The venom secreted by widow spider venom glands is a complex mixture of components with diverse biological functions. Many of them are biologically active proteins and peptides, which play a number of adaptive roles: paralyzing, immobilizing, killing, liquefying prey, and restricting competitors [5]. From early times there have been sporadic reports on the toxicity of the venom of black widow spiders [5,22]. In recent years, the systematic analyses of black widow venoms have deepened our understanding of the spider toxicity. For example, before long, the effect of a Chilean black widow spider (L. mactans) venom on bovine spermatozoa was investigated. The results indicated that the venom increased the Ca²⁺ influx with an EC₅₀ of 6.1 μg/mL and triggered the acrosome reaction in 43.26% of the cells. The application of potassium (10 mM K⁺) or venom (10 μg/mL) did not affect the morphology or DNA stability of the sperm. The effects induced by high K⁺ and venom suggest that direct blocking of K⁺ currents alters the passive properties of the plasma membrane, leading to the entry of Ca²⁺. These results show the importance of functional changes induced by depolarizing the spermatozoa and by the venom. This venom possesses one or more molecules that may be used as pharmacological tools for studies on spermatozoa and have potential applications in reproductive biotechnology [23,24]. When the brown widow spider (L. geometricus) venom was investigated, it was found that the venom damaged the adrenal gland, producing severe alterations on cortex cells and resulting in death by acute adrenal insufficiency. The venom was confirmed to contain fibrinogenolytic and other proteolytic activities, showing specific actions on extracellular matrix proteins such as fibronectin, laminin, collagen type IV, and fibrinogen that might play certain roles in the spider toxicity [25]. Wang et al. [26] employed multiple physiological and biochemical strategies to systematically analyze the electrical stimulation-collected black widow spider (L. tredecimguttatus) venom that was not contaminated with histiocytic proteins and other components and, therefore, was more representative of the pure venom. The venom was demonstrated by gel electrophoresis and mass spectrometry to consist primarily of proteins with molecular masses above 10 kDa, most of which are high-molecular-mass acidic proteins, with fewer proteins and peptides below 10 kDa. The most abundant proteins are distributed around 100 kDa. The venom was demonstrated to be rich in neurotoxins because injection of the venom in mice and P. americana led to obvious poisoning symptoms, with LD₅₀ values of 0.16 mg/kg and 1.87 μg/g, respectively. In addition, the venom could efficiently block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations, and the low-molecular-mass fraction (<10 kDa) of the venom had no obvious effect on the transmission, suggesting that the mammalian toxicity of the venom is primarily based on its larger proteins. Enzymatic analysis indicated that the venom contains multiple kinds of hydrolases, including proteinases, hyaluronidase, alkaline, and acid phosphatases. All of the data demonstrate that the venoms are rich in ion channel modulators and metabolic enzymes, particularly the proteolytic enzymes that can enhance the action of the toxins by breaking down the intercellular reinforcements and basement membrane molecules.

The complexity of black widow spider venom has long been appreciated by researchers in the fields of toxicology and medicine. However, it is the recent advances in protein separation and biological mass spectrometry that allow virtually all venom components to be effectively separated and identified. In 2008, Duan et al. [27] employed a combinative strategy to make a proteomic analysis of the venom collected from living adult spiders (L. tredecimguttatus) by electrical stimulation. A total of 122 non-redundant venom proteins were unambiguously identified, 75 of which had distinct function annotation. Besides the previously reported widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5-like protein, and trypsin inhibitor. These results help to understand the complexity and action mechanism of black widow spider venom. Up until now, the mechanism of action of the black widow spider venom has not been completely clear. It was speculated that the intoxication after a bite by a black widow spider must be related to, besides latrotoxins, other venom components such as hydrolases. Hydrolases have also been identified in the venoms of several snakes and other spider species, and some hydrolases, particularly the proteases such as metalloproteases and serine proteases, were demonstrated to have some participation in the noxious effects of the venoms [28,29,30]. Obviously, the hydrolases and other proteins with special activity in the black widow spider venom should have important potentials in enhancing venom toxicity and extending the venom action scope and model. In addition, there are 47 proteins being matched to predicted protein, hypothetical protein, and protein of unknown function, respectively. These proteins are also potential active components of the venom, which need to be further confirmed.

In an attempt to uncover the dramatic expansion of the black widow toxin arsenal, Haney et al. [31] used a proteomic strategy to determine the venom proteins from the Western black widow spider (L. hesperus). Sixty-one proteins were identified with the mass spectrometry technique from an L. hesperus protein database that matched peptides collected from L. hesperus venom, including 21 latrotoxins, one ICK (inhibitor cystine knot) toxin, and six CRISP (cysteine-rich secretory protein) family toxin proteins. Several types of enzymes were identified in the venom, including hyaluroidases, chitinase, serine proteases, and metalloproteases. These results demonstrate that L. hesperus and L. tredecimguttatus have similarities in the molecular basis of venom toxicity.

