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ORIGINAL RESEARCH
Metabolic differentiation of diploid and triploid European
sea bass juveniles
Cosmas Nathanailides .Dimitris Klaoudatos .Costas Perdikaris .
Spyros Klaoudatos .Markos Kolygas .Fotini Athanassopoulou
Received: 28 November 2018 / Accepted: 6 June 2019
ÓThe Author(s) 2019
Abstract The effects of triploidy were studied on indices of growth and metabolism in juvenile European sea
bass. Ploidy affected flesh quality of Dicentrachus labrax, as protein and water contents were significantly
higher in triploid than in diploid fish and triploid fish exhibited significantly lower lipid content. Compared to
2nfish, triploid fish exhibited 53.4% and 28.6% more DNA and RNA, respectively, 17.2% higher RNA/DNA
ratio and 28.7% more protein/DNA ratio. The activities of the aerobic metabolism enzyme CCO and the
glycolytic LDH of the muscle tissue were significantly higher in the triploid fish. Nevertheless, the ratio of
these two enzymes was lower in the triploids, indicating metabolic difference in the potential for aerobic
metabolism. The increased activity of LDH may reflect a potential shift towards anaerobic metabolism
required under demanding conditions, for example, during burst swimming, confirming the effects of ploidy
on the aerobic swimming capacity of fish. The increased CCO activity of triploids observed in the present
work indicates an effect of ploidy on the capacity for aerobic metabolism of triploid fish.
Keywords Aquaculture Metabolism Triploid Sea bass Polyploidy
Introduction
The nucleic acid content and the concentration of metabolic enzymes are widely used biochemical indices of
aerobic and anaerobic metabolism and fish growth. The total DNA of a tissue can reflect the number of cells,
C. Nathanailides (&)
Faculty of Agriculture, University of Ioannina, 47100 Arta, Greece
e-mail: cosmasfax@yahoo.com
D. Klaoudatos
Hellenic Centre for Marine Research, Institute of Marine Biology, Biotechnology and Aquaculture, Agios Kosmas,
16777 Hellinikon, Athens, Greece
Present Address:
D. Klaoudatos S. Klaoudatos
Department of Ichthyology and Aquatic Environment, University of Thessaly, Fytokou Street, 38 446 Volos, Greece
C. Perdikaris
Department of Fisheries, Regional Unit of Thesprotia, Region of Epirus, 46100 Igoumenitsa, Greece
M. Kolygas F. Athanassopoulou
Laboratory of Ichthyology and Ichthyopathology, Veterinary School, University of Thessaly, 43100 Karditsa, Greece
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Int Aquat Res
https://doi.org/10.1007/s40071-019-0229-6
but is expected to be higher in triploids (Cimino 1974). RNA content may vary according to cell size and
tissue growth (Goolish et al. 1984; Mittakos et al. 2012) and can be used as an index of somatic growth rate in
diploid and triploid fish (Arndt et al. 1994; Suresh and Sheehan 1998). The ratio of RNA/DNA and protein/
DNA can provide some information for the nutritional status and the protein metabolism of growing fish, but
these ratios should be used with caution due to the increased DNA content of 3nfish (Suresh and Sheehan
1998).
The aerobic and anaerobic capacity of cells can be reflected on the mitochondrial enzyme activity and on
enzymes involved in the anaerobic breakdown of glycogen in fish muscle. Cytochrome c oxidase (CCO) is a
mitochondrial enzyme which exhibits higher activity in fast growing fish (Houlihan et al. 1993) and its activity
can be used as an indicator of the aerobic metabolism of fish muscle (Goolish and Adelman 1987), whereas the
activity of the glycolytic enzyme lactate dehydrogenase (LDH) often correlates well with reliance upon
anaerobic glycolysis (Hochachka and Mommsen 1983; Guderley and Gawlicka 1992) and contributes to the
thermal modulation of pyruvate metabolism (Somero 1973).
Triploid fish exhibit some physiological and anatomical differences in the size and shape of their cells. The
cells and nucleus of triploids are larger in size than those of their diploid counterparts (Benfey 1999; Maxime
2008), but the total volume of some organs may remain unchanged, possibly through the reduction in the
number of cells. Ploidy can affect the energy metabolism of growing fish (Gonc¸alves et al. 2018) and their
tolerance to poor water quality (Benfey 1999) indicating reduced tolerance to hypoxic conditions and the
aerobic metabolism of triploid fish. Increased size of cells may result in reduced surface-to-volume ratio
reduced intracellular diffusion of oxygen and exchange of metabolic molecules. In turn, the aerobic and
anaerobic capacity of triploids and diploids may vary, with possible consequences for their metabolic effi-
ciency and growth. For example, triploids may have fewer and larger axial muscle cells and reduced aerobic
swimming capacity (Virtanen et al. 1990). Larger muscle cells may be more suited for anaerobic burst
swimming and their capacity for anaerobic glycolysis of fish can be reflected on increased activity of LDH
(Somero and Childress 1980; Saavedra et al. 2016).
