Journal of Plant Diseases and Protection, 121 (2), 89–95, 2014, ISSN 1861-3829. © Eugen Ulmer KG, Stuttgart J.Plant Dis.Protect. 2/2014 Changes in the expression of mitochondrial cytochrome oxidase subunits due to pyrethroid intoxication in pyrethroid-resistant pollen beetles, Meligethes aeneus (Coleoptera: Nitidulidae) Przemysław Wieczorek 1 , Paweł WĊgorek 2 , Dorota Protasewicz 1 , Joanna Zamojska 2 , Marta Budziszewska 1 , Marek MrówczyĔski 2 & Aleksandra ObrĊpalska-StĊplowska 1,* 1 Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection – National Research Institute, PoznaĔ, Poland 2 Department of Zoology, Institute of Plant Protection – National Research Institute, PoznaĔ, Poland * Corresponding author: olaob@o2.pl or ao.steplowska@iorpib.poznan.pl Received 05 January 2014, accepted 31 January 2014 Abstract Pollen beetle (Meligethes aeneus, Coleoptera: Nitidulidae) is the most important pest of oilseed rape in Europe, causing great yield losses. Due to the heavy use of pyrethroid insec- ticides in controlling Meligethes, a widespread build-up of resistance to pyrethroid active substances has arisen, reported in many countries where the pest occurs. Mutations in the voltage-sensitive sodium channels (VSSC), which constitute the main targets for the pharmacological action, as well as increased oxidative metabolism of pyrethroid active sub- stances in resistant populations are considered, as they are the main molecular mechanisms of the development of pyre- throid resistance. In this study we have analyzed the level of expression of mitochondrial cytochrome oxidase subunit genes (mtCOI, mtCOII) in esfenvalerate-treated populations of M. aeneus collected in 2011 by using the real-time PCR approach. Our results indicate that the esfenvalerate-treated beetles have a significantly higher mtCOI gene expression compared with the untreated ones and that the mtCOII transcript level is also slightly induced. This enhanced expression might, in part, be responsible for the increased oxidative metabolism in pyrethroid-challenged pollen beetle populations. Key words: gene expression induction, insecticide resistance, pyrethroids Introduction Pollen beetle – Meligethes aeneus (Coleoptera: Nitidulidae) is a major insect pest of oilseed rape (Brassica napus). This crop is extensively cultivated and treated with insecticides because of the high density of the pest population. The pro- duction scale of oilseed rape increases throughout Europe every year and Poland is one of the largest oilseed rape pro- ducers. M. aeneus damages the buds and flowers of the plant, reducing the quantity of oilseed rape yields. Chemical plant protection still remains the most important tool in con- trolling pollen beetle and the number of insecticide appli- cations ranges in different countries from 1 to 5 per season (Richardson 2008). Pyrethroids are the main group of insec- ticides that are registered in many countries for pollen beetle control. They target the voltage-sensitive sodium channels (VSSC) in neuronal membranes, causing a dysfunction of their opening state and inhibiting their deactivation (Narahashi 2000). Insects are found to build up resistance against the active substances present in insecticides that are intensively used for pest control. The pyrethroid resistance of M. aeneus popula- tions was first recorded in Poland in the late 1970s (Łąkocy 1977). However, its occurrence was reported in many Euro- pean countries, including France (Derron et al. 2004), Ger- many (Heimbach et al. 2006), and many others (Slater et al. 2011). The recent monitoring of European populations for pollen beetle’s resistance to pyrethroids showed that this phenomenon occurs in 20 out of 21 of the surveyed countries and that it will become more and more widespread. More- over, resistant beetles were found to dominate in Western and Central Europe (Slater et al. 2011). One of the most common molecular basis of this resistance results from the so-called knockdown resistance (kdr), which, in turn, results from the amino acid substitutions caused by mutations in the VSSC gene (Davies et al. 2007, Dong 2007). Other mecha- nisms are based on the activity of detoxification enzymes – oxidases, esterases or glutathione S-transferases. Previously, synergism was found between pyrethroids and piperonyl butoxide (PBO) in the pyrethroid-resistant M. aeneus popu- lations (Moores et al. 2012, Philippou et al. 2011, WĊgorek 2009, WĊgorek et al. 2011). Thus, PBO added to pyrethroids increased their effectiveness in controlling the pollen beetle. This suggested increased activity of cellular oxidases and ester- ases in the pyrethroid detoxication process was demonstrated further (Philippou et al. 2011, WĊgorek 2009, WĊgorek et al. 2011). PBO is known to inhibit the P-450 cytochrome (CYP) oxidation mechanism (Wilkinson et al. 1984). There are sev- eral factors that may be responsible for effective pyrethroid detoxification by oxidative enzymes: altered enzymes in resistant insects or differences in the levels of the studied oxidative systems (P-450 and non-P-450) (Philippou et al. 2011, WĊgorek 2009). The second factor may be a result of the enhanced expression of genes coding oxidative en- zymes. Cytochrome oxidase is a multicomponent enzyme complex whose subunits are encoded by nuclear and mitochondrial genomes. Mitochondrial subunits of DNA sequences (mtCOI and mtCOII) are frequently used in phylogenetic studies and for diagnostic purposes, including studies carried out on insect species (ObrĊpalska-StĊplowska et al. 2008). Recent studies