**6. Application of epigenetics in aquaculture**

Body appearance traits identified include the red body colour excluding normal black pigmen‐ tation in tilapia [168], silvery skin with few spots in rainbow trout [169], albinism in rainbow trout [170] and melanization in threespine sticklebacks (*Gasterosteus aculeatus*) [171]. Genetic traits essential for improving production in fish farming include traits for feed conversion ratio [172], robustness [173], maturation timing [174], cold tolerance [163, 175], high temperature tolerance [176] and salinity tolerance. In anadromous species such as Atlantic salmon, genetic traits for smoltification [177], migration and spawning timing [178] have been determined.

**Crustacean species Trait Method References** Pandad shrimp (*Pandalus latirostris*) Microsatellite [253]

184 Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

Growth traits SNP [160]

Growth traits Transcriptome [161]

Growth traits QTL [157]

High temperature tolerance QTL [157]

Growth traits AFLP [158]

Growth traits QTL [147]

Total and carapace length ALFP [254]

Sex determining loci QTL [255]

Sex determining loci Microsatellite [256]

Body length QTL [159]

Body weight and length QTL [257]

Body length QTL [162]

Body length QTL [158]

Giant freshwater prawn (*Macrobrachium rosenbergii*)

*carinicauda*)

*japonicas*)

*japonicas*)

*japonicas*)

*vannamei*)

*japonicas*)

*monodon*)

*vannamei*)

*chinensis*)

*vannamei*)

*nipponense*)

*japonicas*)

Ridgetail white prawn (*Exopalaemon* 

Kuruma shrimp (*Marsupenaeus* 

Kuruma shrimp (*Marsupenaeus* 

Kuruma shrimp (*Marsupenaeus* 

Pacific white shrimp (*Litopenaeus* 

Kuruma shrimp (*Marsupenaeus* 

Indian black tiger shrimp (*Penaeus* 

Pacific white shrimp (*Litopenaeus* 

Chinese shrimp (*Fenneropenaeus* 

Pacific white shrimp (*Litopenaeus* 

Kuruma shrimp (*Marsupenaeus* 

oriental river prawn (*Macrobrachium* 

The rapid expansion of aquaculture to become one of the leading sources of protein in the world has brought with it an increase in infectious diseases in aquaculture. To reduce the disease

**5.2. Disease resistance and susceptibility traits**

**Table 2.** Growth and performance traits for different crustacean species.

The term 'epigenetics' was first coined by Waddington in 1942 and was defined as changes in the phenotype without inducing changes in the genotype [198, 199]. Studies on chemical modifica‐ tion of DNA bases date as far back as 1948 [200] and by the 1970s, the role of DNA methylation in gene regulation was identified [201]. In subsequent years, the link between DNA methylation and gene expression was established [202] paving way to the discovery of therapeutic drugs such as 5‐azacytidine used to block DNA methylation [203]. In principle, epigenetic changes are regulated by (i) chemical modifications on DNA cytosine residues resulting in DNA methylation and, (ii) histone protein modifications on DNA [204, 205]. Current advances in HTS have refined genomic analyses to base‐pair resolution making it easier to map entire epigenomes of living organ‐ isms enabling us to identify biological markers predictive of the outcome of disease infections, reproduction, growth and adaptation to new environments [206]. As a result of these advances, epigenetics studies in aquaculture have tremendously increased in the last decades with the view to identifying biological markers relevant for improving the production of farmed aquatic organisms. Technologies used for epigenetics analyses in aquaculture include (i) RNA‐seq in Medaka [207] and Nile tilapia [208]; (ii) genome‐wide methylated DNA immunoprecipitation sequencing (MeDIP‐seq) in Nile tilapia [209] and Medaka [207]; (iii) bisulfite sequencing (BS‐seq) in smooth tongue sole (*Cynoglossus semilaevis*) [210, 211], rainbow trout [212] and Nile tilapia [208]; (iv) genetic linkage map analysis using simple sequence length polymorphisms (SSLPs) in medaka [213, 214]; (v) methylation sensitivity amplified polymorphism (MSAP) in Atlantic salmon [18], grass carp [215], brown trout [17], sea urchin (*Glyptocidaris crenularis*) [216] and sea cucumber (*Apostichopus japonicas*) [217]; (vi) 5‐methylcytosine immunolocation in sea lamprey (*Petromyzon marinus*) [218]; (vii) restriction endonuclease hydrolysis of DNA using methylation enzymes in Zebrafish [219] and (viii) bisulfite sequencing PCR in Pacific Oyster (*Crassostrea gigas*) [220] and grass carp [221]. As shown in **Table 3**, epigenetics studies carried out this far include studies on reproduction, growth and adaptation traits. In the case of Atlantic salmon, which is one of the most widely studied species, epigenetic studies have been carried out at different stages of the production cycle as shown in **Figure 2**.