In addition to changes in enzyme activity, other adaptive physiological responses may be employed to
enhance oxygen delivery. For example, at low temperatures, oxygen diffusion is impaired and fish respond to
this by increasing capillarisation (Egginton and Sidell 1989), which is accompanied by increased mito-
chondrial enzyme content (Nathanailides 1996) at least partially compensating for limited oxygen diffusion
and aerobic metabolism. In fact, at cold temperatures, triploid fish can maintain higher metabolic rates than
diploids, but this difference is reversed at warm temperatures, indicating some significant differences between
2nand 3nfish in their capacity for adaptive physiological responses to environmental changes (Atkins and
Benfey 2008).
The specific aim of the present work was to investigate differences in the metabolic specialization of
diploid and triploid D. labrax muscle tissue.
Materials and methods
Production of triploids, somatometrics, and ploidy verification
Triploid (3n)D. labrax were produced by post-fertilization cold shock at 0 °C for 10 min, 5 min after
fertilization (Felip et al. 1997). Larvae of the controls and cold-shocked fish were raised in commercial
hatchery tanks (1.6 m
3
) in duplicates under natural conditions of temperature and photoperiod used for sea
bass fry production.
When the fry reached size above 5 g, 5000 fry of each group were size-graded and evenly splitted in two
circular tanks (3 m
3
water in each) creating two replicates for 2nand 3nfish and reared for a period of
6 weeks. The fish were reared under identical conditions and fed with the same formulated feeds.
After a period of 6 weeks, samples of the diploid and triploid fish were taken and body weight, fillet weight,
and filleting yield, was measured to the nearest 0.1 g in 10 samples from each experimental tank (n= 10 from
each replicate and n= 20 fish in each group).
Daily-specific growth rate was estimated according to the following equation:
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Int Aquat Res
SGR%¼ln BWt2
ðÞln BWt1
ðÞ=days 10:
From each replicate, fish were anaesthetised in with 40 mg l
-1
of clove oil (Perdikaris et al. 2010) and
blood samples (100–200 ll) from 2nto 3nfish (n= 5 fish from each replicate tank, total n= 10 from 3nto
n= 10 from 2nfish) were collected from the caudal vein using heparinized syringes and polyploidy of the
triploid group was confirmed by RBC measurements on blood smears stained with methylene blue-eosine
stain. Nuclear width (nW) and length (nL) were measured using an Olympus microscope fitted with a video
camera and a computer-assisted image analysis system. The nuclear volume (nV) was calculated according to
the ellipsis equation:
Vnucleus ¼4=3pa=2ðÞb=2ðÞ2;
where ais the nL and bis the nW.
Only fish that were confirmed triploids were included in the biochemical analysis (n= 6 for triploid and
n= 10 for diploid).
Biochemical analyses
The DNA and RNA contents of axial muscle tissue were estimated according to the method of Burness et al.
(1999) with some modifications as described by Mittakos et al. (2012). Sample of white epaxial muscle was
obtained and DNA was extracted using the phenol–chloroform extraction procedure. Samples were digested
overnight with 0.1 mg/ml proteinase K. An equal volume of phenol–chloroformisoamyl alcohol (25:24:1) was
added to each digest, and the sample was vortexed and centrifuged for 10 min at 1700g. The upper aqueous
phase was retained and precipitated with 0.5 volumes of ammonium acetate (7.5 M) and 2 volumes of ethanol
(100%) and then centrifuged for 3 min at 1700g. The pellet was washed in 70% ethanol and allowed to dry
and resuspended in 250 ml of distilled water. DNA purity was assessed at 260/280 nm. Absorbance at 260 nm
was used for quantification.
Total RNA was extracted from briefly homogenised tissue in 10 volumes of homogenisation buffer B (4 M
guanidin ethiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, and 15 mM mercaptoethanol) with a Polytron
tissue homogenizer. The extract was frozen at -80 °C until analysis. All subsequent procedures were
conducted at 0°. The samples were suspended in 1 volume of 2 M sodium acetate (pH 4.0), 10 volumes of
buffer-saturated phenol (pH 4.3), and 2 volumes of chloroformisoamyl alcohol solution (49:1) were added to
each homogenate and mixed thoroughly between each step. Subsequently, the homogenates were centrifuged
for 30 min at 3000g. The aqueous phase was retained and mixed with an equal volume of isopropanol and
allowed to precipitate at -20 °C overnight.
