Transcriptomic analysis of the interactions between the Spodoptera exigua midgut and nucleopolyhedrovirus
Graphical abstract
Introduction
The beet armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) is highly polyphagous and considered to be one of the most devastating pests, infesting over 80 crop species grown in fields and greenhouses worldwide and causing significant economic impacts (Smagghe et al., 2003). Due to the rapid development of resistance to multiple agents and the ecological effects of excessive dependence on chemical insecticides for controlling this pest (Osorio et al., 2008), nucleopolyhedrovirus (NPV), a virus affecting insects and causing natural periodic epizootics in insect populations, has received increased attention and is widely acknowledged as an environmentally friendly alternative to chemical insecticides (Moscardi, 1999). NPV-infected larvae always exhibit several typical symptoms, including discoloration, visible swelling with fluid retention, no feeding, various sizes of visibly circular pus blotches on the epidermis, abnormal behavior, and eventually death.
S. exigua nucleopolyhedrovirus (SeNPV), a member of an NPV subfamily, has been successfully applied as a large-scale commercial biological insecticide against S. exigua (Sun and Peng, 2007). Our research group is committed to investigating interactions between SeNPV and S. exigua. We previously found that SeNPV exhibits marked toxicity toward S. exigua larvae, and microscopic observations indicate that both the peritrophic matrix (PM) and midgut cells of S. exigua change greatly after oral infection with SeNPV (Jiang et al., 2011; Guo et al., 2018). Similar to other lepidopteran NPVs, orally administered SeNPV first invades and crosses the insect PM, coming into contact with midgut cells. A substantial infection is then induced, with virions released into intestinal epithelial cells. To defend against infectious viruses, the insect initiates various defense systems, including its potent immune system, to block viral DNA replication (Granados and Lawler, 1981; Sabin et al., 2010). Nonetheless, the molecular mechanisms underlying interactions between S. exigua and SeNPV are largely unknown.
As an acellular structure unique to invertebrates that lines the insect midgut, the PM is primarily assembled by the amorphous proteinaceous matrix and the chitin network, which is formed by the regular or irregular arrangement of chitin fibrils, a crystallized form of chitin (Wang and Granados, 2001). In lepidopteran larvae, the PM can prevent microbial infection and serve as the first barrier to prevent pathogenic microorganisms (including viruses) and toxic compounds from reaching the midgut and body of the insect, where they can infect or exert toxicity (Denecke et al., 2018). Additionally, previous studies have noted that some pathogens or toxins can break through this innate barrier by inhibiting PM formation, altering its permeability or destroying its structure (Wang and Granados, 2001). It is well established that the PM disruption mechanism of the enhancin molecule from granulosis virus degrades insect intestinal mucin, a major PM protein, leading to PM disintegration and markedly enhanced susceptibility of insect larvae to virus infections (Derksen and Granados, 1988; Toprak et al., 2012). Nevertheless, there is little available information regarding SeNPV-induced molecular changes in the PM of S. exigua.
In addition to the action of various pathogen-induced virulence factors, the insecticidal efficiency of pathogens relies on mechanisms developed by the insect host to restrain the course of the infection and/or weaken the impairment resulting from virulence factors (Sun et al., 2019). As the main site for digestion and nutrient absorption, the insect midgut also plays a crucial role in fighting against infection by pathogenic microorganisms, activating numerous innate immune responses, such as the synthesis and utilization of antimicrobial peptides (AMPs) and degradation enzymes, the induction of detoxification and damage repair systems and the activation of immune signaling pathways (Wu et al., 2016). RNAi, apoptosis, autophagy and the JNK, Janus kinase (JAK)/signal transducers and activators of transcription (STAT) and p38 pathways are important defense strategies of insect innate immunity against viruses (Hoffmann, 2003; Tanaka et al., 2008). Furthermore, many protective proteins have been found to have remarkable antiviral activity, such as Bmlipase-1, serine protease-2 (BmSP-2) and alkaline trypsin protein (BmtryP) from the midgut of silkworm Bombyx mori (Ponnuvel et al., 2003; Nakazawa et al., 2004; Ponnuvel et al., 2012).
With advances in molecular genetics technologies, RNA-sequencing (RNA-Seq) has provided important insight into the immune system of many insects, including Drosophila melanogaster (Hoffmann and Reichhart, 2002), Manduca sexta (Kanost et al., 2004) and Meligethes aeneus (Vogel et al., 2014), as well as B. mori (Tanaka et al., 2008). Activation of different responses in S. exigua after exposure to different pathogens or insecticidal proteins has also been reported (Yu et al., 2018; Choi et al., 2012; Hernández-Martínez et al., 2017). Interestingly, a publicly available dataset of the defense/immune-associated genes involved in S. exigua infection with SeNPV was generated by characterizing the transcriptomes of pooled RNA samples from diverse insect colonies and tissues exposed to different pathogenic and nonpathogenic microbial agents (Pascual et al., 2012). Regardless, the defense response of the S. exigua midgut during SeNPV infection remains poorly understood.
Therefore, to investigate the diverse molecular mechanisms underlying interactions between SeNPV and the S. exigua midgut and to provide an adequate theoretical basis for enhancing the utility of SeNPV as a bioinsecticide, the present study addressed the following objectives: (1) ultrastructural changes in the PM in S. exigua after oral infection with SeNPV were assessed by scanning electron microscopy; (2) potential defense-related genes associated with SeNPV-induced midgut responses were examined and identified by transcriptome-based analysis; and (3) the reliability of differentially expressed genes (DEGs) was validated by quantitative real-time PCR. The results provide insight into the mechanisms of insect pest resistance to biopesticides, offer novel targets for improved biopesticides, and stimulate new approaches for understanding, modifying, and utilizing baculoviruses to control insect pests.
Section snippets
Insects and virus
The S. exigua and SeNPV used in this study originated from a field of pesticide-free Chinese cabbage (Brassica rapa) in Zhuanghang, Fengxian District, Shanghai, China (30°53′ N; 121°23′ E, 4.0 m). S. exigua individuals were reared in the laboratory for two years without exposure to any pesticides. Larvae were individually raised on an artificial diet in sterile glass tubes (10 cm high, 2.0 cm diameter) and subsequently maintained in a bioclimatic chamber (Ningbo Jiangnan Instrument Factory,
Influence of SeNPV on the PM structure of S. exigua
The external structure of the PM from larvae after 48 h of feeding on a fresh artificial diet containing sterile deionized water or SeNPV suspension was observed by scanning electron microscopy to investigate the interaction of SeNPV with the S. exigua PM (Fig. 1). In control larvae (Fig. 1A), the surface of the PM was compact and intact, with no ruptures, and flat, with several small ruffles. However, the PMs of larvae fed the SeNPV-treated diet showed an extremely altered structure that was
Discussion
In general, the PM is believed to be the first barrier in insects to protect midgut epithelial cells from viral infection (Denecke et al., 2018). Moreover, changes in morphological structure would be directly described in infected insects when some virus insecticides were used. To provide strong and convincing evidence for determining the exact molecular mechanisms of the response to SeNPV infection, ultrastructural changes in the PM of S. exigua after oral infection with SeNPV were observed in
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China (31672083) and Shanghai Academy of Agriculture Science Program for Excellent Research Team (2018[B-01]). We thank all the members of the Ecological Control of Pests Laboratory of Shanghai Academy of Agriculture Science for their advice, assistance, and technical help.
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