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Abstract [Objectives]This study was conducted to explore the effects of new fly maggot protein feed on the mRNA expression of genes related to the TOR signaling pathway in loaches （Misgurnus anguillicaudatus）. [Methods]Two kinds of test feed with equal nitrogen and energy were prepared by replacing 60% of the fish meal in the control group with the new fly maggot protein feed， i.e.， Diet1 （control group） and Diet2 （60% fish meal replacement group）. The feeding experiment was carried out in an indoor circulating water system， and the breeding period was 60 d. [Results]For the livers， the mRNA levels of TOR and 4EBP1 in the Diet2 group were significantly higher than those in the Diet1 group （P<0.05）， while the expression of 4EBP2 was lower than in the Diet1 group （P<0.05）; and as to the muscles， the mRNA levels of TOR and 4EBP1 in the Diet2 group were significantly lower than those in the Diet1 group （P<0.05）， while there was no significant change in the mRNA level of 4EBP2 between the two groups. [Conclusions]The replacement of fish meal by fly maggot cultures affected the mRNA expression of TOR， 4EBP1 and 4EBP2 in loach livers and muscles.
Key words New fly maggot protein feed; Loach; TOR signaling pathway
The target of rapamycin （TOR） signaling pathway is the main pathway for cells to sense nutrients and regulate growth， and is extremely conservative in evolution. TOR is a serine/threonine kinase that contains two complexes with different structures and functions， namely mTORC1 and mTORC2. After activation of mTORC1， its main downstream factors 4EBPs and S6K are phosphorylated. The phosphorylation of S6K promotes the transcriptional activation and translation initiation and extension of a variety of proteins. The phosphorylated 4EBPs dissociate from the eukaryotic translation initiation factor eIF-4E， which activates the latter， making the 40S ribosomal subunit bind to the 5′ end of the mRNA， which thereby initiates protein translation[1-2].
In mammals， the research of TOR signaling pathway has been very extensive and in-depth. In fish， using zebrafish （Danio rerio） as a model organism， studies have found that the TOR signaling pathway regulates intestinal epithelial cell formation， autophagy， myocarditis， and non-alcoholic fatty liver[3-4]. In addition， the research on the regulation of TOR signaling pathway by the levels of arginine， tryptophan， methionine， leucine， oleic acid and choline in the feed， and soybean meal instead of fish meal and different protein-caloric ratios in turbot （Scophthalmus maximus）[5]， Jian carp （Cyprinus carpio var. Jian）[6]and rainbow trout （Oncorhynchus mykiss）[7]. At present， studies on fish feeding regulation pathways are relatively few and scattered. The macroscopic research only stays in the aspect of feeding rate or digestibility， and the molecular-level research mainly focuses on the gene cloning or mRNA level expression of several feeding regulators. However， there are few in-depth and systematic studies on the signal pathways of fish feeding regulation， and there are no reports on the feeding regulation pathways in loaches （Misgurnus anguillicaudatus）. By studying the effects of the new fly maggot protein feed on the mRNA expression of genes related to the TOR signaling pathway in loaches， the regulation mechanism in loaches under selective feeding using fish meal and the new fly maggot protein feed was explored.
Materials and Methods
Experimental process
The feed of the control group used Peruvian fish meal， soybean meal， and wheat protein powder as the main protein sources， and the composition of other raw materials referred to the formula of loach commercial feed. Two kinds of test feed with equal nitrogen and energy were prepared by replacing 60% of the fish meal in the control group with the new fly maggot protein feed， i.e.， Diet1 （control group） and Diet2 （60% fish meal replacement group）， as shown in Table 1. The new fly maggot protein feed was provided by Xuzhou Aquatic Engineering Technology Research Center. This experiment was carried out in the Aquaculture Laboratory of Xuzhou Aquatic Engineering Technology Research Center. Loach fry were purchased from the Qiligou Farmer's Market in Xuzhou City， with an initial weight of about （2.65±0.02） g. Before the start of the experiment， they were fed with juvenile loach and carp initial feed， and randomly divided after 2 weeks into 2 groups， each with 6 replicates， each with 45 juveniles. After the start of the experiment， feeding was stopped for 24 h， and then the feed was input twice a day， with the feeding amount adjusted according to the actual situation. The experimental period was 60 d. The experimental water was tap water after aeration， and the water was changed every 4 d. The water depth was about 15 cm， and the water tanks contained no soil to ensure clean water quality. The water was heated with heating rods to control the water temperature at 25-27 ℃. After the experiment was completed， the experimental fish were fasted for 24 h， and 6 fish were randomly selected from each barrel， and the livers and muscles were taken and stored at -80 ℃ for the analysis experiment. Total RNA extraction and reverse transcription
The tissue samples were grinded in liquid nitrogen， and the total RNA was extracted from the tissue samples using TaKaRa RNAiso Reagent， and precipitated with isopropanol method. The RNA quality was detected by gel electrophoresis. PrimeScipt RT reagent Kit With gDNA Eraser （Perfect Real Time） （TaKaRa， Japan） was used for reverse transcription. After the reverse transcription， the cDNA was measured for its concentration， diluted to 300 ng/μl， and stored in a refrigerator at -80 ℃.
