Acta Tropica 142 (2015) 149–155
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Inheritance mode, cross-resistance and realized heritability of pyriproxyfen resistance in a field strain of Musca domestica L. (Diptera: Muscidae) Rizwan Mustafa Shah a,∗ , Naeem Abbas a , Sarfraz Ali Shad a,∗ , Marie Varloud b a b
Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Punjab, Pakistan Technical services, Parasitology, Companion Animal, Ceva, France
a r t i c l e
i n f o
Article history: Received 18 October 2014 Received in revised form 23 November 2014 Accepted 24 November 2014 Available online 3 December 2014 Keywords: IGRS Inheritance House fly Cross-resistance Realized heritability
a b s t r a c t Pyriproxyfen is a growth regulator used for the control of different insect pests, including Musca domestica. To assess the risk of resistance and to develop a strategy for resistance management, a field strain of M. domestica was exposed to pyriproxyfen in the laboratory for 30 generations. The inheritance mode, realized heritability of pyriproxyfen resistance and cross-resistance to other insecticides were assessed. Prior to the selection process, the field strain exhibited a resistance ratio (RR) of 25.7, 7.31, 7.67, and 27-fold for pyriproxyfen, methoxyfenozide, cyromazine and lufenuron, respectively, when compared to the pyriproxyfen susceptible strain (Pyri-Sus). After continuous selection with pyriproxyfen, the pyriproxyfen-resistant strain (Pyri-Res) became 206-fold more resistant than the Pyri-Sus strain. The overlapping confidence limits of LC50 values of F1 (Pyri-Res ♂ × Pyri-Sus ♀) and F1 † (Pyri-Res ♀ × Pyri-Sus ♂) suggested an autosomal and completely dominant mode of resistance to pyriproxyfen. Monogenic test of inheritance showed that resistance to pyriproxyfen was governed by multiple genes. The Pyri-Res strain showed very low cross resistance to methoxyfenozide, cyromazine, and lufenuron. The estimated realized heritability was 0.02, 0.05, 0.03 and 0.04 for pyriproxyfen, methoxyfenozide, cyromazine, and lufenuron, respectively. It was concluded that pyriproxyfen resistance in M. domestica was autosomally inherited, completely dominant and polygenic. These results would be helpful for the design of an improved control strategy against M. domestica. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The house fly, Musca domestica L., (Diptera: Muscidae) is a key pest causing significant losses in the livestock industry (Abbas et al., 2014a; Khan et al., 2014). M. domestica is also a probable vector of more than 100 diseases of human and animals (Fasanella et al., 2010; Förster et al., 2007). Moreover, outbreaks of diarrheal diseases have been associated with seasonal abundance of M. domestica in both urban and rural settings of countries including Pakistan (Graczyk et al., 2001; Khan and Akram, 2014). They are also vectors of bird flu virus, causing avian influenza which is a serious problem in poultry throughout the world (Barin et al., 2010; Wanaratana et al., 2011). It has been reported that millions of domestic poultry flocks are affected by avian influenza virus which resulted in more than 150 deaths of humans globally (Otte et al.,
∗ Corresponding authors. Tel.: +92 3416262699. E-mail addresses: [email protected] (R.M. Shah), [email protected] (S.A. Shad). http://dx.doi.org/10.1016/j.actatropica.2014.11.016 0001-706X/© 2014 Elsevier B.V. All rights reserved.
2007). In Punjab, Pakistan livestock farms, manure heaps provides ideal breeding sites for M. domestica. Massive fly densities cause nuisance to the farm workers and animals and reduce the aesthetic value of livestock, leading to economic losses (Acevedo et al., 2009). For decades, synthetic insecticides have been used as a principle control measure for this pest. However, resistance to insecticides in this pest has become a major issue due to the extensive and poorly optimized application of insecticides (Khan et al., 2013). M. domestica is ranked among the top 20 resistant pests of the world (Whalon et al., 2008) and resistance is reported from all over the world against almost all classes of insecticides (Abbas et al., 2014a,b; Acevedo et al., 2009; Bell et al., 2010; Deacutis et al., 2006; Kaufman et al., 2006; Khan et al., 2014; Kristensen and Jespersen, 2003; Shi et al., 2011; Tang et al., 2002). Pyriproxyfen is a juvenile hormone agonist that is an inhibitor of pupal adult metamorphosis (Seng et al., 2008) by causing abnormal growth and ultimately death of the larvae and is most effective at the onset of metamorphosis (Sullivan and Goh, 2008; Wilson et al., 2007). In addition, pyriproxyfen is highly selective for target organisms,
150
R.M. Shah et al. / Acta Tropica 142 (2015) 149–155
has low mammalian toxicity, and serves as a promising agent to be used in integrated management strategies of different pests including M. domestica (Geden and Devine, 2012). However, resistance to pyriproxyfen has already been reported in Bemisia tabaci (Gennadius) (Crowder et al., 2008; Ghanim and Kontsedalov, 2007; Horowitz et al., 2003; Karatolos et al., 2012; Ma et al., 2010) and M. domestica (Li et al., 1998; Pospischil et al., 1996; Zhang et al., 1997). The understanding of the mode of inheritance which characterize insecticide resistance is necessary for the sustainability of pest control (Abbas et al., 2014b; Bouvier et al., 2001). Information relative to dominance and number of genes involved in resistance will aid in our understanding of the development of resistance (Abbas et al., 2014b). The inheritance mode of pyriproxyfen resistance has been previously explored in B. tabaci (Crowder et al., 2008; Horowitz et al., 2003) and M. domestica (Zhang et al., 1997) which have demonstrated different genetics responses or resistance