Environmental DNA Thesis

14 April 2013

Jennifer Mahoney

Humboldt State University

1 Harpst St, Arcata, CA 95521

707/826-3953; 707/826-4060

jrm137@humboldt.edu

RH: Mahoney● Use of environmental DNA to detect unseen species

The Use of Environmental DNA to Detect the Invasive Species Rana catesbeiana within Green Diamond Property in Northern California

JENNIFER MAHONEY, Humboldt State University, Arcata, CA, 95521

ABSTRACT The use of mitochondrial DNA to detect organisms without having to capture them is an advance in wildlife biology that may allow managers to census populations without having to capture and mark individuals. The object of my study will be to use environmental DNA to survey for the presence of the invasive American Bullfrog in four of the ponds within Green Diamond logging property. It was found that this method is useable to sample for American Bullfrogs . This shows that environmental DNA is a usable technique.  

KEY WORDS, American Bullfrog, environmental DNA, PCR, Arcata Marsh, invasive species, Rana catesbeiana

The use of environmental DNA to sample for animals is a new technique that is still in the process of being developed. This technique has been shown to be good for sampling for animals that are evasive or, that require extensive efforts to sample for. This technique has been used to sample for a variety of species like the American bullfrog (Ficetola et al. 2008), the big headed carp, the silver carp (Jerde et al. 2011), the Idaho giant salamander, the Rocky Mountain tailed frog (Goldenberg et al 2011), the crested newt (Thomsen et al. 2012), sturgeon (Dejean et al 2012), and the common carp (Takahara et al 2012).This technique is currently being developed for many other species. The use of environmental DNA allows managers to sample without having to capture and mark animals by testing for their DNA presence in water samples. The water samples have a PCR reaction performed on them to determine if they contain target species DNA.  In many studies it has been shown to be more accurate and effective than traditional sampling methods (Thomsen 2012, Takahara et al 2012).

My study will be performed on timber harvest lands in northern california. This forest is sustainably harvested redwood timber forest and aims to support native fauna. It supports some invasive species like the Himalayan blackberry (Rubus armeniacus) and the American bullfrog (Rana catesbeiana). These invasive species can be detrimental to native species, including the endangered red-legged tree frog (Alford and Richards 1999). Therefore, knowledge of their distribution within the area is valuable information that could help managers better manage for the native fauna.  I explored the extent of the distribution of American bullfrogs by testing water samples from some of the ponds for their mitochondrial DNA.

The methods used for this experiment are replicated from Ficetola et al. 2008 and Goldenburg et al. 2011. I am testing to see if these methods, including the primer selected by Ficetola et al. 2008 for the European range of the frog, can be applied to the Northern California area.

STUDY AREA        

The study will take place at the Green Diamond Resource company property located in Blue Lake, CA 95518. The property is seated on the coast of northern California, 6 miles inland.

This company sustainably harvests Redwood and Douglas fir trees, which comprise a majority of the landscape. The company is required to buffer their tree harvest around streams and, bodies of water that contain vertebrates. My study focuses on four gravel ponds in a clear area that was not forested. The ponds were all adjacent to each other only separated by low banks. These ponds were chosen because they are regularly sampled for frog presence, involving physical capture using box nets. They are regularly used by waterfowl species and, many other species of amphibians have been observed within the ponds including red-legged tree frogs (Rana. Aurora),common tree frogs (Polypedates leucomystax) and, many different species of salamanders. 

