*3.3. ROS and Arthropod Microbiome*

In this review, microbiome refers to the overall genome of microorganisms in a certain niche, which has been shown to shape the phenome [76,77]. Therefore, attaining a balance between the natural microbiota and potential pathogens is very crucial. One way to maintain this balance is through the dual oxidase (Duox)–dependent ROS generation system [16]. Duox is a protein that mainly functions in the generation of ROS; however, the Duox-ROS pathway remains inactive unless proliferation occurs and bacteria come in contact with the mucosal barrier. In this manner, ROS produced from the Duox pathways attack invading pathogens through the mucosal barrier, particularly by H2O2. These attacks can disrupt the tyrosine phosphorylation network of invading pathogens and, thereby, reduce their fitness. Enterobacteria dominate in the midgut by maintaining gut homeostasis [78–80], and these bacterial species are adapted to survive within blood-feeding arthropods (e.g., ticks) as they are resistant to ROS killing. Since enterobacterial species are ROS-generating bacteria, during blood feeding, the gut environment would favor the growth of these bacterial species, overcoming the possible proliferation of *Plasmodium* and other pathogenic organisms [43,81–83]. Challenge with pathogens, therefore, accelerates the expansion of bacterial populations during blood feeding and they escape the constraints of the Duox system. One way for the natural microbiota to escape the Duox system is by avoiding contact with the gut epithelium. To achieve this, bacteria are encaged in a blood bolus during blood feeding. The formation of a dityrosine network (DTN) on the luminal surface of the gut epithelium also makes it difficult for the soluble immune mediators to penetrate the blood bolus; thus, the microbiota avoids activation of the immune responses [84]. The protective effects of this DTN are not only beneficial for the microbiota but also for the *Plasmodium* parasite [85]. The same DTN is also found in ticks and has been proved to maintain *B. burgdorferi* infection.

Furthermore, in ticks, some pathogenic organisms have already adapted to this strategy by altering their transcription mechanism and ameliorating the antioxidant mechanisms, including selenoproteins, to maintain the favorable levels of ROS that allow for their survival and growth. The knockdown of the *SelP* gene increases oxidative stress, thus, decreasing *Ri. parkeri* loads and increasing levels of *Francisella*-like symbionts such as *Candidatus* Midichloria mitochondrii and other bacteria [43,52].

## **4. ROS and Chemical Control of Arthropods**

The metabolism of xenobiotics including acaricides and insecticides also leads to the generation of ROS [16]. Enzymes involved in the detoxification of these acaricides are also known as antioxidants, including glutathione S-transferase, cytochrome P450 monooxygenases, and some esterases. Higher expression or activities of these enzymes are involved with some resistant strains in both mosquitoes and ticks [44,86,87]. Permethrinresistant strains of *Ano. gambiae* were shown to have higher ROS production rates and the increased ROS production in turn resulted in increased *GST* and *catalase* gene expression [44]. The same observations were reported in DDT-resistant *Ano. arabiensis* and *Ano. funestus,* wherein increased catalase and glutathione peroxidase activity was observed, indicating that resistant strains of mosquitoes need a higher capacity for coping with oxidative stress [88]. During every single blood meal, a transient decrease of oxidative stress was observed in mosquitoes. Increased expression and activity of antioxidant enzymes after multiple bouts of blood feeding is essential to maintain the homeostasis of DDT- and permethrin-resistant phenotypes of mosquitoes [88]. Disruption of the oxidative stress, e.g., elevated oxidative stress or reduced capability of minimizing oxidative stress,

increases the susceptibility of DDT-resistant *Ano. gambiae* to DDT [89]. Additionally, some phytochemicals extracted from plants can augment ROS production. When *Culex quinquefasciatus* is exposed to the extracts of the medicinal plant, *Stachytarpheta jamaicensis,* it can lead to the generation of a high amount of ROS, resulting in the death of their larvae [45]. Plant extracts can decrease the levels of antioxidative enzymes in mosquitoes, for example, phytochemicals reduce the levels of carboxylesterases and superoxide dismutases in *Ae. aegypti* larvae (Figure 2) [90,91]. In the ticks, a negative correlation between GST enzyme expression and different phytochemical concentrations was observed, indicating the ability of these phytochemicals to induce oxidative stress in ticks [92].

**Figure 2.** Schematic diagram on the activity of the phytochemicals and its possible mode of activity to cause mortality. Created with Biorender.com (accessed on 10 May 2022).
