Defensible standardized ploidy assessments for Grass Carp (Ctenopharyngodon idella, Cyprinidae) intercepted from the commercial supply chain

Journal of Great Lakes Research 45 (2019) 371–383

Contents lists available at ScienceDirect

Journal of Great Lakes Research journal homepage: www.elsevier.com/locate/jglr

Defensible standardized ploidy assessments for Grass Carp (Ctenopharyngodon idella, Cyprinidae) intercepted from the commercial supply chain Jill A. Jenkins a,⁎, Megan D. Chauvin a, Darren Johnson b, Bonnie L. Brown c,1, Jennifer Bailey d, Anita M. Kelly e, Bryan T. Kinter f a

U.S. Geological Survey, Wetland and Aquatic Research Center, 700 Cajundome Blvd., Lafayette, LA 70506, United States of America Cherokee Nation, 700 Cajundome Blvd., Lafayette, LA 70506, United States of America c Virginia Commonwealth University, Department of Biology, 1000 West Cary Street, Richmond, VA 23284, United States of America d U.S. Fish & Wildlife Service, La Crosse Fish Health Center, Midwest Fisheries Center, 555 Lester Avenue, Onalaska, WI 54650, United States of America e University of Arkansas at Pine Bluff, Lonoke Agriculture Center, 2001 Highway 70 East, Lonoke, AR 72086, United States of America f Ohio Department of Natural Resources, Division of Wildlife, 952 Lima Avenue, Findlay, OH 45840, United States of America b

a r t i c l e

i n f o

Article history: Received 20 April 2018 Accepted 20 November 2018 Available online 16 January 2019 Communicated by: Wendylee Stott Keywords: Triploid Asian carp Flow cytometry Nile Tilapia Standard beads Eyeball

a b s t r a c t Although methods are in place through the U.S. Fish and Wildlife (USFWS) program for ploidy testing of feral caught Grass Carp (Ctenopharyngodon idella) and black carp (Mylopharyngodon piceus), no guidelines exist for carp hauled across state lines. Using 1200 Grass Carp purchased by undercover Ohio law enforcement during 2015–2016, we developed a standardized protocol for discriminating ploidy by using two parameters, nuclear size and DNA content. Bead standards at 2 μm or 4 μm were used to establish nuclear size from Nile Tilapia (Oreochromis niloticus), known diploid (n = 20) and triploid (n = 20) Grass Carp blood, and cells derived from eyeballs of purchased field carp. The control for establishing DNA content was cryopreserved or fresh tilapia blood (2.40 pg). Time postmortem indicated nuclear size was similar over 4 days, but DNA quality from triploids was best at 24 h. Occasionally, only size or DNA content was measurable. Tilapia mean nuclear size (n = 501 samples) was 4.61 μm (SE 0.05) (R2 = 0.94). Known diploid and triploid blood nuclear sizes, compared with tilapia size, were 3.62 μm (SE 0.13) (R2 = 0.96) and 7.58 μm (SE 0.27) (R2 = 0.96), respectively. Mean field carp eye nuclear size was 5.83 μm (SE 0.13). Mean DNA content of cells from field carp eyes (n = 698 fish) was 3.51 pg (SE 0.06). No diploid Grass Carp were detected in the USFWS certified triploid Grass Carp transports. This standard protocol reliably discriminates ploidy and can be used for enforcement of regulations that differ among state jurisdictions. Published by Elsevier B.V. on behalf of International Association for Great Lakes Research.

Introduction Since being imported into the United States in 1963, triploid Grass Carp (Ctenopharyngodon idella) have been produced and widely stocked for biocontrol of nuisance aquatic macrophytes (Malone, 1984; Rasmussen, 2011). Underscoring their importance as an environmental management tool, since the mid-1980s the U.S. Fish and Wildlife Service's National Triploid Grass Carp Inspection and Certification Program (USFWS; NTGCICP) has permitted and certified triploid shipments to states where non-reproductive triploid Grass Carp stocking is ⁎ Corresponding author. E-mail address: [email protected] (J.A. Jenkins). 1 Current address: University of New Hampshire, Department of Biological Sciences, 38 Academic Way, Durham, NH 03824.

allowed (Mitchell and Kelly, 2006; Rasmussen, 2011) (Fig. 1; ESM Table S1). Despite these certifications, diploid Grass Carp have established breeding populations throughout much of the Mississippi River basin and the Trinity River near the Texas Gulf Coast, have recruited within the Lake Erie Basin, and they now occur in Lake Michigan (Chapman et al., 2013; Cudmore et al., 2017; Embke et al., 2016; USFWS, 2015; USGS, 2018; Wieringa et al., 2017). Grass Carp are native to large rivers in temperate latitudes (east Asia), with species distribution models predicting suitable climate in all the Great Lakes, and the Sandusky River within the Lake Erie Basin is a site of natural reproduction (Bain, 1993; Chapman et al., 2013; Wittmann et al., 2014). The Great Lakes fisheries are valued at more than $7 billion annually and the lakes provide drinking water for 40 million people; detrimental ecological effects caused by Grass Carp could influence these ecological services (Baldwin et al., 2018; Buck et al.,

https://doi.org/10.1016/j.jglr.2018.12.004 0380-1330/Published by Elsevier B.V. on behalf of International Association for Great Lakes Research.

372

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

Fig. 1. Type of Grass Carp allowed for each state. Figure modified from MICRA (2015) and Rasmussen (2011).

2010; Embke et al., 2016). Lake Erie is of special concern because it is relatively shallow, warm, and yields more commercial harvest than all the other lakes combined (Rapai, 2016; Baldwin et al., 2018). Ecological impacts of non-native freshwater fish can occur at several levels of biological organization, including the genetic, individual, population, community, and ecosystem levels, with a primary ecosystem effect of Grass Carp being removal of submerged aquatic macrophytes (Cucherousset and Olden, 2011; Chapman et al., 2013). Grass Carp can alter nearshore vegetated areas and wetlands, as well as fish community structure. Native planktivore body condition is reduced, shorelines can erode, and Asian tapeworm (Schyzocotyle acheilognathi) can be transmitted to wild fish (Bettoli et al., 1993; Chapman et al., 2013; Irons et al., 2007; Cucherousset and Olden, 2011). Self-sustaining populations of Grass Carp in North America may be due to escapement during the 1960's, unregulated stocking of diploids in the 1970s, escapees from legal diploid use (MICRA, 2015), or compromises in the certified triploid supply chain. The commercial production of 100% ploidy-tested triploids began with USFWS performing triploid verifications from 1985 to 1995 as a service to the receiving states (Mitchell and Kelly, 2006). In 1995, Public Law 104-40 (109 Stat. 350 approved November 1, 1995 allowed the NTGCICP to charge fees for certification inspections of triploid Grass Carp aquaculture facilities and established non-conformance violation schedules (https://www.fws.gov/warmsprings/FishHealth/frgrscrp. html; accessed 24 January 2018), adding to the incentives for fish producers to abide by federal guidelines. The verification and certification are performed at the fish production facility before shipment by a Coulter counter protocol based on erythrocyte nuclear volume (Wattendorf, 1986), with no further screenings. The USFWS-certified triploid Grass Carp supply chain, or illegal importations occurring outside these shipments, can be investigated by law enforcement. However, because the NTGCICP does not have a law enforcement component, inspection of Grass Carp shipments and enforcement of consequent regulations are dependent upon the receiving states (Conover et al., 2007). Resource managers in states can monitor permitted stockings of nonreproductive triploid Grass Carp, but most often they do not have the resources to provide the law enforcement necessary to confirm the accuracy of the certificates in the haulers' hands or to confiscate fish for ploidy testing to assure no diploid carp occur in the shipment (MICRA, 2015; Schultz et al., 2001). To discriminate feral caught diploids and triploids from public waters, the USFWS performs a uniform, unambiguous procedure that is currently available to all the states (https://www.fws.gov/midwest/ WGL/programs.html; accessed 10 October 2018) (Jenkins and Thomas, 2007). This flow cytometric method is based solely on the DNA content in nuclei of cells originating in the vitreous humor of

