Arachnid venoms are known to be complex chemical cocktails comprised of a mixture of small inorganic and organic molecules, large proteins and enzymes, linear peptides, and cystine-rich peptides (Lüddecke et al., 2022). These components exhibit various functions such as cytolytic, antimicrobial, and insecticidal activities, making them valuable for bio-insecticide and bio-therapeutic discovery (Saez et al., 2010; Windley et al., 2012). Venom peptides usually target various voltage-gated (e.g. sodium, calcium or potassium) or ligand-gated (e.g. acid-sensing) ion channels, glutamate receptors and transient receptor potential channels) as well as other signaling pathways in the central nervous system and neuromuscular junctions (De Lima et al., 2007; Lüddecke et al., 2022; Saez and Herzig, 2019). To assess the potential of venom peptides for therapeutic or agricultural applications, we need to understand how they access their targets, e.g. via traversing biological barriers such as the blood-brain barrier (BBB) or the digestive tract epithelium. Ultimately, this process involves the crossing of cell membranes, which are comprised of a lipid bilayer that compartmentalizes cells (Harayama and Riezman, 2018).

As arachnid venoms are injected into their prey or predators, there was no evolutionary drive to develop oral activity. Nevertheless, some arachnid venom peptides demonstrated oral activity against arthropods (Hardy et al., 2013; Monfared et al., 2022; Mukherjee et al., 2006). The astounding physico-chemical stability of arachnid venom peptides provided by disulfide-rich knottin structures like the inhibitor cystine knot (ICK) motif certainly contributes to their survival in harsh environments like the insect midgut (Pallaghy et al., 1994). However, surviving the midgut is not sufficient for oral activity, the insecticidal peptides also need to traverse epithelial cells lining the insect midgut. Oral activity varies between peptides and insect taxa and only the combination of stability within the midgut and an efficient means of traversing the midgut epithelium will enable oral activity. Although numerous studies recorded the information of protein absorption (hemoglobin, albumin, IgG) in both blood-feeding and non-blood-feeding arthropods (Jeffers and Michael Roe, 2008), very little is known about peptide movement across the insect midgut (Jeffers et al., 2005). The accumulation of orally delivered snowdrop lectin (Galanthus nivalis agglutinin: GNA) was demonstrated in the hemolymph of the lepidopteran Lacanobia oleracea and the hemipteran Nilaparvata lugens (Fitches and Gatehouse, 1998; Fitches et al., 2001; Gatehouse et al., 1998), promoting the application of GNA as vector for insecticidal peptides (Fitches et al., 2002). Common techniques for studying protein and peptide movement across the digestive tract include ELISA, western blots, and immunohistochemistry, comparing the concentration of protein/peptide between midgut and hemolymph (Jeffers and Michael Roe, 2008). In vitro membrane penetration assays using Ussing chambers combined with mass spectrometric detection are also frequently employed to determine peptide quantities on both sides of the midgut epithelium (Denecke et al., 2018; Herzig et al., 2018).

Besides oral insecticidal activity for agricultural applications of arachnid venom peptides, some therapeutic applications might necessitate delivery into the central nervous system (CNS) of vertebrates. The BBB in vertebrates shields the CNS from neurotoxic substances entering the circulatory system, which complicates the delivery of CNS-active drugs into the brain and spinal cord. Traditionally, CNS-active drugs are administrated by invasive delivery strategies, such as intrathecal injection, intraventricular drug infusion or local intracerebral implants (Temsamani et al., 2001). However, due to the inherent risks of physically breaching the BBB, the development of non-invasive therapeutic strategies has received increasing attention. This includes identifying BBB-penetrating peptides or vector-mediated endogenous transport pathways such as carrier or receptor-mediated transport (Zhou et al., 2021). Based on previous data, only 2% of lipophilic small molecules have the capacity of crossing the BBB to reach their therapeutic targets, and nearly 98% of small molecules (<500 Da) and 100% of large molecules including peptides and proteins fail to penetrate the BBB without effective delivery systems (Zhou et al., 2021; Zou et al., 2013). Cell-penetrating peptides, such as rabies virus glycoprotein (RDP) (Fu et al., 2012) and human immunodeficiency virus-1 (HIV-1) (Frankel and Pabo, 1988), are considered efficient carriers for CNS drug delivery (Zou et al., 2013), albeit with distinct mechanisms (El-Andaloussi et al., 2005). Cell penetrating activity is also employed by some venom peptides, although the underlying mechanism remains unclear.

