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Cyromazine resistance in a field strain of house flies, Musca domestica L.: Resistance risk assessment and bio-chemical mechanism
Chemosphere 167 (2017) 308e313
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Cyromazine resistance in a field strain of house flies, Musca domestica L.: Resistance risk assessment and bio-chemical mechanism Hafiz Azhar Ali Khan a, *, Waseem Akram b a b
Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan Department of Entomology, University of Agriculture, Faisalabad, Pakistan
h i g h l i g h t s Rapid development of resistance to cyromazine was observed as a result of selection experiments. Cyromazine resistance in the CYR-SEL strain was unstable. The CYR-SEL strain showed lack of cross-resistance to pyriproxyfen, diflubenzuron, or mehoxyfenozide. Metabolic resistance mechanism responsible in developing cyromazine resistance in the CYR-SEL strain.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 August 2016 Received in revised form 4 October 2016 Accepted 5 October 2016
Developing resistance management strategies for eco-friendly insecticides is essential for the management of insect pests without harming the environment. Cyromazine is a biorational insecticide with very low mammalian toxicity. Resistance to cyromazine has recently been reported in house flies from Punjab, Pakistan. In order to propose a resistance management strategy for cyromazine, experiments were planned to study risk for resistance development, possibility of cross-resistance and bio-chemical mechanisms. A field strain of house flies with 8.78 fold resistance ratio (RR) to cyromazine was reselected under laboratory conditions. After seven rounds of selection (G1-G7), the RR values rapidly increased from 8.8 to 211 fold. However, these values declined to 81fold when the cyromazine selected (CYR-SEL) strain was reared without selection pressure, suggesting an unstable nature of resistance. The CYR-SEL strain showed lack of cross-resistance to pyriproxyfen, diflubenzuron, and methoxyfenozide. Synergism bioassays using enzyme inhibitors: piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF), and metabolic enzyme analyses revealed increased activity of carboxylesterase (CarE) and mixed-function oxidase (MFO) in the CYR-SEL strain compared to the laboratory susceptible (Labsusceptible) strain, suggesting the metabolic resistance mechanism responsible for cyromazine resistance in the CYR-SEL strain. In conclusion, risk of rapid development of cyromazine resistance under consistent selection pressure discourages the sole reliance on cyromazine for controlling house flies in the field. The unstable nature of cyromazine resistance provides window for restoring cyromazine susceptibility by uplifting selection pressure in the field. Moreover, lack of cross-resistance between cyromazine and pyriproxyfen, diflubenzuron, or methoxyfenozide in the CYR-SEL strain suggest that cyromazine could be rotated with these insecticides whenever resistance crisis occur in the field. © 2016 Published by Elsevier Ltd.
Handling Editor: David Volz Keywords: Ecotoxicology Biorational insecticides Risk assessment Urban pest management
1. Introduction The use of insecticides is an integral part of sustainable agriculture and livestock production for the management of insect pests. However, a number of problems arise when insect pests
* Corresponding author. E-mail address: [email protected] (H.A.A. Khan). http://dx.doi.org/10.1016/j.chemosphere.2016.10.018 0045-6535/© 2016 Published by Elsevier Ltd.
develop resistance against insecticides, since the farming community shifts from label recommendations to over-dosage of insecticides for the management of resistant insect pests (Hemingway and Ranson, 2000). Few of the issues which results from this shift of the farming community are: the severity of the insecticide resistance problem, insect pest resurgence, environmental pollution and ill-effects on public health. The most probable solution to all these issues is the wise use of insecticides and
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development of insecticide resistance management programs (Khan et al., 2013a, 2013b). House flies (Diptera: Muscidae) are serious pests of public health importance and transmit a number of deadly diseases like diarrhea, cholera etc. (Malik et al., 2007). Being polyphagous, there are a number of feeding and breeding hosts known for their rapid expansion. Of these, the hosts available at dairies or livestock production units i.e., animal or farmyard manure, have been assumed as the potential multiplication source for house flies, and infestations in nearby communities (Learmount et al., 2002; Khan et al., 2012). In this situation, chemical measures, being quick and easy to apply, become the choice of people facing house flies infestations. However, house flies have the ability to become resistant against insecticides used for their management usually in a short period of time (Kaufman et al., 2010). From the available literature, insecticide resistance to different classes of insecticides (organochlorine, organophosphate, carbamate, pyrethroid, new chemical insecticides) has been reported around the globe (Liu and Yue, 2000; Learmount et al., 2002; Kristensen and Jespersen, 2003, 2004; Kaufman et al., 2006; Acevedo et al., 2009). To combat resistance issue, victim people increase the dosage of insecticide with ultimate effects on public health and environmental pollution. The reaction of people to the above problems is same in Pakistan, and resistance in house flies against different insecticides has been reported the first time in 2013 (Khan et al., 2013a, 2013b), likely a product of excessive insecticide usage. Cyromazine is a biorational insecticide and belongs to Insect Growth Regulator (IGR) class with novel mode of action (Lau et al., 2015). The insecticides belong to IGR class are quite selective in their action, have very low mammalian toxicity, and potentially toxic to target species only (Tunaz and Uygun, 2004). In livestock production units, cyromazine is recommended to control the manure-breeding house flies in two ways: spray application over manure heaps or feed through larvicide (Pinto and do Prado, 2001). Unfortunately, cases of resistance to cyromazine in house flies have also been reported worldwide (Shen and Plapp, 1990; Pinto and do Prado, 2001; Tang et al., 2002; Kristensen and Jespersen, 2003; Acevedo et al., 2009). Recently, a notable level of resistance to cyromazine in house flies has been reported from Pakistan (Khan et al., 2016a); however, there is a lack of information like the rapidity with which cyromazine resistance develops, crossresistance phenomenon, and mechanism of resistance development. All these information are necessary to make an effective cyromazine resistance management plan, and to save the effectiveness of this insecticide for a long period of time. Therefore, the aims of the present study were to explore all these missing information through selection experiments, and to propose a cyromazine resistance management plan. 2. Materials and methods The methodology section has been adapted from the authors' previous work (Khan et al., 2015a, 2015b, 2016a) with some modifications: 2.1. Insects Two strains were used in the present study: a laboratory susceptible (Lab-susceptible) reference strain reared in the laboratory without exposure to any insecticide and a field strain collected from a dairy farm near Lahore (31 320 59 N; 74 200 37 E), Punjab, Pakistan, and was consecutively selected in the laboratory with cyromazine to generate a cyromazine selected strain (CYR-SEL). Both of the strains were reared in the laboratory (25 ± 2 C, 65 ± 5% RH, 12: 12 h (L: D) photoperiod) as discussed previously (Khan et al.,
309
