The Development of Trojan Sex Chromosome Carrying Green Sunfish Lepomis Cyanellus and Red Shiner Cyprinella Lutrensis to Control Their Nuisance Populations

Green Sunfish Lepomis cyanellus and Red Shiner Cyprinella lutrensis are considered highly invasive when introduced outside of their native range. Trojan sex chromosome eradication models have shown the release of YY males (or ZZ females) to be effective at suppressing and eradicating invasive populations of species across genera. Developing Trojan sex chromosome carrying individuals requires aquaculture protocols, knowledge about gonadal development, identification of sex determination systems, and sex reversal methods for the species of interest. We have developed aquaculture protocols for Green Sunfish and Red Shiner. Our spawning methods provided year-round volitional spawns from Green Sunfish and Red Shiner broodstock. Our larval rearing methods resulted in consistent year-round production of Green Sunfish and Red Shiner, allowing us to identify the timing of their gonadal differentiation through histological assessment. Green Sunfish and Red Shiner are gonochoristic, with testes and ovaries differentiating directly from undifferentiated gonads. Green Sunfish’s gonadal differentiation was observed between 39 days post hatch (dph) – 69 dph. Red Shiner’s gonadal differentiation was observed between 45 dph – 105 dph. To investigate these species’ sex determination systems, we used restriction-site associated DNA sequencing (RAD-Seq) for SNP discovery and genotyping of known-sex Green Sunfish and Red Shiner 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. In Green Sunfish, 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 for Green Sunfish, no candidates reliably identified phenotypic sex.The top performing Green Sunfish SNP candidate (ZW_218) correlated with phenotypic sex 63.0% of the time and the presence-absence loci universally amplified in both sexes. Genetic investigations that interrogate a larger fraction of the Green Sunfish genome could uncover a reliable sex identification marker. Additionally, studies on environmental influences on Green Sunfish sex determination systems may be necessary since we observed heavily male-skewed sex ratios of control groups (82.61% male) during aquaculture protocol development and sex reversal trials for this species. For Red Shiner, we used RAD-Seq in addition to a series of breeding experiments with sex reversed males (neofemales) to uncover their 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 a phenotype-genotype concordance score of 77.00% and 84.35%, respectively. These sex-ID markers exhibited a relatively high phenotype-genotype concordance in Red Shiner females (XY_248 = 96.30%, XY_170 = 98.61%) which allowed for selective breeding of neofemales. We observed a 3:1 male to female sex ratio in spawns from neofemales and wild-type males, indicative of a male heterogametic sex determination system (i.e., XY- male/XX-female) within this species. For the sex reversal trials, a low-dose (100 E2 mg per kg of diet) and a high-dose (150 E2 mg per kg of diet) experimental E2 treatment were fed to juvenile Green Sunfish from 30 to 90 dph. Both E2 treatments resulted in 100% feminization, with no morphological or histological differences detected between E2 treated ovaries and those from a control group. Overall, there was no effect of E2 on survival (P = 0.310) and growth rate data suggested no statistical differences (P = 0.0805). However, the growth rate of the high-dose group increased slightly higher after the treatment ended than the other treatments (P = 0.042), suggesting that E2 might suppress growth in Green Sunfish. In addition, the control group did not exhibit a lower mortality rate after the treatment period ended (P = 0.266), whereas both E2 treated groups did (P = 0.0003–0.0050). We found that the low-dose, 100 E2 mg per kg of diet, was sufficient for fully feminizing Green Sunfish if administered during development from 30 to 90 dph and E2 dosages may result in deleterious effects on Green Sunfish’s health and growth. We observed 100% feminization of Red Shiner fed either 50 mg E2 per kg of diet from 2-120 dph or 100 mg E2 per kg of diet from 20-120 dph. Among Red Shiner given the control diet, the 50 mg of E2 per kg of diet, or the 100 mg of E2 per kg of diet from 2-62 dph, the gonadosomatic index and mortality rate were significantly higher in fish given the 100 mg of E2 per kg of diet than the other treatments (P 0.2163). Putative YY Red Shiner that resulted from spawning neofemales with wild-type Red Shiner were viable and differences in fecundity among YY individuals and wild-type individuals were insignificant as exhibited by mean egg counts per day when YY individuals were crossed with wild-type counterparts (P > 0.05). In the 20 spawns observed from crossing either YY-females with YY-males, YY-males with XX-females, or YY- females with XY-males, all the offspring developed into males except for one female that developed from a possibly inbred cross between a putative YY-daughter and an XY-father. The presence of this female may be indicative of autosomally-derived sex modifying genes that produce female phenotypes due to inbreeding homozygosity. Despite the presence of a female from one YY verification cross, the prevalence of all-male progeny from all other YY crosses suggests that the use of a Trojan sex chromosome eradication strategy will be effective at extirpating nuisance Red Shiner populations