Detailed toxin information is provided in the supplementary materials (Supplementary Tables S4–5). Each transcript annotated as a toxin is discussed based on the existing literature, followed by a categorical description of the toxin.
3.3.1. ICK-like spider venom peptides
Cysteine-rich peptides are the best investigated venom components and are believed to exist in most spider venoms. They modulate a broad range of channels and receptors on the membranes of excitable cells (e.g., nerves and muscles) . Ion channels on the membranes of excitable cells are responsible for proper signal transduction. Calcium channels are involved in neurotransmitter release from presynaptic cells, voltage-gated sodium channels enable action potential transmission along excitable cells, and voltage-gated potassium channels are crucial for restoring a resting state in depolarized cells . Toxins interact with these targets to disrupt normal channel function, which can affect breathing or heart function, leading to symptoms such as convulsions, paralysis, and eventually death. Abundant ion channel toxins were identified by annotating the transcripts of S. pengi and T. clavata. The families of the predicted ICK toxins are described in detail below.
Group I (C-C-CC-C-C)
Group I contained three unigenes in T. clavata, two of which (DN1625_c0_g1, DN16263_c0_g1) were similar to Mu-Sparatoxin Hv2 (UniProt A0A088BP94) and one (DN5056_c0_g1) was similar to Kappa-Sparatoxin Hv1a (UniProt P58425) (Fig. 3A). These reference toxins belong to the neurotoxin 10 (Hwtx-1) family. Mu-sparatoxin-Hv2 from Heteropoda venatoria, is insecticidal toxin potently and irreversibly blocks NaV channels in cockroach dorsal unpaired median (DUM) neurons (IC50=833.7 nM) . NaV channels are crucial in the generation and transmission of action potentials in the central nervous system, peripheral nervous system, heart, smooth muscle and skeletal muscle, so the function of NaV channels is crucial, and they are targets of many toxins. There are nine α subtypes, known as NaV1.1-NaV1.9 in mammals, each with different tissue distributions and functions . The reference toxin Kappa-Sparatoxin Hv1a is a potassium and calcium channel blocker. KV channels play an important role in human physiology, including the regulation of neurotransmitter release, heart rate, insulin secretion, nerve cell secretion, skeletal muscle contraction, etc. Numerous channelopathies arising from mutations in these channels or from autoimmune attack on the channels have been characterized [18–19]. Potassium channels are the target of a variety of toxic animal toxins, and the potassium channel inhibitors found in spider venom thus far mainly act on KV1, KV2 and KV4 channels, although a few act on other channels, such as KV3, KV7 and KV11 channels . In S. pengi, Group I contained 9 unigenes (Fig. 3B), two of them showed homology with kappa-sparatoxin-Hv1a and seven were aligned to Mu-sparatoxin-Hv2.
Group II (C-C-CC-CXC-CXC)
In T. clavata, Group II included 5 sequences (Fig. 4A), correspond to U3-lycotoxin-Ls1b (UniProt B6DCQ7), LSTX-D6 (UniProt B6DCU5), U1-lycotoxin-LS1B (UniProt B6DCK2), U19-Lycotoxin-LS1B (UniProt B6DD58) from Lycosa singoriensis and U8-agatoxin Ao1a (UniProt Q5Y4U4) from Agelena orientalis, respectively. The ion channel activity of these reference peptides is not known.
In S. pengi, Group II is composed of 5 sequences (Fig. 4B). One of them resembled Omega-Segestritoxin-Sf1a (UniProt P58605) from Segestria florentina, another resembled U10-Ctenitoxin-Pr1a (UniProt P84000) and the other three resemble U6-Lycotoxin-LS1g (UniProt B6DCV8) from Lycosa singoriensis. Omega-Segestritoxin-Sf1a is potent and selective blocker of N-type voltage-gated calcium channels (Cav2.2/CACNA1B), also blocks vertebrate Cav2.1/CACNA1A (P/Q-type) and Cav1.2/CACNA1C (L-type) channels at very high concentration (2 micromolar) .