2013a). The experiments were conducted at the Institute of Agricultural Sciences, University of the Punjab, Lahore. 2.2. Chemicals Four technical-grade insecticides (cyromazine, diflubenzuron, pyriproxyfen and methoxyfenozide; Chem Service Inc, West Chester PA) were used for toxicological evaluations. In addition, two synergists, S,S,S-tributylphosphorotrithioate (DEF; Chem Service Inc, West Chester PA), and piperonyl butoxide (PBO; Chem Service Inc, West Chester PA), were also used in synergism studies. 2.3. Selection and bioassays The field strain of house flies was selected with cyromazine for seven consecutive generations. Briefly, the selection and insecticidal bioassays were performed by using larval medium. Newly hatched 1st instar larvae were placed on the surface of the media treated with cyromazine (70% mortality level) for seven consecutive generations, and the survivors were used as the parents of the next generation. The numbers of larvae selected in each generation were ranged between 500 and 600 (Table 2). To test the toxicity of IGR insecticides, larval bioassays were performed by following the protocol described by Cetin et al. (2009) with few modifications. In short, 5e7 serial dilutions of each insecticide, with >0% mortality at the lowest dilution and <100% mortality at the highest dilution, were prepared in distilled water. Twenty gram larval media were taken in a plastic cup (z100 mL capacity) and treated with 2.5 mL of serial dilution of IGR insecticide or water alone in control to monitor background mortality. Twenty newly hatched 1st instar larvae were placed on the surface of treated media with the help of a camel hairbrush. The full IGR insecticide bioassay was replicated three times and the number of larvae per dilution was 60. All the bioassays were conducted at 26 ± 2 C, 60 ± 5% RH and continuous light until the emergence of house flies. Final mortality was recorded three-week post-treatment and the larvae/pupae unable to develop into adults were considered dead. To study the stability of cyromazine resistance in the selected strain of house flies, the selection of the strain was stopped at G7 and it was reared for the next seven generations without exposure to any insecticide. Then the strain was bioassayed again with cyromazine and the stability of resistance was assessed by the following formula (Sayyed et al., 2005):
R ¼ ½log ðfinal LC50 Þ log ðinitial LC50 Þ=n Where, n is the number of generations reared without exposure to insecticide. The field strain was bioassayed with pyriproxyfen, diflubenzuron, and metjoxyfenozide before and after the selection experiment (at G0 and G7, respectively) to assess cross-resistance mechanism. All the mortality data were analyzed by Probit analysis using the software SPSS (version 10.0) to determine median lethal concentrations (LC50) and their 95% confidence intervals (CIs). The LC50 values of the respective bioassays were considered significantly different on the basis of non-overlapping of 95% CIs (Litchfield and Wilcoxon, 1949). 3. Cyromazine resistance mechanism To study the mechanism of cyromazine resistance, enzyme inhibitors PBO and DEF diluted in acetone were used at the maximum sublethal level and mixed with insecticide dilutions. The resultant mixture was added in the larval medium for bioassays as stated above. The enzyme analyses were performed as described
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4.3. Cross-resistance analysis
previously (Khan et al., 2015a): Analyses were done by using 3e5-day old female house flies of both Lab-susceptible and CYR-SEL strains, separately. Individual female house flies were separated and homogenized in 1 mL of sodium phosphate buffer (0.1 M; pH 7.8) at 4 C. The homogenates were centrifuged at 10,000g at 4 C for 10 min, and large fragments were then removed (Kristensen, 2005). The supernatant was used immediately for assaying the activities of carboxylesterase (CarE), mixed function oxidase (MFO), and glutathione S-transferase (GST) by using the methodologies of Gao et al. (2014) for CarE and MFO, Yang et al. (2004) for the determination of GST activity, and Bradford (1976) for measuring total proteins.
The field strain before the start of cyromazine resistance was bioassayed against pyriproxyfen, diflubenzuron, and methoxyfenozide. The LC50 values were 0.47, 0.68, and 0.51 mg mL1 for pyriproxyfen, diflubenzuron, and methoxyfenozide, respectively (Table 4). The field strain (CYR-SEL), after seven rounds of selection with cyromazine, was again bioassayed at G7 against pyriproxyfen, diflubenzuron, and methoxyfenozide. The results revealed that the LC50 values against these three insecticides before and after the cyromazine selection experiments were statistically at (overlapping 95% CIs) suggesting lack of cross-resistance.
4. Results
4.4. Synergism bioassays and metabolic enzyme activities
4.1. Toxicity of insecticides against laboratory and field strains before selection experiment
Synergism bioassays were performed by using enzyme inhibitors (PBO and DEF) in combination with cyromazine. The enzyme inhibitors did not increase the toxicity of cyromazine in the Lab-susceptible strain. However, a significant increase in toxicity in the presence of enzyme inhibitors was observed in the CYR-SEL strain, suggesting the involvement of metabolic enzymes in the development of cyromazine resistance. The LC50 values of the CYRSEL strain were 19.03, 4.13, and 5.73 mg mL1 for cyromazine alone, cyromazine þ DEF, and cyromazine þ PBO, respectively (Table 5). Metabolic enzyme analyses revealed significantly increased activity of CarE and MFO in the CYR-SEL strain compared to the Labsusceptible strain, further confirming the involvement of metabolic enzymes in the development of cyromazine resistance (Table 6).
All the tested insecticides showed statistically equal toxicity (95% overlapping CIs) against Lab-susceptible reference strain. The LC50 values for cyromazine, pyriproxyfen, diflubenzuron and methoxyfenozide were 0.09, 0.08, 0.07, and 0.06 mg mL1, respectively (Table 1). However, the field strain showed less susceptibility to these insecticides compared to the Lab-Sus strain. The LC50 values of the field strain for cyromazine, pyriproxyfen, diflubenzuron and methoxyfenozide were 0.79, 0.47, 0.68, and 0.51 mg mL1, respectively. In comparison to the Lab-susceptible strain, the field strain before the selection experiment showed 8.8, 5.9, 9.7, and 8.5 fold resistance (based on RR values) against cyromazine, pyriproxyfen, diflubenzuron, and methoxyfenozide, respectively (Table 1). 4.2. Cyromazine selection experiment The field strain showing 8.78 fold RR to cyromazine was further selected under laboratory conditions (Table 2). The LC50 values significantly increased after seven rounds of selection (Table 3). The LC50 values were 1.22, 1.80, 2.60, 5.25, 7.30, 10.49, and 19.03 mg mL1 from G1 to G7, respectively. There was a rapid increase in the RR values as a result of selection experiment. The RR values jumped from 8.8 to 211fold after seven rounds of selection with cyromazine. The cyromazine selected strain (CYR-SEL) was reared for the next seven generations without exposure to insecticide and bioassayed again at G14 with cyromazine. The result revealed a rapid decline in resistance (the RR values declined from 211 to 81 fold) was observed indicating an unstable nature of cyromazine resistance in the selected strain (Table 3).
5. Discussion Keeping in view the increasing problem of insecticide resistance and the ultimate effect on environmental pollution (Khan et al., 2016b), it's the demand of time to develop resistance management strategies so as to retain the effectiveness of insecticides for long. Cyromazine is an IGR insecticide; however, cases of resistance to cyromazine necessitate studying the nature of resistance and development of management plans. In the present research work, the field strain of house flies before the start of laboratory selection experiments exhibited resistance to cyromazine and three other IGRs which further confirmed the previous report (Khan et al., 2016a) on the field evolved resistance to IGRs in house flies. Resistance to cyromazine has been reported previously in different insect pest like M. domestica (Sheppard et al., 1992; Pinto and do Prado, 2001; Learmount et al., 2002; Kristensen and Jespersen, 2003; Acevedo et al., 2009), Lucilia cuprina (Levot, 2012), Liriomyza trifolii (Ferguson, 2004), and Drosophila melanogaster
Table 1 Toxicity of different insecticides against laboratory susceptible reference and newly collected field strains of house flies. Strain
Lab-susceptible Lab-susceptible Lab-susceptible Lab-susceptible Field (G0) Field (G0) Field (G0) Field (G0)
Insecticide
Cyromazine Pyriproxyfen Diflubenzuron Methoxyfenozide Cyromazine Pyriproxyfen Diflubenzuron Methoxyfenozide
na
420 420 420 420 360 420 360 420
LC50b (95% CIc) (mg mL1)
0.09 0.08 0.07 0.06 0.79 0.47 0.68 0.51
(0.06e0.12) (0.05e0.12) (0.06e0.08) (0.04e0.07) (0.65e0.98) (0.39e0.56) (0.55e0.82) (0.42e0.63)
The same applies to the following tables. a n¼ number of flies used in bioassays and controls. b LC50, median lethal concentration (in microgram per millilitre) estimated to give 50 % mortality. c CI, confidence intervals. d RR, resistance ratio¼ LC50 of field strain /LC50 of Lab-susceptible strain.
