Parasitol Res (2014) 113:1343–1352 DOI 10.1007/s00436-014-3773-4
ORIGINAL PAPER
Cross-resistance, genetics, and realized heritability of resistance to fipronil in the house fly, Musca domestica (Diptera: Muscidae): a potential vector for disease transmission Naeem Abbas & Hafiz Azhar Ali Khan & Sarfraz Ali Shad
Received: 7 November 2013 / Accepted: 10 January 2014 / Published online: 31 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Houseflies, Musca domestica (L.), are ubiquitous pests that have the potential to spread a variety of pathogens to humans, poultries, and dairies. Pesticides are commonly used for the management of this pest. Fipronil is a GABA-gated chloride channel-inhibiting insecticide that has been commonly used for the management of different pests including M. domestica throughout the world. Many pests have developed resistance to this insecticide. A field-collected strain of M. domestica was selected with fipronil for continuous 11 generations to assess the cross-resistance, genetics, and realized heritability for designing a resistance management strategy. Laboratory bioassays were performed using the feeding method of mixing insecticide concentrations with 20 % sugar solutions and cotton soaks dipped in insecticide solutions were provided to tested adult flies. Bioassay results at G12 showed that the fipronil-selected strain developed a resistance ratio of 140-fold compared to the susceptible strain. Synergism bioassay with piperonyl butoxide (PBO) and S,S,S,tributyl phosphorotrithioate (DEF) indicated that fipronil resistance was associated with microsomal oxidase and also esterase. Reciprocal crosses between resistant and susceptible strains showed an autosomal and incompletely dominant resistance to fipronil. The LC50 values of F1 and F′1 strains were not significantly different and dominance values were 0.74 and 0.64, respectively. The resistance to fipronil was completely recessive (DML=0.00) at the highest dose and incompletely dominant at the lowest dose (DML=0.87). The monogenic resistance based on chi-square goodness of fit test N. Abbas (*) : S. A. Shad (*) Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan e-mail: [email protected] e-mail: [email protected] H. A. A. Khan Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan
and calculation of the minimum number of segregating genes showed that resistance to fipronil is controlled by multiple genes. The fipronil resistance strain confirmed very low crossresistance to emamectin benzoate and spinosad while no cross-resistance to chlorpyrifos and acetamiprid when compared to that of the field population. The heritability values were 0.112, 0.075, 0.084, 0.008, and 0.052 for fipronil, emamectin benzoate, spinosad, acetamiprid, and chlorpyrifos, respectively. It was concluded that fipronil resistance in M. domestica was autosomally inherited, incompletely dominant, and polygenic. These findings would be helpful for the better and successful management of M. domestica.
Introduction Houseflies, Musca domestica L. (Diptera: Muscidae), are key pests of poultries, dairies, and human beings (Scott et al. 2000; Kaufman et al. 2001; Deacutis et al. 2006). They are mechanical vectors of more than 100 animal intestinal diseases such as protozoan (amoebic dysentery), bacterial (salmonellosis, shigellosis, and cholera), helminthic (hookworm, roundworm, and tapeworm), and viral infections (Forster et al. 2007; Acevedo et al. 2009; Mishra et al. 2011; Morey and Khandagle 2012; Khan et al. 2012; Kumar et al. 2012, 2013). M. domestica is also known as a potential vector of bird flu virus (avian influenza), posing sporadic threats to poultry industry and humans worldwide (Barin et al. 2010; Wanaratana et al. 2011). The large amount of poultry manure which is exposed to high humidity and temperature can provide an ideal condition for M. domestica growth in poultry farms. High-density population of M. domestica irritates and stresses the poultry workers and hens and influences the economics of poultry products (Acevedo et al. 2009). A number of insecticides from carbamate, organophosphate, pyrethroid, and new chemical groups have been used for the management of M. domestica worldwide (Shi et al. 2011;