mechanisms. Insecticide resistance in natural populations may be monogenic or polygenic with a major phenotypic effect (Abbas et al., 2014b; Sayyed et al., 2008) but the mechanism of insecticide resistance can vary depending upon a type and geographical origin of the pest species (Khan et al., 2014). Moreover, insecticide resistance is not a fixed phenomenon and can change with time and space (Khan et al., 2014; Zhao et al., 2006). The current experiment was planned to investigate the inheritance pattern, cross-resistance to three other insecticides and realized heritability of pyriproxyfen resistance in M. domestica collected from a poultry farm. The focus of this study was to characterize resistance to commercial formulations which were being used for the control of M. domestica in Pakistan. The results will contribute to an optimized program of pyriproxyfen resistance management in M. domestica. 2. Materials and methods 2.1. Insects and rearing A sample population of 100–150 adults of M. domestica was collected by sweep netting from a poultry farm located in Multan, Pakistan (30◦ 5 11N, 71◦ 39 15E). The field collected population designated as Pyri-Field was maintained in meshed plastic jars (34 × 17 cm) in the laboratory. Adults were fed a mixture of powdered milk + sugar (1:1 ratio) and cotton wick soaked with water was provided in a separate Petri dish. Larvae were reared on an artificial media (Abbas et al., 2014a). All the insects were maintained in standard laboratory conditions Abbas et al. (2014a). The susceptible population designated as Pyri-Sus was collected from an area where no insecticides were used and was maintained in the laboratory for more than 40 generations without exposure to any insecticide. 2.2. Chemicals Commercial grade formulated insecticides including pyriproxyfen (Admiral® 10EC, FMC), lufenuron (Match® , 050EC, Syngenta), cyromazine (Trigard® , 75WP, Syngenta) and methoxyfenozide (Runner® , 240SC, Arista Life Sciences) were used. 2.3. Larvicidal bioassays A bioassay based on diet incorporation method (Kristensen and Jespersen, 2003) was used (with some modifications) to determine the toxicities of pyriproxyfen, methoxyfenozide, cyromazine and lufenuron. The larval rearing medium was made with wheat bran, grass meal, yeast, powdered milk, and sugar (20:5:5:1.5:1.5 ratio by weight) and was complemented with water containing
different concentrations of insecticides. Five concentrations for each bioassay were made through serial dilution method and each concentration was replicated three times. Ten first instar larvae collected at G1 were used in each replicate for a total of thirty larvae tested per insecticide concentration. A total of 180 larvae were used for each bioassay, including controls which consisted of only water added to the diet and kept at standard laboratory conditions as described above. Three weeks after the beginning of the larval bioassay, the rate of adult emergence was recorded (Cetin et al., 2006). All the larvae that were unable to develop into adults were considered as dead. 2.4. Selection for resistance Fist instar larvae from Pyri-Field strain (G1 ) were exposed to pyriproxyfen for 30 consecutive generations in the laboratory to generate a pyriproxyfen-resistant strain (Pyri-Res) using the diet incorporation method as described in the bioassay section. On average, 800 larvae were exposed in each generation during the selection experiment. The first generation (G1 ) was exposed to 0.2 g mL−1 of pyriproxyfen and doses were increased gradually for subsequent generations such that the last generation (G30 ) was exposed to 30 g mL−1 . 2.5. Genetic crosses Virgin male and female flies were separated within one day after eclosion to make crosses (Abbas et al., 2014a). Reciprocal crosses of Pyri-Sus and Pyri-Res were made to produce two lines: F1 (12 Pyri-Res ♂ × 12 Pyri-Sus ♀) and F1 † (12 Pyri-Res ♀ × 12 Pyri-Sus ♂). As there was overlapping of LC50 in reciprocal crosses, any line of these crosses was used for back cross. F1 was used for back crossing with the parental populations to generate the two backcross lines: BC1 (12 F1 ♀ × 12 Pyri-Sus ♂) and BC2 (12 F1 ♀ × 12 Pyri-Res ♂). 2.6. Realized heritability (h2 ) Following, the method of Falconer (1989) and Tabashnik (1992), the realized heritability (h2 ) of resistance to pyriproxyfen was estimated as follows: h2 =
R S
In the above equation, R (selection response) was estimated as follows: R=
log final LC50 − log initial LC50 n
where final LC50 means the LC50 of strain after 30 generations of selection, initial LC50 means the LC50 of the Pyri-Field strain before selection and n means number of generations selected with pyriproxyfen. Whereas, S (selection differential) was calculated as follows: S = i × p where i means intensity of selection calculated according to Falconer (1989), p means phenotypic standard deviation calculated as follows: p = [(inital slope + final slope) 0.5]−1 Based on the response of M. domestica to selection in the laboratory, the number of generations required for a tenfold increase in LC50 (G) was calculated as follows: G = R−1
R.M. Shah et al. / Acta Tropica 142 (2015) 149–155
2.7. Statistical analysis
Second, the Lande (1981) method was used to determine the number of genes involved in controlling the pyriproxyfen resistance by using the following formula:
2.7.1. Lethal concentration response data The control mortality was corrected by Abbot’s formula (Abbott, 1925), if occurred and then analyzed with POLO software (Software, 2005) by using probit analysis (Finney, 1971) to determine the LC50 values along with their confidence intervals (CI), slope and its standard error (SE). Resistance ratio (RR) was determined by dividing the LC50 of the Pyri-Res strain with the LC50 of the Pyri-Sus strain.