eyeballs in relation to a known DNA content control (Jenkins et al., 2017; Jenkins and Thomas, 2007). An advantage of flow cytometry over the Coulter counter method includes reliable ploidy determinations through 8 d postmortem using intact cells derived from an immunologically protected site, with the most accurate DNA ploidy assessment technology (Jenkins and Thomas, 2007). This standardized method for determining ploidy, considered as indicative of reproductive status whereby triploids are assumed to be sterile, has been useful for meeting annual federal reporting requirements, tracking fish movements, and forecasting expansion of species distribution (Conover et al., 2007; Cudmore et al., 2017; Fuller and Neilson, 2015; Wittmann et al., 2014). Currently however, no operative and universal ploidy discrimination guidelines are available for state-intercepted shipments of produced triploid carp, typically much smaller than feral fish. Since ploidy discriminations began in 2004, feral Grass Carp have averaged 11 kg (SE 0.5) total weight, over 40 times heavier than the transported fish (Table 1; Fig. 2a). The smaller fish, with eyes b 1/30 the weight of eyes from feral caught Grass Carp (Table 1; Fig. 2a), contain fewer cells for analyses, thus placing methodological constraints on the feral carp USFWS standard operating procedure (SOP). A ploidy testing procedure developed and operated outside the NTGCICP has been recommended as national policy for triploids entering triploid-only states to prevent unintentional and illegal introductions of diploid and triploid Grass Carp (MICRA, 2015). Adaptations of the current USFWS feral carp SOP could be applied to the smaller (starting at ~150 mm; Chapman et al., 2013; Table 1), hauled fish for application in a regulatory context to pinpoint possible breaches in the permitted triploid disseminations. The cytological methods being applied to feral carp are relevant for each of the produced species of Asian carp, the grass- and black carp (Mylopharyngodon piceus), because Grass Carp are, and black carp have been, certified by the NTGCICP for sale and shipment nationally and internationally. Similarly, methods for reliably determining ploidy would be a valuable improvement for intercepted grass- and black carp shipments, but protocols have not yet been instituted. Because laws surrounding permissions on produced Asian carp, as well as the start dates for regulating them, vary among the American states (Fig. 1; Electronic Supplementary Material Table S1), ploidy determinations on carp transported across state lines would allow states to be proactive about lessening the potential effects of invasive carp on ecosystem services, and can be especially effective when directed at the small jurisdictional scale (Peters and Lodge, 2009). Focus could be directed at the land transportation and live sales routes, both pathways with the potential to allow diploid Grass Carp to spread over great distances and across political boundaries (Rasmussen, 2011). The objective of this study, performed in concert with a large-scale investigation into the Grass Carp supply chain, was to develop and delineate a reliable, robust SOP for yielding accurate ploidy discriminations for the small-sized, transported Grass Carp. Working with the Division of Wildlife of the Ohio Department of National Resources, the state of Virginia, and a major Grass Carp producer in Arkansas, standardizations were achieved in determining ploidy from intercepted Grass Carp. Testing known diploid and triploid Grass Carp, as well as 1200 fish from the field, enabled the development of standardized field (Kinter et al., 2018) and laboratory methods, and was supported by robust statistics. Methods Throughout 2015 and 2016, NTGCICP certified triploid Grass Carp were purchased from 16 distributors in Ohio by undercover Ohio Department of Natural Resources law enforcement officers (Kinter et al., 2018). Each year, 40 shipments were sent overnight to the USGS Wetland and Aquatic Research Center (WARC), Lafayette, Louisiana, for a total of 1200 Grass Carp sent for ploidy assessment (n = 15 individuals in a single batch shipment). In spring 2015, for laboratory technique

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

373

Table 1 Comparison of total fish weights, lengths, and eyeball weights (standard error) of feral-caught Asian carpa and NTGCICPb- certified hauled Grass Carp in the state of Ohio (2015–2016). Fish weight (g)

Grass Carp (n = 199) Black carp (n = 57) Ohio carp (n = 1200) a b c

Fish length (mm)

Mean (SE)

Range

11,043 (597) 7333 (935) 255 (10)

272–31,890 7–34,500 31–832

Mean (SE) 894 (15) 801 (37) 289 (4)

Eye weight (g) Range

Mean (SE)

Range

285–1260 95–1380 151–462

8 (0.5) 7 (1) 0.25 (0.01)c

1–14 2–23 0.12–0.42

Data were obtained from the database started at the inception of USFWS feral carp ploidy program, 2004. U.S. Fish and Wildlife Service's National Triploid Grass Carp Inspection and Certification Program (NTGCICP). Eye weights (n = 80) were from known diploid and triploid Grass Carp used in a preliminary experiment that were shipped directly from one of the producers.

optimizations, controlled, preliminary experiments (below) were performed by using known triploid and diploid Grass Carp that had been tested by Coulter counter and sent from producers J.M. Malone and Son, Inc., Lonoke, Arkansas. Tilapia blood control Because Nile Tilapia (Oreochromis niloticus) DNA content value (2.40 pg) falls between the diploid and triploid Grass Carp (Chapman et al., 2013), blood (fresh or preserved) from Nile Tilapia was used as an external control (analyzed in a separate tube) for ploidy

discriminations with each field-collected fish. To reduce resources needed for live tilapia maintenance and containment and to investigate a blood standard for ploidy discriminations for use in locales where temperature windows are unsuitable for tilapia, a cryopreservation method was developed for tilapia blood. Freshly collected tilapia blood in acid citrate dextrose (ACD) was diluted by 55% or 33% with Hank's buffered salt solution (HBSS at 311 mosm/kg) (Jenkins et al., 2017). These cell suspensions were diluted with 15%, 20%, and 25% dimethylsulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO), then maintained at room temperature for 10 min prior to freezing at −20 °C. Upon thawing, each sample at 106 cells/mL was stained and analyzed

Fig. 2. Grass Carp samples obtained from (a) a hauled shipment in Ohio (field-collected for this study) and one feral Grass Carp from a separate study that arrived at the laboratory by overnight shipments on May 4, 2015 for ploidy determinations. The dissected eyeballs of the small hauled carp (arrowhead) and feral carp were of demonstrably different sizes; (b) Blood from hauled Grass Carp was obtained from the lanced isthmus (arrow) and then placed in preservative; (c) An eye from a hauled Grass Carp dissected in the field showed internal bleeding likely due to the dissection process. The needle insertion site for vitreous humor withdrawal is indicated (arrowhead). (d) Ophthalmic blood vessels (arrowhead) the near optic nerve were a source of cells from Grass Carp eyes excised in the field.

374

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

using flow cytometry (below) with fresh koi (Cyprinus carpio) blood as an internal control (analyzed in the same tube) having DNA content of 3.4 pg (SE 0.01) (Tiersch et al., 1989).

Field Sampling. Grass Carp collections in 2015 began April 2 and ended September 28; and in 2016, collections began March 23 and ended October 18. Total fish weights and lengths of the 1200 Grass Carp were recorded prior to processing fish for overnight shipment. Upon arrival to the WARC, chain of custody information was confirmed and shipment conditions were documented. In 2015, the tissue types per fish collected in the field included blood fixed in a formalinized saline, dissected eye in normal saline in a container, and the opposing eye still within the head. After 10 shipments and analyses, only eyes in saline and the eyes within heads were collected from the field (Fig. 2a). Fixed blood To test the potential use of ploidy discriminations using preserved blood samples, between 12.5 and 40 μL of fresh blood was collected from the isthmus of the live carp after wiping the area clean with a 70% isopropyl alcohol pad. The site was nicked with a sterile blood lancet (Nelson, Birkenstrasse, Switzerland) (Fig. 2b), blood collected in a pipette (Microsafe tube, Safe-Tec Clinical Products, Ivyland, PA), then immediately dispensed into a self-standing screw-cap tube with 760 μL of fish physiological saline with formalin (FPSF) [0.1 M NaCl, 1 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 0.6 mM KH2PO4, and 1 mM NaHCO3 made to 5% formalin after adjusting the pH to 8.0 (Brown et al., 2000)]. The fixed blood suspension was vigorously mixed to ensure at least a 1:20 dilution (v/v) and held at or below room temperature until analysis.

