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Use of Environmental DNA to Determine Fantail Darter (Etheostoma flabellare) Density in a Laboratory Setting: Effects of Biomass and Filtration Method

Use of Environmental DNA to Determine Fantail Darter (Etheostoma flabellare) Density in a... Hindawi International Journal of Zoology Volume 2020, Article ID 4731686, 7 pages https://doi.org/10.1155/2020/4731686 Research Article Use of Environmental DNA to Determine Fantail Darter (Etheostoma flabellare) Density in a Laboratory Setting: Effects of Biomass and Filtration Method Ramon A. Guivas and Ben F. Brammell Department of Science and Health, Asbury University, Wilmore, KY 40390, USA Correspondence should be addressed to Ben F. Brammell; ben.brammell@asbury.edu Received 9 December 2019; Accepted 23 March 2020; Published 17 June 2020 Academic Editor: Andrea Galimberti Copyright © 2020 Ramon A. Guivas and Ben F. Brammell. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Estimating fish abundance/biomass holds great importance for freshwater ecology and fisheries management, but current techniques can be expensive, time-consuming, and potentially harmful to target organisms. Environmental DNA (eDNA) has proven an effective and efficient technique for presence/absence detection of freshwater vertebrates. Additionally, recent studies report correlations between target organism density/biomass and eDNA levels, although widespread application of this technique is limited by the number of studies examining this relationship in various species and settings. Additionally, filter clogging is a commonly encountered issue in eDNA studies in environments with significant sediment and/or phytoplankton algae. Fre- quently, a sample must be split into multiple aliquots and filtered separately in order to process the entire sample. 'e present study examines both the relationship between biomass and eDNA and the effects of single versus multiple filter sampling on eDNA concentrations of fantail darters (Etheostoma flabellare) in a laboratory setting. Tank tests were performed in quadruplicate at four environmentally relevant fantail biomass levels. eDNA samples were collected and processed in parallel (one as a whole through a single filter and one in parts through multiple filters). Species-specific primers and a probe were developed for E. flabellare from cytochrome b sequences obtained from locally collected specimens, and real-time quantitative PCR was used to analyze eDNA levels at each biomass. Significant correlations were observed with increasing biomass for both methods, although this relationship was stronger for samples processed by the multiple filter method. 'ese data should be useful in eDNA studies in which turbidity necessitates the use of multiple filters per sample as well as in the use of eDNA to estimate darter populations. (Oncorhynchus tshawytscha) [17], eastern hellbenders 1. Introduction (Cryptobranchus alleganiensis) [18, 19], great crested newts Since its inception in macroinvertebrate studies in 2008 [1], (Triturus cristatus) [20], the Japanese crayfish (Cambaroides environmental DNA (eDNA) has become firmly established japonicas) [21], and the Trinidad golden tree frog (Phyto- as an effective method [2–4] and holds great promise for triades auratus) [22], among others. Single species presence/ increasing the ease and scope of ecological studies. In aquatic absence eDNA monitoring is a well-established and widely systems, single species eDNA detection has primarily been used technique, in some cases enabling detection of cryptic applied to invasive species monitoring and conservation of species where traditional methods were unsuccessful threatened or endangered species [5]. Invasive species [23, 24]. monitored using eDNA include Asian carp (Hypo- Recent studies also reveal correlations between organ- phthalmichthys sp.) [6] and multiple species of molluscs ismal abundance and eDNA levels [25–28], indicating the [7–9], crustaceans [10–12], amphibians [1, 13, 14], and possibility of quantitative eDNA analysis for population reptiles [15, 16]. Rare and endangered taxa eDNA moni- assessment. Laboratory studies with jack mackerels (Tra- toring has included species such as Chinook salmon churus japonicus) [29], adult sea lampreys (Petromyzon 2 International Journal of Zoology aware of only one other darter eDNA study, in which eDNA marinus) [30], round gobies (Neogobius melanostomus) [31], and common carp (Cyprinus carpio) [25] all report positive was used to detect the presence of the federally endangered slackwater darter (Etheostoma boschungi) [53]. 'e present correlations between eDNA levels and fish density or bio- mass. However, larval lamprey densities showed no corre- study provides data useful to approaching darter eDNA lation with eDNA in tank experiments [30]. Field studies density studies. examining the use of eDNA to quantify tadpole populations eDNA offers great promise as a compliment [54] and of the stream dwelling frog Odorrana splendida report a perhaps in some cases a substitute for traditional field studies significant, but not strong, correlation between biomass and where such studies are difficult or impractical. 