E =
8 2 S
3. Results 3.1. Toxicity of different insecticides to Pyri-Sus, Pyri-Field and Pyri-Res strains of Musca domestica
XPyri-Res − XPyri-Sus
Pyriproxyfen and methoxyfenozide were equally toxic to the Pyri-Sus (based on overlapping of 95% CI) but less toxic than cyromazine and lufenuron. Similarly, pyriproxyfen and methoxyfenozide were equally toxic to the Pyri-Field strain (based on overlapping of 95% CI) but less toxic than cyromazine and lufenuron (Table 1). Pyriproxyfen, methoxyfenozide, cyromazine and lufenuron were significantly less toxic to the Pyri-Field strain compared with the Pyri-Sus and led to a RR of 25.7-fold, 7.31-fold, 7.67-fold and 27-fold, respectively. All the tested insecticides were significantly less toxic to the Pyri-Res compared to the Pyri-Field and Pyri-Sus strains. After 30 generations of selection with pyriproxyfen, the Pyri-Res strain developed a RR of 206-fold and 7.92-fold compared to the Pyri-Sus and Pyri-Field strains (Table 1).
where XF1 , XPyri-Res , and XPyri-Sus are log LC50 values of the F1 , PyriRes and Pyri-Sus, respectively. Effective dominance (DML ) was calculated Bourguet et al. (2000) as follows: MTF1 − MTPyri-Sus MTPyri-Res − MTPyri-Sus
where MTPyri-Res , MTF1 , and MTPyri-Sus were the percent mortalities for the Pyri-Res, F1 , and Pyri-Sus strains to a single insecticide tested dose. 2.7.3. Number of genes involved The number of genes involved in controlling pyriproxyfen resistance was estimated by using two approaches. First, the null hypothesis of monogenic resistance based on a chi-square goodness of fit test between expected and observed mortalities was tested as follows: (Sokal and Rohlf, 1981) 2 =
2
In the above equation, 2 B1 , 2 B2 , 2 F1 , 2 XPyri-Sus , and 2 XPyri-Res were variances of the BC1 , BC2 , F1 , Pyri-Sus, and Pyri-Res strains, respectively.
2XF1 − XPyri-Res − XPyri-Sus
DML =
XPyri-Res − XPyri-Sus
where XPyri-Res and XPyri-Sus were the log LC50 of Pyri-Res and Pyri2S = Sus strains, respectively and 2 S was calculated as follows: 2 2 2 2 2 B1 + B2 − F1 + 0.5 XPyri-Sus + 0.5 XPyri-Res
2.7.2. Degree of dominance The dominance (DLC ) value of pyriproxyfen resistance was determined according to Bourguet and Raymond (1998) and Stone (1968) as follows: D=
151
3.2. Cross-resistance to other insecticides in the Pyri-Res strain The Pyri-Res strain at G30 was used to assess the cross-resistance of pyriproxyfen to methoxyfenozide, cyromazine and lufenuron compared to the Pyri-Field strain (Table 1). Results indicated that selection of pyriproxyfen significantly increased the resistance against all the tested insecticides (95% CI did not overlap). The Pyri-Res strain showed cross-resistance against methoxyfenozide (3.34-fold), cyromazine (5-fold) and lufenuron (3-fold) compared with the Pyri-Field strain.
(F − pn)2 pqn
In the above equation, F was the observed mortality in the backcross at a particular dose, n was the numbers exposed to a particular dose, p was the expected mortality at given dose and calculated Georghiou (1969) as 0.5 (number of F1 flies that died + number of Pyri-Sus flies that died)/number of flies exposed in backcross and q was calculated as 1 − p. The null hypothesis of monogenic resistance was rejected on the basis of significant difference between the observed and expected mortalities in backcross.