For the enzyme assays, muscle tissue samples (n= 6 from each group) were homogenated in an ice-cold
medium of 150 mM KCl in 5 mM MOPS buffer (pH 7.4), using a motor-driven glass homogenizer tube fitted
with a Teflon pestle and kept ice cold. Crude homogenates were used for enzyme assays, because the use of
centrifugation, to clarify the homogenate, can result in a significant loss of enzyme activity binding to
subcellular particles (Vezina and Guderley 1991). Prior to enzyme assays, homogenates were treated with
0.05% (v/v) Triton X-100 and each sample was mixed and allowed to stand in ice for about 15 min for full
activation of cytochrome coxidase (Tyler and Nathanailides 1995).
The enzyme assays were performed in duplicate, at 20 °C. The activity of enzymes is given as lmol/min/
mg protein. Cytochrome coxidase (CCO, EC. 1.9.3.1) activity was assayed by following the decrease in
absorbance of reduced cytochrome cat 550 nm, in a medium containing 0.075 M potassium phosphate buffer
pH 6.8, and 0.0025 mM ferrocytochrome c. Ferrocytochrome cwas prepared the day before the assays, in a
solution of 1% (w/v) cytochrome c, 10 mM potassium phosphate buffer and 0.1 mM EDTA (pH 7.0), by
adding 20 mM potassium ascorbate. Potassium ascorbate was removed by dialysis against an ice-cold medium
of Pi-EDTA buffer as above, using three changes of buffer, two during the day and one overnight. The
dialysed solution of ferrocytochrome cprepared in this way contained at least 95% of the total cytochrome cin
reduced form (Tyler and Nathanailides 1995).
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Int Aquat Res
Activity of the glycolytic enzyme lactate dehydrogenase (LDH, EC 1.9.3.1) was assayed in a medium of
50 mM potassium phosphate buffer (pH 7.0) containing 0.15 mM NADH and 0.60 mM sodium pyruvate
(Mittakos et al. 2012).
Total fat contents of fillets were measured in six fish from the 3nfish and 10 fish from the 2nfish according
to the Bligh and Dyer (1959) methodology. Fillet moisture content was determined according to the AOAC
(1998) method. Protein content was estimated using the Folin–Lowry method (Lowry et al. 1951). Student’s
ttest, X
2
, or one-way ANOVA was applied for statistical analyses.
Results
Triploid fish exhibited increased nuclear size. The mean ratio of nL and nW of 3nand 2nfish was similar to
those reported by Felip et al. (1997) for diploid and triploid sea bass, indicating the successful induction of
triploidy in the experimental fish of the present work. The effect of triploidy was magnified when comparing
the nuclear volumes due to the bi-dimensional nature of this trait (Table 1).
The proximate composition of fish flesh was significantly affected by ploidy. Protein and water content
were significantly higher in triploids than in diploids, but 3nfish had lower lipid content (Tables 2and 6).
Growth rate and filleting yield were not affected by ploidy (Table 3).
The nucleic acid content of 3nfish was significantly higher compared to 2nfish, with 3nfish exhibiting
about 53% and 28% higher DNA and RNA content, respectively. Compared to triploids, diploids exhibited
about 17.2% and 28.7% higher RNA/DNA and protein/DNA ratio, respectively (Table 4).
