Nitrogen (N) is one of the most intensively used fertilizers in cropping system and could exert a variety of bottom-up effects on the ecological fitness of herbivores. However, the effects of increased N inputs on insect pesticide tolerance have not been comprehensively understood. Bioassays showed that high N (HN) applied to maize plants significantly increased larval tolerance of Spodoptera litura to multiple insecticides. Activities of detoxification enzymes were significantly higher in the larvae fed on maize plants supplied with HN. RNA-seq analysis showed that numerous GST and cuticle-related genes were induced in the larvae fed on HN maize. RT-qPCR analysis further confirmed four GST genes and larval-specific cuticle gene LCP167. Furthermore, when injected with dsRNA specific to GSTe1, GSTs5, and LCP167, the mortality of larvae treated with methomyl was about 3-fold higher than that of dsGFP-injected larvae. Electron microscope observation showed that cuticle of the larvae fed on HN maize was thicker than the medium level of N. These findings suggest that increased application of N fertilizer enhances insecticide tolerance of lepidopteran pests via induction of detoxification enzymes and intensification of cuticle. Thus, overuse of N fertilizer may increase pest insecticide tolerance and usage of chemical insecticides.

Abuse of chemical fertilizers and insecticides has created many environmental and human health hazards. We hypothesized that high nitrogen (N) in crops changes insect gut microbiota leading to enhanced insecticide tolerance. We investigated the effect of high N in maize on gut microbiota and insecticide tolerance of the polyphagous pest Spodoptera litura. Bioassays showed that high N applied in both maize plants and artificial diets significantly enhanced larval growth but reduced larval sensitivity to the insecticide methomyl. High N promoted the gut bacterial abundance in the genus Enterococcus. Inoculation with two strains (E. mundtii and E. casseliflavus) isolated from the larval guts increased larval tolerance to methomyl. Incorporation of antibiotics in a high-N diet increased the larval sensitivity to methomyl. These findings suggest that excessive application of N fertilizer to crops can increase insecticide tolerance of insect pests via changing gut microbiota, leading to increased use of insecticides worldwide.

With the long-term and large-scale use, herbicides have been well known to influence tritrophic interactions, particularly natural enemies of pests in agro-ecosystems. On the other hand, herbivorous insects, especially the generalist pests, have developed antagonistic interaction to different insecticides, toxic plant secondary metabolites, and even heavy metals. However, whether exposure to herbicides would affect resistance of insects against insecticides is largely unknown, especially in agricultural pests. Here, we first reported that pre-exposure to two widely used herbicides butachlor and haloxyfop-methyl for 48 h can prime the resistance of a generalist agricultural pest Helicoverpa armigera Hübner against insecticide methomyl and fungal toxin aflatoxin B1. In addition, there were no significant differences between control and herbicides-treated caterpillars on weight gain, pupal weight, and pupation rates, suggesting that exposure to herbicides induces resistance of H. armigera accompanied with no fitness cost. Moreover, by determining detoxifying enzyme activities and toxicity bioassay with additional inhibitor of cytochrome P450 piperonyl butoxide (PBO), we showed that exposure to herbicides might prime P450-mediated detoxification of H. armigera against insecticide. Based on these results, we propose that exposure to herbicides prime resistance of H. armigera against insecticide and fungal toxin by eliciting a clear elevation of predominantly P450 monooxygenase activities in the midgut and fat body.

Plants produce secondary metabolites to provide chemical defense against herbivorous insects, whereas insects can induce the expression of detoxification metabolism-related unigenes in counter defense to plant xenobiotics. Tomatine is an important secondary metabolite in tomato (Lycopersicon esculentum L.) that can protect the plant from bacteria and insects. However, the mechanism underlying the adaptation of Spodoptera litura, a major tomato pest, to tomatine in tomato is largely unclear. In this study, we first found that the levels of tomatine in tomatoes subjected to S. litura treatment were significantly increased. Second, we confirmed the inhibitory effect of tomatine on S. litura by adding moderate amounts of commercial tomatine to an artificial diet. Then, we utilized RNA-Seq to compare the differentially expressed genes (DEGs) in the midgut and fat body tissues of S. litura exposed to an artificial diet supplemented with tomatine. In total, upon exposure to tomatine, 134 and 666 genes were upregulated in the S. litura midgut and fat body, respectively. These DEGs comprise a significant number of detoxification-related genes, including 7 P450 family genes, 8 glutathione S-transferases (GSTs) genes, 6 ABC transport enzyme genes, 9 UDP-glucosyltransferases genes and 3 carboxylesterases genes. Moreover, KEGG analysis demonstrated that the upregulated genes were enriched in xenobiotic metabolism by cytochrome P450s, ABC transporters and drug metabolism by other enzymes. Furthermore, as numerous GSTs were induced by tomatine in S. litura, we chose one gene, namely GSTS1, to confirm the detoxification function on tomatine. Expression profiling revealed that GSTS1 transcripts were mainly expressed in larvae, and the levels were the highest in the midgut. Finally, when larvae were injected with double-stranded RNA specific to GSTS1, the transcript levels in the midgut and fat body decreased, and the negative effect of the plant xenobiotic tomatine on larval growth was magnified. These results preliminarily clarified the molecular mechanism underlying the resistance of S. litura to tomatine, establishing a foundation for subsequent pest control.

