The highly conserved transcription factor Pax6 is involved in the development of the eyes, brain, and pancreas in vertebrates and invertebrates, whereas the additional expression pattern in other organs is still elusive. In this study, we cloned and characterized two pax6 homologs in blunt snout bream (Megalobrama amblycephala), named Mapax6a and Mapax6b. The protein alignment and phylogenetic tree showed that Mapax6a and Mapax6b were highly conserved compared with their counterparts in other species. Genomic information analysis revealed that the synteny conservation of Wilms tumor, Aniridia, genitourinary abnormalities, and mental retardation loci was also maintained in this species. By reverse transcription polymerase chain reaction, the expression of Mapax6a was later than that of Mapax6b which was found in the blastula stage, while the expression of Mapax6a started from the somite stage, and both of them persisted in a subsequent stage during the embryonic development. By RNA and protein detection, Mapax6a and Mapax6b were detected in the eye and brain as canonic patterns, and most importantly, they were also enriched in germ cells of the testis and ovary. Therefore, our findings validate the duplication of pax6 in fish, confirm the classical expression patterns in the brain and eye, and, for the first time, present a new acquisition of Mapax6a and Mapax6b in gonadal germ cells in particular. Therefore, our results enrich the expression pattern and evolutionary relationship of pax6 by suggesting that duplicated Mapax6 is involved in gametogenesis in Megalobrama amblycephala.

Topmouth culter (Culter alburnus) is one of the most economically important freshwater fish in China, but intensive aquaculture has caused germplasm degradation and reduced growth performance, while the genetic basis underlying growth variation in this species remains poorly understood. This study aimed to identify major-effect loci and candidate genes associated with growth-related traits to support molecular breeding. Whole-genome resequencing (average depth 11.44×) was performed on 300 individuals derived from random mating among three geographic populations (Danjiangkou, Taihu, and Poyang Lake); 239 individuals with complete phenotypic records were retained for a genome-wide association study (GWAS) of five growth-related traits, including body weight (BW), body weight without viscera (BWW), total length (TL), body length (BL), and body height (BH). After stringent filtering, 7,597,008 high-quality single-nucleotide polymorphisms (SNPs) were obtained, and association analysis was conducted using a linear mixed model, followed by Benjamini–Hochberg false discovery rate correction and 1000-permutation testing for BW and BL. Six genome-wide significant SNPs and 473 suggestive SNPs were identified, with individual significant SNPs explaining over 11% of phenotypic variance, indicating candidate loci of putatively moderate-to-large effect. Significant SNPs were predominantly clustered on chromosomes 16 and 19. Four candidate genes—aig1, cacna1b, pgm5, and bcr—were identified, with functions related to lipid metabolism, muscle structure, and cell proliferation. This first population-level GWAS in topmouth culter provides valuable molecular markers for marker-assisted selection and lays a foundation for accelerated genetic improvement of this species.

To address cryodamage in Culter alburnus sperm, this study evaluated the effects of trehalose supplementation in a conventional cryomedium (D-15 + 10% ethylene glycol). Six experimental groups were established: fresh sperm (G1), a conventional cryomedium (G2), groups supplemented with 10, 100, or 200 mmol/L trehalose (G3–G5), and a control group with extender only (G6). The group with 100 mmol/L trehalose (G4) was associated with improved post-thaw motility parameters (activation rate, movement time, and lifespan) and higher antioxidant (superoxide dismutase and catalase) and energy metabolism (ATPase, succinate dehydrogenase, lactate dehydrogenase) enzyme activities. Ultrastructural damage in G4 included partial plasma membrane rupture and mitochondrial swelling, while G6 exhibited additional damage features including membrane disintegration, mitochondrial disruption, and flagellar fracture. Proteomic analysis revealed that, compared to G1, G4 exhibited higher abundance of proteins (e.g., Histone H2A, cytochrome c oxidase, profilin) involved in structural integrity and energy homeostasis, whereas G6 showed signatures of oxidative stress and metabolic dysfunction (lower abundance of NADH dehydrogenase and higher abundance of calcium-transporting ATPase and glutathione S-transferase). In conclusion, 100 mmol/L trehalose was associated with improved cryopreservation outcomes, and the proteins identified provide a basis for further investigation. This approach offers a framework for refining germplasm conservation strategies in aquaculture.

Procambarus clarkii and Macrobrachium nipponense are two freshwater shrimp species used in rice-farming systems. To guide their cultivation in saline–alkaline waters, two 96 h experiments were conducted on juvenilels of each species using 540 juveniles per species (initial body length: 1.51 ± 0.16 cm for P. clarkii and 0.88 ± 0.03 cm for M. nipponense): combined salinity–pH stress (0/15.0/25.0‰ at pH 7.4/9.0/10.0) and NaHCO₃ alkalinity stress (0/10/20 mmol/L). Under salinity–pH stress, survival of P. clarkii was 100%, 72.23%, and 53.33%, respectively; for M. nipponense, it was 93.33%, 61.13%, and 23.33%. Under alkalinity stress, survival of P. clarkii was 92.22%, 82.22%, and 61.11%; for M. nipponense, it was 91.11%, 65.55%, and 46.67%. In both species, superoxide dismutase, acid phosphatase, and alkaline phosphatase decreased while malondialdehyde increased with stress intensity. Metabolomics revealed species-specific reprogramming: P. clarkii upregulated protective lipids (e.g., glycerophosphoethanolamines, fatty acid esters), whereas M. nipponense showed depletion of critical osmolytes (glycerophosphocholines) and accumulation of apoptosis-related ceramides. KEGG analysis confirmed that energy metabolism, amino acid homeostasis, and ABC transporters were primary pathways affected. In conclusion, extreme stress significantly reduced survival, suppressed immunity, and triggered metabolic reprogramming in both species, with P. clarkii showing substantially higher tolerance. For cultivation, P. clarkii is preferred in moderately high saline–alkaline waters, while M. nipponense requires low-alkalinity freshwater with strict monitoring. These findings support species-specific management of saline–alkaline rice–shrimp systems.