1. Cloacal vs Jaw Swabs: A Novel Technique To
Genetically Determine Shark Diet
Abstract
Methods
Results
Mouth/ Cloacal technique: 1.Place shark in tonic immobility
2.Separate cloaca/mouth opening
3.Enter cloacal or mouth swab
4. Swipe cheek tissue or excrement before placing in vial.
Sharks are known to be carnivorous through the diet that they eat. Outdated diet analysis through gastric lavage is lethal leading to high stress and cardiac arrest. To improve the sampling of larger, specifically chondrichthyes, fish and cut the invasiveness of the common procedure, two other procedures were used: mouth swabs and cloacal swabs. DNA Barcoding was used to analyze the samples. Two white spotted bamboo sharks were proxies for great whites population around the Long Island bay area. DNA barcoding was successful and showed many bands through purification, however through sequencing there weren’t as many promising results.
Ladder
Samples
Control
Control
Shark Anatomy
First dorsal fin
Second dorsal fin
Successful Samples
1.Mark Vials with type of sample, species, and date
2.Samples are preserved in ice
1.Purification of samples
2.Amplification of samples
3.Gel electrophoresis
4.The DNA streams were then put through BLAST, a bioinformatics system, where the DNA is compared to other organisms for the best match.
Introduction
We know the diet of most adult sharks by using an extremely common and well known method known as the gastric lavage, where the stomach would be flushed out with all of the contents in it (Bangley, 2011). Due to lack of systematic or theoretical analysis, marine species are either put through this process, or killed and their stomachs are emptied. Despite success, this procedure induces stress for sharks and can hurt the species during this process. Other procedures that have been created to substitute this stress-inducing methodology are mouth swabs and cloacal swabs (Will, 2014).The gastric lavage method has been proven reliable (Hyslop, 1994) and may have more results to begin identification of stomach content. It is possible that other non-lethal methods can be successful as gastric lavage, and can dispense the same results about the samples.
Gels
Caudal fin
Snout
Lower Anal fin
Analysis
Figure 02: All samples are labeled based on the two sharks sampled A is mouth and B is cloacal. 1 is the first shark sampled, and 2 is the second. BC2 is Bunker fish control, SC2 is shrimp control, and WQ2 is water quality control.
Lower pectoral fin
While data was collected from captured sharks, their sex, body length, as well as species was recorded. For each process of mouth and cloacal swabs, each was measured and recorded from the time that the sample bottle was open, up to when it was closed and contained each swab. In total, two cloacal and two mouth swabs were collected from captured sharks. Controls included the food that these captured sharks were fed. This included shrimp and bunker bait, as well as water quality. Captured sharks underwent both mouth and cloacal procedures. Each shark was put under tonic immobility, ensuring that they were in a still and calm space. Tonic immobility consists of placing the shark on its underside, inducing a state of stupor. Using all seven samples from captured sharks, they were divided into six different rounds for gel electrophoresis. After collecting samples and conducting the first round of gel electrophoresis, bands indicating that we received results from sequencing appeared. Each band represents the DNA captured from liquid that each swab was soaked in, in order to preserve it. When samples were put through sequencing, only 3 of the samples had gotten sufficient results. The samples were then put through BLAST. Two samples seemed to have related to each other by 100 characters. Despite there being no results from BLAST, it is known that two of the samples that were put through sequencing, have some relation to one another. This is compared to Sample_1, which seems to have the least relation to the two. Mouth and cloacal swabs through gel electrophoresis has given us results.
Figure 01: During experimentation, two female spotted Bamboo sharks were tested using mouth and cloacal swabs. Both sharks were scheduled for regular feeding.
Scientific Problems
Which method will reflect more results through gel electrophoresis?
Are these results reflecting the samples that were taken based on what those sharks have eaten?
Figure 04. Samples ran through BLAST. This system takes each and every sample and tries to match them up based on a similarities between each species. Those that are closely related are paired up and the chart displays how many similarities were found.
Background Information
Figure 03. Only 3 out of the 7 samples had enough DNA that would be able to produce a sequence. This may also be reflected in Figure .02. A2C,B1C,B2C and BC2 had the prominent bands.
Basic information concerning average adolescent shark diets in the north east, is needed and mostly unknown. The first method that many scientists commonly resort to has been gastric lavage. Based on past experiments, gastric lavage has proven 50% effective for grass pickerel (Andreasson, 1971), and 60% effective for largemouth bass. Gastric lavage, however, has had effects such as cardiac arrest and aspiration of fluid (Eddleston, 2007).Mouth Swabs, on the contrary, are different in the sense that they may not be as invasive or complicated. Research has been found that there is no digestive enzyme found in the saliva of sharks unlike humans, so it is possible that there can be a natural preservative that can keep samples of their diet in previous days, and there would not be any need to analyze gut contents directly. Cloacal swabs are another great method as well (Will, 2014). This involves using a swab to collect leftover feces from a specimens anal cavity. Since excrement samples have long been used to analyze the diet of several species, this method was highly supported and well suggested (Will, 2014).
Conclusion
While there weren’t many positive results when samples were put through sequencing, two of the three samples were related in some way. When purifying results were received, 4 out of 7 bands appeared. The brighter the bands, the stronger the DNA strand is. Mouth and cloacal swabs according to the purifying chart do pick up DNA based on whatever the sharks have consumed that day but, were not strong enough to go through sequencing. The second round of samples are currently being processed for sequencing.
Figure 06. Sampling was taken place in South Hampton High school. Greg Metzger was able to provide two female spotted bamboo sharks, who will serve as proxies, in place of Great whites.
Figure 05. Gel electrophoresis was used in order to determine if mouth and cloacal swabs will provide enough evidence for discovering the diet of the spotted bamboo sharks.