#### **6.1. Embryogenesis and reproduction traits**

Embryogenesis and reproduction traits determined by epigenetic analyses in aquatic organ‐ isms include sexual dimorphism, embryo development, control of gonadal aromatase and male meiosis [208, 222, 223]. Mhanni and McGowan [219] examined the methylation patterns of the zebrafish genome during early embryogenesis and showed that parental genetic contri‐ butions to the zygote were differently methylated with the sperm being more hypermethyl‐ ated than the oocyte genome. However, immediately after fertilization there was a significant decrease in the embryonic genome methylation, but increased rapidly as the embryo devel‐ oped to normal levels by the gastrulation stage. These observations are consistent with those seen in mouse [224] suggesting that embryo demethylation/re‐methylation is conserved across the vertebrate taxa as of part embryogenesis. As for reproduction traits, Wan et al. [208] found several differentially methylated regions (DMRs) on tilapia chromosomal DNA linked to sex‐ ual dimorphism in which the males had high methylation levels after prolonged exposure to high temperature conditions. Similarly, Navarro‐Martín et al. [222, 223] showed that European seabass juvenile males had double DNA methylation levels than females in the promoter region of gonadal aromatase, the enzyme that converts androgens to estrogens suggesting that methylation levels on gonadal aromatase were predictive of sex determination. Other fish species for which DNA methylation of aromatase has been linked to sex determination include medaka [225] and Japanese flounder (*Paralichthys olivaceus*) [226]. In crustacean, Gómez et al. [227] analysed the post‐translational histone modifications in the testis of *Daphnia magna* and identified cytological markers linked to meiosis progression and the silencing of unsynapsed chromatin. Put together, these studies show that DNA methylation and histone modification can induce reproduction and embryogenesis changes in different aquatic organisms.

#### **6.2. Growth and productivity traits**

Epigenetic factors associated with growth and productivity identified in aquatic organisms include early maturation, regulation of muscle growth and disease resistance. Early matu‐ ration in Atlantic salmon has emerged to be an interesting topic because prior to migration, parr can reach sexual maturity and successfully fertilize adult females. Up to 60% of total paternity in wild populations has been attributed to these precocious male parr or 'sneakers'. To determine the underlying causes of early sexual maturation in parr, Morán and Pérez‐ Figueroa [18] compared genetic and epigenetic differences of two populations of parr and mature fish originating from two different rivers and found no genetic difference between

**Aquatic organism Epigenetic trait References** Zebrafish (*Danio rerio*) Carcinogenesis [258] Zebrafish (*Danio rerio*) Embryo development [219] Zebrafish (*Danio rerio*) Embryonic cardiogenesis [259] Medaka (*Oryzias latipes*) Excision of ToL2 transposal [260] Medaka (*Oryzias latipes*) Control of cardiomyocyte production

in response to stress

reproduction impairment

masculinization of skeletal muscles

Low cadmium exposure [232]

Control of gonadal aromatase [263]

Medaka (*Oryzias latipes*) Hypoxia and transgenerational

*European seabass* (*Dicentrarchus labrax*) Temperature dependent sex ratio shift

Senegalese sole (*Solea senegalensis*) Thermal epigenetic regulation of

European eel (*Anguillarum* 

European eel (*Anguillarum* 

Red eared slider turtle (*Trachemys* 

*anguillarum*)

*anguillarum*)

*scripta elegans*)

Nile tilapia (*Oreochromis niloticus*) Sexual dimorphism [208] Atlantic salmon (*Salmo salar* L.) Early maturation [18]

Tongue sole (Cynoglossidae) Sex reversal [210, 211]

*Daphnia magna* Male meiosis [227] Pacific oyster (*Crassostrea gigas*) Growth [220] Rainbow trout (*Oncorhynchus mykiss*) Glucose intolerance [230]

divergence

Atlantic Cod (*Gadus morhua* L.) Photoperiod influence [228, 229] Grass carp (*Ctenopharyngodon idella)* Individual variations [215] Grass carp (*Ctenopharyngodon idella)* Resistance against grass reovirus [221]

Rainbow trout (*Oncorhynchus mykiss*) Migration‐related phenotypic

**Table 3.** Epigenetics application in aquatic organisms.

muscle growth

Abnormal ovarian DNA methylation‐gonadal

Nile tilapia (*Oreochromis niloticus*) High temperature induced

[214]

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[207]

[209]