Up until now, at least seven different latrotoxins (LTX) have been isolated from the venom of the L. tredecimguttatus spider by means of techniques such as ion exchange chromatography and hydrophobic chromatography [35,36,37] (Table 2). All the studied latrotoxins are large acidic proteins (pI ~5.0–6.0) with molecular masses ranging from 110 to 140 kDa. Most of them are targeted against insects and are called latroinsectotoxins (LITs) (α, β, γ, δ, ε-LIT) [27]. ε-LIT is also highly toxic to C. elegans [38]. α- LTX is the only known venom component that aims specifically at vertebrates [35]. α-Latrocrustatoxin (α-LCT) is active only in crustaceans [37]. To date, the primary structures of four genes respectively encoding four latrotoxins, α-LTX, α-LIT, α-LCT, and δ-LIT, were determined using cDNA or intron-less genomic cDNA [34,39,40,41]. Using electron cryo-microscopy, the three-dimensional (3D) structure of the mature α-LTX monomer was found to contain three regions: the wing (majority of domain II), the body (one quarter of domain II and first 15–16 ankyrin repeats), and the head (ca. 4.5 C-terminal ankyrin repeats) [42]. All the latrotoxins cause massive release of neurotransmitters from the nerve terminals of the respective animals after binding to specific neuronal receptors [5]. In addition, Volkova et al. [43] isolated two low-molecular-mass proteins, LMWP and LMWP2, from black widow spider venom, and the two proteins were inactive to mammals and insects. These low-molecular-mass proteins are called latrodectins and appear to augment the neurotoxicity of latrotoxins, probably by increasing their affinity for the membrane target and reducing vertebrate phyla-specificity such that α-LTX becomes active in insects [8,44,45]. Using phylogenetic analyses and evidence from gene structure, McCowan and Garb [46] showed that latrodectin peptides from black widow spider venom are derived from the ecdysozoan superfamily of neuropeptides containing crustacean hyperglycemic hormones and ion transport peptides. In addition, Akhunov et al. [47,48] studied Kininase and two bradykinin-potentiating peptides of the L. tredecimguttatus venom. They characterized the Kininase as a thiol endopeptidase, which cleaves internal peptide bonds at the proline carboxyl end. The two bradykinin-potentiating peptides prolong depressor effects of bradykinin, stimulate histamine release from cells, and decrease blood pressure in rats. The data available indicate that Latrodectus spp. venoms contain a cocktail of toxins and other biologically active substances; however, many of them wait to be isolated and characterized.

The most studied latrotoxin is α-latrotoxin (α-LTX), which has a molecular mass of about 130 kDa and exerts a lethal effect in vertebrates by inducing a massive neurotransmitter release, acting both in the presence and in the absence of Ca²⁺ [49,50]. The clinical symptoms of latrodectism are mainly due to the presence of α-LTX homologs in the spider venom [51]. Western blotting with polyclonal anti-α-LTX antibody revealed that this 130 kDa band presents in the venoms of five tested Latrodectus species (L. tredecimguttatus, L. hasselti, L. mactans, L. lugubris, and L. hesperus) and these proteins have antigenic similarity. However, Western blotting with the monoclonal antibody derived from L. tredecimguttatus α-LTX found no binding with any proteins in venoms of the other four species, suggesting that there are important structural differences between α-LTXs in the venoms of different Latrodectus species [51]. It is generally accepted that α-LTX is present in the venoms of all Latrodectus species [51,52]. However, it was reported that the venom from the Chilean black widow spider (L. mactans) did not contain α-LTX [53]. This may be incorrect because more recently Garb et al. [4] demonstrated that α-LTX is also present in the venom of the spider by analyzing the molecular evolution of α-LTX. By combining 33 kb of L. hesperus genomic DNA with RNA-Seq, Bhere et al. [54] characterized the α-LTX gene and discovered a paralog 4.5 kb downstream. A 4 kb intron interrupts the α-LTX coding sequence, while a 10 kb intron in the 3′UTR (untranslated region) of the paralog may cause non-sense-mediated decay, which is contrary to previous findings in L. tredecimguttatus that no intron is found in the coding sequence of α-LTX [55]. Phylogenetic analysis confirms these divergent latrotoxins diversified through recent tandem gene duplications. Thus, latrotoxin genes have more complex structures, regulatory controls, and sequence diversity than previously proposed.