'is study eDNA levels [27]. A study examining spawning salmon provides novel data concerning two parameters of great significance to the application of eDNA in field ecology abundance and eDNA found eDNA levels varied with salmon density as well as numerous other factors [28]. studies: Likewise, Lacoursiere-Roussel et al. [32] reported a positive (1) 'e effect of increasing target organism density on but weak correlation between catch per unit effort and lake eDNA levels trout (Salvelinus namaycush) eDNA levels. Additionally, (2) 'e effect of single versus multiple filters to quan- numerous studies have observed seasonal fluctuations in titatively assess eDNA concentrations eDNA, which are often believed to be associated with re- productive behavior [18, 28, 33]. While an emerging un- derstanding supports the quantitative use of eDNA in 2. Methods population assessment, many factors appear to influence 2.1. Fish Collection and Housing. Fantail darters (Etheostoma eDNA release [34] and persistence [35] in the environment, flabellare) were collected from Jessamine Creek (Jessamine and additional studies are needed to validate eDNA as a County, Kentucky) via electrofishing (KYDFW Per- density detection tool across various habitats and species mit#1811153) and acclimated to lab conditions in aged tap [28, 36, 37]. water over seven days prior to transfer to experimental Although quality assurance guidelines concerning tanks. Initial temperature of holding water in the lab eDNA collection and extraction have been established [38], matched stream temperature (6 C) at the time of collection. numerous sample collection and preservation techniques Water temperature was increased 2 C a day for seven days exist, and the technique has been demonstrated to impact ° ° until it reached 20 C, and water was maintained at 20 C detection probability [39–43]. Although both precipitation throughout the remainder of the holding period and the and filtration have been used to capture DNA, filtration has experiment. Fish were housed in a 208-liter tank prior to the been demonstrated to capture more eDNA from water experiment and fed commercially purchased blood worms samples [39, 41, 42] and as such is currently overwhelmingly ad libitum. All applicable international, national, and in- the method of choice in eDNA studies. 'e clogging of filters stitutional guidelines for the ethical care and use of fish were by phytoplankton or suspended sediment is a frequently followed. encountered issue in eDNA collection [25, 38, 44], partic- ularly in turbid, lentic waters [45]. Filter clogging is typically countered by prefiltering [25], using larger filter pore sizes 2.2. eDNA Trials. Four environmentally relevant fantail [25], or by dividing the sample and utilizing several filters to darter biomass levels were calculated based on published minimize clogging [19, 38]. Both prefiltering [46] and in- studies of observed fantail darter densities [55, 56]. Tank creasing filter pore size [42, 47] have been shown to reduce tests for each of the four biomass levels were performed in eDNA recovery. Although dividing a sample among several quadruplicate; a negative control tank was included for each filters is a frequently employed technique [19], we are un- level to assess potential cross contamination between tanks. aware of any studies examining its impact on eDNA efficacy Experimental tanks (38 L) were held in an environmental or detection probability. 'e present study examines the chamber under regulated conditions (12 h day/12 h night, hypothesis that multiple, as opposed to single filter, pro- 20 C, aeration) for 96 hours, and fish were fed blood worms cessing of water samples maintains or improves eDNA ad libitum during the course of the experiment. efficacy. Darters are classified as Etheostomatinae, a subclade of Percidae that contains approximately 250 species endemic to 2.3. eDNA Collection. Takahara et al. [25] and Maruyama eastern North America, comprising more than 20% of North et al. [57] both reported that eDNA initially spiked and then America ichthyofauna [48]. Significant anatomical and reached equilibrium near day four in fish tank tests, so a 96- physiological differences exist between darters and other hour end point was utilized for the present study. At 96 members of Percidae [49, 50]. Darters are frequently in need hours, all darters were removed, and each tank was ho- of study as threatened or endangered species; currently, 27 mogenized by stirring for 1 minute to homogenously dis- darter species are listed as federally threatened or endan- tribute DNA. Two one liter water samples were collected gered on the U.S. Endangered Species List. Fantail darters from each tank using 3.8-liter high-density polyethylene (Etheostoma flabellare), a small, stream dwelling darter, containers not previously exposed to animal DNA and common throughout much of their range in eastern North thoroughly washed with bleach and rinsed with distilled America [51, 52], were selected for this study as easily