1344
Khan et al. 2013a, c). However, extensive and injudicious use of these insecticides has led to develop resistance in the M. domestica (Khan et al. 2013b). Fipronil is a phenylpyrazole insecticide which inhibits the gamma amino butyric acid-gated channel of chloride ions (Cole et al. 1993). This insecticide potently blocks the glutamate-activated chloride channel and inhibits the chloride ion flow in the central nervous system of insects (Hainzl et al. 1998; Ikeda et al. 2003). Fipronil is used for the control of different pest orders including Diptera, Orthoptera, Blattaria, and Lepidoptera (Scott and Wen 1997; Ali et al. 1998; Lecoq and Balanca 1998; Siegfried et al. 1999). Resistance to fipronil has been reported in number of insect pests like Plutella xylostella, Sogatella furcifera, Spodoptera litura (Sayyed and Wright 2004; Ahmad et al. 2008; Tang et al. 2010) including M. domestica (Wen and Scott 1999; Liu and Yue 2000; Kristensen et al. 2004). @Analysis of type of inheritance and measurement of insecticides resistance in pests can give valuable information to develop better strategy for the management of resistance (Ahmad et al. 2007; Zhang et al. 2008). The genetics of resistance to various insecticides in M. domestica have been studied previously (Scott et al. 1984; Liu and Yue 2001; Shono et al. 2002; Tang et al. 2002; Zhang et al. 2008; Shi et al. 2011). However, genetics of fipronil resistance has been reported in few pests like P. xylostella and M. domestica worldwide (Wen and Scott 1999; Sayyed and Wright 2004). The knowledge of the resistance inheritance and mechanisms is essential for the management of pesticide resistance. The type of inheritance of resistance to each chemical needs to be addressed in laboratory for all field use insecticides in all organisms, so that we can make better resistant management plan in the field. In M. domestica, inheritance of crossresistance of pyrethroid to fipronil was documented by Wen and Scott (1999); however, the general characterization of inheritance of resistance to fipronil is still unknown. In the present study, cross-resistance to other insecticide, mechanism, genetics, and realized heritability of M. domestica strains was evaluated. The fipronil-resistant strain was used for cross-resistance to different insecticides. A fipronilresistant strain (FRR) was crossed with fipronil susceptible strain (FSS) in order to determine the genetics (sex linkage, dominance, and number of genes) of resistance to fipronil by using bioassay mortality data.
Materials and methods Insects Adult houseflies were collected by sweep netting from a poultry farm located in Multan (30. 066141N; 71. 68695E), Punjab, Pakistan. The selected poultry farm used pesticides
Parasitol Res (2014) 113:1343–1352
heavily for the management of different poultry pests including M. domestica (personal communication). After collection, flies were brought in meshed plastic jars (34×17 cm) to the laboratory for colonization. The adults were kept in the same jar and fed on powdered milk mixed with sugar 1:1 ratio by weight (in grams), and cotton wick soaked with water was provided in separate Petri dish. The larvae were reared on a medium of powdered milk, sugar, yeast, grass meal, and wheat bran at a ratio of 1.5:1.5:5:5:20 by weight (in grams), respectively, and made a paste with 65 ml water (Bell et al., 2010). All these flies designated as field population were used for start of experiment to generate FRR and unselected (UNSEL) strains. All the insects were reared at 27±2 °C, 60–70 % RH, and 14:10 @(light/dark) photoperiod.
Insecticides and synergists Commercial grade-formulated insecticides used for bioassays included fipronil (Regent® 050EC, Bayer Crop Sciences), spinosad (Tracer® 24SC, Dow Agro-Sciences), emamectin benzoate (Proclaim ® 019EC, Syngenta), acetamiprid (Mospilon® 20WP, Dow Agro-Sciences), and chlorpyrifos (Lorsban® 40EC, Dow Agro-Sciences) piperonyl butoxide (PBO; Sigma Ltd, UK), an inhibitor of cytochrome P450 monooxygenases (microsomal oxidases) and of esterases, and S,S,S-tri-n-butyl phosphorotrithioate (DEF; Sigma Ltd, UK), an esterase-specific inhibitor.