Table 1 Verification of triploidy from RBC haemocytological parameters
Nuclear length, width and volume of RBC (lm) 3n2nStatistics
nW 5.21 (±0.11) 4.04 (±0.02) ttest, p\0.001
nL 7.02 (±0.04) 4.79 (±0.07) ttest, p\0.001
nW/nL 0.74 (±0.01) 0.84 (±0.01) X
2
,p\0.01
nV (lm
3
) 99.27 (±4.57) 40.96 (±0.79) ttest, p\0.001
Ratio of nW (3n)/nW(2n) = 1.31 (±0.03), ratio of nL(3n)/nL(2n) = 1.47 (±0.02)
Table 2 Proximate composition of 3n(n= 5) and 2n(n= 10) D. labrax, axial muscle tissue
3n2nStatistics % of difference in the 3nfish,
compared to 2nfish
Fat (%) 2.81 (±0.23) 3.46 (±0.47) ttest, p\0.05 -23.13%
Protein (%) 10.59 (±0.76) 9.61 (±0.34) ttest, p\0.05 ?10.19%
Water (%) 68.81 (±0.61) 66.87 (±1.22) ttest, p\0.05 ?2.90%
The last column indicates the % of increased or decreased values observed in the 3nfish
Table 3 Somatometric parameters of triploid (n= ?) and diploid (n=?)D. labrax juveniles, after a period of 6 weeks
3n2nStatistics
Tank 1 Tank 2 Tank 1 Tank 2
Initial BW (g) 5.62 (±0.76) 5.52 (±0.35) 5.65 (±0.45) 5.34 (±0.30) ANOVA, p= 0.30
Final BW (g) 18.68 (±0,97) 18.47 (±1.64) 18.31 (±1.19) 18.75 (1.86) ANOVA, p= 0.32
SGR (%/day) 2.85 2.87 2.79 2.98 X
2
,NS
Filleting yield % 41.50 (1.37) 41.24 (1.17) 41.85 (2.51) 40.77 (1.91) X
2
,NS
BW body weight, FY filleting yield (FY = fillet weight W 9100/body weight), SGR specific growth rate
(SGR = LnBW2 -LnBW1 days
-1
)
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Int Aquat Res
The activities of the aerobic metabolism enzyme CCO and the glycolytic LDH of muscle tissue were
significantly higher in the 3nfish. Nevertheless, the ratio of these two enzymes was lower in the 3nfish,
indicating metabolic difference in the potential for aerobic metabolism in the 3nfish (Table 5).
Discussion
There was no significant difference in the growth rate of the two groups. This is in agreement with previously
published data which indicate that, under culture conditions, diploids and triploids may exhibit similar growth
during the first year of their life (Felip et al. 1999). In fact, the maximum triploidy benefits are expected at
least after the onset of the sexual maturation and particularly if larger fish are cultured (e.g., [1 kg) as in
rainbow trout (Felip et al. 2001,2009).
Ploidy had no effect on filleting yield. In other words, the total skeletal muscle tissue content was not
affected by ploidy. Interestingly, ploidy affected the proximate composition of fish flesh. The main effect of
ploidy was on the increased protein (10% higher) and decreased fat (23% less) of triploid fish. Differences in
the protein content between 2nand 3nfish indicate significant differences in the myofibrillar proteins (myosin,
troponin, and tropomyosin). Increased muscle protein and reduced fat in the 3nfish reflects increased total
myofiber content of 3nfillets, but provide no information on the effect of ploidy on the growth dynamics, size,
and number of individual muscle cells which constitute the axial musculature. The nucleic acid content of
skeletal muscle tissue can provide some information for the effects of ploidy on muscle growth. The ratio of
RNA/DNA and protein/DNA can be used as indicators of skeletal muscle growth dynamics (Cheek et al.
1971). DNA content reflects the number of cells and RNA the size. Increased DNA content exhibited in the
present work by the triploid fish reflected the expected increased DNA content (about 1.5 times more) of
3nfish compared to 2n. RNA/DNA was about 17.2% less in the 3nfish. Apparently, increased DNA content of
3nfish is not resulting in elevated RNA levels (Schmidtke et al. 1976). There is some evidence to suggest that
RNA levels are governed by compensatory regulatory mechanism which can reduce transcript levels with
cellular RNA governed by genetic regulatory mechanisms (Pala et al. 2008, Swartz 2016). Under conditions of
similar RNA degradation rates, differences in the RNA/DNA indicate different protein synthetic capacity of
cells, whereas differences in protein/DNA ratios can reflect differences in cell size/growth (Schmidtke et al.
