Research Advances in Gene Regulation and Genetic Improvement of Fish Feeding

Hao LIU, Shengjie LI, Junjie BAI

1. Pearl River Fisheries Research Institute, CAFS/Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation, Ministry of Agriculture, Guangzhou 510380, China;

2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

Generalized feeding habit refers to the kind of food, the way of feeding as well as the feeding place of an animal to take how much food, which belongs to the ecological and morphological category.According to different feeding modes and food contents, the feeding habits of fish can be divided into 4 groups of filter feeding, herbivorous, omnivorous and carnivorous[1]. Feeding habit is subject to the regulation of a variety of factors, which is not only related with the inherent genetic and physiological factors such as appetite, digestive tract structure, but also related with the external nature of food such as protein sources, the palatability of the feed and so on.

With the improvement of people's living standard, the demand for the carnivorous fish is growing, but under the traditional fish culture, ice-preserved low cost fish is the major source of food for the culture, which brings certain influence to the industrial development of carnivorous fish.On the one hand,the quality of the ice-preserved low cost fish is hard to control,and the surplus residual feeds would affect the water quality,making it easy to occur diseases; on the other hand,the use of large amount of ice-preserved low cost fish would bring great pressure to the natural resources.Currently,the most widely used method to make the carnivorous fish that depend on ice-preserved fish and live fish feeding like perch, snakehead and yellow croaker and other sea freshwater fishes feed artificial diets is to domesticate the fishes in technical level to change the feeding habits of fishes.If it is possible to breed the improved variety of well-applied artificial diets, it would not only can promote the large scale culture of carnivorous fish, but it can also reduce the culture cost and improve economic benefits.

The current research related with the genetic improvement of fish feeding habit is mainly concentrated on the following 2 aspects: one is to obtain the offspring that are easy to domesticate to feed artificial feeds through hybridization, such as the hybridization of Channa Argus and Channa maculata can get the F1generationwhich is easy to tame to feed with artificial feed[2]; the other is to breed the improved varieties that are suitable for the application of vegetable protein,such as Oncorhynchus mykiss[3], European seabass[4]. Feeding habit is heritable,and the feeding habit related genes in fish mainly include the appetite associated factor gene and digestive enzyme genes. In this paper,we reported the progress of the polymorphism of genes associated with appetite and its correlation with feeding habits, and summarized the studies on the genetic improvements of fish feeding with the aim to provide references for the study on the genetic improvement of fish feeding habits.

Genetic Regulation of Fish Feeding Habit

Appetite can affect the feeding of fish,making it become the key point in the improvement of fish feeding habit.The feeding domestication of Culter mongolicus found that the fish stocks that are in poor appetite will produce no sensory to the feeding feeds[5]; in addition, the digestive enzymes of fishes with different feeding habits have different activities[6], indicating that there are differences in the digestive enzyme genes among the fishes with different feeding habits.The feeding habit of fish is mainly regulated by the genes associated with appetite regulation as well as the protease and digestive enzymes.

Regulation of fish appetite

The regulation of fish appetite is completed through the delicate interaction of brain and peripheral signals,and the brain, especially the hypothalamus is the major place that produce feeding regulating genes(feeding incitant and feeding suppressant)[7]. The major appetite regulatory factors include neuropeptide Y(NPY)[8],ghrelin[9],cholecystokinin(CCK)[10],orexin[11],and cocaine-amphetamines regulating transcription peptide(CART)[12].

Neuropeptide Y Neuropeptide Y is mainly expressed in the central nervous system,which is the most important known regulatory factor that promotes appetite. The injection of NPY into the ventricle of grass carp showed that 2 -8 h after the injection, the food intake of grass carp improved greatly[13]; after a period of food deprivation,goldfish showed quantative expression of NPY mRNA in hypothalamus, and after injected with NPY, the food intake of goldfish increased significantly[14]. After food deprivation, Silurus meridionalis showed increased amount of NPY expression in the cerebral tissues,and after feeding,the expression amount of NPY decreased significantly, which showed that NPY could regulate the food intake of Silurus meridionalis[15]. Study also has found that if food is given to the body,NPY signal would reduce with the intake of food, and if food is stopped giving, NPY would stay in high secretion levels, indicating that NPY plays an important role in the regulation of starting and maintaining feeding behavior.