METHODS

See table 1 for materials. Water samples were collected on 27 March 2013 between the hours of 1300 and 1700. They were collected by pumping pond water through 0.45 micron pore size cellulose nitrate filters. The pump consisted of a hand pump, a one liter flask, and tubing. Water was sampled using grab bottles sterilized with bleach. The water was poured directly onto the filters and pumping continued until the volume of one liter was reached or the filter became clogged, whichever was reached first. The filters were then placed into a 95% alcohol solution using tweezers sterilized with bleach. Gloves were changed between samples and tweezers re-sterilized with bleach. The samples were then taken back to the lab and placed into the continuous frost freezer (within 6 hours of sampling). The grab bottles were collected and brought back but were not processed for this experiment. DNA was then be extracted using a Qiagen DNA extraction kit(Qiagen 2006). The lab area was sterilized using Rnase away solution. The filters were cut into quarter using scissor sterilized with 95 percent ethanol and fire. All equipment was re-sterilized between samples to prevent contamination. The quarter filter was placed into a 1.5ml centrifuge tube. 180 microliters of ATL buffer and 20 microliters of proteinase K was added to the samples. The samples were then vortexed individually and placed into a thermocycler at 56 degree Celsius for an entire 24 hours. In addition to the filters, I also prepared two negative controls using water. I also prepared a positive control using water that an American Bullfrog was euthanized in (IACUC 12/13B.20-A). I spun this water down in a centrifuge to retrieve a cell pellet from it. These additional tubes were also incubated in the thermocycler for 24 hours. The samples were removed from the thermocycler and vortexed for fifteen seconds. 200 microliters of AL buffer was added to each sample and I vortexed the samples again. 200 microliters 100 percent ethanol was added to each sample and the samples were vortexed again for fifteen seconds. The liquid from the samples were then placed into the Qiagen spin columns. The columns were then placed into the centrifuge and run at 8000rpm for one minute. The samples were then removed and collection tubes were replaced. 500 microliters of AW1 buffer were added and the samples were placed back into the centrifuge at 8000 rpm for one minute. The samples were then removed and, 500 microliters of AW2 buffer was added to each sample and, collection tubes replaced . The samples were then placed back into the centrifuge and run at 14,000 rpm for three minutes. The spin column were then removed and placed into 1.5ml microcentrifuge collection tubes. 200 microliters of buffer AE was added to each sample. The samples were placed into the centrifuge and run at 8000 rpm for one minute to elute the DNA. This last step was repeated to elute the maximum amount of DNA product. The DNA was diluted using five microliters of DNA to 45 microliters of water. A PCR reaction was performed on the resulting DNA. The Polymerase chain reaction (PCR) amplification was performed with primers 5′-TGCCAACGGAGCATCATTC-3′ and 5′-ATAAAGGTAGGAGCCGTAGT-3′ especially designed for this experiment. These primers amplify a 79bp segment of mitochondrial cyt-b, which is monomorphic in all 397 individuals analyzed by population genetic studies covering the whole native and European range of the species (Ficetola et al. 2008). According to Ficetola et al. (2008) this sequence does not match any other closely related amphibian species so it should not provide any false positives. The primers will be mixed with the sample DNA, DNA polymerase, dinucleotide triphosphates, dyes and various buffers required for the reaction. The DNA will be placed in a thermo-cycler at 95 degrees Celsius for ten minutes. It was then dropped to sixty one degrees Celsius for thirty seconds. It was then placed at seventy two degrees Celsius for 90 seconds. This was repeated fifty five times but, all repeat steps will only be held at the ninety five degree step for thirty seconds. The samples were then pipetted into a 2% agarose gel and electrophoresis was performed on them until the positive control band was visible (Ficetola et al 2008).

RESULTS

See Figure 1. The PCR reaction yielded a 79bp fragment of DNA from the positive control. This shows that the methods did work correctly and, can positively identify species presence in a water sample collection. None of the sites produced a band of DNA 79 bp in length. This indicated that the ponds do not have American bullfrog presence. This matches trap efforts that have been performed on the pond from January to April which have not reported American bullfrog presence since January.

DISCUSSION

This study verified the PCR methods for identifying species presence in the environment but, there was no verification of the filters and their usage. More research must be done to verify these primers and verify they do not cross amplify for other species in this area that are closely related. With additional research this tool can also be expanded to detect not only distribution but population size, by performing a quantitative PCR reaction and analyzing the amount of DNA found in the sample. I hope to expand this research to perfect the use of qPCR by performing environmental DNA assays for known population sizes and developing a ratio formula(amount of DNA retrieved/ amount of animals present in a habitat) that can be used by manager and biologist to lessen the necessity for mark-recapture sampling efforts.

MANAGEMENT IMPLICATIONS

This study may provide the managers of the Green Diamond Timber Company with valuable information on the distribution of an invasive species that could be more accurate than call surveys performed in the area and less labor intensive and much less invasive than trap efforts. There are many species of amphibians which are secretive and require extremely destructive and invasive methods to sample for population distribution. Some species of salamanders burrow many feet down and require extremely invasive digging to sample their distributions.  Bodies of water on timberlands must be surveyed for any wildlife to be classified before logging can take place. This tool can streamline that process and minimize the amount of disturbance added to the streams, which occurs during these surveys.

ACKNOWLEDGMENTS

I would like to acknowledge Barbra Clucas for mentoring me through this project. I would also like to thank Lowell Diller for telling me about this technique and, assisting me with access to Green Diamond’s property. I would also like to thank John Reiss and Timothy Girod for providing me with the water used as a positive control. All materials were provided by Anthony Baker from the Humboldt State University Biology Department Core Facility and, he also mentored me through this entire project so, he gets the largest acknowledgment of all.

LITERATURE CITED

Alford, R.A. and Richards S.J. “Global Amphibian Declines: A problem in Applied Ecology”                  Annual Review of Ecology and Systematics, Vol. 30, (1999), pp. 133-165 Accessed:                     `April 15, 2013 http://www.jstor.org/stable/221682

Darling, J.A., and Mahon, A.R., 2011, “From molecules to management— Adopting DNA-based            methods for monitoring biological invasions in aquatic environments”. Environmental          Research, v. 111, iss. 7, p. 978–988, doi:10.1016/j.envres.2011.02.001.

Dejean, T., Valentini, A., Duparc, A., Pellier-Cuit, S., Pompanon, F., Taberlet, P., and Miaud, C.,             2011, “Persistence of environmental DNA in freshwater ecosystems”: PLoS ONE, v. 6, no. 8, e23398, doi:10.1371/journal.pone,0023398.

