| The sex of fish is very plastic,and sex determination in Nile tilapia is both controlled by genetic factors and affected by temperature.During the critical period of sex determination,high-temperature（36°C）can induce the sex reversal from XX genetic female to physiological male.Therefore,the sex determination mechanism of Nile tilapia is GSD+TE type（Genetic sex determination + temperature effect）.However,the molecular mechanism of how temperature affects sex determination in Nile tilapia remains unclear.It has been shown that certain key sex determination genes were regulated by alternative splicing in insects,reptiles,mammals and other animals.In this study,we investigated the effect and possible mechanisms of hightemperature on alternative splicing of pre-m RNA in the gonads of Nile tilapia,the results are as follows:1.Pac Bio Iso-Seq（Isoform-sequencing）analysis of high-temperature treated gonads.The Nile tilapia genetic females（XX）of 9 dpf were treated at 36℃ for 30 d（39 dpf）.The gonadal tissues from the control female group（FC）and high-temperature treated female group（FT）were sampled for Pac Bio Iso-Seq.4,478 and 5,504 alternative splicing genes were identified in FC and FT,accounting for 47.78% and 50.10% of the encoding genes,respectively.10,649 and14,302 alternative splicing events were identified in FC and FT,respectively.Retention intron（RI）events accounted for the largest proportion.The gene numbers of 7 alternative splicing events that were unique and shared by FC and FT were analyzed.RI events had the largest shared number,and alternative first exon（AF）events had the largest unique number.Alternative splicing analysis of important sex determination and differentiation-related genes revealed that high-temperature treatment had increased the numbers of alternative splicing events of 11 sex differentiation-related genes such as Kdm6 bb.2.Combined analysis of Pac Bio Iso-Seq and RNA-Seq screened differentially expressed transcripts（DETs）between FT and FC.A total of 122 DETs were identified,of which 65 were up-regulated and 57 were down-regulated.The 122 DETs corresponded to a total of 115 genes.108 genes were one-to-one correspondence with the DETs,and the other 7 genes were all corresponding to 2 DETs.A total of 298 differentially expressed genes（DEGs）were screened by RNA-Seq of gonadal tissue of 21 dpf Nile tilapia.The genes corresponding to DETs overlapped with DEGs,including Armc4,Vcl,Dmrt1,Kiaa0319,Cdk20,Tcf7l1-c,Rec8 and Ermap.3.Analysis of alternative splicing events in Kdm6 bb under high-temperature.Hightemperature had the greatest effect on the frequency of RI5 events of Kdm6bb（FC: 68.42%,FT:5.00%）,therefore,RI5 was the main event of alternative splicing in Kdm6 bb affected by hightemperature.SQ-PCR showed that the splicing ratio of intron 5 was significantly higher in tilapia brain,kidney,muscle,skin,spleen,intestine and gonadal tissues in FT than in FC.The splicing ratio of intron 5 was 34.30 ± 1.06%,92.11 ± 3.28% and 59.30 ± 1.71% in the gonads of FC,FT and normal males,respectively.4.To screen for key cis-acting elements that may regulate splicing of Kdm6 bb intron5.The p CI-neo-Kdm6 bb plasmid was constructed and transfected into Nile tilapia embryonic stem cells（TES1）and Zebrafish embryonic cells（ZF4）,both of which can not express Kdm6 bb.Only RI5 and Δ5 bands were detected in both cell lines after transfection in TES1 cells,and the transfection efficiency in TES1 cells was significantly higher than that in ZF4 cells.Therefore,TES1 cells were selected to study the splicing pattern of Kdm6 bb intron5 in vitro.Intron 5 in p CI-neo-Kdm6 bb was deleted in a unit of 18 bases and 17 p CI-neo-Kdm6 bb deletion plasmids were constructed.All deletants were transfected into TES1 cells,and the results showed that compared to p CI-neo-Kdm6bb（WT）,the splicing ratio of intron 5 had significantly increased in deletants D181-198 and D253-269（P < 0.05）.The splicing ratio had significantly decreased in deletant D37-54（P < 0.01）.It is tentatively suggested that the 181-198 and 253-269 regions in intron 5 may be intronic splicing silencer（ISS）that regulates splicing of Kdm6 bb intron 5,and the 37-54 region may be an intronic splicing enhancer（ISE）.5.To screen for splicing factors that may regulate splicing of Kdm6 bb intron 5.Analysis of splicing factors that can bound on and regulate intron splicing on Kdm6 bb intron 5 and flanking exon sequences using cat RAPID revealed that it contained eight MBNL1（Muscleblind like splicing regulator 1）binding sites.I-V were in intron 5 and VI-VIII were in exon 6.The MBNL1 binding site mutant fragments were obtained by PCR amplification using p CI-neoKdm6 bb as a template,and 4 p CI-neo-Kdm6 bb mutant plasmids were constructed and transfected into TES1 cells.SQ-PCR has shown that compared with WT,the splicing ratio of Kdm6 bb intron 5 showed a significant increase in M Ⅰ-VIII（P < 0.01）.The splicing ratio in MⅠ-V was also significantly increased（P < 0.01）,and it was higher than that in M Ⅰ-Ⅷ.However,the splicing ratio in M VI-VIII was significantly reduced（P < 0.01）,indicating that MBNL1 s can bind to the relevant sites to regulate splicing of intron 5 and may function in a positiondependent manner.Thus,MBNL1 is likely to be a regulator of splicing of Kdm6 bb intron 5.In summary,the present study found that high-temperature treatment significantly had altered the splicing pattern of pre-m RNA in the gonads of Nile tilapia,increased the number of alternative splicing events,and screened the alternative splicing gene,Kdm6 bb,involved in sex determination.RI5 is the main event that high-temperature affected Kdm6 bb alternative splicing.The present study found that high-temperature can up-regulate the splicing ratio of Kdm6 bb intron 5 in most tissues,identified ISS,ISE and upstream splicing protein MBNL1 that regulate the splicing of intron 5,and preliminarily elucidated the molecular mechanism of hightemperature regulating splicing of Kdm6 bb intron 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