Tachykinin-related peptide signalling is important for the immune response of the mealworm beetle Tenebrio molitor L.

Introduction: Insects possess a well-developed innate immune system, which encompasses both cellular and humoral mechanisms. On the basis of the similarities in neuropeptide actions between insects and vertebrates, we assume that neuropeptides such as tachykinin-related peptides (TRPs) regulate insect immune responses and are themselves modulated following infection.

Methods: In this study, we examined how immune activation affects the expression of genes encoding TRP precursors and receptors (TRP and TRPR) and whether TRPs directly modulate selected immune mechanisms in the pest species Tenebrio molitor. In our experiment, we combined cytokine stimulation, genetic knockdown, pharmacological inhibition, immune profiling, and survival analysis to dissect neuropeptide function.

Results: Our results revealed two important insights. First, after activation of the immune system, TRP and TRPR genes were significantly downregulated in the nervous system and immune-related cells. These changes are closely correlated with the changes of the expression level of immune genes. We then show, using Spantide II, a potent antagonist of TRPR, and RNAi knock down of TRP and TRPR, the modulation of key processes of the T. molitor humoral response. This includes the over expression of genes encoding antimicrobial peptides and the important arthropod immune effector phenoloxidase activity.

Discussion: Our findings highlight a compelling association between the TRP and immune regulation in Tenebrio and provide insights into the hormonal regulation of physiological processes in insects. Our research also provides novel insights that can contribute to the development of sustainable pest control strategies amid increasing insecticide resistance.

1 Introduction

Insects are the most diverse taxon on earth, and they have a significant impact on our lives. Their roles in pollination and sanitation, for example, are crucial to human well-being. But insects as pests also cause substantial economic losses (1). This situation necessitates the development of new, biosafe, and targeted agents for pest control. Paradoxically, the mass rearing of certain pest species, such as Tenebrio molitor, can help meet the growing food demands of the human population (2). For this reason, a better understanding of the immunology of T. molitor is essential not only for developing new pest control strategies but also for improving insect mass-rearing practices.

Insects have an innate immune system, which is composed of cellular and humoral responses, resulting in effective protection against infection (3). The cellular response entails processes such as phagocytosis, nodulation, and encapsulation, in which haemocytes - the main cells of the insect haemolymph - are involved. The humoral response comprises the activity of proteins and enzymes such as lysozyme, antimicrobial peptides (AMPs), and phenoloxidase (PO), an important enzyme in arthropod immune function that generates cytotoxic substances but also contributes to the sclerotization of the cuticle (4). However, the activation and coordination of these responses require precise regulation. Emerging evidence suggests that this regulation is at least partially mediated by neuroendocrine factors, including neuropeptides that can directly interact with immune cells (3, 5). Neuropeptides are key regulators of physiological homeostasis and are involved in all aspects of insect biology, including development, metabolism, behaviour, and reproduction. Importantly, recent studies indicate that they may also act as immunomodulators, highlighting their role in the cross-talk between the neuroendocrine and immune systems (6, 7). These small signalling molecules are primarily produced and secreted by neurosecretory cells (NSCs) in the insect central nervous system, but endocrine cells in peripheral tissues such as the gut and reproductive system also contribute to their production (8).

One of the largest and most important families of neuropeptides identified in insects is the tachykinin-related peptides (TRPs). They show high structural and functional homology with tachykinins (TKs) present in vertebrates (9). Tachykinins such as vertebrate substance P (SP) have pleiotropic effects and take part in nociception, stress responses, and the regulation of muscle contractions (9). These functions are consistent with the action of TRPs in insects (9, 10). Moreover, both neuropeptide families interact with specific G protein-coupled receptors (GPCRs), leading to the activation of signalling pathways affecting cell activity (11). Our previous studies indicated that TRPs can influence both the cellular response by modulating haemocyte morphology and activity and the humoral response by regulating the expression levels of immune-related genes in insects (4, 12). This finding is in line with the effect of SP on the activity of the vertebrate immune system (13). Furthermore, our previous studies showed that human SP influences the immune system activity of Tenebrio molitor beetles, which supports the hypothesis that the immunomodulatory role of the TK system is evolutionarily conserved (12). Given the key role that neuropeptides play in signalling within the nervous system and other tissues, it seems likely that TRPs influence the process of pathogen recognition and modulate the overall immune response. The mechanisms, however, by which TRPs regulate the immune response remain largely unknown. Therefore, here, we study two essential questions to investigate the immunoregulatory role of TRPs in insects. First, whether the TRP pathway is modulated during the immune response against bacterial components or after cytokines administration? Second, are TRPs directly involved in the regulation of the insect immune system?

The first part of our research focused on evaluating changes in the expression patterns of genes encoding TRP precursor (TRP) and receptor (TRPR) in different phases of immune system activation. To obtain a more complete picture of the changes observed in the TRP system, the T. molitor immune system was activated via different activators, including one of the most important insect cytokines, Spätzle-like (TmSpz-like), a key ligand of the Toll receptor. The use of TmSpz-like will allow us to better support our previous finding that cytokines are modulators of the neuroendocrine system in insects, similar to humans (7). Because of the nature of neuropeptides, which are most likely stored in neuroendocrine cells and released during a specific physiological state, molecular analysis of their expression levels was supported by immunocytochemical analysis of the presence of TRP precursors in the Tenebrio nervous system. This new finding strongly suggests that insect cytokines can affect neuropeptide levels in insect neuroendocrine cells.

In the second part of the research, we focused on the direct effect of TRP on the T. molitor immune system on the basis of the expression of immune-related genes and phenoloxidase. For this purpose, we used RNAi gene knock down and Spantide II, a potent antagonist of TRPR. Using both dsRNA to selectively disrupt TRP-related pathways and Spantide II to block TRPRs will provide a comprehensive understanding of TRP function and its impact on immune system modulation. Additionally, we identified the combination that most significantly affect T. molitor immune system function and then tested the survival of these groups following an additional application of Escherichia coli, which, under normal conditions, significantly reduces beetle survival.

Our research advances the understanding of how the TRP system influences immune regulation in insects. By uncovering how hormonal pathways affect immunity, we identify potential targets for pest management and ways to enhance the health and productivity of farmed insects used in food and feed.

2 Results

2.1 Expression patterns of the TRP and TRPR genes after activation of the Tenebrio immune system

To evaluate the potential participation of TRP signalling in the immune response after T. molitor immune system activation, the expression levels of TRP and TRPR genes were analysed in the nervous system (brain and ventral nerve cord (VNC)) and immune-related cells (fat body and haemocytes). The analysis of three time points (3, 6, and 24 hours) revealed changes in TRP and TRPR gene expression at different stages of the immune response (7, 14).

To activate different signalling pathways involved in immune system activation, insects were injected with lyophilized E. coli (Ec), and peptidoglycan of Staphylococcus aureus (PG). Additionally, to evaluate the potential role of insect cytokines in the modulation of the insect immune system, beetles were also injected with TmSpz-like, a key cytokine and ligand of the Toll receptors in insects (15). The molecular analysis was preceded by survival experiments, which revealed significant differences between beetles injected with different activators of the immune system (Figure 1A). After Ec treatment, the survival of T. molitor males was significantly lower than that of control individuals injected with physiological saline (PS) (Gehan–Breslow–Wilcoxon test, p ≤ 0.01, n=50 per treatment). Moreover, statistically significant differences between survival curves were reported between TmSpz-like treatment and Ec (Gehan-Breslow-Wilcoxon test, p ≤ 0.05, n=50 per treatment).

