Research Article pubs.acs.org/journal/ascecg
Application of Sustainable Natural Bioesources in Crop Protection: Insight into a Podophyllotoxin-Derived Botanical Pesticide for Regulating Insect Vestigial Wing of Mythimna separata Walker Zhiqiang Sun,†,§ Min Lv,†,§ Xiang Yu,‡ and Hui Xu*,†,‡ †
Research Institute of Pesticidal Design & Synthesis, College of Plant Protection, Northwest A&F University, 22 Xinong Road, Yangling 712100, Shaanxi Province, P.R. China ‡ College of Chemistry & Pharmacy, Northwest A&F University, 22 Xinong Road, Yangling 712100, Shaanxi Province, P.R. China S Supporting Information *
ABSTRACT: In continuation of our program for integrated application of podophyllotoxin (isolated from Juniperus Sabina) as a forest sustainable natural resource in crop protection, an indepth study on the mechanism of action of podophyllotoxin derivatives as botanical pesticides was necessary. On the basis of our previous results, here the transcriptional response of vestigial wing in Mythimna separata Walker (a crop-threatening insect pest) to a podophyllotoxin-derived insecticidal agent was analyzed by using RNA-Seq. This is the first study to explore the vestigial wing behavior of insect pests caused by xenobiotics. These results suggested that this agent could suppress wing-related development pathways, such as the insulin signaling pathway, juvenile hormone biosynthesis, wing disc morphogenesis, wing disc development, and imaginal disc-derived wing morphogenesis; it markedly repressed wing development-related genes of insulin receptor, insulin-like precursor polypeptide D, juvenile hormone, engrailed-like, vestigial-like, serrate homologue, notch, and distalless homebox, and activated wing development-related genes of indian hedgehog and spalt major-like, validated by qRT-PCR. Our results will pave the way for a future application of this sustainable forest natural bioresource as a crop protection agent to control insect pests damage in agriculture. KEYWORDS: Podophyllotoxin, Sustainable forest resource, Crop protection agent, Vestigial wing, RNA-Seq
■
INTRODUCTION The oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae), widely distributed in China, Japan, Southeast Asia, India, Eastern Australia, New Zealand, and some Pacific Islands, is a polyphagous and gluttonous lepidopteran pest, and harmful to maize, wheat, rice, and some crucifer crops.1 Due to its long-distance migratory behavior, it is hard to control. In 2012, the intermittent outbreaks of third-generation larvae of M. separata widely occurred in China, and about 4 million hectares of crops (e.g., corn, rice, wheat, etc.) were a terribly complete loss.2 Currently, the primary method for dealing with M. separata outbreaks is by using chemical pesticides; however, the increasing and extensive application of synthetic agrochemicals has resulted in resistance in pest populations, and a negative impact on human and environmental health.3 Thus, development of the potential alternatives from the sustainable natural bioresources to control insect pests is highly desirable.4 In continuation of our program to discover new natural-productbased insecticidal agents,5 by using podophyllotoxin (1, Figure 1a), a sustainable naturally occurring cyclolignan isolated from Juniperus Sabina (widely distributed in northwestern China), as © XXXX American Chemical Society
a lead compound, we prepared a series of 4α-acyloxy-2′(2′,6′)(di)halogenopodophyllotoxin derivatives (2, Figure 1a).6 They mainly showed the potent growth inhibitory activity against third-instar larvae of M. separata as we reported other naturalproduct-based insecticidal agents such as obacunone and fraxinellone.7,8 When compared with that of the blank control (33 days), the growth duration from the third-instar larva to moth was shortened to 30 days in the treated groups. Interestingly, many malformed moths with imperfect wings in the treated groups appeared during the stage of adult emergence (Figure 1b).6 To insects, wings serve not only as organs of flight, but also as protective covers, sound producers, and visual cues for species recognition. The studies on how the insect wings are developed can also help us to understand the insect migratory and evolution.9 It is noteworthy that M. separata, with vestigial wings, cannot efficiently find their mates to copulate with each other properly to produce the next Received: December 22, 2016 Revised: April 9, 2017 Published: April 11, 2017 A
