Putative Sugar Transporters of the Mustard Leaf Beetle <i>Phaedon cochleariae</i>: Their Phylogeny and Role for Nutrient Supply in Larval Defensive Glands

<div><p>Background</p><p>Phytophagous insects have emerged successfully on the planet also because of the development of diverse and often astonishing defensive strategies against their enemies. The larvae of the mustard leaf beetle <i>Phaedon cochleariae</i>, for example, secrete deterrents from specialized defensive glands on their back. The secretion process involves ATP-binding cassette transporters. Therefore, sugar as one of the major energy sources to fuel the ATP synthesis for the cellular metabolism and transport processes, has to be present in the defensive glands. However, the role of sugar transporters for the production of defensive secretions was not addressed until now.</p><p>Results</p><p>To identify sugar transporters in <i>P. cochleariae</i>, a transcript catalogue was created by Illumina sequencing of cDNA libraries. A total of 68,667 transcripts were identified and 68 proteins were annotated as either members of the solute carrier 2 (SLC2) family or trehalose transporters. Phylogenetic analyses revealed an extension of the mammalian GLUT6/8 class in insects as well as one group of transporters exhibiting distinctive conserved motifs only present in the insect order Coleoptera. RNA-seq data of samples derived from the defensive glands revealed six transcripts encoding sugar transporters with more than 3,000 counts. Two of them are exclusively expressed in the glandular tissue. Reduction in secretions production was accomplished by silencing two of four selected transporters. RNA-seq experiments of transporter-silenced larvae showed the down-regulation of the silenced transporter but concurrently the up-regulation of other SLC2 transporters suggesting an adaptive system to maintain sugar homeostasis in the defensive glands.</p><p>Conclusion</p><p>We provide the first comprehensive phylogenetic study of the SLC2 family in a phytophagous beetle species. RNAi and RNA-seq experiments underline the importance of SLC2 transporters in defensive glands to achieve a chemical defense for successful competitive interaction in natural ecosystems.</p></div

Heatmap of the variance stabilization transformed data (vsd) of ds<i>Pcsut2</i>-injected <i>versus</i> ds<i>gfp</i>-injected samples.

<p>Samples derived from glandular tissue. For further explanation see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084461#pone-0084461-g008" target="_blank">Figure 8</a>.</p

Number of assembled transcripts and average length after assembly and reassembly showing the usefulness of reassembling.

<p>Number of assembled transcripts and average length after assembly and reassembly showing the usefulness of reassembling.</p

Differential expression analysis using DESeq package.

<p>baseMeanA: mean of normalized counts value of ds<i>gfp</i>-injected samples. baseMeanB: mean of normalized counts values of dsRNA-<i>gfp</i>-injected, dsRNA-<i>Pcsut1</i>-injected, dsRNA-<i>Pcsut2</i>-injected, dsRNA-<i>Pcsut6</i>-injected samples. Fold-change: baseMeanA compared to baseMeanB. Log₂fold-change: logarithm (to base 2) of fold-change values. Pval: p-value for the statistical significance of this change. Padj: p-value adjusted for multiple testing with Benjamini-Hochberg procedure which controls false discovery rate.</p

Characterized trehalose as well as glucose/fructose transporters.

<p>They are listed with their description, accession number and organism. Those transporters were added to <i>P. cochleariae</i>’s chosen sequences to calculate phylogenetic trees.</p

The phylogenetic tree of the nine sequences derived from <i>P. cochleariae</i> belonging to the purple branch (see <b>Figure 3</b> and <b>4</b>, Figure S4) and homologous sequences derived from the whole tree of life, especially from <i>Dendroctonus ponderosae</i> (Dc) as well as from <i>Tribolium castaneum</i> (Tc), was calculated using RaxML.

<p>Indicated in purple, it can be seen that the beetles’ sequences build up a separate branch.</p

Heatmap of the variance stabilization transformed data (vsd) of ds<i>Pcsut6</i>-injected <i>vs</i>. ds<i>gfp</i>-injected samples.

<p>Samples derived from glandular tissue. For further explanation see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084461#pone-0084461-g008" target="_blank">Figure 8</a>.</p

Distribution of mRNA levels of putative SLC2 transporters in various tissues of juvenile <i>P. cochleariae</i> by using quantitative real-time PCR.

<p>Distribution of mRNA levels of putative SLC2 transporters in various tissues of juvenile <i>P. cochleariae</i> by using quantitative real-time PCR.</p

Heatmap of the variance stabilization transformed data (vsd) of ds<i>Pcsut1</i>-injected <i>vs</i>. ds<i>gfp</i>-injected samples.

<p>Samples derived from glandular tissue. For this, the transcript counts of the sugar transporters of each sample after dsRNA-injection have been normalized to the effective library size and the variance over all samples has been stabilized by applying the DESeq package. For each heatmap, the 30 most abundant sugar transporter transcripts are shown. Ds<i>gfp</i>-injected samples are the same in each heatmap.</p

Tissue-Specific Transcript Profiling for ABC Transporters in the Sequestering Larvae of the Phytophagous Leaf Beetle <i>Chrysomela populi</i>

<div><p>Background</p><p>Insects evolved ingenious adaptations to use extraordinary food sources. Particularly, the diet of herbivores enriched with noxious plant secondary metabolites requires detoxification mechanisms. Sequestration, which involves the uptake, transfer, and concentration of occasionally modified phytochemicals into specialized tissues or hemolymph, is one of the most successful detoxification strategies found in most insect orders. Due to the ability of ATP-binding cassette (ABC) carriers to transport a wide range of molecules including phytochemicals and xenobiotics, it is highly likely that they play a role in this sequestration process. To shed light on the role of ABC proteins in sequestration, we describe an inventory of putative ABC transporters in various tissues in the sequestering juvenile poplar leaf beetle, <i>Chrysomela populi</i>.</p><p>Results</p><p>In the transcriptome of <i>C. populi</i>, we predicted 65 ABC transporters. To link the proteins with a possible function, we performed comparative phylogenetic analyses with ABC transporters of other insects and of humans. While tissue-specific profiling of each ABC transporter subfamily suggests that ABCB, C and G influence the plant metabolite absorption in the gut, ABCC with 14 members is the preferred subfamily responsible for the excretion of these metabolites <i>via</i> Malpighian tubules. Moreover, salicin, which is sequestered from poplar plants, is translocated into the defensive glands for further deterrent production. In these glands and among all identified ABC transporters, an exceptionally high transcript level was observed only for <i>Cpabc35</i> (<i>Cpmrp</i>). RNAi revealed the deficiency of other ABC pumps to compensate the function of <i>Cp</i>ABC35, demonstrating its key role during sequestration.</p><p>Conclusion</p><p>We provide the first comprehensive phylogenetic study of the ABC family in a phytophagous beetle species. RNA-seq data from different larval tissues propose the importance of ABC pumps to achieve a homeostasis of plant-derived compounds and offer a basis for future analyses of their physiological function in sequestration processes.</p></div