2009).
Insecticides and bioassays
Two commercial grade insecticides, Bifenthrin (Hornet 20.5 % SC) and Dimethoate (Hi Grade, 40% EC) were used for residual bioassay. A residual bioassay was performed and filter paper size was set according to WHO recommendations (12×14 cm). Insecticide treated and control group were formed and each susceptibility tube contains 15 flies. Sugar, condensed milk and water paste were provided to both groups till satiation. In the treated groups flies were given exposure to insecticides impregnated filter paper while in the control groups flies were given exposure to distilled water impregnated filter paper. Flies were given exposure for one hour after this they were transferred to clean bioassay susceptibility tubes for 24 hours observation. Mortality data was recorded with four hour interval till 24 hours. The flies that survived after 24 hours of insecticide exposure were considered resistant. According to Kaufman et al. (2010) water containing 10 % sugar was provided to flies during 24 hours observation periods. Three replicated experiments were performed for both insecticides.
Solutions
Four concentrations i-e: field rate, intermediate field rate, half-field and half of half field rate were prepared for each insecticide. For Bifenthrin, field rate (1.4 ml/2ml), intermediate field rate (0.9 ml/2ml), half-field (0.4 ml/2ml) and half of half field rate (0.2 ml/2ml) were prepared. For Dimethoate, field rate (8.4 ml/2ml), intermediate field rate (4.53 ml/2ml), half-field (0.67 ml/2ml) and half of half field rate (0.28 ml/2ml) were prepared. The control group was treated with only water. Concentrations for Bifenthrin and Dimethoate were prepared according to WHO recommendations (WHO, 2006). The solutions were prepared in 10ml of distilled water.
Data analysis
The data of three replicates was analyzed by probit analysis by the use of SPSS software, (Version 10.0 for windows, SPSS Inc., Chicago, USA) to determine median lethal concentrations (LC50) and data can be corrected by the use of Abbott’s formula (Abbott, 1925). LC50 values were calculated and after this these values were compared with values of Lab susceptible strain and in result resistance ratios were obtained. These resistance ratios were scaled dependent on following criterion: resistance ratio (RR) = 1, showed no resistance; RR = 2–10, very low resistance; RR = 11–20, low resistance; RR = 21–50, moderate resistance; RR = 51–100, high resistance; and RR =[100, very high resistance (Saleem et al. 2008; Ahmed and Arif 2009).
Chemicals for estimation of detoxifying enzymes
Following chemicals were used for this purpose
- Ethanol
- Sodium phosphate buffer
- Beta naphthyl acetate (Substrate B)
- Methanol
- Alpha naphthyl acetate (Substrate A)
- Sodium dodecyl Sulphate (SDS)
- Fast blue B (FBB) salt
- (GSH) Reduced glutathione
- (TMBZ) 3,3,5’,5’-Tetramethyl benzidine
- (CDNB) 1-chloro-2,4-dinitrobenezene
- Phosphoric acid (85%)
- (Coomassie brilliant blue G-250) Bradford dye reagent
- Beta naphthol
- Potassium phosphate buffer
- Alpha naphthol
- Bovine serum albumin (BSA)
- H2O2 (3%)
- Cytochrome c
- Sodium acetate buffer.
Enzyme preparation
Protocol reported by Naseem et al. (2013) was followed for enzyme preparation. Maggots were kept freezed at -20˚ⅽ. Each maggot was separately placed in eppendorf containing 600 µl Sodium phosphate buffer and was homogenized. The homogenated solution was then centrifuged at 15682 G (for five minutes). The pellet formed after centrifugation was discarded while the crude extract (supernatant) was transfer to new Eppendorf. Then microarray plates were prepared for estimation of non-specific esterases (α & β), monooxygenases, total proteins and Glutathione-S-transferases (GSTs) was done using aliquots as source of enzymes.