ac- water. Water samples were processed through a 47 mm cessible members of this wide ranging subclade. We are diameter glass microfiber filter (VWR, 0.42 mm thickness International Journal of Zoology 3 and 0.7 μm pore size) in a manner similar to previous studies study. Environmental DNA was quantified using a StepO- TM [6, 42, 58] with vacuum filtration immediately using one of nePlus Real-Time PCR system (Life Technologies, the two methods: one sample from each tank was filtered Carlsbad, CA, USA) in optical 96-well PCR plates. Each plate through a single filter and the other was divided into three contained no fish control tank samples to assess contami- 333 ml aliquots, each of which was filtered separately. Filters nation and fantail tissue samples as a positive control. Each were extracted immediately. 20 μl reaction contained the following: TaqMan EMM (10 μL), nuclease free water (7 μL), eDNA extract (2 μL), and E. flabellare primer/probe mix (1 μL). 'ermocycler con- 2.4. DNA Extraction. eDNA extraction was performed using ° ° ditions were as follows: 95 C for 10 min, 55 cycles of 95 C for a DNeasy blood and tissue kit (Qiagen), demonstrated to 15 s, and 60 C for one minute. provide superior yields relative to other extraction methods DNA extractions from E. flabellare fin clips were used to [59], and a modified version of a published protocol [54]. generate a standard as standards for the qPCR analysis. We Briefly, whole filters were cut into 30–40 pieces and incu- used a Qubit 4 Fluorometer ('ermo Fisher) and a Qubit 1X bated at 56 C overnight in 720 μl ATL buffer and 80 μl dsDNA HS Assay Kit (Cat. No. Q32850) to quantify the Proteinase K. Final elutions were performed twice into DNA concentrations from the tissue extract. We then di- 400 μl of AE buffer, and the extracted DNA was stored at luted a 7.88 ng/μl fantail darter DNA extract to five levels to −20 C until analysis. use as a standard curve: 0.5 pg/l, 5 pg/l, 50 pg/l, 500 pg/l, and Tissue DNA extraction was performed using a DNeasy 5,000 pg/l. 'ese dilutions cover the range of DNA con- blood and tissue kit (Qiagen) according to the provided centrations that were observed in tank water-extracted protocol. Tissue was lysed overnight at 56 C in proteinase K eDNA in this study. and eluted twice to increase DNA yield. 2.7. Statistical Analysis. Type I linear regression was used to 2.5. Primer Design. Cytochrome b was sequenced (GenBank: examine the relationship of darter biomass and eDNA KT880219.1) from locally collected fantail darters (Jessamine concentration within a single filter method following the Creek, Jessamine County, KY: 37.859380, −84.630755) using previously published methods [44]. Both eDNA concen- published primers [60]. Using this sequence, a species- tration and fish biomass were log transformed following the specific primer probe assay was designed that amplifies a previously published methods [44, 62]. All tests were per- 118 bp amplicon within E. flabellare cytochrome b: formed using IBM SPSS Statistics 25. Forward primer: 5′-AAGCGAAGAAGCGAGTTAGG-3′ 3. Results Reverse primer: 5′-GGTGCTACGGTCATCACTAATC- 3′ Data from the single filter method trial indicated a positive Probe: 5′-6 FAM/CCCACATAA/ZEN/G GCACTG- correlation between darter biomass and eDNA concentra- CAGAGAGT/3IABkFQ-3′ tion (Figure 1(a), p � 0.326, r � 0.08). Interestingly, the multiple filter method demonstrated a significant 'ree other species of the family Percidae occurring (p � 0.034) and stronger correlation (r � 0.30) between sympatrically with E. flabellare in mid-sized central Ken- these same two variables in parallel analysis (Figure 1(b)). tucky streams were considered in the design of the primers Amplification was observed in every tank containing and probes. We sequenced cytochrome b from locally darters, but no amplification was observed in any of the no collected (Jessamine Creek, Jessamine County, KY: fish control tanks, included in each trial to assess potential 37.859380, −84.630755) sympatric specimens using the same transfer of DNA between tanks. PCR efficiency was 97.2% as primer set [60] and designed primers and probes with a determined using a standard curve of diluted E. flabellare minimum of three mismatches in each oligonucleotide (f. tissue. primer, r. primer, and probe) based on reported require- ments for specificity [61]. Sympatric species considered 4. Discussion include Etheostoma caeruleum (KT880220.1), Etheostoma blennioides (KT880218.1), and Percina caprodes 'e few laboratory studies that have been completed ex- (KT880217.1). Nonspecific amplification was tested using amining eDNA and biomass utilize biomass ranges up to 200- tissue extracted DNA from each of these species. fold, much greater than the more environmentally realistic 2-, 4-, and 8-fold differences examined in the present study. In 2.6. eDNA Analysis. Dilution of tank-extracted samples tank studies (200 L) with Japanese mackerel (Trachurus showed evidence of inhibition based on reduced product japonicus) with an average of 6.9 g, 40.2 g, and 319.5 g of total DNA yields despite the use of TaqMan Environmental biomass per tank, eDNA shedding rates differed between all ° ° Master Mix 2.0. Nondiluted samples amplified 9.8% of the groups at 13 C but not at 18, 23, or 28 C [29]. 