Figure 1

Figure 1. Expression patterns of the TRP and TRPR genes after activation of the Tenebrio immune system according to additional analyses. (A) Survival curve of T. molitor males after injection of physiological saline (control, green line) or peptidoglycan of Staphylococcus aureus (PG, 1 mg/mL, OD₆₀₀ = 1, violet line), a suspension of lyophilized Escherichia coli K12 (Ec, 1 mg/mL, OD₆₀₀ = 1; red line), or a suspension of TmSpz-like at a concentration of 10^–7 M (black line). The colours in the table indicate an increase (red) or decrease (blue) in survival relative to the group in the top row of the table. The values are presented as the means ± SEMs. Table – Statistical comparison of estimated survival curves based on the Gehan-Breslow–Wilcoxon test; ns – nonsignificant difference, *p ≤ 0.05, **p ≤ 0.01, n = 50 per research variant. B-E. Heatmaps showing the changes in the expression levels of genes encoding TRP precursor (B) and TRP receptor (TRPR) (C) in the brain, ventral nerve cord (VNC), fat body, and haemocytes and the changes in the expression levels of immune-related genes in the fat body (D) and haemocytes (E) 3, 6 and 24 hours after the application of physiological saline (control), Ec, PG or TmSpz-like protein. The values are expressed as log2fold values, shades of red indicate upregulation, shades of blue indicate downregulation, *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001, n = 3 per research variant, one biological repetition was samples collected from at least 20 (B, VNC, haemocytes) or 10 (fat body) individuals. (F) - Correlation matrix showing the dependencies between the expression levels of TRP and TRPR genes in the brain (B), ventral nerve cord (VNC), fat body (FB), and haemocytes (Hem). These changes were correlated with the expression levels of immune-related genes (Toll, Cactus (Cac), Dorsal (Dor), Relish, Domeless (Dom), Stat92E (Stat), Attacin 2 (Att2), and Tenecin 3 (Ten3)) in the fat body (FB) and haemocytes (Hem). To estimate the correlation of the data, the Pearson correlation coefficient method was used. The matrix was generated via SRplot software (https://www.bioinformatics.com.cn/srplot). Size of the dot - level of the r value. The r value is presented in the middle of the dot. Different colours indicate different r values. Red shading indicates positive correlations (r ≥ 0), and blue shading indicates negative correlations (r ≤ 0). Circles indicate statistically significant correlations (p ≤ 0.05).

The analysis of the expression patterns of TRP revealed that the significant changes in expression level were mostly visible at the first tested time point (Figure 1, Supplementary Figure S1). After 3 h, a significant decrease in the expression level of the TRP gene in the fat body and brain of beetles injected with TmSpz-like protein was observed (Figure 1B, Supplementary Figure S1). Significant downregulation of TRP was also observed in the brain after Ec application. Significant overexpression of TRP was only visible in the VNC after TmSpz-like protein injection. At later time point, 6 h after the application of immune system activators, no significant changes were observed. After 24 h, the only significant increase that occurred was in the fat body after TmSpz-like protein treatment.

In the case of TRPR, a greater number of significant changes were observed (Figure 1C, Supplementary Figure S2). After 3 h, a decrease in the expression level of the TRPR gene was detected in the brain (after the application of PG), VNC (Ec and PG), fat body (all groups), and haemocytes (Ec and TmSpz-like protein). The only increase in the expression level was detected in VNC 6 hours after the TmSpz-like protein. However, after 24 hours, there was a decrease in the expression level of TRPR in the fat body (after TmSpz-like protein) and haemocytes (after Ec and PG application).

Next, we examined changes in the expression levels of selected immune-related genes in cells directly involved in the immune response (fat body cells and haemocytes) and correlated with changes in the expression levels of the TRP and TRPR genes after immune activation (Figures 1D, E, Supplementary Figures S3-Supplementary Figures S6). We found a significant positive correlation between the expression of TRP in VNC and the TRPR in the brain and VNC. The same correlation also occurred in TRPR between the brain and VNC (Figure 1F). Interestingly, some dependencies were detected between the expression levels of genes encoding TRP, TRPR and immune-related genes (Figure 1F). A significant positive correlation has been reported between the expression of Toll in the fat body and TRPR in the brain, VNC, and fat body. Moreover, the expression level of Domeless was positively correlated with the expression of TRP in the VNC and TRPR in the brain. Conversely, the expression of Domeless in haemocytes is negatively correlated with TRP expression in the fat body. On the other hand, in haemocytes, a negative correlation between the expression level of TRPR and that of Attacin 2 was also detected.

Significant correlations were also reported between the expression of genes related to immune system activity (Figure 1F).

2.2 Immunolocalization of TRP precursor in the nervous system after activation of the immune system

To analyse changes in the distribution of the TRP precursor following immune system activation, immunolocalization was performed. The immunocytochemical results revealed differences in the distribution and abundance of the TRP precursor in both the brain and different ganglions of the VNC of T. molitor. These changes were time- and immune activator dependent. After 3 h (Figure 2A), a relative decrease in the intensity and abundance of the fluorescent signal associated with the immunolocalization of TRP was observed in all the parts of the VNC after Ec injection. A weaker fluorescence signal also occurred in the second abdominal ganglion after the application of PG and the TmSpz-like protein and in the terminal abdominal ganglion (TAG) after the application of the TmSpz-like protein. After 6 h in the group activated with Ec, a decrease of fluorescent signal was observed in all the examined tissues, including the brain (Figure 2B). After PG application, the signal intensity and abundance were relatively greater than those in the control group. Additionally, in the brain after the injection of TmSpz-like protein, such an increase occurred with a simultaneous decrease in the relative amount of the precursor in the second abdominal ganglion and TAG. Long-term effects (24 h incubation) were observed only after Ec application in the second terminal ganglion and the second abdominal ganglion (Figure 2C).

Figure 2

Figure 2. Micrographs showing the distribution of TRP precursor in the brain; second terminal ganglion (2nd T), second abdominal ganglion (2nd A) and terminal abdominal complex (TAG) 3 (A), 6 (B) and 24 h (C) after the application of physiological solution (control); Escherichia coli (Ec), Staphylococcus aureus peptidoglycan (PG) and Spätzle-like protein (TmSpz-like). The orange colour shows a 3D projection of the presented structure, which was superimposed on the pictures to show the distribution of the TRP precursor. A 3D projection was created with AMIRA 3D software on the basis of the obtained Z-stack files of the whole analysed structure (threshold 75–255 for fluorescent intensity). Sample examination was performed on the same day under the same conditions and microscope setup, which allowed comparison of the obtained micrographs. Scale (white line) 100 µm.

2.3 Evaluation of the potential direct effects of TRP on insect immune mechanisms.

In previous experiments, we reported that the expression of immune-related genes in T. molitor was associated with changes in the expression levels of TRP and TRPR. However, it remains unclear whether the observed immune responses are directly dependent on TRP signalling and thus whether TRPs play a direct role in regulating immune function in insects. Therefore, we used Spantide II to pharmacologically inhibit TRPR activity. We also applied dsRNA to knockdown the genes encoding the TRP precursor and receptor (consequently, dsTRP and dsTRPR). This combined pharmacological and molecular approach enabled us to assess the direct involvement of TRP signalling in modulating immune gene expression and insect survival. Moreover, to confirm the specificity and effectiveness of TRP pathway inhibition, we readministered synthetic TRP (Tenmo-TRP-7, amino acid sequence: MPRQSGFFGMRa which possesses a sequence similar to the native TRP identified in T. molitor) to selected groups.

2.3.1 Survival experiment after Spantide II and dsRNA treatment

First we focused on the effects of the tested compounds on the survival of T. molitor males. Physiological saline, Tenmo-TRP-7, and Spantide II did not affect the survival of T. molitor males (Figure 3A). In these cases, around 90% of individuals survive. Simultaneous injection of Tenmo-TRP-7 and Spantide II significantly influences beetle mortality. Until the last day of observation (21^st day), only 2.63% of the tested individuals survived. The comparison of survival curves revealed significant differences between beetles simultaneously injected with Tenmo-TRP-7 and Spantide II and the remaining variants (Gehan-Breslow-Wilcoxon test, p ≤ 0.0001, in all cases, n=50 per treatment).