Verification of primer amplification efficiency
The primer design method of fluorescence quantitative PCR referred to the method of Zhang[8]， as shown in Table 2. After verification， RPSD （DQ848899.1） was selected as the reference gene. The consistency of the amplification efficiency between the target sequence and the internal reference sequence was verified by making a concentration standard curve.
Results and Analysis
For the livers， the mRNA levels of TOR and 4EBP1 in the Diet2 group were significantly higher than those in the Diet1 group （P<0.05）， while the expression of 4EBP2 was lower than that in the Diet1 group （P<0.05）， as shown in Fig. 1. As to the muscles， the expression levels of TOR and 4EBP1 in the Diet2 groups were significantly lower than those in the Diet1 group （P<0.05）， while the expression of 4EBP2 was not significantly different between the two groups （P>0.05）， as shown in Fig. 2.
Haitao ZHANG et al. Effect of New Fly Maggot Protein Feed on mRNA Expression of TOR Signaling Pathway-related Genes in Loaches （Misgurnus anguillicaudatus）
Conclusions and Discussion
This study found that the replacement of fish meal with the new fly maggot protein feed affected the expression of TOR， 4EBP1 and 4EBP2 mRNA in the livers and muscles of loaches. The results of this study showed that in the livers， the mRNA levels of TOR and 4EBP1 in the replacement group were significantly higher than those in the control group， while the expression of 4EBP2 was lower than in the control group; and in the muscles， the mRNA levels of TOR and 4EBP1 in the replacement group were lower than those in the control group， while there was no significant change in the mRNA level of 4EBP2 in the replacement group. Similar to this study， the studies in Jian carp found that the level of choline level regulates the mRNA expression of TOR and 4EBP2 in livers， muscles， spleens and kidneys[9]. Xu et al.[10]found that the replacement of fish meal with soybean meal affected the expression of 4EBP1 mRNA in the intestines of juvenile turbot fish， and the expression of 4EBP1 mRNA was affected by the sampling time point after ingestion. The above studies indicate that the mRNA expression of TOR and 4EBPs in Jian carp and turbot juveniles is regulated by feed nutrients. In the study of Zhang[8]， the levels of branched-chain amino acids did not affect the mRNA expression of TOR， 4EBP1 and 4EBP2 in muscles of turbot juveniles; and similar to the study of Zhang Kaikai， Luo et al.[11]showed that the TOR mRNA expression in juvenile cobia was not regulated by the level of feed corn gluten meal， which might be due to the interspecies difference in the regulation of TOR and 4EBPs by feed nutrients. In addition， the mechanism of action of different feed nutrients on TOR and 4EBPs in the same species of fish may also be different. The mechanism of feed nutrients on the mRNA expression of TOR and 4EBPs in fish needs to be further studied. Studies in humans have shown that 4EBP1 and 4EBP2 have very similar gene sequences and crystal structures， and the amino acid sequence of 4EBP2 is only different from 4EBP1 at positions 60 to 63[12]. 4EBP1 and 4EBP2 have the same physiological functions， and both can bind to the eukaryotic translation promoter： eIFs， and inhibit the activity of the latter. In fish， there are relatively few comparative studies on 4EBP1 and 4EBP2， and the difference between the two under the regulation by feed nutrients needs further study. References
[1]GINGRAS AC， GYGI SP， RAUGHT B， et al. Regulation of 4EBP1 phsphorylation：a novel two-step mechanism[J]. Gene& Development， 1999， 13（11）： 1422-1437.
[2]BANKO J L， FRANCIS P， LINGFEI H， et al. The translation repressor 4EBP2 is critical for e IF4F complex formation，synaptic plasticity， and memory in the hippocampus[J]. Journal of Neuroscience the Official Journal of the Society for Neuroscience， 2005， 25（42）： 9581-9590.
