Figures
Abstract
Background
Spinosad is important in pest management strategies of multiple insect pests. However, spinosad resistance is emerging in various pest species. Resistance has in some species been associated with alterations of the target-site receptor, but in others P450s seems to be involved. We test the possible importance of nine cytochrome P450 genes in the spinosad-resistant housefly strain 791spin and investigate the influence of spinosad on P450 expression in four other housefly strains.
Results
Significant differences in P450 expression of the nine P450 genes in the four strains after spinosad treatment were identified in 40% of cases, most of these as induction. The highly expressed CYP4G2 was induced 6.6-fold in the insecticide susceptible WHO-SRS females, but decreased 2-fold in resistant 791spin males. CYP6G4 was constitutively higher expressed in the resistant strain compared to the susceptible strain. Furthermore, CYP6G4 gene expression was increased in susceptible WHO-SRS flies by spinosad while the expression level did not alter significantly in resistant fly strains. Expression of CYP6A1 and male CYP6D3 was constitutively higher in the resistant strain compared to the susceptible. However, in both cases male expression was higher than female expression.
Conclusion
CYP4G2, CYP6A1, CYP6D3 and CYP6G4 have expressions patterns approaching the expectations of a hypothesized sex specific spinosad resistance gene. CYP4G2 fit requirements of a spinosad resistance gene best, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin. Differential expression of P450s between sexes is more a rule than an exception. Noteworthy differences between spinosad influenced expression of P450 genes between a field population and established laboratory strains were shown.
Citation: Højland DH, Jensen K-MV, Kristensen M (2014) Expression of Xenobiotic Metabolizing Cytochrome P450 Genes in a Spinosad-Resistant Musca domestica L. Strain. PLoS ONE 9(8): e103689. https://doi.org/10.1371/journal.pone.0103689
Editor: John Vontas, University of Crete, Greece
Received: January 23, 2014; Accepted: July 5, 2014; Published: August 28, 2014
Copyright: © 2014 Højland et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by a mobility stipend to Dorte H. Højland from the Danish Agency for Science Technology and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Spinosad is a mixture of two macrocyclic lactones; spinosyn A and spinosyn D isolated from the actinomycete bacteria, Saccharopolyspora spinosa and has been developed as a commercial insecticide [1]. Its mode of action is unique as its primary target site appears to be a subtype of the nicotinic acetylcholine receptors (nAChRs) with also a secondary target site suggested to be the γ-aminobutyric acid (GABA)-gated chloride channel [2]. Spinosad has been introduced for insect control initially in 1997 and plays an essential role for the control of Lepidoptera and Diptera pests as well as planthoppers, spider mites, ticks, fleas and head lice. There are more than 30 examples of resistance to spinosad and half of these were selected in the field [3]. The mechanism of spinosad resistance has been implied to involve changes of target site and metabolism [4].
A key element in preventing development of resistance as well as resistance management is the understanding of the molecular mechanisms potentially responsible for resistance. Prior to the introduction a decade ago of bait-formulated spinosad for housefly control in Denmark we established a baseline for spinosad toxicity of Danish houseflies. Field populations of houseflies collected from livestock farms throughout Denmark showed up to 7.5-fold resistances (compared to the susceptible reference strain WHO-SRS) to spinosad in feeding bioassays, which was considered to reflect the natural variation of spinosad toxicity in field populations [5]. Additional studies with insecticide-resistant laboratory strains showed that there was no cross-resistance to the major insecticide classes in the housefly [5].
Spinosad resistance in the laboratory housefly strain rspin [6] selected for high spinosad resistance, is inherited as a recessive trait linked to autosome 1. The resistance in the rspin strain does apparently not involve P450-mediated metabolism as resistance level is unchanged upon pretreatment with the xenobiotic metabolism inhibitor piperonyl butoxide (PBO) [6]. Likewise, spinosad resistance in a Chinese housefly strain was autosomal, although incompletely dominant, controlled by multiple genes and not influenced by PBO [7].
Spinosad resistance in the multi-resistant Danish field population (791a) has been shown to be associated with P450 activity in a study implementing PBO based bioassays as well as gene expression studies [8]. In the spinosad-selected strain 791spin strain, originating from 791a, the male determining factor are located on autosome III [9]. The housefly CYP4G2 is also located on chromosome III [10]. Insect CYP4G is remarkable by having orthologs distributed across Insecta [11]. The CYP4G enzymes function as oxidative decarbonylases, catalysing the terminal step in insect hydrocarbon production synthesizing alkanes making up a large part of the cuticular hydrocarbons [12].
If spinosad influence expression of xenobiotic genes including P450 genes, we hypothesize that variation of this response is a selectable trait in adaptation to an environment influenced by spinosad, thus having a resistance potential. Additionally, the coordination of P450 gene expression in response to spinosad can elucidate important new insights into the general response to a xenobiotic compound. This paper thus investigates the constitutive and spinosad-induced expression levels of nine resistance associated housefly P450 genes. CYP6A1 assists in the metabolism of organochlorine- and organophosphate-insecticides [13], [14] and is constitutively over-expressed in an organophosphate-resistant strain [15], [16] as well as in neonicotinoid-resistant strains [17]. CYP6A36 is constitutively overexpressed in a permethrin-resistant strain, whereas no difference was found for CYP6A37 [18]. CYP6D1 is constitutively over-expressed in pyrethroid-resistant houseflies [19], [20] and in neonicotinoid-resistant strains [17]. CYP6D3 is constitutively over-expressed in pyrethroid-resistant houseflies and previously reported involved in insecticide resistance in the housefly [20]. CYP6G4 is the potential housefly orthologue of Drosophila melanogaster CYP6G1, where over-expression is correlated with DDT and neonicotinoid resistance [21], [22]. The mitochondrial CYP12A1 and CYP12A2 metabolize a variety of insecticides and xenobiotics and are constitutively over-expressed in the diazinon-resistant Rutgers strain [23].