Estimation of activity of detoxifying enzymes
- Non-Specific Esterases
Protocol given by Van Asperen (1962) was followed to measure activity of both esterases. Same procedure was followed for α and β esterases estimation except substrate. Alpha naphthyl acetate (Substrate A) was used to estimate α esterases while Beta naphthyl acetate was used as (Substrate B) for estimation of β esterases. The microarray plates were prepared contain of 9 µl supernatant, 9 µl substrate solution (0.1M) and 120 µl Sodium phosphate buffer (100 mM) in each well. The reference solution mixture consists of 9 µl substrate solution and 129 µl Sodium phosphate buffer and then at 37˚C (incubation temperature) for 30 minutes. 42 µl 1% FBB solution and 107 µl Sodium dodecyl sulphate solution (SDS) were added after incubation to stop the reaction. The optical densities (OD) were measured at 620 nm and 545nm for α and β esterases, respectively by using microarray reader. The OD of reference solution was subtracted from the OD of supernatant solution. The stock solutions of α and β naphthol were prepared for α and β esterases, respectively. These solutions were made for standard curves in order to compare with the resulting optical densities. The product concentrations were estimated by comparing them with the resulting optical densities (mM/min/mg of protein) was used to express enzyme activity.
- Monooxygenases
The method followed for of monooxygenases estimation was put forth by Vulule et al. (1999). Reference solution contained 150 µl 3,3’,5,5’-Tetramethylbenzidine (TMBZ), 90 µl potassium phosphate buffer (PBB) and 23 µl 3% hydrogen peroxide. Reaction mixture consists of 15 µl supernatant, 150 µl 3,3’,5,5’-Tetramethylbenzidine (TMBZ), 75 µl potassium phosphate buffer (PBB) and 23 µl 3% H2O2. The OD were recorded at 620nm after five minutes using microarray reader. Standard curve of cytochrome c was made to measure the quantity of monoxygenases.
- Total Protein Assay
Dye binding protocol of Bradford (1976) was used to estimate total protein contents. Reference solution mixture contained 106 µl sodium phosphate buffer and 107 µl Bradford dye reagent. Reaction mixture consists of 10 µl supernatant, 96 µl sodium phosphate buffer and 107 µl Bradford dye reagent and plate was shaken to mix them thoroughly. The plate was enclosed in aluminum foil for 15 min incubation 30˚C. After incubation, for five minutes the plate (containing solution) were left to develop color and after this absorbance was recorded at 595nm. The protein content was estimated by comparing it with the standard curve of BSA.
- Glutathione S-Transferases
By using the protocol of Habig et al. (1974), the GST activity for CDNB was assessed. Reference solution contained 19 µl CDNB, 38 µl reduced Glutathione and 956 µl sodium phosphate buffer. The mixture consists of 19 µl supernatant, 38 µl reduced Glutathione, 19 µl CDNB and 937 µl sodium phosphate buffer. These mixtures were prepared in Eppendorf and then 250 µl were transfered in plate. After five minutes, the absorbance was recorded at 340nm using microarray reader and the values of absorbance were converted to units of concentration using molar extinction coefficient (ɛ) (9.6 Mm cm1- ) for CDNB-GSH conjugate
CDNB-GSHconjugate =
Preparation of solutions used
| Sr. No | Solution | Preparation |
| 1 | Sodium phosphate buffer (100 Mm) | Na2HPO4 (8.9 g) was dissolved in distilled water (500mL) to prepare solution A. NaH2PO4 (7.8 g) was dissolved in distilled water (500mL) to prepare solution B. Then both solutions were mixed and adjusted up to PH 7.0 followed by adding100 µl of Triton X-100. |
| 2 | Alpha naphthyl acetate (0.1 M) | Solution of 0.1 M was prepared by dissolving Alpha Naphthyl acetate (1.86 g) in methanol (100ml) |
| 3 | Beta naphthyl acetate (0.1 M) | Solution of 0.1 M was prepared by dissolving Beta naphthyl-acetate (1.86 g) in methanol (100ml). |
