This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.
Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.
Venoms are animal secretions predominantly injected into another animal for the purposes of predation or defense, and also have important biological applications with biomedical relevance1–3. Only certain animals synthesize venom, but its production is taxonomically widespread across invertebrates (e.g., cnidarians, cone snails, scorpions, and spiders) and vertebrates (e.g., snakes, some fish and mammals), because it has independently evolved multiple times1,4. Biochemical characterization of venoms typically show they are composed of a wide variety of proteins and peptides, which largely act on the circulatory and nervous systems of injected animals to rapidly deliver constituent toxins1. A small fraction of venomous animals may pose a threat to humans, particularly species with synanthropic distributions5. The study of venom composition and functional activities has played an important role in the functional characterization of vertebrate cellular components (particularly neuronal ion channels)6 and in elucidating fundamental cellular processes (e.g. neurosecretion)7. Moreover, select venom peptides have useful properties in biomedical contexts, including their uses as treatments for cancer and pain8,9, and are mined for candidate drug leads and antivenom development. Venoms also play prominent roles in ecological and evolutionary research1,4,10,11, thus the collection of these hazardous secretions has many utilities.
There are >40,000 described species of spider (Order Araneae), and all but one of the more than 100 spider families possess paired venom glands that terminate in fangs12. The high species diversity of spiders suggests that they represent the largest clade of venomous organisms. However, biochemical characterization of spider venoms has largely concentrated on a small number of species most often associated with human envenomation. Recent proteomic and transcriptomic studies of spider venoms indicate they typically contain many unique proteins and peptides2,13,14. Advances in high-throughput cDNA sequencing and mass spectrometry peptide fingerprinting has greatly facilitated the discovery of these venom proteins15. Nevertheless, such work begins with the collection of sufficient venom and/or venom glands from the spiders, and detailed documentation for such techniques are few16.
This paper presents a protocol for the collection of venom and venom glands from black widow spiders, which can be applied to similarly sized spiders. The collection of venom separately from the glands enables identification of proteins that are secreted into the venom as opposed to proteins performing other cellular functions. Black widows and other Latrodectus species are widely recognized as being among the most hazardous spiders due to their highly neurotoxic venom, which causes severe pain in humans, which is sometimes accompanied by profuse sweating, muscle contractions, hypertension, difficulty breathing and patchy paralysis5. The venom collection protocol presented here uses electro-stimulation to deliver electrical current to anesthetized spiders to elicit muscular contractions and release of venom. Venom droplets are quickly collected with micro-capillaries and dispensed into tubes for freezer storage. Because the protocol involves hazardous procedures, it should only be performed by well-trained individuals and caution is urged at key steps. The collected venom has numerous uses, such as characterization and isolation of constituent molecules2, for physiological experiments or functional assays18, and to stimulate venom gene expression11. The protocol concludes with a description of venom gland dissection and preservation useful for the cloning of venom-specific genes, the expression of which has been shown to occur 2-3 days following venom depletion in different spiders10,11.
1. Preparation of Electro-stimulator Apparatus
2. Preparation of Immobilizing Forceps and Other Materials
3. Immobilization of Spider in Forceps
4. Venom Collection
5. Venom Gland Dissections
The collection of venom and venom gland dissections are frequently performed to characterize venom proteins and peptides at the sequence level10,11,15. Collected venom may also be used in physiological assays to determine their functional activities18. The collection of venom will stimulate venom gland gene expression, thereby facilitating the cloning of specific toxin transcripts via RT-PCR10,11. Identification of venom proteins can also be accomplished in a high-throughput manner by integrating standard mass spectrometry techniques with sequence databases generated from venom gland cDNA libraries15,21. An example of such work, starting from venom and glands collected using the protocol detailed above, demonstrating its effectiveness, is illustrated in Figure 1.
