Elsevier

Gene

Volume 557, Issue 2, 25 February 2015, Pages 215-221
Gene

Identification of microRNAs by small RNA deep sequencing for synthetic microRNA mimics to control Spodoptera exigua

https://doi.org/10.1016/j.gene.2014.12.038Get rights and content

Highlights

  • 127 conserved miRNAs were identified in Spodoptera exigua.

  • 3 miRNAs were expressed at relatively higher levels during the larval development.

  • Oversupply of the 3 miRNAs caused reduced larval growth.

  • Sex-miR-4924 reduced the expression of chitinase 1 and caused abortive molting.

  • Diets containing miRNA mimics can be used to control S. exigua development.

Abstract

Beet armyworm, Spodoptera exigua, is a major pest of cotton around the world. With the increase of resistance to Bacillus thuringiensis (Bt) toxin in transgenic cotton plants, there is a need to develop an alternative control approach that can be used in combination with Bt transgenic crops as part of resistance management strategies. MicroRNAs (miRNAs), a non-coding small RNA family (18–25 nt), play crucial roles in various biological processes and over-expression of miRNAs has been shown to interfere with the normal development of insects. In this study, we identified 127 conserved miRNAs in S. exigua by using small RNA deep sequencing technology. From this, we tested the effects of 11 miRNAs on larval development. We found three miRNAs, Sex-miR-10-1a, Sex-miR-4924, and Sex-miR-9, to be differentially expressed during larval stages of S. exigua. Oral feeding experiments using synthetic miRNA mimics of Sex-miR-10-1a, Sex-miR-4924, and Sex-miR-9 resulted in suppressed growth of S. exigua and mortality. Over-expression of Sex-miR-4924 caused a significant reduction in the expression level of chitinase 1 and caused abortive molting in the insects. Therefore, we demonstrated a novel approach of using miRNA mimics to control S. exigua development.

Introduction

Spodoptera exigua, commonly known as beet armyworm, is an important agricultural pest that originated in Asia. Its worldwide presence affects a wide host range that includes vegetables, flowers, and field crops. The larvae feed on both foliage and fruit and cause serious defoliation of cotton, ultimately resulting in severe economic loss to growers. Over the last decade, there have been major successes in using genetically engineered cotton plants with enhanced resistance or tolerance to insect pests in commercial planting (Guo et al., 2011). Transgenic Bt cotton is considerably effective in controlling lepidopteran pest species by decreasing the usage of chemical insecticide sprays and reducing the loss of beneficial arthropods (Wu and Guo, 2005). In recent years, there have been growing concerns about the development of Bt resistance in several lepidopteran families including S. exigua and further spread of Bt resistance in the environment (Guo et al., 2011). Organisms that are tolerant of Bt may contribute to the development and spread of Bt resistance. Thus, development of resistance in S. exigua to Bt toxin is a major issue in cotton transgenic technology and pest control. There is a need for an alternative and novel approach that can replace or be used in combination with Bt crops as part of pest management strategies to stem the occurrence of resistant pests.

MicroRNAs (miRNAs) are small RNAs (sRNAs) widely present in animals and plants involved in post-transcriptional regulation of gene transcripts (Rao et al., 2012). These small endogenous regulatory RNAs are ~ 22 nt in length and their precursors can fold into stem-loop structures (Bartel, 2004). Through mRNA cleavage or translational repression, miRNAs are known to have important roles in numerous physiological processes including growth, development, metabolism, behavior, and apoptosis (Carrington and Ambros, 2003). A single miRNA can target the mRNA of several genes, or several miRNAs may be required to regulate a single mRNA. One-third of human genes known to be regulated by miRNAs encode transcriptional or developmental factors (Lewis et al., 2005).

Several studies have shown the effects of miRNAs on the growth and development of insects. By altering the expression of miRNAs, researchers were able to interfere with the normal development of insects leading to potentially fatal consequences (Chawla and Sokol, 2011). In Drosophila melanogaster, the loss of let-7 and miR-125 led to severe metamorphic defects (Caygill and Johnston, 2008). In Bombyx mori, the temporal and spatial expressions of several miRNAs have been suggested to play vital roles in larval development, pupal maturation, and adult emergence (Yu et al., 2008). The prevention of Dicer-1, a key enzyme involved in miRNAs biogenesis, has led to delay in Blattella germanica nymphal features (Gomez-Orte and Belles, 2009). The oral feeding of chemically synthesized sRNA targeting acetylcholinesterase (AChE gene) resulted in mortality and growth inhibition in Helicoverpa armigera (Kumar et al., 2009). Similarly, oversupply of Har-miR-2002b in H. armigera by oral feeding led to larval mortality and reduction in fecundity (Jayachandran et al., 2013). Herein, we demonstrate a novel approach to controlling S. exigua development by the use of a miRNA mimic which interferes with larval growth and development.

Section snippets

Identification of conserved miRNAs

Using high-throughput sequencing, we obtained 17,183,630 sRNA reads from S. exigua. After removing the redundant sequences, we obtained 2,166,090 unique sequences. As seen in Fig. 1, the distributions of the lengths of these sRNAs varied, ranging from 19 nt to 30 nt with a bimodal distribution. One of the peaks, approximately 21–23 nt in length, represents miRNAs described by Zhang et al. (2009). The other one at approximately 26–27 nt in length appeared to be the piRNA-like sRNAs as reported (Sai

Discussion

In recent years, the roles of miRNAs in insect growth and development have been extensively studied (Rao et al., 2012, Sokol et al., 2008, Liu et al., 2010, He et al., 2008). For example, miR-184 is expressed ubiquitously in Drosophila at all stages of its life cycle: embryo, larva, and adult. Moreover, the expression pattern dynamically changes during the development of the embryo, especially in the central nervous system, and has been shown to be an important factor in development (Li et al.,

Insect strains

S. exigua was provided by Dr. Zhigang Zhang (National Biopesticide Engineering Research Center, Hubei Academy of Agricultural Science, Wuhan, China). A colony of the insects was maintained at the Institute of Tropical Bioscience and Biotechnology (Haikou, China) on a diet consisting of 5 g vitamin C, 40 g yeast extract, 50 g barley powder, 60 g soybean powder, 14 mL ethylic acid (36%), 20 g agar powder, 1 g benzoic acid, and 3 g sodium benzoate in 1000 mL water. The insects were reared at 28 °C under a 14

Competing interests

The authors have declared that no competing interest exists.

Acknowledgments

The authors thank Dr. Zhigang Zhang for providing the insect colony, technical guidance and bioassays (National Engineering Research Center for Biological Pesticides, Hubei, China). This work was partially funded by the National Natural Science Foundation (31101501 and 31201486) and the Special Fund for Agro-scientific Research in the Public Interest of the People's Republic of China (Grant No. 201403075). The funders had no role in study design, data collection, analysis or interpretation, and

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    These authors contributed equally to this work.

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