| 4 | FBB Solution (1%) | Solution 1% FBB salt was obtained by dissolving FBB Salt (1 g) in distilled water (100ml). |
| 5 | Sodium Dodecyl Sulphate Solution (5%) | Solution of 5% SDS was obtained by dissolving Sodium Dodecyl Sulphate (5 g) in distilled water (5 g). |
| 6 | Reduced Glutathione (1mM) | By dissolving reduced glutathione (3.1 mg) in sodium phosphate buffer (10ml), the required concentration was prepared. |
| 7 | 1-Chloro-2, 4-dinitrobenzidine (CDNB) (1mM) | By dissolving 1-Chloro-2, 4-dinitrobenzidine (2.1 mg) in Methanol (10ml), the required concentration was prepared. |
| 8 | 3, 3’, 5, 5’-Tetramethyl Benzidine (TMBZ) | By dissolving TMBZ (0.01 g) in Methanol (5ml) and sodium acetate buffer (15 ml) having 0.25 M (pH 5.0). |
| 9 | Bradford Dye Reagent (Coosmassie brilliant blue G-250) | By adding Coosmaisse brilliant blue G-250 (100 mg) in 95% Ethanol (50 ml), then (85%) phosphoric acid (100 ml) was dissolved in it. Distilled water was added in the mixture in order to dilute the solution up to volume of one litre. |
| 10 | Potassium phosphate buffer (0.625 M) | K2HPO4 (2.7 g) added in (distilled water 25 ml) to prepare solution A. KH2PO4 (2.1 g) was added in distilled water to prepare solution B. After preparing solution A and B, both solutions were mixed (at pH 7.0). |
| 11 | Sodium acetate buffer (0.25 M)
|
Two solutions A and B were mixed (at pH 5.4). CH3COONa (2 g)mixed in deionized water (100 ml) to make solution A. CH3COOH (1.4 ml) was mixed in deionized water (100 ml) to make solution B. |
Statistical analysis
One way ANOVA was applied for assessment of activity of different detoxifying enzymes using software GraphPad Prism (version 8.3.1).
RESULTS
Residual bioassay
The results of residual bioassays revealed that in case of bifenthrin, mortality at field rate (1.4ml/2ml) for L 1 and L 2 was 100 % and 100 %, respectively. At intermediate field rate (0.9ml/2ml), the mortality for L1 and L2 was 77.7% and 73.3%, respectively. At half field rate (0.4ml/2ml) it was 66.6% and 60% for L1 and L2, respectively. At half of half field rate (0.2ml/2ml), the mortality for L1 and L2 was 57.7 % and 44.4 %. In case of dimethoate, mortality at field rate (8.4ml/2ml) for L 1 and L 2 was 100 % and 100 %, respectively. At intermediate field rate (4.53ml/2ml), the mortality for L1 and L2 was also 100% and 100 %, respectively. At half field rate (0.67ml/2ml) it was 82.2% and 77.7% for L1 and L2, respectively. At half of half field rate (0.33ml/2ml), the mortality for L1 and L2 was 71.1 % and 68.8% . Chak no. 07 SB population was relatively more resistant than Chak no.02 NB population as less mortality was recorded in Chak no. 07 SB population (Tables 1—4).
In the case of lab strain flies were reared for three generations and then exposed to insecticide impregnated filter paper to determine their resistance against bifenthrin and dimethoate. In the case of lab strain mortality rate was very high, for bifenthrin at field rate it was 100%, at intermediate field rate it was 95.5% , at half field it was 91.1 % and at half of half field rate it was 88.8%. In the case of dimethoate at field rate it was 100%, at intermediate field rate it was 100% , at half field it was 95.5 % and at half of half field rate it was 91.1%.
Results show that LC 50 for lab strain of bifenthrin was 10.976 µgm/L and LC for lab strain of dimethoate was 8.879 µgm/L. LC 50 of bifenthrin field strain bhalwal location 1 and 2 was 91.295 µgm/L and 135.776 µgm/L respectively. LC 50 of dimethoate field strain bhalwal location 1 and 2 was 74.340 µgm/L and 84.531 µgm/L respectively. Resistance ratio of bifenthrin was 8.317 fold for field strain of location 1 and 12.370 fold for field strain of location 2. Slopes of both tested populations against bifenthrin were steeper than that of the susceptible population.