[3]DING YH， SUN XJ， XU XL. TOR-autophagy signaling in adult zebrafish models of cardiomyopathy[J]. Autophagy， 2012， 8（1）： 142-143.
[4]DING Y， SUN X， REDFIELD M， et al. Target of rapamcyin （TOR）-based therapeutics for cardiomyopathy [J]. Cell Cycle， 2012， 11（3）： 428-429.
[5]XU DD， HE G， MAI KS， et al. Postprandial nutrient-sensing and metabolic responses after partial dietary fish-meal replacement by soyabean meal in turbot （Scophthalmus maximus L.） [J]. British Journal of Nutrition， 2015， 115（3）： 1-10.
[6]WU P， FENG L， KUANG SY， et al. Effect of dietary choline on growth， intestinal enzyme activities and relative expressions of target of rapamycin and e IF4E-binding protein2 gene in muscle， hepatopancreas and intestine of juvenile Jian carp （Cyprinus carpio var. Jian） [J]. Aquaculture， 2011， 317（1-4）： 107-116.
[7]SEILIEZ I， PANSERAT S， LANSARD M， et al. Dietary carbohydrate-to-protein ratio affects TOR signaling and metabolism-related gene expression in the liver and muscle of rainbow trout after a single meal [J]. American Journal of Physiology-Regulatory Integrative and Comparative Physiology， 2011， 300（3）： R733-R743.
[8]ZHANG KK. Studies on nutritional metabolism of several functional amino acids for juvenile turbot （Scophthalmus maximus L.）[D]. Thesis for Doctor of Science. Ocean University of China， Qingdao.2014.
[9]WU P， JIANG J， LIU Y， et al. Dietary choline modulates immune responses， and gene expressions of TOR and eIF4E-binding protein2 in immune organs of juvenile Jian carp （Cyprinus carpio var. Jian） [J]. Fish & Shellfish Immunology， 2013， 35（3）：697-706.
[10]XU DD. Preliminary study on the mechanisms of nutri-ent sensing and responses of different protein sources in juvenile turbot （Scophthalmus maximus L.）[D]. Qingdao： Ocean University of China， 2014.
[11]LUO YW， AI QH， MAI KS， et al. Effects of dietary corn gluten meal on growth performance and protein metabolism in relation to IGF-I and TOR gene expression of juvenile cobia （Rachycentron canadum） [J]. Journal of Ocean University of China， 2012， 12（3）： 418-426.
[12]FUKUYO A， YASUKO I， ISHIDA T， et al. Structural scaffold for e IF4E binding selectivity of 4E-BP isoforms： crystal structure of e IF4E binding region of 4E-BP2 and its comparison with that of 4E-BP1[J]. Journal of Peptide Science， 2011， 17（9）： 650-657.
Key words New fly maggot protein feed; Loach; TOR signaling pathway
The target of rapamycin （TOR） signaling pathway is the main pathway for cells to sense nutrients and regulate growth， and is extremely conservative in evolution. TOR is a serine/threonine kinase that contains two complexes with different structures and functions， namely mTORC1 and mTORC2. After activation of mTORC1， its main downstream factors 4EBPs and S6K are phosphorylated. The phosphorylation of S6K promotes the transcriptional activation and translation initiation and extension of a variety of proteins. The phosphorylated 4EBPs dissociate from the eukaryotic translation initiation factor eIF-4E， which activates the latter， making the 40S ribosomal subunit bind to the 5′ end of the mRNA， which thereby initiates protein translation[1-2].
In mammals， the research of TOR signaling pathway has been very extensive and in-depth. In fish， using zebrafish （Danio rerio） as a model organism， studies have found that the TOR signaling pathway regulates intestinal epithelial cell formation， autophagy， myocarditis， and non-alcoholic fatty liver[3-4]. In addition， the research on the regulation of TOR signaling pathway by the levels of arginine， tryptophan， methionine， leucine， oleic acid and choline in the feed， and soybean meal instead of fish meal and different protein-caloric ratios in turbot （Scophthalmus maximus）[5]， Jian carp （Cyprinus carpio var. Jian）[6]and rainbow trout （Oncorhynchus mykiss）[7]. At present， studies on fish feeding regulation pathways are relatively few and scattered. The macroscopic research only stays in the aspect of feeding rate or digestibility， and the molecular-level research mainly focuses on the gene cloning or mRNA level expression of several feeding regulators. However， there are few in-depth and systematic studies on the signal pathways of fish feeding regulation， and there are no reports on the feeding regulation pathways in loaches （Misgurnus anguillicaudatus）. By studying the effects of the new fly maggot protein feed on the mRNA expression of genes related to the TOR signaling pathway in loaches， the regulation mechanism in loaches under selective feeding using fish meal and the new fly maggot protein feed was explored.