3.3. Sex linkage or maternal effect The pyriproxyfen resistance in Pyri-Res strain decreased from 206-fold to 160-fold and 163-fold for F1 and F1 † , respectively
Table 1 Toxicity of insecticides to Pyri-Sus, Pyri-Field and Pyri-Res strains of Musca domestica. Insecticide Pyriproxyfen Methoxyfenozide Cyromazine Lufenuron Pyriproxyfen Methoxyfenozide Cyromazine Lufenuron Pyriproxyfen Methoxyfenozide Cyromazine Lufenuron * **
Strain
LC50 [95% CI] g mL−1
Slope ± SE
Pyri-Sus
0.01 (0.006–0.015) 0.04 (0.01–0.15) 0.003 (0.002–0.005) 0.001 (0.001–0.002) 0.26 (0.16–0.36) 0.29 (0.16–0.42) 0.02 (0.01–0.05) 0.03 (0.02–0.04) 2.06 (1.46–2.93) 0.97 (0.62–2.05) 0.10 (0.06–0.24) 0.09 (0.06–0.16)
1.39 0.60 1.39 1.44 1.51 1.23 0.94 1.12 1.53 1.11 1.12 1.31
Pyri-Field
Pyri-Res
± ± ± ± ± ± ± ± ± ± ± ±
0.28 0.25 0.28 0.27 0.28 0.27 0.25 0.26 0.27 0.26 0.27 0.28
Resistance ratio, calculated as LC50 of Pyri-Res, or Pyri-Field strain/LC50 of Pyri-Sus strain. Resistance ratio, calculated as LC50 of Pyri-Res strain/LC50 of Pyri-Field strain.
N
df
2
P
RR*
RR**
180 180 180 180 180 180 180 180 180 180 180 180
4 4 4 4 4 4 4 4 4 4 4 4
0.23 0.30 1.72 0.16 0.43 3.59 0.92 0.61 0.30 0.33 0.78 0.62
0.99 0.99 0.79 0.99 0.98 0.46 0.92 0.96 0.99 0.99 0.94 0.96
1 1 1 1 25.7 7.31 7.67 27 206 24.9 10.8 86
7.92 3.34 5 3
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R.M. Shah et al. / Acta Tropica 142 (2015) 149–155
Table 2 Toxicity of pyriproxyfen to Pyri-Sus, Pyri-Res, F1, F1† , BC1 and BC2 strains of Musca domestica. Strain
N*
LC50 g mL−1 [95% CI]
Slope ± SE
Pyri-Sus Pyri-Res F1 (Pyri-Res ♂ × Pyri-Sus ♀) F1 † (Pyri-Res ♀ × Pyri-Sus ♂) BC1 (F1 ♀ × Pyri-Sus ♂) BC2 (F1 ♀ × Pyri-Res ♂)
180 180 180 180 180 180
0.01 (0.006–0.02) 2.06 (1.46–2.93) 1.60 (1.08–2.26) 1.63 (0.87–2.71) 1.23 (0.71–1.81) 1.24 (0.87–1.65)
0.99 1.53 1.46 0.99 1.27 1.81
* ** ***
± ± ± ± ± ±
0.25 0.27 0.27 0.25 0.27 0.30
2
df
P
RR**
D***
0.21 0.30 0.25 2.05 0.68 0.47
4 4 4 4 4 4
0.99 0.98 0.99 0.73 0.95 0.98
1 206 160 163 123 124
– – 0.95 0.96 – –
Number of larvae used in the bioassay including control. Resistance ratio, calculated as LC50 of Pyri-Res, F1 , F1 † , BC1 , BC2 strains/LC50 of Pyri-Sus strain. Degree of dominance, ranged from 0 (completely recessive) to 1 (completely dominant).
Table 3 Effective dominance of resistance to pyriproxyfen in Pyri-Res strain of Musca domestica. Dose (g mL−1 )
Strain
Mortality (%)
DML *
0.5
Pyri-Sus Pyri-Res F1 Pyri-Sus Pyri-Res F1 Pyri-Sus Pyri-Res F1 Pyri-Sus Pyri-Res F1 Pyri-Sus Pyri-Res F1
100 16.7 29.2 100 33.3 58.3 100 50 75 100 63.3 83.3 100 83.3 95.5
0.75 Incompletely dominant
1
2
4
8
Table 4 Direct test of monogenic inheritance for pyriproxyfen resistance by chi-square analysis.