Eyes One eye (Fig. 2c) was removed by cutting the tissues around the circumference, using both straight and curved blade scalpels, taking care not to puncture the eye. The optic nerve was cut to leave several mm remaining (Fig. 2d) because a blood vessel parallel to the nerve was a source of cells for analyses at WARC. Eyes were placed in individual, wide-mouth sterile plastic 100 mL containers with 0.9% sodium chloride isotonic saline (w/v) (Ricca Chemical Company, Arlington, TX) filled to the top to minimize shaking during shipment. The container and corresponding fish head were shipped together in a double sealed plastic bag with the fish identifying information. Batches were shipped overnight to WARC on ice at 4 °C–8 °C in coolers stuffed with paper in empty spaces to minimize sample jostling. Because solution osmolality may influence cell condition, saline osmolality was measured in 2016 (carp collections #41–80) upon arrival by using a vapor pressure osmometer (Wescor Corp., Logan, UT).

Preliminary experiments In June 2015, samples from 20 verified diploid- and 20 verified triploid Grass Carp were shipped overnight to WARC. The shipment included fish heads, fresh carp blood collected in ACD then placed in Streck Cell Preservative™ (Streck, Omaha, NE) (Olivier and Jenkins, 2015), and fixed carp blood in FPSF. For flow cytometry (see below), eyes were dissected and cells from vitreous humor prepared and incubated at 24 °C for 20 min (Jenkins and Thomas, 2007); the fixed blood was processed (Brown et al., 2000) and incubated at 37 °C for 20 min, or overnight at 4 °C. Each of the cell sources from the known diploid and triploid fish were collected per day, over four days, to simulate potential shipping scenarios with variable arrival times and the consequent potential changes to sample DNA quality and nuclear size.

Laboratory processing No more than five field samples were processed simultaneously, with four flow cytometry tubes assigned per eye: external tilapia control, blood from optic nerve vessel, and two replicates of cells from the vitreous humor (see ESM Appendix S1). Thus, individuals were assigned an external control of tilapia blood (either cryopreserved or freshly collected) with a known genome size of 2.40 pg, to which each of the three carp replicates was compared. Blood from the optic nerve blood vessel (Fig. 2d) was collected by using a 1–10 μL pipette, and eye cells were collected by using an insulin needle and syringe (27 G × 5/8″; Becton Dickinson, Franklin Lakes, NJ) after injecting 50–100 μL of both air and ACD containing 0.2% Triton-X100 (Sigma Aldrich, St. Louis, MO) near the rim of the sclera (Fig. 2c). To dislodge cells, eyeballs were vigorously vortexed in a 5 mL screw top vial containing HBSS for 5 s, then held for 5 min at 24 °C; cells were removed with the same syringe used for injection and placed in 125 μL propidium iodide staining solution (PI) (Jenkins and Thomas, 2007). A second withdrawal was used for the third replicate. To each tube, 0.5 μL of either 4.0 μm or 2.0 μm flow cytometry size calibration beads (F13838; Invitrogen, Carlsbad, CA) were added. The tubes were gently vortexed and incubated at 24 °C for 25 min in the dark and then analyzed by flow cytometry (see ESM Appendix S1). Flow cytometry FACSCalibur (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, CA) calibration was performed with DNA QC Particles and FACSComp (BDIS), and fluorochrome excitation was with a 488 nm laser. Cells in PI solution were run at low speed, with no more than 300 events per second and approximately 10,000 total nuclei per replicate were analyzed (Jenkins and Thomas, 2007; see ESM Appendix S1). The SSC (side scatter) parameter was set to allow visualization of bead singlets. The primary threshold parameter for event collection was FSC (forward scatter, or size), and the secondary was FL2-A (area of nuclei; fluorescence 585 nm/42 band pass), and data were collected in doublet discrimination mode with linear parameters. Peak DNA channels (FL2A), showing the maximum number of events in a histogram, were determined for tilapia and carp nuclei, whereby carp DNA content (pg) can be calculated relative to the known tilapia value at 2.40 pg (Chapman et al., 2013). The nuclear size parameter in arbitrary units was recorded for the 2 μm and 4 μm bead standards as well as the main populations of tilapia- and carp nuclei (Fig. 3). Size data (i.e., μm) were recorded with x-axis geometric (Geo) means, preferable for non-homogeneous populations such as elliptical nuclei. Data were acquired, and main nuclei populations gated by using CellQuest (BDIS) and analyzed by using CellQuest and FlowJo software (TreeStar, Ashland, OR); histograms of DNA content from known diploid and triploid cells in the preliminary experiment (Fig. 4), and selected field sample replicates were analyzed by using Modfit LT software (v 2.0) (Verity Software House, Topsham, ME) to calculate peak width, or coefficient of variation (CV), with wider peaks indicative of fragmentation level (Jenkins, 2011). Statistical analyses DNA content was calculated by comparing histogram peak channels of corresponding tilapia (2.40 pg) to carp peak channels. In order to determine if the DNA parameter was influenced by the amount of time through 4 days in cold storage from eye collection to flow cytometric analysis, data from the preliminary experiment were used; the CVs from each eye were rank transformed. All models were checked for homogeneity and normality. Tukey's Studentized Range Test was performed to compare means. Similarly, to determine if nuclear size was influenced by the amount of time in cold storage, regression analyses of ploidy (diploid or triploid) was run with the responses of carp Geo

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

375

Fig. 3. Flow cytograms of external control Nile Tilapia Oreochromis niloticus blood (a and b) stained for DNA fluorescence level by using propidium iodide solution showing a histogram peak CV of 2.89 (a) and nuclear size (thin arrow) at 222 Geo mean compared with 2 μm (arrowhead) and 4 μm (thick arrow) beads (b). Cells from eyes of a hauled Grass Carp (c and d) stained for DNA fluorescence showing a histogram peak CV of 2.57 (c) and nuclear size at 444 Geo mean compared with beads (d) as in above (b). Both DNA histogram peak and nuclear size exemplified a triploid Grass Carp (c and d); more cellular debris gathered at the origin from eye cells (d) than blood (b). Comparing the peak channels of (a) and (c), the DNA content of the triploid carp cells was 3.22 pg.

mean on the independent variables of time and tilapia nuclear size (having been calculated at 4.6076 μm ± 0.0523 (SE); see below), and their interaction. All statistical analyses were performed with the Statistical Analysis System 9.4 (SAS Institute, 2013) at a significance level α = 0.05. Inclusion of the standard beads as internal controls per tube was implemented during the field-collected carp analyses. Statistics were performed first for establishing tilapia nuclear size, and this estimate was used to determine nuclear size of known diploid and triploid carp blood. Next, nuclear size was calculated using the cells from carp eyes from the field collections. For the field experiment, the explanatory (independent X) variables were bead size (2 μm and 4 μm) and tilapia DNA content (2.40 pg), and the response (dependent Y) variables included carp nuclear size, tilapia nuclear size, and carp DNA content. Homogeneity of variance and normality of residuals were examined using simple linear regression analyses (PROC REG) and general linear models (PROC GLM). All regressions and general linear models were fit through the origin. Flow cytometrically determined Geo means of the size parameter were recorded for each carp nuclei, tilapia nuclei, and beads. Specifically, regression coefficients were generated (PROC REG)

between the following pairs of dependent and independent variables: (field carp Geo mean, bead size 2 μm), (field carp Geo mean, bead size 4 μm), (tilapia Geo mean, bead size 2 μm), (tilapia Geo mean, bead size 4 μm), and (field carp Geo mean, tilapia Geo mean). Likewise, regression coefficients were generated for triploid carp Geo mean and diploid carp Geo mean with the tilapia Geo mean, with size of tilapia nuclei having been calculated by using 4 um beads (Table 2), and time (Days 1–4). Descriptive summary calculations of the nuclear sizes of observed diploid-, triploid-, and field Grass Carp are presented (Table 3). All data are provided (Jenkins et al., 2018) Results The tilapia blood control that was cryopreserved with 15% DMSO and an initial cell dilution at 55% yielded DNA content closest to that of fresh tilapia blood, and with the lowest peak CVs, to yield the target value of 2.40 pg (Fig. 5a). The initial cell concentration contributed to the stained nuclei quality; the comparable 15% DMSO treatment with more concentrated source tilapia cells resulted in less distinct peaks and a DNA content of 2.45 pg (Fig. 5b). The DNA values of all other