Resistance ratio of dimethoate was 8.372 fold for field strain of location 1 and 9.520 fold for field strain of location 2. Very low to low level of resistance to bifenthrin were determined in field-collected strains with resistance ratios ranging from 8.317-12.370. Very low level of resistance to dimethoate were determined in both field-collected strains with resistance ratios ranging from 8.372-9.520. Slopes of both tested populations against dimethoate were steeper than that of the susceptible population.
|
Bifenthrin concentrations in (ml/2ml) |
Percent mortality of L1 population |
||||
| Observation time in hours | |||||
| 4 | 8 | 12 | 16 | 24 | |
| C =0.00 | 0 | 0 | 0 | 0 | 0 |
| 1/2 HF = 0.20 | 34.4 | 42.2 | 49.9 | 54.4 | 57.7 |
| HF = 0.40 | 41.1 | 52.2 | 55.5 | 61.1 | 66.6 |
| Int = 0.9 | 47.7 | 58.8 | 67.7 | 72.2 | 77.7 |
| F = 1.4 | 62.2 | 78.8 | 86.6 | 92.2 | 100 |
Table 1: Percent mortality of L1 population against different concentrations of Bifenthrin
|
Bifenthrin concentrations in (ml/2ml) |
Percent mortality of L2 population |
||||
| Observation time in hours | |||||
| 4 | 8 | 12 | 16 | 24 | |
| C = 0.00 | 0 | 0 | 0 | 0 | 0 |
| 1/2HF = 0.20 | 31.1 | 35.0 | 38.8 | 42.2 | 44.4 |
| HF = 0.40 | 39.9 | 51.1 | 54.4 | 57.7 | 60.0 |
| Int = 0.9 | 45.5 | 55.5 | 64.4 | 71.1 | 73.3 |
| F = 1.4 | 59.9 | 76.6 | 84.4 | 91.1 | 100 |
Table 2: Percent mortality of L2 population against different concentrations of Bifenthrin
|
Dimethoate concentrations in (ml/2ml) |
Percent mortality of L1 population |
||||
| Observation time in hours | |||||
| 4 | 8 | 12 | 16 | 24 | |
| C = 0.00 | 0 | 0 | 0 | 0 | 0 |
| ¼ F = 0.33 | 42.2 | 51.1 | 58.8 | 66.6 | 71.1 |
| HF = 0.67 | 48.8 | 67.7 | 73.3 | 79.9 | 82.2 |
| Int = 4.53 | 100 | 100 | 100 | 100 | 100 |
| F = 8.4 | 100 | 100 | 100 | 100 | 100 |
Table 3: Percent mortality of L1 population against different concentrations of Dimethoate
|
Dimethoate concentrations in (ml/2ml) |
Percent mortality of L2 population |
||||
| Observation time in hours | |||||
| 4 | 8 | 12 | 16 | 24 | |
| C = 0.00 | 0 | 0 | 0 | 0 | 0 |
| 1/2HF = 0.33 | 39.9 | 48.8 | 56.8 | 62.2 | 68.8 |
| HF = 0.67 | 44.4 | 62.2 | 67.7 | 72.2 | 77.7 |
| Int = 4.53 | 100 | 100 | 100 | 100 | 100 |
| F = 8.4 | 100 | 100 | 100 | 100 | 100 |
Table 4: Percent mortality of L2 population against different concentrations of Dimethoate
| Bifenthrin
concentrations in (ml/2ml)
|
Percent mortality of Lab Strain |
||||
| Observation time in hours | |||||
| 4 | 8 | 12 | 16 | 24 | |
| C = 0.00 | 0 | 0 | 0 | 0 | 0 |
| 1/2HF = 0.20 | 54.4 | 73.0 | 79.9 | 85.5 | 88.8 |
| HF = 0.40 | 66.6 | 75.5 | 80.0 | 87.7 | 91.1 |
| Int = 0.9 | 77.7 | 85.5 | 89.9 | 92.2 | 95.5 |
| F = 1.4 | 79.9 | 81.1 | 88.8 | 97.7 | 100 |
Table 5: Percent mortality of lab strain against different concentrations of Bifenthrin
|
Dimethoate concentrations in (ml/2ml) |