Figure 3

Figure 3. Survival curves of T. molitor males after injection of a potent antagonist of the TRP receptor (TRPR) Spantide II (A), knockdown of the TRP and TRPR genes (B), and double knockdown of the TRP and TRPR genes (C). In graph A, control represents individuals injected with physiological saline; TRP represents individuals injected with Tenmo-TRP-7 at a concentration of 10^–5 M; Spantide II represents beetles injected with Spantide II at a concentration of 10^–3 M; and Spantide + TRP represents simultaneous injection of Spantide II and Tenmo-TRP-7 at a concentration of 10^-5 M. In graph B, Control individuals were injected with dsRNA targeted to Galleria mellonella lysozyme (GmLys), while gene knockdowns were performed using dsTRP for the TRP gene and dsTRPR for the TRP receptor gene (TRPR). The dsControl + PS represents insects injected with dsGmLys and physiological saline; dsControl + TRP represents individuals injected with dsGmLys and Tenmo-TRP-7 at a concentration of 10^–5 M; dsTRPR + PS represents insects injected with dsTRPR and physiological saline; dsTRPR + TRP represents insects injected with dsTRPR and Tenmo-TRP-7 at a concentration of 10^–5 M. In Graph C, Control 2KD individuals were injected with a double dose of dsLysGm, while dsTRP/TRPR was used for the double knockdown of TRP and TRPR. The colours in the table indicate an increase (red) or decrease (blue) in survival relative to the group in the top row of the table. The values are the means ± SEMs. Table – Statistical comparison of estimated survival curves based on the Gehan-Breslow–Wilcoxon test; ns, nonsignificant differences, *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, n = 50 per research variant.

The survival experiment after dsRNA treatment revealed significant differences (Gehan-Breslow-Wilcoxon test, p ≤ 0.05, n=50 per research variant) (Figure 3B). After TRP knockdown, a significant reduction in T. molitor mortality compared with that in the control group was observed (Gehan-Breslow-Wilcoxon test, p ≤ 0.05, n=50 per research variant). Interestingly, a comparison of survival curves after knockdown of the TRP gene revealed significant differences compared with the groups treated with dsGmLys (dsControl) and Tenmo-TRP-7. Significant differences compared to dsTRP were also observed with groups injected with dsTRPR + physiological saline, and dsTRPR and Tenmo-TRP-7 (Gehan-Breslow-Wilcoxon test, p ≤ 0.05 and p ≤ 0.001, respectively, n=50 per treatment). Compared with the control, double knockdown (dsTRP/TRPR) did not result in any significant differences (Figure 3C).

2.3.2 Changes in the expression levels of immune-related genes

Our previous transcriptomic analysis revealed that the application of Tenmo-TRP-7 led to significant changes in the expression patterns of a wide spectrum of immune-related genes (6). For this reason, the next step in our research was the determination of the effects of Spantide II, dsTRP and dsTRPR on the expression levels of selected immune genes in the fat body and haemocytes. For analysis of basic immune pathways involved in the stress response, Toll (receptor of the Toll signalling pathway), Dorsal (the NF-κB transcription factor in the Toll pathway), Cactus (the IκB inhibitor that sequesters Dorsal), Relish (transcription factor of the Imd signalling pathway), Domeless (receptor of the JAK/STAT signalling pathway) and Stat92E (the key transcription factor in the JAK/STAT pathway) were chosen. Moreover, the expression of genes encoding selected AMPs (Attacin 2 and Tenecin 3) was analysed. AMP gene selection was based on transcriptomic changes occurring after Tenmo-TRP-7 application (6).

RT–qPCR analyses revealed that changes in the expression levels of immune-related genes after the application of Spantide II and dsRNA differed depending on the tested immune tissues (Figures 4A–D, Supplementary Figures S7-S12). In the fat body, the application of Tenmo-TRP-7 decreases the expression of Toll, Domeless and Attacin 2. Injection of Spantide II and blocking of TRPR elicited effects opposite to those of Tenmo-TRP-7 injection, and Toll overexpression was observed. Nevertheless, Spantide II injection led to downregulation of the Attacin 2 gene, and upregulation of Tenecin 3 gene. The application mixture of Tenmo-TRP-7 and Spantide II abolished the effect of separate injection of Tenmo-TRP-7 and Spantide II. Interestingly, this mixture led to a decrease only in the Attacin 2 expression level (Figure 4A). Knockdown of the precursor and the receptor individually did not induce any changes; only the double knockdown led to a marked decrease in the expression levels of Toll, Dorsal and Relish. Insects treated with dsGmLys (dsControl) followed by Tenmo-TRP-7 injection showed that non-specific dsRNA did not strongly affect the action of Tenmo-TRP-7, and a significant reduction in the expression levels of Toll, Relish, Domeless, and Tenecin 3 was observed. Interestingly, after receptor silencing and Tenmo-TRP-7 application, Dorsal, Domeless and Attacin 2 were overexpressed (Figure 4B, Supplementary Figure S11).

Figure 4

Figure 4. Changes in the humoral response of T. molitor after the agonist of TRP receptor (TRPR) - Spantide II Spantide II and the knockdown of the TRP and TRPR genes. A-D. Heatmaps showing changes in the expression levels of immune-related genes after Spantide II (A, C) or dsRNA treatment (B, D) in the fat body and haemocytes, respectively. Expression levels of genes tested in the fat body and haemocytes after the application of physiological saline (control), Tenmo-TRP-7 (TRP) at a concentration of 10^–5 M, Spantide II at a concentration of 10^–3 M, or a mixture of Tenmo-TRP-7 and Spantide II (A, C). Expression levels of immune-related genes after the injection of dsRNA-targeted genes encoding lysozyme in Galleria mellonella (GmLys, dsControl) or genes related to TRP signalling (genes encoding the TRP precursor (dsTRP) or receptor (dsTRPR)) (B, D). The effects of double injection of dsRNA and physiological saline or Tenmo-TRP-7 (TRP) at a concentration of 10^–5 M were also tested. In addition, the effects of both TRP and TRPR knockdown were analysed. The control for the double-knockdown experiment (dsControl 2KD) was the injection of a double dose of dsRNA-targeted GmLys. The values are expressed as log2fold values, shades of red indicate upregulation, shades of blue indicate downregulation, *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001, n = 3 per research variants, as one biological repetition is considered the samples collected from 10 (fat body) or 20 (haemocytes) individuals. (E-H). Phenoloxidase (PO) activity in T. molitor haemolymph after Spantide II and a mixture of Tenmo-TRP-7 treatment (E), knockdown of TRP and TRPR (F), double injection of dsRNA and physiological saline or TRP (G) or double knockdown of the tested genes (H). A – Phenoloxidase activity in the haemolymph of T. molitor after the application of physiological saline (Control), Tenmo-TRP-7 (TRP) at a concentration of 10^–5 M, Spantide II at a concentration of 10^–3 M, or a mixture of Tenmo-TRP-7 and Spantide II. (F)– PO activity after injection of dsRNA-targeted Galleria mellonella lysozyme (GmLys) (dsControl) or dsRNA-targeted genes related to TRP signalling (genes for TRP precursor (dsTRP) or receptor (dsTRPR)). (G) – PO activity after application of dsRNA and additional injection with physiological saline or Tenmo-TRP-7 (TRP) at a concentration of 10^–5 M. (H) – PO activity determined after injection of a double dose of dsGmLys (dsControl 2KD) or double knockdown of TRP and TRPR. The values are presented as the percentage change relative to the control group. The results are presented as the means ± SDs, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n ≥ 10 per research variant.

