Diploid Grass Carp Research Articles

Assessing Invasive Carp in the Neosho River‐Grand Lake System of Kansas and Oklahoma

Invasive carp populations have purported a negative influence on native biota at high densities. These invasive fishes (i.e., bighead carp Hypophthalmichthys nobilis, silver carp Hypophthalmichthys molitrix, grass carp Ctenopharyngodon idella, and black carp Mylopharyngodon piceus) each exhibit similar life history characteristics. In the Neosho River‐Grand Lake system (i.e., John Redmond Reservoir, Kansas, downstream to Grand Lake O’ the Cherokees, Oklahoma), only bighead carp and grass carp have been documented to date. The distribution and status of bighead carp throughout this system were previously unknown due to limited historical data and low abundance. While few bighead carp are encountered within this system, grass carp exhibited relatively higher abundance a were used to provide insights into bighead carp. Captures of both species were used to inform management and suppression efforts. Sampling locations (n = 18) were established for environmental DNA analyses throughout the Neosho River‐Grand Lake system. We sampled 13 sites using a suite of gears for standardized targeted fish sampling. All invasive carp were measured, sexed, and otoliths removed for ageing and microchemical analysis. Grass carp were processed for ploidy testing following the U.S. Fish and Wildlife Service protocol. Environmental DNA analyses generated positive results for the eDNA presence of bighead carp and silver carp. Otolith age estimates suggest fish are long lived and supported by multiple year classes. Additionally, a sampled two‐year‐old grass carp demonstrates spawning and recruitment potential. Otolith microchemistry suggests largescale broad movement patterns. Ploidy testing confirmed the first documented evidence of diploid grass carp in the Neosho River‐Grand Lake system and revealed reproductive viability. Our results may provide future insights into locations for containment, removal, and/or eradication.

Grass Carp Reproduction in Small Tributaries of Harry S. Truman Reservoir, Missouri: Implications for Establishment in Novel Habitats

AbstractSubstantial work has been conducted to estimate the river length required for recruitment of invasive Grass Carp Ctenopharyngodon idella and bigheaded carps (Bighead Carp Hypophthalmichthys nobilis and Silver Carp H. molitrix); however, the distance upstream and size of stream required for spawning remain unclear. Adult Grass Carp are regularly captured in Harry S. Truman Reservoir, Missouri, although there have been only unconfirmed sightings of Silver Carp. Fertile diploid Grass Carp are used legally for vegetation control in Missouri; however, it is uncertain whether Grass Carp in Harry S. Truman Reservoir are the result of natural reproduction or escapees. We used ichthyoplankton tows and environmental DNA (eDNA) to investigate which carp species were present in the reservoir basin and to determine whether these carps were spawning in reservoir tributaries. Grass Carp eggs were collected in four of five tributaries during June 2014. We did not detect eggs, larvae, or eDNA of Bighead Carp or Silver Carp. The occurrence of Grass Carp eggs generally coincided with rapid increases in discharge and turbidity. These tributaries may be the smallest rivers worldwide in which spawning of Grass Carp has been documented. We infer from the developmental stages captured and the distance from the reservoir that sufficient river length exists for egg survival. These results have broad implications concerning the establishment of Grass Carp in other novel habitats, including other reservoirs and lake basins, smaller rivers, and the Laurentian Great Lakes ecosystem.

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

Abstract 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.

Assessing the risk of diploid grass carp Ctenopharyngodon idella in the certified triploid supply chain in Ohio