0.46 Incompletely recessive 0.50 Incompletely recessive
Concentration (g mL−1 )
Number exposed
Observed dead (%)
Expected dead (%)
2 (df = 1)
P*
0.5 1 2 4 8
30 30 30 30 30
7 (0.23)** 14 (0.47) 18 (0.60) 20 (0.83) 21 (0.93)
6 (0.20) 11 (0.37) 15.5 (0.52) 20 (0.68) 25.5 (0.85)
6.93 15.93 29.63 56.07 160.79
0.01
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Inheritance mode, cross-resistance and realized heritability of pyriproxyfen resistance in a field strain of Musca domestica L. (Diptera: Muscidae) Rizwan Mustafa Shah a,∗ , Naeem Abbas a , Sarfraz Ali Shad a,∗ , Marie Varloud b a b
Department of Entomology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Punjab, Pakistan Technical services, Parasitology, Companion Animal, Ceva, France
a r t i c l e
i n f o
Article history: Received 18 October 2014 Received in revised form 23 November 2014 Accepted 24 November 2014 Available online 3 December 2014 Keywords: IGRS Inheritance House fly Cross-resistance Realized heritability
a b s t r a c t Pyriproxyfen is a growth regulator used for the control of different insect pests, including Musca domestica. To assess the risk of resistance and to develop a strategy for resistance management, a field strain of M. domestica was exposed to pyriproxyfen in the laboratory for 30 generations. The inheritance mode, realized heritability of pyriproxyfen resistance and cross-resistance to other insecticides were assessed. Prior to the selection process, the field strain exhibited a resistance ratio (RR) of 25.7, 7.31, 7.67, and 27-fold for pyriproxyfen, methoxyfenozide, cyromazine and lufenuron, respectively, when compared to the pyriproxyfen susceptible strain (Pyri-Sus). After continuous selection with pyriproxyfen, the pyriproxyfen-resistant strain (Pyri-Res) became 206-fold more resistant than the Pyri-Sus strain. The overlapping confidence limits of LC50 values of F1 (Pyri-Res ♂ × Pyri-Sus ♀) and F1 † (Pyri-Res ♀ × Pyri-Sus ♂) suggested an autosomal and completely dominant mode of resistance to pyriproxyfen. Monogenic test of inheritance showed that resistance to pyriproxyfen was governed by multiple genes. The Pyri-Res strain showed very low cross resistance to methoxyfenozide, cyromazine, and lufenuron. The estimated realized heritability was 0.02, 0.05, 0.03 and 0.04 for pyriproxyfen, methoxyfenozide, cyromazine, and lufenuron, respectively. It was concluded that pyriproxyfen resistance in M. domestica was autosomally inherited, completely dominant and polygenic. These results would be helpful for the design of an improved control strategy against M. domestica. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The house fly, Musca domestica L., (Diptera: Muscidae) is a key pest causing significant losses in the livestock industry (Abbas et al., 2014a; Khan et al., 2014). M. domestica is also a probable vector of more than 100 diseases of human and animals (Fasanella et al., 2010; Förster et al., 2007). Moreover, outbreaks of diarrheal diseases have been associated with seasonal abundance of M. domestica in both urban and rural settings of countries including Pakistan (Graczyk et al., 2001; Khan and Akram, 2014). They are also vectors of bird flu virus, causing avian influenza which is a serious problem in poultry throughout the world (Barin et al., 2010; Wanaratana et al., 2011). It has been reported that millions of domestic poultry flocks are affected by avian influenza virus which resulted in more than 150 deaths of humans globally (Otte et al.,
∗ Corresponding authors. Tel.: +92 3416262699. E-mail addresses: [email protected] (R.M. Shah), [email protected] (S.A. Shad). http://dx.doi.org/10.1016/j.actatropica.2014.11.016 0001-706X/© 2014 Elsevier B.V. All rights reserved.
2007). In Punjab, Pakistan livestock farms, manure heaps provides ideal breeding sites for M. domestica. Massive fly densities cause nuisance to the farm workers and animals and reduce the aesthetic value of livestock, leading to economic losses (Acevedo et al., 2009). For decades, synthetic insecticides have been used as a principle control measure for this pest. However, resistance to insecticides in this pest has become a major issue due to the extensive and poorly optimized application of insecticides (Khan et al., 2013). M. domestica is ranked among the top 20 resistant pests of the world (Whalon et al., 2008) and resistance is reported from all over the world against almost all classes of insecticides (Abbas et al., 2014a,b; Acevedo et al., 2009; Bell et al., 2010; Deacutis et al., 2006; Kaufman et al., 2006; Khan et al., 2014; Kristensen and Jespersen, 2003; Shi et al., 2011; Tang et al., 2002). Pyriproxyfen is a juvenile hormone agonist that is an inhibitor of pupal adult metamorphosis (Seng et al., 2008) by causing abnormal growth and ultimately death of the larvae and is most effective at the onset of metamorphosis (Sullivan and Goh, 2008; Wilson et al., 2007). In addition, pyriproxyfen is highly selective for target organisms,
150
R.M. Shah et al. / Acta Tropica 142 (2015) 149–155
has low mammalian toxicity, and serves as a promising agent to be used in integrated management strategies of different pests including M. domestica (Geden and Devine, 2012). However, resistance to pyriproxyfen has already been reported in Bemisia tabaci (Gennadius) (Crowder et al., 2008; Ghanim and Kontsedalov, 2007; Horowitz et al., 2003; Karatolos et al., 2012; Ma et al., 2010) and M. domestica (Li et al., 1998; Pospischil et al., 1996; Zhang et al., 1997). The understanding of the mode of inheritance which characterize insecticide resistance is necessary for the sustainability of pest control (Abbas et al., 2014b; Bouvier et al., 2001). Information relative to dominance and number of genes involved in resistance will aid in our understanding of the development of resistance (Abbas et al., 2014b). The inheritance mode of pyriproxyfen resistance has been previously explored in B. tabaci (Crowder et