Spider Genomics

PowerPoint

DNA Variation of Sheet Web, Orb and Ground Dwelling Spiders

Meggen Meyer
Bellarmine University
Louisville Kentucky
Dr. Bill Tietjen : Senior Research Advisor

Abstract

A combination of Sheet Web, Orb and Ground Dwelling spiders where studied at the Molecular level. Genomic DNA was extracted from a various number of spiders. The concentration of DNA was a calculated by a Flurometer and calculations from these runs indicated the proper dilutions to be used when running a RAPD-PCR. From previous research it was found that primer three worked best with spider DNA. The resulting DNA was then run on a 2% argarose gel which was stained with ethidium bromide. The gels where then photographed and bands were measured and compared to a 100bp ladder. Differences were analyzed first by comparison in a chart based on molecular weights and second by using a dendrogram analysis.

Introduction

There are more than 30,000 kinds of spiders in the world and scientists believe there may be up to 50,000 to 100,000 kinds. Some are smaller than the head of a pin but some are larger than a person's hand. Approximately 3,000 of the 30,000 variations are located in North America. Currently, spiders are classified on physical characteristics such as, coloration, spinnerets, and number and arrangement of eyes.  The goal of this research was to take the current physical identifications and compare the types of spiders at the genomic level in order to detect an evolutionary relationship. 

 The current way of classifying spiders is through physical characteristics. Orb -weaving spiders are typically classified by their shape, color and size.  Orb-weavers belong to the Araneidae family. These spiders are approximately 1/16” to 1 and 1/8” long.  Many times it is common for the male to be smaller than the female.   Orb spiders have eight eyes arranged in two horizontal rows of four.  The chelicerae normally have a small bump in the outer portion. Their webs are generally large and are in a circular fashion that radiate out from the center with a spiral catch area in the center.  Many species of spiders replace there webs daily because they are very delicate and are easily damaged by wind, rain or prey. When replacing their webs they often use pieces of the old web within their new web.

            Ground dwellers are in the Family Lycosidae.  They have 8 dark eyes of unequal size arranged in three rows.  The abdomen and cephalothoraxes are usually long. The average size is about ½”. They have dark, mottled colors and are usually brown to camouflage them among dead leafs and stones.  Except for one species, these spiders do not spin webs. The webs that are spun are constructed funnels on the inner edge of leaves or crevices.

            Sheet- web builders belong to the Family Linyphiidae. Sheet- web builders have long slender legs with short strong bristles.  There are generally 8 eyes arranged in 2 parallel rows.  They are typically smaller in size around 1/16” to 3/8” long.  Their webs are entirely non-sticky and are relatively flat bowl or hammock shaped. They hide beneath their webs until prey are trapped.  They also use this dome shape to escape from predators.

By looking at the above classifications, it could be hypothesized that the sheet- and orb-weaving spiders would be more closely related evolutionarily.   This is because both build a web to live and prey on, whereas ground dwellers do not.   In order to get genetic proof of their relationship a RAPD was run on the genomic DNA of all three types of these spiders.

            Previous research has examined the relationship of kin in spiders.  The basis for the research was to examine the evolution of cooperation and group-living in spiders from subsocial family groups and whether they were constrained of their cannibalistic nature.  A group of social-living spiders were observed when placed together and starved for 7-10 days that social group was then compared to a group of nonkin spiders that were placed together and starved.   The results showed that nonkin spiders were more likely to result to cannibalism than the kin spiders did.   There wasn’t any information found that RAPD’s have been performed on spiders to look at evolutionary relationships.

Materials and Methods

               Spider Collection.  A variety of spiders were collected at Joe Creason Park in Louisville, Kentucky. Of these spiders twenty-one were used consisting of Orb, Sheet and Ground dwelling species. Orb-weavers were Araneus angulatus, and Neriene radiata. Sheet- web weavers included Frontinella pyramitela, and Floinda coccinea.  Ground-dwelling species were Kukulcania hibernalis, Angelena labyrinthica, Tidarren sisyphoide and Lycosa rabida.