Methods
Houseflies
Housefly breeding followed standard laboratory conditions. Egg laying was performed on crumpled filter paper soaked in whole milk. Breeding jars (5 L plastic buckets) containing 4 L of medium were seeded with 200 mg of eggs, corresponding to 2700 eggs. The breeding medium consisted of wheat bran 400 g, lucerne meal 200 g, bakers yeast 10 g, malt extract 15 mL, whole milk 500 mL and water 500 mL. For adult feeding, cube sugar and water were given continuously. Feeding started after emergence with whole-milk powder mixed with icing sugar (1∶1 w/w) [24].
The insecticide-susceptible standard reference strain WHO-SRS was received in 1988 from the Department of Animal Biology, University of Pavia, Italy.
The unselected neonicotinoid resistant laboratory population 766b was collected in 2005 in Denmark. Female and male 766b flies were 140-fold and 130-fold resistant to imidacloprid at LC50, respectively [25]. The 766b strain is susceptible to spinosad [8].
The multi-resistant 791a laboratory population was collected in the context of a resistance survey in 1997 in Denmark. The strain has never been selected after collection. The strain was highly resistant to pyrethroid, anti-cholinesterase and showed some resistance to the chitin synthesis disrupting larvicides as well as fipronil [26]–[28]. Female and male 791a flies were 20-fold and 22-fold resistant to imidacloprid at LC50, respectively [17]. 791a females were 27-fold spinosad-resistant at LC50, whereas 791a male houseflies were susceptible (5-fold resistant) [8].
The spinosad selected 791spin strain was created by selection of the spinosad resistant field strain 791a. The initial selection of 791spin was made by 24 h non-choice feeding sugar impregnated with 71 µg spinosad g−1 sugar; males (N = 573, 9% survival) and females (N = 406, 32% survival). Selection was repeated in generations 2, 5, 7, 10, 13, 18 and 22 after the initial selection, with increasing concentrations of spinosad. 791spin females were 21-fold spinosad-resistant at LC50, whereas 791spin male houseflies were 6-fold resistant which is considered to be within the natural variation in spinosad toxicity in susceptible Danish field populations [5]. Female and male 791spin flies were considered susceptible to imidacloprid having 3-fold and 4-fold resistance factors at LC50, respectively [8]. The strain is retained by regular annually selections with spinosad-impregnated sugar: male flies 0.4 mg spinosad per g sugar and female flies 3.2 mg spinosad per g sugar, for a maximum 72 hours.
The field population 845b (unselected) was collected August 23. 2011 at a dairy farm located at Nykøbing Mors, Denmark (56°53′51.07″N, 8°48′42.81″E). The flies were collected on private land with consent of the owner. The field collection did not involve endangered or protected species. The strain could be characterized as a good representative for Danish field populations with no or a low level of resistance towards neonicotinoids and pyrethroids [29]. F1 and F2 flies from the 845b field population were used for experiments testing resistance level as well as the influence of spinosad on expression on P450 genes.
Treatment of houseflies for gene expression analysis
Five to seven days old, adult male and female flies were subjected to a non-choice feeding test with spinosad (88%, 76.1% spinosyn A and 11.9% spinosyn D, DOW AgroSciences). The insecticide were diluted with analytic-grade acetone and impregnated on. Susceptible male and female flies were given spinosad in dose 0.063 mg per g sugar and male and female flies from the field populations 766b and 845b were given spinosad in dose 0.25 mg and 0.5 mg per g sugar, respectively. Males and females of multi-resistant strain 791a were given respectively 0.5 mg and 2 mg spinosad per g sugar [17].
All flies had access to water, milk and sugar ad libitum before trials. Flies, which were alive at day 5 (130–500 flies) were placed in cages with full access to water and were given excess of spinosad-treated granular sugar in a small petri-dish as the only food. The feeding tests were carried out at 25–26°C, 60–65% RH in continuous light. Twenty-four hours upon test start living and fresh looking flies were collected by vacuum suction, immediately sedated by cold and killed at –20°C. The flies were hereafter kept on –80°C until RNA extraction.
Parallel experimental set-ups were performed with resistant and susceptible flies, with the one exception that the sugar offered were left acetone-coated. These flies served as reference for examinations of constitutive gene expression levels.
Primer designs for quantitative real-time PCR
Gene specific primer pairs were designed based on sequences obtained from the NCBI GenBank with the exception of CYP4G2, which was extracted from Zhu et al. [10]. Gao et al. [30] mentioned CYP6G4 (Musca domestica) to be an ortholog to CYP6G1 (Drosophila melanogaster). The CYP6G1 gene was blasted against the GenBank database (blastn, http://blast.ncbi.nlm.nih.gov/) with 65% identity.