Considering the significant potential of arachnid venom peptides in pharmacology and agronomy, the mechanism of how they cross biological membranes deserves further attention. The present review will therefore shed some light on the capacity and characteristics of arachnid venom peptides penetrating biological membranes with a detailed focus on the underlying mechanisms.

Cell membranes consist of a lipid bilayer, which is a biological membrane composed of two layers of lipid molecules, each containing a hydrophilic head and a hydrophobic tail. Mammalian membrane lipids mainly include phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol, phosphatidylserine (PS), glycolipids, and sphingolipids (Cockcroft, 2021). In contrast, the membrane composition in insect cells differs significantly from that of mammalian cells. Insect cells have shorter fatty chains: a higher content of PE and phosphatidylinositol (PI) and lower levels of PS, glycolipids, sphingolipids, and cholesterol (Alberts B and Lewis, 2002). Additionally, insect cell membranes contain a lower saturation of fatty acid chains. It is generally believed that insects have different membrane lipid compositions to adapt to lower body temperatures (Marheineke et al., 1998).

Cell-penetrating peptides (CPPs) have been shown to cross cell membranes either via endocytosis or direct permeation (Jiao et al., 2009). Most known CPPs are typically rich in positively charged residues like arginine and lysine (Duchardt et al., 2007; Skotland et al., 2015; Trabulo et al., 2010). The guanidinium group in arginine and the amine group in lysine play important roles in interacting with negatively charged surfaces on the cell membrane, as well as forming hydrophobic alpha helical structures in membrane bilayers. Cationic CPPs can enter the cell by directly penetrating cell membranes or by triggering endocytosis-independent uptake (Green et al., 1989; Nakase et al., 2004). In case of transfer by endocytosis, CPPs are packed into endosomes and must escape from endosomes before being transported to lysosomes for degradation. The high content of cationic amino acids in CPPs was shown to modify the cytomembrane pH, resulting in endosomal escape of CPPs (Magzoub et al., 2005; Takechi-Haraya and Saito, 2018). Amphipathic peptides comprise another group of CPPs, which translocate through the cell membrane by forming multi-peptide complexes with the hydrophobic domains facing outward and the hydrophilic domains facing inward, resulting in direct translocation through the bilayer (Deshayes et al., 2006).

The efficiency of cationic CPP entry is largely influenced by the lipid composition of the membrane (Islam et al., 2018). Experiments using a mouse endothelioma cell line revealed that longer fatty acid chains and higher cholesterol content result in less stable endosomes, leading to a reduced rate of CPP entry (Islam et al., 2018). Therefore, the shorter fatty acid chains in insect cell membranes may lead to higher permeability for CPPs compared to mammalian cell membranes. However, further experiments are required to confirm how the lipid composition in insects might affect the selectivity and efficiency of CPPs.

The vertebrate blood-brain barrier (BBB) is composed of endothelial cells which form the walls of the capillaries, surrounded by a second layer of the endothelial basement membrane and a third layer of the astrocytic glia limitans ( Figure 2 ) (Abbott et al., 2010). The BBB plays a critical role in maintaining a stable fluid microenvironment for the vertebrate central nervous system (CNS) by protecting it from macromolecules and neurotoxic substances, by segregating neurotransmitters into separated pools, by regulating the concentration of specific ions and by facilitating the absorption of necessary nutrients and metabolites for nervous tissue (Abbott et al., 2010). Tight junctions significantly restrict the permeability of ions and polar solutes and block macromolecules from transversing via the paracellular diffusion pathway between endothelial cells from the blood to the brain fluid (Abbott et al., 2010). Lipid-mediated free diffusion is a common pathway for lipid soluble small molecule drugs with a molecular weight less than 400 Da to cross the BBB, while other water-soluble drugs may be transported within the BBB via carrier-mediated transport (CMT) system (Pardridge, 2012). In comparison, transcytosis via endocytic mechanisms provides the main route for proteins and peptides entering the CNS, which can be further divided into receptor-mediated transcytosis (RMT) and adsorptive-mediated transcytosis (AMT) (Abbott et al., 2010). In some particular pathological conditions, such as inflammation, the permeability of the BBB could be increased (Zhao et al., 2022).