Generalist phytophagous insects adapt to adventurous chemical environment in a wide variety of host plants by extraordinary detoxifying metabolic abilities. However, how polyphagous insect cope with the diversity of plant defenses remains largely unknown and only a few counter-defense genes detoxifying a wide range of toxic secondary metabolites have been well characterized. Here, we identify a cytochrome P450 gene (CYP6AB60) from tobacco cutworm (Spodoptera litura) in response to three different plant's defense metabolites. After being exposed to artificial diet supplemented with coumarin (COU), xanthotoxin (XAN) or tomatine (TOM), activities of P450 and CYP6AB60 transcript levels in both midgut and fat body tissues were significantly increased. Developmental expression analysis revealed that CYP6AB60 was expressed highly during the larval stages, and tissue distribution analysis showed that CYP6AB60 was expressed extremely high in the midgut, which correspond to the physiological role of CYP6AB60 from S. litura larvae in response to plant allelochemicals. Furthermore, when larvae are injected with double-stranded RNA (dsRNA) specific to CYP6AB60, levels of this transcript in the midgut and fatbody decrease and the negative effect of plant's defense metabolites on larval growth is magnified. These data demonstrate that the generalist insect S. litura might take advantage of an individual detoxificative gene CYP6AB60 to toxic secondary metabolites from different host plants. The CYP6AB60 can be a potential gene to carry out RNAi-mediated crop protection against the major polyphagous pest S. litura in the future.

Silicon improves plant resistance to a wide range of biotic and abiotic stresses. However, available silicon as a plant nutrient in soil is scarce. A continuous supply of available silicon is vital for plants. Earthworms are important detritivores in many terrestrial ecosystems. Therefore, we hypothesized that earthworms harbor silicate solubilizing bacteria in their guts and play a key role in silicon weathering and dynamics. Silicate solubilizing bacteria were isolated from the gut of geophagous endogeic earthworm (Pheretima guillelmi) and their effects on silicon weathering and availability to maize (Zea mays) were examined. Significantly more silicate solubilizing bacteria were observed in the earthworms gut than those in the surrounding soil in all sampling locations. The 16S rRNA analysis showed a higher silicate solubilizing bacteria diversity with 20 bacterial strains isolated from the gut walls, belonging to the genera of Aeromonas, Bacillus, Cellvibrio, Ensifer, Flavobacterium, Microbacterium, Paracoccus, Pseudomonas, Rhizobium, and Streptomyces. Of these bacteria, only three species were found in the surrounding soil. Inoculation with the three fastest-growing strains in liquid culture increased soluble silicon released from the feldspar and quartz powder in the media. Inoculation with the 3C1 strain in the potting soil significantly increased soluble silicon content in the soil, enhanced silicon uptake and accumulation in maize plants and promoted seedling growth. Taken together, these results not only highlight the importance of earthworm gut microbes in soil silicon weathering but also indicate a new approach in searching for silicate solubilizing bacteria to increase soil silicon availability in agriculture.

The major yolk protein precursors (YPP) gene, vitellogenin (Vg), usually considered as a reproductive indicator and molecular marker for evaluating insect fecundity, is controlled by insect hormone (mainly ecdysteroids and juvenile hormone), transcription factors and many other fecundity-related genes. To better understand the underlying molecular regulation mechanisms of the NlVg in the brown planthopper Nilaparvata lugens (N. lugens), the correlation between one early ecdysone response gene E74 and one important fecundity-related gene angiotensin converting enzyme (ACE) on the regulation of Vg gene expression, was investigated. We first showed that the mRNA expression level of NlACE were significantly higher in a high-fecundity population (HFP) than a low-fecundity population (LFP) at different development stages, and knockdown of NlACE expression by RNA interference (RNAi) results in a reduced level of NlVg expression and N. lugens fecundity. Subsequently, we analyzed the promoter of NlACE and found an E74A binding site, which was also differentially expressed in HFP and LFP. Then a gene putatively encoding E74A, namely NlE74A, predominant in the ovary and fat body was cloned and characterized. Furthermore, the developmental profile during female adult and the tissue-specific expression pattern of NlACE and NlE74A were similar to the expression pattern of NlVg gene, implying that both NlACE and NlE74A may be involved in regulating the expression of NlVg. Finally, after injecting the dsRNA of NlE74A, the NlACE expression levels were significantly reduced simultaneously at 24 h and 48 h post-injection, and the NlVg expression level was significant reduced at 24 h post-injection and the downswing was more significant at 48 h post-injection. These results imply that regulation of NlE74A on NlVg transcription might be mediated by NlACE through the E74 binding site at the NlACE promoter region in N. lugens.