Percent mortality of Lab Strain |
||||
| Observation time in hours | |||||
| 4 | 8 | 12 | 16 | 24 | |
| C = 0.00 | 0 | 0 | 0 | 0 | 0 |
| 1/2HF = 0.33 | 62.2 | 71.1 | 78.8 | 89.9 | 93.3 |
| HF = 0.67 | 66.6 | 71.1 | 83.3 | 91.1 | 95.5 |
| Int = 4.53 | 100 | 100 | 100 | 100 | 100 |
| F = 8.4 | 100 | 100 | 100 | 100 | 100 |
Table 6: Percent mortality of lab strain against different concentrations of Dimethoate
Table. 7: Toxicity of Bifenthrin and dimethoate insecticides to adults of Musca domestica from Bhalwal,Sargodha, Pakistan.
| Insecticide | Population | LC50[µgm/L](95%Cl) | Slope (± SE) | X2 | df | RR (95%) |
| Bifenthrin | Lab Strain | 10.976 (5.456-18.567) | 1.17±0.51 | 1.568 | 2 | |
| Field Strain Bhalwal L 1 (Chak No. 07 SB) | 91.295 (68.286-134.654) | 1.6 ± 0.35 | 7.844 | 2 | 8.317 | |
| Field Strain Bhalwal L 2 (Chak No. 02 NB) | 135.776 (91.215-169.295) | 1.93±0.34 | 8.738 | 2 | 12.370 | |
| Dimethoate | Lab Strain | 8.879 (4.162-15.792) | 1.18±0.66 | 0.491 | 2 | |
| Field Strain Bhalwal L 1 (ChakNo.07 SB) | 74.340 (53.112-105.467) | 1.68±0.45 | 1.256 | 2 | 8.372 | |
| Field Strain Bhalwal L 2 (Chak No. 02 NB) | 84.531(53.220-136.09) | 1.65±0.4 | 2.197 | 2 | 9.520 |
Comparative activity of different enzymes in control and treated groups
Activity of alpha-esterases
The possible mechanism involved in resistance of housefly against insecticides may be due to the activity of detoxifying enzymes. Activity of alpha-esterases in bifenthrin treated field strain from location 1 and 2 was significant (p<0.05) as compare to lab strain. While activity of alpha-esterases in dimethoate treated field strain from location 1 and 2 was non-significant (p > 0.05) as compare to lab strain.
Activity of beta-esterases
Activity of beta-esterases in bifenthrin treated field strain from location 1 and 2 was significant (p < 0.05) as compare to lab strain. While activity of beta-esterases in dimethoate treated field strain from location 1 and 2 was found non-significant (p > 0.05) as compare to lab strain.
Activity of monooxygenases
Activity of monooxygenases in bifenthrin treated field strain from location 1 and 2 was significant (p < 0.05) as compare to lab strain. While activity of monooxygenases in dimethoate treated field strain from location 1 and 2 was found significant (p < 0.05) as compare to lab strain.
GST Activity
Activity of GST in bifenthrin treated field strain from location 1 and 2 was non-significant (p > 0.05) as compare to lab strain. While activity of GST in dimethoate treated field strain from location 1 and 2 was found significant (p < 0.05) as compared to lab strain.
Protein activity
Activity of protein in bifenthrin treated field strain from location 1 and 2 was (p > 0.05) as compare to lab strain. While activity of protein in dimethoate treated field strain from location 1 and 2 was found significant (p > 0.05) as compare to lab strain.