[222, 223]

[261]

[262]

[212]


**Table 3.** Epigenetics application in aquatic organisms.

Medaka [207] and Nile tilapia [208]; (ii) genome‐wide methylated DNA immunoprecipitation sequencing (MeDIP‐seq) in Nile tilapia [209] and Medaka [207]; (iii) bisulfite sequencing (BS‐seq) in smooth tongue sole (*Cynoglossus semilaevis*) [210, 211], rainbow trout [212] and Nile tilapia [208]; (iv) genetic linkage map analysis using simple sequence length polymorphisms (SSLPs) in medaka [213, 214]; (v) methylation sensitivity amplified polymorphism (MSAP) in Atlantic salmon [18], grass carp [215], brown trout [17], sea urchin (*Glyptocidaris crenularis*) [216] and sea cucumber (*Apostichopus japonicas*) [217]; (vi) 5‐methylcytosine immunolocation in sea lamprey (*Petromyzon marinus*) [218]; (vii) restriction endonuclease hydrolysis of DNA using methylation enzymes in Zebrafish [219] and (viii) bisulfite sequencing PCR in Pacific Oyster (*Crassostrea gigas*) [220] and grass carp [221]. As shown in **Table 3**, epigenetics studies carried out this far include studies on reproduction, growth and adaptation traits. In the case of Atlantic salmon, which is one of the most widely studied species, epigenetic studies have been carried out at different stages of

186 Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

Embryogenesis and reproduction traits determined by epigenetic analyses in aquatic organ‐ isms include sexual dimorphism, embryo development, control of gonadal aromatase and male meiosis [208, 222, 223]. Mhanni and McGowan [219] examined the methylation patterns of the zebrafish genome during early embryogenesis and showed that parental genetic contri‐ butions to the zygote were differently methylated with the sperm being more hypermethyl‐ ated than the oocyte genome. However, immediately after fertilization there was a significant decrease in the embryonic genome methylation, but increased rapidly as the embryo devel‐ oped to normal levels by the gastrulation stage. These observations are consistent with those seen in mouse [224] suggesting that embryo demethylation/re‐methylation is conserved across the vertebrate taxa as of part embryogenesis. As for reproduction traits, Wan et al. [208] found several differentially methylated regions (DMRs) on tilapia chromosomal DNA linked to sex‐ ual dimorphism in which the males had high methylation levels after prolonged exposure to high temperature conditions. Similarly, Navarro‐Martín et al. [222, 223] showed that European seabass juvenile males had double DNA methylation levels than females in the promoter region of gonadal aromatase, the enzyme that converts androgens to estrogens suggesting that methylation levels on gonadal aromatase were predictive of sex determination. Other fish species for which DNA methylation of aromatase has been linked to sex determination include medaka [225] and Japanese flounder (*Paralichthys olivaceus*) [226]. In crustacean, Gómez et al. [227] analysed the post‐translational histone modifications in the testis of *Daphnia magna* and identified cytological markers linked to meiosis progression and the silencing of unsynapsed chromatin. Put together, these studies show that DNA methylation and histone modification

can induce reproduction and embryogenesis changes in different aquatic organisms.

Epigenetic factors associated with growth and productivity identified in aquatic organisms include early maturation, regulation of muscle growth and disease resistance. Early matu‐ ration in Atlantic salmon has emerged to be an interesting topic because prior to migration,

the production cycle as shown in **Figure 2**.

**6.2. Growth and productivity traits**

**6.1. Embryogenesis and reproduction traits**

parr can reach sexual maturity and successfully fertilize adult females. Up to 60% of total paternity in wild populations has been attributed to these precocious male parr or 'sneakers'. To determine the underlying causes of early sexual maturation in parr, Morán and Pérez‐ Figueroa [18] compared genetic and epigenetic differences of two populations of parr and mature fish originating from two different rivers and found no genetic difference between

increase in mRNA expression of myogenic regulatory factors (*Myog* and *Myf‐5*) and *Pax7* in fast muscle. Overall, these studies show that DNA methylation and histone modification of chromosomal DNA play an important role in regulating muscle growth, disease resistance

