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تعداد صفحات این فایل: ۲۱ صفحه

بخشی از مقاله انگلیسیعنوان انگلیسی:Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts~~en~~

Abstract

The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest

۱ Introduction

The banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is considered one of the most invasive and destructive pests of banana worldwide [1]. The larvae of C. sordidus are a severe constraint on banana and plantain production in most areas where these crops are cultivated, especially in Africa [2–۵] where this insect pest has been associated with rapid plantation decline [6] and with a phenomenon called “yield decline syndrome” in West Africa. The larvae of the banana weevil, which are the most destructive stage of the insect, is responsible for considerable damage of the plant corm, interfering with root initiation, nutrient and water uptake and plant development [6]. When a severe weevil infestation occurs, crop losses of up to 100% have been reported [7]. It is well known that chemical control of this insect pest is not only undesirable but also expensive. Options for biological control are limited and pheromone-based insect trapping results in either low or ineffective captures [8, 9]. Many basic advances have been made by studying the banana weevil, including, studies regarding pest resistance [10], insect resistant germplasm [2, 11, 12], plant antifeedants [13], cultural control practices [14] and biological control [15]. Despite extensive and recent biochemical and physiological studies, limited genomic information exists, especially for important tissues such as the midgut. The availability of transcriptome sequences from insect midgut tissues will facilitate identification of genes that are expressed in the intestinal tract and their respective metabolic and functional roles. It is well known that the curculionids are the largest family of beetles [16], which in general are important plant tissue damaging pests such as the banana weevil C. sordidus[1]. The rapid growth of next-generation DNA sequencing technologies such as 454-based pyrosequencing [17, 18] have allowed the characterization of the transcriptome of many important, non-model insect species [19–۲۳], thus providing valuable and unprecedented opportunities to increase our knowledge of expressed genes, especially in those insect pests where little or no genomic resources exist [24]. In this study, we used a 454-based pyrosequencing platform to sequence the C. sordidus larval midgut transcriptome allowing the characterization of transcripts encoding different genes associated with metabolic functions and potential insecticide targets. Many of these transcripts were protease-like genes from different digestive enzyme families, mainly associated with aminopeptidases, carboxypeptidases, serine proteases and cysteine proteases. The C. sordidus transcriptome represents an important contribution to understanding the biology of this insect pest and for the identification of potential target genes involved in protein digestion and many other metabolic pathways.

Materials and Methods

The experiments were carried out under a standard protocol in the lab and no specific permissions were required for these locations/activities. In addition, these study did not involve any endangered or protected species.

Insect dissection and of midgut RNA extraction

C. sordidus larvae were collected from corms obtained at a plantain field near Manizales, Colombia (1058 m, 5° ۴’ ۱۳۲” N, 75° ۴۱’ ۷۷” O). Collected larvae were inspected under a stereoscope and the fourth instar larvae were selected based on the size of the head capsule as described by [25] and then used for midgut dissection (Fig 1). Gut tissue was obtained by dissecting in DEPC-treated distilled water. The gut content and peritrophic matrix were removed and the washed midgut tissue was flash-frozen using liquid nitrogen and stored at -80°C. RNA extraction was performed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA was then purified using the RNeasy MinElute Cleanup Kit (Qiagen, Chatsworth, CA) after removing genomic DNA contamination using the TURBO DNA-free™ Kit (Ambion, Carlsbad, CA) according to manufacturer’s instructions.

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