Abstract Non-native grass carp (Ctenopharyngodon idella) have been stocked in the United States for vegetation control since the 1970s, and recent evidence suggests some natural reproduction in the Great Lakes basin. Despite all states and provinces bordering Lake Erie either banning grass carp or requiring imports of only sterile, U.S. Fish and Wildlife Service (USFWS)-certified triploids, the majority of grass carp captured and analyzed from Lake Erie are diploid, or reproductively viable. Potential sources of diploid grass carp include illegal importation, compromises in the USFWS-certified triploid shipments, migration from legal diploid states, or natural reproduction resulting from diploid stockings prior to the 1988 Ohio law requiring only certified triploids. The goal of this study was to explore the risk that diploid grass carp occur in the USFWS-certified triploid supply chain destined for Ohio. During 2015 and 2016, undercover Ohio Department of Natural Resources-Division of Wildlife law enforcement purchased 1200 grass carp from 16 distributors, and overnighted 80 dissected grass carp head and eyeball sample shipments (n = 15 fish per shipment) for ploidy analysis by flow cytometry. Standardized methods for both field and laboratory processing were established. No diploid grass carp were detected in these collections, indicating fidelity of the USFWS-certified triploid grass carp supply chain in Ohio. Thus, these shipments are not a likely source of diploid grass carp in Lake Erie. This study is the first large-scale evaluation of the potential for ecological risk from diploid grass carp occurrence in USFSW-certified shipments of triploids for national distribution.

First direct confirmation of grass carp spawning in a Great Lakes tributary

Abstract Grass carp (Ctenopharyngodon idella), an invasive species of Asian carp, has been stocked for many decades in the United States for vegetation control. Adult individuals have been found in all of the Great Lakes except Lake Superior, but no self-sustaining populations have yet been identified in Great Lakes tributaries. In 2012, a commercial fisherman caught four juvenile diploid grass carp in the Sandusky River, a major tributary to Lake Erie. Otolith microchemistry and the capture location of these fish permitted the conclusion that they were most likely produced in the Sandusky River. Due to this finding, we sampled ichthyoplankton using paired bongo net tows and larval light traps during June–August of 2014 and 2015 to determine if grass carp are spawning in the Sandusky River. From the samples collected in 2015, we identified and staged eight eggs that were morphologically consistent with grass carp. Five eggs were confirmed as grass carp using quantitative Polymerase Chain Reaction for a grass carp-specific marker, while the remaining three were retained for future analysis. Our finding confirms that grass carp are naturally spawning in this Great Lakes tributary. All eggs were collected during high-flow events, either on the day of peak flow or 1–2 days following peak flow, supporting an earlier suggestion that high flow conditions favor grass carp spawning. The next principal goal is to identify the spawning and hatch location(s) for the Sandusky River. Predicting locations and conditions where grass carp spawning is most probable may aid targeted management efforts.

Persistence of Triploid Grass Carp in Devils Lake, Oregon

Abstract Grass carp Ctenopharyngodon idella are sometimes used as a biological tool for managing aquatic vegetation in reservoirs. Sterile, triploid fish were stocked in Devils Lake, Oregon, during 1986, 1987, and 1993 to control aquatic vegetation. We present a case study for using multiple measures on the same fish to determine whether illegal stocking of fertile, diploid grass carp occurred. An investigation into the estimated age of a dead grass carp found in Devils Lake suggested that it was significantly younger than would otherwise be expected, given the only stocking events occurred during 1986, 1987, and 1993. To determine whether illegal stocking or reproduction by presumed sterile grass carp had occurred in Devils Lake, we conducted a study that balanced the needs of lethally sampling grass carp for biological measures with the socially and politically sensitive sentiment of the pro–grass carp citizenry of Devils Lake. These considerations, in combination with a low catch per-unit effort, resulted in a modest sample size for grass carp. We sampled grass carp and recorded multiple measures for each fish. Ploidy testing of blood samples indicated the grass carp were all triploid. Based on gonadal histopathology, six fish were male, two were female, and two were sex-indeterminate with severe gonadal dysgenesis. Age estimates from lapillus otoliths were consistent with fish originating from the legal stocking events in Devils Lake. The grass carp were 21–30 y old, and we were unable to find published reports of grass carp anywhere else in the world that are older. The grass carp were significantly smaller than much younger fish from other regions. The small size of these grass carp relative to their age in Devils Lake suggests food limitations that stunted growth. The dead grass carp that was the impetus for this study was aged by anatomical structures that we have since found to be unreliable. This suggests that the dead grass carp was probably in fact older and originated from the legal stockings. The use of multiple biological measurements on a modest sample size of grass carp, combined with the knowledge that no juvenile grass carp have been observed since legal stocking occurred, lead us to conclude that the grass carp in Devils Lake are sterile fish that originated from legal stocking events.

