International Journal of Marine Science, 2025, Vol.15, No.3, 167-178 http://www.aquapublisher.com/index.php/ijms 174 studies have increased the expression of endogenous HSP24 in abalone through heat induction, which is equivalent to a "overexpression" effect. It was found that the survival rate of pre-heat shock-treated abalone significantly increased the subsequent fatal high temperature stress ((Wan et al., 2012)). This functionally reflects the protective effect of small molecule heat shock proteins in anti-heat. Although this is strictly not a genetically modified overexpression, it provides functional evidence. To achieve true stable overexpression, strategies such as shellfish virus vectors can be used. 6.2.3 Exploration of the function of reproductive development regulatory genes Compared with growth and immunity, the functional research on genes related to abalone reproduction is relatively weak, but there have been some explorations in recent years. To verify the role of the abalone IGFBP-5 gene mentioned above in reproduction, researchers conducted gene interference experiments. After knocking down IGFBP-5 in the neural tissue of female abalone, it was observed that the secretion of its gonadotropin mature hormone (presumably GnRH-like substance) was reduced, resulting in a delay in ovarian development by about 10 days, delaying egg laying than the normal group. This result supports a model of the role of IGF signaling in regulating gonad development (Wang et al., 2016). Similarly, functional studies have also been conducted for possible key hormone receptor genes in abalone reproduction, such as estrogen-associated receptor (ERR) genes. Some studies have used antagonists to inhibit ERR activity, which is equivalent to loss of function. It was found that the expression of yolk proteogen gene in abalone gonads has decreased and egg development is blocked, indicating that ERR signal may be involved in yolk synthesis and oocyte maturation. Furthermore, some studies have focused on meiotic regulatory genes during abalone gamete generation, such as Cdc25. Through drug inhibition of Cdc25, it was found that abalone oocytes were stagnant in the first premeiosis phase and could not mature, thus demonstrating the conservative role of cell cycle regulation in abalone egg maturation (Figure 2) (Ferencova et al., 2022). Although these functional studies are fragmented, they suggest several gene targets that can affect fertility. Figure 2 Cdc25b+/− oocytes resume meiosis but partially arrest in MI (Adopted from Ferencova et al., 2022) 6.3 Challenges and prospects in building a functional verification model system Although technologies such as RNAi and CRISPR have been tried in abalone, there are still multiple challenges to establishing a mature functional gene verification model system. In terms of experimental materials: Abalone embryos and larvae are demanding and fragile to the environment, and it is not easy to obtain a large number of synchronously developed embryos for microscopy operations. Currently, most functional tests remain in the juvenile stage and have not developed a pattern that can cultivate edited individuals into adults and observe traits. Secondly, the abalone generation cycle is relatively long and generally has sexual maturity of 2 to 3 years, making it time-consuming and labor-intensive to establish a stable genetic modification genealogy. This is in sharp contrast to the rapid iteration of model organisms such as zebrafish and mice. Therefore, developing alternative
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