'e treatments DNA yield observed at the 1 : 20 dilution that was used for in this study represent a 6-fold and 46-fold increase in bio- analysis (data available in Appendix A). A 1 : 20 dilution was mass in the upper two treatments compared to the lower. selected based on previous studies indicating optimal yields Trials with sea lampreys (Petromyzon marinus) reported observed at 1 : 20 dilutions [16] and the lower biomass to significant increases in biomass with 0, 2, 20, and 200 adults water ratios utilized in the present study relative to that per 2000 L tank but not with 0, 1, 5, and 25 larva in 28 L 4 International Journal of Zoology y = 7.3448x + 15.146 y = 3.6611x + 23.72 R = 0.3005 R = 0.0802 0.2 0.7 1.2 1.7 2.2 0.2 0.7 1.2 1.7 2.2 Darter biomass (g/l) Darter biomass (g/l) (a) (b) Figure 1: eDNA concentrations at various biomass levels for single filter method (p � 0.326) (a) and multiple filter method (p � 0.034) (b). aquaria [30]. Assuming homogenous weight of the adult sea 'e positive correlation between biomass and eDNA has lampreys, these treatments exceeded biomass levels in the been previously observed, but not in a small, stream dwelling present study in all but the lowest treatment. Juvenile carp species at environmentally relevant densities. Doi et al. [26] (Cyprinus carpio) trials in 9 L tanks found significant cor- reported positive correlations between Plecoglossus altivelis relations between 16 g, 79 g (5-fold), and 158 g (10-fold) densities as estimated from snorkeling surveys and eDNA biomass and eDNA levels [25], representing similar biomass levels. Klymus et al. [68] observed a positive correlation in level treatments to those utilized in the present experiment. eDNA and biomass in two species of bighead carp (Hypo- 'e results of the present experiment represent the lowest phthalmichthys nobilis and Hypophthalmichthys molitrix). biomass/eDNA relationships tested to date and are consistent Our work builds on these previous efforts by comparing with the results of previous studies, most of which reported environmentally relevant biomass levels with eDNA con- positive correlations between biomass and eDNA. centrations and providing data supporting the validity of 'e relationship between biomass and eDNA levels was utilizing multiple, versus single, filters in field studies. significant and positive for both filter methods, although the r values were not large (0.29 and 0.49). 'ese values are similar to Data Availability those observed by Iwai et al. [27]. When comparing tadpole 'e data used to support the findings of this study are abundance (0.32) and biomass (0.29) to eDNA copy number in available upon request to the corresponding author. lotic samples sites, they observed large variations in the bio- mass-eDNA relationship within a single stream. Likewise, Conflicts of Interest Baldigo et al. [44] reported r values of 0.44 (density) and 0.25 (biomass) in Adirondack mountain headwater brook trout 'e authors declare that they have no conflicts of interest. (Salvelinus fontinalis) populations. 'ese data support a growing data set indicating a loose correlation between or- Acknowledgments ganism abundance and eDNA concentrations. 'e results of the present study support the premise that 'is project was supported by an Undergraduate Research multiple filters improve the resolving capacity of eDNA as Grant (#9118243-Guivas) from the Kentucky Academy of the combined filter method (333 ml per filter) demonstrated Science. a significant relationship between biomass and eDNA, while the same samples processed in a single filter (1 L per filter) Supplementary Materials did not. Hunter et al. [63] report a 10% increase in DNA yield utilizing multiple as opposed to single filters; although Table 1: nondiluted eDNA concentrations for nondiluted this was not visible in our study, it is possible that a larger samples. 'ese data were not used in the publication because sample size would have revealed a similar trend. Filtering they appear to be inhibited relative to 1 : 20 diluted samples. larger volumes of water leads to a greater concentration of Table 2: eDNA concentrations for samples diluted 1 : 20 inhibitors [64], which perhaps could be linked to greater following DNA extraction. Table 3: CT values for samples inconsistency in DNA detection in single filter samples if following a 1 : 20 dilution of extracted DNA. Table 4: CT inhibitors are removed more effectively in the multiple filter values for nondiluted samples. Figure 1: eDNA concentra- process as less inhibitor would be present in each filter tions at various biomass levels for single filter method (A) during the extraction process. Regardless of the mechanism, (p � 0.031) and multiple filter method (B) (p � 0.002) for these results provide evidence that the use of multiple filters nondiluted samples. 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Use of Environmental DNA to Determine Fantail Darter (Etheostoma flabellare) Density in a Laboratory Setting: Effects of Biomass and Filtration Method

