The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) is among the most destructive pests of stored grains, with global distribution [1]. It is a primary feeder and infests a variety of stored products and related commodities [2], which are essential for human nutrition and global food security [1, 3]. Moreover, it is a primary colonizer, thus larvae and adults can easily penetrate the kernels even at low moisture content and complete their life cycle in intact whole grain kernels [4, 3, 2]. As a result, most life stages, especially the larvae, are unaffected by contact insecticides that are applied on the external part of the grain kernel [1]. Crucially, R. dominica has a rapid population growth resulting in devastating infestation levels, especially at elevated temperatures [1, 5]. Management of R. dominica in stored grain and other commodities have been investigated thoroughly around the globe [1, 6]. In general, its control is currently based on two broad categories of insecticides, the fumigants [7] and contact insecticides [8]. However, it is now well-established that populations of R. dominica have developed resistance to both chemical and non-chemical treatments. In particular, high levels of resistance to phosphine [9, 10, 11], pirimiphos-methyl [12], deltamethrin [7, 13] have been reported in many parts of the world, such as Australia, USA and Brazil [9, 10, 11]. At the same time, this species cannot be easily controlled by some “traditional” contact insecticides that are applied directly on grains, such as the organophosphorous compound pirimiphos-methyl [12] and the pyrethroid deltamethrin [7, 13]. Moreover, it is well-established that R. dominica is less susceptible than other major stored product insect species to non-chemical control methods, such as diatomaceous earths [14], which poses serious challenges to grain industry towards management of this species. Therefore, there is a demand to identify newer, reduced risk compounds that can be effectively used in controlling this notorious pest.
One of the newer active ingredients that have been registered in many countries for the control of R. dominica is the juvenile hormone analogue (JHA) s-methoprene, [15]. JHAs target and disrupt the endocrine system of insects by causing abnormal larval-pupal or nymphal-pupal development and/or even death [16]. In general, s-methoprene has many desirable characteristics, such as good environmental profile and extremely low mammalian toxicity [17, 18] and it is currently considered as a good alternative to many other conventional contact insecticides [15]. In the case of R. dominica, s-methoprene has been proved very effective against R. dominica in different commodities and conditions [15, 19, 20, 21, 22]. Progeny production of R. dominica could be even contrοlled when applied in parts of the grain mass [22]. It also exhibits a considerable residual efficacy on stored grains, thus holds high potential as a grain protectant for long-term treatment [15, 23]. Further, s-methoprene has many desirable characteristics, such as good environmental profile and extremely low mammalian toxicity [17, 18], and thus, it is currently being considered as a good alternative to many other contact insecticides [15, 24].
Although resistance to JHAs is rare and not frequent, resistance to pyriproxifen in the house fly Musca domestica L. (Diptera: Muscidae) and the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) [25], as well as s-methoprene in mosquitoes [16] have been reported, suggesting that resistance may develop in the case of other species, including R. dominica. An s-methoprene resistant population of R. dominica showed that required a very high dose (40 mg kg− 1) for its complete control in wheat grain [26]. This dose rate is approximately 67 times higher than the registered rate applied in Australia, questionning the usage of this insecticide as a grain protectant and demanding the need to overcome this problem. Moreover, resistance to s-methoprene may jeopardize the resistance managment strategies to phosphine and neurotoxic insecticides [27], on which the inclusion of a JHA, e.g. on a rotation basis, is a key element.
Piperonyl butoxide (PBO), has been used extensively either alone or in combination with other active ingredients as a synergist in crop protection, especially to break resistance to specific group of insecticides such as pyrethroids that exhibits toxicity through mixed function oxidases including P450s [28]. Several studies reported the interaction of PBO with cytochrome P450s [28, 29]. The mechanism through which PBO inhibits P450s is mostly unknown but it has been proposed that carbine interacts with haem iron of the P450 and carbene, thus leading to its inactivation [28, 30, 31]. In the case of stored product protection, PBO has been applied in conjunction with diatomaceous earths with very good results against different species, including R. dominica [32, 33, 34]. Moreover, the simultaneous application of PBO with pyrethroids increased their efficacy, as compared with the application of pyrethroids alone [35, 36]. For example, PBO improved the insecticidal effect of deltamethrin in protecting grains against the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) [37].
The molecular mechanism of s-methoprene resistance has not been fully elucidated yet. In the fruit fly, Drosophila melanogaster Meigen (Diptera: Drosophilidae), the absence of mutation of a so-called methoprene tolerant (MET) gene results in s-methoprene resistance [38, 39]. The protein (MET) encoded by the MET gene belongs to the family of basic helix-loop-helix (bHLH)-PAS transcriptional regulators that bind JH with high affinity [40]. In the absence of JH-III, the juvenile hormone synthesized in most insects, or a synthetic mimic, MET forms homodimers (Gce in D. melanogaster forming heterodimer), whereas their presence leads to dissociation of the MET dimer and thus binding of the ligand. Computational analysis and ligand binding assays of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) MET protein indicated that the mutations V280F and V297F abolish JH-III binding. Additionally, ligand-dependent protein assays where both MET monomers carry the V297F mutation are resistant in presence of s-methoprene, thus do not dissociate, in contrast to their wild type counterparts [40]. Also, functional assays by knocking down MET in T. castaneum, render the insects resistant to natural JH and as well as methoprene [41]. Alternatively, resistance to s-methoprene in other species has been associated with high activity of P450 monooxygenases and esterases, which probably also contribute to resistance to s-methoprene and other JHAs [42, 43]. However, detailed research revealing the exact relationship between s-methoprene and P450s is not established, but it has been shown that P450s can metabolize JHAs, as in the case of pyriproxifen [44], which consists an indication of the same phenomenon may occur in the case of s-methoprene.
Resistance to s-methoprene has not been analysed yet in R. dominica, largely due to the lack of genomic resources for this pest species. RNA sequencing technologies have evolved rapidly in the last years [45]. They allow the study of transcriptomes without necessarily relying on a reference genome, thus greatly facilitating the study of several non-model species. Subsequently, comparison of gene transcription levels between insecticide resistant and insecticide-susceptible insect populations can lead to candidate genes that could play a role in the observed resistant phenotype. Such analysis has been performed in several insects and mites [46–49], providing not only a better understanding of insecticide resistance, but also valuable genomic resources that prove useful for studying different aspects of the biology of arthropods that constitute the most diverse animal clade [50–52].
In this regard, the aim of the present work was to investigate, for the first time, the mechanisms underlying s-methoprene resistance in the lesser grain borer, R. dominica. We useds- methoprene-resistant and susceptible strains and compared their response to s-methoprene alone, but also in combination with PBO, with the perspective of using the latter to break resistance. The bioassays showed that the combined use of s-methoprene + PBO increased the efficiency of the former, thereby suggesting a possible involvement of CYPs in the resistance mechanism. Subsequently, we sequenced the transcriptomes of s-methoprene-resistant and susceptible strains and identified the Cytochrome P450 (CYP) genes. Interestingly, their analysis revealed that a number of them were significantly up-regulated in the s-methoprene-resistant strain and are thus worth of further investigation to determine their role in insecticide resistance.