The overall goal of this procedure is to isolate the seven distinct silk producing glands from the spider's, abdomen, and use these tissues for molecular and biochemical studies. This is accomplished by first anesthetizing the spider. The second step of the procedure is to separate the cephalic, thax, and the abdomen and pin the abdomen down with insect pins in a small dissection dish.
The third step of the procedure is to make incisions in the exoskeleton to expose the silk producing glands. The final step of the procedure is removal of the different silk producing glands. Ultimately, results can be obtained that demonstrate that the different silk producing glands express distinct silk mRNAs or proteins through molecular and biochemical approaches, such as quantitative real-time PCR, or mass spectrometry respectively.
This method will provide important insight into spider biology research revealing proteins that are stored within the silk producing glands. These proteins have applications that affect the military and medicine as well as many commercial and industrial applications. We first got the idea to do this particular study when we needed to learn more about the proteins that are stored within the silk producing glands.
The main advantage of this technique is the female black widow spiders have very large abdomens making it very easy to isolate the silk producing glands. Individuals new to this method may have difficulty because oftentimes the glands rupture during dissection. There are also certain anatomical structures that are difficult to isolate.
Visual demonstration is critical for a good dissection. Demonstrating the procedure will be Kobe LaMattina, Tiffany Tootin, Blasingame, and Y Shaw Prior to beginning dissection. Prepare 500 milliliters of dissecting buffer solution composed of 0.1 molar sodium chloride 0.015 molar sodium citrate, and 0.1%ethyl bicarbonate.
Then prepare a cardboard box with plastic lining to contain the spider and prevent it from crawling out. We typically obtain black widow spiders from wood piles, pushes or garages, and use glass jars to house them in the lab. Once obtained, transfer the spider from a glass jar into the box while handling the spider.
Wear two pairs of latex or gardening gloves for safety. Then with a small plastic culture vial, approach the spider from the backside and slide the spider into the vial and immediately plug the vial. Introduce carbon dioxide gas into the vial at five to 10 PSI for 10 minutes to anesthetize the spider initial dissection of the abdomen from the cephalic thax should be performed within five minutes while the spider is still under anesthesia.
Place the anesthetized spider into a small dissection dish. Clip the pedestal, the narrow stalk, connecting the cephalic thax in the abdomen to release the abdomen. The cephalic thax contains the fangs of the spider and requires proper disposal.
Carefully remove the cephalic thax placed into a Petri dish and freeze overnight. Before discarding, locate the hole in the spider's abdomen made from the pedes cell clipping by identifying the site of fluid emerging from the abdomen. Using a single dissecting pin, insert the pin through the hole and into the abdomen to fasten the abdomen to the dissection tray, orienting the spider so the pedestal is furthest away from the dissector, and the spint is closest to the dissector.
Using a pair of micro scissors, start in the hole from pedes cell clipping and cut laterally on each side through the exoskeleton until the dorsal side is reached. Using forceps, peel back the anterior exoskeleton segment and insert a second pin at this location. Next, turn the dissecting tray at a 90 degree angle from the initial lateral cuts.
Continue cutting until the spints are reached. The incision will be perpendicular or vertical to the initial lateral cut. The spints are below the hourglass and have a circular appearance.
Do not cut through the spints when the vertical incision is made. Submerge the abdomen in dissecting buffer solution. Peel the remaining exoskeleton back starting from the initial lateral incision.
When this is completed, the fatty tissue whiteish to yellowish in appearance should be clearly visible using forceps. Begin teasing away the fat layer until most of the fat is removed and the silk glands are clearly visible. If eggs are present, they should be removed.
At this point, dissect the glands in the following order. Tubular form, major ate flagella formm, aggregate minor ate assif, formm, and then the piriform. We will describe each gland in the order of dissection.
The tubular form glands sit on top of all other glands and have three pairs of very long cylindrical tubes that are abundant in the abdominal cavity. Carefully tease these glands off of the rest of the glands. Allow them to float free, but remain attached to the spinner reds by their ducts.
Next, tease away the two major ampule glands also found in pears. These glands are large and crescent shaped with long, convoluted distal tails and proximal ducks that decrease in width. When nearing the spiders spints, remove the major ampule glands and place them into sterile 1.5 milliliter micro centrifuge tubes.
Place the glands on ice after their removal. Next, remove the flagella formm gland. The flagella formm gland is also present as a pear.
It is around multilobular structure with a small cylindrical zigzag duct that extends down toward the spints after their removal placed them on ice. Following this, the minor alet is found very similar in morphology to the major alet, but significantly smaller. The aggregate gland found in pairs is large and multilobular, and has a very large excretory duct with large irregular lobes.
The sino formm glands occur in large numbers and resemble short, small finger-like projections. The piriform gland is the smallest gland in the abdomen and has many compacted cylindrical ducts that resemble a fan with many small excretory ducts that extend down to the spints. Now that the tubular formm gland is the only gland left free floating, remove it by pinching the duct with forceps and carefully pulling until it breaks away from the exit point by the spint After dissection, flash, freeze all of the dissected glands by placing the tube until liquid, nitrogen until frozen and store at minus 80 degrees Celsius for storage.
Alternatively, the glands can be frozen immediately after they're independently removed during the dissection procedure. This figure shows the isolation of silk gene, TUSP one mRNA levels in the different silk producing glands, excluding the piriform gland using quantitative real-time PCR analysis following isolation of total RNA from the glands. The tubular formm glands contain the greatest abundance of TUSP one mRNA.
This figure shows a typical result of the amino acid composition profile of the proteins extracted from the tubular formm gland. Blue coloration reflects amino acids with polar side chain groups, whereas red coloration represents amino acid residues with non-polar side chain groups. This figure displays mass spectrometry analysis of protein extracts obtained from the piriform gland following insolution triptych.
Digestion here times two represents twofold magnification of spectrum intensity. Spectrum peptide ion masses marked with the symbol pound indicate peptides derived from the silk fibroin PYSP one After its development. This technique paved the way for researchers in the soak biology community.
It was used to explore chemical and molecular processes involved in soak fiber production, silk fiber assembly, and extrusion. If this technique is completed correctly, it can be accomplished in one and a half to three hours. While attempting this procedure, it's important to wear two pairs of gloves until the spider fangs are removed.
At the conclusion of this method, the silk producing glands can be used to extract RNA and protein. These materials can be used for downstream applications such as quantitative real-time PCR, as well as mass spectrometry. The information that can be gathered from these studies will help us better understand the silk assembly and silk extrusion pathway.
After watching this video, you should have a good understanding of how to isolate the distinct seven silk producing glands from the female Black widow spider.