RRd
Fit of probit line Slope (SE)
2
c
df
p
2.36 2.29 2.34 2.55 1.82 1.99 1.85 1.73
7.67 9.67 3.38 3.63 1.50 4.70 1.73 2.46
4 4 4 4 3 4 3 4
0.11 0.06 0.50 0.45 0.68 0.32 0.63 0.65
(0.19) (0.19) (0.20) (0.22) (0.21) (0.18) (0.20) (0.16)
8.8 5.9 9.7 8.5
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Table 2 Selection concentrations and survival rate of the field strain in cyromazine selections on successive generations. Selected generation
Concentration (mg mL1)
No. larvae selected
G1 G2 G3 G4 G5 G6 G7
544 500 580 600 510 500 550
Survival (number)
1.55 2.24 3.47 4.82 12.50 21.22 37.81
Male
Female
88 71 80 94 75 76 86
96 90 83 110 78 85 90
Table 3 Resistance selection of the field strain of house flies with cyromazine. Strain
Field (G0) G1 G2 G3 G4 G5 G6 G7(CYR-SEL) G14
LC50 (95% CI) (mg mL1)
n
360 360 420 420 360 420 420 420 420
0.79 (0.65e0.98) 1.22 (1.01e1.52) 1.80 (1.48e2.22) 2.60 (2.14e3.25) 5.25 (4.10e7.50) 7.30 (5.74e10.05) 10.49 (8.47e13.53) 19.03 (15.50e24.15) 7.31 (6.22e8.55)
Fit of probit line
RR
Slope (SE)
c
2
df
p
1.82 1.99 1.84 1.95 1.98 1.67 1.71 1.76 2.41
1.50 2.23 0.60 3.56 3.15 3.84 6.71 3.94 4.92
3 3 4 4 3 4 4 4 4
0.68 0.53 0.96 0.47 0.37 0.43 0.15 0.42 0.29
(0.21) (0.22) (0.17) (0.20) (0.27) (0.19) (0.18) (0.18) (0.20)
8.8 13.6 20 28.9 58.8 81 116.6 211 81
G7 ¼ Cyromazine selected strain after seven generations of continuous selection. G14 ¼ CYR-SEL strain after seven generations without any insecticide exposure.
Table 4 Cross-resistance analysis of the CYR-SEL strain of house flies to other insecticides. Insecticide
LC50 (95% CI) (mg mL1)
n
Cyromazine (G0) Cyromazine (CYR-SEL) Pyriproxyfen (G0) Pyriproxyfen (CYR-SEL) Diflubenzuron (G0) Diflubenzuron (CYR-SEL) Methoxyfenozide (G0) Methoxyfenozide (CYR-SEL)
360 420 420 420 360 420 420 420
Fit of probit line
0.79 (0.65e0.98) 19.03 (15.50e24.15) 0.47 (0.39e0.56) 0.54 (0.46e0.64) 0.68 (0.55e0.82) 0.76 (0.63e0.94) 0.51 (0.42e0.63) 0.55 (0.44e0.69)
RR
Slope (SE)
c
2
df
p
1.82 1.76 1.99 2.21 1.85 1.73 1.73 1.62
1.50 3.94 4.70 5.20 1.73 3.52 2.46 1.28
3 4 4 4 3 4 4 4
0.68 0.42 0.32 0.27 0.63 0.48 0.65 0.87
(0.21) (0.18) (0.18) (0.19) (0.20) (0.16) (0.16) (0.16)
8.78 211.44 5.88 6.75 9.71 10.85 8.50 9.17
Table 5 Effect of enzyme inhibitors on the toxicity of cyromazine against house flies strains. Strain
Laboratory
CYR-SEL (G7)
a
Compound
n
Cyromazine Cyromazine Cyromazine Cyromazine Cyromazine Cyromazine
þ DEF þ PBO þ DEF þ PBO
420 420 420 420 420 420
LC50 (95% CI) (mg mL1)
0.09 (0.06e0.12) 0.08 (0.07e0.10) 0.10 (0.08e0.11) 19.03 (15.50e24.15) 4.13 (3.39e4.93) 5.73 (4.63e6.97)
SRa
Fit of probit line Slope (SE)
c
2
df
p
2.36 1.91 1.86 1.76 2.10 1.71
7.67 3.53 3.62 3.94 3.56 5.51
4 4 4 4 4 4
0.11 0.47 0.46 0.42 0.47 0.24
(0.19) (0.17) (0.17) (0.18) (0.20) (0.16)
1.13 0.90 4.61 3.32
SR ¼ LC50 of cyromazine alone/LC50 of cyromazine along with an enzyme inhibitor.
Table 6 Metabolic enzyme activities in the laboratory susceptible and CYR-SEL strains of house flies. Strain
Lab-susceptible CYR-SEL a
CarE
MFO
Activity ±SE nmol min1 mg1
Ratioa
Activity ±SE pmol min1 mg1
Ratioa
133.83 ± 10.11 212.67 ± 5.75
1.59
68.99 ± 7.20 176.67 ± 9.29
2.56
Enzyme activity in the CYR-SEL strain/enzyme activity in the laboratory susceptible strain.
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(Daborn et al., 2000). There could be different reasons for the development of the field evolved resistance to cyromazine. For example, Acevedo et al. (2009) reported that regional chemical use strategies, and over dosing other than the label recommendations by the farming community and lack of monitoring surveys were the main reasons for the cyromazine resistance in house flies in Argentina. Similarly, Khan et al. (2016a) reported that the excessive sprays of IGRs in cropping areas around the dairy facilities was the main reason for the occurrence of cyromazine resistance in house flies from Punjab, Pakistan. Whereas, treatment of animal manure with cyromazine rather than feedthrough application for a long time was the main reason for the development of resistance to cyromazine in Denmark (Kristensen and Jespersen, 2003). In the present study, the field strain of house flies with 8.8 fold resistance to cyromazine was re-selected under the laboratory conditions in order to study the mechanism of resistance and crossresistance potential to other insecticides. The results revealed a rapid development of resistance to cyromazine after seven rounds of selection and the RR values increased from 8.8 to 211fold. Laboratory selection with cyromazine in different insects has been studied in the past in an attempt to study the rapidity of resistance development. For example, L. cuprina from New South Wales Monaro was selected under the laboratory conditions for 13 generations, but the development of resistance was very slow i.e., the RR values increased from 2.3 to 8.1 fold (Levot, 2013). This might be due to the spatio-temporal variations, insect species in question, insecticide exposure history or the combination of all these factors (Khan et al., 2015a, 2015b). In our study, resistance to cyromazine was unstable in the selected strain when it reared without selection pressure. The resistance was declined from 211 to 81 fold over a period of seven generations. The unstable nature of cyromazine resistance has also been reported in the leafminer, L. trifolii (Ferguson, 2004), when it was reared in the absence of selection pressure. There are a number of cases where resistance have been reverted when the selected pests were reared in the absence of pesticide selection pressure (Flexner et al., 1989; Kristensen et al., 2000; Khan et al., 2015b). Fitness cost and initial gene frequencies have been assumed responsible for the reversion of resistance in insects under laboratory conditions (Roush and Mckenzie, 1987; Khan et al., 2014); however, further studies are required to confirm the phenomenon of fitness cost responsible for the reversion of resistance in the cyromazine selected strain of house flies. The cross-resistance potential of the insecticide selected strains is a valuable tool in order to screen out alternate insecticides for rotational use when resistance crisis become severe in the field. In this way, the insecticides to which selected strains don't show cross-resistance can be a part of resistance management strategy to a particular insecticide (Georghiou and Taylor, 1977). In the present research work, the CYR-SEL strain of house flies didn't show cross resistance to diflubenzuron, pyriproxyfen, and methoxyfenozide. Before the start of selection experiment, the LC50 values were 0.47, 0.68, and 0.51 mg mL1 for pyriproxyfen, diflubenzuron, and methoxyfenozide, respectively. Whereas, after the selection experiment, the CYR-SEL strain showed statistically non-significant change in the LC50 values to the above mentioned three insecticides. These results indicate the above insecticides could be used in rotation to cyromazine for the effective management of house flies. Previously, the lack of cross-resistance in the cyromazine selected strains of D. melanogaster (Wilson, 1997), L. cuprina (Levot, 2013), and M. domestica (Khan et al., 2016a) has been reported. Cross-resistance between different insecticide could be expected either due to the structural similarity or common resistance mechanism (Kristensen and Jespersen, 2003). Being in different insecticide classes, the cross-resistance between