First evidence of grass carp recruitment in the Great Lakes Basin

We use aging techniques, ploidy analysis, and otolith microchemistry to assess whether four grass carp Ctenopharyngodon idella captured from the Sandusky River, Ohio were the result of natural reproduction within the Lake Erie Basin. All four fish were of age 1+. Multiple lines of evidence indicate that these fish were not aquaculture-reared and that they were most likely the result of successful reproduction in the Sandusky River. First, at least two of the fish were diploid; diploid grass carp cannot legally be released in the Great Lakes Basin. Second, strontium:calcium (Sr:Ca) ratios were elevated in all four grass carp from the Sandusky River, with elevated Sr:Ca ratios throughout the otolith transect, compared to grass carp from Missouri and Arkansas ponds. This reflects the high Sr:Ca ratio of the Sandusky River, and indicates that these fish lived in a high-strontium environment throughout their entire lives. Third, Sandusky River fish were higher in Sr:Ca ratio variability than fish from ponds, reflecting the high but spatially and temporally variable strontium concentrations of southwestern Lake Erie tributaries, and not the stable environment of pond aquaculture. Fourth, Sr:Ca ratios in the grass carp from the Sandusky River were lower in their 2011 growth increment (a high water year) than the 2012 growth increment (a low water year), reflecting the observed inverse relationship between discharge and strontium concentration in these rivers. We conclude that these four grass carp captured from the Sandusky River are most likely the result of natural reproduction within the Lake Erie Basin.

Induced Gynogenesis in Grass Carp (Ctenopharyngodon idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Grass carp (Ctenopharyngodon idellus) eggs were activated by UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (♀) × common carp (♂) and then duplicated by cold shock in 4-6°C water for 10-12 min. Different cold shock initiation times resulted in two types of diploid gynogenetic grass carp: meiotic gynogenetic (meiG) and mitotic gynogenetic (mitG). Over a 5-year period, a total of 17,170 meiG and 1,080 mitG fry were produced and 6,862 meiG and 372 mitG grass carp survived. The gynogenetic fish were confirmed by morphological characteristics, chromosome examination, and microsatellite DNA analysis. The morphological traits of the gynogenetic grass carp were similar to those of wild diploid grass carp. Normal gynogenetic fish were identified as diploid with 48 chromosomes by chromosomal metaphases examination, while nonviable abnormal embryos were detected as haploid with 24 chromosomes. Microsatellite DNA analysis indicated that after one generation of gynogenesis, the genetic purity of meiG and mitG grass carp was significantly increased over that of wild grass carp. In addition, both meiG and mitG grass carp groups were 100% female, and 88% of these showed normal ovary development. Thus, the sex determination mechanism in female grass carp was homogamety. The ability to establish pure all-female groups of meiG and mitG grass carp should be a valuable contribution to both fish genetics and grass carp breeding.

Construction and Characterization of Grass Carp (Ctenopharyngodon idellus) Fosmid Library

Grass carp (Ctenopharyngodon idellus) genomic fosmid library cotaining 129 014 clones was constructed and characterized from one diploid grass carp. The average insert size of the fosmid library was determined to be 35 kb by pulsed field gel electrophoresis, which is 4.1-fold coverage of the grass carp genome. To demonstrate the probability of picking the functional genes from the library, eleven functional genes were screened by three-dimensional PCR technique. The number of positive clones of these genes was from 1 to 6. So, this library may screen any useful genes from grass carp. This grass carp genome fosmid library will be integrated in the presently ongoing efforts to determine the sequence of the grass carp genome.

