Sequencing and Characterization of the Invasive Sycamore Lace Bug <i>Corythucha ciliata</i> (Hemiptera: Tingidae) Transcriptome

<div><p>The sycamore lace bug, <i>Corythucha ciliata</i> (Hemiptera: Tingidae), is an invasive forestry pest rapidly expanding in many countries. This pest poses a considerable threat to the urban forestry ecosystem, especially to <i>Platanus</i> spp. However, its molecular biology and biochemistry are poorly understood. This study reports the first <i>C</i>. <i>ciliata</i> transcriptome, encompassing three different life stages (Nymphs, adults female (AF) and adults male (AM)). In total, 26.53 GB of clean data and 60,879 unigenes were obtained from three RNA-seq libraries. These unigenes were annotated and classified by Nr (NCBI non-redundant protein sequences), Nt (NCBI non-redundant nucleotide sequences), Pfam (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Swiss-Prot (A manually annotated and reviewed protein sequence database), and KO (KEGG Ortholog database). After all pairwise comparisons between these three different samples, a large number of differentially expressed genes were revealed. The dramatic differences in global gene expression profiles were found between distinct life stages (nymphs and AF, nymphs and AM) and sex difference (AF and AM), with some of the significantly differentially expressed genes (DEGs) being related to metamorphosis, digestion, immune and sex difference. The different express of unigenes were validated through quantitative Real-Time PCR (qRT-PCR) for 16 randomly selected unigenes. In addition, 17,462 potential simple sequence repeat molecular markers were identified in these transcriptome resources. These comprehensive <i>C</i>. <i>ciliata</i> transcriptomic information can be utilized to promote the development of environmentally friendly methodologies to disrupt the processes of metamorphosis, digestion, immune and sex differences.</p></div

Selection and evaluation of reference genes for expression analysis using quantitative real-time PCR in the Asian Ladybird <i>Harmonia axyridis</i> (Coleoptera: Coccinellidae)

<div><p><i>Harmonia axyridis</i> (Coleoptera: Coccinellidae) is a polyphagous insect that is an important biological agent used to control agricultural and forestry pests. The role of functional genes in <i>H</i>. <i>axyridis</i> based on quantitative real-time PCR (qRT-PCR) is increasingly well understood to investigate biology, physiology, feeding behavior and the role of important genes in physiological processes. Quantitative real-time PCR (qRT-PCR) is a powerful and reliable technique to quantify gene expression. Using qRT-PCR, expression levels of target genes are determined based on the levels of internal reference genes; therefore, reference genes need to be stably expressed under specific experimental conditions. However, there have been no studies on the stability of reference genes used in <i>H</i>. <i>axyridis</i>. In this study, we systematically investigated expression profiles of nine candidate reference genes from <i>H</i>. <i>axyridis</i>, including β-actin (<i>ACTIN</i>); elongation factor 1 α (<i>EF1A</i>); ribosomal proteins L10, L18, L28, S13, and S15 (<i>RPL10</i>, <i>RPL18</i>, <i>RPL28</i>, <i>RPS13</i> and <i>RPS15</i>); glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>); and superoxide dismutase (<i>SOD</i>). Four analytical methods (geNorm, BestKeeper, NormFinder, and the ΔCt method) were used to evaluate the suitability of these genes as internal reference genes for three biotic factors (developmental stage, tissue, and sex) and two abiotic treatments (temperature and photoperiod). RefFinder, a comprehensive evaluation platform integrating the four analytical methods, was used to rank the overall stability of these reference genes. Among the nine candidate genes, different reference genes were identified as having the most stable expression across biotic and abiotic factors. Genes encoding ribosomal proteins typically had the most stable expression, though <i>EF1A</i> was the most stable across developmental stages and photoperiods. To validate the suitability of these reference genes, heat shock protein 90 (<i>HSP90</i>) was chosen as a target. Significant up-regulation in <i>HSP90</i> expression level in response to both low and high temperature was observed when using the most suitable reference genes but not when using an arbitrarily selected reference gene. The reference genes identified in this study will provide the basis for future functional genomics research in <i>H</i>. <i>axyridis</i> and will also facilitate the establishment of a standardized qRT-PCR program for other related insects.</p></div

Summary of functional annotations of <i>C</i>. <i>Ciliata</i> unigenes.

<p>Summary of functional annotations of <i>C</i>. <i>Ciliata</i> unigenes.</p

Output statistics from AM, AF and Nymphs of <i>C</i>. <i>Ciliata</i>.

<p>Output statistics from AM, AF and Nymphs of <i>C</i>. <i>Ciliata</i>.</p

Stability of reference gene expression across abiotic conditions.

<p>Stability of reference gene expression across abiotic conditions.</p

Recommended <i>H</i>. <i>axyridis</i> reference genes for various experimental conditions.

<p>Recommended <i>H</i>. <i>axyridis</i> reference genes for various experimental conditions.</p

Coding sequence predictions of <i>C</i>. <i>Ciliata</i> transcriptome by BLASTx and ESTScan.

<p>(<b>a</b>) Length distribution of CDs using BLASTx (E-value < 10⁻⁵); (<b>b</b>) Length distribution of proteins using BLASTx; (<b>c</b>) Length distribution of CDs predicted by ESTScan, and <b>(d</b>) Length distribution of proteins using ESTScan.</p

Sequences and amplicon characteristics of qRT-PCR primers for nine candidate reference genes and one target gene.

<p>Sequences and amplicon characteristics of qRT-PCR primers for nine candidate reference genes and one target gene.</p

Stability of reference gene expression across biotic conditions.

<p>Stability of reference gene expression across biotic conditions.</p

Volcano plots of differentially expressed unigenes.

<p>The abscissa represents the expressed levels fold change of unigenes in three different samples. The ordinate indicates the statistically significant difference degree. The lower and higher -log10 (p-adj) values mean greater differences. The scatters in diagram stand for each gene, the blue dot represents there was no significant difference of genes. The up-regulated and down-regulated genes were indicated by a red dot and green dot, respectively.</p