cyromazine and diflubenzuron, pyriproxyfen or methoxyfenozide was not expected (Kristensen and Jespersen, 2003; Committee, 2007; Sparks and Nauen, 2015). Cyromazine is a moulting disruptor and belongs to IRAC main group #17; diflubenzuron is an inhibitor of chitin biosynthesis and belongs to the IRAC main group # 15; pyriproxyfen is a juvenile hormone mimic and belongs to IRAC main group # 05; methoxyfenozide is an ecdysone agonist and belongs to IRAC main group # 18. In an attempt to find out the mechanism of cyromazine resistance in the CYR-SEL strain, cyromazine toxicity was assessed in the presence of an enzyme inhibitor (PBO or DEF). A significant increase in toxicity in the presence of enzyme inhibitors was observed in the CYR-SEL strain, suggesting the involvement of metabolic enzymes in the development of cyromazine resistance. This was further confirmed by enzyme analyses which revealed enhanced activities of CarE and MFO in the CYR-SEL strain of house flies. Previously, cyromazine resistance in house flies from Denmark has been reported to be linked with increased activities of cytochrome P450 monooxygenase and glutathione S-transferase (Kristensen and Jespersen, 2003). In conclusion, the study reveals a rapid rate of resistance development to cyromazine in the selected strain of house flies. Resistance developed under the continuous selection pressure which pointed out the possibility of resistance development in the field if spray applications or selection pressure of cyromazine persist for long. However, the unstable nature of cyromazine resistance provides the opportunity to restore its toxicity by discontinuing its use for a certain period of time, if resistance crisis occur in the field. Moreover, lack of cross-resistance to the other insecticides tested (pyriproxyfen, diflubenzuron and methoxyfenozide) suggest that these products could be used in rotation and/or as an alternate to reduce the possibility of resistance development to cyromazine in the field. References Acevedo, G.R., Zapater, M., Toloza, A.C., 2009. Insecticide resistance of house fly, Musca domestica (L.) from Argentina. Parasitol. Res. 105, 489e493. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Cetin, H., Erler, F., Yanikoglu, A., 2009. Survey of insect growth regulator (IGR) resistance in house flies (Musca domestica L.) from southwestern Turkey. J. Vector Ecol. 34, 329e337. Committee, I.R.A., 2007. IRAC Mode of Action Classification (Updated July). iraconline.org. Daborn, P.J., McKenzie, J.A., Batterham, P., 2000. A genetic analysis of cyromazine resistance in Drosophila melanogaster (Diptera : Drosophilidae). J. Econ. Entomol. 93, 911e919. Ferguson, J.S., 2004. Development and stability of insecticide resistance in the leafminer Liriomyza trifolii (Diptera: Agromyzidae) to cyromazine, abamectin, and spinosad. J. Econ. Entomol. 97, 112e119. Flexner, J., Theiling, K., Croft, B., Westigard, P., 1989. Fitness and immigration: factors affecting reversion of organotin resistance in the twospotted spider mite (Acari: Tetranychidae). J. Econ. Entomol. 82, 996e1002. Gao, C.-F., Ma, S.-Z., Shan, C.-H., Wu, S.-F., 2014. Thiamethoxam resistance selected in the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae): cross-resistance patterns, possible biochemical mechanisms and fitness costs analysis. Pestic. Biochem. Physiol. 114, 90e96. Georghiou, G.P., Taylor, C.E., 1977. Genetic and biological influences in evolution of insecticide resistance. J. Econ. Entomol. 70, 319e323. Hemingway, J., Ranson, H., 2000. Insecticide resistance in insect vectors of human disease. Annu. Rev. Entomol. 45, 371e391. Kaufman, P.E., Gerry, A.C., Rutz, D.A., Scott, J.G., 2006. Monitoring susceptibility of house flies (Musca domestica L.) in the United States to imidacloprid. J. Agric. Urban Entomol. 23, 195e200. Kaufman, P.E., Nunez, S.C., Mann, R.S., Geden, C.J., Scharf, M.E., 2010. Nicotinoid and pyrethroid insecticide resistance in houseflies (Diptera: Muscidae) collected from Florida dairies. Pest Manag. Sci. 66, 290e294. Khan, H.A., Akram, W., Iqbal, J., Naeem-Ullah, U., 2015a. Thiamethoxam resistance in the house fly, Musca domestica L.: current status, resistance selection, crossresistance potential and possible biochemical mechanisms. PLoS One 10, e0125850. Khan, H.A., Akram, W., Iqbal, N., 2015b. Selection and Preliminary mechanism of
H.A.A. Khan, W. Akram / Chemosphere 167 (2017) 308e313 resistance to profenofos in a field strain of Musca domestica (Diptera: Muscidae) from Pakistan. J. Med. Entomol. 52, 1013e1017. Khan, H.A., Akram, W., Shad, S.A., 2013a. Resistance to conventional insecticides in Pakistani populations of Musca domestica L. (Diptera: Muscidae): a potential ectoparasite of dairy animals. Ecotoxicology 22, 522e527. Khan, H.A., Akram, W., Shad, S.A., 2014. Genetics, cross-resistance and mechanism of resistance to spinosad in a field strain of Musca domestica L. (Diptera: Muscidae). Acta Trop. 130, 148e154. Khan, H.A., Shad, S.A., Akram, W., 2012. Effect of livestock manures on the fitness of house fly, Musca domestica L. (Diptera: Muscidae). Parasitol. Res. 111, 1165e1171. Khan, H.A., Shad, S.A., Akram, W., 2013b. Resistance to new chemical insecticides in the house fly, Musca domestica L., from dairies in Punjab. Pak. Parasitol. Res. 112, 2049e2054. Khan, H.A.A., Akram, W., Arshad, M., Hafeez, F., 2016a. Toxicity and resistance of field collected Musca domestica (Diptera: Muscidae) against insect growth regulator insecticides. Parasitol. Res. 115, 1385e1390. Khan, H.A.A., Akram, W., Khan, T., Haider, M.S., Iqbal, N., Zubair, M., 2016b. Risk assessment, cross-resistance potential, and biochemical mechanism of resistance to emamectin benzoate in a field strain of house fly (Musca domestica Linnaeus). Chemosphere 151, 133e137. Kristensen, M., 2005. Glutathione S-transferase and insecticide resistance in laboratory strains and field populations of Musca domestica. J. Econ. Entomol. 98, 1341e1348. Kristensen, M., Jespersen, J.B., 2003. Larvicide resistance in Musca domestica (Diptera: Muscidae) populations in Denmark and establishment of resistant laboratory strains. J. Econ. Entomol. 96, 1300e1306. Kristensen, M., Jespersen, J.B., 2004. Susceptibility of spinosad in Musca domestica (Diptera: Muscidae) field populations. J. Econ. Entomol. 97, 1042e1048. Kristensen, M., Knorr, M., Spencer, A.G., Jespersen, J.B., 2000. Selection and reversion of azamethiphos-resistance in a field population of the housefly Musca domestica (Diptera: Muscidae), and the underlying biochemical mechanisms. J. Econ. Entomol. 93, 1788e1795. Lau, K.W., Chen, C.D., Lee, H.L., Norma-Rashid, Y., Sofian-Azirun, M., 2015. Evaluation of insect growth regulators against field-collected Aedes aegypti and Aedes albopictus (Diptera: Culicidae) from Malaysia. J. Med. Entomol. 52, 199e206. Learmount, J., Chapman, P., MacNicoll, A., 2002. Impact of an insecticide resistance strategy for house fly (Diptera : Muscidae) control in intensive animal units in the United Kingdom. J. Econ. Entomol. 95, 1245e1250.