Discussion
Houseflies act as major public health pest because these are mechanical carriers of more than 100 diseases of humans and animals (Forster et al., 2007; Malik et al., 2007; Scott et al., 2009). Different methods are used for the control of houseflies but insecticides are main weapon for the quick and effective control of houseflies. However due to extensive use of insecticides problem of resistance to insecticides are produce in houseflies (Azam & Hussein, 2002; Jin & Feng, 2001; White et al., 2007). Different enzyme such as specific or non-specific esterases, monooxygenases and transferases play important role in resistance development because they help in insecticide detoxification.
This present study was conducted to check the resistance status of houseflies collected from two locations of Bhalwal Punjab, Pakistan against bifenthrin (pyrethriod) and dimethoate (organophosphate). The results of residual bioassays showed different resistance levels in both house fly populations. The resistance level was higher in bifenthrin treated house flies as compare to the dimethoate treated house flies. But populations should not be considered resistant until a tenfold resistance ratio is observed (Valles et al., 1997).
In this study, very low to low level resistance was observed for bifenthrin with resistance ratios ranging from 8.317-12.370. In Pakistan different pyrethroid insecticides have been utilized for control of various pests of crops, dairies and poultries for example bifenthrin, lambda-cyhalothrin, cypermethrin and deltamethrin (Muhammad et al., 2008; Saleem et al., 2008; Khan et al., 2013). In numerous insect pests such as S. litura (Saleem et al., 2008; Abbas et al., 2014), spotted bollworm, Earias vittella (Fabricius) (Ahmad and Arif 2009), diamondback moth, Plutella xylostella (L.) (Sayyed et al., 2005), C. pipiens (Daaboub et al., 2008), codling moth, Cydia pomonella (L.) (Sauphanor et al., 2000), and American bollworm, Helicoverpa armigera (Hubner) (Kranthi et al., 2001, Faheem et al., 2013) has been reported resistance to pyrethroids. Rigorous use for the management of poultry pests like ticks, mite, lice, 30 fleas and flies in Pakistan is the fundamental source of resistance to pyrethroid insecticides.
Formerly, a study of dairy farmer’s was directed to decide perspectives, information and practices (producers use) for M. domestica control. This study showed that producers use pesticides on their own encounters without talking with entomologists (Khan et al., 2013). Very low level of resistance was seen against dimethoate. Various organophosphate insecticides have been utilized for the control of different pests in Punjab, Pakistan and high resistance levels in M. domestica have been recorded by various researchers from Punjab, Pakistan (Saleem et al., 2008; Khan et al., 2011; Shad et al., 2012; Khan et al., 2013) and also globally (Cheikh et al., 2009; Wang et al., 2010). But dimethoate is not commonly used for the control of houseflies in Punjab, Pakistan, that why very low level of resistance was shown by houseflies. Another factor may be involved in keeping resistance level low was the dilution of the resistant population with susceptible flies was undoubtedly very important (Meyer et al., 1987).
Biochemical estimation of insecticide detoxifying enzymes was performed to measure their modified activities in field flies in comparison to lab strain. Elevated activity of alpha and beta esterases was found against bifenthrin in both populations. The result close to our finding were also in accordance of other authors like Farnsworth et al. (2010) and Szalanski et al., (1995) they reported elevated esterases in pyrethroid resistant insect pest. Contradictory results were found by Campbell et al., (1998) and Claudianos et al., (1999) as they observed decreased activity of esterases in treated insect. The activity of alpha and beta esterases showed non significance difference in both field and lab strain of dimethoate. These results were closed to the findings of Taskin and Kence (2004), who reported that organophosphates field strains of houseflies had low activity of non-specific esterases than lab strain.