It was found that α-LTX creates Ca²⁺-permeable channels in lipid bilayers, which involves toxin assembly into homotetrameric complexes that harbor a central channel [56]. The Ca²⁺ influx through the channels induced by α-LTX in the presynaptic membrane accounts for a large part of its effects. Three proteins in the plasma membrane, neurexin (NRX), latrophilin (LPH or CIRL), and protein tyrosine phosphatase σ (PTPσ), have been found to function as the receptors for α-LTX. After binding a receptor, α-LTX can exert its effects through two major mechanisms: (i) Ca²⁺-dependent action involving α-LTX insertion into the plasma membrane and pore formation; and (ii) Ca²⁺-independent action based on receptor-mediated signaling. The advances in the study on the structure and functions of latrotoxins, particularly α-LTX, greatly improved our understanding of the mechanisms regulating neurotransmitter release. For earlier reviews see Ushkaryov et al. [5,57], Rohou et al. [58], and Silva et al. [59].

Although much effort has been focused on the research of α-LTX, the mechanism of action of α-LTX has not been completely clear and remains the research hotspot in the related field. In order to further probe and distinguish the two action mechanisms of α-LTX, Deak et al. [60] employed a combination of gene knockout, electrophysiology, and the other related techniques to investigate the effects of α-LTX on the synaptic exocytosis of hippocampal neurons and showed that the Ca²⁺-independent release mechanism of α-LTX requires the synaptic SNARE (soluble N-ethylmaleimide-sensitive protein receptor) proteins synaptobrevin/VAMP (synaptic vesicle-associated membrane protein) and SNAP-25 (synaptosomal-associated protein-25), and, at least partly, the synaptic active zone protein Munc13-1. In contrast, the Ca²⁺-dependent mechanism of α-LTX-induced release utilizes a novel pathway of membrane fusion that does not require the classical synaptic fusion machinery, and thus differs from the physiological action potential-induced, Ca²⁺-triggered release pathway. Their data characterize two independently Ca²⁺-triggered pathways of synaptic vesicle fusion at central synapses that likely perform distinct physiological functions.

Of the three specific receptors for α-LTX, neurexin binds to α-LTX only in the presence of extracellular Ca²⁺, while latrophilin and protein tyrosine phosphatase σ bind to α-LTX even in the absence of extracellular Ca²⁺. Because the contribution of protein tyrosine phosphatase σ is small [5], latrophilin is a key receptor of Ca²⁺-independent secretion by α-LTX. The main exogenous ligand of latrophilin 1 is α-LTX [61]. The precise mechanism of latrophilin-mediated exocytosis in response to α-LTX is not well understood [62]. Hiramatsu et al. [63] transfected mast cells, typical non-neuronal secretory cells, with latrphilin and investigated the effects of α-LTX on exocytotic release from the transfected mast cells. It was found that α-LTX caused intracellular Ca²⁺ to increase and lead to exocytosis in the presence of extracellular Ca²⁺. However, neither Ca²⁺ increase nor exocytosis was observed in the absence of extracellular Ca²⁺. These observations indicate that, in the presence of extracellular Ca²⁺, latrophilin-bound α-LTX can work as a Ca²⁺ ionophore. In addition, α-LTX was found to affect the signal transduction in mast cells by phosphorylating SNARE proteins including SNAP-23, syntaxin-4, and VAMP-8 through PKC (protein kinase C)-dependent and PKC-independent pathways. These results demonstrate that, in the presence of extracellular Ca²⁺, the latrophilin-mediated toxic effect of α-LTX can be exerted through the elevation of intracellular Ca²⁺ and the phosphorylation of SNARE proteins, which expands our understanding of the latrophilin-mediated action mechanism of α-LTX.

In the last decades, latrotoxins, particularly α-LTX, from the venom of the black widow spider have been extensively employed as agent tools to investigate the molecular mechanism involved in the regulation of neurotransmitter release. The resulting achievements have greatly improved our understanding of the synaptic transmission [5]. Besides, Mesngon and McNutt [64] have probed the possibility of using α-LTX as a therapeutic agent. They developed embryonic stem cell-derived neurons (ESNs) as a therapeutic research platform and evaluated the potential for α-LTX to antagonize botulinum poisoning. The botulinum neurotoxins (BoNTs) are the most poisonous substances known and exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. It was demonstrated that α-LTX attenuated the severity or duration of BoNT-induced paralysis in neurons. Treatment of BoNT-intoxicated ESNs with α-LTX rescued full-length SNAP-25 expression. This is the first demonstration of a successful therapeutic application of α-LTX and suggests that α-LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication.