CYP4G2, F: 5′-cgaggaggatgatgaaataagcaagc-3′, R: 5′-ttggacatggccatcatggcatct-3′; CYP6A1 (GenBank: M25367), F: 5′-aattttgccaatcgtggtctg-3′, R: 5′-tccaccattaccaagtggcc-3; CYP6A36 (DQ642009), F: 5′-aaaggcatggccgttgttat-3′, R: 5′-acttgagaagcggcaaaatg-3′; CYP6A37 (DQ642010), F: 5′-atgcaaatcctcatccccg-3′, R: 5′-ccgtgactttgtcatgggaga-3′; CYP6D1 (U22366), F: 5′-gcaaatgcactcaggatttcc-3′, R: 5′-tgcccaagagggagatgataa-3′; CYP6D3 (AF200191), F: 5′-tgccccataagggaggct-3′, R: 5′-agaccattgactggtactaaaaccg-3′; CYP6G4 (FJ911556), F: 5′-gctgcaaagcaaattggg-3′, R: 5′-actacgcaccacattcag-3′; CYP12A1 (U86618), F: 5′-atccgttgaccttgggaaatg-3′, R: 5′-tcatcctgcagcaaacctgtt-3′; CYP12A2 (U94698), F: 5′-cctatttgagggcctgcat-3′, R: 5′-tgggaacacgatagccact-3′; GAPDH (DQ386609) F: 5′-ccggtatctccctcaacg-3′, R: 5′-tgacacggttggagtaaccga-3′.
The primer pairs used were designed not to span introns since the present study used gDNA for external standards in real-time PCR runs. To avoid non-specific amplification all RNA samples were routinely treated with DNase before use. Upon optimization forward and reverse primers were used in optimal concentration 150 nM. Amplicon sequence specificity was verified dissociation curves giving rise to single peaks at the specific melting temperature of the products. A full summary on primer design, optimization and validation is given by Markussen and Kristensen [17].
RNA and DNA extraction
Total RNA from whole bodies of houseflies was extracted using the RNeasy Maxi kit (Qiagen, Ballerup, Denmark). Pools of flies (approx. 1.2 g equivalent to 60 flies) were thoroughly grinded with liquid nitrogen, a mortar and pestle and homogenized with buffer added β-mercaptoethanol supplied by the RNeasy kit according to the manufacturer’s protocol. Isolated RNA was DNase-treated and concentrated using the RNeasy MinElute kit (Qiagen). Gel electrophoresis and spectrophotometry (Nanodrop; NanoDrop Technologies, Wilmington, USA) was performed to assess the integrity and the concentration of each RNA sample, which was dissolved in RNase-free water and stored at −20°C until use.
Extraction of gDNA used for external standards was performed according to the manufacturer’s protocol for the DNeasy kit (Qiagen). Genomic DNA was stored as stocks of 125 ng µL−1 at −20°C corresponding to ∼120,000 copies of a single-copy gene. The mass of the haploid housefly genome (the C-value; http://www.genomesize.com) is ∼1.04 pg therefore 1 ng of gDNA from M. domestica contains approx. 962 copies of a single-copy gene. A fresh 10-fold serial dilution at five quantities ranging from 125 ng (∼120000 gene copies) to 0.0125 ng (∼12 gene copies) was prepared for each real-time PCR run.
RT reaction and real-time PCR
First-strand cDNA was synthesized from RNA using the High Capacity Reverse Transcription kit (ABI). In a single RT reaction, 1 µg tRNA was mixed with 10 µL RT buffer, 10 µL random primers [10X], 4 µL dNTPs [25X], 5 µL Multiscribe Reverse Transcriptase [5 U µL−1] and RNase free water (Qiagen) to a final volume of 100 µL. The reaction was incubated at 25°C for 10 min for primer-RNA binding, followed by reverse transcription at 37°C for 120 min and 85°C for 5 min.
PCR of 20 µL reactions were performed using 20 ng of the cDNA samples, SYBR Green PCR master mix (ABI) and 150 nM of primers specific for the CYP4G2, CYP6A1, CYP6A36, CYP6A37, CYP6D1, CYP6D3, CYP6G4, CYP12A1, CYP12A2 and GAPDH genes. All samples and the external standards were run in four technical replicates. The strain variance is accounted for by randomization of the flies selected for RNA purification as well as the number of flies used; approx. 60 per sample in addition to 2–4 biological replicas depending on the availability of houseflies. Absence of gDNA was confirmed through PCR runs of no-RT controls in replicates of two.
The PCR runs were performed on ABI PRISM 7500 HT Sequence Detection Systems with Sequence Detection system software version 1.4 (ABI) initiated by a 2 min activation step at 50°C followed by a polymerase activation step for 10 min at 95°C. Amplification was obtained by 40 cycles of 15 s at 95°C with a 1 min anneal and extending step at 60°C. A final dissociation stage at 95°C for 15 sec, 60°C for 15 sec and 95°C for 15 sec was added to generate a melting curve for verification of amplification product specificity. The PCR data in Table 1 and 2 are presented as the mean copy number per 20 ng of RNA ± standard deviation of 2–4 biological replicates. Statistical analysis was undertaken using pairwise Wilcoxon Ranking test, where a P-value less than 0.05 was considered to be statistically significant [31].
Results and Discussion
The xenobiotic response of P450s is known to play an important role in the development of insecticide resistance. Enhancement of insecticide detoxification has been associated with increased P450 expression of genes leading to resistance [11]. The present experimental setup was selected in order to elucidate which, if any, of the nine P450 genes associated with resistance in the housefly could be responsible or partly responsible for spinosad resistance in the 791spin strain.