DOI: 10.1021/acssuschemeng.6b03145 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Figure 1. Chemical structures of podophyllotoxin (1) and its derivatives (2), and representative malformed moth of M. separata produced by derivatives 2. (a) Isolation of compound 1 from Juniperus Sabina, followed by structural modifications to produce 2. (b) The symptoms for the treated M. separata (b-f) during the stage of adult emergence (CK: a). photoperiod, and they had been cultured without exposure to any agrochemicals. In this experiment, a podophyllotoxin-derived insecticidal agent 2a was used, which was diluted to 1 mg/mL in acetone. For the treated group, 240 pre-third-instar larvae (10 larvae per dish) were used. Fresh wheat leaves were dipped into the corresponding solution for 3 s, and then taken out and dried in a room. Leaves treated with acetone alone were used as a blank control group (120 pre-third-instar larvae (10 larvae per dish) were used). Several treated leaves were kept in each dish. If the treated leaves were consumed, additional treated ones were added to the dish. After 48 h, untreated fresh leaves were added to all dishes until adult emergence. The experiment was carried out at 25 ± 2 °C and relative humidity (RH) 65−80% and with the 12 h/12 h (light/dark) photoperiod.6 During the stage of adult emergence, malformed moths of M. separata produced by compound 2a and normal moths of M. separata in the blank control group were collected, snap-frozen immediately in liquid nitrogen, and stored at −80 °C. Transcriptomics and Sequencing. Approximately 12 malformed moths (12 normal moths as the control) were ground in liquid nitrogen. Total RNA was isolated using TRIzol reagent (invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and treated with RNase-free DNase I (Takara, China).14 After extraction of total RNA and treatment with DNase I, oligo(dT) species were used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA was fragmented. Then, cDNA was synthesized using the mRNA fragments as templates. Short fragments were purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments were connected with adapters. The suitable fragments were selected for the PCR amplification. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used in quantification and qualification of the sample library. Then, the library was sequenced using Illumina HiSeq 4000. Two biological replicates for each strain were prepared. De Novo RNA-seq Assembly and Annotation. After sequencing, FastQC was employed to check the quality distribution of the raw data. The raw reads were preprocessed by filtering against low-quality reads. Clean reads were de novo assembled with Trinity pipeline. The resulting sequences of Trinity are called transcripts. To reduce redundancy, Tgicl was used to cluster gene family to get final
generation. Thus, the number of insect pests will be drastically decreased, and their damage to the crops will be relieved. On the other hand, the transcriptome is the whole set of transcripts and their quantity in a cell, tissue, organ, or whole organism under special physiological conditions.10 The advancement in massively parallel RNA-Seq technologies provides a cost-effective way for a better understanding of the complex changes of organisms.11 The transcriptome profiling has been applied to various insect species as a means of analyzing gene expressing patterns and associating these with toxicity of insecticide.12 Furthermore, transcriptional profiling of individual stages within the insect life-cycle provides evidence for key genes up- and downregulated during the developmental process.13 To explore the mechanism about the growth inhibitory activity of derivatives 2, we assessed the transcriptional response of vestigial wing in M. separata to the insecticidal agent 2a (Figure 1a; the final mortality rates of 1 and 2a at 1 mg/mL were 37.0% and 70.4%, respectively), one of the most potent compounds from 2.6 By comparing the changes in gene expression, we identified an overlapping set of genes involved in insect wing development. The RNA-Seq data for selected genes were validated using quantitative reverse transcription PCR (qRT-PCR). To the best of our knowledge, this is the first study to explore the vestigial wing behavior of insect pests treated by the natural-product-based insecticidal agent. These results may facilitate the elucidation of the molecular mechanism of the vestigial wing of M. separata after treatment with the podophyllotoxin-derived compounds.
■
MATERIALS AND METHODS
Sample Preparation. The oriental armyworm (Mythimna separata) strain was originally collected from a maize field located at the Northwest A&F University, Yangling, China, and maintained in the laboratory for nearly four years. The insects were reared at 25 ± 2 °C on 5−6 cm wheat seedlings on 12 h/12 h (light/dark) B
DOI: 10.1021/acssuschemeng.6b03145 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Figure 2. Transcriptome analysis of M. separata. (a) Species-specific distribution. (b) Number of DEGs. (c) Enriched KEGG pathways for the DEGs in the transcriptome of M. separata treated by compound 2a. Unigenes based on pairwise sequence similarities. If there is more than one sample, we would execute Tgicl again with each sample’s Unigene to get the final Unigene for downstream analyses.15 The Unigenes would be divided into two classes. One was clusters, in which the prefix was CL with the cluster id behind it, and the other one were singletons, in which the prefix was Unigene. After assembly, we performed functional annotation with 7 functional databases (NR, NT, GO, COG, KEGG, Swissprot, and Interpro) for all unigenes.16 Blast was used to align unigenes to NT, NR, COG, KEGG, and SwissProt to get the annotation, with a cutoff P-value of