376

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

Fig. 4. Flow cytograms of known diploid and triploid Grass Carp whose ploidy had been previously verified by using Coulter counter. Focusing on nuclear DNA content (a and b) and size (c – f) using cells from vitreous humor; each carp sample was compared with an external Nile Tilapia Oreochromis niloticus blood control with known DNA content of 2.40 pg (purple peaks; a and b), to which cells a known diploid Grass Carp (a; green peak channel 164) and triploid (b; peak channel 276) were compared (tilapia peak channels 192 (a) and 196 (b)). From the histograms, DNA content for the diploid was calculated to be 2.05 pg, and the triploid value was 3.40 pg. Flow cytometric density plots of the same blood samples comparing the diploid and triploid Grass Carp eye cells nuclear sizes of 4.70 μm and 9.03 μm, (d and f) respectively, with the paired external tilapia controls calculated at 4.6076 μm (0.0523 SE) (c and e). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

treatments resulted in a DNA content range of 2.43 pg–2.50 pg. Thus, tilapia blood diluted 55% with HBSS and frozen with 15% DMSO was selected to serve as an external control in Grass Carp ploidy assessments, in addition to tilapia blood freshly drawn in ACD.

The 2015 Grass Carp from Ohio exhibited wider ranges in fish lengths and weights than in 2016 (Table 1). Overall, the intercepted carp were on average 1/3 the length and over 43 times lighter in weight than the feral Grass Carp from the USFWS feral carp ploidy

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383 Table 2 Summary data (standard error) from the regression models for nuclear size using blood from Nile Tilapia, beads standards, and cells from vitreous humor of Grass Carp eyes after analysis by flow cytometry. Sample

Nuclear size (μm) (SE) a

Tilapia vs 4 μm beads Tilapia vs 2 μm beadsa Diploid carp vs tilapiab Triploid carp vs tilapiab Field carp vs 4 μm beads Field carp vs 2 μm beads

4.61 (0.52) 4.44 (0.10) 3.62 (0.13) 7.57 (0.27) 5.91 (0.05) 5.96 (0.09)

N 501 226 20 20 499 211

a

Nuclear size of tilapia was established in the field experiments due to bead use. Known diploid and triploid carp were analyzed with tilapia only, thus sizes were back-calculated from measurements using field data. b

determination program (Table 1) (ACRCC, 2017; USFWS, 2015). The eyeballs of feral caught grass- and black carp were 28 to over 30 times heavier than Ohio eye weights, respectively (Table 1). The number of fish in the 48 field collections statistically analyzed for the 2015–2016 field seasons was 720 (from batch numbers 29, 31–34, 36–50, 52–56, and 58–80; analyzed with bead standards) (Fig. 3). The batches of samples that supported statistical analyses were not N24 h in shipping, eyes or cells were not aberrant in any way, and sample replicates included standard sized beads. In 2015, one individual was missing from the shipments. Ploidy of six individuals was indeterminate, with one fish yielding too few cells for an assessment, five were degraded, and the remainder (n = 593) were triploid. Thirteen ploidy assessments were dependent on the FPSF-fixed blood because the fresh sample did not yield adequate results. In 2016, every Grass Carp was triploid. Quality of samples was influenced by punctures of eyes on dissection (n = 6, thus the corresponding eye in shipped heads not yet dissected were used for the ploidy determinations), harshness of eye removal during dissection resulting in bruising (Fig. 2c), as well as the length of time in cold storage prior to analysis. Variable time periods spent in shipping and holding from dissection to flow cytometry occurred more frequently at the start of the 2015 field season, with one shipment period at 5 days, three batches at 4 days, seven at 2 days, and the remaining 69 batches taking one day. From the preliminary experiment, time of eyes in cold storage had no influence on DNA CVs of diploids, but triploid CVs were significantly different from each other over the four days (P = 0.0397) (Day 4 ≥ Day3 ≥ Day2 ≥ Day1). Regarding nuclear size, the time eyes were held in cold storage had no influence on either diploid or triploid ploidy determination; both regression models showed that time and the time interaction with tilapia size were not significant, with R2 = 0.2289 (P = 0.2135; P = 0.2187) and R2 = 0.1254 (P = 0.0726; P = 0.5481) for diploids and triploids, respectively. All regressions passed the homogeneity and normality tests. The regression coefficients generated between the 4 μm beads and the flow cytometer determined tilapia blood nuclear size resulted in an average estimate of 4 × 1.15019 = 4.6076 μm ± 0.0523 (SE), with R2 = 0.9392 (Table 2; Fig. 3b). Similarly, using 2 μm beads to estimate the tilapia nuclear size, the value 4.4429 μm ± 0.1000 (SE) was obtained, with R2 = 0.8972 (Table 2; Fig. 3b). After having determined the size of tilapia nuclei in relation to standard beads in the field fish analyses (Table 2), to generate an estimate of nuclear size of known diploid carp blood nuclei, regression coefficients were generated with an Table 3 Descriptive, observed dataa on nuclear sizes from cells from vitreous humor from known diploid and triploid Grass Carp and Grass Carp purchased from the supply chain Ohio in 2015 and 2016.

Field carp Triploid carp Diploid carp a

Nuclear size (μm) (SE)

Range (μm)

5.83 (0.13) 8.71 (0.29) 4.21 (0.15)

4.27–7.77 5.71–11.06 2.63–7.06

Nuclear sizes were calculated using tilapia size being 4.61 μm.