Mitochondrial DNA Dosage Effects in Triploid Grass Carp

AbstractThe grass carp Ctenopharyngodon idella is used widely in aquaculture for aquatic weed control. Historically, concern about the proliferative growth of nonnative diploid grass carp populations led to development of the hydrostatic pressure shock technique for the production of sterile triploids. Despite the success of this technique in accomplishing management goals, little is known about the cellular physiology of triploids. In particular, few studies have explored whether the induction of triploidy via hydrostatic pressure shock affects mitochondrial DNA (mtDNA) copy number in addition to that of chromosomal DNA. While the nuclear DNA (nDNA): mtDNA ratio is conserved in polyploid yeast, there are few data suggesting that vertebrate polyploids have nDNA: mtDNA ratios consistent with those of their diploid counterparts. In this study, two novel quantitative polymerase chain reaction assays were used to explore gene copy number in the nuclear and mitochondrial genome of diploid and triploid grass carp and to determine whether nuclear–mitochondrial copy number differences can be exploited to determine the ploidy of individual fish. The results of this study suggest that the ratio of mtDNA to overall cellular DNA is conserved in vertebrate polyploids and that real‐time assays of mtDNA copy number are not a feasible alternative to current methodologies for identifying triploid grass carp.

Control of macrophytes by grass carp (Ctenopharyngodon idella) in a Waikato drain, New Zealand

Hornwort (Ceratophyllum demersum L.) and other aquatic macrophytes have historically been mechanically removed from the Rangiriri drain and Churchill East drain to maintain drain efficiency. As an alternative control method for the high plant biomass that accumulates at the end of summer, the effect of stocking diploid grass carp (Ctenopharyngodon idella L.) on the aquatic vegetation was evaluated in these Waikato drainage systems. At the start of the trial, both drains had a low diversity of aquatic macrophytes, and of the nine species (including the emergents), seven were exotic. Two months after grass carp were released to Churchill East drain (the treated drain) the four submerged and floating macrophyte species became scarce in the main drain. Over the same period, these species increased in biomass in Rangiriri drain (the untreated drain), where hornwort became dense and surface‐reaching and remained so for the duration of the trial. However, grass carp did not control submerged vegetation in smaller side drains or the shallow, upper parts of the main drain, or the marginal sprawling species and emergent species. The cost of leasing the grass carp was similar to the cost of clearing the drains mechanically, but grass carp provided continuous weed control. However, subsequent to this trial, 62 dead grass carp were found in Churchill East drain in February 2001, and weed cover subsequently increased. This illustrates that grass carp management in New Zealand agricultural drains can be problematic due to periodic fish kills.

Molecular Genetic Aspects of Tetraploidy in the Common Carp Cyprinus carpio

Genome duplications are believed to have occurred on multiple occasions in vertebrate evolution. Studies of duplicate gene loci in tetraploid animals may reveal important general aspects of gene duplication, an important mode of gene evolution in metazoans. The common carp Cyprinus carpio has twice as many chromosomes as most other cyprinid fishes due to tetraploidization previously estimated to have occurred 50 Myr ago. Our sequence analyses of duplicate carp loci suggest that the tetraploidization took place less than 16 Myr ago. This is further supported by sequence comparisons with the diploid grass carp, which seems to have diverged from the common carp approximately 19 Myr ago. Duplicate loci appear to remain expressed for millions of years and may accumulate mutations leading to drastic amino acid replacements as shown here for somatotropin. Therefore, both loci should always be characterized in molecular studies of tetraploid animals such as goldfish, salmonid fishes, and Xenopus laevis. The long life of duplicate genes may explain the occurrence of numerous large multigene families in higher metazoans.