The P450s in this study were selected based on the documentation of their involvement in resistance and/or degradation of xenobiotics. Additionally CYP4G2 were selected because spinosad resistance possibly is linked to chromosome III [9], where CYP4G2 is located. The house-keeping gene GAPDH was added as a control of expression levels.
The four additional housefly strains of this study comes with different histories; an insecticide-susceptible reference strain (WHO-SRS) kept in laboratory breeding for decades, a neonicotinoid-resistant and spinosad-susceptible field population (766b) kept in breeding for 8 years without selection, a multi-resistant (including spinosad) field population (791a) kept in breeding without selection for 15 years and a newly collected spinosad-susceptible field population (845b).
Gene expression of a house-keeping gene in Danish houseflies
The house-keeping gene GAPDH was included in this study to assess the influence of spinosad on general gene expression. For the most part, GAPDH gene expression remained constant within resistant laboratory strains (Table 2). The newly-collected 845b strain had a lower level of GAPDH gene expression than the other four strains. Short-term spinosad exposure did not alter expression significantly in any of the five strains tested here (P>0.15), with the exception of the insecticide-susceptible WHO-SRS strain. Here expression was increased 1.8- and 1.9-fold in males and females, respectively (P<0.0006).
791spin P450 gene expression
A comparison of P450 expression with or without spinosad exposure of 791spin and WHO-SRS showed multiple deviations from neutrality (Figure 1 and 2). Differential expression is mostly up-regulation for both strains, but a few cases of down-regulation were also observed for 791spin (Figure 2). Spinosad exposure in 791spin males caused minor effects (2-fold or less) in both directions. In females, no decreasing effect was observed for spinosad exposure, while the CYP6A36, CYP6G4 and CYP12A1 were up-regulated 4-, 8.3- and 3.4-fold, respectively (Table 1).
The line represents no effect of spinosad exposure. Genes in right-lower and left-upper corner are down-regulated and up-regulated by spinosad, respectively. Points marked with * are significantly different from the separation line by α = 0.05.
The line represents no effect of spinosad exposure. Genes in right-lower and left-upper corner are down-regulated and up-regulated by spinosad, respectively. Points marked with * are significantly different from the separation line by α = 0.05.
Most of the genes tested here had a higher constitutive expression in 791spin than in the susceptible WHO-SRS strain for both males and females (Table 1). CYP6A36 had the lowest level of expression of the P450s investigated, while the highest expression was that of CYP4G2 for both sexes.
The 791spin strain has a sex-dependent resistance profile with regard to spinosad. Females were resistant, while males were considered within the range of susceptibility. This could indicate that the responsible gene(s) would be highly expressed in females compared to males in this strain due to the sex-specific resistance pattern. This was only the case for one single gene, CYP4G2 (P<0.0014). Sex determination in 791spin is due to a factor on autosome III [9], which led to a hypothesis of the spinosad resistance being due to a factor on the corresponding copy of autosome III. CYP4G2 is located on autosome III, and is induced by permethrin in the permethrin-resistant ALHF houseflies [18]. Multiple carbon-oxygen bonds, including aldehydes, are present in spinosad [1]. Spinosad or initial degradation products of spinosad could be substrates for the CYP4G2 enzyme, which is a decarbonylase enzyme catalyzing cleavage of long-chain aldehydes to hydrocarbons with release of CO2 [12]. The increased constitutive expression in females compared to males fits with CYP4G2 as a contributor to spinosad resistance in 791spin. Crossover in male houseflies is rare and it could be suggested that the male factor is connected to a certain low–expressed allele of the CYP4G2 gene, located on the same chromosome. Then females might have another version of the allele, which is expressed at a higher level. Then females would have two highly-expressed copies of CYP4G2, while males have one highly-expressed copy and one low-expressed copy (linked with the M factor). This could explain the sex-specific difference in spinosad resistance.
CYP6G4 is a possible ortholog of the CYP6G1 gene in D. melanogaster and constitutive overexpression of CYP6G1 is causing DDT and neonicotinoid resistance in the fruit fly [21], [22]. A similar role for CYP6G4 in houseflies could be suggested. Recently, CYP6G4 has shown to be over-expressed in a pyrethroid resistant housefly strain from China, but no causal link was established [30]. In this study, CYP6G4 expression was higher in the 791spin strain compared to the susceptible strain, 2-fold in males and almost 15-fold in females. However, CYP6G4 gene expression in 791spin was lower than that of the spinosad susceptible strains. CYP6G4 gene expression was not significantly different between sexes, regardless of treatment (Table 1). Spinosad treatment caused a gene expression increase in males of the 791spin strain; whereas spinosad treatment decreased CYP6G4 expression in females, but neither of the effects were significant (P<0.70). This is contradicted by the other spinosad susceptible strains having a higher expression level of CYP6G4 than 791spin, so a possible role of CYP6G4 seems to be minor. Further investigations, especially description of the CYP6G4 alleles, are needed to elucidate the role of CYP6G4 in this strain as well as its potential role in xenobiotic metabolism and its importance as a housefly insecticide resistance gene.