Orexin Orexin is composed of 2 peptides of orexin-A and orexin-B,which are expressed in the hypothalamus. After injection of orexin in animals and goldfish, the food intake increased significantly, and the effect of orexin-A is stronger than that of orexin-B[16]. After the food deprivation of goldfish, the number of orexin like immunoreactive cells in most area of the bottom part of hypothalamus significantly increased, and after the injection of glucose, the activity of orexin like immunoreactivity decreased, indicating that orexin is mainly to stimulate the appetite[17]. After the perineural injection of orexin, Halichoeres ornatissimus showed the stimulation in appetite and an increase in the motion activity[18].The deprivation of food could improve the prepro-orexin mRNA level in the brains of zebra fish and gadus[19-20].Therefore, orexin could improve the appetite of fish and thereby regulating the feeding behavior of fish.

Ghrelin Ghrelin is an endogenous ligand for the growth hormone receptor. It was first extracted from the stomach of rats in 1999 by Kojima et al.[9]. It has been proved in mammals that the ghrelin genes could not only promote the growth hormone secretion, but also can be involved in the regulation of gastrointestinal physiological activities through the peripheral and central pathways. The ghrelin genes of fish are mainly expressed in the stomach, but showed low level of expression in the brain. The injection of goldfish or human ghrelin could promote the food intake of goldfish, and the continuous feeding of ghrelin proteins could result in the increase of appetite of Tilapia mossambica.Hunger could increase the expression level of ghrelin genes in the gastric tissues of jewfish[21-22].The study shows that the regulation effect of orexin on appetite is regulated by the independent NPY and Orexin pathway[23].

Cholecystokinin For fish, when the food enters into the intestines, it begins to release CCK and influences the appetite. CCK could strongly promote the fish to feed by combining with the corresponding receptors, which would also stimulate the secretion of gastric acid and pancreatic enzyme as well as increase gastrointestinal motility[10].CCK is a kind of multi-functional braingut peptide, and the food deprivation of grass carp can result in the downregulated expression of this gene.However,after feeding,the expression level of this gene would increase significantly, indicating that this gene is the major gene controlling the appetite of grass carp[24]. The CCK level in the plasma of rainbow trout fed with highfat diet was significantly higher than that fed with low-fat diet[25]. In goldfish,central and peripheral injection of sulfated CCK-8 could inhibit food intake[26]; after the intracerebroventricular injection of CCK, the channel catfish showed an inhibition of food intake, while the rainbow trout treated with CCK antagonist showed an increase in food intake[27]. In addition,food deprivation would increase the mRNA level of CCK gene in the livers of amberjack and east-ray, and the CCK mRNA levels in the brain of goldfish and the blind gut of amberjack also increased significantly after feeding,indicating that CCK is an important factor involved in the regulation of appetite and food intake.

Regulation of cocaine-amphetamine on transcript peptide Research on fish has found that CART as well as its mRNA are mainly distributed in the brain and some peripheral tissues, such as liver, kidney, heart,intestine,stomach,gills and reproductive system; after feeding, the mRNAof CART gene in the brain of goldfish increased,while during food shortage,the mRNA level decreased[28].Food deprivation could reduce the mRNA level of CART gene in channel catfish and Atlantic salmon[29-30]. The intracerebroventricular injection of human CART could inhibit the goldfish feeding, and the expression level of CART gene decreased significantly after food deprivation, which indicated that the CART polypeptide could play a role in the regulation of food intake[31]. Research has shown that leptin and CART have a synergistic effect in the goldfish[27].

Effects of digestive enzymes on fish feeding

The digestive enzymes related with food intake mainly include pepsin,lipoprotein lipase,amylase and trypsin,and the feeding habit of fish matches with the organization structure and digestible function as well as the distribution of digestive organs. The activities of digestive enzymes are different with different organization structure and digestive functions of digestive organs[6].In general,the digestive tract of the grass feeding fish is longer than that of the carnivorous fish, and the related enzyme activity is also stronger, while the protease activity of the carnivorous fish is stronger. The study on the activity of digestive enzymes in mandarin fish, black carp,grass carp, common carp, crucian carp and silver carp found that the protease activity was related with feeding habit,and the protease activity of carnivorous fish was significantly higher than that of omnivorous[32].HUANG et al.[33]pointed out that when the protein mass fraction in feed was in the range of 32%-40%, the proteinase activity in liver-pancreas or intestine would increase with the increase of protein mass fraction. The study of Kawai also proved that the proteinase activity in the amphibious catfish and carp also increased with the increase of protein content in feed.Therefore, in a certain range, the protease activity increases with the increase of protein content in the diet,and the variety,distribution and activity of the digestive enzymes are also significantly related with the feeding habit of fish.

