Use of Eyeballs for Establishing Ploidy of Asian Carp

Abstract

Grass carp Ctenopharyngodon idella, silver carp Hypophthalmichthys molitrix, and bighead carp H. nobilis are now established and relatively common in the Mississippi and Atchafalaya rivers. Commercial fishers of Louisiana's large rivers report recurrent catches of grass carp, and the frequency of bighead carp and silver carp catch is increasing. Twelve black carp Mylopharyngodon piceus were recently captured from the Mississippi and Atchafalaya River system, and 10 were analyzed for ploidy. By using the methods described herein, all 10 fish were determined to be diploid. Such correct identifications of ploidy of feral Asian carp species, as well as other species, would provide science-based information constructive for meeting reporting requirements, tracking fish movements, and forecasting expansion of species distribution. To investigate the postmortem period for sample collection and to lessen demands on field operations for obtaining samples, a laboratory study was performed to determine the length of time for which eyeballs from postmortem black carp could be used for ploidy determinations. Acquiring eyes rather than blood is simpler and quicker and requires no special supplies. An internal DNA reference standard with a documented genome size, including erythrocytes from diploid black carp or Nile tilapia Oreochromis niloticus, was analyzed simultaneously with cells from seven known triploid black carp to assess ploidy through 12 d after extraction. Ploidy determinations were reliable through 8 d postmortem. The field process entails excision of an eyeball, storage in a physiological buffer, and shipment within 8 d at refrigeration temperatures (48C) to the laboratory for analysis by flow cytometry.

Comparison of Diploid and Triploid Saugeye Recruitment, Growth, and Condition in Kansas Impoundments

“…), which consisted of 0.2% Triton X‐100 (Sigma Aldrich, St. Louis, Missouri), 0.05‐g/L propidium iodide (Sigma Aldrich), and 2.0‐g/L RNAse A (BioBasic, Inc., Markham, Ontario) in sterile, 0.45‐μm‐filtered phosphate‐buffered saline. After incubation on ice for 30 min, samples were analyzed for DNA content using a FACScan flow cytometer (BD Biosciences, San Jose, California) set to collect 10,000 particle events at 488 nm under doublet discrimination mode (Jenkins and Thomas ; Fetherman et al. ).…”

Mitochondrial DNA Dosage Effects in Triploid Grass Carp

“…However, because the contribution of mtDNA molecules to the overall cellular DNA appears to be conserved in triploid individuals, the methodology described herein is wholly ineffective for use as a retrospective diagnostic. As such, it is recommended that one of the previously described techniques be used to distinguish between diploid and triploid grass carp instead (Beck et al 1980;Wattendorf 1986;Bonar et al 1988;Jenkins and Thomas 2007).…”

“…The alternative hypothesis evaluated was that the typical nDNA : mtDNA ratio of diploid carp is disrupted in triploids (that is, the contribution of mtDNA to whole cellular DNA is reduced), such that there are significant measurable effects on the relationships between nDNA and mtDNA C t . In the case of the alternative hypothesis, the difference in nDNA : mtDNA ratio between diploids and triploids can be used to supplement the already-established techniques for ploidy determination in grass carp (Beck et al 1980;Wattendorf 1986;Bonar et al 1988;Jenkins and Thomas 2007).…”

Grass carp in the Great Lakes region: establishment potential, expert perceptions, and re-evaluation of experimental evidence of ecological impact

“…For ploidy analysis, grass carp eyeballs were excised in the field, stored up to 11 days in sterile biological Hank's balanced salt solution at a pH of 7.4 and 315 milliosmoles·kg -1 at 4°C until analysis by flow cytometry. See Jenkins and Thomas (2007) and Thomas et al (2011) for a detailed description of methodology. Cell types were determined as triploid, diploid, or mosaic (in which individuals contained both diploid and triploid cells).…”

“…Improved predictions of geographic areas at risk for invasion through the use of species distribution models (SDMs) have been achieved with methodological advances (Gallien et al 2010;Jiménez-Valverde et al 2011) and increases in the quantity and quality of species occurrence databases (e.g., Global Biodiversity Information Facility) and environmental data layers (e.g., WorldClim). Combined with recent assessments of invasion pathways (Wilson et al 2009) and propagule pressure (Lockwood et al 2005), improvements in detection technologies such as environmental DNA (eDNA; Ficetola et al 2008;Jerde et al 2011) and fish ploidy assessment (Jenkins and Thomas 2007) have increased the capacity to improve other Asian carp risk assessments, prevent species introductions, and efficiently monitor areas of species presence and dispersal (Nico et al 2005;Kolar et al 2007).…”