Transcriptome Analysis in Cotton Boll Weevil (<i>Anthonomus grandis</i>) and RNA Interference in Insect Pests

<div><p>Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, <i>Anthonomus grandis</i>, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, <i>A. grandis</i>, was analyzed. The <i>A. grandis</i> transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to <i>Tribolium castaneum</i> sequences. A comparison of <i>A. grandis</i>, <i>Drosophila melanogaster</i> and <i>Bombyx mori</i> protein families’ data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with <i>T. castaneum</i> SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to <i>A. grandis</i> female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, <i>A. grandis</i>. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.</p> </div

Contigs length distribution showing the major number ranging from 350 to 1000 pb.

<p>The average contig length was 676pb. </p

Knock-down of <i>Meloidogyne incognita</i> proteases affects nematode reproduction and egg viability (after 45 DAI).

<p>(A) Number of eggs per gram of root. (B) Total egg hatching ratio. Experiments were repeated twice. Different letters mean statistical significance through one-way ANOVA and Tukey test (<i>p</i>≤0.05).</p

Relative abundance of specific protease gene transcripts in <i>Meloidogyne incognita</i>.

<p>Real-time qRT-PCR analysis of <i>M. incognita</i> proteases transcript levels at different stages of the nematode life cycle. (A) Cathepsin D-like aspartic proteinase (<i>Mi-asp-1</i>, Accession: DQ360827). (B) Chymotrypsin-like serine proteinase (<i>Mi-ser-1</i>, AY714229). (C) Cathepsin L cystein proteinase (<i>Mi-cpl-1</i>, AJ557572). Each bar represents the mean of duplicate assays repeated twice. Standard errors are shown. Different letters mean statistical difference (<i>p</i>≤0.05) according to the iteration test (Rest 2009 Software). The results are presented as fold change in comparison to the stage that had the smaller relative expression value that was arbitrarily designed as 1.</p

<i>In</i><i>silico</i> analyses of all <i>Meloidogyne incognita</i> aspartic, serine and cysteine proteases ESTs present in EST data bank dbEST.

<p>Representation of <i>M. incognita</i> proteases expressed sequence tags (ESTs) in databanks. Bars show the percentage of proteases EST number relative to the total number of EST available for each developmental stage. ESTs from proteases were retrieved from NCBI-dbEST (<a href="http://www.ncbi.nlm.nih.gov/dbest/index.html" target="_blank">http://www.ncbi.nlm.nih.gov/dbEST/index.html</a>) and their representation was assessed by the number of ESTs relative to the total number of ESTs available for the developmental stage considered. The developmental stages considered were; eggs (14,671 ESTs), freshly hatched J2s (33,835 ESTs), mixed parasitic stages (3,133 ESTs) and females (4,427 ESTs). The distribution of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ESTs is indicated for comparison. </p

Effect of protease knock-down on progeny virulence of <i>M. incognita</i>.

<p>Hatched J2 from eggs that were laid by females that feed on transgenic and control plants were inoculated in wild-type tobacco. (A) Relative number of galls per plant at 45 DAI; (B) Relative number of egg masses per plant at 45 DAI; Experiments were repeated twice. Different letters mean statistical significance through one-way ANOVA and Tukey test (P≤0.05).</p

Gene cloning and transgenic tobacco plant generation for host-derived RNA-interference of <i>Meloidogyne incognita</i> proteases.

<p>(A) Regions of proteinases genes used in RNAi experiments. Numbers indicate nucleotide positions. (B) Schematic representation of the pK7GWIWG2(I) (Karimi et al. 2002) hairpin double-stranded RNA (dsRNA) constructs containing the sense and antisense coding regions fragments of <i>Mi-asp-1</i>, <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> separately and together. (C) Characterization of RNAi lines for silencing of <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> and the fragments in tandem of <i>Mi-asp-1</i>, <i>Mi-ser-1</i> and <i>Mi-cpl-1</i> (Fusion), by PCR. Attempts for generate ds-<i>Mi-asp-1</i> lines were not successful. Sense (S) fragment, anti-sense (AS) fragment, undistinguishable fragment (Sense or Anti-sense) (F). (D) RT-PCR of the single-stranded pK7GWIWG2(I) intron (spacer) of the hairpin dsRNA was used to confirm the expression of <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> and fusion dsRNAs in seedlings of independent transgenic tobacco lines at 15 d post-germination.</p

Genes involved in RNAi mechanism found in <i>A. grandis</i> transcriptome.

<p>The comparison with genes of <i>C. elegans</i>, <i>T. castaneum</i>, and <i>D. melanogaster</i> suggested that RNAi mechanism is well conserved in insects (A, B, C, D), including lack of amplification (E). No gene involved in dsRNA degradation was found (F). The number of contigs found in <i>A. grandis</i> transcriptome for each gene class is shown. </p

Distance neighbor-joining tree showing the phylogeny of a SID-like contig of <i>A. grandis</i> (A_grandis_454_c2889) and SID-like proteins of the insects <i>T. castaneum</i>, <i>B. impatiens</i>, <i>A. mellifera</i>, <i>L. migratoria</i>, <i>B. mori</i>, <i>A. gossypii</i>, <i>H. saltator</i>, <i>Camponotus floridanus</i>.

<p>The percentage of percentage of bootstrap confidence values is shown at the nodes. </p

Comparison of dicer and argonaute PAZ domains.

<p>Two cotton boll weevil contigs were aligned to five species sequences: <i>D. melanogaster</i> (Dm_Dicer-1, Dm_AGO1C, Dm_AGO2)<i>, C. elegans</i> (Ce_Dicer1, Ce_Alg1, Ce_Alg2)<i>, Homo sapiens</i> (Hs_Dicer-1, Hs_Ago1)<i>, A. thaliana</i> (At_Dicer-like-1, At_AGO, At_AGO1) and <i>Schizosaccharomyces pombe</i> (Sp_AGO1). The sequence IDs are the same found in the NCBI Protein Database. Secondary structures within the domain are indicated as α-helices and β structures. The highlighted residues are responsible for the stabilization of the dsRNA-binding region. In yellow, a subdomain of aromatic residues. Along with a cysteine residue (blue), preceded by a proline and a glutamate (yellow), some invariant residues (red) create a hydrophobic subdomain that interacts with RNA. Residues that differ in dicer and argonaute PAZ domains are shown in brown. </p