Materials and Methods
Experimental process
The feed of the control group used Peruvian fish meal， soybean meal， and wheat protein powder as the main protein sources， and the composition of other raw materials referred to the formula of loach commercial feed. Two kinds of test feed with equal nitrogen and energy were prepared by replacing 60% of the fish meal in the control group with the new fly maggot protein feed， i.e.， Diet1 （control group） and Diet2 （60% fish meal replacement group）， as shown in Table 1. The new fly maggot protein feed was provided by Xuzhou Aquatic Engineering Technology Research Center. This experiment was carried out in the Aquaculture Laboratory of Xuzhou Aquatic Engineering Technology Research Center. Loach fry were purchased from the Qiligou Farmer's Market in Xuzhou City， with an initial weight of about （2.65±0.02） g. Before the start of the experiment， they were fed with juvenile loach and carp initial feed， and randomly divided after 2 weeks into 2 groups， each with 6 replicates， each with 45 juveniles. After the start of the experiment， feeding was stopped for 24 h， and then the feed was input twice a day， with the feeding amount adjusted according to the actual situation. The experimental period was 60 d. The experimental water was tap water after aeration， and the water was changed every 4 d. The water depth was about 15 cm， and the water tanks contained no soil to ensure clean water quality. The water was heated with heating rods to control the water temperature at 25-27 ℃. After the experiment was completed， the experimental fish were fasted for 24 h， and 6 fish were randomly selected from each barrel， and the livers and muscles were taken and stored at -80 ℃ for the analysis experiment. Total RNA extraction and reverse transcription
The tissue samples were grinded in liquid nitrogen， and the total RNA was extracted from the tissue samples using TaKaRa RNAiso Reagent， and precipitated with isopropanol method. The RNA quality was detected by gel electrophoresis. PrimeScipt RT reagent Kit With gDNA Eraser （Perfect Real Time） （TaKaRa， Japan） was used for reverse transcription. After the reverse transcription， the cDNA was measured for its concentration， diluted to 300 ng/μl， and stored in a refrigerator at -80 ℃.
Verification of primer amplification efficiency
The primer design method of fluorescence quantitative PCR referred to the method of Zhang[8]， as shown in Table 2. After verification， RPSD （DQ848899.1） was selected as the reference gene. The consistency of the amplification efficiency between the target sequence and the internal reference sequence was verified by making a concentration standard curve.
Results and Analysis
For the livers， the mRNA levels of TOR and 4EBP1 in the Diet2 group were significantly higher than those in the Diet1 group （P<0.05）， while the expression of 4EBP2 was lower than that in the Diet1 group （P<0.05）， as shown in Fig. 1. As to the muscles， the expression levels of TOR and 4EBP1 in the Diet2 groups were significantly lower than those in the Diet1 group （P<0.05）， while the expression of 4EBP2 was not significantly different between the two groups （P>0.05）， as shown in Fig. 2.