In haemocytes, Tenmo-TRP-7 injection significantly reduced the expression level of Toll, whereas TRPR blocking through treatment with Spantide II elicited opposite effects, with a significant upregulation of most of the tested genes observed (except Cactus and Dorsal) (Figure 4C, Supplementary Figure S9). A mixture of Spantide II and Tenmo-TRP-7 abolished the effects observed with separate injections of these compounds and did not cause any changes in the expression levels of the selected immune-related genes. The use of dsRNA targeted to TRP causes a significant decrease in the expression of Toll, Dorsal and Stat92E in haemocytes. Knockdown of TRPR significantly decreases the expression levels of Toll, Cactus, Dorsal and Tenecin 3 (Supplementary Figures S9, 10). In contrast, simultaneous silencing of both TRP and TRPR resulted in significant overexpression of Attacin 2 and Tenecin 3. Similar to a single injection of Tenmo-TRP-7, the application of dsGmLys and Tenmo-TRP-7 decreased the levels of Toll and Domeless, whereas in insects injected with dsTRPR, the administration of either physiological saline or Tenmo-TRP-7 led to Cactus and Tenecin 3 overexpression (Supplementary Figures S12).

2.3.3 Phenoloxidase activity

Our previous studies revealed that Tenmo-TRP-7 significantly affects PO system activity. This was expressed as changes in PO activity and changes in the expression levels of genes related to the PO system (4). For this reason, the next step was to analyse changes in the activity of this enzyme after Spantide II and dsRNA application. Like in previous studies, Tenmo-TRP-7 application significantly increased PO activity (Dunnett’s post hoc test, p ≤ 0.01, n≥ 10 per research variant). Blocking the receptor with Spantide II and using a mixture of Spantide II and Tenmo-TRP-7 also led to increased PO activity (Dunnett’s post hoc test, p ≤ 0.001 and p ≤ 0.01, respectively, n≥ 10 per research variant) (Figure 4E).

Compared with the control, the use of dsRNA directed against the TRP precursor and simultaneous knockdown of TRP and TRPR resulted in a significant decrease in PO activity (Dunnett’s post hoc test, p ≤ 0.05; Student’s t test, t = 2.90, p ≤ 0.01, respectively, n≥ 10 per research variant) (Figures 4F, H). However, significant differences were also observed in PO activity between the groups in which TRP and TRPR were knocked down (Dunnett’s post hoc test, p ≤ 0.001, n≥ 10 per research variant). Interestingly, compared with the other treatments, the injection of physiological saline after dsTRPR treatment caused a significant increase in PO activity (Dunnett’s post hoc test, p ≤ 0.001, all cases, n≥ 10 per research variant) (Figure 4G).

2.4 Survival assessment following TRP system suppression and immune activation with Escherichia coli

On the basis of previous analyses, we selected the experimental variants that had the strongest impact on the immune system activity of T. molitor to assess how the modulation of TRP signalling affects insect survival during immune system activation. For this purpose, we conducted a survival experiment in which Ec injection, which significantly affected Tenebrio survival (see above). The results revealed statistically significant changes in survival only in the group of insects with double knockdown of TRP and TRPR genes. In this group, survival was significantly greater than that in the groups injected with physiological saline, Tenmo-TRP-7, or Spantide II (Gehan-Breslow–Wilcoxon test, p ≤ 0.05, in all cases, n=50 per research variants) (Figure 5).

Figure 5

Figure 5. Survival curves of T. molitor males after injection of Tenmo-TRP-7 (TRP), the potent TRP receptor antagonist Spantide II, and double knockdown (dsTRP/TRPR), followed by immune system activation via Escherichia coli (Ec). Control represents individuals injected with physiological saline; TRP represents individuals injected with Tenmo-TRP-7 at a concentration of 10^–5 M; Spantide II represents beetles injected with Spantide II at a concentration of 10^–3 M. After 2 hours, the groups were injected with Ec. For the double-knockdown control (Control 2KD) individuals were injected with a double dose of dsLysGm, while dsTRP/TRPR was used for the double knockdown of TRP and TRPR. After 7 days when the efficient knockdown of TRP and TRPR was achived, individuals were injected with Ec. The colours in the table indicate an increase (red) in survival relative to the group in the top row of the table. The values are the means ± SEMs. Table – Statistical comparison of estimated survival curves based on the Gehan-Breslow–Wilcoxon test; ns, nonsignificant difference, *p ≤ 0.05, n = 50 per research variant.

3 Discussion

Although our previous studies explored the influence of TRPs on immune responses, it remains unclear whether these effects are direct or indirect, highlighting the need to clarify how TRP signalling interacts with immune activation. Therefore, we first examined how the expression of genes encoding the TRP precursor and receptor changed after activation of the immune system. In humans, SP levels fluctuate depending on the physiological state of the body (16). Given the structural and functional similarities between SP and TRP, we hypothesized that analogous changes may occur in insects. What is important, overall, the main changes were significant downregulation of TRPR gene in the nervous system and immune-related cells. The downregulation of TRP was also noted, but to a lesser extent. These findings suggest that once the immune response is triggered, TRP-related signalling can be inhibited. This notion may be associated with our previous studies looking at the immunomodulatory effect of TRP in T. molitor. Research by Urbański et al. (4, 6) revealed that the application of Tenmo-TRP-7 significantly downregulated some immune-related genes and decreased the antimicrobial activity of T. molitor haemolymph. Considering these results, we may assume that TRP signalling is inhibited at the initial stage of immune system activation, probably to elicit maximal activation of immune mechanisms. Moreover, TRP and TRPR expression are significantly correlated across different parts of the nervous system. This positive correlation in the nervous system may be due to regulatory mechanisms aimed at maintaining homeostasis and ensuring proper functioning of both the nervous and immune systems.

By contrast, and because of the nature of neuropeptides, the analysis of TRP and TRPR gene expression was enriched by examining the relative abundance of TRP precursor in the T. molitor nervous system during immune system activation. Generally, neuropeptides are synthesized and stored in neuroendocrine cells, from which they are released in response to specific physiological triggers (17). Our study revealed that the significant decrease in the expression level of TRPR is associated with a lower abundance of TRP expressed as an immunofluorescent signal in the nervous system, especially at the initial stage of immune system activation. This observation may suggest that TRP can be released into the haemolymph. Thus, we cannot exclude the possibility that the significant downregulation of TRPR at this time point is a possible adaptive mechanisms that can decrease cell sensitivity to the presence of neuropeptide (18). Similar regulations occur in vertebrates. For example, increased levels of insulin in the bloodstream lead to a decrease in the number of associated receptors. This mechanism helps maintain homeostasis and prevents detrimental effects from excessive receptor activation, paralleling how insects may modulate TRPR expression to manage immune responses effectively (19). This hypothesis is also supported by the fact that the downregulation of TRPR is observed not only in the nervous system but also in the fat body and haemocytes. Nevertheless, in the brain, the changes in TRP abundance are less pronounced, potentially due to the role of TRP as a neuromodulator (20).