Constitutive CYP12A1 expression was 3-fold higher in male houseflies compared to females for both WHO-SRS and 791spin. Furthermore, 791spin flies had an expression level of approximately 10-fold that of WHO-SRS. The sex-dependent difference in CYP12A1 gene expression remained after spinosad exposure (P<0.0022) in 791spin, but was eliminated in WHO-SRS (Table 1). Spinosad increased CYP12A1 expression in 791spin males (P<0.0003) and females (P<0.0001) 1.5- and 3.4-fold, respectively (Table 1). The high constitutive expression of CYP12A1 in 791spin flies could indicate importance in spinosad resistance, but the fact that male expression is 3-fold higher than female constitutive expression indicates the opposite.
In 791spin males, the constitutive CYP12A2 expression was increased 1.3-fold by spinosad exposure (P<0.0091), while in the resistant females, no effect of short-term spinosad exposure was observed (P>0.95). Expression in 791spin is similar to WHO-SRS expression, and lower than the other strains (Table 1 and 2), indicating no specific role for CYP12A2 in spinosad resistance in 791spin.
Increased expression of CYP6A36 has like CYP6D1 been associated with pyrethroid resistance in the USA, whereas CYP6A37 did not differ between ALHF and a susceptible strain [10], [18]. No significant effect of spinosad treatment on CYP6A36 gene expression was observed for any of the five strains (P>0.072), with the exception of 791spin females. Here spinosad caused a 4-fold increase in CYP6A36 gene expression. Furthermore, 791spin had a low CYP6A36 expression compared to the other strains of this study irrespective of sex (Table 2). The very low expression of CYP6A36 in 791spin disqualifies CYP6A36 as responsible for spinosad resistance, while the specific female induction points in the other direction. Spinosad exposure caused a significant decrease (P<0.0019) in CYP6A37 gene expression for male 791spin flies (Figure 2). Independent of treatment, CYP6A37 expression was significantly lower in both WHO-SRS (P<0.0034) and 791spin (P<0.0007) females than in males (Table 1). This suggests no role for CYP6A37 in sex-specific spinosad resistance in 791spin.
The epoxidation activity of the cytochrome P450 CYP6A1 enzyme is possibly linked to organochlorine and organophosphate resistance, and also has juvenile hormone I and III as substrate [32]. The data for CYP6A1 expression show a higher expression level in 791spin flies compared to WHO-SRS flies. Spinosad exposure increases expression levels in WHO-SRS 2.9- and 1.7.fold in males and females, respectively (P<0.041). A similar effect is observed for 791spin males, where expression was induced 1.7-fold (P<0.020). However, female expression remained at a similar level (P>0.54). The induction of CYP6A1 gene expression by spinosad in susceptible WHO-SRS flies, combined with the lack of effect in 791spin females, indicate a possible role for CYP6A1. However, in 791spin male constitutive expression is higher than female expression, which indicates no role for CYP6A1 in 791spin.
Prior results of feeding bioassays and gene expression studies in 791spin and its parental 791a strain suggest an involvement of cytochrome P450 enzymes in housefly spinosad resistance, which seems to be specifically female-linked [8], [17]. These synergist and gene expression studies indicated a minor role of cytochrome P450 genes CYP6D1 and CYP6D3 in spinosad resistance in male and female houseflies, respectively. CYP6D1 is over-expressed in flies resistant to pyrethroids and neonicotinoids [17], [19], [20]. A wide range of substrates is known for the CYP6D1 enzyme including the organophosphate chlorpyrifos and the synthetic pyrethroids deltamethrin and cypermethrin [33]–[35]. CYP6D3 has been used as a reference to CYP6D1 and is also located on chromosome I [36]. In this study, male constitutive expression of CYP6A1 and CYP6D3 was higher in 791spin than in WHO-SRS flies. Likewise, female constitutive expression of CYP6A1 was also higher in resistant flies compared to susceptible. This correlates with the pervious study of CYP6A1, CYP6D1 and CYP6D3 [8]. However, here male CYP6D1 and female CYP6D3 expression in 791spin did not differ from WHO-SRS. Markussen et al. (2012) suggested a role for CYP6D1 and CYP6D3 in male and female resistance, respectively. However, here the opposite is indicated by a high CYP6D1 and CYP6D3 expression in 791spin females and males, respectively. This further complicates the determination of the importance of these two genes in spinosad resistance.
Increased expression has been linked to elevated resistance by increased enzymatic degradation of insecticide e.g. degradation of deltamethrin by CYP6D1 [19], [20]. But, what does down-regulation signify? It could be hypothesized that down-regulation is due to reduced energy costs. Perhaps down-regulation is a part of a coupled system, so that groups of P450 genes are up-regulated and others are down-regulated. An example of this is a transcription factor pathway in Drosophila where 20% of differential expressed genes (including multiple resistance associated P450s) are genes targeted by the transcription factor CncC [37].
Alternatively the action is more direct and a given P450 enzyme activates the insecticide, making it more toxic. This is e.g. shown by bioassay experiments with neonicotinoids. The addition of the synergist PBO to feeding test with imidacloprid increased toxicity 9-fold, whereas toxicity of thiamethoxam decreased in male houseflies [17]. Thiamethoxam has also been shown as a pro-insecticide in plants [38]. Down-regulation will thus have a direct survival effect.