al., 2008; Horowitz et al., 2003) and M. domestica (Zhang et al., 1997) which have demonstrated different genetics responses or resistance mechanisms. Insecticide resistance in natural populations may be monogenic or polygenic with a major phenotypic effect (Abbas et al., 2014b; Sayyed et al., 2008) but the mechanism of insecticide resistance can vary depending upon a type and geographical origin of the pest species (Khan et al., 2014). Moreover, insecticide resistance is not a fixed phenomenon and can change with time and space (Khan et al., 2014; Zhao et al., 2006). The current experiment was planned to investigate the inheritance pattern, cross-resistance to three other insecticides and realized heritability of pyriproxyfen resistance in M. domestica collected from a poultry farm. The focus of this study was to characterize resistance to commercial formulations which were being used for the control of M. domestica in Pakistan. The results will contribute to an optimized program of pyriproxyfen resistance management in M. domestica. 2. Materials and methods 2.1. Insects and rearing A sample population of 100–150 adults of M. domestica was collected by sweep netting from a poultry farm located in Multan, Pakistan (30◦ 5 11N, 71◦ 39 15E). The field collected population designated as Pyri-Field was maintained in meshed plastic jars (34 × 17 cm) in the laboratory. Adults were fed a mixture of powdered milk + sugar (1:1 ratio) and cotton wick soaked with water was provided in a separate Petri dish. Larvae were reared on an artificial media (Abbas et al., 2014a). All the insects were maintained in standard laboratory conditions Abbas et al. (2014a). The susceptible population designated as Pyri-Sus was collected from an area where no insecticides were used and was maintained in the laboratory for more than 40 generations without exposure to any insecticide. 2.2. Chemicals Commercial grade formulated insecticides including pyriproxyfen (Admiral® 10EC, FMC), lufenuron (Match® , 050EC, Syngenta), cyromazine (Trigard® , 75WP, Syngenta) and methoxyfenozide (Runner® , 240SC, Arista Life Sciences) were used. 2.3. Larvicidal bioassays A bioassay based on diet incorporation method (Kristensen and Jespersen, 2003) was used (with some modifications) to determine the toxicities of pyriproxyfen, methoxyfenozide, cyromazine and lufenuron. The larval rearing medium was made with wheat bran, grass meal, yeast, powdered milk, and sugar (20:5:5:1.5:1.5 ratio by weight) and was complemented with water containing
different concentrations of insecticides. Five concentrations for each bioassay were made through serial dilution method and each concentration was replicated three times. Ten first instar larvae collected at G1 were used in each replicate for a total of thirty larvae tested per insecticide concentration. A total of 180 larvae were used for each bioassay, including controls which consisted of only water added to the diet and kept at standard laboratory conditions as described above. Three weeks after the beginning of the larval bioassay, the rate of adult emergence was recorded (Cetin et al., 2006). All the larvae that were unable to develop into adults were considered as dead. 2.4. Selection for resistance Fist instar larvae from Pyri-Field strain (G1 ) were exposed to pyriproxyfen for 30 consecutive generations in the laboratory to generate a pyriproxyfen-resistant strain (Pyri-Res) using the diet incorporation method as described in the bioassay section. On average, 800 larvae were exposed in each generation during the selection experiment. The first generation (G1 ) was exposed to 0.2 g mL−1 of pyriproxyfen and doses were increased gradually for subsequent generations such that the last generation (G30 ) was exposed to 30 g mL−1 . 2.5. Genetic crosses Virgin male and female flies were separated within one day after eclosion to make crosses (Abbas et al., 2014a). Reciprocal crosses of Pyri-Sus and Pyri-Res were made to produce two lines: F1 (12 Pyri-Res ♂ × 12 Pyri-Sus ♀) and F1 † (12 Pyri-Res ♀ × 12 Pyri-Sus ♂). As there was overlapping of LC50 in reciprocal crosses, any line of these crosses was used for back cross. F1 was used for back crossing with the parental populations to generate the two backcross lines: BC1 (12 F1 ♀ × 12 Pyri-Sus ♂) and BC2 (12 F1 ♀ × 12 Pyri-Res ♂). 2.6. Realized heritability (h2 ) Following, the method of Falconer (1989) and Tabashnik (1992), the realized heritability (h2 ) of resistance to pyriproxyfen was estimated as follows: h2 =
R S
In the above equation, R (selection response) was estimated as follows: R=
log final LC50 − log initial LC50 n
where final LC50 means the LC50 of strain after 30 generations of selection, initial LC50 means the LC50 of the Pyri-Field strain before selection and n means number of generations selected with pyriproxyfen. Whereas, S (selection differential) was calculated as follows: S = i × p where i means intensity of selection calculated according to Falconer (1989), p means phenotypic standard deviation calculated as follows: p = [(inital slope + final slope) 0.5]−1 Based on the response of M. domestica to selection in the laboratory, the number of generations required for a tenfold increase in LC50 (G) was calculated as follows: G = R−1
R.M. Shah et al. / Acta Tropica 142 (2015) 149–155
2.7. Statistical analysis
Second, the Lande (1981) method was used to determine the number of genes involved in controlling the pyriproxyfen resistance by using the following formula:
2.7.1. Lethal concentration response data The control mortality was corrected by Abbot’s formula (Abbott, 1925), if occurred and then analyzed with POLO software (Software, 2005) by using probit analysis (Finney, 1971) to determine the LC50 values along with their confidence intervals (CI), slope and its standard error (SE). Resistance ratio (RR) was determined by dividing the LC50 of the Pyri-Res strain with the LC50 of the Pyri-Sus strain.