In addition,the encoding genes of taste receptors like Tas1r1, PKD2L1,PKD1L3 and CD36[34], obesity related genes such as FTO, APOA2[35]can affect the fish’s food intake to a certain extent. It can provide theoretical basis for the genetic of the feeding habit of fish by selecting the above genes related with appetite regulation and digestive enzymes genes as the candidate functional gene to study the polymorphism of genes associated with appetite and its correlation with feeding habits of fish.

Study on the Polymorphism of Genes Related with Fish Feeding Habit and Its Relation with Fish Feeding Habit

After the crossbreed of female mandarin fish with male Siniperca scherzeri,the hybrid F1generation was raised in 2 different groups.One group is fed with dead fish, while the other group fed with no dead fish. The transcriptome sequencing and digital gene expression profiling analysis found that there were differences expression genes, and data analysis showed that there was significant difference in the abundance in the appetite regulated gene SNP loci in the 2 groups of fish[36],indicating that the polymorphism of genes related with fish feeding may have an influence on fish feeding habit and food intake.

Polymorphism of pepsin gene

The feeding habit of mandarin fish food is unique that after active food intake, it would live on live fish for life,which also limits the further development of the varieties of mandarin fish.In the study on the molecular basis of the phenotypic difference of different feeding habits domestication of mandarin fish,pepsinogen(PEP)was used as the candidate gene that affected the feeding habit of mandarin fish. In the PEP exons of the 2 groups of mandarin fish that was easiest to tame and most difficult to tame, 2 single nucleotide polymorphisms (SNP) were found. The diplotype analysis results showed that there was significant difference in the diplotype of Dip1 and Dip5 in the 2 feeding habits domesticated groups (P ＜0.05)[37]. The study concluded that PEP gene was a major gene or linked with the major gene that affected the feeding habits of mandarin fish, making it possible to be used as the candidate genetic marker for the analysis on the domestication of mandarin fish feeding habits.

Polymorphism of NPY gene

As the main appetite promoting factor,NPY plays a key role in starting and maintaining the feeding behavior.The study on the NPY gene of Pelteobagrus fulvidraco found there was a mutation in the second exon of NPY gene, and the correlation analysis on different genotype and the major grow properties of the maximum group and minimum group in the 6-month group,12-month group and 12-month group of P. fulvidraco showed that in the male group and all 6-month group,the AA genotype weights were significantly higher than that of AB genotype and BB genotype(P＜0.05)[38].From the increase of weight, it could find that the mutant AA genotype showed significant advantages, which can be used as the genotype for high quality breeding. Thereby, the feasibility of other feeding incitant genes and feeing suppressant genes to be used as the candidate improved genes can be explored based on similar study.

Polymorphism of lipoprotein lipase gene

Lipoprotein lipase (LPL)is one of the key enzyme of lipoprotein metabolism,and its physiological function is to decompose chylomicrons and triglyceride with very low density of lipoprotein into glycerol and fatty acids to provide the energy for tissue oxidation and storage. LIANG et al.[39]used the direct sequencing of PCR products method to detect and analyze the genetic polymorphism of SNPs in the lipoprotein lipase gene of mandarin fish, and the results showed that 3 SNPs loci were detected. And the diplotype analysis on the mutation sites showed that and there were significant differences in the diplotype of Dip2 in the domesticated and undomesticated groups(P＜0.05),indicating that the LPL gene could be used as the candidate gene affecting the domestication of the feeding habits of mandarin fish. In the future, the mutation site that lead to the phenotypic dif-ferences in feeding habit domestication can be used as the genetic marker to breed the mandarin fish varieties with improved feeding habits that can accept dead fish or artificial feed by combining molecular marker associated breeding technique.

Polymorphism of amylase and trypsin gene

Amylase (AMY) plays an important role in digesting starch and other polysaccharide substances in food,and the activity of amylase has an direct effect on the digestive ability of fish to starch and glycogen in food.Trypsin (TRY) is mainly to hydrolyze protein and activate the other zymogen secreted by pancreas to realize the regulation on the digestion of food.The single nucleotide polymorphism detection and analysis on AMY gene and TRY gene of mandarin fish showed that there was no significant difference in the distribution of each gene in the mutation sites among the group easy to domesticate and the group hard to domesticate(P＞0.05)[40].Similar study can also be carried out among other fish, especially herbivorous fishes which have relatively higher content of starch in food, to explore the possibility of using AMY gene and TRY gene as the candidate gene for the genetic improvement of the feeding habits of fish.