According to our hypothesis, different immune activators seem to be a key variable for TRP pathway modulation. Here, we found differences in the changes in TRP and TRPR expression levels, as well as in the relative abundance of TRP in the nervous system after the application of different bacterial components and TmSpz-like cytokine. In general, the application of Ec and TmSpz-like elicited more significant changes in the expression levels of genes related to TRP signalling and potential TRP abundance in the nervous system than PG did. Furthermore, Ec significantly decreased the survival rate of the tested T. molitor. These differences may suggest a relationship between TRP signalling and the Imd pathway, but also Toll pathway, in which the TmSpz-like cytokine is an important component (21). TmSpz-like proteins play a key role in the immune response. They are known to interact with Toll receptors, initiating immune system signalling cascades that enhance defence mechanisms. TmSpz-like proteins appear later in the immune response, which may classify them as late response indicators (21). Interestingly, our previous research also suggested that TmSpz-like action can be linked to the regulation of the T. molitor neuroendocrine system (7). In vertebrates, different cytokines regulate innate and adaptive immunity, activate inflammatory responses, and direct leukocyte trafficking (22). These inflammatory factors can also affect the expression of genes related to the function of the neuroendocrine system. For example, when cytokines are present, the expression of SP is significantly increased (16). Our results seem to confirm the existence of similar mechanisms in insects. Three hours after TmSpz-like protein injection, a noticeable decrease in the abundance of the immunofluorescent signal for TRP in the VNC was observed. To our knowledge, this is the first evidence that insect cytokines may affect the abundance of neuropeptides in insect neuroendocrine cells and probably also lead to neuropeptide release to the haemocoel. Nevertheless, a significant increase in TRP expression in the VNC was observed at this time point. This observation can be explained by a compensatory response to the initial depletion of the TRP precursor pool in neuroendocrine cells. These findings suggest that TmSpz-like is not only a regulator of immune system function but also a neuroendocrine regulator. However, to fully confirm this hypothesis, further research is needed.

The potential relationships between TRP signalling and immune system activity are also apparent in the correlation of the expression levels of TRP, TRPR, and immune-related genes in fat body. For a better understating role of TRP signalling and insect immune system the most important seems to be a significant positive correlation between the expression level of TRPR and the expression level of Toll and Domeless. Since TRPs can regulate processes related to immunomodulation and can also act as stress mediators, there may be coregulation of the expression of their receptors and those responsible for the immune response. This crosstalk between signalling pathways can help respond effectively to changing environmental conditions (6, 23). The results of further experiments seem to confirm connection between TRP and the Toll pathway because injection of the TRPR agonist Spantide II elicits the opposite effect (upregulation) on the expression level of the Toll gene, as does the application of Tenmo-TRP-7 (downregulation). Importantly, simultaneous injection of Tenmo-TRP-7 and Spantide II did not affect the expression level of the Toll gene. On the other hand, opposite results were observed after dsRNA treatment. Injection of dsTRPR together with Tenmo-TRP-7 resulted in overexpression of Dorsal, which is a main transcription factor of the Toll pathway. This can be explained by the compensation process in signal networks: when the main route is blocked, the system activates other regulatory mechanisms. The administration of Tenmo-TRP-7 can no longer act through the standard TRPR pathway, which has been silenced; thus the cell can activate alternative signalling pathways, leading to increased Dorsal expression. Interestingly simultaneous knockdown of TRP and TRPR led to significant downregulation of the Toll gene, as well as Dorsal, providing additional evidence that TRP signalling is tightly interconnected with Toll pathway regulation. The differences between these experiments may be associated with the different impacts of these methods on the TRP system. By blocking TRP receptors, Spantide II allows us to assess how the inhibition of TRP signalling affects the immune response. On the other hand, dsRNA-mediated gene knockdown targets specific genes involved in TRP pathways, providing insights into how the downregulation of these genes impacts gene expression patterns and immune signalling. However, dsRNA treatment did not completely inhibit TRP signalling because, owing to the nature of neuropeptides, some portions of TRPs may be present in the neuroendocrine system, and the knockdown of TRPR did not exclude the presence of active TRPR on the cell surface. For this reason, the use of Spantide II and dsRNA enables the observation of both physiological and molecular changes, offering a comprehensive approach to studying the role of TRP signalling in immune processes.

The interaction between TRP signalling and the Toll pathway in the fat body involves both immunity and metabolism (24). Toll activation induces a metabolic shift by suppressing insulin-like peptide (ILP) signalling, reducing lipid stores, and limiting growth (25, 26). TRPs also modulate feeding via adipokinetic hormone (AKH) signalling (27, 28), and both ILPs and AKHs have immunomodulatory roles (29, 30). Additional evidence of neuropeptide crosstalk comes from the colocalization of allatotropin (AT) and TRP neurons (31) and correlations between AT and TRP components in T. molitor (Supplementary Figure S13) (7). Overall, the data indicate a close link between TRP signalling and Toll pathway regulation, potentially reinforced by interactions with other neuropeptides.

The activation of the immune system also revealed a significant correlation between TRP system elements and the expression of Domeless genes in fat body, which are key components of the JAK/STAT pathway. This pathway regulates immune responses and metabolism during infection. In Drosophila, cytokines activate JAK/STAT via Domeless in tissues such as muscles, promoting immune functions and reallocating energy by suppressing ILPs, lowering glycogen, and increasing glucose (32). The correlation between Domeless expression in the fat body and TRP/TRPR in nervous tissue suggests cooperation in immune and metabolic regulation. However, Spantide II and dsRNA treatment only partially confirmed the direct dependencies of the TRP system on JAK/STAT in the fat body. The injection of Tenmo-TRP-7 significantly downregulated the Domeless gene, and the simultaneous injection of Tenmo-TRP-7 with Spantide II abolished this effect. Interestingly, the application of dsControl and the administration of Tenmo-TRP-7 had effects similar to those of Tenmo-TRP-7 alone, whereas the knockdown of the expression of the TRPR gene and Tenmo-TRP-7 injection caused the overexpression of Domeless. This result may indicate that TRP-related signalling is effectively blocked. However, after double knockdown, significant changes in Domeless expression have not been reported.

Only weak correlations were found between TRP signalling genes and Relish, a key Imd pathway gene, in the fat body during immune activation in T. molitor. Kamareddine et al. (33) showed that the Imd pathway can be involved in the regulation of host metabolism during infection, probably via TRP signalling. Despite the small number of correlations between genes associated with TRR signalling and Relish, Spantide II treatment and dsRNA application led to interesting results. Tenmo-TRP-7, Spantide II, or their combination did not affect Relish, but all the treatments downregulated Attacin 2, an AMP mostly linked to Imd pathway (34). Double knockdown of TRP and TRPR decreased Relish expression without affecting Attacin 2, whereas the combination of Tenmo-TRP-7 and dsTRPR led to Relish overexpression. These results can be related to the newest finding which indicate that the Attacins synthesis may be also co-regulated via Toll signalling, which is strongly affected by TRP (14). To gain a deeper understanding of the relationship between TRP signalling and Imd pathways in the fat body, further research is needed.

Conducting analyses not only on the fat body but also on haemocytes confirms that TRP signalling may be crucial for modulating the activity of these cells. This is indicated, among other things, by a significant correlation between genes associated with TRP signalling and key immune genes. Similar to fat body, a positive correlation was observed between the expression of TRPR in haemocytes and the expression of Toll, Domeless and Stat92E, the key transcription factor in the JAK/STAT pathway. However, the positive correlation was observed also in case of expression level TRPR and Tenecin 3. The strong link between TRP receptors and the expression of immune-related genes in haemocytes suggests that TRP signalling may be directly involved in modulating immune signalling pathways in haemocytes. In particular, the correlation between TRPR and the transcription factor Stat92E appears to support this conclusion, as Stat92E plays a central regulatory role in JAK/STAT-dependent immune activation. These results support our previous findings because the application of Tenmo-TRP-7 significantly affects haemocyte morphology (4). Additionally, the effects of Spantide II and dsRNA treatments further confirmed the importance of TRP signalling in haemocyte function. The application of Spantide II and blocking TRP receptor led to the upregulation of almost all immune-related genes in haemocytes. Moreover, a mixture of Tenmo-TRP-7 and a TRPR antagonist abolished these effects, which point to direct effect of Tenmo-TRP-7 on the expression of tested genes in haemocytes.