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Levot, G., 2012. Cyromazine resistance detected in Australian sheep blowfly. Aust. Vet. J. 90, 433e437. Levot, G., 2013. Response to laboratory selection with cyromazine and susceptibility to alternative insecticides in sheep blowfly larvae from the New South Wales Monaro. Aust. Vet. J. 91, 61e64. Litchfield, J.a., Wilcoxon, F., 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96, 99e113. Liu, N., Yue, X., 2000. Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae). J. Econ. Entomol. 93, 1269e1275. Malik, A., Singh, N., Satya, S., 2007. House fly (Musca domestica): a review of control strategies for a challenging pest. J. Environ. Sci. Health Part B 42, 453e469. Pinto, M.C., do Prado, A.P., 2001. Resistance of Musca domestica L. populations to cyromazine (insect growth regulator) in Brazil. Mem. Inst. Oswaldo Cruz 96, 729e732. Roush, R.T., Mckenzie, J.A., 1987. Ecological genetics of insecticide and Acaricide resistance. Annu. Rev. Entomol. 32, 361e380. Sayyed, A.H., Gatsi, R., Ibiza-Palacios, M.S., Escriche, B., Wright, D.J., Crickmore, N., 2005. Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac. Appl. Environ. Microbiol. 71, 6863e6869. Shen, J., Plapp, F., 1990. Cyromazine resistance in the house Fly (Diptera: Muscidae): genetics and cross-resistance to difluhenzuron. J. Econ. Entomol. 83, 1689e1697. Sheppard, D.C., Gaydon, D.M., Miller, R.W., 1992. Resistance in house-flies (Diptera, Muscidae) selected with 50 Ppm feed-through cyromazine. J. Agric. Entomol. 9, 257e260. Sparks, T.C., Nauen, R., 2015. IRAC: mode of action classification and insecticide resistance management. Pestic. Biochem. Physiol. 121, 122e128. Tang, J.D., Caprio, M.A., Sheppard, D.C., Gaydon, D.M., 2002. Genetics and fitness costs of cyromazine resistance in the house fly (Diptera: Muscidae). J. Econ. Entomol. 95, 1251e1260. Tunaz, H., Uygun, N., 2004. Insect growth regulators for insect pest control. Turkish J. Agric. For. 28, 377e387. Wilson, T.G., 1997. Cyromazine toxicity to Drosophila melanogaster (Diptera: Drosophilidae) and lack of cross-resistance in natural population strains. J. Econ. Entomol. 90, 1163e1169. Yang, Y., Wu, Y., Chen, S., Devine, G., Denholm, I., Jewess, P., Moores, G., 2004. The involvement of microsomal oxidases in pyrethroid resistance in Helicoverpa armigera from Asia. Insect biochem. Mol. Biol. 34, 763e773.
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Cyromazine resistance in a field strain of house flies, Musca domestica L.: Resistance risk assessment and bio-chemical mechanism Hafiz Azhar Ali Khan a, *, Waseem Akram b a b
Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan Department of Entomology, University of Agriculture, Faisalabad, Pakistan
h i g h l i g h t s Rapid development of resistance to cyromazine was observed as a result of selection experiments. Cyromazine resistance in the CYR-SEL strain was unstable. The CYR-SEL strain showed lack of cross-resistance to pyriproxyfen, diflubenzuron, or mehoxyfenozide. Metabolic resistance mechanism responsible in developing cyromazine resistance in the CYR-SEL strain.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 August 2016 Received in revised form 4 October 2016 Accepted 5 October 2016
Developing resistance management strategies for eco-friendly insecticides is essential for the management of insect pests without harming the environment. Cyromazine is a biorational insecticide with very low mammalian toxicity. Resistance to cyromazine has recently been reported in house flies from Punjab, Pakistan. In order to propose a resistance management strategy for cyromazine, experiments were planned to study risk for resistance development, possibility of cross-resistance and bio-chemical mechanisms. A field strain of house flies with 8.78 fold resistance ratio (RR) to cyromazine was reselected under laboratory conditions. After seven rounds of selection (G1-G7), the RR values rapidly increased from 8.8 to 211 fold. However, these values declined to 81fold when the cyromazine selected (CYR-SEL) strain was reared without selection pressure, suggesting an unstable nature of resistance. The CYR-SEL strain showed lack of cross-resistance to pyriproxyfen, diflubenzuron, and methoxyfenozide. Synergism bioassays using enzyme inhibitors: piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF), and metabolic enzyme analyses revealed increased activity of carboxylesterase (CarE) and mixed-function oxidase (MFO) in the CYR-SEL strain compared to the laboratory susceptible (Labsusceptible) strain, suggesting the metabolic resistance mechanism responsible for cyromazine resistance in the CYR-SEL strain. In conclusion, risk of rapid development of cyromazine resistance under consistent selection pressure discourages the sole reliance on cyromazine for controlling house flies in the field. The unstable nature of cyromazine resistance provides window for restoring cyromazine susceptibility by uplifting selection pressure in the field. Moreover, lack of cross-resistance between cyromazine and pyriproxyfen, diflubenzuron, or methoxyfenozide in the CYR-SEL strain suggest that cyromazine could be rotated with these insecticides whenever resistance crisis occur in the field. © 2016 Published by Elsevier Ltd.
Handling Editor: David Volz Keywords: Ecotoxicology Biorational insecticides Risk assessment Urban pest management
1. Introduction The use of insecticides is an integral part of sustainable agriculture and livestock production for the management of insect pests. However, a number of problems arise when insect pests
* Corresponding author. E-mail address: [email protected] (H.A.A. Khan). http://dx.doi.org/10.1016/j.chemosphere.2016.10.018 0045-6535/© 2016 Published by Elsevier Ltd.