377

estimate of 4 × 0.9052 = 3.6208 μm ± 0.1309 (SE), resulting in R2 = 0.9550 (Table 2). Similarly, for known triploid carp, the size estimate was 4 × 1.8948 = 7.5792 μm ± 0.2695 (SE), with R2 = 0.9588 (Table 2). The regression coefficients generated between the 4 μm bead (Table 2; Fig. 3) and the flow cytometrically determined carp Geo mean resulted in an average size estimate of 4 × 1.4782 μm = 5.9126 μm ± 0.05224 (SE), with R2 = 0.9625 (Table 2; Fig. 3d). Similarly, using the 2 μm beads (Fig. 3), the nuclear size was 5.9604 μm ± 0.0907(SE), with R2 = 0.9534 (Table 2; Fig. 3d). All regressions passed the homogeneity test and were significant (P b 0.0001). Normality was assumed, because the residuals were unimodal and symmetric. The mean measured sizes of known diploid and triploid eye nuclei (Table 2; Fig. 4e, f) compared with the external control's tilapia nuclear size (4.6094 μm) (Fig. 4c, d) regressed from the standard 4 μm bead size were 4.21 μm (SE 0.15) and 8.71 μm (SE 0.29), respectively (Table 3). The sample size for field carp DNA content determinations over the two years was 698 eyes. The DNA parameter from the remaining field carp eyes was not reliable. Mean genome sizes of those samples only one day in shipping was 3.51 pg (SE 0.06), with a range of 2.84–4.42 pg. The osmolality of shipment saline ranged from 239 to 308 mosm/kg, with a mean of 265.6 mosm/kg (SE 1.4). The cells from the first replicate from the ophthalmic vessel routinely yielded good quality DNA peaks (low CVs) (e.g., Fig. 3c), with mostly consistent triploid Grass Carp DNA content estimates. However, the nuclear sizes of blood from the ophthalmic vessel were smaller than the nuclei from the vitreous humor, thus the opthalmic replicates nuclear size parameter was not used for ploidy determinations. For the replicates of cells derived from vitreous humor from field samples, the flow cytograms indicated that the size of carp nuclei was consistently larger than the size of the tilapia blood nuclei (Fig. 3b, d). Ploidy determinations of fish from the first 30 field batches were made with the results of flow cytometric analyses that did not include the standard sized beads; empirical verifications of ploidy were based on DNA content compared with the external tilapia blood control and on the relative size of carp nuclei from vitreous humor compared with tilapia nuclei (Figs. 3–5). In 2016, one batch (# 57; 8 of 15 fish) indicated polyploidy of triploidy/diploidy in nuclear size, but not in DNA content obtained from vitreous humor (Fig. 6). This doublet Geo mean abnormality (Fig. 6a, c, d) was not detected in any other batches. Of those 8 Grass Carp, the range of the percentages of nuclei in each subpopulation representing the diploid nuclei was 22.2%–70.2%, with the average in the diploid and triploid subpopulations at 53.3% (SE 4.8%) and 46.7% (SE 4.8%), respectively. Only one DNA peak was prominent (Fig. 6b) for each of the 15 fish in the batch, indicating no detectable differences in relative DNA content among the differently sized nuclei. As with these abnormal 8 Grass Carp samples, other scenarios underscored the necessity of using the two biological endpoints ̶ nuclear size and DNA content ̶ for a scrupulous determination of ploidy. In some cases, only the size or the DNA parameter was measurable (Fig. 7). For example, when too few cells were obtained per eye, or when debris was profuse (Fig. 7a), the main population of nuclei for a size estimate was not detectable, yet the corresponding DNA peak was instructive (Fig. 7b). In some cases, DNA was degraded for reasons such as time in shipment (Fig.7d), thus the nuclear size was the definitive parameter for determining ploidy of that sample (Fig. 7c). In most all eye samples shipped, however, enough cells in good condition were obtained from the vitreous humor to generate usable data for both relative nuclear size and DNA content to enable a reliable ploidy determination (Fig. 7e and f). For the fixed blood samples, time in FPBS influenced DNA peak location (Fig. 8), in that a longer time (48 h) in the FPBS increased the condensation of DNA, while the DNA content between the diploid blood fixed for 24 h or 48 h appeared to differ due to the amount of staining that can occur. After blood was stored in FPBS for 24 h, the relative ploidy after incubation at 37 °C could be ascertained because the diploid peak values were lower than the triploid peaks. However, the tilapia

378

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

Fig. 5. Cryopreserved tilapia blood (left peaks in a and b) compared with koi blood (right peaks) stained with propidium iodide and flow cytometrically analyzed in choosing a suitable storage method for Nile Tilapia blood. Tilapia blood was diluted either 55% (a) or 33% (b) with Hank's balanced salt solution (311 mosm/kg) and added to 15% (v/v) dimethylsulfoxide, incubated at 24 °C for 20 min, then frozen at −20 °C for use as an external control for assessing Grass Carp ploidy. The CVs of the tilapia and koi nuclear distributions were 2.98 and 3.22, respectively in (a), and 4.95 and 9.59 in (b). The genome size of the tilapia was estimated at 2.40 pg, similar to fresh tilapia blood (a) and 2.45pg (b).

Fig. 6. Eight individuals from Batch #57 of the hauled Grass Carp showed a bimodal geometric mean for the nuclear size parameter (a, c, d), without two corresponding peaks of differing DNA content (b). Fish 1 (c) displayed 22.2% of the nuclei as diploid and Fish 4 displayed 70.2% as diploid, exemplifying variable percentages in the subpopulations. Beads of 4 μm size are shown (arrowhead) in relation to the diploid and triploid-sized nuclei (arrows)(a).

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

379

Fig. 7. Typical results of the standard protocol for flow cytometry analysis of cells from eyes from hauled Grass Carp, showing density plots of carp nuclear size and 4 um bead standards (arrows) (a, c, e) with its corresponding DNA histogram after staining with propidium iodide solution (b, d, f). In (a) and (b), DNA was the biological parameter that was diagnostic of ploidy, with a peak coefficient of variation (CV; peak width at half maximal height) of 4.00 with 416 nuclei in the peak (b); no clearly detectable population of nuclei was apparent for measuring size by geometric mean (a). In (c) and (d), nuclear size compared with bead size allowed for determination of triploidy as opposed to DNA having been degraded, showing no clear histogram peak channel (d). In (e) and (f), either biological parameter of nuclear size or DNA, with a peak CV of 1.74, could be used to determine ploidy of that fish. The external tilapia controls are not shown.

380

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

Fig. 8. The relative DNA content of carp blood preserved with physiological saline with formalin (FPSF) was influenced by the amount of time the samples remained in FPSF and the temperature of sample incubation prior to flow cytometric analysis. When incubated at 37 °C for 20 min, primary peaks from propidium iodide-stained blood having been fixed for 24 h (a and b) and for 48 h (c and d) showed peaks allowing distinction between diploids (a and c) and triploids (b and d) whereby the primary diploid peak occurred to the left of the primary triploid peak. However, more than one peak occurred in triploid samples at both the 37 °C for 20 min (d) and overnight at 4 °C incubations (f). Ploidy discernments were not comparable among treatments and aberrant second peaks (d and f) in triploids from degradation would disallow use of FPSF under the warmer incubations.

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

blood control would also need to be preserved similarly for 24 h due to comparable effects of the FPBS on DNA condensation and consequent staining with propidium iodide. Two DNA peaks occurred more often in triploids than in diploids, disallowing ploidy determination (Fig. 8d and f). Incubation in the cold overnight resulted in DNA peaks with the lowest CVs. Incubation methods and fixation times influenced results; comparable and usable results were obtained within treatments only when samples were not degraded. Discussion Reproductively competent diploids can lessen ecosystem services or the livelihood of the other taxa, especially of concern in the Great Lakes if Grass Carp not only survive but establish in a warming climate scenario (Cudmore et al., 2017; Wittmann et al., 2014). Determining the entry point of an invasive species into the Great Lakes can help policy makers decide on management actions (Rapai, 2016). In Lake Erie, the most productive, shallow, and warmest of the Great Lakes, over 70% of the ~112 Grass Carp captured and analyzed for ploidy through January 2018 have been diploid (Wieringa et al., 2017; J. Bailey, USFWS, personal communication). Coordinated and integrated actions among stakeholders would help to prevent introductions of diploids. The variable status and dynamic history of the states' regulations and policies on Grass Carp are far less united than those efforts on behalf of states primarily along the country's northernmost border (ESM Table S1; Fig. 1), and those of the Great Lakes Fishery Commission which fosters cross-border communication with Canada. Generally, state and federal regulations directed at Asian carps in North America have been slow to evolve, and applications of regulations is influenced by political and economic considerations; some states maintain strict policies and regulations regarding Grass Carp possession and use, but neighboring states may not ESM Table S1; Fig. 1 (Peters and Lodge, 2009; Rasmussen, 2011). Equivalent approaches for certifying, shipping, stocking, and regulating triploids would safeguard aquatic resources by preventing accidental or illegal introductions. This study focused on a single component of the triploid Grass Carp supply chain within a single state. Currently, transporting triploid Grass Carp is allowed by 30 states (MICRA, 2015); Ohio is a state that mandates only NTGCICP-certified transports. The NTGCICP is effective because of standardized protocols, procedures, inspections, and consequences (MICRA, 2015). By virtue of our work to investigate the biosecurity of the USFWS-certified commercial supply chain, SOPs were developed for shipping, handling, and dissection (Kinter et al., 2018), and for processing eyeballs from hauled fish for discriminating diploidy from triploidy (see ESM Appendix S1). Robust methods underlying Grass Carp shipment inspections would help to mitigate the weak link problem that occurs with inconsistent rules regionally (Peters and Lodge, 2009), and would pinpoint diploids prior to their introductions. Until now, no protocols have supported ploidy determinations for use in court proceedings regarding Grass Carp shipments. Our results showed that size of the nucleus from cells derived from the vitreous humor, in addition to the nuclear DNA content, were both important determinants for reliably discriminating ploidy. Generally, the analytical approach for differentiating between diploid and triploid animals is based on distinct cellular characteristics. As nuclear size increases with chromosome number, triploid cells are larger and the nucleo-cytoplasmic ratio is maintained (Swarup, 1959), with triploid DNA content being 1.5 times the diploid level (Jenkins et al., 2017; Jenkins and Thomas, 2007). The size of cells and their nuclei have been used to identify triploids, with the erythrocyte being the most common cell type (Purdom, 1993). The differences in elliptical nucleus dimensions between diploid and triploid fish were first noted by Swarup (1959); and since then microscopic, image analyses, and Coulter counter measures of cell and nuclear volume have been decisive (Cormier et al., 1993; Flajshans, 1997; McCarter, 1988). One microscopic technique intended for the field could be useful for law