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Copyright © 2020 Ramon A. Guivas and Ben F. Brammell. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi International Journal of Zoology Volume 2020, Article ID 4731686, 7 pages https://doi.org/10.1155/2020/4731686 Research Article Use of Environmental DNA to Determine Fantail Darter (Etheostoma flabellare) Density in a Laboratory Setting: Effects of Biomass and Filtration Method Ramon A. Guivas and Ben F. Brammell Department of Science and Health, Asbury University, Wilmore, KY 40390, USA Correspondence should be addressed to Ben F. Brammell; ben.brammell@asbury.edu Received 9 December 2019; Accepted 23 March 2020; Published 17 June 2020 Academic Editor: Andrea Galimberti Copyright © 2020 Ramon A. Guivas and Ben F. Brammell. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Estimating fish abundance/biomass holds great importance for freshwater ecology and fisheries management, but current techniques can be expensive, time-consuming, and potentially harmful to target organisms. Environmental DNA (eDNA) has proven an effective and efficient technique for presence/absence detection of freshwater vertebrates. Additionally, recent studies report correlations between target organism density/biomass and eDNA levels, although widespread application of this technique is limited by the number of studies examining this relationship in various species and settings. Additionally, filter clogging is a commonly encountered issue in eDNA studies in environments with significant sediment and/or phytoplankton algae. Fre- quently, a sample must be split into multiple aliquots and filtered separately in order to process the entire sample. 'e present study examines both the relationship between biomass and eDNA and the effects of single versus multiple filter sampling on eDNA concentrations of fantail darters (Etheostoma flabellare) in a laboratory setting. Tank tests were performed in quadruplicate at four environmentally relevant fantail biomass levels. eDNA samples were collected and processed in parallel (one as a whole through a single filter and one in parts through multiple filters). Species-specific primers and a probe were developed for E. flabellare from cytochrome b sequences obtained from locally collected specimens, and real-time quantitative PCR was used to analyze eDNA levels at each biomass. Significant correlations were observed with increasing biomass for both methods, although this relationship was stronger for samples processed by the multiple filter method. 'ese data should be useful in eDNA studies in which turbidity necessitates the use of multiple filters per sample as well as in the use of eDNA to estimate darter populations. (Oncorhynchus tshawytscha) [17], eastern hellbenders 1. Introduction (Cryptobranchus alleganiensis) [18, 19], great crested newts Since its inception in macroinvertebrate studies in 2008 [1], (Triturus cristatus) [20], the Japanese crayfish (Cambaroides environmental DNA (eDNA) has become firmly established japonicas) [21], and the Trinidad golden tree frog (Phyto- as an effective method [2–4] and holds great promise for triades auratus) [22], among others. Single species presence/ increasing the ease and scope of ecological studies. In aquatic absence eDNA monitoring is a well-established and widely systems, single species eDNA detection has primarily been used technique, in some cases enabling detection of cryptic applied to invasive species monitoring and conservation of species where traditional methods were unsuccessful threatened or endangered species [5]. Invasive species [23, 24]. monitored using eDNA include Asian carp (Hypo- Recent studies also reveal correlations between organ- phthalmichthys sp.) [6] and multiple species of molluscs ismal abundance and eDNA levels [25–28], indicating the [7–9], crustaceans [10–12], amphibians [1, 13, 14], and possibility of quantitative eDNA analysis for population reptiles [15, 16]. Rare and endangered taxa eDNA moni- assessment. Laboratory studies with jack mackerels (Tra- toring has included species such as Chinook salmon churus japonicus) [29], adult sea lampreys (Petromyzon 2 International Journal of Zoology aware of only one other darter eDNA study, in which eDNA marinus) [30], round gobies (Neogobius melanostomus) [31], and common carp (Cyprinus carpio) [25] all report positive was used to detect the presence of the federally endangered slackwater darter (Etheostoma boschungi) [53]. 'e present correlations between eDNA levels and fish density or bio- mass. However, larval lamprey densities showed no corre- study provides data useful to approaching darter eDNA lation with eDNA in tank experiments [30]. Field studies density studies. examining the use of eDNA to quantify tadpole populations eDNA offers great promise as a compliment [54] and of the stream dwelling frog Odorrana splendida report a perhaps in some cases a substitute for traditional field studies significant, but not strong, correlation between biomass and where such studies are difficult or impractical. 