Bioscience Methods 2024, Vol.15, No.6, 302-314 http://bioscipublisher.com/index.php/bm 305 3.2 Mechanism of action of AMPs against bacteria, fungi, and viruses AMPs employ various mechanisms to combat bacteria, fungi, and viruses. The primary mode of action involves the disruption of microbial membranes. This can occur through several mechanisms, including the barrel-stave, carpet, and toroidal pore models. In the barrel-stave model, AMPs insert themselves into the membrane, forming a pore that disrupts membrane integrity. The carpet model involves AMPs covering the membrane surface, leading to membrane disintegration. The toroidal pore model combines elements of both, where AMPs induce curvature in the membrane, forming transient pores (Pasupuleti et al., 2012; Corrêa et al., 2019). In addition to membrane disruption, AMPs can interfere with intracellular targets. For instance, they can bind to nucleic acids, inhibiting DNA, RNA, and protein synthesis. Some AMPs also generate reactive oxygen species (ROS), leading to oxidative stress and cell death. These multifaceted mechanisms make it difficult for pathogens to develop resistance against AMPs (Huan et al., 2020; Dijksteel et al., 2021). 3.3 Gene regulation and expression of AMPs in earwigs The expression of AMPs in earwigs is tightly regulated by the immune system. Upon infection, pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs), triggering signaling pathways that lead to the activation of AMP genes. Key signaling pathways involved include the Toll and Imd pathways, which are analogous to the Toll-like receptor and TNF receptor pathways in mammals. These pathways activate transcription factors such as NF-κB, which bind to the promoter regions of AMP genes, initiating their transcription (Zhang and Gallo, 2016; Shanmugaraj et al., 2021). Environmental factors, such as microbial exposure and stress, can also influence AMP expression. For example, earwigs exposed to bacterial infections show a rapid upregulation of AMP genes, enhancing their ability to combat the invading pathogens. Additionally, epigenetic modifications, such as DNA methylation and histone acetylation, play a role in the long-term regulation of AMP gene expression, ensuring a swift and robust immune response upon subsequent exposures (Patel and Akhtar, 2017; Guryanova and Ovchinnikova, 2022a). In summary, AMPs in earwigs are a vital component of their innate immune system, providing a robust defense against a wide range of pathogens through diverse mechanisms and tightly regulated gene expression. 4 RNA Interference (RNAi) Mechanisms in Pathogen Defense 4.1 Overview of RNAi in invertebrate immunity RNA interference (RNAi) is a crucial antiviral defense mechanism in invertebrates, providing RNA-based protection against viral infections. Unlike vertebrates, which rely on the interferon (IFN) system for antiviral defense, invertebrates utilize RNAi to degrade viral RNAs in a sequence-specific manner. This mechanism is activated by the recognition of viral double-stranded RNA (dsRNA), which is produced during viral replication. The RNAi pathway then processes these dsRNAs into small interfering RNAs (siRNAs) that guide the degradation of viral RNA, thereby restricting viral replication and dissemination (Nayak et al., 2013; Wang and He, 2019). 4.2 RNAi pathways in earwigs and their role in viral suppression In earwigs, as in other invertebrates, the RNAi pathway plays a pivotal role in antiviral immunity. The pathway involves several key steps: the recognition of viral dsRNA, its processing into siRNAs by the enzyme Dicer, and the incorporation of these siRNAs into the RNA-induced silencing complex (RISC). The RISC, guided by the siRNAs, targets and degrades complementary viral RNAs, effectively suppressing viral replication. This mechanism has been observed to be highly effective against various types of viruses, including negative-strand RNA viruses, which do not produce easily detectable amounts of dsRNA but still trigger a potent RNAi response (Mueller et al., 2010; Guryanova and Ovchinnikova, 2022b). 4.3 Molecular regulation of RNAi-mediated defense The regulation of RNAi-mediated defense in earwigs involves a complex interplay of molecular components. Key regulatory proteins include Dicer, which processes viral dsRNA into siRNAs, and Argonaute, a core component of
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