Effect of spinosad in a new field strain
The field population 845b was included in this study to examine a neonicotinoid- and spinosad-susceptible field population. In general, high levels of P450 gene expression was found in this newly acquired field population for eight out of nine P450 genes in both males and females. Female expression of CYP6D3 was more than 30-fold higher in 845b than in WHO-SRS (Table 2). This was despite 845b proving susceptible to pyrethrin synergized by PBO, imidacloprid and spinosad by test with discriminating doses [29]. Short-term spinosad exposure had no effect on gene expression of P450 genes with a few exceptions. In females, CYP6A1 expression was decreased 2.5-fold (P<0.0021) and CYP12A1 expression was increased 1.8-fold (P<0.0020). None of the other seven P450 genes were affected by spinosad in females (P>0.13). Furthermore, spinosad caused no effect in 845b males for any of the nine P450 genes tested here (P>0.10).
The high gene expression levels observed in this newly-collected strain could be due to the energy requirements involved in maintaining an alert detoxification system. The 845b strain still represent life in the field, while the other strains used for this study have adapted to the laboratory, properly losing some of the defense mechanisms involved in being a wild fly. The pattern of decreased expression in strains adapted to the laboratory was observed for the 845b strain in a two-year study of gene expression [39].
Effect of spinosad in an insecticide-susceptible reference strain
The highest constitutive P450 expression in the susceptible reference strain WHO-SRS was observed for CYP4G2 in both sexes, whereas CYP6A36 and CYP6A1 were the lowest expressed genes (Table 1).
In general, spinosad exposure caused an increase in gene expression of the nine P450 genes described here for both males and females (Figure 1). GAPDH was doubled by short-term spinosad exposure (Table 1). When taking that into consideration and normalizing to GAPDH levels, only a few P450 genes were affected by spinosad. In males, the induction of gene expression was no higher than 3-fold with the non-normalized data, while female induction ranged from 1.7-fold to almost 7-fold. In no case did spinosad exposure decrease the expression level significantly.
CYP4G2 expression was increased 6.6-fold by spinosad in females (P<0.0050). A similar increase (P<0.0043) was observed in expression of CYP6G4, while CYP12A1 and CYP12A2 were increased approximately 2-fold in females (P<0.0006).
Furthermore, spinosad caused an increase in expression of CYP6A1 (P<0.041), CYP6A37 (P<0.0034) and CYP6D3 (P<0.0006) for both males and females. However, no effect of spinosad was observed for male CYP6D1, CYP12A1 and CYP12A2 expression as well as for CYP6A36 expression.
The most remarkable effect by spinosad was observed for CYP6G4, where WHO-SRS female expression was increased almost 7-fold (P<0.0043) and male expression 2.6-fold (P<0.0012) as a result of short-term spinosad exposure. This could indicate a role for CYP6G4 in spinosad resistance or at least a role in xenobiotic response. In most cases, gene expression of P450 genes was induced in the susceptible strain, suggesting spinosad affecting several genes, not all responsible for the resistance mechanism.
Effect of spinosad in a neonicotinoid-resistant laboratory strain
In the neonicotinoid-resistant laboratory population 766b the highest constitutive gene expression was observed for CYP4G2 and CYP6D1, for both males and females (Table 2). Noteworthy is the 7-fold and 15-fold higher constitutive expression of CYP6G4 in males and females, respectively, compared to WHO-SRS. The 766b female CYP4G2, CYP6D3 and CYP12A2 expression increased (1.4- to 4.8-fold), upon spinosad treatment (P<0.0043). Male CYP6D3 gene expression was not affected by spinosad (P>0.065), while CYP4G2 and CYP12A2 expression increased in females 1.6-fold (P<0.0087) and 2.8-fold (P<0.0006), respectively. Furthermore, CYP6A1, CYP6D1 and CYP6G4 were induced in males (P<0.015).
Furthermore, spinosad treatment caused gene expression in males to increase 2-fold for CYP12A1 (P<0.0041), whereas females didn’t change CYP12A1 expression (P>0.96).
CYP6D1 appears to be involved in male neonicotinoid resistance while female resistance seems to be linked to over-expression of the CYP6D3 gene [17]. Both CYP6D1 and CYP6D3 expression proved higher in 766b than in WHO-SRS. CYP6D1 in males was induced by spinosad, while CYP6D3 was induced in females, indicating possible roles for these genes in resistance.
The high CYP6G4 gene expression observed in 766b in comparison with WHO-SRS could indicate a role of CYP6G4 in 766b neonicotinoid resistance, but not in spinosad resistance, since 766b is susceptible to spinosad. Additionally, previous studies has shown that neonicotinoid resistance in strain 766b responded to PBO, indicating metabolism associated resistance as well as reduced expression of the nicotinic acetylcholine-receptor subunit Mdα2 [17], [40].
Effect of spinosad in a multi-resistant laboratory strain
In the multi-resistant laboratory strain 791a the CYP6A1 gene was expressed at the lowest level of the genes described in this strain, whereas CYP4G2 expression was expressed at the highest level, regardless of treatment (Table 2). Spinosad decreased CYP4G2 gene expression (P<0.0021). Likewise, the CYP12A2 gene was decreased by spinosad treatment in males, while the other seven genes were unaffected by spinosad (P>0.10).
Conclusion
It can be concluded that there is very clear difference between the expression of P450 genes in male compared to female houseflies. Most of the genes tested where spinosad caused an effect were up-regulated by spinosad exposure. In the present investigation a marked difference between a true field strain (F1) and the laboratory reared strains was observed.