Protection of neural tissue from disruption by toxins and other harmful substances forms the basis of the abundance and diversity of blood-brain barrier (BBB) types across different taxa (Dunton et al., 2021). While the traditional view is that the vertebrate BBB evolved from the invertebrate BBB, it is far more likely that these barriers developed convergently with different morphological structures and similar functions (Dunton et al., 2021). All four major arthropod subphyla (Hexapoda, Myriapoda, Chelicerata and Crustacea) have some form of BBB with further morphological disparity but generally containing the neural lamella, the perineurium, and the subperineurium ( Figure 2 ) (Dunton et al., 2021). The neural lamella is a basement membrane of connective tissue, sitting between insect hemolymph and the perineurium (Dunton et al., 2021). The perineurium is a discontinuous layer of perineurial glia cells lined by gap junctions, which supports the neural lamella maintenance (Dunton et al., 2021). Overlayed by the perineurium, the subperineurium is formed by 4-6 sided polyploid subperineurial glia cells and their adjoining junctions (septate junctions, SJs) and functions as barrier, obstructing neurons from ions, molecules and polar solvents and blocking paracellular passage to neurons (Dunton et al., 2021; Limmer et al., 2014). The SJs can lead to increased resistance of insects against pesticides, as Drosophila larvae with higher expression of SJs showed higher resistance to insecticides from the Avermectin class (Chen et al., 2023).

The insect glia cells that are located between the subperineurium and neurons share many morphological and functional similarities with mammalian glia cells, albeit the neurons of insects and mammals are quite different (Spong et al., 2016). For example, insect neurons can withstand long periods of oxygen deprivation, allowing insects to survive adverse environmental conditions (Spong et al., 2016). Insect axons also lack myelination, although this does not apply to all other invertebrates, and the main excitatory transmitter of insect neurons is acetylcholine as compared to glutamate in vertebrates (Spong et al., 2016). The majority of arthropod BBB functional analysis was performed in the 1970’s and 80’s and largely focused on Drosophila (Dunton et al., 2021). However, Drosophila are somewhat unique in that pleated-sheet SJs are the dominant junction type and they lack tight junctions in their subperineurium (Dunton et al., 2021). Other Diptera, such as Musca (house fly) and Calliphora (blowfly) have both septate and tight junctions, while the lepidopteran Manduca sexta (hawkmoth) only have tight junctions (Dunton et al., 2021).

The insect digestive tract consists of three regions: foregut, midgut, and hindgut, each with distinct features (Caccia et al., 2019; Li et al., 2018). The midgut, as shown in Figure 2 , is comprised of two parts, gastric caeca and ventriculus, and is vital for digestion and nutrient absorption (Li et al., 2018). Gastric caeca are finger-like projections in the initial midgut section, serving as additional sites for digestion and absorption. Ventriculus is the primary digestion and absorption site, lined with columnar cells featuring apical microvilli for secretion and scattered goblet cells for pH regulation. Goblet cells are abundant in lepidopteran larvae midguts, maintaining an alkaline environment to neutralize plant toxins (Berenbaum, 1980; Pentzold et al., 2014). Besides, endocrine cells also intercalate in columnar cells, and are responsible for secreting bioactive peptides (Caccia et al., 2019). In some insects, the peritrophic matrix (PM) is present that serves as a non-cellular barrier surrounding the inner side of the midgut, protecting the epithelium from mechanical and chemical damage and pathogen infection and improving the absorption of diluted nutrients (Lehane, 1997). The midgut is also believed to be the major site for penetration by most insecticidal compounds and orally active arachnid venom peptides (Chapman, 1998).