Venom collected from L. hesperus adult females was digested with trypsin and subjected to an in solution MuDPIT analysis, which links HPLC (high performance liquid chromatography) to tandem mass spectrometry (MS/MS). Here the MuDPIT analysis was performed by the University of Arizona Proteomics Consortium. Masses of detected peptides and their dissociated fragments (daughter ions, inferred from spectra) were compared to peptide sequences theoretically predicted from translations of cDNA sequences obtained from a gene expression library constructed from L. hesperus venom glands (glands were acquired by the dissection protocol described above)21. In this experiment, 36 proteins were detected from all collected spectra, at the 99.9% probability threshold, and contained at least one detected peptide. One of the detected proteins is supported by 43 total spectra (exemplar spectra shown in Figure 1A), corresponding to three exclusive peptides, covering 35% of the protein sequence (Figure 1B). The detected protein (translated from a collected cDNA) has a top BLASTp hit to latrodectin from L. tredecimguttatus (GenBank Accession P49125.1), with an e-score of 2e-46, and shares 80% identity at the amino acid sequence level with the protein detected in this experiment. Latrodectin, which is also known as alpha-latrotoxin low molecular weight protein, is a recognized venom component of black widow spiders22,23, verifying the effectiveness of the presented protocol. Some proteins identified from the venom correspond to sequences for which no BLAST hit is found. It is unclear whether such a result is due to the limited genomic resources available for spiders as well as the limited functional information for their proteins, or because the unknown protein represents a venom contaminant. Nevertheless, gene or protein expression analyses examining the abundance of such a novel protein in venom glands relative to other tissues should reveal whether it is a genuine venom component.
Figure 1: Latrodectin peptide identified from black widow venom using mass spectrometry. (A) Representative spectra (one of 43) detected via MS/MS portion of MuDPIT analysis of L. hesperus venom which was assigned to predicted translation of a collected cDNA sequence shown in part B. Spectra shows mass to charge ratio (m/z) of detected daughter ions on the horizontal axis produced by fragmenting parent peptide (CGEEDFGEEIVK), with letters at top indicating the peptide sequence based on daughter ion masses. (B) Protein sequence to which spectra in part A was assigned, showing in red, bold and underlined text the corresponding sequence from the spectra shown in part A, additional red text (not bold) represents another peptide in this protein detected with other collected spectra. The protein sequence is translated from a cDNA sequence obtained from dissected venom glands.
Venoms represent an important source of physiologically reactive proteins, peptides and other molecules with applications for drug discovery, as well as for fundamental aspects of cellular and ecological research1–3. However, the collection of venom, particularly from dangerous or small animals, is a challenging task. This protocol demonstrates how venom and venom glands can be collected from black widow spiders, and confirms the success of this approach via a combination of MuDPIT analysis of the venom and a protein database derived from cDNAs cloned from venom glands21. While this protocol works well for black widows and medium sized spiders, other venom collection techniques have been employed for larger mygalomorph (tarantula-like) spiders, such as direct aspiration of venom from fangs into glass pipettes e.g., 24. This latter approach, however, will not work well for smaller-sized spiders that are not aggressive.
One especially critical aspect of the venom collection protocol described here is the initial phases of preparation and optimization of the collection process so that it becomes more routine, consistent and quicker. The protocol is initially challenging to master, but with repeated trials, it becomes easier and faster. Caution is also urged at all critical phases involving the handling of hazardous spiders, the use of electrical current, fine-point glass micro capillaries, and syringe needles. It is important to wear appropriate personal protective equipment such as nitrile gloves, a lab coat, long pants and closed shoes, as well as eyewear when shaping micro capillaries.
Another challenging aspect to venom collection is the small amount of venom produced by any one spider, particularly Latrodectus species, from which the amounts collected may be limited to 1-2 microliters per individual at best. Obtaining sufficient venom for downstream applications, such as protein gels or functional assays may require the combination of venom from multiple individuals into one tube. In such cases, venom should only be combined from individuals of the same sex, ontogenetic stage, and population given the recognition of intersexual, developmental and geographic variability in some venoms25,26. Spiders may also exhibit considerable variation in the amount of venom produced among individuals, where lesser amounts may reflect the recent depletion of the gland. Thus it may be advisable to collect venom several days after their last feeding. If little venom is released, excessive current should not applied to the spider, which may cause the cuticle to rupture, leading to contamination of the venom with hemolymph or death.
Contamination of venom samples with spider silk or human sources should also be avoided through the use of sterile or clean equipment. Despite these challenges, collection of pure venom, leaving the spider alive, is preferable to methods that obtain venom from gland homogenates (which do not separate venom components from other cellular proteins) and kill the spider. It is also critical to ensure that samples are quickly frozen to prevent protein degradation.