develop resistance against insecticides, since the farming community shifts from label recommendations to over-dosage of insecticides for the management of resistant insect pests (Hemingway and Ranson, 2000). Few of the issues which results from this shift of the farming community are: the severity of the insecticide resistance problem, insect pest resurgence, environmental pollution and ill-effects on public health. The most probable solution to all these issues is the wise use of insecticides and
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development of insecticide resistance management programs (Khan et al., 2013a, 2013b). House flies (Diptera: Muscidae) are serious pests of public health importance and transmit a number of deadly diseases like diarrhea, cholera etc. (Malik et al., 2007). Being polyphagous, there are a number of feeding and breeding hosts known for their rapid expansion. Of these, the hosts available at dairies or livestock production units i.e., animal or farmyard manure, have been assumed as the potential multiplication source for house flies, and infestations in nearby communities (Learmount et al., 2002; Khan et al., 2012). In this situation, chemical measures, being quick and easy to apply, become the choice of people facing house flies infestations. However, house flies have the ability to become resistant against insecticides used for their management usually in a short period of time (Kaufman et al., 2010). From the available literature, insecticide resistance to different classes of insecticides (organochlorine, organophosphate, carbamate, pyrethroid, new chemical insecticides) has been reported around the globe (Liu and Yue, 2000; Learmount et al., 2002; Kristensen and Jespersen, 2003, 2004; Kaufman et al., 2006; Acevedo et al., 2009). To combat resistance issue, victim people increase the dosage of insecticide with ultimate effects on public health and environmental pollution. The reaction of people to the above problems is same in Pakistan, and resistance in house flies against different insecticides has been reported the first time in 2013 (Khan et al., 2013a, 2013b), likely a product of excessive insecticide usage. Cyromazine is a biorational insecticide and belongs to Insect Growth Regulator (IGR) class with novel mode of action (Lau et al., 2015). The insecticides belong to IGR class are quite selective in their action, have very low mammalian toxicity, and potentially toxic to target species only (Tunaz and Uygun, 2004). In livestock production units, cyromazine is recommended to control the manure-breeding house flies in two ways: spray application over manure heaps or feed through larvicide (Pinto and do Prado, 2001). Unfortunately, cases of resistance to cyromazine in house flies have also been reported worldwide (Shen and Plapp, 1990; Pinto and do Prado, 2001; Tang et al., 2002; Kristensen and Jespersen, 2003; Acevedo et al., 2009). Recently, a notable level of resistance to cyromazine in house flies has been reported from Pakistan (Khan et al., 2016a); however, there is a lack of information like the rapidity with which cyromazine resistance develops, crossresistance phenomenon, and mechanism of resistance development. All these information are necessary to make an effective cyromazine resistance management plan, and to save the effectiveness of this insecticide for a long period of time. Therefore, the aims of the present study were to explore all these missing information through selection experiments, and to propose a cyromazine resistance management plan. 2. Materials and methods The methodology section has been adapted from the authors' previous work (Khan et al., 2015a, 2015b, 2016a) with some modifications: 2.1. Insects Two strains were used in the present study: a laboratory susceptible (Lab-susceptible) reference strain reared in the laboratory without exposure to any insecticide and a field strain collected from a dairy farm near Lahore (31 320 59 N; 74 200 37 E), Punjab, Pakistan, and was consecutively selected in the laboratory with cyromazine to generate a cyromazine selected strain (CYR-SEL). Both of the strains were reared in the laboratory (25 ± 2 C, 65 ± 5% RH, 12: 12 h (L: D) photoperiod) as discussed previously (Khan et al.,
309
2013a). The experiments were conducted at the Institute of Agricultural Sciences, University of the Punjab, Lahore. 2.2. Chemicals Four technical-grade insecticides (cyromazine, diflubenzuron, pyriproxyfen and methoxyfenozide; Chem Service Inc, West Chester PA) were used for toxicological evaluations. In addition, two synergists, S,S,S-tributylphosphorotrithioate (DEF; Chem Service Inc, West Chester PA), and piperonyl butoxide (PBO; Chem Service Inc, West Chester PA), were also used in synergism studies. 2.3. Selection and bioassays The field strain of house flies was selected with cyromazine for seven consecutive generations. Briefly, the selection and insecticidal bioassays were performed by using larval medium. Newly hatched 1st instar larvae were placed on the surface of the media treated with cyromazine (70% mortality level) for seven consecutive generations, and the survivors were used as the parents of the next generation. The numbers of larvae selected in each generation were ranged between 500 and 600 (Table 2). To test the toxicity of IGR insecticides, larval bioassays were performed by following the protocol described by Cetin et al. (2009) with few modifications. In short, 5e7 serial dilutions of each insecticide, with >0% mortality at the lowest dilution and <100% mortality at the highest dilution, were prepared in distilled water. Twenty gram larval media were taken in a plastic cup (z100 mL capacity) and treated with 2.5 mL of serial dilution of IGR insecticide or water alone in control to monitor background mortality. Twenty newly hatched 1st instar larvae were placed on the surface of treated media with the help of a camel hairbrush. The full IGR insecticide bioassay was replicated three times and the number of larvae per dilution was 60. All the bioassays were conducted at 26 ± 2 C, 60 ± 5% RH and continuous light until the emergence of house flies. Final mortality was recorded three-week post-treatment and the larvae/pupae unable to develop into adults were considered dead. To study the stability of cyromazine resistance in the selected strain of house flies, the selection of the strain was stopped at G7 and it was reared for the next seven generations without exposure to any insecticide. Then the strain was bioassayed again with cyromazine and the stability of resistance was assessed by the following formula (Sayyed et al., 2005):
R ¼ ½log ðfinal LC50 Þ log ðinitial LC50 Þ=n Where, n is the number of generations reared without exposure to insecticide. The field strain was bioassayed with pyriproxyfen, diflubenzuron, and metjoxyfenozide before and after the selection experiment (at G0 and G7, respectively) to assess cross-resistance mechanism. All the mortality data were analyzed by Probit analysis using the software SPSS (version 10.0) to determine median lethal concentrations (LC50) and their 95% confidence intervals (CIs). The LC50 values of the respective bioassays were considered significantly different on the basis of non-overlapping of 95% CIs (Litchfield and Wilcoxon, 1949). 3. Cyromazine resistance mechanism To study the mechanism of cyromazine resistance, enzyme inhibitors PBO and DEF diluted in acetone were used at the maximum sublethal level and mixed with insecticide dilutions. The resultant mixture was added in the larval medium for bioassays as stated above. The enzyme analyses were performed as described
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4.3. Cross-resistance analysis
previously (Khan et al., 2015a): Analyses were done by using 3e5-day old female house flies of both Lab-susceptible and CYR-SEL strains, separately. Individual female house flies were separated and homogenized in 1 mL of sodium phosphate buffer (0.1 M; pH 7.8) at 4 C. The homogenates were centrifuged at 10,000g at 4 C for 10 min, and large fragments were then removed (Kristensen, 2005). The supernatant was used immediately for assaying the activities of carboxylesterase (CarE), mixed function oxidase (MFO), and glutathione S-transferase (GST) by using the methodologies of Gao et al. (2014) for CarE and MFO, Yang et al. (2004) for the determination of GST activity, and Bradford (1976) for measuring total proteins.
The field strain before the start of cyromazine resistance was bioassayed against pyriproxyfen, diflubenzuron, and methoxyfenozide. The LC50 values were 0.47, 0.68, and 0.51 mg mL1 for pyriproxyfen, diflubenzuron, and methoxyfenozide, respectively (Table 4). The field strain (CYR-SEL), after seven rounds of selection with cyromazine, was again bioassayed at G7 against pyriproxyfen, diflubenzuron, and methoxyfenozide. The results revealed that the LC50 values against these three insecticides before and after the cyromazine selection experiments were statistically at (overlapping 95% CIs) suggesting lack of cross-resistance.
4. Results
4.4. Synergism bioassays and metabolic enzyme activities
4.1. Toxicity of insecticides against laboratory and field strains before selection experiment
Synergism bioassays were performed by using enzyme inhibitors (PBO and DEF) in combination with cyromazine. The enzyme inhibitors did not increase the toxicity of cyromazine in the Lab-susceptible strain. However, a significant increase in toxicity in the presence of enzyme inhibitors was observed in the CYR-SEL strain, suggesting the involvement of metabolic enzymes in the development of cyromazine resistance. The LC50 values of the CYRSEL strain were 19.03, 4.13, and 5.73 mg mL1 for cyromazine alone, cyromazine þ DEF, and cyromazine þ PBO, respectively (Table 5). Metabolic enzyme analyses revealed significantly increased activity of CarE and MFO in the CYR-SEL strain compared to the Labsusceptible strain, further confirming the involvement of metabolic enzymes in the development of cyromazine resistance (Table 6).
All the tested insecticides showed statistically equal toxicity (95% overlapping CIs) against Lab-susceptible reference strain. The LC50 values for cyromazine, pyriproxyfen, diflubenzuron and methoxyfenozide were 0.09, 0.08, 0.07, and 0.06 mg mL1, respectively (Table 1). However, the field strain showed less susceptibility to these insecticides compared to the Lab-Sus strain. The LC50 values of the field strain for cyromazine, pyriproxyfen, diflubenzuron and methoxyfenozide were 0.79, 0.47, 0.68, and 0.51 mg mL1, respectively. In comparison to the Lab-susceptible strain, the field strain before the selection experiment showed 8.8, 5.9, 9.7, and 8.5 fold resistance (based on RR values) against cyromazine, pyriproxyfen, diflubenzuron, and methoxyfenozide, respectively (Table 1). 4.2. Cyromazine selection experiment The field strain showing 8.78 fold RR to cyromazine was further selected under laboratory conditions (Table 2). The LC50 values significantly increased after seven rounds of selection (Table 3). The LC50 values were 1.22, 1.80, 2.60, 5.25, 7.30, 10.49, and 19.03 mg mL1 from G1 to G7, respectively. There was a rapid increase in the RR values as a result of selection experiment. The RR values jumped from 8.8 to 211fold after seven rounds of selection with cyromazine. The cyromazine selected strain (CYR-SEL) was reared for the next seven generations without exposure to insecticide and bioassayed again at G14 with cyromazine. The result revealed a rapid decline in resistance (the RR values declined from 211 to 81 fold) was observed indicating an unstable nature of cyromazine resistance in the selected strain (Table 3).
