## 4 Discussion
Many protein families depend on Cu, such as chaperones, responsible for control in different systems of the organism, e.g., digestive, reproductive, and nervous systems, with interchangeable functions [58]. Another example of a Cu-dependent metalloprotein is pro-phenyloxidases (PPOs). These tyrosinases aid in the humoral immune system, sclerotization, and melanization of injuries in insects, and their structures contain Cu binding sites. It is known that the application of growth-disrupting insecticides affects the general development of stalk bugs. One of the symptoms of its action is the irregular deposition of chitin in the cuticle and different melanization patterns, such as accentuations in the joints of the legs and color reduction in other regions [59; 60]. While the observed Cu increase in _E. heros_ males might be suggestive of a sex-specific sublethal toxic effect, the correlation between the Cu concentration and immune response warrants further investigation. Although Ni is found in crucial metalloenzymes in many prokaryotes, fungi, algae, and plants [61], it is highly toxic to the mammalian reproductive system [62] and insects. In _Spodoptera litura_, Ni caused a reduction in the spermatic capacity, weight, and volume of the testicles and increased the lipid peroxidation levels analyzed in testicular tissues [63], but it also led to a biphasic immune response, where low concentrations stimulated immune activity, but higher concentrations inhibited it across generations [64]. Some works on phytophagous insects feeding on Ni-hyperaccumulator plants show that these individuals may tolerate and store this micro-element in parts of the organisms [65]. Still, no associated biological functions were observed. Moreover, considering a Ni-hyperaccumulator host plant, adding Ni to the diet of lab-fed mijud bugs, _Melanotrichus boydi_ (Hemiptera: Miridae), did not influence its feeding preference [66]. Despite this, Ni's biological function in insects remains unclear, especially concerning stalk bugs. Our findings suggest the need for future studies to explore Ni accumulation in _E. heros_ and its possible implications, particularly when considering interactions with pyriproxyfen. In addition to the classic mode of action of pyriproxyfen linked to agonism with the juvenile hormone, Zn deficiencies caused in _E. heros_ females and treated males may indicate indirect hormonal dysregulation. Some proteins known as metalloendopeptidatases (MEPs) rely on Zn to cleave insect-specific hormones, such as AKH, which relies on the MEP neprilysin to cleave the Asn3-Phe4 peptide bond [67]. Additionally, Zn (II) is a structural component of G-protein-coupled receptors (GPCRs), sites where this and other peptide hormones bind to trigger specific activation [67; 68]. The observed Zn deficiency suggests it may be a limiting factor in the final nymphal development of _E. heros_. Future research should focus on understanding the role of AKH and other related MEPs in stalk bugs exposed to pyriproxyfen to better correlate these findings. Understanding the balance of element homeostasis in insects is crucial for predicting how environmental changes, such as insecticide exposure and metal pollution, might affect insect health and their interactions with beneficial organisms [69]. Based on the observed data on the dynamics of circulating metabolites and micro-elements in _E. heros_, we presume that pyriproxyfen, directly and indirectly, has activity affecting different physiological routes that might be related to its primary mode of action and other secondary effects. The alterations observed in these components are responses to the functionality of different physiological systems associated with factors such as sex, age, and physiological homeostasis. Further research is needed to explore these dynamics, particularly in the context of pest management and environmental contamination.