'is study eDNA levels [27]. A study examining spawning salmon provides novel data concerning two parameters of great significance to the application of eDNA in field ecology abundance and eDNA found eDNA levels varied with salmon density as well as numerous other factors [28]. studies: Likewise, Lacoursiere-Roussel et al. [32] reported a positive (1) 'e effect of increasing target organism density on but weak correlation between catch per unit effort and lake eDNA levels trout (Salvelinus namaycush) eDNA levels. Additionally, (2) 'e effect of single versus multiple filters to quan- numerous studies have observed seasonal fluctuations in titatively assess eDNA concentrations eDNA, which are often believed to be associated with re- productive behavior [18, 28, 33]. While an emerging un- derstanding supports the quantitative use of eDNA in 2. Methods population assessment, many factors appear to influence 2.1. Fish Collection and Housing. Fantail darters (Etheostoma eDNA release [34] and persistence [35] in the environment, flabellare) were collected from Jessamine Creek (Jessamine and additional studies are needed to validate eDNA as a County, Kentucky) via electrofishing (KYDFW Per- density detection tool across various habitats and species mit#1811153) and acclimated to lab conditions in aged tap [28, 36, 37]. water over seven days prior to transfer to experimental Although quality assurance guidelines concerning tanks. Initial temperature of holding water in the lab eDNA collection and extraction have been established [38], matched stream temperature (6 C) at the time of collection. numerous sample collection and preservation techniques Water temperature was increased 2 C a day for seven days exist, and the technique has been demonstrated to impact ° ° until it reached 20 C, and water was maintained at 20 C detection probability [39–43]. Although both precipitation throughout the remainder of the holding period and the and filtration have been used to capture DNA, filtration has experiment. Fish were housed in a 208-liter tank prior to the been demonstrated to capture more eDNA from water experiment and fed commercially purchased blood worms samples [39, 41, 42] and as such is currently overwhelmingly ad libitum. All applicable international, national, and in- the method of choice in eDNA studies. 'e clogging of filters stitutional guidelines for the ethical care and use of fish were by phytoplankton or suspended sediment is a frequently followed. encountered issue in eDNA collection [25, 38, 44], partic- ularly in turbid, lentic waters [45]. Filter clogging is typically countered by prefiltering [25], using larger filter pore sizes 2.2. eDNA Trials. Four environmentally relevant fantail [25], or by dividing the sample and utilizing several filters to darter biomass levels were calculated based on published minimize clogging [19, 38]. Both prefiltering [46] and in- studies of observed fantail darter densities [55, 56]. Tank creasing filter pore size [42, 47] have been shown to reduce tests for each of the four biomass levels were performed in eDNA recovery. Although dividing a sample among several quadruplicate; a negative control tank was included for each filters is a frequently employed technique [19], we are un- level to assess potential cross contamination between tanks. aware of any studies examining its impact on eDNA efficacy Experimental tanks (38 L) were held in an environmental or detection probability. 'e present study examines the chamber under regulated conditions (12 h day/12 h night, hypothesis that multiple, as opposed to single filter, pro- 20 C, aeration) for 96 hours, and fish were fed blood worms cessing of water samples maintains or improves eDNA ad libitum during the course of the experiment. efficacy. Darters are classified as Etheostomatinae, a subclade of Percidae that contains approximately 250 species endemic to 2.3. eDNA Collection. Takahara et al. [25] and Maruyama eastern North America, comprising more than 20% of North et al. [57] both reported that eDNA initially spiked and then America ichthyofauna [48]. Significant anatomical and reached equilibrium near day four in fish tank tests, so a 96- physiological differences exist between darters and other hour end point was utilized for the present study. At 96 members of Percidae [49, 50]. Darters are frequently in need hours, all darters were removed, and each tank was ho- of study as threatened or endangered species; currently, 27 mogenized by stirring for 1 minute to homogenously dis- darter species are listed as federally threatened or endan- tribute DNA. Two one liter water samples were collected gered on the U.S. Endangered Species List. Fantail darters from each tank using 3.8-liter high-density polyethylene (Etheostoma flabellare), a small, stream dwelling darter, containers not previously exposed to animal DNA and common throughout much of their range in eastern North thoroughly washed with bleach and rinsed with distilled America [51, 52], were selected for this study as easily ac- water. Water samples were processed through a 47 mm cessible members of this wide ranging subclade. We are diameter glass microfiber filter (VWR, 0.42 mm thickness International Journal of Zoology 3 and 0.7 μm pore size) in a manner similar to previous studies study. Environmental DNA was quantified using a StepO- TM [6, 42, 58] with vacuum filtration immediately using one of nePlus Real-Time PCR system (Life Technologies, the two methods: one sample from each tank was filtered Carlsbad, CA, USA) in optical 96-well PCR plates. Each plate through a single filter and the other was divided into three contained no fish control tank samples to assess contami- 333 ml aliquots, each of which was filtered separately. Filters nation and fantail tissue samples as a positive control. Each were extracted immediately. 