            DNA extraction.  The Wizard Genomic DNA purification kit (protocol E of Promega) was used for extraction. This protocol is designed for plant material, but it showed isolation of genomic DNA from arachnids as well.  Spiders were crushed with frozen CO₂ by sterile plastic Epindorf tissue grinders. After most of the CO₂ had evaporated, 600µl of nuclei lyses solution was added and vortexed for 3 seconds, just enough to wet the tissue and was then incubated at 65° C for 15 minutes.  Next, 3µl of Rnase was added and mixed by inversion followed by 15 minute incubation at 37° C.  Following incubation the samples where cooled to room temperature before moving forward.  Two hundred µl of protein precipitation solution was added and the samples were vortexed vigorsley for 20 seconds to extract as much of the protein as possible.  Tubes were then centrifuged at 13,000-16,000 g for 15 minutes and the supernatant containing the DNA was transferred to a sterile 1.5 microcentrifuge tube, leaving behind the precipitated protein pellet. The supernatant was then added to 600µl of room-temperature isopropanol. The solution was mixed by inversion and centrifuged again at 13,000-16,000 g for 5 minutes.  The supernatant was carefully decanted and 600µl of 70% ethanol was added to wash the DNA and then centrifuged again for 1 minute.  Ethanol was then dried for approximately 45 minutes with a Savant speed vacuum.  Finally 100µl of DNA rehydration solution was added and the samples were incubated at 65°C for 60 min with periodic gentle mixing.  The DNA was then stored at 2-8°C.  

Fluorometer.  The fluorometer was first calibrated to zero with a high range DNA assay solution of filtered water, 10 x TAE and H33258 stock solution. This solution was run because it was found that spiders have a higher concentration of DNA. After it was calibrated, 2000µl of assay solution plus 2µl of sample calf thymus DNA where ran. After the fluorometer was calibrated the spider DNA was run. 2000µl of assay solution was placed in a cuvette and then zeroed. 2µl of spider DNA was mixed into the cuvette and place in the flurometer to get the exact concentration.  

RAPD reaction.  Extracted DNA was used in a RAPD reaction to get product that could be analyzed by gel electrophoresis.  A Ready-To-Go RAPD kit was used (Promega).   In this kit were beads that provide the reagents for RAPD reactions in a handy ambient-temperature-stable bead.  These beads contain thermo stable polymerases, dNTPs, BSA, and buffer.  The primer for RAPD analysis consists of a single oligonucleiotide.  Primer three was found to work best for this research (5’ –d[GTAGACCCGT]-3’).   Sterile procedure was observed for this reaction.  It was checked that the bead was at the bottom of the tube.  Five µl of a single RAPD primer was added. The loading of the sample was dependent on the concentration of DNA with an addition of distilled water to bring the sample to 25µl in total, including the primer.  A short, gentle vortex is then required to thoroughly mix the solution.  The tubes where then placed in a Stratagene Robocyler and  cycled at 95° C for 5 minutes, 36°C for 1 minute, and 72°C for 2 minutes, for a total of 45 repeated cycles.

Gel Electrophoresis.  Electrophoresis was carried out with a 2% agarose gel. Two µl of ethidium bromide was mixed into the gel solution after the agarose had been completely melted in the microwave. The gels were ran for approximately two hours or until the yellow band ran completely off the gel. Examine Figure 1 and 2 for these gels.

Photography.  A Nikon digital camera was used to photograph the gels on a Fotodyne UV illuminator. The images where then exported to the Kodak Scientific Imaging System. Once the image was captured it was saved and printed out for further exploration.

Data Analysis.  DNA fragments for each spider were measured on agarose gels.  The distance each band migrated from the well was measured with respect to a standard 100bp DNA ladder from Promega.  Using Log graphing paper and the curve of the standard molecular weight ladder, the base length of the bands were calculated.   The RAPD products from the gel electrophoresis are shown in Figures 1 and 2.  The bands were recorded (Table 1,2) and then scored as positive or negative for each spider species.  The bands were recorded in excel spreadsheets for a better visual comparison.  Table 1 shows the bands observed of the sheet spiders.  Table 2 compares the bands of the sheet web builders and the orb weavers.

 Results and Discussion           

By analysis of the molecular weight bands there was no conclusive data that any species were closely related.  It was seen that some species have similar banding patterns, but not enough to state that they were of the same guild. In turn it was proven that species of spiders vary in different population regions. Since the data was so skewed, a dendogram analysis was not able to be performed. Previous research had claimed that you could tell species variation through genetic testing. This research now proves that genetic testing cannot be used in telling different guilds of spiders.

Figure 1: Sheet and Ground Weavers Gel

Table 1: Sheet and Ground Weaver Bands

 Figure 2: Orb and Sheet Web Gel