Dejean, T., Valentini, A., Miquel, C., Taberlet, P., Bellemain, E., and Miaud, C., 2012, Improved             detection of an alien invasive species through environmental DNA barcoding—The   example of the American bullfrog Lithobates catesbeianus: Journal of Applied Ecology,           v. 49, iss. 4, p. 953–959, doi: 10.1111/j.1365-2664.2012.02171.x.

Ficetola, G.F., Miaud, Claude, Pompanon, François, and Taberlet, Pierre, 2008, Species    detection using environmental DNA from water samples: Biology Letters, v. 4, p. 423–      425, doi:10.1098/rsbl.2008.0118.

Goldberg, C.S., Pilliod, D.S., Arkle, R.S., and Waits, L.P., 2011, Molecular detection of   vertebrates in stream water—A demonstration using Rocky Mountain tailed frogs and    Idaho giant salamanders: PLoS ONE v. 6, no. 7, e22746,doi:10.1371/journal.pone.                  0022746.

Jerde, C.L., Mahon, A.R., Chadderton, W.L., and Lodge, D.M., 2011, “Sight-unseen” detection of rare aquatic species using environmental DNA: Conservation Letters, v. 4, iss. 2, p.       150–157, doi:10.1111/j.1755-263X.2010.00158.x.National Center for Biotechnology       Information, 2012, NCBI: U.S. National Laboratory of Medicine, National Center for            Biotechnology Information database, accessed October 4, 2012, at             http://www.ncbi.nlm.nih.gov/genbank/.

Pilliod, D.S., Goldberg, C.S., Laramie M.B., and Waits, L.P. “Application of Environmental        DNA for Inventory and Monitoring of Aquatic Species”, January 2013, U.S. Department                 of the Interior, U.S. Geological Survey Fact sheet 2012-3146 January 2013

Qiagen, DNeasg Blood and Tissue Handbook July 2006

Takahara, T., Minamoto, T., Yamanaka, H., Doi, H., and Kawabata, Z., 2012, Estimation of fish biomass using environmental DNA: PLoS One, v. 7, iss. 4, e35868, doi:10.1371/   journal.pone.0035868.

Thomsen, P.F., Kielgast, J., Iversen, L.L., Wiuf, C., Rasmussen, M., Gilbert, M.T.P., Orlando,      L., and Willerslev, E., 2012, Monitoring endangered freshwater biodiversity using       environmental DNA: Molecular Ecology, v. 21, iss. 11, p. 2565–2573, doi:10.1111     /j.1365-294X.2011.05418.x.

 Waits, L.P., and Paetkau, D., 2005, Noninvasive genetic sampling tools for wildlife biologists— Review of applications and recommendations for accurate data collection: Journal of Wildlife Management, v. 69, iss. 4, p. 1419–1433, doi:10.2193/0022-           541X(2005)69[1419:NGSTFW]2.0.CO;2.

Figure 1. Electrophoresis results

Figure 1. Electrophoresis results. Mahoney” The Use of Environmental DNA to Detect the Invasive Species Rana catesbeiana within Green Diamond Property in Northern California”. 14 April 2013

Table 1. Required Materials

Product ID	Description
Fisher Scientific Cat. #: 02‐893D Vendor Cat. #: 2105‐0032	1‐L Nalgene Bottle
Fisher Scientific Cat. #:10 182 50B Vendor Cat. #: DS4101‐1000	1000ml* Nalgene Polypropylene Vacuum Flask with Tubulation
Fisher Scientific Cat. #:14 135M	#8 Fisherbrand Rubber Stopper* (stopper must be drilled with ½” drill bit to accommodate stem of filter funnel)
Fisher Scientific Cat. #: 13 310 110 Vendor Cat. #: 96410 15	1Masterflex Platinum‐cured Silicone Tubing (size L/S 15)
Fisher Scientific Cat. #: 13 310 110 Vendor Cat. #: 96410 15	Whatman Disposable Filter Funnel with 47mm diameter Cellulose Nitrate (WCN 0.45um pore diameter) Filter Paper
Fisher Scientific Cat. #:09 875 19 Vendor Cat. #: 1920‐7001	Whatman Disposable Filter Funnel with 47mm diameter Cellulose Nitrate (WCN 0.45um pore diameter) Filter Paper
Fisher Scientific Cat. #: S90724A Vendor Cat. #: 7‐206‐1	2Hand Vacuum Pump
Fisher Scientific Cat. #: 09‐753‐50	Fisherbrand Filter Forceps
5′-TGCCAACGGAGCATCATTC-3′ and 5′-ATAAAGGTAGGAGCCGTAGT-3′	Primers
PCR Materials	Qiagen DNeasy KIT DNA polymerase DNTP Buffers 2% agarose gel
N/A	Thermocyler
N/A	6Fume‐Hood Vacuum Line (available in many laboratories)

Table 1. Required Materials Mahoney.” The Use of Environmental DNA to Detect the Invasive Species Rana catesbeiana within Green Diamond Property in Northern California”. 14 April 2013

Contains list of services for current record