By using multiple physiological and biochemical strategies, Yan et al. [65] characterized the aqueous extract of the eggs of L. tredecimguttatus. The eggs were demonstrated to be rich in high-molecular-mass proteins and the peptides were below 5 kDa, showing multiple hydrolase activities and neurotoxicities towards mammals and insects. For investigating the possible relationship of proteins in the eggs with the egg toxicity, Li et al. [66] analyzed the protein composition of the eggs using proteomic strategies and compared it with that of the spider’s venom. The proteins of eggs were shown to be primarily distributed in the molecular mass range of higher than 55 kDa as well as around 34 kDa, having high abundance proteins with molecular masses of about 60 kDa and 130 kDa. A total of 157 proteins were identified from the egg extract which are involved in important cellular functions and processes including catalysis, transport, and metabolism regulation. Comparison indicated that the protein composition of eggs is more complex than that of venom, and there are only a few similarities between the protein compositions of the two materials. No known typical black widow spider venom proteins were found in the egg extract, suggesting that the eggs have their own distinct toxic mechanism (Table 3).

For obtaining more detailed information on the egg toxicity of the black widow spider, much earlier efforts had been made to purify and characterize the toxic components from the spider eggs; however, the work was unsuccessful or incomplete [20,21]. Recently, four proteinaceous bioactive components were purified and preliminarily characterized from the eggs of the black widow spider (L. tredecimguttatus); they were named Latroeggtoxin-I to Latroeggtoxin-IV [67,68,69]. Latroeggtoxin-I has a molecular mass of 23.8 kDa and can block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm preparations completely in a reversible manner. BLAST analysis using the determined N-terminal sequence of the protein demonstrated that no significant similarity to the existing proteins was found, indicating that Latroeggtoxin-I is a novel neurotoxic protein purified from the eggs of the black widow spider [67]. After being abdominally injected into mice and P. americana, Latroeggtoxin-II, with a molecular mass of 28.7 kDa, made the animals, especially P. Americana, display a series of symptoms of poisoning, including becoming depressed, moving slowly, and lagging in response. Preliminary electrophysiological experiments demonstrated that Latroeggtoxin-II selectively inhibits tetrodotoxin-resistant Na⁺ channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na⁺ channel currents. Using multiple proteomic strategies, Latroeggtoxin-II was shown to have only a few similarities to the existing proteins in the databases, suggesting that it is also a novel protein isolated from the eggs of the black widow spider [68]. Preliminary analyses indicate that Latroeggtoxin-III has a molecular mass of about 36.0 kDa and exhibits neurotoxicity against cockroaches but has no obvious effect on mice, suggesting that Latroeggtoxin-III, different from Latroeggtoxin-I and -II, is an insect-specific toxin. When utilizing the determined N-terminal sequence of Latroeggtoxin-III to perform a homology analysis using the protein BLAST program, Latroeggtoxin-III was demonstrated to be a proteolytically cleaved product of vitellogenin [69]. Different from Latroeggtoxin-I to -III, Latroeggtoxin-IV is a broad-spectrum antibacterial peptide of 3.6 kDa, showing inhibitory activity against all the five tested species of bacteria (S. aureus, S. typhimurium, B. subtilis, E. coli, P. aeruginosa), with the highest activity against S. aureus [69].

The early preliminary studies found that the spiderlings of the black widow spider (L. tredecimguttatus) had obvious toxicity to animals [20,21]. To further probe the black widow spiderling toxicity, Peng et al. [70] performed a systematical analysis of the aqueous extract of newborn black widow spiderlings. The extract was shown to contain 69.42% of proteins varying in molecular masses and isoelectric points. Abdominal injection of the extract into mice and cockroaches caused obvious poisoning symptoms as well as death, with LD₅₀ being 5.30 mg/kg in mice and 16.74 μg/g in P. americana. Electrophysiological experiments indicated that the extract at a concentration of 10 μg/mL could completely block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm preparations within 21.0 ± 1.5 min, and 100 μg/mL extract could inhibit a certain percentage of voltage-activated Na⁺, K⁺, and Ca²⁺channel currents in rat dorsal root ganglion neurons. These results demonstrate that the spiderlings are rich in neurotoxic and other bioactive components, which play important roles in the spiderling toxicity.

Why the spiderlings and even the eggs of the black widow spider, like the venom glands, have evolved toxic components is an interesting question. It is speculated that the existence of such components provides a certain protection from some greedy arthropods, which is supported by the report of Russell et al. [71]. They demonstrated that Latrodectus egg toxins have deleterious effects on the web-building activity of A. diadematus. The web-building activity of the spiders receiving 3–5 g/kg body weight was abnormal and one spider receiving 1 g/kg body weight died 6 h after feeding. On the other hand, antimicrobial components may play a dual role in spider-prey interaction, functioning both in the prey capture strategy as well as in protecting the spider from potentially infectious organisms arising from prey ingestion. The adult female black widow spiders usually suspend their egg sacs from the ceiling deep in the retreat, where it is often dark and humid and suitable for the breeding of pathogenic microorganisms. There must be specific mechanisms to protect the eggs and spiderlings. Thus, it is speculated that the antibacterial components may play important roles in protecting the eggs and newborn spiderlings from some pathogenic microorganisms.