5. Discussion Keeping in view the increasing problem of insecticide resistance and the ultimate effect on environmental pollution (Khan et al., 2016b), it's the demand of time to develop resistance management strategies so as to retain the effectiveness of insecticides for long. Cyromazine is an IGR insecticide; however, cases of resistance to cyromazine necessitate studying the nature of resistance and development of management plans. In the present research work, the field strain of house flies before the start of laboratory selection experiments exhibited resistance to cyromazine and three other IGRs which further confirmed the previous report (Khan et al., 2016a) on the field evolved resistance to IGRs in house flies. Resistance to cyromazine has been reported previously in different insect pest like M. domestica (Sheppard et al., 1992; Pinto and do Prado, 2001; Learmount et al., 2002; Kristensen and Jespersen, 2003; Acevedo et al., 2009), Lucilia cuprina (Levot, 2012), Liriomyza trifolii (Ferguson, 2004), and Drosophila melanogaster
Table 1 Toxicity of different insecticides against laboratory susceptible reference and newly collected field strains of house flies. Strain
Lab-susceptible Lab-susceptible Lab-susceptible Lab-susceptible Field (G0) Field (G0) Field (G0) Field (G0)
Insecticide
Cyromazine Pyriproxyfen Diflubenzuron Methoxyfenozide Cyromazine Pyriproxyfen Diflubenzuron Methoxyfenozide
na
420 420 420 420 360 420 360 420
LC50b (95% CIc) (mg mL1)
0.09 0.08 0.07 0.06 0.79 0.47 0.68 0.51
(0.06e0.12) (0.05e0.12) (0.06e0.08) (0.04e0.07) (0.65e0.98) (0.39e0.56) (0.55e0.82) (0.42e0.63)
The same applies to the following tables. a n¼ number of flies used in bioassays and controls. b LC50, median lethal concentration (in microgram per millilitre) estimated to give 50 % mortality. c CI, confidence intervals. d RR, resistance ratio¼ LC50 of field strain /LC50 of Lab-susceptible strain.
RRd
Fit of probit line Slope (SE)
2
c
df
p
2.36 2.29 2.34 2.55 1.82 1.99 1.85 1.73
7.67 9.67 3.38 3.63 1.50 4.70 1.73 2.46
4 4 4 4 3 4 3 4
0.11 0.06 0.50 0.45 0.68 0.32 0.63 0.65
(0.19) (0.19) (0.20) (0.22) (0.21) (0.18) (0.20) (0.16)
8.8 5.9 9.7 8.5
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Table 2 Selection concentrations and survival rate of the field strain in cyromazine selections on successive generations. Selected generation
Concentration (mg mL1)
No. larvae selected
G1 G2 G3 G4 G5 G6 G7
544 500 580 600 510 500 550
Survival (number)
1.55 2.24 3.47 4.82 12.50 21.22 37.81
Male
Female
88 71 80 94 75 76 86
96 90 83 110 78 85 90
Table 3 Resistance selection of the field strain of house flies with cyromazine. Strain
Field (G0) G1 G2 G3 G4 G5 G6 G7(CYR-SEL) G14
LC50 (95% CI) (mg mL1)
n
360 360 420 420 360 420 420 420 420
0.79 (0.65e0.98) 1.22 (1.01e1.52) 1.80 (1.48e2.22) 2.60 (2.14e3.25) 5.25 (4.10e7.50) 7.30 (5.74e10.05) 10.49 (8.47e13.53) 19.03 (15.50e24.15) 7.31 (6.22e8.55)
Fit of probit line
RR
Slope (SE)
c
2
df
p
1.82 1.99 1.84 1.95 1.98 1.67 1.71 1.76 2.41
1.50 2.23 0.60 3.56 3.15 3.84 6.71 3.94 4.92
3 3 4 4 3 4 4 4 4
0.68 0.53 0.96 0.47 0.37 0.43 0.15 0.42 0.29
(0.21) (0.22) (0.17) (0.20) (0.27) (0.19) (0.18) (0.18) (0.20)
8.8 13.6 20 28.9 58.8 81 116.6 211 81
G7 ¼ Cyromazine selected strain after seven generations of continuous selection. G14 ¼ CYR-SEL strain after seven generations without any insecticide exposure.
Table 4 Cross-resistance analysis of the CYR-SEL strain of house flies to other insecticides. Insecticide
LC50 (95% CI) (mg mL1)
n
Cyromazine (G0) Cyromazine (CYR-SEL) Pyriproxyfen (G0) Pyriproxyfen (CYR-SEL) Diflubenzuron (G0) Diflubenzuron (CYR-SEL) Methoxyfenozide (G0) Methoxyfenozide (CYR-SEL)
360 420 420 420 360 420 420 420
Fit of probit line
0.79 (0.65e0.98) 19.03 (15.50e24.15) 0.47 (0.39e0.56) 0.54 (0.46e0.64) 0.68 (0.55e0.82) 0.76 (0.63e0.94) 0.51 (0.42e0.63) 0.55 (0.44e0.69)
RR
Slope (SE)
c
2
df
p
1.82 1.76 1.99 2.21 1.85 1.73 1.73 1.62
1.50 3.94 4.70 5.20 1.73 3.52 2.46 1.28
3 4 4 4 3 4 4 4
0.68 0.42 0.32 0.27 0.63 0.48 0.65 0.87
(0.21) (0.18) (0.18) (0.19) (0.20) (0.16) (0.16) (0.16)
8.78 211.44 5.88 6.75 9.71 10.85 8.50 9.17
Table 5 Effect of enzyme inhibitors on the toxicity of cyromazine against house flies strains. Strain
Laboratory
CYR-SEL (G7)
a
Compound
n
Cyromazine Cyromazine Cyromazine Cyromazine Cyromazine Cyromazine
þ DEF þ PBO þ DEF þ PBO
420 420 420 420 420 420
LC50 (95% CI) (mg mL1)
0.09 (0.06e0.12) 0.08 (0.07e0.10) 0.10 (0.08e0.11) 19.03 (15.50e24.15) 4.13 (3.39e4.93) 5.73 (4.63e6.97)
SRa
Fit of probit line Slope (SE)
c
2
df
p
2.36 1.91 1.86 1.76 2.10 1.71
7.67 3.53 3.62 3.94 3.56 5.51
4 4 4 4 4 4
0.11 0.47 0.46 0.42 0.47 0.24
(0.19) (0.17) (0.17) (0.18) (0.20) (0.16)
1.13 0.90 4.61 3.32
SR ¼ LC50 of cyromazine alone/LC50 of cyromazine along with an enzyme inhibitor.
Table 6 Metabolic enzyme activities in the laboratory susceptible and CYR-SEL strains of house flies. Strain
Lab-susceptible CYR-SEL a
CarE
MFO
Activity ±SE nmol min1 mg1
Ratioa
Activity ±SE pmol min1 mg1
Ratioa
133.83 ± 10.11 212.67 ± 5.75
1.59
68.99 ± 7.20 176.67 ± 9.29
2.56
Enzyme activity in the CYR-SEL strain/enzyme activity in the laboratory susceptible strain.