Interestingly, the most pronounced effects of dsRNA-mediated silencing in haemocytes were observed at the level of transcription factors, which highlights the central regulatory position of TRP signalling in immune gene expression. Single knockdown of either TRP or TRPR mainly affected transcription factors, with decreased expression of Dorsal and Stat92E following dsTRP treatment, and reduced levels of Cactus and Dorsal after dsTRPR silencing. Given that Cactus functions as the cytoplasmic inhibitor of Dorsal, its simultaneous reduction together with Dorsal implies a broader suppression of the Toll cascade rather than a simple compensatory mechanism.

In contrast, only simultaneous knockdown of TRP and TRPR evoked effects such as those observed in the case of Spantide II, and significant upregulation of the genes encoding Attacin 2 and Tenecin 3 was reported. However, in the variants subjected to double injection (knockdown + physiological saline or Tenmo-TRP-7), Cactus and Tenecin 3 were also significantly upregulated. In these cases, we cannot exclude the possibility that this can be an effect of repeated mechanical injury associated with the injection, but also from the physiological consequences of TRP/TRPR disruption itself, which may trigger compensatory activation of immune signalling pathways similar to that observed under receptor blockade with Spantide II.

The relationship between TRP signalling and Tenebrio haemocyte activity can be supported by the results of the analysis of PO activity, the enzyme produced and secreted by haemocytes in proenzyme form (prophenoloxidase, proPO). Silencing of the gene encoding the TRP precursor and simultaneous knockdown of TRP and TRPR expression resulted in the inhibition of PO activity. However, in some cases, an increase in PO activity was reported, for example, after the receptor was suppressed through Spantide II and after the mixture of Spantide II and Tenmo-TRP-7 was used. These findings are consistent with transcriptomic data indicating that TRP signalling may differentially modulate the expression of PO-related genes (6). These results suggest a complex, potentially dual role of TRP signalling in regulating PO activity, encompassing both immune-related functions and cuticular melanization processes, which may be mediated by the involvement of TRP in maintaining water balance (35–37). Moreover, similar to the fat body, TRP signalling action on haemocytes also may be directly related to the modulation of haemocyte metabolism, which will be the subject of our future research.

Despite the undeniable influence of the TRP system on T. molitor immunity, from a physiological perspective, the most critical question is how TRP modulation affects the lifespan of the tested beetles. Results of the survival experiments further underscore the complexity and multidimensional nature of this issue. While Tenmo-TRP-7 and Spantide II alone had no effect, their combination reduced T. molitor lifespan. These results may be related to cumulative dysregulation of TRP signalling - potentially resulting from antagonist-induced blockade of constitutive receptor activity, non-physiological accumulation of exogenous TRP, and/or activation of compensatory pathways. Although constitutive activity of GPCRs is well established, in the context of insect TRP signalling this explanation remains hypothetical, and the precise mechanism underlying this effect requires further investigation. What is important, TRP knockdown decreased mortality, suggesting that partial inhibition of the TRP system might support longevity probably by modulating stress response, immunity, and metabolism. However, this effect was not detected with TRPR or combined TRP/TRPR knockdown, which requires further study. Additionally, considering that Ec activates the Imd and Toll pathway, particularly triggering antimicrobial peptide expression, and reduce the beetle survival it is important to assess whether the modulation of TRP signalling has any measurable effect on insect survival under such immune-challenging conditions. Gehan-Breslow-Wilcoxon analyses revealed no significant differences between groups after Tenmo-TRP-7, Spantide II, or double-knockdown application and immune system activation via Ec. These results may suggest that the effect of TRP is subtle or compensated for by the activation of signalling pathways related to other neuropeptides. However, strong suppression of TRP signalling by double-knockdown of TRP and TRPR reduce T. molitor mortality after Ec injection, which is a significant confirmation close relationship between TRP signalling and T. molitor immune system. Our supposition support also fact that, injection of Tenmo-TRP-7 reduced beetle survival ratio, especially during the first days after Ec administration.

Our results indicate that the TRP system in T. molitor plays an immunomodulatory role. We demonstrate that both TRP and TRPR expression are closely correlated with key immune-related genes, particularly those involved in the Toll and JAK/STAT pathways. Functional assays using Spantide II and RNAi-mediated gene silencing further confirm that TRP signalling can modulate immune gene expression in fat body and haemocytes, and the activity of phenoloxidase. Importantly, we show for the first time that TRP signalling is influenced by insect cytokines such as TmSpz-like. Although some effects may be secondary and linked to roles in energy mobilization and stress responses through other neuropeptides, this highlights the broader neuroendocrine context in which TRP operates. These investigations are important because they not only increase our understanding of insect physiology but also have practical implications for agriculture and pest management. By elucidating the roles of TRPs in immune and metabolic processes, we may identify novel targets for pest control strategies that exploit these pathways, potentially leading to more sustainable and effective approaches to managing populations of pest insects. On the other hand, some variants significantly enhance the expression of AMP genes and reduce beetle mortality, which is desirable for optimizing insect mass-rearing processes.

Future studies should adopt a more mechanistic approach to dissect the intracellular consequences of TRP/TRPR disruption. Although the current work provides pathway-level evidence through transcriptional profiles of Dorsal, Cactus, and Stat92E, clarifying how TRP signalling influences downstream cascades requires further targeted experiments. A priority will be the immunocytochemical localization of Dorsal and Stat92E. In addition future work should investigate the intracellular signalling mechanisms linking TRP activity to immune and metabolic outputs. Given that TRP receptors are GPCRs, and that similar neuropeptide pathways interact with nutrient-sensing mechanisms, one important direction will be examining whether TRP signalling converges on mTOR, MAPK, or other conserved metabolic regulators. Finally, expanding this research beyond TRPR alone to the broader TRP system will be essential. Our ongoing work on transient receptor potential channels (TRP channels) channels provides an opportunity to determine whether peptide signalling interacts with ion channel-mediated responses in insect immunity and stress physiology. Parallel approaches involving simultaneous silencing of multiple neuropeptides (e.g., TRP, ILP, AKH) will help uncover how these regulatory networks integrate metabolic and immune functions.

4 Materials and methods

4.1 Insects

The model organisms used in the experiment were 7–8-day-old adult males of T. molitor to avoid effects related to aging of the immune system and hormonal changes associated with oogenesis in females. Insects were reared at the Department of Animal Physiology and Developmental Biology (Adam Mickiewicz University, Poznań, Poland) in MIR 154-PE incubators (PHCbi, Singapore, Republic of Singapore) in full darkness at 28 °C and 50–60% humidity. The larvae were kept in boxes with oatmeal, to which apple slices were added 3 times a week, and the pupae were removed. The adult insects used in the experiment were kept in sterile dishes with oatmeal and a piece of apple.

4.2 Injection and sample collection

Before the injection of the test compounds and sample collection, the insects were anaesthetized for 7 min with endogenous carbon dioxide and then washed in ethanol and distilled water. Then, with a microliter syringe (Hamilton Company, Reno, NV, USA), individuals were injected under the coxa of the third pair of legs with 2 µL of the tested solutions. Since T. molitor commonly experiences injuries and the injection of sterile saline has minimal impact (38), the study did not include a control group of unmanipulated males. Anaesthesia and the injection procedure does not cause mortality in T. molitor adults, and no death was observed in any of the groups.

Selected insect tissues were collected via a Zeiss Stemi 504 microscope (Zeiss, Jena, Germany) under sterile conditions. To collect the haemolymph with haemocytes, the tibia of the first pair of legs was cut to obtain a sample without contamination from other cells (7).