Elevated activities of monoxygenases were found in bifenthrin field strain as compare to lab strain at both locations. Significant difference between the field and lab strain indicated the role of monooxygenases in pyrethroid resistance. Similar results are reported by many other authors they reported increased level of monooxygenases in pyrethroid resistant houseflies (Kasai & Scott 2000; Liu & Pridgeon 2002; Wheelock & Scott 1992). The activity of monooxygenases showed significance difference in both field and lab strain of dimethoate. Evidence for increased in activity of monooxygenases against organophosphates in houseflies has also been reported in different areas of the world (Kasai & Scott, 2000; Scott & Zhang, 2003).
It is commonly thought that monooxygenases are involved in detoxification of all insecticides (Hodgson, 1985). The non-significant difference was observed between both field and lab strain of bifenthrin at both locations in the activity of GST. Many researchers have reported that pyrethroids did not act as substrate for GSTs (Grant & Matsumura, 1989; Reidy et al., 1990; Yu & Nguyen, 1996). High activity of GSTs was found in dimethoate treated field strain as compare to lab strain at both locations. The similar results were also observed by Franciosa and Berge (1995) and Taskin and Kence (2004) who found high activity of GSTs in organophosphate resistant field strain of housefly.
The process of resistance development can be slowed down by environment sanitation and proper disposal of waste and by doing this feeding and breeding sites of flies can be eliminated (Khan et al., 2012; Learmount et al., 2002; Malik et al., 2007).
To aware the public about the hazard associated with house flies awareness campaign on health and hygiene should be started in the under develop areas of Pakistan. Insecticides having different mode of action should be alternatively used. Over and under use of insecticide cause resistance so the type and dose should be decided after consulting with entomologist. WHO recommended dose of insecticides should be used.
SUMMARY
Housefly is a pest of public health concern that act as vector of various diseases. Insecticides are one of the novel strategies used for effective management of these pests. The purpose of the current study was to assess the resistance status of housefly populations against two insecticides. Flies were collected from two different localities i.e: Chak no 07 SB Bhalwal (32°07’16.1″N 72°38’25.3″E) and Chak no 02 NB Bhalwal (32°05’45.0″N 72°39’27.1″E). The housefly populations were reared in laboratory by cotton oilcakes soaked in water until the foul smell was developed. The flies were then transferred to the rearing media and adult flies were used for bioassay. Two insecticides i.e: Bifenthrin and Dimethoate were selected to perform bioassay by using residual bioassay technique.
Susceptibility or resistance against four concentrations of each insecticide was assessed. The doses for Bifenthin and Dimethoate were prepared according to recommendations by WHO. Bioassay were performed on adult flies . One hour exposure of insecticides were given and then the filter paper was removed. Mortality rate was observed for 24 hours and those flies that survived 24 hours post-exposure to insecticides were considered resistant. The percent mortality of houseflies was higher among Dimethoate treatments as compared to Bifenthrin in both localities. The resistant flies were then kept freezed in order to immobilize them. Enzyme preparation for biochemical estimation of detoxifying enzymes was done and microarray plate were made. The standard protocols were followed for the estimation of biochemical enzymes.
The activity of detoxifying enzymes like non-specific esterases and monooxygenases was higher in field strain of bifenthrin when compared with lab strain of bifenthrin in both populations of housefly. However, there was a non-significant difference in activity of GST in field strain of bifenthrin when compared with lab strain of bifenthrin in both populations of housefly. The activity of detoxifying enzymes like non-specific esterases and monooxygenases had non -significant difference in field strain of dimethoate when compared with lab strain of dimethoate in both populations of housefly. However, there was significant difference in activity of GST in field strain of dimethoate when compared with lab strain of dimethoate in both populations of housefly.
CONCLUSION
The present study indicated that houseflies not showed high resistance against both insecticides from the selected areas. Bifenthrin indicated high resistance as compared to Dimethoate in both localities. The activity of detoxifying enzymes like non-specific esterases and monooxygenases was higher in field strain of bifenthrin when compared with lab strain of bifenthrin in both populations of housefly. However, there was significant difference in activity of GST in field strain of dimethoate when compared with lab strain of dimethoate in both populations of housefly.