4.1.6. Effects on the Immune System

As opposed to vertebrates, insects do not have a developed acquired immunity. Instead, they have a robust innate immunity that can be subclassified into cellular and humoral responses [88]. Cellular immunity is characterized by the action of defense cells (hemocytes) in aggregation, phagocytosis and encapsulation processes. The humoral immune response comprises the activation

of antimicrobial peptides, of reactive oxygen species (ROS), and of enzyme complexes that regulate melanization and the coagulation cascade, among them the phenoloxidase (PO) [93,94].

In this context, previous data from our group indicated that JBU and the ubiquitous Soybean Urease (uSBU) are able to induce activation of the insect immune response in *R. prolixus* [48,95]. Defferrari and coworkers demonstrated that JBU is capable of activating both, the cellular and humoral immune responses. The activation of cellular aggregation induced by JBU is mediated by the cyclooxygenase (COX) pathway and required extracellular Ca++ ions. JBU also elicited the darkening of the hemolymph, an immune response associated with the melanization reaction triggered by the PO. At cellular level, immunolocalization assays demonstrated that the toxin is capable of inducing cytoskeleton damage and nuclear condensation in hemocytes [95]. Additionally, Martinelli and collaborators reported that uSBU in vivo and in vitro is also capable of inducing hemocyte aggregation in *R. prolixus* [48].

Based on these studies, cellular and biochemical approaches were carried out in order to evaluate the effects of Jaburetox and Soyuretox on the immune response of *R. prolixus*. Like JBU and uSBU, Jaburetox and Soyuretox induced Ca++-dependent aggregation of hemocytes in vivo and in vitro, mediated by the COX pathway [64,82]. Despite the aggregation, Fruttero et al. and Moyetta et al. demonstrated that the phagocytic capacity of hemocytes is not altered by the toxin [77,82]. In addition, Jaburetox also generated chromatin condensation, cytoskeleton disorganization and caspase 3 activation in the hemocytes, indicating the induction of apoptosis by the toxin [82]. The interaction of Jaburetox with the hemolymphatic cells was also seen upon in vivo and in vitro treatments, and the peptide was found in different subcellular locations [77].

Besides affecting the cellular immune response, Jaburetox also modulates the humoral immunity. In *R. prolixus*, the toxin induced an increment in the PO activity in vivo, without altering the activity of other effectors, such as the antibacterial cecropins and lysozymes [82]. Jaburetox triggered in hemocytes the increment of NOS gene expression. NO produced by the enzyme is known to induce the formation of free radicals that aid in immune defenses. However, these changes in gene expression were not accompanied by the corresponding modifications in protein levels in hemocytes or in enzymatic activity of NOS assayed in vitro, after the exposure to the toxin [77,82]. Through fluorescence assays with specific probes, it was observed that cells aggregated in the presence of Jaburetox had a greater local production of NO [77,82]. In 2020, Grahl et al. demonstrated in cultured hemocytes that a high dose of Jaburetox (6 µM) induced a significant increase of ROS production without altering cell viability [83].

When the Jaburetox-treated insects were injected with the pathogenic bacterium *Staphylococcus aureus*, the bacterial clearance was significantly reduced, indicating an immunosuppressive effect. Thus, the cellular and humoral immune activations triggered by Jaburetox do not protect the insect against posterior bacterial challenges [82]. These responses are similar to those elicited by bacterial and protozoan pathogens, raising the possibility that Jaburetox is recognized by the innate insect immunity as a pathogen-associated molecular pattern.

Another important immune response is the release of extracellular nucleic acid traps [96]. This immune mechanism of vertebrates and invertebrates is characterized by ROS-dependent release of chromatin into the cytoplasm, promoting the association of the nuclear material with antimicrobial proteins. Thereafter, this complex is released to the extracellular medium to withstand infections [96,97]. In this context, considering the changes in gene expression and nuclear condensation induced by Jaburetox in *R. prolixus*, experiments were designed to evaluate the impact of Jaburetox on the interactions of nucleic acids (DNA and RNA) extracted from the same insect species and used to mimic extracellular nucleic acid traps. It was observed that injection of the toxin together with RNA caused an increase in hemocyte aggregation, however when the toxin is injected together with DNA, no aggregation was seen. Concerning humoral responses, Jaburetox plus RNA yielded an increased PO activity only 6 h after injection, while Jaburetox plus DNA sustained an augmented humoral response both at 6 and 18 h after injection [83].

The effect of extracellular nucleic acids on the Jaburetox-induced immunosuppressive effect against pathogenic bacteria was also studied. Immunocompetence assays injecting Jaburetox alone, or Jaburetox plus DNA or RNA before the injection of bacteria, demonstrated that both RNA and DNA counteracted Jaburetox effects, and restored the bacterial-clearance capacity of the insects [83].

Finally, to better understand the immunological modulation caused by nucleic acids, the toxin ability to induce the release of extracellular nucleic acid traps was evaluated. It was seen that Jaburetox was not able to induce the release of RNA or DNA, either upon in vitro or in vivo treatments. The incapacity of the insects to release extracellular nucleic acids after Jaburetox treatment could partly explain the immunosuppressive effect of the peptide and the weakened response of the treated insects against a bacterial challenge [83]. Since *R. prolixus* has been an instrumental model to understand the effects of the urease-derived peptides, we have summarized all our findings in the Figure 3.