4.3 Activation of T. molitor immune system

The immune system activation procedure was performed according to a method previously described by Konopińska et al. (7). To activate different signalling pathways involved in immune system activation, insects were injected with 2 μL of a suspension of lyophilized E. coli K12 (Ec, 1 mg/mL, OD₆₀₀ = 1; Sigma Aldrich, Saint Louis, MO, USA) or peptidoglycan of S. aureus (PG, 1 mg/mL, OD₆₀₀ = 1; Sigma Aldrich, Saint Louis, MO, USA) in physiological saline (PS, 128 mM NaCl, 18 mM CaCl₂, 1.3 mM KCl, 2.3 mM NaHCO₃). Moreover, to evaluate the effects of cytokines, the TmSpz-like protein was also used. The protein was synthesized by Biomatik (Kitchener, Canada) on the basis of available sequences (15) and transcriptomic data (https://www.ncbi.nlm.nih.gov). Owing to the EC₅₀ value, the TmSpz-like solution in PS was injected at a concentration of 10^-7 M, which effectively activated the Toll pathway while avoiding toxicity and nonspecific effects (39). As the total volume of Tenebrio haemolymph is approximately 18 µL, the final concentration in the insect’s body was 10^–8 M (40). Controls were injected with PS. After 3, 6, or 24 hours, the brain, VNC, haemolymph, and fat body were isolated. The time variants were selected on the basis of previous studies and the available literature (7, 14).

4.3.1 Survival experiment

The survival experiments were performed as described by Urbański et al. (6) After the injection of immune activators, 10 males were kept in a plastic box for 21 days. The beetles were kept under the same conditions as the other individuals used in this study. The number of dead and living individuals was checked every day during the experiment. Dead individuals were removed from the boxes. Ten individuals kept in plastic boxes were considered one biological replicate. Each research variant was repeated at least five times (5 × 10 individuals = 50 beetles per treatment).

4.3.2 Expression levels of tested genes after activation of the T. molitor immune system

At selected time points after activation of the Tenebrio immune system, the brain, VNC, haemolymph, and fat body were isolated from each individual and transferred to separate Eppendorf tubes containing 200 µL of lysis buffer (Zymo Research, Irvine, CA, USA). The collected samples were frozen in liquid nitrogen and stored at -80°C. Before RNA isolation, the samples were homogenized for 1 min via a pellet homogenizer (Kimble Chase, Vineland, NJ, USA). RNA isolation was performed with a Quick-RNA Mini-prep kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. To eliminate DNA residues, the samples were subsequently incubated with a Turbo DNA-free kit (Thermo Fisher Scientific, Waltham, MA, USA). The quality and quantity of the obtained RNA were analysed with a DS-11 spectrophotometer (DeNovix, Inc., Wilmington, DE, USA).

cDNA was synthesized from equal concentrations of RNA templates (100 ng for brain, VNC, fat body, and 50 ng for haemocytes) via a LunaScript^® RT SuperMix Kit (New England Biolabs, Ipswich, MA, USA). Additional quality control (no RT control) was also performed. For RT–qPCR analysis, the expression levels of genes encoding TRP precursor and TRPR were analysed. Moreover, in both experiments, the expression levels of selected immune-related genes were estimated. The primers used in the experiment were synthesized by the Institute of Biochemistry and Biophysics of the Polish Academy of Science (Warsaw, Poland). To confirm our results, the amplicons were sequenced by the Molecular Biology Techniques Laboratory (Faculty of Biology, Adam Mickiewicz University, Poznań, Poland) and compared with data available in a public database (https://www.ncbi.nlm.nih.gov). The expression of selected genes was normalized on the basis of the expression level of the gene encoding T. molitor ribosomal protein L13a (TmRpL13a) (41). The sequences of the primers used are available in Supplementary Table S1. The efficiency of the primers used ranged from 92–108%. RT–qPCR analysis was performed on a QuantStudio™ 3 Real-Time PCR System, 96-well, 0.2 mL (Applied Biosystems™, Waltham, MA, USA) and a Corbett Research RG-6000 Real-Time PCR Thermocycler (Qiagen, Hilden, Germany). Three biological replicates and at least two technical repeats were used for each repeat. In one biological replication, tissues from at least 20 individuals (for the brain, VNC, and haemolymph) or 10 individuals (for the fat body, owing to its specific structure) were collected for each sample. The relative expression was calculated via the method described by Plaffl (42).

4.3.3 Immunolocalization of TRP precursor in the nervous system after activation of the immune system

Analysis of changes in the distribution and abundance of the TRP precursor after immune system activation was performed according to the method described by Urbański et al. (43). The immune system of the insects was activated as described in section 2.4. The control was PS-injected individuals. After 3, 6, and 24 h, the brain and VNC were isolated. The isolated structures were left in 2% paraformaldehyde for 24 h. Then, the samples were washed (4 × 1 h) in phosphate-buffered saline (PBS) and incubated overnight at 4°C in a solution containing 4% Triton-X 100 (PBSTX) (Sigma–Aldrich, St. Louis, MO, USA), 2% normal goat serum (NGS, Jackson ImmunoResearch Lab., West Grove, PA, USA) and 2% bovine serum albumin (BSA, Sigma–Aldrich, St. Louis, MO, USA). Next, they were incubated with primary antibodies (1:500, anti-Aedae-TRP-2, gifted from Prof. J. Veenstra) (44) with 2% NGS, 2% BSA, and 0.4% Triton-X at 4 °C for 72 hours. The samples were washed again in PBSTX (4 × 1 h), and then secondary antibodies in PBSTX (1:200) were added and incubated for 24 h at 4°C in the dark. The samples were washed with PBS (4 × 15 min) and sealed on a microscope slide in 90% glycerol with DABCO. In addition, two negative controls were made, one without the primary antibodies and one without secondary antibodies. At least two biological replicates were performed for each variant. The samples were analysed with a confocal microscope (LSM 510, Axiovert 200 M, Carl Zeiss, Oberkochen, Germany). The samples related to the selected time points were collected at the same time, including the control individuals. Additionally, sample examination was performed on the same day under the same conditions and microscope setup, which allowed comparison of the obtained micrographs. For a detailed analysis of the tested parts of the Tenebrio nervous system, each of the structures was examined via a Z-stack module. Z-stack files based on the fluorescence intensity (threshold 75–255) were used for 3D visualization of the distribution and abundance of fluorescence-positive signals related to the presence of the TRP precursor. 3D visualization was performed with AMIRA 3D software (Thermo Fisher Scientific, Waltham, MA, USA).

4.4 Effects of Spantide II and dsRNA on selected immune mechanisms of T. molitor

4.4.1 Antagonist of TRPR – Spantide II

Spantide II (Sigma–Aldrich, St. Louis, MO, USA) was chosen for its higher affinity for NK1 and insect TRPR than Spantide I and Spantide III (45). On the basis of the literature data, in the experiment, insects were injected with a solution of Spantide II in PS at a concentration of 10^–3 M. On the basis of the literature concerning the total haemolymph volume of adult T. molitor (approx. 18 μL), the final concentration of Spantide II was 10^–4 M (40, 46). In the experiment with Spantide II, the effect of Tenmo-TRP-7 (amino acid sequence: MPRQSGFFGMRa) on selected immune bioassays was also tested as an internal control of the effect of TRP signalling activation. Tenmo-TRP-7 was synthesized by Creative Peptides (Shirley, NY, USA; purity >95% HPLC) and was used at concentrations of 10^–5 M in PS. The concentrations were chosen on the basis of literature data and our previous studies (4, 12). Moreover, simultaneous injection of Tenmo-TRP-7 and Spantide II was also evaluated. Previously, the strongest immunomodulatory effect of Tenmo-TRP was observed 24 hours after its injection. For this reason, the effects of Spantide II and Tenmo-TRP-7 were evaluated at this time point (4, 12). The negative control was injected with PS.

