Expression of xenobiotic metabolizing cytochrome P450 genes in a spinosad-resistant Musca domestica L. strain.

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.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.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

Adaptation of <i>Musca domestica</i> L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

<div><p>Background</p><p>The housefly, <i>Musca domestica</i>, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain.</p><p>Results</p><p>We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F₁, F₁₃ and F₂₉ to test how gene expression changes following laboratory adaption. Four (<i>CYP6A1</i>, <i>CYP6A36</i>, <i>CYP6D3</i>, <i>CYP6G4</i>) of five investigated P450 genes adapted to breeding by decreasing expression. <i>CYP6D1</i> showed higher female expression in F₂₉ than in F₁. For males, about half of the genes accessed in the global gene expression were up-regulated in F₁₃ and F₂₉ in comparison with the F₁ population. In females, 60% of the genes were up-regulated in F₁₃ in comparison with F₁, while 33% were up-regulated in F₂₉. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F₁₃ flies in comparison with F₁. Gene expression then increased from F₁₃ to F₂₉ in males and decreased further in females.</p><p>Conclusion</p><p>The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains.</p></div

P450 gene expression of insecticide-susceptible and -resistant housefly strains upon sugar (constitutive) and spinosad exposure measured by quantitative real-time PCR.

<p>P450 gene expression of insecticide-susceptible and -resistant housefly strains upon sugar (constitutive) and spinosad exposure measured by quantitative real-time PCR.</p

Number of genes up-regulated and down-regulated as an effect of laboratory adaption in male and female 845b houseflies.

<p>Sequences which were found in less than ten copies in F₁ flies were considered background noise. This left 19,756 and 19,150 sequences for males and females, respectively. Values of F₁₃ and F₂₉ above 1.2-fold F₁ fly expression were considered up-regulated, while values below 0.8-fold F₁ were considered down-regulated. Values above 0.8-fold and below 1.2-fold were characterized as ‘unchanged’ from the F₁ flies.</p

Constitutive P450 gene expression of the housefly field strain 845b over 21 months of laboratory adaption measured by quantitative real-time PCR.

<p>Mean mRNA transcript copy number ×1000 is per 20 ng of total RNA. Ranking of significance levels (5%) between comparisons of fly generations were assigned a, b and c, to indicate significance.</p

P450 gene expression over time using qPCR and transcriptome analysis in 845b males and females.

<p>Data is normalized to gene expression for F₁ males and females, respectively. Copies of <i>CYP6A1</i> were not observed in the transcriptome analysis, and <i>CYP6A1</i> is denoted not applicable. Trans: transcriptome data.</p

Total gene expression of 845b F₁₃ and F₂₉ male and female as a function of the F₁ gene expression.

<p>The line represents no difference from the F₁ flies. Square represents F₁₃ and cross represents F₂₉. Genes in right-lower and left-upper corner are down-regulated and up-regulated over time, respectively. A few genes surpassed 20 and were omitted from the figure.</p

WHO-SRS male (A) and female (B) spinosad-influenced gene expression as a function of the constitutive gene expression.

<p>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.</p