Learning And Memory,Epigenetic Modification,and Molecular Markers In The Food Habit Domestication Of Mandarin Fish

Mandarin fish is one of the most commercially important freshwater fishes for aquaculture in China.Once the fry of mandarin fish start feeding,they only accept live prey fish in the wild and refuse dead prey fish or artificial diets under cultivation,which contribute to its unique feeding habits.At present,the source of feed in the intensive culture of mandarin fish is almost cultivated live prey fish,which not only leads to extremely high cultivation costs,so it is accompanied by the problem that supply cannot be guaranteed all the year round,but also disease prevention is very difficult in the cultivation process.The technique of artificial diets domestication in mandarin fish is a problem that needs to be solved in the field of aquaculture.Mandarin fish refuse dead prey fish or artificial diets and can be trained to transform their inborn feeding habit.Feeding habit is a congenital behavior characteristic,which might be attributed to the integration of natural inheritance and acquired learning and memory.Learning and memory play an important role in adjusting feeding behavior according to environmental changes to improve feeding success rate,which mandarin fish is able to learn socially.In the present study,to investigate the effect of learning and memory on feeding habit transformation,we compared the success rate of feeding habit transformation from live prey fish to dead prey fish,the food intake of dead prey fish,the standard learning times of biting or swallowing dead prey fish,the number of robbing actions,and the occurrence rate of robbing action of mandarin fish in control group(without pre-trained demonstrator)and experimental group(with pre-trained demonstrator).The behavioral parameters,expression of genes involved in learning and memory,and de novo transcriptome sequencing was performed in mandarin fish during food habit demonstrations with or without pre-trained demonstrator,respectively.Then,the genes and signaling pathways involved in whether to participate in social learning that led to differences in the individual domestication ability were compared.The mandarin fish in the experimental group(with pre-trained demonstrator)had higher food intake(19.38±1.22)and success rate of feeding habit transformation(86%)than those in the control group(without pre-trained demonstrator)(8.92±0.71,65%)(P<0.05),and lower standard learning times(3.46±0.24)than those in the control group(5.89±0.30)(P<0.05).There are more competitive behaviors among individuals in the experimental group,the number of robbing actions(12.31±1.35)and the occurrence rate of robbing action(78%)were significantly increased in the experimental group than those in the control group(5.42±0.65,41%)(P<0.05).Gene expression of CCAAT enhancer binding protein D(cebpd),early growth response 1(egr1,also known as zif268),and fos-related antigen 2(fosl2,also known as fra2),proto-oncogene c-Fos(c-fos),and synaptotagmin-4(syt4)were significantly increased(P<0.05)in the mandarin fish brains of the experimental group(with pre-trained demonstrator).Therefore,the enhanced transcription of these genes might play important roles in memory consolidation after social learning.Compared with the undomesticated mandarin fish in the control group(without pre-trained demonstrator),12472 genes were significantly up-regulated and 12347 genes were significantly down-regulated in the undomesticated mandarin fish in the experimental group(with pre-trained demonstrator).Enrichment analysis of differentially expressed genes shows 13 signal pathways related to changes in learning and memory in the food habit demonstrations of mandarin fish: long-term potentiation,long-term depression,circadian rhythm,circadian entrainment,c AMP signaling pathway,neurotrophin signaling pathway,retrograde endocannabinoid signaling,glutamatergic synapse,cholinergic synapse,serotonergic synapse,GABAergic synapse,dopaminergic synapse and taste transduction.The related differentially expressed genes are closely related to feeding habit transformation caused by whether the mandarin fish is fully engaged in social learning(with or without pre-trained demonstrator).It is speculated that the feeding habit transformation is related to the long-term potentiation and long-term inhibition associated with the learning and memory,as well as the circadian rhythm and taste signal transduction associated with the feeding process.The results showed that mandarin fish has the ability of social learning in domestication.The undomesticated mandarin fish individuals in the experimental group rapidly adjusted their feeding behavior,developed new food preferences,improved the efficiency and success rate of food domestication,and accelerated the feeding habit transformation by learning and imitating the pre-trained demonstrators.Social learning process might be the mechanisms underlying group domestication method.Learning and memory could plastically shorten the time of feeding habit transformation,and they play an important role on domestication of mandarin fish.In the present study,to investigate the effect of memory on feeding habit transformation,we compared the reaction time to dead prey fish and the success rate of feeding habit transformation from live prey fish to dead prey fish of mandarin fish in the1 st experimental group(trained once)and the 2nd experimental group(trained twice).The behavioral parameters,expression of genes involved in learning and memory,and DNA methylation levels in the Cp Gs of the taste receptor type 1 member 1(t1r1)gene involved in feed identification were examined in mandarin fish.The mandarin fish in the2 nd group had higher success rate of feeding habit transformation(100%)than those in the 1st group(67%),and shorter reaction time to dead prey fish(<1 s)than those in the1 st