Four requirements were purposed to help identify possible resistance genes in this study; 1) higher constitutive expression in 791spin compared to WHO-SRS, 2) spinosad induction of P450 expression in WHO-SRS or other spinosad-susceptible strains, while 3) no significant effect was observed in 791spin, 4) female constitutive expression in 791spin is higher than male expression.
CYP4G2, CYP6A1, CYP6D3 and CYP6G4 have an expression pattern somewhat fitting the requirements and therefore the resistance profile of 791spin, but only CYP4G2 fit all four requirements significantly, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and e.g. pointing to a single spinosad resistance gene. Small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin.
Analyzing the expression of metabolic detoxification genes rarely gives a clear and unambiguous answer to which enzymes are involved in resistance [41]. Furthermore, the P450s ability to attack the highly complicated spinosad molecule is still unresolved. There is an indication of CYP6G4 as an insecticide-resistance gene, where involvement in spinosad resistance cannot be rejected. The high expression levels of P450 genes in flies from a field population compared to established laboratory strains presented in this study questions the use of laboratory strains beyond resistance gene identification and warrants studies of P450 expression in field populations, since only the heterogeneous nature of field populations would allow for the selection of the rare variants corresponding to resistance alleles likely to trigger control failure [42]. Laboratory strains can only be used for assessing field control issues if the starting genetic variation is present in the field strain adapted to the laboratory. Differential expression is both up- and down-regulated. Increased expression has been functionally linked to elevated resistance, but it is still puzzling what down-regulation signifies. Is it a part of an interconnected regulatory network? Is it linked to the overall energy budget? Or is it linked to toxicology dynamics of the P450 system?
The differences in expression of minor and major insecticide resistance genes are some of the important tools in pesticide resistance management aiming to limit or prevent development of resistance by controlling factors, which may lead to resistance. This study serves as a stepping stone for the dissection of P450 expression in houseflies in relation to xenobiotics.
Acknowledgments
The authors would like to thank Folmer Jensen, Lars Damberg and Claus Dahl for providing helpful technical assistance during housefly sampling and breeding.
Author Contributions
Conceived and designed the experiments: DHH KMVJ MK. Performed the experiments: DHH. Analyzed the data: DHH KMVJ MK. Wrote the paper: DHH KMVJ MK.
References
- 1. Sparks TC, Crouse GD, Durst G (2001) Natural products as insecticides: the biology, biochemistry, and quantitative structure-activity relationships of spinosyns and spinosoids. Pest Management Science 57: 896–905.
- 2. Watson GB (2001) Actions of insecticidal spinosyns on gamma-aminobutyric acid responses from small-diameter cockroach neurons. Pesticide Biochemistry and Physiology 71: 20–28.
- 3. Sparks TC, Dripps JE, Watson GB, Paroonagian D (2012) Resistance and cross-resistance to the spinosyns - A review and analysis. Pesticide Biochemistry and Physiology 102: 1–10.
- 4. Scott JG (2008) Unraveling the mystery of spinosad resistance in insects. Journal of Pesticide Science 33: 221–227.
- 5. Kristensen M, Jespersen JB (2004) Susceptibility of spinosad in Musca domestica (Diptera: Muscidae) field populations. Journal of Economic Entomology 97: 1042–1048.
- 6. Shono T, Scott JG (2003) Spinosad resistance in the housefly, Musca domestica, is due to a recessive factor on autosome 1. Pesticide Biochemistry and Physiology 75: 1–7.
- 7. Shi J, Zhang L, Gao XW (2011) Characterisation of spinosad resistance in the housefly Musca domestica (Diptera: Muscidae). Pest Management Science 67: 335–340.
- 8. Markussen MDK, Kristensen M (2012) Spinosad resistance in female Musca domestica L. from a field-derived population. Pest Management Science 68: 75–82.
- 9.
Højland DH, Scott JG, Jensen KM, Kristensen M (2013) Autosomal male determination in a spinosad-resistant housefly strain from Denmark. Pest Manag Sci.
- 10. Zhu F, Li T, Zhang L, Liu N (2008) Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica. BMC Physiology 8: 18.
- 11.
Feyereisen R (2012) Insect CYP Genes and P450 Enzymes. In: Gilbert LI, editor. Insect Molecular Biology and Biochemistry: Elsevier, Amsterdam. 236–316.
- 12. Qiu Y, Tittiger C, Wicker-Thomas C, Le Goff G, Young S, et al. (2012) An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 109: 14858–14863.
- 13. Andersen JF, Utermohlen JG, Feyereisen R (1994) Expression of House-Fly Cyp6a1 and Nadph-Cytochrome P450 Reductase in Escherichia-Coli and Reconstitution of an Insecticide-Metabolizing P450 System. Biochemistry 33: 2171–2177.
- 14. Feyereisen R, Andersen JF, Carino FA, Cohen MB, Koener JF (1995) Cytochrome-P450 in the House-Fly - Structure, Catalytic Activity and Regulation of Expression of Cyp6a1 in an Insecticide-Resistant Strain. Pesticide Science 43: 233–239.
- 15. Carino FA, Koener JF, Plapp FW, Feyereisen R (1994) Constitutive Overexpression of the Cytochrome-P450 Gene Cyp6a1 in a House-Fly Strain with Metabolic Resistance to Insecticides. Insect Biochemistry and Molecular Biology 24: 411–418.
- 16. Sabourault C, Guzov VM, Koener JF, Claudianos C, Plapp FW, et al. (2001) Overproduction of a P450 that metabolizes diazinon is linked to a loss-of-function in the chromosome 2 ali-esterase (Md alpha E7) gene in resistant houseflies. Insect Molecular Biology 10: 609–618.