Spiracles in the insect integument have been reported as an alternative route for insecticidal compounds to reach the insect hemolymph and CNS (Sugiura et al., 2008; Sumita et al., 2016). Spiracles are the external openings of the trachea located in the insect exoskeleton and thereby provide a means for entry into the insect respiration system ( Figure 3 ). For peptides to penetrate the spiracles, they first need to cross a filter apparatus composed of cuticular setae that prevents the entry of dust and the loss of water. This is followed by the atrium and the delicate valve lids protected by the cuticular frame (Wasserthal and Fröhlich, 2017). The valve lids are lateral folds extending from the exterior tracheal wall, connecting a larger tracheal space (vestibulum) which then further splits up into a dorsal and a ventral tracheal trunk, a network of tracheoles and eventually terminal tracheoles, where the aerosol gets into contact with the hemolymph (Burrows, 1980; Hayashi and Kondo, 2018; Wasserthal and Fröhlich, 2017). The mesothoracic spiracles are likely the primary entry sites for some insecticides such as pyrethroids, as it provides the quickest route for insecticides to the CNS (Sumita et al., 2016). The commercialized spider venom peptide insecticide Spear^®-T was designed as a contact foliar spray and is presumed to enter the insects through their respiratory system (King, 2019; Sukiran et al., 2023).

CPPs usually consist of positively charged amino acids, such as lysine (K) and arginine (R), and have a considerable level of amphipathicity and cationicity, which provides CPPs with high affinity for the negatively charged lipid membranes (Rádis-Baptista, 2021). Previous research has demonstrated that cell surface binding for arginine-rich CPPs is more efficient than for lysine-rich CPPs (Amand et al., 2008, 2012; Pan et al., 2021a). Additionally, Chen et.al, found that arginine-rich peptides are able to penetrate insect cell membranes efficiently (Chen et al., 2012). However, although arginine residues dominate in most cationic non-arachnid CPPs, arachnid venom CPPs on the other hand preferentially utilize lysine over arginine residues (Madani et al., 2011). Additionally, most arachnid-derived BBB penetrating peptides and midgut penetrating peptides also contain more lysine than arginine residues, with only a few exceptions such as the scorpion peptide chlorotoxin, and the spider peptides Sp1a, JFTX- 23 and GS‐ω/κ‐HXTX‐Hv1a. Overall, lysine plays a dominant role in 18 of the 23 membrane-crossing arachnid peptides listed in Table 1 . The underlying reason for the prevalence of lysine in these peptides, however, remains unclear. One potential explanation is that, although the absorption of arginine-rich peptides is higher than that of lysine-rich peptides during endocytosis, lysine-rich peptides are more potent in causing liposome leakage. This increased leakage allows lysine-rich peptides to more easily reach the hemolymph compared to arginine-rich peptides (Strömstedt et al., 2009). Additionally, lysine residues may contribute to the toxicity of arachnid venom peptides. For example, lysine substitutions can enhance the activity of scorpion antimicrobial venom peptides, which are the critical defensive peptides in host innate immunity (Li et al., 2022). Another possible explanation could be due to the differences in composition of invertebrate vs. vertebrate membranes (Alberts B and Lewis, 2002; Marheineke et al., 1998), which lysine or arginine being more effective in either one or the other type of membrane.

Besides the distribution of positively charged amino acids, the unique disulfide-rich secondary structure is also believed to contribute to the activity of the venom neurotoxins, providing toxins with extraordinary thermal and chemical stability (Herzig and King, 2015). All the BBB-penetrating peptides in Table 1 are disulfide-rich, providing them with high enzymatic stability in vertebrate serum and improving their half-life in the CNS (Saez et al., 2010). Nevertheless, 3 of 8 CPPs and 2 of 8 orally active peptides ( Table 1 ) are linear peptides with no disulfide bonds. Unfortunately, in vitro experiments to determine the membrane-permeating ability of venom peptides are generally conducted in the absence of proteases, renal clearance or off-target binding. It therefore remains to be determined whether the membrane-permeating peptides listed in Table 1 are also suitable in vivo. For orally active peptides, stability towards enzymatic degradation is crucial because they are required to survive for a sufficient time in both insect midgut and hemolymph. Surprisingly, two linear orally active peptides, lycotoxin I variant and checacin1 exhibited moderate oral insecticidal activity. Both linear orally active peptides not only show insecticidal activity but also antimicrobial/cytotoxic activity as pore-forming peptides (Krämer et al., 2022; Yan and Adams, 1998). Therefore, it is possible that the oral activity of the lycotoxin I variant and checacin1 is due to their pore-forming ability in midgut epithelial cells. Apart from these two linear pore-forming peptides, the other six orally activity peptides are all disulfide-rich (five of them being ICK peptides).