group(>1 s).After the first training,gene expression of cebpd,egr1,and c-fos were significantly increased(P<0.05)in the mandarin fish brains of the 1st experimental group.Compared with the 1st experimental group,the expression levels of creb1,bdnf,cebpd,fosl2,and c-fos involved in long-term memory formation were significantly increased(P<0.05)while the expression levels of egr1 and t1r1(involved in feeding habit formation)were significantly reduced(P<0.05)in the 2nd experimental group after repeated training.Compared with the control group,the gene expression of syt4 was significantly increased(P<0.05)in the 2nd experimental group after two trainings.Therefore,the enhanced transcription of these genes might play important roles in memory consolidation of accepting dead prey fish.No significant changes in DNA methylation levels were detected in the promoter regions of creb1,bdnf,cebpd,egr1,fosl2,c-fos and syt4.DNA methylation levels at five candidate Cp G(cytosine–guanine)sites contained in the predicted Cp G island in the 5′-flanking region of t1r1 were significantly decreased(P<0.05)in brains of the 2nd group compared with that of the 1st group.The methylation level of the Cp G island in the t1r1 gene was positively correlated with the expression level.These results indicated that the repeated training can improve the feeding habit transformation through the memory formation of accepting dead prey fish.DNA methylation of the t1r1 might be a regulatory factor for feeding habit transformation from live prey fish to dead prey fish in mandarin fish.This study improves the understanding of molecular mechanisms of learning and memory and the epigenetic regulation during the unique feeding habit transformation in mandarin fish.Based on the existing research,this study analyzed the transcriptome data related to learning and memory of demonstration in mandarin,and the differences in expression of pepsinoge(pep)gene and growth hormone(gh)gene were detected.Gene pep and gh have been considered as candidate genes for food habit domestication traits in mandarin fish.In this study,polymorphic SNPs on candidate genes were genotyped in dead prey fish/artificial diets feeder group and nonfeeder group,and evaluated for associations with food preference traits in 103 mandarin fish individuals.Five single nucleotide polymorphisms(SNPs)were totally identified in pep and gh genes.The effective allele numbers ranged from 1.1959 to 1.7001,with a mean of 1.4274 alleles per locus.The observed(Ho)and expected heterozygosities(He)values ranged from 0.1800 to 0.3585,and from 0.1655 to 0.4160,respectively.The value of polymorphism information content(PIC)was 0.2477,which is a classification of the median polymorphism level in the mandarin fish population.These results showed that the two genotypes TT and CT in the pep-A T/C of the SNP locus were significantly correlated with the high level of the food preference traits in mandarin fish(P<0.05).The three genotypes of Genotype1(CT,CC/CT/TT,AA,AA,TT),Genotype2(TT,CC/CT/TT,AA,AA,TT)and Genotype3(TT,CC,AA,AA,TT)were also significantly associated with high levels of the food preference traits(P<0.05).These genotypes affect the traits of food habit domestication in mandarin fish,and Genotype2 has the highest correlation,which can be selected as the optimal genotype individuals.In conclusion,our results demonstrated that these effective SNP molecular markers in pep and gh genes could influence food habit domestication performance of mandarin fish and could be used for accelerating the molecular marker-assisted selective breeding in mandarin fish.In this study,a number of highly polymorphic microsatellite(SSR)molecular markers were developed in the Siniperca chuatsi,S.scherzeri and S.roulei(first time).A total of 149 EST microsatellite markers(EST-SSRs)were mined from the transcriptome sequences,selected for primer designed and successfully amplified in S.scherzeri and S.roulei respectively.Then 98 pairs of primers were screened in S.scherzeri,resulting in 26 polymorphic SSRs with polymorphism ratio of 26.5%;51 pairs of primers were screened in S.roulei,resulting in 11 polymorphic SSRs with polymorphism ratio of 21.6%.Microsatellite-enriched libraries were constructed using magnetic bead enrichment.A total of 194 genome microsatellite markers(G-SSRs)were selected for primer designed and successfully amplified in S.chuatsi and S.scherzeri respectively.Then 144 pairs of primers were characterized in S.chuatsi,resulting in 53 polymorphic SSRs with polymorphism ratio of 36.8%;50 pairs of primers were characterized in S.scherzeri,resulting in 14 polymorphic SSRs with polymorphism ratio of 28.0%.In total,40 polymorphic loci were developed in the S.scherzeri,of which G-SSR showed a higher level of polymorphism than EST-SSR.The food habit of S.scherzeri and S.roulei are more easily domesticated than S.chuatsi.In conclusion,these microsatellite markers obtained in S.scherzeri and S.roulei will lay the theoretical foundation for the breeding of new hybrid mandarin fish breeds.And specific microsatellite molecular markers that can be used to identify well the S.chuatsi,S.scherzeri and its hybrids,which could avoid unkown hybridization and introgression for germplasm resources and provide reliable molecular markers for accelerating the anticipated selective breeding in these species.Based on the studies of the learning and memory function and epigenetic mechanism of food preference transformation,SNPs association analysis of candidate genes with food habit domestication traits,polymorphic SSRs characterization and molecular classification identification of hybrids in mandarin fish,our findings could provide theoretical foundation and technology method for solving the problem of refusing dead prey fish or artificial diets due to its specific feeding habit and the molecular marker-assisted selective breeding in mandarin fish.