Toxicological bioassay A feeding bioassay was used to know the toxicities of the five abovementioned insecticides (Kaufman et al. 2006). A range of five serial solutions (causing >0 and 0.05) when their CI overlaps (Litchfield and Wilcoxon 1949). Resistance ratio (RR) was determined as follows: LC50 of the FRR strain=LC50 of the FSS strain: Degree of dominance The dominance (DLC) value of fipronil resistance was determined according to Stone (1968) and Bourguet and Raymond (1998) while effective dominance (DML) was estimated according to Bourguet et al. (2000) as the following formula: DML ¼ ðMTRS −MTSS Þ=ðMTRR −MTSS Þ: In the above equation, MTRR, MTRS, and MTSS were the percent mortalities to a single insecticide dose for the FRR, F1,
1346
Parasitol Res (2014) 113:1343–1352
First, the hypothesis of monogenic resistance was tested statistically using a chi-square goodness of fit test. According to Sokal and Rohlf (1981), the null hypothesis of monogenic resistance based on chi-square goodness of fit test was calculated as the following formula:
fipronil and emamectin benzoate but higher than that of acetamiprid. Fipronil, acetamiprid and chlorpyrifos were significantly less toxic to the UNSEL (95 % CI did not overlap, P
ORIGINAL PAPER
Cross-resistance, genetics, and realized heritability of resistance to fipronil in the house fly, Musca domestica (Diptera: Muscidae): a potential vector for disease transmission Naeem Abbas & Hafiz Azhar Ali Khan & Sarfraz Ali Shad
Received: 7 November 2013 / Accepted: 10 January 2014 / Published online: 31 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Houseflies, Musca domestica (L.), are ubiquitous pests that have the potential to spread a variety of pathogens to humans, poultries, and dairies. Pesticides are commonly used for the management of this pest. Fipronil is a GABA-gated chloride channel-inhibiting insecticide that has been commonly used for the management of different pests including M. domestica throughout the world. Many pests have developed resistance to this insecticide. A field-collected strain of M. domestica was selected with fipronil for continuous 11 generations to assess the cross-resistance, genetics, and realized heritability for designing a resistance management strategy. Laboratory bioassays were performed using the feeding method of mixing insecticide concentrations with 20 % sugar solutions and cotton soaks dipped in insecticide solutions were provided to tested adult flies. Bioassay results at G12 showed that the fipronil-selected strain developed a resistance ratio of 140-fold compared to the susceptible strain. Synergism bioassay with piperonyl butoxide (PBO) and S,S,S,tributyl phosphorotrithioate (DEF) indicated that fipronil resistance was associated with microsomal oxidase and also esterase. Reciprocal crosses between resistant and susceptible strains showed an autosomal and incompletely dominant resistance to fipronil. The LC50 values of F1 and F′1 strains were not significantly different and dominance values were 0.74 and 0.64, respectively. The resistance to fipronil was completely recessive (DML=0.00) at the highest dose and incompletely dominant at the lowest dose (DML=0.87). The monogenic resistance based on chi-square goodness of fit test N. Abbas (*) : S. A. Shad (*) Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan e-mail: [email protected] e-mail: [email protected] H. A. A. Khan Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan
and calculation of the minimum number of segregating genes showed that resistance to fipronil is controlled by multiple genes. The fipronil resistance strain confirmed very low crossresistance to emamectin benzoate and spinosad while no cross-resistance to chlorpyrifos and acetamiprid when compared to that of the field population. The heritability values were 0.112, 0.075, 0.084, 0.008, and 0.052 for fipronil, emamectin benzoate, spinosad, acetamiprid, and chlorpyrifos, respectively. It was concluded that fipronil resistance in M. domestica was autosomally inherited, incompletely dominant, and polygenic. These findings would be helpful for the better and successful management of M. domestica.
Introduction Houseflies, Musca domestica L. (Diptera: Muscidae), are key pests of poultries, dairies, and human beings (Scott et al. 2000; Kaufman et al. 2001; Deacutis et al. 2006). They are mechanical vectors of more than 100 animal intestinal diseases such as protozoan (amoebic dysentery), bacterial (salmonellosis, shigellosis, and cholera), helminthic (hookworm, roundworm, and tapeworm), and viral infections (Forster et al. 2007; Acevedo et al. 2009; Mishra et al. 2011; Morey and Khandagle 2012; Khan et al. 2012; Kumar et al. 2012, 2013). M. domestica is also known as a potential vector of bird flu virus (avian influenza), posing sporadic threats to poultry industry and humans worldwide (Barin et al. 2010; Wanaratana et al. 2011). The large amount of poultry manure which is exposed to high humidity and temperature can provide an ideal condition for M. domestica growth in poultry farms. High-density population of M. domestica irritates and stresses the poultry workers and hens and influences the economics of poultry products (Acevedo et al. 2009). A number of insecticides from carbamate, organophosphate, pyrethroid, and new chemical groups have been used for the management of M. domestica worldwide (Shi et al. 2011;
