Tachykinin-related peptides modulate immune-gene expression in the mealworm beetle Tenebrio molitor L

Abstract

Tachykinins (TKs) are a group of conserved neuropeptides. In insects, tachykinin-related peptides (TRPs) are important modulators of several functions such as nociception and lipid metabolism. Recently, it has become clear that TRPs also play a role in regulating the insect immune system. Here, we report a transcriptomic analysis of changes in the expression levels of immune-related genes in the storage pest Tenebrio molitor after treatment with Tenmo-TRP-7. We tested two concentrations (10^-8 and 10^-6 M) at two time points, 6 and 24 h post-injection. We found significant changes in the transcript levels of a wide spectrum of immune-related genes. Some changes were observed 6 h after the injection of Tenmo-TRP-7, especially in relation to its putative anti-apoptotic action. Interestingly, 24 h after the injection of 10^-8 M Tenmo-TRP-7, most changes were related to the regulation of the cellular response. Applying 10^-6 M Tenmo-TRP-7 resulted in the downregulation of genes associated with humoral responses. Injecting Tenmo-TRP-7 did not affect beetle survival but led to a reduction in haemolymph lysozyme-like antibacterial activity, consistent with the transcriptomic data. The results confirmed the immunomodulatory role of TRP and shed new light on the functional homology between TRPs and TKs.

Figure 1

Survival curve of T. molitor males after the injection of physiological saline (control) and Tenmo-TRP-7 at concentrations of 10^–7 and 10^–5 M (final concentrations in insect bodies of 10^–8 and 10^–6 M, respectively). The values are given as the means ± SEM.

Figure 2

Gene ontology (GO) analysis based on the transcriptomic data of fat body and haemocytes from Tenebrio molitor was performed using the PANTHER database, and it included biological processes and molecular functions (https://www.pantherdb.org/).

Figure 3

GO enrichment dot plot presenting changes in the expression level of different sets of genes observed 6 h after Tenmo-TRP-7 treatment at a concentration of 10^–8 M. The identification of the most representative GO terms representing biological process and molecular function using REVIGO (http://revigo.irb.hr/) was performed (cut off < 0.05). The size of the dots represents the number of genes in the significantly differentially expressed genes (DEGs). Gene ratio’ is the percentage of total DEGs in the given GO term. For the graphical presentation of the obtained data, ggplot2 (https://ggplot2.tidyverse.org) for RStudio was used (http://www.rstudio.com)^,,.

Figure 4

GO enrichment dot plot presenting changes in the expression level of different sets of genes observed 6 h after Tenmo-TRP-7 treatment at a concentration of 10^–6 M. The identification of the most representative GO terms of biological process and molecular function using REVIGO (http://revigo.irb.hr/) was performed (cut off < 0.05). The size of the dots represents the number of genes in the significantly differentially expressed genes (DEGs). Gene ratio’ is the percentage of total DEGs in the given GO term. For the graphical presentation of the obtained data, ggplot2 (https://ggplot2.tidyverse.org) for RStudio was used (http://www.rstudio.com)^,,.

Figure 5

Reverse transcription quantitative PCR (RT-qPCR) analysis of the expression level of genes encoding selected antimicrobial peptides ((A) attacin 2; (B) tenecin 3) and the Toll receptor (C) in immune-related tissues (pooled fat body and haemocytes) 6 h after Tenmo-TRP-7 application at concentrations of 10^–8 and 10^–6 M. The values are the means ± SEM.

Figure 6

GO enrichment dot plot presenting a comparison of transcriptomic data between control individuals injected with physiological saline and 24 h after Tenmo-TRP-7 treatment at a concentration of 10^–8 M. The identification of the most representative GO terms of biological process and molecular function using REVIGO (http://revigo.irb.hr/) was performed (cut off < 0.05). The size of the dots represents the number of genes in the significantly differentially expressed genes (DEGs). Gene ratio' is the percentage of total DEGs in the given GO term. For the graphical presentation of the obtained data, ggplot2 (https://ggplot2.tidyverse.org) for RStudio was used (http://www.rstudio.com)^,,.

Figure 7

GO enrichment dot plot presenting changes in the expression levels of different sets of genes observed 24 h after Tenmo-TRP-7 treatment at a concentration of 10^–6 M compared to the control. The identification of the most representative GO terms for biological process and molecular function using REVIGO (http://revigo.irb.hr/) was performed (cut off < 0.05). The size of the dots represents the number of genes in the significantly differentially expressed genes (DEGs). Gene ratio’ is the percentage of total DEGs in the given GO term. For the graphical presentation of the obtained data, ggplot2 (https://ggplot2.tidyverse.org) for RStudio was used (http://www.rstudio.com)^,,.

Figure 8

Reverse transcription quantitative PCR (RT-qPCR) analysis on the expression levels of genes encoding selected antimicrobial peptides ((A) attacin 2; (B) tenecin 3) and the Toll receptor (C) in immune-related tissues (pooled fat body and haemocytes) 24 h after applying Tenmo-TRP-7 at concentrations of 10^–8 and 10^–6 M. Values are the means 土 SEM. *p ≤ 0.05; **p ≤ 0.01.

Figure 9

Lysozyme-like antimicrobial activity of T. molitor haemolymph against Micrococcus luteus when collected from control individuals injected with physiological saline (Control) or a solution of physiological saline and Tenmo-TRP-7 at concentrations of 10^–7 and 10^–5 M (for final concentrations of 10^–8 and 10^–6 M, respectively). L 0.1 mg, positive control, solution of physiological saline and lysozyme at a concentration of 0.1 mg/mL. To activate the immune system, beetles were injected with attenuated Staphylococcus aureus 2 h after Tenmo-TRP-7 administration. For better visualization of the observed differences, the obtained values were reversed. Asterisks indicate statistically significant differences compared to the control individuals; *p ≤ 0.05; ****p ≤ 0.0001; values are given as the means ± SEM.