Groups III (C-CC-CC-CC-C-C-C), IV (C-C-CC-C-C-CXC-C-C), V (C-C-CC-C-CC-C-C-C) and Ⅵ(C-C-CC-CXC-CXC-CC)
In T. clavata, Group III contained only one sequence (DN18027_c0_g1) (Fig. 5), which had 66.3% identity with Omega-agatoxin-1A from Agelenopsis aperta. Omega-agatoxins are antagonists of voltage-gated calcium channels, subtypes of omega-agatoxins are distinguished as type I (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IC), type II (omega-Aga-IIA and omega-Aga-IIB), and type III (omega-Aga-IIIA and omega-Aga-IIIB). All except type III toxins block calcium channels in insect motor nerve terminals and in neuronal cell bodies at nanomolar concentrations . No sequence from S. pengi in this family.
In T. clavata, group IV contained 9 unigenes (Fig. 6A), 5 of which displayed similarity to toxin CSTX-20 from Cupiennius salei (UniProt B3EWT5). One of which presented identity to U19-ctenitoxin-Pn1a (UniProt P83997) from Phoneutria nigriventer. Three other sequences were similar to U3-aranetoxin-Ce1a3 (UniProt Q8MTX1) from Caerostris extrusa. In S. pengi, one sequence is similar to U33-theraphotoxin-Cg1b (UniProt B1P1J0) from Chilobrachys guangxiensis and one to U8-theraphotoxin-Hhn1d (UniProt D2Y2E4) from Cyriopagopus hainanus, the other 5 unigenes correspond to CSTX-20 (Fig. 6B).
In T. clavata, group V consisted of 4 unigenes (Fig. 7), two of them showed homology with U20-lycotoxin-Ls1c (UniProt B6DD62) from Lycosa singoriensis. The other two sequences were similar to U15-lycotoxin-Ls1a (UniProt B6DD43) from Lycosa singoriensis and U7-agatoxin-Ao1a (UniProt Q5Y4V9) from Agelena orientalis, respectively. No sequence from S. pengi in this family.
In S. pengi, group Ⅵ contained only one sequence (Fig. 8), the sequence had homology to U16-lycotoxin-Ls1b (UniProt B6DD53C) from Lycosa singoriensis. No sequence from T. clavata in this family.
Group Ⅶ (C-C-C-CC-CXC-CXC-C-C-C)
In T. clavata, Group Ⅶ was represented by only two sequences (DN21003_c0_g1, DN51867_c0_g1), which had identity with U9-agatoxin-Ao1a (UniProt Q5Y4U3) from Agelena orientalis. No sequence from S. pengi in this family (Fig. 9).
Enzymes are very common components of the venom of poisonous animals such as poisonous snakes, scorpions and centipedes. Proteases are enzymes that hydrolyze the amide bonds of the peptide units of polypeptides and proteins. The overall purpose of such an enzyme arsenal co-injected with toxins into a prey’s tissue seems clear: by destroying the barriers imposed by the extracellular matrix and cell membranes, the toxins can quickly reach their targets . Additionally, the proteolytic activity of some of these enzymes facilitates subsequent preoral digestion. As proteins play important roles in the maintenance of homeostasis, proteases are vital regulators of physiological processes. Many pathological conditions of humans and animals have been linked to the malfunctioning of this category of enzymes; as a result, they are seen as attractive targets for drug discovery .
Unigenes related to metalloproteinases were found in S. pengi and T. clavata. The role of metalloproteinases in venoms is not fully understood, but it may be a diffusion factor promoting the diffusion of other venoms. It may be involved in proteolysis of other venomous toxins, or it may aid extra-oral digestion of prey .
In S. pengi, we found unigenes may encoding acetylcholinesterase. Acetylcholine-mediated neurotransmission is fundamental for nervous system function. Acetylcholinesterase (AChE) hydrolyses and inactivates acetylcholine, thereby regulating the concentration of the transmitter at the synapse . In venom, the toxic role of this enzyme is unclear; it could reduce musculatory control by rapidly hydrolyzing acetylcholine, or it works synergistically with alkaline phosphatase to paralyze prey through hypotension.