Haitao ZHANG et al. Effect of New Fly Maggot Protein Feed on mRNA Expression of TOR Signaling Pathway-related Genes in Loaches （Misgurnus anguillicaudatus）
Conclusions and Discussion
This study found that the replacement of fish meal with the new fly maggot protein feed affected the expression of TOR， 4EBP1 and 4EBP2 mRNA in the livers and muscles of loaches. The results of this study showed that in the livers， the mRNA levels of TOR and 4EBP1 in the replacement group were significantly higher than those in the control group， while the expression of 4EBP2 was lower than in the control group; and in the muscles， the mRNA levels of TOR and 4EBP1 in the replacement group were lower than those in the control group， while there was no significant change in the mRNA level of 4EBP2 in the replacement group. Similar to this study， the studies in Jian carp found that the level of choline level regulates the mRNA expression of TOR and 4EBP2 in livers， muscles， spleens and kidneys[9]. Xu et al.[10]found that the replacement of fish meal with soybean meal affected the expression of 4EBP1 mRNA in the intestines of juvenile turbot fish， and the expression of 4EBP1 mRNA was affected by the sampling time point after ingestion. The above studies indicate that the mRNA expression of TOR and 4EBPs in Jian carp and turbot juveniles is regulated by feed nutrients. In the study of Zhang[8]， the levels of branched-chain amino acids did not affect the mRNA expression of TOR， 4EBP1 and 4EBP2 in muscles of turbot juveniles; and similar to the study of Zhang Kaikai， Luo et al.[11]showed that the TOR mRNA expression in juvenile cobia was not regulated by the level of feed corn gluten meal， which might be due to the interspecies difference in the regulation of TOR and 4EBPs by feed nutrients. In addition， the mechanism of action of different feed nutrients on TOR and 4EBPs in the same species of fish may also be different. The mechanism of feed nutrients on the mRNA expression of TOR and 4EBPs in fish needs to be further studied. Studies in humans have shown that 4EBP1 and 4EBP2 have very similar gene sequences and crystal structures， and the amino acid sequence of 4EBP2 is only different from 4EBP1 at positions 60 to 63[12]. 4EBP1 and 4EBP2 have the same physiological functions， and both can bind to the eukaryotic translation promoter： eIFs， and inhibit the activity of the latter. In fish， there are relatively few comparative studies on 4EBP1 and 4EBP2， and the difference between the two under the regulation by feed nutrients needs further study. References
[1]GINGRAS AC， GYGI SP， RAUGHT B， et al. Regulation of 4EBP1 phsphorylation：a novel two-step mechanism[J]. Gene& Development， 1999， 13（11）： 1422-1437.
[2]BANKO J L， FRANCIS P， LINGFEI H， et al. The translation repressor 4EBP2 is critical for e IF4F complex formation，synaptic plasticity， and memory in the hippocampus[J]. Journal of Neuroscience the Official Journal of the Society for Neuroscience， 2005， 25（42）： 9581-9590.
[3]DING YH， SUN XJ， XU XL. TOR-autophagy signaling in adult zebrafish models of cardiomyopathy[J]. Autophagy， 2012， 8（1）： 142-143.
[4]DING Y， SUN X， REDFIELD M， et al. Target of rapamcyin （TOR）-based therapeutics for cardiomyopathy [J]. Cell Cycle， 2012， 11（3）： 428-429.
[5]XU DD， HE G， MAI KS， et al. Postprandial nutrient-sensing and metabolic responses after partial dietary fish-meal replacement by soyabean meal in turbot （Scophthalmus maximus L.） [J]. British Journal of Nutrition， 2015， 115（3）： 1-10.
[6]WU P， FENG L， KUANG SY， et al. Effect of dietary choline on growth， intestinal enzyme activities and relative expressions of target of rapamycin and e IF4E-binding protein2 gene in muscle， hepatopancreas and intestine of juvenile Jian carp （Cyprinus carpio var. Jian） [J]. Aquaculture， 2011， 317（1-4）： 107-116.
[7]SEILIEZ I， PANSERAT S， LANSARD M， et al. Dietary carbohydrate-to-protein ratio affects TOR signaling and metabolism-related gene expression in the liver and muscle of rainbow trout after a single meal [J]. American Journal of Physiology-Regulatory Integrative and Comparative Physiology， 2011， 300（3）： R733-R743.
[8]ZHANG KK. Studies on nutritional metabolism of several functional amino acids for juvenile turbot （Scophthalmus maximus L.）[D]. Thesis for Doctor of Science. Ocean University of China， Qingdao.2014.
[9]WU P， JIANG J， LIU Y， et al. Dietary choline modulates immune responses， and gene expressions of TOR and eIF4E-binding protein2 in immune organs of juvenile Jian carp （Cyprinus carpio var. Jian） [J]. Fish & Shellfish Immunology， 2013， 35（3）：697-706.
[10]XU DD. Preliminary study on the mechanisms of nutri-ent sensing and responses of different protein sources in juvenile turbot （Scophthalmus maximus L.）[D]. Qingdao： Ocean University of China， 2014.
[11]LUO YW， AI QH， MAI KS， et al. Effects of dietary corn gluten meal on growth performance and protein metabolism in relation to IGF-I and TOR gene expression of juvenile cobia （Rachycentron canadum） [J]. Journal of Ocean University of China， 2012， 12（3）： 418-426.
[12]FUKUYO A， YASUKO I， ISHIDA T， et al. Structural scaffold for e IF4E binding selectivity of 4E-BP isoforms： crystal structure of e IF4E binding region of 4E-BP2 and its comparison with that of 4E-BP1[J]. Journal of Peptide Science， 2011， 17（9）： 650-657.