381

enforcement purposes; the method is based on the predominance of abnormally shaped triploid erythrocyte nuclei, and secondarily the larger size of cell and nuclei (Krynak et al., 2015). In our study, eight individuals from one shipment contained nuclei of obvious abnormal shape identified by flow cytometry (Fig. 6), perhaps an abnormal metaphase II division. Erythrocyte nuclear measurements of diploids and triploids can overlap (Wolters et al., 1982), thus misclassifications can occur by microscopy. In flow cytometry, relative measures of size are generated by particles suspended in a fluid stream passing through a laser beam, whereby forward angle light scatter (FSC) is determined by the amount of the beam passing around the cell. Estimates of size have been performed with such diverse biological particulates as liposomes (Vorauer-Uhl et al., 2000), cyanobacteria (Olson et al., 1990), and bacteria (Koch et al., 1996). Unknown size can be determined in relation to uniform microspheres (Olson et al., 1990). In our preliminary experiment that generated flow cytometric estimates of nuclear size from cells obtained from within the eye derived from known diploids and triploids, an overlap of nuclear size ranges occurred (Table 3). With the field-collected Grass Carp, the measured mean nuclear size of 5.83 μm (0.13 SE) fell in between the measured values from known diploids and triploids from the preliminary experiment (Table 3). When this field value of 5.83 μm (0.13 SE) was compared with the regressed values for carp of known ploidy (using tilapia at 4.61 μm), the calculated field carp nuclear size estimate was 1.74 μm smaller than the triploid value, yet 2.21 μm larger than the diploid value. However, this measured average field value was just 0.08 μm smaller than the regressed value of 5.91 μm (Table 2). The elliptical nature of carp nuclei influenced size results, as did the shipping conditions (i.e., field eyes in saline versus whole heads). Also, laser light wavelengths and the ranges of angles optically detected are among some of the limitations for absolute size generation by flow cytometry (Shapiro, 1995). Within the field sample containers, the blood cells derived from the ophthalmic vessel experienced different shipping conditions than those from the vitreous humor, even those from the same eye. The first replicate tubes for the flow cytometry analysis contained blood derived from this vessel on the outside of the eye (Fig. 2d), being proximate to saline of a particular osmolality. Upon visualization of the flow cytometry data from these replicates, the main population of Geo means generally coincided with the external tilapia control Geo mean, thus the nuclear size was not used as a determinant for assigning ploidy. Cellular homeostasis is controlled by the plasma membrane; and, if a buffer's osmolality is hypotonic, as in this field study with the osmolality of shipment saline averaging 265.6 mosm/kg (SE 1.4), water can enter the cell (Olivier and Jenkins, 2015) or cytoplasmic contents can diffuse to the more hypotonic environment. In this case, carp blood osmolality of ~305–311 mosm/kg was likely consistent among fish, thus a more hypertonic shipping buffer would have allowed better equilibration of the blood in the vessel and the buffer, lessening the influence on cell and nuclear size. Osmolality adjustments in the future will allow the size parameter from blood cells derived from the ophthalmic vessel to be used for ploidy discriminations. The eye, an immunologically protected site whereby good cell quality is maintained, supports an acceptable postmortem period for ploidy determinations (Jenkins and Thomas, 2007). Nuclear size was a robust metric as shown by no changes of diploid and triploid nuclei over 4 days in cold storage in the preliminary study. The field sample DNA CVs and relative locations of peak channels compared to the external tilapia control were more variable than the nuclear size parameter. This could be due to the fragility of the DNA and its potential for degrading, as well as cell type variability slightly influencing staining characters. Cells obtained from the internal eye environment included pigmented cells, erythrocytes especially when an eye was bruised (Fig. 2c), and the preferred cells from the clear vitreous humor. For feral carp, cells of good quality for flow cytometric analysis based on the DNA content were available through 12 days postmortem

382

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383

(Jenkins and Thomas, 2007). In this study, eyes from purchased Grass Carp weighed approximately 30 times less than feral carp eyes, so the available cells for analysis were fewer and the buffering capacity of the vitreous humor was much less due to lower volumes. The results indicated that triploid DNA CVs were lowest at 24 h as opposed to days 2–4 (P = 0.0397); but again the time of eyes in cold storage, through 4 days, had no influence on nuclear size. This illustrates the usefulness of two complementary biological endpoints for ploidy discrimination, as there is potential variability in sample condition and numbers of readable events in a replicate (Fig. 7). As with other hematologically characterized polyploid fish (Gao et al., 2007), our flow cytometric calculations of cell sizes from diploid carp, tilapia, and triploid carp (Table 2) are parallel to their increase in DNA content from diploid Grass Carp at 2.0 pg (0.01 SD) (Tiersch et al., 1989), tilapia at 2.40 pg (Chapman et al., 2013), to triploid Grass Carp at 3.0 pg (0.01 SD) (Tiersch et al., 1989) and triploid black carp at ~3.2 pg (Jenkins and Thomas, 2007). In this study, the mean genome sizes of close to 700 field samples was 3.51 pg (SE 0.06), above the absolute triploid Grass Carp measure. Again, this value reflects fragility of DNA with fragmentation allowing increases in staining. To obtain the best quality data, the logistics of sample collection, handling, and transport from remote locations needs to be considered in the study design (see Kinter et al., 2018; Olivier and Jenkins, 2015). Using FPSF to fix samples allows blood to be shipped for analysis later (Brown et al., 2000; Schultz et al., 2001). The chemical components of the field kits for use with fixed blood are stable at room temperature, facilitating ploidy analyses up to 1 month following collection; this procedure has been routinely used in the state of Virginia (Brown et al., 2000). In this study, analysis of field samples occurred within 5 days after sample collection and primarily after only one day in shipment. After 10 batches had been mailed, field crews halted the bleeding (Fig. 3) and blood fixation due to reliability of data from fresh eyes and because fish head and eye removal was the quickest and easiest method for sample processing by field personnel. Regardless, 13 of those 150 fixed samples informed the ploidy determinations in 2015 (Fig. 8). Objectively derived data with robust sample sizes supports scientific results, and thus could be used in a court of law if ploidy regulations need enforcement. The flow cytometric event number typically collected per sample for this study was 10,000 (Fig. 3–8), meaning that size and DNA content parameters are collected on each of the 10,000 nuclei. Because data are collected for such DNA studies at 300 cells per second, thus approximately 33 s was needed for flow cytometric analysis per replicate. This sample size (3 replicates of 10,000 nuclei each), and the low amount of time for analyses, contrasts with the method using 200 cells measured by microscopy (Krynak et al., 2015). Another advantage of flow cytometric technology is that the primary histogram peak indicating triploidy compared to the external tilapia control can be unmistakable with extremely low events counts, demonstrated with 416 nuclei (Fig. 7b) out of a total of 10,051 events (Fig. 7a). In the absence of rigorous state inspection programs and law enforcement, consistent state regulations prohibiting diploids and restricting the use of triploid Grass Carp to USFWS certified triploids would provide the greatest protections to prevent the accidental or illegal introductions of diploid Grass Carp (MICRA, 2015). To that end, use of nuclear size as well as DNA content as co-determinants for reliable ploidy analysis of Grass Carp collected from interstate transport shipments is now available for use, within a bracketed postmortem time (~24 h) and a sample-handling regime. Once a sample is obtained by a flow cytometrist with standard protocols in hand, results can be obtained within an hour. This standard protocol (ESM Appendix S1) is applicable for state-specific purposes, with results of interest binationally. The SOP for use with eyeballs also may be particularly useful for enforcement of laws regarding Grass Carp ploidy that often differ among state and within-state jurisdictions. Lastly, results from this study