Genetic Improvement of Fish Feeding Habit

The feeding habit of fish is heritable, and the food recognition mechanism is thought to be related with the development of its sensory organs and digestive tract. For the Micropterus salmoides fed with artificial feed under the same environment, the varieties with different genetic background showed differences in food intake,which is thought to be related with the hereditary of feeding habit.

At present, research related with the genetic improvement of fish feeding habit is mainly concentrated on 2 aspects: one is to obtain the offspring that are easy to domesticate to feed artificial feeds through hybridization;the other is to breed the improved varieties that are suitable for the application of vegetable protein. There have many examples on obtaining the variety with improved feeding habits through hybridization. For example,Channa argus is a kind of ferocious carnivorous fish, while Channa asiatica is a kind of omnivorous fish like animals.Compared with Channa argus,it is easier for Channa asiatica to take artificial feed. In culture production, it needs to match with ice-preserved low cost fish to feed Channa argus.In production,the filial generation of Channa argus and Channa asiatica shows better culture efficiency in taking artificial feed than the 2 parents.Using Channa argus as male parent and Channa maculata as female parent, it can obtain the first filial generation with obvious heterosis through hybridization,which is much easier to domesticate to feed with artificial feed in character. In current culture production, the hybrid snakehead can be cultured with artificial feed instead of ice-preserved fish.MAO et al.[41]compared the digestive enzyme activity of hybrid snakehead and its parents, and the results showed that the AMY activity of hybrid snakehead was significantly higher than that of its parents in liver and duodenum(P＜0.05),showing better feeding effect. There was differentiation in the feeding habits of easily domesticated individuals and hard domesticated individuals in the offspring of the hybridization of Siniperca chuatsi and Siniperca scherzeri.LIANG et al. studied the relationship of the food intake of Siniperca chuatsi, Siniperca scherzeri and their crossbred offspring, metabolic regulation of gene and as well as its SNP polymorphism with the food intake and using efficiency of artificial feed as well as the growth speed of mandarin fish.Moreover, with the selected offspring of Siniperca chuatsi female parent and the offspring of Siniperca scherzeri as male parent, they cultured the hybrid mandarin fish that was easy to domesticate with artificial feed, grew fast and had strong disease-resistance,which showed significant heterosis in using artificial feed. Mircopterus salmoides is one of the most important varieties cultures in fresh water in China,but its dependence on ice-preserved fish affects its large-scale culture.Domesticated from active food intake,the domesticated group could be fed with artificial feed, and using the domesticated group as the parents to carry out multi-generation breeding can produce the fine varieties that have stable heredity and can be fed with artificial feed better.

Another method to improve the feeding habit of fish is to carry out the genetic improvement to the varieties that can be fed with plant protein,which can use plant protein instead of fish meal as the protein source[42]. On the one hand, it can limit the consumption amount of fish meal, which can protect the marine fishery resources and ensure the sustainable development of marine fishery.On the other hand, it can pick out the group which can use plant protein feed best as the parents to breed the varieties with fine characters like stable inheritance,using plant protein better.

Carter et al.raised young rainbow trout in different ponds but under the same state and fed the fish with soya bean meal instead of fish meal of different proportions,and the data analysis showed that when the soya bean meal replaced 26.9% of fish meal, the growth rate of young rainbow trout reached the maximum[43]; then it can pick out the individuals with excellent growth from the groups fed with the optimal replaced feed, which could make it possible to produce the rainbow trout varieties that can better use plant protein through continuous multigeneration hybridization. The study found that the European seabass could be fed with total plant protein without affecting its growth and nitrogen utilization rate, which is of great significance for the improvement on the feeding habits of carnivorous varieties like the Chinese Micropterus salmoides.

The results of the study on the replacement of fish feeding protein source are generally believed that the influence of plant proteins on the experimental fish is mainly determined by the source of plant protein and the proportion in the feed.In a certain proportion range, replacing fish meal with plant protein shows no significant negative effects on the growth rate, feed digestibility coefficient, feed conversion rate and active feeding of the ex-perimental fish, but it would obviously inhibit the growth rate and active food intake out of the certain range.It is predicted that the significant reduce on the growth rate of the experimental fish that has improved feeding habit is mainly caused by the improper plant protein proportion which results in the decrease of the food intake amount of the experimental fish (appetite reduced). Therefore, the study on the food intake mechanism of fish is of great significance to the improvement of fish feeding habits, and enhancing the appetite of fish is one of the key factors in improving fish feeding habits.

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