1344
Khan et al. 2013a, c). However, extensive and injudicious use of these insecticides has led to develop resistance in the M. domestica (Khan et al. 2013b). Fipronil is a phenylpyrazole insecticide which inhibits the gamma amino butyric acid-gated channel of chloride ions (Cole et al. 1993). This insecticide potently blocks the glutamate-activated chloride channel and inhibits the chloride ion flow in the central nervous system of insects (Hainzl et al. 1998; Ikeda et al. 2003). Fipronil is used for the control of different pest orders including Diptera, Orthoptera, Blattaria, and Lepidoptera (Scott and Wen 1997; Ali et al. 1998; Lecoq and Balanca 1998; Siegfried et al. 1999). Resistance to fipronil has been reported in number of insect pests like Plutella xylostella, Sogatella furcifera, Spodoptera litura (Sayyed and Wright 2004; Ahmad et al. 2008; Tang et al. 2010) including M. domestica (Wen and Scott 1999; Liu and Yue 2000; Kristensen et al. 2004). @Analysis of type of inheritance and measurement of insecticides resistance in pests can give valuable information to develop better strategy for the management of resistance (Ahmad et al. 2007; Zhang et al. 2008). The genetics of resistance to various insecticides in M. domestica have been studied previously (Scott et al. 1984; Liu and Yue 2001; Shono et al. 2002; Tang et al. 2002; Zhang et al. 2008; Shi et al. 2011). However, genetics of fipronil resistance has been reported in few pests like P. xylostella and M. domestica worldwide (Wen and Scott 1999; Sayyed and Wright 2004). The knowledge of the resistance inheritance and mechanisms is essential for the management of pesticide resistance. The type of inheritance of resistance to each chemical needs to be addressed in laboratory for all field use insecticides in all organisms, so that we can make better resistant management plan in the field. In M. domestica, inheritance of crossresistance of pyrethroid to fipronil was documented by Wen and Scott (1999); however, the general characterization of inheritance of resistance to fipronil is still unknown. In the present study, cross-resistance to other insecticide, mechanism, genetics, and realized heritability of M. domestica strains was evaluated. The fipronil-resistant strain was used for cross-resistance to different insecticides. A fipronilresistant strain (FRR) was crossed with fipronil susceptible strain (FSS) in order to determine the genetics (sex linkage, dominance, and number of genes) of resistance to fipronil by using bioassay mortality data.
Materials and methods Insects Adult houseflies were collected by sweep netting from a poultry farm located in Multan (30. 066141N; 71. 68695E), Punjab, Pakistan. The selected poultry farm used pesticides
Parasitol Res (2014) 113:1343–1352
heavily for the management of different poultry pests including M. domestica (personal communication). After collection, flies were brought in meshed plastic jars (34×17 cm) to the laboratory for colonization. The adults were kept in the same jar and fed on powdered milk mixed with sugar 1:1 ratio by weight (in grams), and cotton wick soaked with water was provided in separate Petri dish. The larvae were reared on a medium of powdered milk, sugar, yeast, grass meal, and wheat bran at a ratio of 1.5:1.5:5:5:20 by weight (in grams), respectively, and made a paste with 65 ml water (Bell et al., 2010). All these flies designated as field population were used for start of experiment to generate FRR and unselected (UNSEL) strains. All the insects were reared at 27±2 °C, 60–70 % RH, and 14:10 @(light/dark) photoperiod.
Insecticides and synergists Commercial grade-formulated insecticides used for bioassays included fipronil (Regent® 050EC, Bayer Crop Sciences), spinosad (Tracer® 24SC, Dow Agro-Sciences), emamectin benzoate (Proclaim ® 019EC, Syngenta), acetamiprid (Mospilon® 20WP, Dow Agro-Sciences), and chlorpyrifos (Lorsban® 40EC, Dow Agro-Sciences) piperonyl butoxide (PBO; Sigma Ltd, UK), an inhibitor of cytochrome P450 monooxygenases (microsomal oxidases) and of esterases, and S,S,S-tri-n-butyl phosphorotrithioate (DEF; Sigma Ltd, UK), an esterase-specific inhibitor.
Toxicological bioassay A feeding bioassay was used to know the toxicities of the five abovementioned insecticides (Kaufman et al. 2006). A range of five serial solutions (causing >0 and 0.05) when their CI overlaps (Litchfield and Wilcoxon 1949). Resistance ratio (RR) was determined as follows: LC50 of the FRR strain=LC50 of the FSS strain: Degree of dominance The dominance (DLC) value of fipronil resistance was determined according to Stone (1968) and Bourguet and Raymond (1998) while effective dominance (DML) was estimated according to Bourguet et al. (2000) as the following formula: DML ¼ ðMTRS −MTSS Þ=ðMTRR −MTSS Þ: In the above equation, MTRR, MTRS, and MTSS were the percent mortalities to a single insecticide dose for the FRR, F1,
1346
Parasitol Res (2014) 113:1343–1352
First, the hypothesis of monogenic resistance was tested statistically using a chi-square goodness of fit test. According to Sokal and Rohlf (1981), the null hypothesis of monogenic resistance based on chi-square goodness of fit test was calculated as the following formula:
fipronil and emamectin benzoate but higher than that of acetamiprid. Fipronil, acetamiprid and chlorpyrifos were significantly less toxic to the UNSEL (95 % CI did not overlap, P