E =
8 2 S
3. Results 3.1. Toxicity of different insecticides to Pyri-Sus, Pyri-Field and Pyri-Res strains of Musca domestica
XPyri-Res − XPyri-Sus
Pyriproxyfen and methoxyfenozide were equally toxic to the Pyri-Sus (based on overlapping of 95% CI) but less toxic than cyromazine and lufenuron. Similarly, pyriproxyfen and methoxyfenozide were equally toxic to the Pyri-Field strain (based on overlapping of 95% CI) but less toxic than cyromazine and lufenuron (Table 1). Pyriproxyfen, methoxyfenozide, cyromazine and lufenuron were significantly less toxic to the Pyri-Field strain compared with the Pyri-Sus and led to a RR of 25.7-fold, 7.31-fold, 7.67-fold and 27-fold, respectively. All the tested insecticides were significantly less toxic to the Pyri-Res compared to the Pyri-Field and Pyri-Sus strains. After 30 generations of selection with pyriproxyfen, the Pyri-Res strain developed a RR of 206-fold and 7.92-fold compared to the Pyri-Sus and Pyri-Field strains (Table 1).
where XF1 , XPyri-Res , and XPyri-Sus are log LC50 values of the F1 , PyriRes and Pyri-Sus, respectively. Effective dominance (DML ) was calculated Bourguet et al. (2000) as follows: MTF1 − MTPyri-Sus MTPyri-Res − MTPyri-Sus
where MTPyri-Res , MTF1 , and MTPyri-Sus were the percent mortalities for the Pyri-Res, F1 , and Pyri-Sus strains to a single insecticide tested dose. 2.7.3. Number of genes involved The number of genes involved in controlling pyriproxyfen resistance was estimated by using two approaches. First, the null hypothesis of monogenic resistance based on a chi-square goodness of fit test between expected and observed mortalities was tested as follows: (Sokal and Rohlf, 1981) 2 =
2
In the above equation, 2 B1 , 2 B2 , 2 F1 , 2 XPyri-Sus , and 2 XPyri-Res were variances of the BC1 , BC2 , F1 , Pyri-Sus, and Pyri-Res strains, respectively.
2XF1 − XPyri-Res − XPyri-Sus
DML =
XPyri-Res − XPyri-Sus
where XPyri-Res and XPyri-Sus were the log LC50 of Pyri-Res and Pyri2S = Sus strains, respectively and 2 S was calculated as follows: 2 2 2 2 2 B1 + B2 − F1 + 0.5 XPyri-Sus + 0.5 XPyri-Res
2.7.2. Degree of dominance The dominance (DLC ) value of pyriproxyfen resistance was determined according to Bourguet and Raymond (1998) and Stone (1968) as follows: D=
151
3.2. Cross-resistance to other insecticides in the Pyri-Res strain The Pyri-Res strain at G30 was used to assess the cross-resistance of pyriproxyfen to methoxyfenozide, cyromazine and lufenuron compared to the Pyri-Field strain (Table 1). Results indicated that selection of pyriproxyfen significantly increased the resistance against all the tested insecticides (95% CI did not overlap). The Pyri-Res strain showed cross-resistance against methoxyfenozide (3.34-fold), cyromazine (5-fold) and lufenuron (3-fold) compared with the Pyri-Field strain.
(F − pn)2 pqn
In the above equation, F was the observed mortality in the backcross at a particular dose, n was the numbers exposed to a particular dose, p was the expected mortality at given dose and calculated Georghiou (1969) as 0.5 (number of F1 flies that died + number of Pyri-Sus flies that died)/number of flies exposed in backcross and q was calculated as 1 − p. The null hypothesis of monogenic resistance was rejected on the basis of significant difference between the observed and expected mortalities in backcross.