4.4.2 Synthesis of specific dsRNA-targeted genes encoding TRP precursor and receptor

dsRNAs directed against the precursor and TRP receptor were synthesized via a modified method described by Zanchi et al. and Keshavarz et al. (47, 48). The first step of specific dsRNA synthesis was the extraction of total RNA from adult T. molitor and larvae of Galleria mellonella (control) with the Quick-RNA Mini-prep Kit (Zymo Research, Irvine, CA, USA). As an internal control, dsRNA based on G. mellonella lysozyme cDNA, which has no sequence homology with any known gene of T. molitor, was used. The prepared samples were subsequently incubated with a TurboDNA-free kit (Thermo Fisher Scientific, Waltham, MA, USA). The RNA concentrations and quality were checked with a DS-11 spectrophotometer (DeNovix, Inc., Wilmington, DE, USA). The cDNA was subsequently synthesized on an RNA template via the LunaScript^® RT SuperMix Kit (New England Biolabs, Ipswich, MA, USA), and the quality of the RNA was checked via a no-RT control. The fragments were amplified via PCR with the KAPA2G Fast ReadyMix PCR Kit (KAPA Biosystems, Sigma–Aldrich, St. Louis, MO, USA) via a template derived from synthesized cDNA. The primer sequences are available in Supplementary Table S1. To check the product quality, the obtained amplicons were analysed via electrophoresis via a 2% TAE agarose gel with ethidium bromide. Next, the samples were cleaned with the PCR/DNA Clean-Up DNA Kit (EURx, Gdańsk, Poland), and dsRNA synthesis was initiated via the HighYield T7 RNA Synthesis Kit (Jena Bioscience GmbH, Jena, Germany) according to the manufacturer’s protocol. The incubation time at 37°C was extended to 4 hours. To remove DNA residues, the samples were additionally incubated with a TurboDNA-free kit. For RNA hybridization, the samples were heated to 95°C and left in a thermocycler to cool overnight. The synthesized dsRNA was then washed with a 5 M NH₄OAc (Thermo Fisher Scientific, Waltham, MA, USA) and ethanol gradient (Bioultra, molecular grade) (70%-99%). The dsRNA pellet was then resuspended in nuclease-free water and kept at -20°C until further use. For the suppression of the targeted genes, beetles were injected with 2 μg of prepared dsRNA (1 μg/μL in 2 μL of water). For double-knockdown, beetles were injected with 4 μg of prepared dsRNA (2 μg/μL of dsRNA GmLys in 2 μL of water) as a control or with 4 μg of a mixture of dsRNA-targeted genes encoding TRP precursor (2 μg) and receptor (2 μg).

Before the experiments, the knockdown efficiency was estimated by extraction of total RNA (n=3, pools of three adults per day) at different time points after exposure (Figure 6). The samples were then processed as described above. The relative expression was evaluated via RT–qPCR as described in section 4.3.2. On the basis of the obtained results, the day on which the studied genes were silenced was selected.

Figure 6

Figure 6. Efficiency of TRP and TRPR knockdown at selected time points. (A, B) – Single knockdown of TRP (A) and TRPR (B) at 8th day after dsRNA injection. (C, D) – Expression of TRP (C) and TRPR (D) 7 days after simultaneous injection of dsRNA-targeted genes encoding TRP precursors and TRP receptors (2 KD, double knockdown). Control – The mRNA quantity of TRP and TRPR were measured in relation to Galeria melonella lysozyme (GmLys) as a negative control by RT-qPCR. The values are presented as the means ± SDs; *p ≤ 0.05, **p ≤ 0.01.

For the double-injection variant, individuals were injected with dsRNA directed against the gene encoding Galleria lysozyme or TRPR. After 8 days, the insects were injected with physiological saline or Tenmo-TRP-7 at a concentration of 10–⁵ M. The control group was injected with dsGmLys and physiological saline.

4.4.3 Survival experiment after injection of Tenmo-TRP-7, Spantide II or dsRNA

A survival experiment involving the different variants with Tenmo-TRP-7, Spantide II and dsRNA treatment was performed according to the methods described in section 4.3.1.

4.4.4 The expression levels of immune-related genes after the modulation of TRP signalling

The determination of changes in the expression levels of immune-related genes after the modulation of TRP signalling via Spantide II and dsRNA in the fat body and haemocytes was performed according to the methods described in section 4.3.2.

4.4.5 The activity of phenoloxidase in T. molitor haemolymph after the modulation of TRP signalling

Phenoloxidase activity was measured via a modified method published by Sorrentino et al. and Urbański et al. (49, 50). A haemolymph sample (1 µL) was transferred to a paper filter (Whatman No. 52, Sigma–Aldrich, St. Louis, MO, USA) soaked with DL-DOPA (Sigma–Aldrich, St. Louis, MO, USA) solution (2 mg/1 mL) in 10 mM phosphate buffer. Two technical repeats were performed for each individual. At least 10 biological replicates were performed in each group. The samples were then incubated for 30 min in the dark. After drying, they were scanned with a SHARP AR 153 EN (600 dpi, 8 bits, grayscale) and analysed via ImageJ software (version 2). For each sample, a ‘mean pixel value’ was measured in its central part (40 pixels). To better visualize the changes occurring after the application of the neuropeptide under study, the results are presented as a percentage of change relative to control individuals.

4.5 Survival assessment following TRP system suppression and immune activation with E. coli

Insects were injected with physiological saline, Tenmo-TRP-7 at a concentration of 10^–5 M, or Spantide II at a concentration of 10^–3 M. After 2 hours, the immune system was activated. E. coli was chosen to trigger the immune response because of its strong impact on T. molitor survival. Additionally, to examine how the simultaneous silencing of TRP and TRPR affects the ability of the beetle to survive immune system activation, 8 days after dsRNA injection, the insects were injected with E. coli. The rest of the experiment followed the procedure described in section 4.3.1.

4.6 Statistical analyses

Statistical analysis was performed via GraphPad Prism 9 software (Adam Mickiewicz University licence). The outliers were identified via the ROUT method. The normality of the distribution was tested via the Shapiro–Wilk test. Depending on the number of tested research variables, results consistent with the normality of the distribution were analysed by one-way ANOVA with Dunnett’s post hoc test or Student’s t test (depends on the number of analysed groups). The results with a nonnormal distribution were analysed with the Kruskal–Wallis test with Dunn’s post hoc test and the Mann–Whitney U test (depends on the number of analysed groups). The correlation of the data was analysed via the Pearson correlation coefficient method in SRplot software (https://www.bioinformatics.com.cn/srplot). In the manuscript, only statistically significant correlations were described. Survival curves were estimated on the basis of the Kaplan–Meier estimator. The differences between survival curves were calculated via the Gehan-Breslow-Wilcoxon test.

Data availability statement

Data associated with the research are available on Mendeley Data (10.17632/cygy9j9ty7.2), https://data.mendeley.com/datasets/cygy9j9ty7/2.

Author contributions

NK: Writing – review & editing, Writing – original draft, Formal analysis, Visualization, Data curation, Conceptualization, Methodology. KW: Formal analysis, Data curation, Writing – review & editing. GN: Methodology, Writing – review & editing. MK: Methodology, Writing – review & editing. SC: Writing – review & editing, Methodology, Data curation. JR: Writing – review & editing, Conceptualization. AU: Supervision, Data curation, Methodology, Visualization, Formal analysis, Conceptualization, Funding acquisition, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This research was supported by Grant No. 2021/41/B/NZ9/01054 from the National Science Centre (Poland). Natalia Konopińska is a scholarship holder of the Adam Mickiewicz University Foundation for the academic year 2025/2026.

Acknowledgments

Special thanks go to Prof. J. A. Veenstra for sharing the anti-TRP antibodies. We would also like to thank Prof. Guy Smagghe for his valuable comments during data interpretation, and Natalia Bylewska, Radosław Gmyrek, Karolina Kozłowska, and Sara Tchórzewska for their help in rearing T. molitor. During the preparation of this work, the authors used Rubriq software (American Journal Experts (AJE)) for language correction. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Conflict of interest

Author GN was employed by genXone S.A.

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that Generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2026.1725225/full#supplementary-material

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