- 17. Markussen MDK, Kristensen M (2010) Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L. Pesticide Biochemistry and Physiology. 98: 50–58.
- 18. Zhu F, Feng JN, Zhang L, Liu N (2008) Characterization of two novel cytochrome P450 genes in insecticide-resistant house-flies. Insect Molecular Biology 17: 27–37.
- 19. Liu N, Scott JG (1998) Increased transcription of CYP6D1 causes cytochrome P450 mediated insecticide resistance in house fly. Insect Biochemistry and Molecular Biology 28: 531–535.
- 20. Kasai S, Scott JG (2000) Overexpression of cytochrome P450CYP6D1 is associated with monooxygenase-mediated pyrethroid resistance in house flies from Georgia. Pesticide Biochemistry and Physiology 68: 34–41.
- 21. Daborn P, Boundy S, Yen J, Pittendrigh B, Ffrench-Constant R (2001) DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid. Molecular Genetics and Genomics 266: 556–563.
- 22. Daborn PJ, Yen JL, Bogwitz MR, Le Goff G, Feil E, et al. (2002) A single P450 allele associated with insecticide resistance in Drosophila. Science 297: 2253–2256.
- 23. Guzov VM, Unnithan GC, Chernogolov AA, Feyereisen R (1998) CYP12A1, a mitochondrial cytochrome P450 from the house fly. Archives of Biochemistry and Biophysics 359: 231–240.
- 24. Kristensen M, Huang J, Qiao CL, Jespersen JB (2006) Variation of Musca domestica L. acetylicholinesterase in Danish housefly populations. Pest Management Science 62: 738–745.
- 25. Kristensen M, Jespersen JB (2008) Susceptibility to thiamethoxam of Musca domestica from Danish livestock farms. Pest Management Science 64: 126–132.
- 26. Kristensen M, Jespersen JB (2003) Larvicide resistance in Musca domestica (Diptera: Muscidae) populations in Denmark and establishment of resistant laboratory strains. Journal of Economic Entomology 96: 1300–1306.
- 27. Kristensen M, Jespersen JB, Knorr M (2004) Cross-resistance potential of fipronil in Musca domestica. Pest Management Science 60: 894–900.
- 28. Kristensen M, Spencer AG, Jespersen JB (2001) The status and development of insecticide resistance in Danish populations of the housefly Musca domestica L. Pest Management Science. 57: 82–89.
- 29.
Højland DH, Jensen KM, Kristensen M (2013) A comparative study of P450 gene expression in field and laboratory Musca domestica L. strains. Pest Manag Sci.
- 30. Gao Q, Li M, Sheng CF, Scott JG, Qiu XH (2012) Multiple cytochrome P450s overexpressed in pyrethroid resistant house flies (Musca domestica). Pesticide Biochemistry and Physiology 104: 252–260.
- 31.
R Development Core Team (2012) R: A Language and Environment for Statistical Computing. In: Computing RFfS, editor.
- 32. Scott JG, Wen ZM (2001) Cytochromes P450 of insects: the tip of the iceberg. Pest Management Science 57: 958–967.
- 33. Wheelock GD, Scott JG (1992) The Role of Cytochrome-P450ipr in Deltamethrin Metabolism by Pyrethroid-Resistant and Susceptible Strains of House-Flies. Pesticide Biochemistry and Physiology 43: 67–77.
- 34. Wheelock GD, Scott JG (1992) Anti-P4501pr Antiserum Inhibits Specific Monooxygenase Activities in Lpr House-Fly Microsomes. Journal of Experimental Zoology 264: 153–158.
- 35. Zhang ML, Scott JG (1996) Cytochrome b(5) is essential for cytochrome P450 6D1-mediated cypermethrin resistance in LPR house flies. Pesticide Biochemistry and Physiology 55: 150–156.
- 36. Kasai S, Scott JG (2001) Expression and regulation of CYP6D3 in the house fly, Musca domestica (L.). Insect Biochemistry and Molecular Biology 32: 1–8.
- 37. Misra JR, Lam G, Thummel CS (2013) Constitutive activation of the Nrf2/Keap1 pathway in insecticide-resistant strains of Drosophila. Insect Biochem Mol Biol 43: 1116–1124.
- 38. Nauen R, Ebbinghaus-Kintscher U, Salgado VL, Kaussmann M (2003) Thiamethoxam is a neonicotinoid precursor converted to clothianidin in insects and plants. Pesticide Biochemistry and Physiology 76: 55–69.
- 39.
Højland DH, Jensen KMV, Kristensen M (2014) Adaptation of Musca domestica L. field population to laboratory breeding causes transcriptional alterations Plos One.
- 40. Markussen MDK, Kristensen M (2010) Low expression of nicotinic acetylcholine receptor subunit Md alpha 2 in neonicotinoid-resistant strains of Musca domestica L. Pest Management Science. 66: 1257–1262.
- 41. Saavedra-Rodriguez K, Suarez AF, Salas IF, Strode C, Ranson H, et al. (2012) Transcription of detoxification genes after permethrin selection in the mosquito Aedes aegypti. Insect Molecular Biology 21: 61–77.
- 42. Ffrench-Constant RH (2013) The Molecular Genetics of Insecticide Resistance. Genetics 194: 807–815.