1976). The raised ratio of protein/DNA in the 2nfish (28.7% higher) can be partially explained by the raised
DNA content of 3nfish (about 53% higher), which was paralleled by a much smaller increase (about 28.6%) in
Table 4 Nucleic acid (RNA, DNA) content of triploid (n= 6 fish) and diploid (n= 10) D. labrax axial muscle tissue
nucleic acid 3n2nStatistics % of difference in the 3nfish,
compared to 2nfish
RNA (lg
-1
mg
-1
tissue) 17.10 (±1.98) 13.30 (±1.19) ttest p\0.001 ?28.6%
DNA (lg
-1
mg
-1
tissue) 9.36 (±0.72) 6.10 (±0.8) ttest p\0.001 ?53.44%
RNA/DNA 1.83 (±0.06) 2.21 (±0.27) X
2
p\0.05 -18.2%
Protein/DNA 1.14 (±0.11) 1.60 (±0.23) X
2
p\0.01 -28.7%
The last column indicates the % of increased or decreased values observed in the 3nfish in reference to the 2nfish
Table 5 Enzyme activities (n= 6 in each group) of cytochrome coxidase (CCO) and lactate dehydrogenase (LDH) from 3nand
2n D. labrax, axial muscle tissue
3n2nStatistics
CCO 4.22 (±0.12) 3.89 (±0.13) ttest p\0.001
LDH 118.57 (±4.44) 99.20 (±11.16) ttest p\0.001
CCO/LDH 0.036 (±0.008) 0.040 (±0.006) X
2
p\0.01
Enzyme activity for CCO is given in ı
`moles of ferrocytochrome coxidized min
-1
mg protein
-1
, and for LDH as reduction of
NADH lmoles min
-1
mg protein
-1
. Differences assessed by Student’s ttest (*p\0001). Data were arc-sin transformed prior to
statistical analysis. Numbers in parentheses indicate standard deviation
123
Int Aquat Res
RNA content. In conclusion, differences in the ratio of RNA/DNA reflect the effects of ploidy on DNA
content as well differences in myofibril umber and size between the two groups. Assuming that RNA levels
are governed by compensatory regulatory mechanism in the 3nfish (Pala et al. 2008, Swartz 2016), the
increased RNA content of triploid sea bass provides indirect evidence of muscle hypertrophy in this group
with larger cells requiring more RNA (Schmidtke et al. 1976).
In addition to ploidy effects on the nucleic acid content, evidence of ploidy effects on skeletal muscle
metabolism was observed in enzyme activities assayed in the present work. Differences in the activity of CCO
and LDH indicate differences in the capacity for aerobic and anaerobic metabolism, respectively (Childress
and Somero 1979). The activities of CCO and LDH of 2nand 3nfish indicate metabolic specialization
differences between the two groups, which may offer some advantages under certain conditions to one of the
two groups. For example, the increased activity of LDH may reflect a potential shift towards anaerobic
metabolism required under demanding conditions, for example, during burst swimming with higher capacity
for anaerobic swimming of triploid fish, but this advantage can be associated with negative consequences on
the sustained routine swimming speeds (Virtanen et al. 1990; Marras et al. 2013). Furthermore, summer high
water temperatures, coupled with reduced dissolved oxygen levels, conditions which are expected to worsen
by the on-going climate change, may create unfavourable conditions for triploids in the summer. This
potential seasonal disadvantage of 3nsea bass may be reversed during winter time. The increased CCO
activity of 3nfish observed in the present work is in agreement with the reported effects of ploidy on the
capacity for cold acclimation of triploid fish (Atkins and Benfey 2008). Increased mitochondrial enzyme
content is considered a frequently observed physiological compensatory response of fish cells, responding to
limited oxygen diffusion and reaction rates at cold temperatures (Nathanailides et al. 1996). In conclusion,
ploidy affected nucleic acid, enzyme content, and proximate composition in D. labrax (Table 6).
The results of the present work indicate significant differences in metabolic specialization of 2nand 3nD.
labrax skeletal muscle. The European sea bass is widely cultivated at floating sea cages and fish are exposed to
challenging winter lows and summer high temperatures. At temperatures below 11 °C, the mitochondrial
enzyme content, feeding, and growth of sea bass is reduced and it became lethargic (Trigari et al. 1992;
Nathanailides et al. 2010). It would be interesting to investigate the potential ploidy effect on the capacity for
thermal acclimation at low winter and high summer seasonal temperatures during the entire production cycle
of Mari-cultured European sea bass.
Table 6 Differences (%) in the biochemical parameters of triploid fish compared to diploids
Biochemical parameter % of increase (?)/ decrease(-) in the 3nfish
Numbers in parenthesis indicate the magnitude of difference
RNA (lg
-1
mg
-1
tissue) ?28.57%
(91.3)
DNA (lg
-1
mg
-1
tissue) ?53.44
(91.5)
RNA/DNA -18.2
(90.8)
Protein/DNA -28.75
(90.7)
Fat (%) -23.13
(90.7)
Protein (%) ?10.19
(90.9)
Water (%) ?2.90
(91.0)
CCO ?7.82
LDH ?16.33
CCO/LDH -11.11
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Int Aquat Res
Compliance with ethical standards
Ethical approval All animal procedures were in strict accordance to the fish welfare recommendations of the Faculty of
Veterinary Medicine, University of Thessaly.
Conflict of interest There is no conflict of interest between authors in the publication of this paper.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided
you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if
changes were made.
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