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(Daborn et al., 2000). There could be different reasons for the development of the field evolved resistance to cyromazine. For example, Acevedo et al. (2009) reported that regional chemical use strategies, and over dosing other than the label recommendations by the farming community and lack of monitoring surveys were the main reasons for the cyromazine resistance in house flies in Argentina. Similarly, Khan et al. (2016a) reported that the excessive sprays of IGRs in cropping areas around the dairy facilities was the main reason for the occurrence of cyromazine resistance in house flies from Punjab, Pakistan. Whereas, treatment of animal manure with cyromazine rather than feedthrough application for a long time was the main reason for the development of resistance to cyromazine in Denmark (Kristensen and Jespersen, 2003). In the present study, the field strain of house flies with 8.8 fold resistance to cyromazine was re-selected under the laboratory conditions in order to study the mechanism of resistance and crossresistance potential to other insecticides. The results revealed a rapid development of resistance to cyromazine after seven rounds of selection and the RR values increased from 8.8 to 211fold. Laboratory selection with cyromazine in different insects has been studied in the past in an attempt to study the rapidity of resistance development. For example, L. cuprina from New South Wales Monaro was selected under the laboratory conditions for 13 generations, but the development of resistance was very slow i.e., the RR values increased from 2.3 to 8.1 fold (Levot, 2013). This might be due to the spatio-temporal variations, insect species in question, insecticide exposure history or the combination of all these factors (Khan et al., 2015a, 2015b). In our study, resistance to cyromazine was unstable in the selected strain when it reared without selection pressure. The resistance was declined from 211 to 81 fold over a period of seven generations. The unstable nature of cyromazine resistance has also been reported in the leafminer, L. trifolii (Ferguson, 2004), when it was reared in the absence of selection pressure. There are a number of cases where resistance have been reverted when the selected pests were reared in the absence of pesticide selection pressure (Flexner et al., 1989; Kristensen et al., 2000; Khan et al., 2015b). Fitness cost and initial gene frequencies have been assumed responsible for the reversion of resistance in insects under laboratory conditions (Roush and Mckenzie, 1987; Khan et al., 2014); however, further studies are required to confirm the phenomenon of fitness cost responsible for the reversion of resistance in the cyromazine selected strain of house flies. The cross-resistance potential of the insecticide selected strains is a valuable tool in order to screen out alternate insecticides for rotational use when resistance crisis become severe in the field. In this way, the insecticides to which selected strains don't show cross-resistance can be a part of resistance management strategy to a particular insecticide (Georghiou and Taylor, 1977). In the present research work, the CYR-SEL strain of house flies didn't show cross resistance to diflubenzuron, pyriproxyfen, and methoxyfenozide. Before the start of selection experiment, the LC50 values were 0.47, 0.68, and 0.51 mg mL1 for pyriproxyfen, diflubenzuron, and methoxyfenozide, respectively. Whereas, after the selection experiment, the CYR-SEL strain showed statistically non-significant change in the LC50 values to the above mentioned three insecticides. These results indicate the above insecticides could be used in rotation to cyromazine for the effective management of house flies. Previously, the lack of cross-resistance in the cyromazine selected strains of D. melanogaster (Wilson, 1997), L. cuprina (Levot, 2013), and M. domestica (Khan et al., 2016a) has been reported. Cross-resistance between different insecticide could be expected either due to the structural similarity or common resistance mechanism (Kristensen and Jespersen, 2003). Being in different insecticide classes, the cross-resistance between
cyromazine and diflubenzuron, pyriproxyfen or methoxyfenozide was not expected (Kristensen and Jespersen, 2003; Committee, 2007; Sparks and Nauen, 2015). Cyromazine is a moulting disruptor and belongs to IRAC main group #17; diflubenzuron is an inhibitor of chitin biosynthesis and belongs to the IRAC main group # 15; pyriproxyfen is a juvenile hormone mimic and belongs to IRAC main group # 05; methoxyfenozide is an ecdysone agonist and belongs to IRAC main group # 18. In an attempt to find out the mechanism of cyromazine resistance in the CYR-SEL strain, cyromazine toxicity was assessed in the presence of an enzyme inhibitor (PBO or DEF). A significant increase in toxicity in the presence of enzyme inhibitors was observed in the CYR-SEL strain, suggesting the involvement of metabolic enzymes in the development of cyromazine resistance. This was further confirmed by enzyme analyses which revealed enhanced activities of CarE and MFO in the CYR-SEL strain of house flies. Previously, cyromazine resistance in house flies from Denmark has been reported to be linked with increased activities of cytochrome P450 monooxygenase and glutathione S-transferase (Kristensen and Jespersen, 2003). In conclusion, the study reveals a rapid rate of resistance development to cyromazine in the selected strain of house flies. Resistance developed under the continuous selection pressure which pointed out the possibility of resistance development in the field if spray applications or selection pressure of cyromazine persist for long. However, the unstable nature of cyromazine resistance provides the opportunity to restore its toxicity by discontinuing its use for a certain period of time, if resistance crisis occur in the field. Moreover, lack of cross-resistance to the other insecticides tested (pyriproxyfen, diflubenzuron and methoxyfenozide) suggest that these products could be used in rotation and/or as an alternate to reduce the possibility of resistance development to cyromazine in the field. References Acevedo, G.R., Zapater, M., Toloza, A.C., 2009. Insecticide resistance of house fly, Musca domestica (L.) from Argentina. Parasitol. Res. 105, 489e493. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Cetin, H., Erler, F., Yanikoglu, A., 2009. Survey of insect growth regulator (IGR) resistance in house flies (Musca domestica L.) from southwestern Turkey. J. Vector Ecol. 34, 329e337. Committee, I.R.A., 2007. IRAC Mode of Action Classification (Updated July). iraconline.org. Daborn, P.J., McKenzie, J.A., Batterham, P., 2000. A genetic analysis of cyromazine resistance in Drosophila melanogaster (Diptera : Drosophilidae). J. Econ. Entomol. 93, 911e919. Ferguson, J.S., 2004. Development and stability of insecticide resistance in the leafminer Liriomyza trifolii (Diptera: Agromyzidae) to cyromazine, abamectin, and spinosad. J. Econ. Entomol. 97, 112e119. Flexner, J., Theiling, K., Croft, B., Westigard, P., 1989. Fitness and immigration: factors affecting reversion of organotin resistance in the twospotted spider mite (Acari: Tetranychidae). J. Econ. Entomol. 82, 996e1002. Gao, C.-F., Ma, S.-Z., Shan, C.-H., Wu, S.-F., 2014. Thiamethoxam resistance selected in the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae): cross-resistance patterns, possible biochemical mechanisms and fitness costs analysis. Pestic. Biochem. Physiol. 114, 90e96. Georghiou, G.P., Taylor, C.E., 1977. Genetic and biological influences in evolution of insecticide resistance. J. Econ. Entomol. 70, 319e323. Hemingway, J., Ranson, H., 2000. Insecticide resistance in insect vectors of human disease. Annu. Rev. Entomol. 45, 371e391. Kaufman, P.E., Gerry, A.C., Rutz, D.A., Scott, J.G., 2006. Monitoring susceptibility of house flies (Musca domestica L.) in the United States to imidacloprid. J. Agric. Urban Entomol. 23, 195e200. Kaufman, P.E., Nunez, S.C., Mann, R.S., Geden, C.J., Scharf, M.E., 2010. Nicotinoid and pyrethroid insecticide resistance in houseflies (Diptera: Muscidae) collected from Florida dairies. Pest Manag. Sci. 66, 290e294. Khan, H.A., Akram, W., Iqbal, J., Naeem-Ullah, U., 2015a. Thiamethoxam resistance in the house fly, Musca domestica L.: current status, resistance selection, crossresistance potential and possible biochemical mechanisms. PLoS One 10, e0125850. Khan, H.A., Akram, W., Iqbal, N., 2015b. Selection and Preliminary mechanism of
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