20 μl reaction contained the following: TaqMan EMM (10 μL), nuclease free water (7 μL), eDNA extract (2 μL), and E. flabellare primer/probe mix (1 μL). 'ermocycler con- 2.4. DNA Extraction. eDNA extraction was performed using ° ° ditions were as follows: 95 C for 10 min, 55 cycles of 95 C for a DNeasy blood and tissue kit (Qiagen), demonstrated to 15 s, and 60 C for one minute. provide superior yields relative to other extraction methods DNA extractions from E. flabellare fin clips were used to [59], and a modified version of a published protocol [54]. generate a standard as standards for the qPCR analysis. We Briefly, whole filters were cut into 30–40 pieces and incu- used a Qubit 4 Fluorometer ('ermo Fisher) and a Qubit 1X bated at 56 C overnight in 720 μl ATL buffer and 80 μl dsDNA HS Assay Kit (Cat. No. Q32850) to quantify the Proteinase K. Final elutions were performed twice into DNA concentrations from the tissue extract. We then di- 400 μl of AE buffer, and the extracted DNA was stored at luted a 7.88 ng/μl fantail darter DNA extract to five levels to −20 C until analysis. use as a standard curve: 0.5 pg/l, 5 pg/l, 50 pg/l, 500 pg/l, and Tissue DNA extraction was performed using a DNeasy 5,000 pg/l. 'ese dilutions cover the range of DNA con- blood and tissue kit (Qiagen) according to the provided centrations that were observed in tank water-extracted protocol. Tissue was lysed overnight at 56 C in proteinase K eDNA in this study. and eluted twice to increase DNA yield. 2.7. Statistical Analysis. Type I linear regression was used to 2.5. Primer Design. Cytochrome b was sequenced (GenBank: examine the relationship of darter biomass and eDNA KT880219.1) from locally collected fantail darters (Jessamine concentration within a single filter method following the Creek, Jessamine County, KY: 37.859380, −84.630755) using previously published methods [44]. Both eDNA concen- published primers [60]. Using this sequence, a species- tration and fish biomass were log transformed following the specific primer probe assay was designed that amplifies a previously published methods [44, 62]. All tests were per- 118 bp amplicon within E. flabellare cytochrome b: formed using IBM SPSS Statistics 25. Forward primer: 5′-AAGCGAAGAAGCGAGTTAGG-3′ 3. Results Reverse primer: 5′-GGTGCTACGGTCATCACTAATC- 3′ Data from the single filter method trial indicated a positive Probe: 5′-6 FAM/CCCACATAA/ZEN/G GCACTG- correlation between darter biomass and eDNA concentra- CAGAGAGT/3IABkFQ-3′ tion (Figure 1(a), p � 0.326, r � 0.08). Interestingly, the multiple filter method demonstrated a significant 'ree other species of the family Percidae occurring (p � 0.034) and stronger correlation (r � 0.30) between sympatrically with E. flabellare in mid-sized central Ken- these same two variables in parallel analysis (Figure 1(b)). tucky streams were considered in the design of the primers Amplification was observed in every tank containing and probes. We sequenced cytochrome b from locally darters, but no amplification was observed in any of the no collected (Jessamine Creek, Jessamine County, KY: fish control tanks, included in each trial to assess potential 37.859380, −84.630755) sympatric specimens using the same transfer of DNA between tanks. PCR efficiency was 97.2% as primer set [60] and designed primers and probes with a determined using a standard curve of diluted E. flabellare minimum of three mismatches in each oligonucleotide (f. tissue. primer, r. primer, and probe) based on reported require- ments for specificity [61]. Sympatric species considered 4. Discussion include Etheostoma caeruleum (KT880220.1), Etheostoma blennioides (KT880218.1), and Percina caprodes 'e few laboratory studies that have been completed ex- (KT880217.1). Nonspecific amplification was tested using amining eDNA and biomass utilize biomass ranges up to 200- tissue extracted DNA from each of these species. fold, much greater than the more environmentally realistic 2-, 4-, and 8-fold differences examined in the present study. In 2.6. eDNA Analysis. Dilution of tank-extracted samples tank studies (200 L) with Japanese mackerel (Trachurus showed evidence of inhibition based on reduced product japonicus) with an average of 6.9 g, 40.2 g, and 319.5 g of total DNA yields despite the use of TaqMan Environmental biomass per tank, eDNA shedding rates differed between all ° ° Master Mix 2.0. Nondiluted samples amplified 9.8% of the groups at 13 C but not at 18, 23, or 28 C [29]. 