point to the fidelity of the supply chain of USFWS-certified triploid Grass Carp. Acknowledgements The authors respectfully recognize the longstanding collaboration with J.M. Malone and Son, Inc., a primary producer of USFWS-certified triploid Grass Carp. The authors thank Dr. R. Dale for statistical consultation, and C. Matkin and H. Olivier for laboratory assistance. The authors thank the ODNR-Division of Wildlife law enforcement and fish management personnel for their multiple efforts in the field. This project was in part supported by a grant from the Great Lakes Restoration Initiative to the Ohio Department of Natural Resources, Division of Wildlife (USGS Agreement #15SETAASC009021), and by the U.S. Geological Survey Invasive Species Program and Ecosystems Mission Area. The authors declare no competing financial interest. The following agencies are gratefully acknowledged for their contributions: AL Dept. of Conservation and Natural Res.; AK Dept. of Fish and Game; AZ Game and Fish Dept.; CO Parks and Wildlife; CT Dept. of Energy and Environmental Protection; DE Div. of Fish and Wildlife, Dept. of Natural Resources and Environmental Control; FL Fish and Wildlife Conservation Commission; Univ. of GA; GA Dept. of Natural Resources; HI Dept. of Land and Natural Resources; HI Dept. of Ag.; IA Dept. of Natural Resources; IL Dept. of Natural Resources; IN Dept. of Natural Resources; LA Dept. of Wildlife and Fisheries; MA Dept. of Fish and Game; MD Dept. of Natural Resources; ME Dept. of Inland Fisheries and Wildlife; MI Dept. of Natural Resources; MO Dept. of Conservation; NC Wildlife Resources Commission; ND Game and Fish Dept.; Univ. of NE; NE Game and Parks Commission; NH Fish and Game Dept.; NJ Dept. of Environmental Protection; NM Dept. of Fish and Game; NY Dept. of Environmental Conservation; OK Dept. of Wildlife Conservation; OH Dept. of Natural Resources; OR Dept. of Fish and Wildlife; PA Fish and Boat Commission; RI Dept. of Environmental Management; SC Dept. of Natural Resources; TN Wildlife Resources Agency; U.S. Fish and Wildlife Service – Grand Junction Fish and Wildlife Conservation Office; U.S. Fish and Wildlife Service – Large Rivers Coordination Office; WA Dept. of Fish and Wildlife; VA Dept. of Game and Inland Fisheries; VT Dept. of Environmental Conservation; WA Dept. of Fish and Wildlife; Wisconsin Department of Natural Resources; WY Game and Fish Department. Data from this study are available in a USGS data release https://doi.org/10.5066/F7QC02D4 (Jenkins et al., 2018). Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jglr.2018.12.004. References Asian Carp Regional Coordinating Committee, 2017. Asian Carp Action Plan for Fiscal Year 2017. , pp. 1–260. http://www.asiancarp.us/. Bain, M.B., 1993. Assessing impacts of introduced aquatic species: Grass Carp in large systems. Environ. Manag. 17, 211–224. Baldwin, N.A., Saalfeld, R.W., Dochoda, M.R., Buettner, H.J., Eshenroder, R.L., O'Gorman, R., 2018. Commercial Fish Production in the Great Lakes 1867–2015 [online]. Available from. http://www.glfc.org/great-lakes-databases.php, Accessed date: 3 August 2018. Bettoli, P.W., Maceina, M.J., Noble, R.L., Betsill, R.K., 1993. Response of a reservoir fish community to aquatic vegetation removal. N. Am. J. Fish Manag. 3, 110–124. Brown, B.L., Schultz, S.L.W., White, F.K.H., 2000. A convenient field method of tissue preservation for flow cytometric ploidy assessment of Grass Carp. Trans. Am. Fish. Soc. 129, 1354–1359. Buck, E.H., Upton, H.F., Stern, C.V., Nichols, J.E., 2010. Asian carp and the Great Lakes region. Congressional Research Service Reports, Paper 12. University of Nebraska, Lincoln, NE (23 pp). Chapman, D.C., Davis, J.J., Jenkins, J.A., Kocovsky, P.M., Miner, J.G., Farver, J., Jackson, P.R., 2013. First evidence of Grass Carp recruitment in the Great Lakes Basin. J. Great Lakes Res. 39, 547–554. https://doi.org/10.1016/j.jglr.2013.09.019. Conover, G., Simmonds, R., Whalen, M. (Eds.), 2007. Management and Control Plan for Bighead, Black, Grass, and Silver Carps in the United States. Asian Carp Working

