Developing detailed rearing methods and describing the onset of gonadal differentiation in Red Shiners Cyprinella lutrensis could facilitate the development of novel techniques to control or enhance populations, enable toxicology studies, and help construct bioassays. In this study, we develop and report aquaculture practices for Red Shiner that ensure consistent year‐round production in laboratory settings and evaluate the timing of sexual differentiation via histological gonad examinations. Our methods resulted in a mean of 56.00% (SD = 8.98%) survival through the larval stages of development, and we obtained spawns from captive‐reared Red Shiners 138 d posthatch. Red Shiners are gonochoristic, and both ovaries and testes differentiate directly from undifferentiated gonads. Ovaries begin to differentiate in females 45 d posthatch, while testes begin differentiating in males 105 d posthatch. This study provides in‐depth protocols for the closed‐cycle aquaculture of Red Shiners and describes the gonadal differentiation and development of both sexes.

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The Red Shiner Cyprinella lutrensis is of increasing management interest as an invasive species that negatively impacts many native fishes throughout North America. Trojan sex chromosome (TSC)‐carrying individuals could theoretically control invasive fish populations by skewing the sex ratio to 100% male. The efficacy of TSC‐based control programs requires an understanding of a population's sex determination system, yet such information is lacking for Red Shiner. We used single‐digest restriction site‐associated DNA sequencing to discover sex‐linked single‐nucleotide polymorphisms (SNPs), and we conducted a series of breeding experiments to uncover the sex determination system. All candidate sex‐linked SNPs that fit our selection criteria exhibited a pattern of male heterogamety. We developed two sex‐identification (sex‐ID) marker assays, XY_248 and XY_170, which showed phenotype–genotype concordance scores of 77.00% and 84.35%, respectively. These sex‐ID markers exhibited relatively high phenotype–genotype concordance in females (XY_248 = 96.30%; XY_170 = 98.61%), which allowed for selective breeding of phenotypically feminized genetic males. We observed a 3:1 male : female sex ratio in spawns from feminized males crossed with wild‐type males, indicative of a male heterogametic sex determination system (i.e., XY male/XX female). The discovery of a male heterogametic sex determination system, in combination with our two markers, increases the likelihood of developing an effective TSC eradication strategy for invasive Red Shiner populations.

We used restriction‐site associated DNA sequencing for SNP discovery and genotyping of known‐sex green sunfish Lepomis cyanellus DNA samples to search for sex‐diagnostic single nucleotide polymorphisms (SNPs) and restriction‐site associated sequences present in one sex and absent in the other. The bioinformatic analyses discovered candidate SNPs and sex‐specific restriction‐site associated sequences that fit patterns of male or female heterogametic sex determination systems. However, when primers were developed and tested, no candidates reliably identified phenotypic sex. The top performing SNP candidate (ZW_218) correlated with phenotypic sex 63.0% of the time and the presence‐absence loci universally amplified in both sexes. We recommend further investigations that interrogate a larger fraction of the L. cyanellus genome. Additionally, studies on the effect of temperature and rearing density on sex determination, as well as breeding of sex‐reversed individuals, could provide more insights into the sex determination system of L. cyanellus.

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