Egg fertility, early development and survival from crosses of diploid female × triploid male grass carp ( Ctenopharyngodon idella)

Ova obtained from diploid grass carp females were inseminated with sperm from diploid and triploid males, during three consecutive spawning seasons (1986, 1987, 1988), with reproducible results. Fertilization success was significantly lower in diploid × triploid crosses (27, 38 and 25%) compared with diploid × diploid crosses (49, 84 and 82%). Hatching embryos from diploid × triploid crosses had significantly ( P < 0.05) smaller body and yolk sac areas, and utilized their yolk more slowly than embryos from diploid × diploid crosses. Most diploid × triploid embryos surviving to hatch, suffered from notochord deformities and died before exogenous feeding began. Survival of juveniles from diploid × triploid crosses from hatching to 5 months was 0.21% and 0.125% for 1987 and 1988, compared with 95% and 84%, for diploid × diploid crosses. Coulter Counter ploidy determination for nine (five in 1987, four in 1988) diploid × triploid juvenile survivors revealed a diploid genome.

Chicken Erythrocytes as an Internal Reference for Analysis of DNA Content by Flow Cytometry in Grass Carp

Abstract We estimated the nuclear DNA content of unfixed erythrocytes of the grass carp Ctenopharyngodon idella by fluorescence flow cytometry using propidium iodide as a fluorochrome. Frozen erythrocytes from the domestic chicken Gallus gallus were thawed and used as an internal reference for simultaneous analysis with grass carp samples. The nuclear DNA content of erythrocytes from diploid grass carp (mean ± SD) was 2.00 ± 0.01 pg/cell, whereas triploids possessed 2.97 ± 0.03 pg/cell. Variations in the nuclear DNA content ofdifferent chickens were standardized in relation to the DNA content of fresh human leukocytes (7.00 pg/cell). The value for DNA content of the chicken erythrocytes (around 2.5 pg/cell) occupied a position intermediate between the two grass carp values, and thus provided a clear reference for discrimination between diploids and triploids.

Evaluation of the separation of triploid and diploid grass carp, Ctenopharyngodon idella (Valenciennes), by external morphology

The external morphology of diploid and triploid grass carp was studied to determine whether the two types could be separated by differences in external characteristics. In a comparison of 27 measurements, six scale counts and five fin formulae from two stocks of diploid and triploid fish obtained from commercial producers, analysis of covariance and discriminant analysis indicated a classification accuracy of only 65–85%. Clearly, triploid grass carp differentiated by external morphology alone should not be stocked into waters where diploid fish are illegal or would cause management problems.

Growth Comparisons of Diploid and Triploid Grass Carp under Varying Conditions

Abstract Growth of fry and fingerling diploid and triploid grass carp (Ctenopharyngodon idella) was evaluated under a variety of experimental situations that involved high and low stocking densities and various levels of food availability. Diploid grass carp grew faster and had significantly (P < 0.05) higher condition factors in every situation where they were grown together with triploid grass carp. When diploid and triploid grass carp were grown in separate pools and fed to satiation with duckweed, there were no significant differences (P < 0.05) in growth, condition factors, food conversions, or rates of food consumption. The difference in the rate of growth between diploid and triploid grass carp can be exploited to presegregate diploid from triploid fish, thus reducing the time spent certifying ploidy with a Coulter Counter (R).

Rapid Identification of Triploid Grass Carp with a Coulter Counter and Channelyzer

Abstract A method of electronically estimating ploidy in fishes has been developed to maximize the number of fish that can be analyzed per hour while minimizing costs. This technique uses a Coulter Counter Model ZM and a C-1000 Channelyzer to analyze distribution of erythrocyte nuclear volumes. Diploid grass carp (Ctenopharyngodon idella) possess mean nuclear volumes of 10.06 μm3 (SD = 0.60) while mean triploid nuclear volumes were 14.82 μm3 (SD = 0.96). The technique proved effective for fish that were 27 d to 3 years old, 19-905 mm total length, and rapidly growing or nutritionally stressed. With the modifications and techniques suggested, 1,600-2,400 fish can be evaluated by a three-person team in an 8-h period. Use of this technique allowed triploid grass carp to be efficiently selected and certified prior to stocking for weed control purposes.