B. xylophilus is highly destructive to pines. Until now, there is a lack of effective prevention and control of it. So, genes in the xenobiotic metabolism were very important to speculate the target for the control of B. xylophilus. In eukaryotes, genes in the xenobiotic metabolism can remove drugs from cells, resulting in a decrease in intracellular drug concentration, contributing for drug resistance. Several studies have already shown the multilayer detoxification of B. xylophilus against plant defences, which involves the detoxification and xenobiotic metabolic pathways of the nematode. Even there is a study on the effect of emamectin benzoate in B. xylophilus with the same approach. So, it is necessary to study the function of xenobiotic metabolism in order to provide theoretical basis for the development of LH control methods and targets. LH is a broad-spectrum, high-efficiency, and low-toxic nematicide, which has a good killing effect on chicken, sheep, and human gastrointestinal nematodes. The effect of LH, xenobiotic detoxification and pathogenic mechanism of B. xylophilus has important value and provides a theoretical basis for prevention B. xylophilus.
Nine RNA libraries directly reflected the effect of LH on the genetic activity of B. xylophilus. Among the 32 common Gene Ontology (Fig. 3A), antioxidant activity, catalytic activity, detoxification, immune system process, molecular transducer activity, synapse, synapse part, transporter activity, signal transducer activity showed that LH treatment had a significant effect on the neural transmission and immune system of B. xylophilus. The single-organism process was only in the group of YDvsYG, includes a 3'5'-cyclic nucleotide phosphodiesterase and 3 Cuticle collagen, which were significantly up-regulated from low concentration to high concentration. The Cuticle collagen, an extracellular collagen matrix that surrounds C. elegans, is the first barrier against environmental damage, protecting nematodes from pathogens, dryness, and other stress[24–26]. The up-regulation of the high concentration of Cuticle collagen indicated that the integument of B. xylophilus thickened, the resistance of nematodes was enhanced. Xenobiotic detoxification pathways with drug metabolism - cytochrome P450 (ko00982) and metabolism of xenobiotics by cytochrome P450 (ko00980) were found, including most of the up-regulation FMO, UDT, GST, ADH and a Juvenile hormone epoxide hydrolase down-regulations (Fig. 3B) (Additional file 4). Compared with C. elegans, B. xylophilus has specific gene up-regulation in FMOs branch (FMO-5, FMO-10), which has a function similar to cytochrome P450 and adds molecular oxygen to insoluble xenobiotic organisms. Many insects and microorganisms can metabolize chemicals into less toxic or more soluble compounds. They use cytochrome P450 or FMO to oxidize it to alcohols, then ADHs to oxidize alcohols to aldehydes/ketones, and finally ALDHs to oxidize these products to acids for subsequent metabolism or transport. ADH is one of the key genes regulating the dynamic behavior of B. xylophilus. P450, UGT, glutathione -S transferase, Redox, dehydrogenase, and reductase of C. elegans under five different Benzimidazole drugs increased. UGT are enzymes that are glucuronidated the substrates include drugs, environmental toxins, and endogenous compounds. And the products are water-soluble and readily excreted. UGT inhibitors resulted in a decrease of metabolite production in nematode and the decrease of drug resistance. UGT inhibitor increased the sensitivity of wild type H. contortus to naphtalophos (anthelmintic). GST can catalyze the binding of glutathione with electrophilic groups of chemical substances, and eventually form mercapturic acid to be excreted in vitro. And 3 GST and P450 were upregulated contributed to mitigating tetrabromobisphenol A-induced toxicity on the C. elegans. Thus, based on the results and previous studies, UDT, GST, FMO, ADH, Juvenile hormone epoxide hydrolase were considered as one of the main factors of B. xylophilus detoxification ability.
Sets of profiles were predefined which were expected in the process of genetic changes. These sets of profiles cover all of the possible gene expression changes, and each represents a single drug concentration expression pattern. Profile significance levels were determined by gene enrichment, and significant profiles indicate the most common functions of co-expressed genes, with the main biological character[33–35]. Xenobiotic detoxification pathways, such as Glycolysis/ Gluconeogenesis, Glutathione metabolism, Metabolism of xenobiotics by cytochrome P450, Drug metabolism-cytochrome P450 were significant enrichment. Also, Lysosome, Phagosome, Peroxisome, and other immune-related pathways were significantly enriched (Fig. 5B Fig. 6B), indicating that the innate immune defense of B. xylophilus increased, such as Lysosome, Cysteine proteinase, and transthyretin-like proteins. On the other hand, it has been reported that LH has the function of enhancing immune response by enhancing phagocytosis[37–39]. LH is a typical deworming drug that activates nematode nAChR, which acts as a powerful agonist in muscle contraction and movement and is a key target[36, 40]. NAChR transfers the ion signal to disorganized muscle protein through Sodium/Chloride and causes gene up-regulation, produce body-wall muscle twitchin, paralysis, and ultimately death in B. xylophilus (Additional file 9). This is one of the direct causes of the death of B. xylophilus, but how LH acts on the nervous system signal transduction process of B. xylophilus awaits further exploration (Fig. 10) (Additional file 10 A).
Genes in the same biological pathway are more likely to be co-expressed to synchronize an array of biochemical reactions[41, 42]. We studied the connectivity patterns of drug metabolism-cytochromeP450 (ko00982) and metabolism of xenobiotics by cytochrome P450 (ko00980) pathways in profile1and profile 6 gene expression data. In Profiles 6, we observed that 54 genes were associated with the correction of Pearson, that is, the corresponding P-value was < 0.05. FMO (BUX. Gene. S00600.44, BUX. Gene. S00713.800) and ADH (BUX. gene. S01281.195, BUX. gene. S01147.198) were significant correlation in the center gene (Fig. 7), which were highly consistent with the previous conjecture that its play important role in xenobiotics metabolism. Among them, highly correlated Cuticle collagen(BUX.gene.s 00540.1); Lipase (BUX.gene.s 01661.12); ALDH (BUX.gene.s01144.247) all produced proteins related to nematode immunity. Pyruvate dehydrogenase E1 component subunit beta (BUX.gene.s01066.55) is highly related to the TCA cycle and mitochondrial energy metabolism, providing energy for the growth and development of nematodes[38, 39, 43]. We speculate that pyruvate dehydrogenase was significantly up-regulated because of aldehyde and ketone oxidase oxidated intermediate (disulfide). In profiles1, we observed that cuticle collagen (BUX.gene.s00252.94); Zinc metalloproteinase (BUX.gene.s00579.211) as zinc matrix metalloproteinases can improve the resistance to high temperature and bacteria in C. elegans; Cystathionine beta-synthase (BUX.gene.s00083.35) provides cysteine, which is important for the formation of the stratum corneum in C. elegans. As well as, it produces neuromodulator and smooth muscle relaxant hydrogen sulfide in muscle and subcutaneous tissue[45–47].
In conclusion, from the transcriptomes of B. xylophilus treated with 2.5 mg /mL,3.5 mg/mL LH, and DEPC-H2O, with STEM and Spearman correlation, pathogenesis, xenobiotic detoxification pathways, and candidate detoxification genes were identified. We propose that B. xylophilus response to LH was varied and complex which facilitated its survival in LH environment (Fig. 10) (Additional file 10 B). However, this is the first time to study the effect of LH on B. xylophilus and to explore the resistance mechanism of B. xylophilus and these findings contribute in several ways to our understanding of B. xylophilus under LH. Lays the groundwork for future research into LH could also be conducted to determine the effectiveness of control of B. xylophilus.