High content of lysine and arginine are crucial for enhancing the cell membrane permeability of cationic CPPs. Therefore, poly-lysine or poly-arginine tags have been suggested as carrier vectors to facilitate the permeability of cargo through the cell membrane (Chen et al., 2012; Pan et al., 2021a; Yaroslavov et al., 2003).

To enhance midgut penetration rate of the arachnid venom peptides, delivery vectors with well-known membrane penetration mechanisms have been used such as carrier proteins (i.e. GNA) (Fitches et al., 2004) and insect-specific entomopathogens (e.g. viruses: luteoviruses or baculoviruses; fungi: Beauveria or Metarhizium) (Bonning et al., 2014; Rajput et al., 2023). The big advantage of insect-specific entomopathogens is their phyletic selectivity which limits their effects to the targeted pests (e.g. the fungus Metarhizium acridum is selective for grasshoppers of the family Acrididae) (Herzig et al., 2014; Lomer et al., 2001). Co-application of venom peptides with Cry toxins isolated from the bacteria Bacillus thuringiensis (Bt) is also commonly used to enhance the oral toxicity, as Cry toxins can directly damage the lining of the gut and form pores, which helps venom peptides to pass through (Pan et al., 2021b). Genetically modified crops expressing Cry toxins have been globally introduced (Gassmann and Hutchison, 2012), and pyramiding of arachnid venom peptides with Cry toxins could be another option delivering peptides into the hemolymph (Moar and Anilkumar, 2007). Nanoformulation provides a new approach for peptide delivery (Wei et al., 2022), as well as a novel nano-vehicles containing only viral coat protein without genetical materials, which is considered safer for transporting insecticidal peptides compared to the intact virus (Xue et al., 2024).

The BBB permeability can also be enhanced by using different delivery vectors, such as cationized albumin that reaches the brain via adsorptive-mediated endocytosis and mAb OX26 that recognizes transferrin receptors expressed on brain capillaries (Temsamani et al., 2001). Nanoparticles, such as liposomes are also considered an attractive vector because of their unique physicochemical characteristics compatible with hydrophilic, lipophilic, and hydrophobic therapeutic agents for delivery across the BBB (Lai et al., 2013). Cell-penetrating peptides (i.e. R11, TD, TAT, CTP and LNP) can also be employed as carriers via co-engineering with venom peptides for crossing the BBB (Li et al., 2017; Yao et al., 2015; Zou et al., 2013).

The multifaceted potential applications of arachnid venom peptides in pharmacology and agronomy underscore the importance of understanding both their ability and the underlying mechanisms for crossing biological barriers, including cell membranes, the midgut and respiratory epithelium and the BBB. Evidence suggests that cationic arachnid venom peptides permeate cell membranes via the endocytosis pathway. Within the midgut epithelium, venom peptides may utilize either transcellular pathways via endocytosis or paracellular pathways via septate junctions. Similarly, in crossing the BBB, arachnid venom peptides demonstrate diverse mechanisms, including transcytosis via endocytosis pathways. Notably, lysine and arginine residues appear to play significant roles in membrane interactions, with lysine-rich peptides being predominant in arachnid peptides penetrating cell membranes, the midgut epithelium and the BBB. For peptides with no inherent membrane-permeating capabilities, utilizing delivery vectors, such as nanoparticles, entomopathogens or cell-penetrating peptides presents promising strategies to enhance their membrane permeability. Overall, elucidating the intricate mechanisms of arachnid venom peptides in penetrating lipid bilayer membranes not only enhances our understanding of venom biology, but also holds immense promise for the development of novel therapeutic drugs and bioinsecticides.