3.3. Sex linkage or maternal effect The pyriproxyfen resistance in Pyri-Res strain decreased from 206-fold to 160-fold and 163-fold for F1 and F1 † , respectively
Table 1 Toxicity of insecticides to Pyri-Sus, Pyri-Field and Pyri-Res strains of Musca domestica. Insecticide Pyriproxyfen Methoxyfenozide Cyromazine Lufenuron Pyriproxyfen Methoxyfenozide Cyromazine Lufenuron Pyriproxyfen Methoxyfenozide Cyromazine Lufenuron * **
Strain
LC50 [95% CI] g mL−1
Slope ± SE
Pyri-Sus
0.01 (0.006–0.015) 0.04 (0.01–0.15) 0.003 (0.002–0.005) 0.001 (0.001–0.002) 0.26 (0.16–0.36) 0.29 (0.16–0.42) 0.02 (0.01–0.05) 0.03 (0.02–0.04) 2.06 (1.46–2.93) 0.97 (0.62–2.05) 0.10 (0.06–0.24) 0.09 (0.06–0.16)
1.39 0.60 1.39 1.44 1.51 1.23 0.94 1.12 1.53 1.11 1.12 1.31
Pyri-Field
Pyri-Res
± ± ± ± ± ± ± ± ± ± ± ±
0.28 0.25 0.28 0.27 0.28 0.27 0.25 0.26 0.27 0.26 0.27 0.28
Resistance ratio, calculated as LC50 of Pyri-Res, or Pyri-Field strain/LC50 of Pyri-Sus strain. Resistance ratio, calculated as LC50 of Pyri-Res strain/LC50 of Pyri-Field strain.
N
df
2
P
RR*
RR**
180 180 180 180 180 180 180 180 180 180 180 180
4 4 4 4 4 4 4 4 4 4 4 4
0.23 0.30 1.72 0.16 0.43 3.59 0.92 0.61 0.30 0.33 0.78 0.62
0.99 0.99 0.79 0.99 0.98 0.46 0.92 0.96 0.99 0.99 0.94 0.96
1 1 1 1 25.7 7.31 7.67 27 206 24.9 10.8 86
7.92 3.34 5 3
152
R.M. Shah et al. / Acta Tropica 142 (2015) 149–155
Table 2 Toxicity of pyriproxyfen to Pyri-Sus, Pyri-Res, F1, F1† , BC1 and BC2 strains of Musca domestica. Strain
N*
LC50 g mL−1 [95% CI]
Slope ± SE
Pyri-Sus Pyri-Res F1 (Pyri-Res ♂ × Pyri-Sus ♀) F1 † (Pyri-Res ♀ × Pyri-Sus ♂) BC1 (F1 ♀ × Pyri-Sus ♂) BC2 (F1 ♀ × Pyri-Res ♂)
180 180 180 180 180 180
0.01 (0.006–0.02) 2.06 (1.46–2.93) 1.60 (1.08–2.26) 1.63 (0.87–2.71) 1.23 (0.71–1.81) 1.24 (0.87–1.65)
0.99 1.53 1.46 0.99 1.27 1.81
* ** ***
± ± ± ± ± ±
0.25 0.27 0.27 0.25 0.27 0.30
2
df
P
RR**
D***
0.21 0.30 0.25 2.05 0.68 0.47
4 4 4 4 4 4
0.99 0.98 0.99 0.73 0.95 0.98
1 206 160 163 123 124
– – 0.95 0.96 – –
Number of larvae used in the bioassay including control. Resistance ratio, calculated as LC50 of Pyri-Res, F1 , F1 † , BC1 , BC2 strains/LC50 of Pyri-Sus strain. Degree of dominance, ranged from 0 (completely recessive) to 1 (completely dominant).
Table 3 Effective dominance of resistance to pyriproxyfen in Pyri-Res strain of Musca domestica. Dose (g mL−1 )
Strain
Mortality (%)
DML *
0.5
Pyri-Sus Pyri-Res F1 Pyri-Sus Pyri-Res F1 Pyri-Sus Pyri-Res F1 Pyri-Sus Pyri-Res F1 Pyri-Sus Pyri-Res F1
100 16.7 29.2 100 33.3 58.3 100 50 75 100 63.3 83.3 100 83.3 95.5
0.75 Incompletely dominant
1
2
4
8
Table 4 Direct test of monogenic inheritance for pyriproxyfen resistance by chi-square analysis.
0.46 Incompletely recessive 0.50 Incompletely recessive
Concentration (g mL−1 )
Number exposed
Observed dead (%)
Expected dead (%)
2 (df = 1)
P*
0.5 1 2 4 8
30 30 30 30 30
7 (0.23)** 14 (0.47) 18 (0.60) 20 (0.83) 21 (0.93)
6 (0.20) 11 (0.37) 15.5 (0.52) 20 (0.68) 25.5 (0.85)
6.93 15.93 29.63 56.07 160.79
0.01