J.A. Jenkins et al. / Journal of Great Lakes Research 45 (2019) 371–383 Group Aquatic Nuisance Species Task Force, Washington, D.C. (223 pp.). http:// asiancarp.us/documents/Carps_Management_Plan.pdf. Cormier, S.M., Neiheisel, T.W., Williams, D.E., Tiersch, T.R., 1993. Natural occurrence of triploidy in a wild brown bullhead. Trans. Am. Fish. Soc. 122, 390–392. https://doi. org/10.1577/1548-8659(1993)122b0390:NOOTIAN2.3.CO;2. Cucherousset, J., Olden, J.D., 2011. Ecological impacts of non-native freshwater fishes. Fisheries 36, 215–230. Cudmore, B., Jones, L.A., Mandrak, N.E., Dettmers, J.M., Chapman, D.C., Kolar, C.S., Conover, G., 2017. Ecological risk assessment of Grass Carp (Ctenopharyngodon idella) for the Great Lakes Basin. DFO Can. Sci. Advis. Sec. Res. Doc. 2016/118 (Vi + 115 pp). Embke, H.S., Kocovsky, P.M., Richter, C.A., Pritt, J.J., Mayer, C.M., Qian, S.S., 2016. First direct confirmation of Grass Carp spawning in a Great Lakes tributary. J. Great Lakes Res. 42, 899–903. https://doi.org/10.1016/j.jglr.2016.05.002. Flajshans, M., 1997. A model approach to distinguish diploid and triploid fish by means of computer-assisted image analysis. Acta Vet. Brno 66, 101–110. Fuller, P., Neilson, M.E., 2015. The U.S. geological survey's nonindigenous aquatic species database: over thirty years of tracking introduced aquatic species in the United States (and counting). Manag. Biol. Invasions 6, 159–170. https://doi.org/10.3391/ mbi.2015.6.2.06. Gao, Z., Wang, W., Abbas, K., Zhou, X., Yang, Y., Diana, J.S., Wang, H., Wang, H., Li, Y., Sun, Y., 2007. Haematological characterization of loach Misgurnus anguillicaudatus: comparison among diploid, triploid and tetraploid specimens. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 147, 1001–1008. https://doi.org/10.1016/j.cbpa.2007.03.006. Irons, K.S., Sass, G.G., McClelland, M.A., Stafford, J.D., 2007. Reduced condition factor of two native fish species coincident with invasion of non-native Asian carps in the Illinois River, U.S.A. Is this evidence for competition and reduced fitness? J. Fish Biol. 71 (Suppl. D), 258–273. Jenkins, J.A., 2011. Male germplasm in relation to environmental conditions: synoptic focus on DNA. In: Tiersch, T.R., Green, C.C. (Eds.), Cryopreservation in Aquatic Species, 2nd edn. World Aquaculture Society, Baton Rouge, LA, pp. 227–239. Jenkins, J.A., Thomas, R.G., 2007. Use of eyeballs for establishing ploidy of Asian carp. N. Am. J. Fish Manag. 27, 1195–1202. https://doi.org/10.1577/M06-261.1. Jenkins, J.A., Draugelis-Dale, R.O., Glennon, R.P., Kelly, A.M., Brown, B.L., Morrison, J.R., 2017. An accurate method for measuring triploidy of larval fish spawns. N. Am. J. Aquac. 79, 224–237. https://doi.org/10.1080/15222055.2017.1296517. Jenkins, J.A., Johnson, D., Kinter, B.T., 2018. Establishing a Standard Ploidy Assessment Method Using Grass Carp in Ohio, 2015–2016: U.S. Geological Survey Data Release. https://doi.org/10.5066/F7QC02D4. Kinter, B.T., Jenkins, J.A., Tyson, J.T., 2018. Assessing the risk of diploid Grass Carp Ctenopharyngodon idella in the certified triploid supply chain in Ohio. J. Great Lakes Res. 44, 1093–1099. https://doi.org/10.1016/j.jglr.2018.07.004. Koch, A.L., Robertson, B.R., Button, D.K., 1996. Deduction of the cell volume and mass from forward scatter intensity of bacteria analyzed by flow cytometry. J. Microbiol. Methods 27, 49–61. Krynak, K.L., Oldfield, R.G., Dennis, P.M., Durkalec, M., Weldon, C., 2015. A novel field technique to assess ploidy in introduced Grass Carp (Ctenopharyngodon idella, Cyprinidae). Biol. Invasions 17, 1931–1939. https://doi.org/10.1007/s10530-0150856-9. Malone, J.M., 1984. Triploid white Amur. Fisheries 92, 36. McCarter, N.H., 1988. Verification of the production of triploid Grass Carp (Ctenopharyngodon idella) with hydrostatic pressure. N. Z. J. Mar. Freshw. Res. 22, 501–505. Mississippi Interstate Cooperative Resource Association (MICRA), 2015. The use of Grass Carp (Ctenopharyngodon idella) in the United States: Production, triploid certification, shipping, regulation, and stocking recommendations for reducing spread throughout the United States. Report to the U.S. Fish and Wildlife Service. Agreement #F12AP00630 , p. 69. http://www.micrarivers.org/resource-materials/micra-documents/category/15-micra-reports.html.

383

Mitchell, A.J., Kelly, A.M., 2006. The public sector role in the establishment of Grass Carp in the United States. Fisheries 31, 113–122. Olivier, H.M., Jenkins, J.A., 2015. Proper handling of animal tissues from the field to the laboratory supports reliable biomarker endpoints. In: Alford, J.B., Peterson, M.S., Green, C.C. (Eds.), Impacts of Oil Spill Disasters on Marine Habitats and Fisheries in North America. CRC Press, Boca Raton, FL, pp. 81–93. Olson, R.J., Chisholm, S.W., Zettler, E.R., Armbrust, E.V., 1990. Pigments, size, and distribution of Synechococcus in the North Atlantic and Pacific oceans. Limnol. Oceanogr. 35, 45–58. Peters, J.A., Lodge, D.M., 2009. Invasive species policy at the regional level: a multiple weak links problem. Fisheries 34, 373–380. https://doi.org/10.1577/1548-844634.8.373. Public Law 104-40, 1995. An Act to Authorize the Collection of Fees for Expenses for Triploid Grass Carp Certification Inspections, and for Other Purposes (109 Stat. 350, S. 268). Purdom, C.E., 1993. Chromosome engineering. Genetics and Fish Breeding. Chapman and Hall, New York, pp. 204–222. Rapai, W., 2016. Lake Invaders: Invasive Species and the Battle for the Future of the Great Lakes. Wayne State University Press, Detroit, MI 978-0-8143-4124-7 (170 pp.). Rasmussen, J.L., 2011. Regulations as a tool in Asian carp management. In: Chapman, D.C., Hoff, M.H. (Eds.), Invasive Asian Carps in North America. American Fisheries Society Symposium 74. American Fisheries Society, Bethesda, MD, pp. 175–189. SAS Institute I, 2013. SAS/STAT 9.4. SAS Institute, Inc, Cary, NC. Schultz, S.L.W., Steinkoenig, E.L., Brown, B.L., 2001. Ploidy of feral Grass Carp in the Chesapeake Bay watershed. N. Am. J. Fish Manag. 21, 96–101. https://doi.org/10.1577/ 1548-8675(2001)02.1. Shapiro, H.M., 1995. Practical Flow Cytometry. Wiley-Liss, New York (542 pp). Swarup, H., 1959. Effect of triploidy on the body size, general organization and cellular structure in Gasterosteus aculeatus (L.). J. Genet. 56, 143–155. Tiersch, T.R., Chandler, R.W., Wachtel, S.S., Elias, S., 1989. Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. Cytometry 10, 706–710. U.S. Geological Survey, 2018. Nonindigenous Aquatic Species Database (Grass Carp). Online at. https://nas.er.usgs.gov/viewer/omap.aspx?SpeciesID=514, Accessed date: 3 August 2018. USFWS, 2015. 2015 Annual Report to Congress: Second Annual Summary of Activities and Expenditures to Manage the Threat of Asian Carp in the Upper Mississippi and Ohio River Basins: July 2014 Through September 2015. A Report to Congress Pursuant to the Water Resources Reform and Development Act of 2014 (PL 113-121). , p. 62. https://www.fws.gov/midwest/fisheries/asian-carp/WRRDA2015.pdf. Vorauer-Uhl, K., Wagner, A., Borth, N., Katinger, H., 2000. Determination of liposome size distribution by flow cytometry. Cytometry 39, 166–171. Wattendorf, R.J., 1986. Rapid identification of triploid Grass Carp with a Coulter counter and channelyzer. Prog. Fish Cult. 48, 125–132. https://doi.org/10.1577/1548-8640 (1986)48b125:RIOTGN2.0CO;2. Wieringa, J.G., Herbst, S.J., Mahon, A.R., 2017. The reproductive viability of Grass Carp (Ctenopharyngodon idella) in the western basin of Lake Erie. J. Great Lakes Res. 43, 405–409. https://doi.org/10.1016/j.jglr.2016.12.006. Wittmann, M.E., Jerde, C.L., Howeth, J.G., Maher, S.P., Deines, A.M., Jenkins, J.A., Whitledge, G.W., Burbank, S.R., Chadderton, W.L., Mahon, A.R., Tyson, J.F., Gantz, C.A., Keller, R.P., Drake, J.M., Lodge, D.M., 2014. Grass Carp in the Great Lakes region: establishment potential, expert perceptions, and re-evaluation of experimental evidence of ecological impact. Can. J. Fish. Aquat. Sci. 71, 1–8. https://doi.org/10.1139/cjfas-2013-0537. Wolters, W.R., Chrisman, C.L., Libey, G.S., 1982. Erythrocyte nuclear measurements of diploid and triploid channel catfish, Ictalurus punctatus (Rafinesque). J. Fish Biol. 20, 253–258.