'e treatments DNA yield observed at the 1 : 20 dilution that was used for in this study represent a 6-fold and 46-fold increase in bio- analysis (data available in Appendix A). A 1 : 20 dilution was mass in the upper two treatments compared to the lower. selected based on previous studies indicating optimal yields Trials with sea lampreys (Petromyzon marinus) reported observed at 1 : 20 dilutions [16] and the lower biomass to significant increases in biomass with 0, 2, 20, and 200 adults water ratios utilized in the present study relative to that per 2000 L tank but not with 0, 1, 5, and 25 larva in 28 L 4 International Journal of Zoology y = 7.3448x + 15.146 y = 3.6611x + 23.72 R = 0.3005 R = 0.0802 0.2 0.7 1.2 1.7 2.2 0.2 0.7 1.2 1.7 2.2 Darter biomass (g/l) Darter biomass (g/l) (a) (b) Figure 1: eDNA concentrations at various biomass levels for single filter method (p � 0.326) (a) and multiple filter method (p � 0.034) (b). aquaria [30]. Assuming homogenous weight of the adult sea 'e positive correlation between biomass and eDNA has lampreys, these treatments exceeded biomass levels in the been previously observed, but not in a small, stream dwelling present study in all but the lowest treatment. Juvenile carp species at environmentally relevant densities. Doi et al. [26] (Cyprinus carpio) trials in 9 L tanks found significant cor- reported positive correlations between Plecoglossus altivelis relations between 16 g, 79 g (5-fold), and 158 g (10-fold) densities as estimated from snorkeling surveys and eDNA biomass and eDNA levels [25], representing similar biomass levels. Klymus et al. [68] observed a positive correlation in level treatments to those utilized in the present experiment. eDNA and biomass in two species of bighead carp (Hypo- 'e results of the present experiment represent the lowest phthalmichthys nobilis and Hypophthalmichthys molitrix). biomass/eDNA relationships tested to date and are consistent Our work builds on these previous efforts by comparing with the results of previous studies, most of which reported environmentally relevant biomass levels with eDNA con- positive correlations between biomass and eDNA. centrations and providing data supporting the validity of 'e relationship between biomass and eDNA levels was utilizing multiple, versus single, filters in field studies. significant and positive for both filter methods, although the r values were not large (0.29 and 0.49). 'ese values are similar to Data Availability those observed by Iwai et al. [27]. When comparing tadpole 'e data used to support the findings of this study are abundance (0.32) and biomass (0.29) to eDNA copy number in available upon request to the corresponding author. lotic samples sites, they observed large variations in the bio- mass-eDNA relationship within a single stream. Likewise, Conflicts of Interest Baldigo et al. [44] reported r values of 0.44 (density) and 0.25 (biomass) in Adirondack mountain headwater brook trout 'e authors declare that they have no conflicts of interest. (Salvelinus fontinalis) populations. 'ese data support a growing data set indicating a loose correlation between or- Acknowledgments ganism abundance and eDNA concentrations. 'e results of the present study support the premise that 'is project was supported by an Undergraduate Research multiple filters improve the resolving capacity of eDNA as Grant (#9118243-Guivas) from the Kentucky Academy of the combined filter method (333 ml per filter) demonstrated Science. a significant relationship between biomass and eDNA, while the same samples processed in a single filter (1 L per filter) Supplementary Materials did not. Hunter et al. [63] report a 10% increase in DNA yield utilizing multiple as opposed to single filters; although Table 1: nondiluted eDNA concentrations for nondiluted this was not visible in our study, it is possible that a larger samples. 'ese data were not used in the publication because sample size would have revealed a similar trend. Filtering they appear to be inhibited relative to 1 : 20 diluted samples. larger volumes of water leads to a greater concentration of Table 2: eDNA concentrations for samples diluted 1 : 20 inhibitors [64], which perhaps could be linked to greater following DNA extraction. Table 3: CT values for samples inconsistency in DNA detection in single filter samples if following a 1 : 20 dilution of extracted DNA. Table 4: CT inhibitors are removed more effectively in the multiple filter values for nondiluted samples. Figure 1: eDNA concentra- process as less inhibitor would be present in each filter tions at various biomass levels for single filter method (A) during the extraction process. Regardless of the mechanism, (p � 0.031) and multiple filter method (B) (p � 0.002) for these results provide evidence that the use of multiple filters nondiluted samples. 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International Journal of ZoologyHindawi Publishing Corporation

Published: Jun 17, 2020

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