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189

Epigenetic factors shown to induce adaptation changes in cultured aquatic organisms include nutrition, migration, salinity and photoperiod exposure. Several nutritional studies have shown that rainbow trout displays persistent hyperglycaemia when fed high carbo‐ hydrate (HighCHO) diets. To underpin the underlying causes, Marandel et al. [230] exam‐ ined the liver of rainbow trout fed HighCHO diets and found global DNA hypomethylation and hypoacetylation of histone H3K9 resembling hyperglycaemic and diabetes conditions in zebrafish and mammals. They also showed that *g6pcb2* ohnologs that encode the glucose‐6‐ phosphatase (G6pc) enzyme involved in gluconeogenesis catalysis were hypomethylated at specific CpG sites indicating that the hepatic epigenetic landscape of rainbow trout can be affected by dietary carbohydrates. As for migration traits, Baerwald et al. [212] identi‐ fied several DMRs between migratory smolts and resident rainbow trout juveniles in which most DMRs encoded proteins associated with migration showing that epigenetic variations were linked to migration traits in anadromous fish. Their findings were in concordance with Morán et al. [17] who found genome‐wide methylation differences between hatchery reared and seawater brown trout. In addition, Morán et al. [17] showed that salt diets used during the seawater phase triggered genome‐wide methylation changes when administered in fresh‐ water reared trout indicating that DNA methylation could play a vital role in enabling anad‐ romous fish acclimatize to seawater after transfer from freshwater. DNA methylation and histone modification have also been associated with adaptation changes induced by adverse environmental conditions as shown in Nile tilapia exposed to industrial pollutions [231], eels to cadmium exposure [232], sea urchin (*G. crenularis*) exposure to perfluoroctane sulfonate (PFOS) [216] and the three‐spine stickleback (*G. aculeatus*) hexabromocyclododecane (HBCD)

mary, these studies demonstrate that DNA methylation and histone modification contribute to nutritional, environmental and photoperiod adaptation in different aquatic organisms and

Although teleost fish are the largest known vertebrate group with more than 27,000 species [8], they account for a small proportion of vertebrate species whose whole genomes have been fully sequenced and characterized. The pufferfish genome is one of the earliest fish genome to be sequenced and characterized by 2002 [234], which raised interests to sequence the genomes of other fish species. The zebrafish (*Danio rerio*) whole genome sequencing proj‐ ect was started by Welcome Trust Sanger Institute in 2001 [235] while the Medaka genome was sequenced in 2007 [236]. Thus, Zebrafish and medaka are not only among the earliest

that these factors could have an influence on improving production in aquaculture.

**7. Whole genome sequencing of aquatic organisms**

) and 5‐aza 2′ deoxycytidine (5AdC) pollutants [233]. In sum‐

and sex maturation in fish.

**6.3. Adaption epigenetic traits**

exposed to 17‐β oestradiol (E<sup>2</sup>

**Figure 2.** The cycle shows the use of different aspects of functional genomics to improve the production of Atlantic salmon at different stages of the production‐cycle. Note that genetics and epigenetics studies are focused on identifying important traits in fish while metagenomics studies are mostly focused on environmental identification of infectious pathogens. Fish from different growth stages are also evaluated for the mucosal microbiota investigations using metagenomics analyses. Nutrigenomics is mostly applied at the outgrower stage. Growth stages are depicted from spawning (A), embryogenesis (B), hatching (C), fingerlings and fry stage (D), Parr stage (E), post‐smolts (F), outgrower stage (G) and broodstock (H). Nutrigenomics are after through the feeding stages while the timing of most vaccinations is the parr (D) stage in order to enable fish develop protective antibodies by the post‐smolt (E) stage and outgrower stage when they are most vulnerable to stress‐related infectious diseases. (X): Depicts the migration of adult fish from seawater into freshwater for spawning. (Z): depicts migration from freshwater to seawater at the parr stage.

parr and mature fish. However, epigenetic analysis showed significant single‐locus varia‐ tions in the gonads followed by the brain and liver between parr and mature fish suggesting that early maturation in Atlantic salmon parr was mediated by epigenetic processes and not genetic differences. As for disease resistance, Shang et al. [221] showed that CpA/CpG methylation of grass carp *Ctenopharyngodon idella* melanoma differentiation associated gene 5 (MDA5) (CiMDA5) was tightly associated with resistance against GCRV. In their findings, they found CpA/CpG methylation sites in the CiMDA5 genome that consisted of putative densely methylated elements (DMEs) that were significantly higher in GCRV susceptible fish than in the resistant fish. In terms of muscle growth, Giannetto et al. [228] found a correlation between DNA (cytosine‐5)‐methyltransferases (DNMTs) increase in fast muscle with prolonged exposure to light indicating that photoperiod influence may be involved in the DNMTs regulation of muscle growth in Atlantic cod. Similarly, Nagasawa et al. [229] found high histone methyltransferases levels of the mixed‐lineage leukaemia (MLL) gene in fast muscle of Atlantic cod subjected to prolonged light exposure, which corresponded with increase in mRNA expression of myogenic regulatory factors (*Myog* and *Myf‐5*) and *Pax7* in fast muscle. Overall, these studies show that DNA methylation and histone modification of chromosomal DNA play an important role in regulating muscle growth, disease resistance and sex maturation in fish.