The extraction of venom promotes subsequent venom production, thereby stimulating venom gene expression in the venom gland. Thus, because this protocol allows for spiders to survive venom depletion, their glands may be dissected several days later (killing the spider) at a point where venom gene expression is expected to be sufficient for genetic studies, such as transcript cloning10,11. Several important precautions must also be taken in venom gland dissections. Emphasis should be placed on using lab equipment and reagents that are free of RNases that degrade RNA. Thus it is recommended to wipe forceps and other non-disposable equipment and surfaces with solutions that eliminate RNase and DNA contamination. The dissections should be performed as quickly as possible and directly frozen to further ensure RNA integrity of the tissue. Finally, dissections should only be performed on anesthetized spiders, after their cephalothorax and abdomen are quickly separated.
In conclusion, this article provides a verified protocol to obtain spider venom and venom glands. Venom and venom glands allow for the isolation and characterization of their protein and peptide components using proteomic and transcriptomic approaches. In addition, venom samples may represent the starting point of functional assays, which determine the biomedical and pharmacological potential of their constituent molecules. Nearly all spiders produce venom, and the wide diversity of venom components synthesized by individual species suggests a vast diversity of venom molecules are yet to be discovered13. Accordingly, this protocol provides tools to investigate the rich source of biologically active molecules present in spider venoms.
The authors have nothing to disclose.
I thank the following individuals for their assistance in the development of this protocol: Chuck Kristensen, Greta Binford, Alex K. Lancaster, Konrad Zinsmaier, and Mays Imad. Mass spectrometry and proteomics data were acquired by the Arizona Proteomics Consortium supported by NIEHS grant ES06694 to the SWEHSC, NIH/NCI grant CA023074 to the AZCC and by the BIO5 Institute of the University of Arizona. Funding for this work was provided from the National Institutes of Health (National Institute of General Medicine) from grants 1F32GM83661-01 and 1R15GM097714-01 to Jessica E. Garb.
Nerve and muscle stimulator (electro-stimulator) | Grass Technologies | SD9 | http://www.grasstechnologies.com/products/stimulators/stimsd9.html |
Voltmeter | RadioShack | 22-223 | any generic voltmeter/multimeter can be substituted |
Pointed Featherweight Forceps | Bioquip | 4748 | |
Plasti Dip | Performix | Available at Ace Hardware | |
21 G X 1 1/2in Precision Glide hypodermic syringe needle | BD Medical | 305190 | |
Vacuum filter flask 1L | Nalgene | DS4101-1000 | smaller flask sizes may also work |
Buchner Two Piece funnel, 90 mm | Nalgene | 4280-0900 | |
5 microlitter Capillary Bores (Micro capillaries) | VWR | 53508-375 | |
Mounting putty strip | Loctite | Available at Ace Hardware | |
Fisherbrand* General-Purpose Extra-Long Forceps Length: 11-13/16 in. | Fisher | 10-316C | |
SSC Buffer, 20X (pH 7.0), Molecular Grade | Promega | V4261 | can be made from stock chemicals (150 mM NaCl, 15 mM sodium citrate) |
Eppendorf safe-lock tubes 0.5 mL tubes (cryogenic safe) | Eppendorf | 22363611 | |
Nalgene Dewar, 1L, HDPE, Liquid nitrogen benchtop flask | Thermo Scientific | 4150-1000 | |
Rnase Away | VWR | 53225-514 | |
Ultra Fine Tweezers (dissecting forceps) | EMS | 78310-0 | similar high-quality fine point forceps can be substituted |
Foot switch/pedal | Linemaster Switch Corp. | 491-S | |
Two-pronged extension clamp, with vinyl covered sleeves, 8.5 inches | VWR | 21570-007 | similar models could be substituted |
Clamp Holder | VWR | 89084-746 | similar models could be substituted, must accommodate diameter of extension clamp rod |
Magnetic base (holds extension clamp via clamp holder) | VWR | 300042-270 | similar apparatus able to securely hold extension clamp in fixed position may be substituted |
Plastic Collecting Vials (large/40 dram) | Bioquip | 8940 | |
Cotton sewing thread | Threadart.com | THRCOT9 | similar product could be substituted |