Transcriptome Analysis Reveals Early Hemocyte Responses upon In Vivo Stimulation with LPS in the Stick Insect Bacillus rossius (Rossi, 1788)

Abstract

Despite a growing number of non-model insect species is being investigated in recent years, a greater understanding of their physiology is prevented by the lack of genomic resources. This is the case of the common European stick insect Bacillus rossius (Rossi, 1788): in this species, some knowledge is available on hemocyte-related defenses, but little is known about the physiological changes occurring in response to natural or experimental challenges. Here, the transcriptional signatures of adult B. rossius hemocytes were investigated after a short-term (2 h) LPS stimulation in vivo: a total of 2191 differentially expressed genes, mostly involved in proteolysis and carbohydrate and lipid metabolic processes, were identified in the de novo assembled transcriptome and in-depth discussed. Overall, the significant modulation of immune signals-such as C-type lectins, ML domain-containing proteins, serpins, as well as Toll signaling-related molecules-provide novel information on the early progression of LPS-induced responses in B. rossius.

Keywords: European stick insect; RNAseq; hemocytes; insect physiology; metabolic processes; protease cascade.

Figure 1

Scatter plot of log₁₀-transformed gene expression levels observed in the pre- and post-LPS stimulated samples, calculated as TPMs. Differentially expressed genes are marked in red.

Figure 2

Bayesian phylogeny of insect CTL domain-containing proteins, including representatives from Drosophila melanogaster (Diptera), Tribolium castaneum (Coleoptera), Apis mellifera (Hymenoptera), and Bombyx mori (Lepidoptera), plus the 11 CTLs identified in B. rossius. The full unrooted tree, which depicts the complex evolutionary relationships among the three major structural classes of CTLs (i.e., CTL−S, IML, and CTL−X) is displayed in panel (A). The branch that includes all B. rossius CTLs (highlighted with a yellow background) are detailed in panel (B). The numbers shown close to each node represent posterior probability support values. Poorly supported nodes (i.e., <0.5) were collapsed. Dm: D. melanogaster; Am: A. mellifera; Tc: T. castaneum; Bm: B. mori. Panel (C) reports the domain architectures and gene expression trends following LPS stimulation of B. rossius CTLs. The portion of BrCTL7 included in a dashed box indicates a missing, unassembled region. D1 and D2 indicate the two CLECT domains of IMLs.

Figure 3

Bayesian phylogeny (A), domain architecture and gene expression trend following LPS stimulation (B), and multiple sequence alignment of B. rossius ML domain-containing proteins (C). The sequences from B. rossius are highlighted in yellow in the phylogenetic tree. Homsap: Homo sapiens; Dromel: Drosophila melanogaster; species name of the Tinema genus are expanded in the figure. The numbers shown close to each node represent posterior probability support values. Poorly supported nodes (i.e., <0.5) were collapsed. Asterisks indicate highly conserved residues in the MSA. The putative signal peptide of BrML6, which did not meet the significance threshold for being detected by SignalP, is indicated by a dashed circle.

Figure 4

Schematic general overview of the immune pathways leading to the LPS-dependent modulation of NF-kB signaling in B. rossius. Colored background of shapes denotes modulation of molecules by the 2 h LPS stimulation (black).

Figure 5

Scatterplot for significantly enriched Biological Process GO IDs against log10-transformed FDR-corrected p-values. Dot size is proportional to the number of DEGs mapping to that GO term. The reliability of statistical estimates increases from left to right along the x-axis.