#### **6.3. Adaption epigenetic traits**

parr and mature fish. However, epigenetic analysis showed significant single‐locus varia‐ tions in the gonads followed by the brain and liver between parr and mature fish suggesting that early maturation in Atlantic salmon parr was mediated by epigenetic processes and not genetic differences. As for disease resistance, Shang et al. [221] showed that CpA/CpG methylation of grass carp *Ctenopharyngodon idella* melanoma differentiation associated gene 5 (MDA5) (CiMDA5) was tightly associated with resistance against GCRV. In their findings, they found CpA/CpG methylation sites in the CiMDA5 genome that consisted of putative densely methylated elements (DMEs) that were significantly higher in GCRV susceptible fish than in the resistant fish. In terms of muscle growth, Giannetto et al. [228] found a correlation between DNA (cytosine‐5)‐methyltransferases (DNMTs) increase in fast muscle with prolonged exposure to light indicating that photoperiod influence may be involved in the DNMTs regulation of muscle growth in Atlantic cod. Similarly, Nagasawa et al. [229] found high histone methyltransferases levels of the mixed‐lineage leukaemia (MLL) gene in fast muscle of Atlantic cod subjected to prolonged light exposure, which corresponded with

into freshwater for spawning. (Z): depicts migration from freshwater to seawater at the parr stage.

188 Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

**Figure 2.** The cycle shows the use of different aspects of functional genomics to improve the production of Atlantic salmon at different stages of the production‐cycle. Note that genetics and epigenetics studies are focused on identifying important traits in fish while metagenomics studies are mostly focused on environmental identification of infectious pathogens. Fish from different growth stages are also evaluated for the mucosal microbiota investigations using metagenomics analyses. Nutrigenomics is mostly applied at the outgrower stage. Growth stages are depicted from spawning (A), embryogenesis (B), hatching (C), fingerlings and fry stage (D), Parr stage (E), post‐smolts (F), outgrower stage (G) and broodstock (H). Nutrigenomics are after through the feeding stages while the timing of most vaccinations is the parr (D) stage in order to enable fish develop protective antibodies by the post‐smolt (E) stage and outgrower stage when they are most vulnerable to stress‐related infectious diseases. (X): Depicts the migration of adult fish from seawater Epigenetic factors shown to induce adaptation changes in cultured aquatic organisms include nutrition, migration, salinity and photoperiod exposure. Several nutritional studies have shown that rainbow trout displays persistent hyperglycaemia when fed high carbo‐ hydrate (HighCHO) diets. To underpin the underlying causes, Marandel et al. [230] exam‐ ined the liver of rainbow trout fed HighCHO diets and found global DNA hypomethylation and hypoacetylation of histone H3K9 resembling hyperglycaemic and diabetes conditions in zebrafish and mammals. They also showed that *g6pcb2* ohnologs that encode the glucose‐6‐ phosphatase (G6pc) enzyme involved in gluconeogenesis catalysis were hypomethylated at specific CpG sites indicating that the hepatic epigenetic landscape of rainbow trout can be affected by dietary carbohydrates. As for migration traits, Baerwald et al. [212] identi‐ fied several DMRs between migratory smolts and resident rainbow trout juveniles in which most DMRs encoded proteins associated with migration showing that epigenetic variations were linked to migration traits in anadromous fish. Their findings were in concordance with Morán et al. [17] who found genome‐wide methylation differences between hatchery reared and seawater brown trout. In addition, Morán et al. [17] showed that salt diets used during the seawater phase triggered genome‐wide methylation changes when administered in fresh‐ water reared trout indicating that DNA methylation could play a vital role in enabling anad‐ romous fish acclimatize to seawater after transfer from freshwater. DNA methylation and histone modification have also been associated with adaptation changes induced by adverse environmental conditions as shown in Nile tilapia exposed to industrial pollutions [231], eels to cadmium exposure [232], sea urchin (*G. crenularis*) exposure to perfluoroctane sulfonate (PFOS) [216] and the three‐spine stickleback (*G. aculeatus*) hexabromocyclododecane (HBCD) exposed to 17‐β oestradiol (E<sup>2</sup> ) and 5‐aza 2′ deoxycytidine (5AdC) pollutants [233]. In sum‐ mary, these studies demonstrate that DNA methylation and histone modification contribute to nutritional, environmental and photoperiod adaptation in different aquatic organisms and that these factors could have an influence on improving production in aquaculture.
